Citation
Genetic, developmental, and molecular characterization of a high oleic acid peanut (Arachis hypogaea L.)

Material Information

Title:
Genetic, developmental, and molecular characterization of a high oleic acid peanut (Arachis hypogaea L.)
Creator:
Moore, Kim M., 1951-
Copyright Date:
1990
Language:
English

Subjects

Subjects / Keywords:
DNA ( jstor )
Enzymes ( jstor )
Fatty acids ( jstor )
Gels ( jstor )
Genotypes ( jstor )
Nonesterified fatty acids ( jstor )
Peanut oil ( jstor )
Peanuts ( jstor )
Petroleum ether ( jstor )
Species ( jstor )

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
AHJ9787 ( ltuf )
22913688 ( oclc )
0023046164 ( ALEPH )

Downloads

This item has the following downloads:


Full Text












GENETIC, DEVELOPMENTAL, AND MOLECULAR CHARACTERIZATION OF A HIGH OLEIC ACID
PEANUT (Arachis hvpociaea L.)












By

KIM M. MOORE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY




UNIVERSITY OF FLORIDA


1990













ACKNOWLE DGEMENTS

The author expresses his most sincere appreciation to his major advisor, Dr. David Knauft, for his advice, encouragement, and expert editorial assistance in completing this manuscript. Appreciation is also extended to Dr. Al Norden for his initial encouragement to pursue this particular research project. A special thanks is extended to Dr. Sherlie West for his support, advice, and understanding that led to the completion of this project.

Appreciation is also extended to the other members of his advisory committee, Dr. Ken Buhr, Dr. Paul Lyrene, and Dr. Ken Quesenberry, for their thoughtful suggestions in this dissertation.

A very special thanks is extended to Dr. Rex Smith who, although not a member of the advisory committee, provided excellent support and invaluable input. Thanks is also extended to Dr. Kamal Chowdhury for his assistance on the analysis of RFLPs.

Thanks is also extended for the technical assistance of Karen Bedigian, Harry Wood, Jeff Seib, and Natalie McGill.

The author also wishes to acknowledge both the Florida Foundation Seed Association and Proctor and Gamble, Incorporated, for their funding of portions of this research.















TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS.11

LIST OFTABLES.V LIST OF FIGURES.viii ABSTRACT.xi


CHAPTERS


I INTRODUCTION. . . . . . .

II THE INHERITANCE OF HIGH OLEIC ACID
IN PEANUT. . . . .

Introduction. . . . .
Materials and Methods. . . .
Results and Discussion .
Saponification Versus Direct
Esterification .
Cross with F78114. . .
Cross with F519-9. . .
Cross with PI 262090. . .


suimmary.

VARIATION IN FATTY ACID COMPOSITION IN DEVELOPING SEED OF Arachis hvpocraea L. Introduction . Materials and Methods. . . . Results and Discussion .


RESTRICTION FRAGMENT LENGTH POLYMORPHISM IN THE GENUS Arachis. . . .

Introduction . Materials and Methods. . . .
DNA Extraction .
Extraction 1 (CTAB). . .

iii


. . . 17


III










Extraction 1 (OTAB).73 Extraction 2 (potassium acetate). 75 Southern Blotting.77 Probe Preparation.78 Radio-labeling Probes .80
Prehybridization and Hybridization of
Blots.81 Results and Discussion.82

v. . . . .109

REFERENCES.112

BIOGRAPHICAL SKETCH.120














LIST OF TABLES


Table Page

1-1 Oleic and linoleic acid content of
vegetable oils .

2-1 Oleic and linoleic acid content of the four
peanut breeding lines used in crossing. . . 21

2-2 A comparison of two methods of fatty acid
analysis, saponified versus nonsaponified.
Palmitic, stearic, oleic, linoleic,
arachidic, and benhenic acid contents are
shown from four different oil sources.
Also shown are values of tcatc . . 25

2-3 A comparison of total areas integrated on
chromatographs of saponified oils versus
nonsaponified oils. Four different oil sources
are included. Also included is the percent
of the total fatty acids measured when
saponification is performed that are
measured when saponification is not
performed . . .27

2-4 F, progeny from the cross between high
(F435) and normal (F519-9), (F78114), and (PI
262090) oleic acid phenotypes . . .28

2-5 Segregation data for F2 progeny from the
cross between high (F435) and normal
(F78114) oleic acid lines . . .31

2-6 BC1 oleic acid phenotypic segregation ratios
for two peanut lines (F78114 and F519-9) crossed to a high-oleic-acid peanut line
(F435) . . . 32

2-7 Phenotypic segregation of F3 families from
crosses between high (F435) and normal
(78114 or F519-9) oleic acid peanut lines . . 33









Table Pacre

2-8 Phenotypic segregation of oleic acid content
for F progeny from the cross between high
(F4351 and normal (F519-9) oleic acid peanut
lines .36

2-9 Segregation data for F2 progeny from the
cross between high (F435) and normal
(PI 262090) oleic acid lines . .38

3-1 Oleic and linoleic acid content of three
peanut breeding lines. .44

3-2 R 2 values of individual plants and for a
composite of all plants for each genotype
for maturity versus dry matter .47

3-3 R 2 values for regressions of the percent
oleic acid with maturity rating as the independent variable compared with dry
matter as the independent variable for three
genotypes, F519-9, F435, and F78114. .51

3-4 The percent oleic acid and standard errors
for high, moderate, and low oleic acid peanut
genotypes sampled at various stages of dry
matter deposition .60

3-5 The percent palmitic acid and standard errors
for high, moderate, and low oleic acid peanut
genotypes sampled at various stages of dry
matter deposition .63

3-6 The percent linoleic acid and standard errors
for high, moderate, and low oleic acid peanut
genotypes sampled at various stages of dry
matter deposition .64

4-1 Absorbances of DNA extracts from four
A. hvpocraea lines and four perennial Arachis
species at two wavelengths. Extraction was
method 1 (CTAB) using young mature leaf
tissue. .83

4-2 Absorbances of DNA extracts from four
A. hvpocraea lines and four perennial Arachis
species at two wavelength. Extraction was
method 2 (potassium acetate) using young
mature leaf tissue. .84











4-3 Absorbances of DNA extracts from four
A. hypogaea lines and four perennial Arachis
species at two wavelengths. Extraction was
method 1 (CTAB) using immature leaf tissue. .88

4-4 Genomic DNA clones, library cell locations,
and approximate sizes of inserts isolated for
production of radio-labeled probes . .92

4-5 Gene clones used as radio-labeled probes . . . 93

4-6 Pair-wise indices of genetic similarity of
four A. hvpogaea lines and four Arachis
species. The similarity index was calculated by dividing the total number of DNA fragments
common between two genotypes by the total
number of unique fragment sizes represented
by the paired genotypes .107


vii


Table


Page














LIST OF FIGURES


Figure PAge

1-1 End use of peanuts as percent of the
total 1984 U. S. production . . . . . . . . . 4

1-2 The fatty acid percentages of the total
fatty acid composition of peanut oil . . . . . 6

1-3 The currently proposed biochemical pathway
for desaturation of oleic acid to linoleic
acid in higher plants . . . . . . . . . . . . 14

2-1 Frequency distribution of number of F2
offspring in phenotypic classes based on oleic
acid content. Data has been pooled for all
families from the cross of F78114 and F435. 30

2-2 Frequency distribution of number of F2 offspring in phenotypic classes based on
oleic acid content. Data has been pooled for all families from the cross of F519-9
and F435 . . . . . . . . . . . . . . . . . . . 35

2-3 Frequency distribution of number of F2 offspring in phenotypic classes based on
oleic acid content. Data has been pooled
for all families from the cross of PI 262090
and F435 . . . . . . . . . . . . . . . . . . . 39

3-1 Regression plot of the percent dry matter versus maturity classification of peanut seed
sampled from all four plants of line F519-9 48

3-2 Regression plot of the percent dry matter versus maturity classification of peanut seed
sampled from all four plants of line F435 52

3-3 Regression plot of the percent dry matter versus maturity classification of peanut seed
sampled from all four plants of line F78114 . 53


viii








Figure


Pacfe


3-4 Regression plot of the percent oleic acid
versus maturity classification of peanut seed
sampled from all four plants of line F519-9 54

3-5 Regression plot of the percent oleic acid
versus maturity classification of peanut seed
sampled from all four plants of line F435 55

3-6 Regression plot of the percent oleic acid
versus maturity classification of peanut seed
sampled from all four plants of line F78114 . 56

3-7 Regression plot of the percent oleic acid
versus the percent dry matter of peanut seed
sampled from all four plants of line F519-9 . 57

3-8 Regression plot of the percent oleic acid
versus the percent dry matter of peanut seed
sampled from all four plants of line F435 . . 58

3-9 Regression plot of the percent oleic acid
versus the percent dry matter of peanut seed
sampled from all four plants of line F78114 . 59

3-10 Regression plot of the percent palmitic acid
versus the percent dry matter of peanut seed
sampled from all four plants of line F519-9 . 62

3-11 Regression plot of percent linoleic acid
versus percent dry matter of peanut seed
sampled from all four plants of line F435 . . 65

3-12 Regression plot of percent linoleic acid
versus percent dry matter of peanut seed
sampled from all four plants of line F519-9 . 66

3-13 Regression plot of percent linoleic acid
versus percent dry matter of peanut seed
sampled from all four plants of line F78114 . 67

4-1 DNA extracts from eight peanut genotypes
using extraction method 1 on mature leaf
tissue . . . . . . . . . . . . . . . . . . . . 86

4-2 DNA extracts from eight peanut genotypes
using extraction method 2 on mature leaf
tissue . . . . . . . . . . . . . . . . . . . . 87








Figure

4-3



4-4

4-5


definition.


4-6


Autoradiograph of probe


genotypes with 4-7 Autoradiograph
genotypes with 4-8 Autoradiograph
genotypes with 4-9 Autoradiograph
genotypes with 4-10 Autoradiograph
genotypes with 4-11 Autoradiograph
genotypes with 4-12 Autoradiograph
genotypes with 4-13 Autoradiograph
genotypes with 4-14 Autoradiograph
genotypes with 4-15 Autoradiograph
genotypes with


fragment of probe fragment of probe fragment of probe fragment of probe fragment of probe fragment of probe fragment of probe fragment of probe fragment of probe fragment


HPI16 on peanut sizes in kilobases.

HPI6 on peanut size in kilobases

atP6 on peanut sizes in kilobases.

coxI on peanut sizes in kilobases.

HP167 on peanut sizes in kilobases.

HP172 on peanut sizes in kilobases.

HP158 on peanut sizes in kilobases.

HP152 on peanut sizes in kilobases.

rrn5-rrnl8 on peanut sizes in kilobases. .

HP154 on peanut sizes in kilobases. .


DNA extracts from eight peanut genotypes using extraction method 1 on immature leaf tissue . .

Clones separated from pUC . .

Autoradiograph of probe atpa on peanut genotypes illustrating an unacceptable autoradiograph based on poor fragment


Page


. . . . . . . . . . . . . . . 94


95


96 98


99 100 101


102 103 104


105














Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


GENETIC, DEVELOPMENTAL, AND MOLECULAR CHARACTERIZATION OF A HIGH OLEIC ACID
PEANUT (Arachis hvpociaea L.)

By

Kim M. Moore

May 1990

Chairman: David A. Knauft
Major Department: Agronomy

Shelf life and nutritional value are important factors affecting the quality of peanuts and peanut products. These factors are directly related to the chemical composition of the oil. Peanut oil is composed of 95% triacylglycerides which vary in their composition by the relative proportions of component fatty acids. Typically, 90% of the total fatty acid composition is made up of three fatty acids, palmitic, oleic, and linoleic acids. Oleic acid has been shown to be more desirable for both stability and nutrition than palmitic or linoleic acids. In 1987, a peanut line high in oleic acid was identified. This peanut line was crossed with four other peanut lines and cultivars to determine the mode of inheritance of the oil character. Segregating F2

populations were analyzed along with F1, F3, and backcross








generations. Among the f our crosses, three F2 crosses produced segregating ratios of 3 normal to 1 high oleic acid phenotype. One of the crosses produced a segregating ratio of 15 normal to 1 high oleic acid phenotype. Analysis of

the F3 and backcross data further supported a hypothesis of inheritance by two genes, each with a dominant and recessive allele.

Further investigations were conducted to determine if

variation in gene action occurred during development. Seeds of three peanut genotypes that were low (F78114), moderate (F519-9), and high (F435) in oleic acid were analyzed for fatty acids at different developmental stages. Oleic acid content increased in the early stages of development of all three genotypes. Linoleic acid content was relatively unchanged during development of F519-9 and F78114 but declined during development of the high oleic acid line. Early stages of development may be the best time to isolate mRNA to produce cDNA for screening to characterize the genes.

The high oleic acid genotype along with F519-9, F78114, PI 262090, and four species of perennial peanut were compared by variation in DNA fragment length polymorphism. The four A. hypogaea lines were uniform and showed few polymorphic fragments. Polymorphisms were more readily detected among the four perennial peanut species.


xii














CHAPTER I

INTRODUCTION

The peanut, Arachis hypogaea L., is a native legume of South America. It is a member of the family Lequminosae, tribe Aeschynomeneae, subtribe Stylosanthyenae. The genus, Arachis, is divided into seven sections based on morphology and cross-compatibility. The sectional arrangement of the genus follows the ecological and geographic features of its continent of origin, South America. The currently accepted center of origin for the genus is the Mato Grosso area of Brazil, located just north and east of Paraguay (Wynne and Halward, 1989).

The cultivated peanut, A. hypogaea, is a member of the section Arachis. This section shows considerable diversity in the area west of the Paraguay River through northern Bolivia to the Andes mountains. Arachis hvpoqaea can be divided into two subspecies, based on morphological differences. These subspecies are hvpogaea and fastigiata. The subspecies hVpogaea is subdivided into variety hypogaea, also known as the Virginia type, and variety hirsuta, also known as the Peruvian runner type. The subspecies fastigiata is subdivided into variety fastigiata, commonly










known as Valencia type, and variety vulgaris, also known as Spanish type (Stalker and Moss, 1987).

Peanut was cultivated before the European exploration of the Americas but has been limited in range to warmer regions of the western hemisphere (Hammons, 1982). The earliest dated peanuts found in association with human activities were estimated at 3800 years old by radiocarbon dating (Hammons, 1982). These samples originated from an archeological site near Las Haldas, Peru. By the time of the European exploration and colonization of the Americas, peanut was grown throughout the warmer regions of the western hemisphere, including the islands of the Caribbean. It was probably on the island of Hispaniola where Europeans first encountered peanut culture. Peanut was then spread by European explorers and travelers to Asia, the Pacific Islands, Europe, southeastern U.S., and both coasts of Africa (Hammons, 1982).

Currently, there are twenty-four countries that each produce more than three million kg of peanuts annually. China leads the world in total production with 6,400,000 metric tons produced in 1987. India is second with 4,350,000 metric tons and the United States third with 1,620,000 metric tons. World production in 1987 was over nineteen million metric tons (Commodity Research Bureau, 1988). These production numbers are elevated considerably, relative to twenty years ago, due to the increasing value of










peanut as a source of high-quality edible oil (McGill, 1973). Unlike most countries, where peanut is grown for oil, only 24% of the 1984 U. S. production of shelled peanuts were crushed for oil, as shown in Figure 1-1 (Commodity Research Bureau, 1984). The typical oil content of peanut is approximately 52% (Cobb and Johnson, 1973). With this high level of oil, any factors affecting the quality of the oil will in turn affect the total product quality. The quality of the oil is of particular interest not only to the oil producer, but also to the processors and roasters of peanuts. A number of methods have been developed to measure oil quality quantitatively. Peroxide value, iodine number, and fatty acid content have all been used to quantify oil quality, and all are directly related to the chemical composition of the oil (Cobb and Johnson, 1973). The two principal quality characteristics most affected by the chemical composition are the storage stability of the oil and the nutritional value or liability. Both of these factors are directly related to the degree of unsaturation of the oil and more specifically related to the fatty acid composition.

Peanut oil, like other vegetable oils, is composed of

monoacylglycerides, diacylglycerides, triacylglycerides, and free fatty acids. An acylglyceride is a glycerol molecule, C 3H 5(OH)3, to which organic acids (fatty acids) are bound, substituting for one, two, or all three of the hydroxyl




























P 24.


14.9 % Candy

IN Salted

Butte
0.97 XRoasted

Crushed for Oil

EOther 0ox


7.0%
Figure 1-1. End use of peanuts as percent of the total 1984 U. S. production.


15.9 %


37.0 XC








5

groups, e.g., mono-, di-, and triacylglycerides. In peanut oil, triaclyglcerides account for more than 95% of the total lipids (Sanders, 1980a). The remainder is made up of approximately 1.7% diacylglycerides, 0.3% monoacylglycerides, 0.7% free fatty acids, and the remaining

2.3% is made up of polar lipids, sterols, and hydrocarbon sterol esters. The fatty acid composition of the triacylglycerides is variable. Typically, however, 90% of the fatty acid composition of peanut oil is made up of three fatty acids: palmitic, oleic, and linoleic (Cobb and Johnson, 1973). Palmitic acid is a 16-carbon, completely saturated, fatty acid. Oleic acid and linoleic are both 18carbon chains with one and two double bonds, respectively. A generalized breakdown of peanut oil fatty acid composition is given in Figure 1-2.

Of the oil quality factors dependent on fatty acid

composition, storage-stability is most directly related to the degree of fatty acid saturation. The most common cause of oil degradation in storage is oxidation, and the result is termed oxidative rancidity. Although some fatty acids are more prone to degradation than others, regardless of the degree of saturation, the loss of wholesomeness is most commonly caused by oxidative rancidity. This type of rancidity is directly related to the degree of unsaturation of an oil. Oxidation of the double bond in the triacylglycerides and free fatty acids results in the













Palmitic Stearic 52.0 X 11.0 1

Oleic

1.1 D Linoleic
S2.5 XCI/

1.41X Arochidic

SBehonic

* Lignoceric 28.0 X Figure 1-2. The fatty acid percentages of the total fatty acid composition of peanut oil.








formation of peroxide groups at or near the double bond. 7 The peroxides then decompose to form acids, alcohols, aldehydes, ketones, and other hydrocarbons that result in the odors commonly associated with rancidity (St. Angelo and Ory, 1973). Therefore, the advantage of more saturation is more stability.

The second quality factor, nutritional composition, is also important in the establishment of edible oil quality. Fat chemical composition and level of dietary fat intake have been found to affect level and composition of serum cholesterol (Gustafsson et al., 1985; Bronsgeest-Schoute et al., 1979; Kuusi et al., 1985; Schonfeld et al., 1982). Cholesterol and cholesterol fatty esters are components of the atherosclerotic plaques that restrict arterial blood flow and contribute to heart disease. By altering dietary fat intake both in level and/or composition, serum cholesterol levels can be reduced. A compositional change in dietary fat that has recently been shown to reduce serum cholesterol is a high monounsaturated diet. In a study conducted in 1986, it was concluded that diets high in monounsaturates, i.e., oleic acid, were as effective in reducing serum cholesterol levels as low-fat diets (Grundy, 1986).

Since the degree of unsaturation is critical to both nutritional quality and storage-stability, relative proportions of fatty acids in an oil are important in determining total oil quality. oils higher in










monounsaturated fatty acids would be desirable for both nutrition quality and storage stability. In Table 1-1, a comparison of seven different vegetable oils is shown with their respective oleic acid (monounsaturate) and linoleic acid (polyunsaturate) content (USDA, 1975). As shown in the table, peanut is second only to olive oil in oleic acid content.

Variation of fatty acid composition in peanut oil has

been shown to be influenced by several factors. It may vary according to variety, location, year-to-year variation, environmental variation, and physiological maturity (Bovi, 1982; Jamieson et al., 1921; Knauft et al., 1986; Worthington et al., 1972; Hartzook, 1969; Norden et al., 1987; Rachmeler, 1988; Worthington and Hammons, 1971; Worthington, 1969; Young et al., 1974).

In a comparison of peanut genotypes grouped as Virginia Runner, Virginia Bunch, and Spanish Bunch with respect to growth habit, it was found that oleic and linoleic acid contents varied significantly among groups (Raheja et al., 1987). Total oil content of the three genotypes was very uniform, ranging only from 48.9% to 49.8%. Oleic acid content was much more varied, ranging from 37.6% as a low in the Virginia Bunch to 54.7% as a high in the Virginia Runner type. Linoleic acid was also varied both within and among















Table 1-1. Oleic and linoleic acid content of vegetable oils.


Oil Source %Oleic Acid %Linoleic Acid

Corn 28 53
Cottonseed 21 50
olive 76 7
Peanut 47 29
Safflower 15 72
Sesame 38 42
Soybean 20 52


(USDA, 1975)








10

peanut types, ranging as low as 29.7 in the Virginia Runner to 46.7% in the Virginia Bunch.

In a survey of 110 genotypes, some assayed over more than one growing season, wide variation in fatty acid composition was observed (Worthington and Hammons, 1971). The genotypes consisted of Virginia, Spanish, and runner market types and plant introductions. Palmitic, oleic, and linoleic were the fatty acids with the most variability. Palmitic acid ranged from 6.7% to 13.7%, and the average of all 110 genotypes was 10%. Oleic acid ranged from 35.8% to 71.4%, and with an average of 45.0%. Linoleic acid ranged from 11.1% to 40.1% with an average of 29.9%. The oleic and linoleic acid contents had a strong negative correlation and the genotype with the highest oleic acid level also had the lowest linoleic content. In the Worthington and Hammons study and the Raheja et al. study, the Spanish varieties consistently showed lower oleic acid and higher linoleic content than the Virginia and runner varieties. Higher peroxide values, indicating less stability, have been previously reported for Spanish varieties compared with runner and Virginia varieties (Picket and Holley, 1951) (Fore et al., 1953).

In another study, 82 peanut genotypes were tested for their fatty acid composition (Worthington et al., 1972). The genotypes represented a wide variation in genetic background. The three major fatty acids varied as follows:










palmitic 7.4-12.9; oleic 35.7-68.5; and linoleic 14.1-40.3. Another study of 40 peanut cultivars also included 12 plant introductions representing all four A. hypogaea botanical types and two other Arachis species, A. monticola and A. nambyquarae (Treadwell et al., 1983). Palmitic acid ranged from 7.5-11.8%; oleic acid ranged from 39.3-56.6%; and linoleic acid ranged from 26.0-38.9%. These values are well within the range recorded throughout the literature.

The highest oleic and lowest linoleic values recorded for peanut are 80% oleic and 2% linoleic (Norden et al., 1987). The variant fatty acid levels were found in an experimental breeding line, F435, of Spanish botanical type. The highest oleic acid level previously published was 71.4% and the lowest linoleic acid level was 11.1%. Earlier evaluations of F435 were found to have typical Spanish botanical type values for oleic and linoleic, 50% oleic and 26% linoleic. The oleic and linoleic acid contents of F435 deviated sufficiently from previously established ranges within Arachis to justify a genetic study of the character.

Variant fatty acid phenotypes have been identified in other oil seed crops and some have been found to be controlled by major genes. In a mutant soybean [Glycine max

(L.) Merr.] line, high linoleic acid was found to be controlled by two alleles at one locus (Wilcox and Cavins, 1985). In flax (Linum usitatissimum L.) two mutant lines were found to have increased linoleic and reduced linolenic










acid in the seed oil. Analysis of progeny from crosses between the mutants and normal parental lines showed that the variation in linoleic and linolenic was controlled by two unlinked genes with additive gene action (Green, 1986). In a high oleic acid sunflower (Helianthus annuus L.) line the phenotype was found to be controlled by two genes, one with partial dominance (Miller et al., 1987). For the high oleic acid character to be expressed one gene must have at least one dominant allele present.

When inheritance is simple it is sometimes possible to trace the character to a single protein or enzyme in a biochemical pathway. In the production of plant storage fats and oils, many enzymes are involved. Fat synthesis begins in the plastids, using translocated sugars as the carbon source. The synthesis of fats from carbohydrates proceeds by the esterification of fatty acids onto a glycerol backbone. The glycerol is derived from glycolysis and is formed by the reduction of dihydroxyacetone phosphate (Salisbury and Ross, 1985). The fatty acids are synthesized from molecules of acetyl CoA adding two carbons at a time to the chain. More than 99% of the fatty acids in peanut oil are made of even-numbered carbon chains. These evennumbered fatty acid chains are formed through the fatty acid synthase system. Fatty acid synthase catalyzes a series of reactions where one molecule of acetyl-CoA and seven molecules of a three-carbon compound, malonic acid in the










form of its CoA thioester malonyl-CoA, are linked to form palmitic acid (16:0). The reaction evolves seven molecules of carbon dioxide and requires the reducing power of 14 NADPH (nicotinamide adenine dinucleotide phosphate) (Lehninger, 1982). Palmitic acid is formed in plastids, where it is also lengthened by two carbons forming stearic acid (18:0). Stearic acid, still in the plastid, is then desaturated to oleic acid (18:1) with the enzyme stearoylACP desaturase. This system is well understood and established up to the formation of oleic acid. The mode of introduction of the second and third double bonds are not so clearly defined. The substrates are not firmly established and the enzyme system appears to be membrane bound (Stumpf, 1989). The most probable mechanism is outlined in Figure 13. One carrier protein and at least three important enzymes that have not been characterized may affect the protein structure of the enzymes or carrier protein, which will also affect the activity of these species. Changes in the activities of the enzymes will affect the formation of the fatty acids and ultimately alter the final ratios of the various fatty acids. Experimental data have shown that the system is also affected by the type of tissue examined, the temperature at which the tissue is grown, the light regime to which the tissue was exposed, and the age of the tissue (Stumpf, 1989).









NADH



NAD + NADH:Carrier Reductase


Carrier (oxid) 2-Linoleoyl PC/PE



Carrier (red) o 2-Oleoyl PC/PE (oxid) A 12
2-Lyso PC/PE


CoA

Oleoyl transferase Oleoyl CoA


NAD reduced nicotinamide adenine dinucleotide
NADH = oxidized nicotinamide adenine dinucleotide
CoA = coenzyme A
red = reduced oxid = oxidized
PC/PE = phosphotidylcholine/phosphotidylethanolamine
Figure 1-3. The currently proposed biochemical pathway for desaturation of oleic acid to linoleic acid in higher plants.










Variation in fatty acid composition has been shown to

be related to the maturity of the peanut seeds tested, which corroborates in vitro experimental data. In 1969, Worthington reported varying fatty acid contents of peanuts of four different maturity classes. The maturity classes were measured in weeks from gynophore penetration of the soil. Oleic acid content ranged from 41.2% in the earliest maturity class to 52.1% in the most mature class. A reduction in linoleic acid content also occurred over maturity, ranging from 32.3% in the most immature to 28.9% in the most mature. In another study, a comparison was made between the fatty acid composition of the triacylglycerides versus the free fatty acids over varying maturity classes of 'Florunner' peanut oil (Sanders, 1980b). Some fatty acids, palmitic and oleic, varied over maturity in both triacylglyceride and free fatty acid forms. Linoleic acid was relatively stable over maturity in both triacylglyceride and free fatty acid forms.

If the high oleic acid character in peanut is

controlled by major genes, the mutant line may be helpful in determining the pathways of fatty acid synthesis. Since it is known that the rate of synthesis of both oleic and linoleic acid varies during seed maturation in peanut, a comparison of rates between a normal peanut line and the mutant (F435 line) may be valuable in determining the










biochemical pathway of oleic acid desaturation to linoleic acid.

To date, simply inherited characters of economic value have not been identified in peanut. If the high oleic acid character is controlled by only a few major genes, it could be an important subject for gene isolation and transformation into other peanut cultivars and possibly other oil seed species as well. Preliminary work necessary for molecular transformation would require the development or adaptation of protocol compatible with genus Arachis and ultimately A. hvpogaea.

The objectives of this dissertation research were

threefold. The initial objective was to determine the mode of inheritance of the high oleic acid character. The second objective was to determine if the rate of formation of oleic acid or linoleic acid varied in the high oleic acid line in relation to a particular stage of seed development. The third objective was to develop or adapt molecular genetic protocol that could lay the ground work for the gene isolation and molecular transformation of the high oleic acid character.














CHAPTER II

THE INHERITANCE OF HIGH OLEIC ACID IN PEANUT Introduction

Fatty acid composition is an important determinant of quality in edible oils. oil stability and nutritional quality are both dependent on the relative proportions of the saturated and unsaturated fatty acids that constitute the oil. Oxidative rancidity increases with increased levels of polyunsaturated fatty acids. Oxidation of the carbon double bonds of fatty acids produces acids, aldehydes, ketones, and other hydrocarbons that cause odors and flavors commonly associated with rancidity (St. Angelo and Ory, 1973). Therefore, the total amount of unsaturation is inversely proportional to the keeping quality of the oil. Fats with more saturation are less prone to oxidation during storage and processing than polyunsaturates. From a nutritional standpoint, polyunsaturates have been desirable for their role in lowering plasma cholesterol levels. However, a recent study showed that human diets containing oils high in monounsaturates were as effective in lowering serum cholesterol levels as were low-fat diets (Grundy, 1986). It was also demonstrated that beef cattle and swine fed diets high in monounsaturates produced meats with










significantly higher levels of unsaturation (St. John et al., 1987).

Peanut oil varies in both quantity and relative

proportion of fatty acids. Although there are eight fatty acids in peanut found in quantities greater than 1.0%, palmitic (16:0), oleic (18:1), and linoleic (18:2) constitute approximately 90% of the total fatty acid composition (Cobb and Johnson, 1973). Generally, palmitic acid constitutes nearly 10%, and the oleic and linoleic acid proportions together make up 80% of the fatty acid composition in peanut oil (Ahmed and Young, 1982). The variation in composition has been related to maturity, temperature, planting date, location, market grade, and peanut genotype (Bovi, 1982; Harris and James, 1969; Holaday and Pearson, 1974; Knauft et al., 1986; Mozingo et al., 1988; Norden et al., 1987; Young et al., 1972; Young et al., 1974). In 1987, Norden et al. reported a peanut line, F435, with 80% oleic acid and 2% linoleic acid. This line extended the known variability of these two fatty acids, which had been reported to range from 36% to 71% for oleic acid and from 11% to 43% for linoleic acid (Bovi, 1982; Norden et al., 1987; Treadwell et al., 1983).

The F435 line is a Spanish botanical type (A. hypoQaea ssp. fastiqiata var. vulqaris). This botanical type accounts for only 10.8% of the total U.S. peanut acreage. The Virginia botanical type (A. hypogaea ssp. hvpogaea var.










hypociaea) is the predominant botanical type grown in the U.S., with over 80% of the total U.S. acreage (Holbrook and Kvien, 1989). Spanish lines are generally lower yielding than Virginia types and are not as well adapted to the principal peanut production regions of the U.S., i.e., the warm, humid Southeast. Besides being a Spanish botanical type, F435 is a breeding line with a pod-splitting characteristic that has made it unsuitable for release as a cultivar. To improve the oil quality of the more widely cultivated botanical types, it would be necessary to transfer this high oleic acid characteristic from the Spanish F435 breeding line to Virginia and runner peanut cultivars and adapted lines.

An understanding of the mode of inheritance will allow most efficient transfer of this trait to adapted breeding lines and runner and Virginia market type cultivars. The following study was conducted to elucidate the genetic basis of the high oleic/low linoleic acid character.

Materials and Methods

In the spring of 1986, seed from the high oleic acid line, F435, were planted in a greenhouse along with seed from F78114, a Virginia market type, and F519-9, a component line of the runner market type cultivar, 'Sunrunner' (Norden et al., 1985). The F78114 had an oleic acid content of 45.4%, which is lower than the midpoint of the range of peanut (Table 2-1). F519-9 had an oleic acid content of










53.6%, which is near the midpoint of the range of oleic in peanut (Table 2-1). Crosses were made between F435 and F78114 and between F435 and F519-9. In both cases

reciprocal crosses were made. Seed from the F, generation were planted in the field at the University of Florida Agronomy Farm, near Gainesville, in July 1986. These F,

plants produced F2 seed that were harvested in December of the same year. These F2 seed were analyzed for fatty acid composition. Fatty acid analysis was also performed on all parents used in crossing.

Fatty acid determinations were made by first extracting oil from the seeds, esterifying the oil, and using gas chromatography to determine the relative proportions of the various fatty acids. Saponification prior to esterification was omitted since the relative proportions of free fatty acids were found to be the same as relative proportions of total fatty acids. This was determined by comparing fatty acid analysis with and without saponification on four different peanut oil sources, F435, F519-9, F78114, and commercial cold press peanut oil. Saponification was performed for comparison by adding 2 ml of 10% KOH in methanol/water (4:1 v/v) to the reaction vials containing the oil extracts and heating the vials to 80*C for 90

minutes. The vials were then cooled and 1 ml of 1. 8 M H2S04 added to each. One ml of petroleum ether was added to each vial and vigorously shaken. The vials were allowed to stand














Table 2-1. Oleic and linoleic acid content of the four peanut breeding lines used in crossing.


Fatty Acid

% Oleic % Linoleic

Genotype Mean Rancre Std. Err. Mean Rancre Std. Err.

F78114 45.4 43.3-46.4 0.42 34.3 32.6-36.8 0.55

F519-9 55.6 50.9-61.5 1.08 25.9 21.1-30.4 0.99

PI 262090 59.8 52.8-63.9 1.56 22.5 19.3-28.3 0.93

F435 80.1 72.6-82.3 1.01 2.2 1.2-3.6 0.22


Based on a 10-seed sample from each line.








22

for a phase separation, but in some cases centrifugation was necessary. The upper petroleum ether phase was pipetted off, and this procedure was repeated.

Prior to saponification and/or esterification, the oil was extracted by cutting approximately 0.1 g slices from the end of the cotyledons of an individual seed and soaking these slices in 2 ml of petroleum ether in a 13X100 mm culture tube overnight. Embryo ends of the seeds were saved for later planting to produce the subsequent generations. The petroleum ether was then pipetted into a 5 ml reaction vial. For saponification, the petroleum ether was removed by evaporation, leaving the oil extracted from the seed sample. The oil samples in the reaction vials were then ready for saponification. When saponification was not performed, oil was esterified immediately following extraction. Boron-trifluoride in methanol (BF 3) was added directly to the vials containing the extracted oil in petroleum ether (Metcalfe and Schmitz, 1961). This mixture was shaken briefly and then heated to 1000C for 3 minutes. After heating, the reaction vials were cooled and 1 ml of deionized HO2 added to each of the vials. The vials were shaken vigorously and then allowed to stand for clearing and a phase separation. The upper petroleum ether phase containing the fatty acid methylesters was sampled and a 1 gl aliquot injected into a gas chromatograph (GC). The first 1000 samples were run on a Varion 3700 with manual










injection and strip chart recorder. All subsequent determinations were performed on a Hewlett-Packard 3690a with automatic sampler, integrator, and flame ionization detector. Both the injector and detector temperatures were 2500C. The oven temperature was programed for an initial temperature setting of 19O*C for 3 minutes, then increasing at the rate of 3*C per minute until reaching a final temperature of 2200C. The column was a 2 mn glass column packed with 10% cyanosilicone (Supelco SP2330) on 100/120 Chromosorb WAW. The detector, column, and settings were the same for both instruments. The relative proportions of the fatty acids were calculated as the percent of the total area under the recorded peaks.

Frequency distributions of the fatty acid phenotypes were recorded. Individuals were arranged by percent oleic acid, in classes of 4% increments. Segregating ratios were tested to determine the goodness-of-fit to proposed genetic ratios using the chi-square test.

The crosses F435 X F78114 and F435 X F519-9 were

repeated in the summer of 1987 and an additional cross of

F435 X PI 262090 was also included. The F1 seed produced from these crosses were analyzed using the same aforementioned procedure. The embryo ends of these seeds were saved and planted to produce subsequent generations.

The F1, F2, F3, and backcross generations were produced in a greenhouse at the University of Florida Agronomy Farm near










Gainesville. Seed of the F2 generation were analyzed for fatty acid composition with same technique, and the embryo ends were planted in the same greenhouse. The F3 seed produced were also analyzed for fatty acid composition. Backcrosses were also made between F, progeny from the F435 X F78114 cross and each parent, and backcrosses between F, progeny of the F435 X F519-9 cross and each of those parents. All seed were analyzed for fatty acid composition using the method previously described.

Results and Discussion

Saponification Versus Direct Esterification

Saponification of oil breaks the ester linkages that bind fatty acids to glycerol. Therefore, GC analysis of methyl ester preparations from saponified oil would represent total fatty acid content. However, if the relative proportions of free fatty acids and fatty acids released by the esterification reagent (BF3) were found to be in the same proportions as total fatty acids, saponification would not be necessary. When fatty acid profiles of four different peanut oil sources saponified versus nonsaponified were compared using a t-test, there were no differences in palmitic, stearic, oleic, linoleic, arachidic, nor behenic acid contents between the two procedures (Table 2-2). During esterification with BF3, some fatty acids may be dissociated from glycerol. Total










Table 2-2. A comparison of two methods of fatty acid analysis, saponified versus nonsaponified. Palmitic, stearic, oleic, linoleic, arachidic, and benhenic acid contents are shown from four different oil sources. Also shown are values of tcatc.


F435
non
Fatty Acid sapon sapon

Palmitic 6.7% 9.2%

Stearic 2.0% 3.2%

Oleic 80.8% 79.%

Linoleic 2.5% 2.9%

Arachidic 1.1% 2.5%

Behenic 1.4% 2.5%

* tcalc compares the means of each


F519-9 F78114 Commerc
non non
sapon sapon sapon sapon sapon

9.3% 10.6% 8.2% 10.1% 10.6% 2.0% 1.8% 3.7% 2.6% 1.9% 54.2% 53.9% 48.2% 47.6% 47.3% 26.5% 26.7% 35.8% 35.2% 32.1% 1.2% 1.0% 0.4% 0.5% 1.0% 1.4% 0.9% 0.3% 0.4% 1.5%

fatty acid saponified versus nonsaponified.


ial oil non
sapon *t atc 11.5% 1.70 1.7% 0.30 47.4% 0.04 33.4% 0.03 0.9% 0.80 1.0% 0.04










area integrated on the chromatograph is an indicator of sample concentration. If samples were measured approximately equal prior to saponification and/or esterification, then the difference in area integrated between the saponified and nonsaponified samples should be equal to the difference between the total fatty acid content and the free fatty acid content of peanut oil. It has been reported that more than 95.0% of peanut oil is made up of triacylglycerides and less than 0.05% free fatty acid (Sanders, 1980a). The difference in integrated areas on the chromatograph between saponified and nonsaponified is not as great as the difference between percent triacylglycerides and percent free fatty acids (Table 2-3). This difference indicates that some saponification is occurring but that the BF 3 is not strong enough for complete saponification. However, the degree of saponification by BF3 is apparently sufficient to produce a representative sample of the total fatty acid content in peanut oil. Since saponification was found to be unnecessary to determine the content of the fatty acids of interest in this study, the saponification step was omitted.

Crosses with F78114

When the high oleic acid line, F435, was crossed with

F78114, all F1seed had oleic acid levels similar to the F78114 parent (Table 2-4), regardless of the genotype on

which the seed were borne. F2 seed f rom this family















Table 2-3. A comparison of total areas integrated on chromatographs of saponified oils versus nonsaponified oils. Four different oil sources are included. Also included is the percent of the total fatty acids measured when saponification is performed that are measured when saponification is not performed.


Area Integrated

Source Saponified NonSaponified % Nonsaponified*

F435 3,048,100 374,215 12.3%

519-9 3,999,500 374,530 7.3%

F78114 3,567,899 289,789 8.1%

Commercial oil 4,699,600 604,060 12.9%


* calculated by area nonsaponified divided by area saponified, times 100.





Oleic Acid Classification


Phenotypic Range


Cross Normal High % Oleic Linoleic

F78114 X F435 37 0 38.0-65.1 17.2-40.3
F435 X F78114 30 0 36.7-60.4 25.8-36.7

F519-9 X F435 9 0 44.3-70.5 11.4-36.7
F435 X F519-9 20 0 42.0-71.4 11.3-39.3

PI 262090 X F435 13 0 61.4-67.9 14.9-20.7
F435 X PI 262090 31 0 61.2-69.8 11.4-19.2


Table 2-4. F1 progeny from the cross between high (F435) and normal (F519-9), (F78114), and (PI 262090) oleic acid phenotypes.










displayed bimodal distributions (Figure 2-1), and were classified as either normal oleic acid (less than 70%) or high oleic acid (70% or greater). This division between high and normal oleic acid content was established from parental phenotypes (Table 2-1) used in the crosses. There was a well defined break in the phenotypic distributions of

the F2 populations which allowed for definitive grouping of individuals into either high oleic acid or normal oleic acid classes. A test of homogeneity was conducted on all F2 families and there were no differences among families (Table

2-5). In the F2 segregating population the proportion of seed in the two categories (Table 2-5) was consistent with a 15:1 ratio, indicating that two recessive genes were responsible for the high oleic acid characteristic. F1 plants used as both male and female parents in crosses with F78114 produced all normal oleic acid seed in the BC1 generation (Table 2-6), and the backcross to F435 produced a phenotypic distribution consistent with the expected ratio of 3:1 (normal to high oleic acid seed). F2 embryo ends of sampled seed were used to generate F3 families. Three F3 families (Table 2-7), derived from high oleic acid seed, consisted entirely of the high oleic acid phenotype. F3 families derived from normal oleic acid seed, consisted of either all normal oleic acid seed or normal to high oleic acid phenotypes in 15:1 or 3:1 ratios.










30


~25 232425
1 2 0
>20 17 17


15 13 15

o10- 10 10
E
6 R
Z 5 5 45 3 3 3 3






Percent Oleic Acid

Figure 2-1. Frequency distribution of number of F2 offspring in phenotypic
classes based on oleic acid content. Data has been pooled for all families from
the cross of F78114 and F435.

















Table 2-5. Segregation data for F2 progeny from the cross between high (F435) and normal (F78114) oleic acid lines.


Oleic Acid
Classification
Family Cross Normal High X2 P
(15:1)
1 F78114 X F435 49 3 0.02 .90-.75
2 F78114 X F435 32 2 0.13 .75-.50
3 F435 X F78114 56 5 0.77 .50-.25
4 F435 X F78114 40 0 2.67 .25-.10
5 F435 X F78114 28 3 0.62 .50-.25
6 F435 X F78114 26 3 0.77 .50-.25
7 F435 X F78114 18 1 0.03 .90-.75


Pooled 249 17 0.01 .95-.90
Homogeneity 5.00 .75-.50





















Table 2-6. BC1 oleic acid phenotypic segregation ratios for two peanut lines (F78114 and F519-9) crossed to a high oleic acid peanut line (F435).


observed Expected
Low High ratio
Cross Oleic Oleic Low:Hicrh P

F435 X F78114
BC (F1 X F78114) 17 0 1:0
BC (F1 X F78114) 37 0 1:0
BC (F1 X F435) 11 1 3:1 .25

F435 X F519-9
BC (F1 X F435) 7 4 1:1 .25
BC (F1 X F435) 6 5 1:1 .59
















Table 2-7. Phenotypic segregation of F 3 families from crosses between high (F435) and normal (F78114 or F519-9) oleic acid peanut lines.


Oleic Acid Composition
F', Observed F 3 Expected
Cross F,2 Normal Hicih Ratio

F78114 X F435 High 0 54 all high
F78114 X F435 High 0 42 all high
F78114 X F435 High 0 40 all high
F78114 X F435 Normal 47 3 15:1

F519-9 X F435 High 0 45 all high
F519-9 X F435 Normal 20 0 all normal
F519-9 X F435 Normal 20 0 all normal
F519-9 X F435 Normal 16 0 all normal
F519-9 X F435 Normal 20 5 3:1
F519-9 X F435 Normal 20 5 3:1
F519-9 X F435 Normal 35 15 3:1
F519-9 X F435 Normal 39 11 3:1
F519-9 X F435 Normal 36 14 3:1










Cross with F519-9

F1 seed from the cross of the 'Sunrunner' component

line F519-9 with F435 also showed no high oleic acid types,

nor were there any reciprocal -cross differences. F2 seed from this cross also showed a bimodal distribution (Figure 2-2), with seed containing between 45% and 70% oleic acid classified as normal and those containing between 70% and 85% oleic acid classified as high. Classification ambiguities were resolved by considering the proportion of linoleic acid in the seed. All seed classified as high oleic acid had less than 5% linoleic acid, whereas all seed classified as normal oleic acid had more than 10% linoleic

acid. F2 seed from this cross segregated in a 3:1 ratio (Table 2-8) of normal to high oleic acid level, indicating that a single-recessive-gene difference between F435 and F519-9 was responsible for the high oleic acid, with the homozygous recessive condition required for expression of the high oleic acid character.

The embryo-ends of the sampled F2 generation seed were

planted to produce F3 families. In the families analyzed, the F3 data were consistent with the single-recessive-gene hypothesis (Table 2-7). High oleic acid seed were expected to be homozygous recessive and would breed true in the subsequent generation. The normal oleic acid seed were expected to be either homozygous dominant or heterozygous and either breed true for normal oleic acid or segregated in












~30 29
-o 26 2425
5 B
20 20
c20

o 15 15 14 15
12 12 13
10 10 .
z8





Percent Oleic Acid

Figure 2-2. Frequency distribution of number of F2 offspring in phenotypic classes based on oleic acid content. Data has been pooled for all families from the cross of F519-9 and F435.



























Oleic Acid
Composition
Family Cross Normal High x2 P
(3:1)


1 F519-9 X F435 65 19 0.25 .75-.50
2 F519-9 X F435 32 8 0.53 .50-.25
3 F519-9 X F435 34 13 0.18 .25-.10
4 F519-9 X F435 28 12 0.48 .50-.25
5 F519-9 X F435 9 4 0.23 .75-.50
6 F435 X F519-9 9 5 0.86 .50-.25
7 F435 X F519-9 30 6 1.33 .25-.10
8 F435 X F519-9 25 5 1.11 .50-.25

Pooled 232 72 4.07 .90-.75
Homogeneity 0.90 .99


Table 2-8. Phenotypic segregation of oleic acid
content f or F2 progeny f rom the cross between high (F435) and normal (F519-9) oleic acid peanut lines.










a ratio of 3:1, normal oleic acid to high oleic acid content.

When F, plants from the F519-9 X F435 cross were

backcrossed to the F435 parent, offspring fit a 1:1 ratio of high to normal oleic acid (Table 2-6). This is again consistent with a single-gene hypothesis, where a homozygous recessive (F435) is crossed to a heterozygote (F519-9) and result in a phenotypic ratio of 1:1. Cross with PI 262090

F1 seed from the cross of F435 with the PI 262090 were found to have oleic and linoleic acid contents similar to the normal oleic acid parent (PI 262090) (Table 2-4). All F2 families from this cross segregated in a 3:1 ratio of normal to high oleic acid phenotypes (Table 2-9) (Figures 23).

Summary

Simple inheritance of fatty acid variants have been reported in other oilseed crop species. In sunflower, (Helianthus annuus L.) it has been reported that a single partially dominant gene is responsible for a high oleic acid phenotype (Urie, 1985). In soybean [Glycine max (L.) Merr.], two additive alleles at a single locus were found to control linoleic acid content (Wilcox and Cavins, 1985). Induced mutants in rapeseed (Brassica napus L.) produced high linoleic acid and low linolenic acid oil by the effects of two additive alleles at each of two independent loci















Table 2-9. Segregation data for F2 progeny from the cross between high (F435) and normal (PI 262090) oleic acid lines.


Oleic Acid
Composition
Family Cross Normal High X2 P
(3:1)


1 PI 262090 X F435 19 7 0.05 .90-.75
2 PI 262090 X F435 18 6 0.0 1.00
3 F435 X PI 262090 18 6 0.0 1.00
4 F435 X PI 262090 27 6 0.82 .50-.25
5 F435 X PI 262090 23 7 0.04 .90-.75

Pooled 105 32 0.20 .75-.50
Homogeneity 0.71 .50-.25











20 19
18
o 16
-v 15



w- 10 9
0 8 8 8 8 8
L . 7
5
E 5 4

1 5

0
%u ,,b+ bb+ b"+ bg. e,%+ roo+ ( f3+ o r I+ (.C)+1('' '

Percent Oleic Acid

Figure 2-3. Frequency distribution of number of F2 offspring in phenotypic
classes based on oleic acid content. Data has been pooled for all families from
the cross of PI 262090 and F435.








40

(Brunklaus-Jung and Robbelen, 1987). Results presented here indicated major genes control the oleic and linoleic

acid content in peanut. Together, the F1, F2, F3, and BC, generation data from all three peanut crosses reported, support the hypothesis that the high oleic acid character is controlled by two recessive genes. While the combination of the two genes has not been reported previously, the current study showed one recessive gene to be present in two separate lines (F519-9 and PI 262090). This information about the simple inheritance of high oleic acid in peanut will facilitate genetic improvement of the nutritional quality and storage stability of peanut oil. Transfer of this high oleic acid characteristic to desirable lines and cultivars may be accomplished by traditional backcross breeding. Also, with the development of protocol appropriate to peanut, it may be possible to move this character within Arachis and to other species through molecular genetic methods.














CHAPTER III

VARIATION IN FATTY ACID COMPOSITION IN DEVELOPING SEED OF Arachis hvpocraea L.
Introduction

A number of studies have been conducted to assess

changes in fatty acid composition of developing peanut seed (Holaday and Pearson, 1974; Sanders, 1980a; Sanders, 1980b; Sanders et al., 1982; Worthington, 1969; Young et al., 1968). In these studies, seed from several peanut cultivars commonly cultivated in the southeastern U. S. were assayed for fatty acid and lipid class composition over the course of seed development. The general conclusion of past studies and of principal interest in this study is that the oleic acid content of the oil increased during seed development. It was also noted that palmitic acid decreases as seed matures, and that the linoleic acid composition was relatively stable, though some researchers report a slight decline (Sanders et al., 1982; Young et al., 1968).

The high level of oleic acid in peanut line F435 was

established in Chapter II to be controlled by two recessive genes. Those two genes are assumed to produce a pronounced alteration of one or more enzymes involved in fatty acid synthesis. Since previous work has shown that oleic acid varies over the course of development in common cultivars, 41










it was thought that there may be additional variation in oleic acid production or rate of production in the developing seeds of line F435.

If a unique pattern of oleic and/or linoleic acid production was observed in the F435 line as compared to normal lines, this information could be of considerable value in understanding the biochemical control of linoleic acid synthesis. This information could also be important in the molecular isolation of the gene through further understanding of the fatty acid synthase system.

An impediment to developmental studies in peanut has

been the lack of a uniform method to determine physiological maturity. Days from pegging is unreliable since the rate of maturation is affected by the location of the peg on the plant. Proposed methods for determination of maturity have included visual examination of the color and structural characteristics of the pod mesocarp (Williams and Drexler, 1981), visual classification of reproductive growth stages (Boote, 1982), dry matter deposition, and level of free arginine in the seed (Tai and Young, 1977). Two of these methods were employed in the current study, the nondestructive method by Williams and Drexler and dry matter deposition.










Materials and Methods

On February 3, 1989, seeds of three peanut lines were planted in pots in a greenhouse. The lines planted were F78114, F519-9, and F435. F78114 is low in oleic acid; F519-9 is moderate in oleic acid; and F435 is high is oleic acid (Table 3-1). Five seed of each line were planted in each of four pots. one week after emergence, plants were thinned to one plant per pot. After 122 days, plants were removed from the pots and ten pods from each plant were sampled. Due to the indeterminate nature of peanut, a sample of ten pods was considered adequate to represent the greatest variation in maturity possible for each individual plant.

Maturity was evaluated using the non-destructive method by Williams and Drexler (1981). This rating system consists of seven developmental stages. Within each stage, there are four distinct subclasses. For the purposes of this study the developmental stages and subclasses were numbered consecutively from 1 to 28 with 1 corresponding to stage 1 subclass a and 28 corresponding to stage 7 subclass d.

A total of forty seed per genotype, ten seed per plant, was sampled for analysis. The maturity rating was recorded and the seed immediately sampled for dry matter. In each pod, the seed proximal to the peg was used as the sample













Table 3-1. Oleic and linoleic acid content of three peanut breeding lines.



Fatty Acid
% Oleic % Linoleic

Genotype Mean Ranae Std. Err. Mean Range Std. Err.

F78114 45.4 43.3-46.4 0.42 34.3 32.6-36.8 0.55

F519-9 55.6 50.9-61.5 1.08 25.9 21.1-30.4 0.99

F435 80.1 72.6-82.3 1.01 2.2 3.6-1.2 0.22



Based on a 10-seed sample from each line.








45

seed. These seed were weighed and placed in a temperaturecontrolled oven at 1100C. After at least 17 hours the seeds were removed and allowed to cool in a desiccator, then weighed again. The percent dry matter was calculated from these weights.

After dry matter was determined, the dried whole seeds were chopped and soaked overnight in approximately 2 ml of petroleum ether. The petroleum ether was then pipetted into a 5 ml reaction vial. The petroleum ether was removed by evaporation, leaving the oil extracted from the seed sample. The oil samples in the reaction vials were then ready for saponification. To the reaction vials containing the oil extracts, 2 ml of 10% KOH in methanol/water (4:1 v/v) was added and the vials heated in a water bath to 800C for 90 minutes. The vials were then cooled and 1 ml of 1. 8 M H2S04 added to each. one ml of petroleum ether was added to each vial and vigorously shaken. The vials were allowed to stand for a phase separation but in some cases centrifugation was necessary. The upper petroleum ether phase was pipetted of f and this extraction procedure repeated.

The free fatty acids in the 2 ml sample of petroleum ether from each sample were then esterified as previously described (Chapter II) using boron trifluoride in methanol as the esterification reagent. Samples of the fatty acid methyl esters in petroleum ether were then injected into a Hewlett-Packard model 5890a gas chromatograph, also as










previously described (Chapter II). Fatty acid percentages were calculated based on percent area by a Hewlett-Packard 3392 integrator.

Dry matter, palmitic acid, oleic acid, and linoleic acid percentages were collected and recorded along with maturity ratings. Linear and non-linear regression analyses were performed on the data and the corresponding correlation coefficients were calculated. Correlation comparisons were made between maturity rating and dry matter accumulation for each genotype. Correlation comparisons for each genotype were also made between dry matter and percent oleic acid, dry matter and percent palmitic acid, and dry matter and percent linoleic acid.

Results and Discussion

The method of maturity rating by visual examination of pod mesocarp was initially performed to get a quick estimate of maturity prior to chemical analysis. Dry matter deposition was expected to be the preferred method of measuring maturity because of its objectivity over the more subjective visual rating. The two methods had not been previously compared. Non-linear regression analysis showed that a logarithmic function most accurately described the correlation between dry matter and maturity rating for all

genotypes. The highest R2 values f or dry matter versus maturity rating were found in the analysis of the F519-9

seeds (Table 3-2) (Figure 3-1). The highest R2 value for the





Table 3-2. R 2 values of individual plants and for a composite of all plants for each genotype for maturity versus percent dry matter.



Genotype Plant ii2
F519-9 1 0.87
2 0.84
3 0.46
4 0.92
1-4 0.64
F435 1 0.47
2 0.47
3 0.69
4 0.37
1-4 0.43
F78114 1 0.59
2 0.54
3 0.59
4 0.55
1-4 0.49











% Dry Matter
so

70 , . .
0-50' E3
60 . J:) . . I., .

1" 13
50 . 12 C30 -- . .

40 . .
0
30 1- .
i 1
20 . .

101
0 5 10 15 20 25 30
Maturity Rating X R%=Matter
R2 . 0.64 N.S
Y - 34.1 + 10.510

Figure 3-1. Regression plot of the percent dry matter versus maturity classification of peanut seed sampled from all four plants of line F519-9.
00








49

F519-9 was 0.92 for plant 4. The overall R2 for F519-9 was

0.64. This value was the highest of the three genotypes. The original work on the maturity rating system used in this study was performed on 'Florunner'. Since F519-9 is closely related to 'Florunner', a different botanical type from F435, and a different market type from F78114, it may be more likely to conform to the 'Florunner' maturity

classification. Therefore, the highest R2 values might be expected for F519-9.

The value of R2 for dry matter versus maturity rating on the F78114 Virginia market type was lower than for the F519-9 runner line (Table 3-3, Figure 3-2). The F435 R2

value was slightly lower than the F78114 R2 values (Figure 3-3). Dry matter has been concluded to indicate level of maturity in peanut (Tai and Young, 1977). However, in the same study, it was also shown that the rate of accumulation and total dry matter at maturity may vary among genotypes. Nevertheless, dry matter appears to be a more definitive measure of maturity than the color and morphology of the mesocarp. Maturity based on color and morphological characteristics can be subjective, besides being genotype dependent. Although there was a correlation between percentage dry matter and maturity rating, dry matter was used as the independent variable in the regression analysis of the fatty acids, palmitic, oleic, and linoleic acids. The maturity rating was used only as the independent








50

variable in regressions on percentage of oleic acid for the

three genotypes. By comparison, the R2 values associated with maturity rating were found to be lower than the R2 values of regressions where dry matter was the independent variable (Table 3-3, Figures 3-4 through 3-9).

The relative oleic acid percentage increased in all

genotypes as dry matter increased from 10% to 50% (Table 34). In line F78114, the oleic acid content increased from 36.2% to 44.1%; in line F519-9, the oleic acid content increased from 25.6% to 53.3%; and in line F435, the oleic

acid content increased from 73.7% to 76.7%. The R2 value for F519-9 was the highest of the three genotypes at 0.62.

The R2 for F78114 was 0.24 and for F435 was only 0.13. Though the R2 values are low, examination of the figures reveals that there was only one sample of F435 that had less than 40% dry matter and even that sample was well within the range of the oleic acid distribution for that genotype. In all genotypes, however, the samples under 40% dry matter had the lowest oleic acid contents. Previous studies have shown that oleic acid increases with maturity (Sanders, 1980b). If more immature seeds had been sampled, under 40% dry matter, with correspondingly lower oleic acid contents, more

of a trend may have been established and R2 values may have been higher. It may be necessary to sample more seeds to show this in all genotypes.















Table 3-3. R 2 values for regressions of the percent oleic acid with maturity rating as the independent variable compared with percent dry matter as the independent variable for three genotypes, F519-9, F435, and F78114.




R 2

Genotype Maturity Dry Matter

F519-9 0.30 0.62

F435 0.12 0.13

F78114 0.21 0.24











% Dry Matter _______

80 [







7 . G. 3. .

C3 0


Maturity Rating
X Dry Matter


R2- 0.43 Y - 39.74 + 9.7In0


Figure 3-2. Regression plot of the percent dry matter versus maturity classification of peanut seed sampled from all four plants of line F435















. . 8 . . I . .

13 a
. . . . 11. !!!!
E3

. . . . .


. . . M .


. . . I .
a


% Dry 70


60


50


40 30


20


Maturity Rating
x w


It 0.49 Y 21.74 + 12.431nX


Figure 3-3. Regression plot of the percent dry matter versus maturity classification of peanut seed sampled from all four plants of line F78114.


Matter











% Oleic Acid
"'M


JI J


60



50 40, 30,


Maturity Rating
X OtIec Add
a


R 2 0.30 Y - 42.0 + 0.1I1nX


Figure 3-4. Regression plot of the percent oleic acid versus maturity classification of peanut seed sampled from all four plants of line F519-9.


-D
. . I _


!












% Oleic Acid 82


so . . . M .


78, 76


74


. . E3 . . 13 . CI . q . 0 .

E3
. . . . 13 .


. Id . d . .


Maturity Rating
X Oleic Acid
a


R 2- 0.12 Y - 74.95 + 1.061nX


Figure 3-5. Regression plot of the percent oleic acid versus maturity classification of peanut seed sampled from all four plants of line F435.


































Maturity Rating
X Oleic Acid
-a-


Figure 3-6. Regression plot of the percent oleic acid versus maturity classification of peanut seed sampled from all four plants of line F78114.


% Oleic Acid
501


4540, 354 30


D 0







E30
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I. . . . . . . .


R 2-O0.21 Y - 6O7





. . I . I . a . MM . .

E3 a
C3 0



. . I . . . I., .
13

. . . I .



0 20 30 40 50 60 70 a
% Dry Matter


% Oleic Acid 701


60, 50,


40 30, 0% 0%


X Oleic Acid
a


R 2. 0.62 Y - a.32 + MGM


Figure 3-7. Regression plot of the percent oleic acid versus the percent dry matter of peanut seed sampled from all four plants of line F519-9.












% Oleic Acid 82 ,


a
13 E3
. . .
C7 13

13
. . . . 3 . 6 % . M . .
E3

. . .
. . a CE33 13 C3 C3


78 76


74


To' Dry Matter X Oleic Acid
a


R2 _ 0.13 Y - 61.3 + 3.910


Figure 3-8. Regression plot of the percent oleic acid versus the percent dry matter of peanut seed sampled from all four plants of line F435.












)Ieic Acid


)


Rt 0.24 Y -18.3 + 0.22X em=


40 50
%o Dry Matter X 0191c Acid
-a-


Figure 3-9. Regression plot of the percent oleic acid versus the percent dry matter of peanut seed sampled from all four plants of line F78114.


50



45 40 35


30'
2'


C3 0






E3
03 1

. . . . . . . . . . . . . . . . .9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
















Table 3-4. The percent oleic acid and standard errors for high, moderate, and low oleic acid peanut genotypes sampled at various stages of dry matter deposition.


Percent Oleic Acid and Standard Error Genotypes
Percent F78114 F519-9 F435
Dry Matter Mean *SE Mean SE Mean SE

10-29 36.2 -1 25.6 -

30-39 36.8 1.50 45.9 9.64 73.3

40-49 44.1 1.04 53.3 1.49 76.7 0.90

50-59 42.7 0.49 56.5 2.70 77.5 0.51

60-69 - - 55.3 0.37 77.5 0.40

70-79 - - 77.0 0.96


Standard error of the mean.
Insufficient data.










The proportion of palmitic acid was found to be

unchanged over seed development in the F5l9-9 line (Figure 3-10). However, there were only three seeds sampled that had less than 40% dry matter. Both the F435 and F78114 showed a decline in percent palmitic acid as dry matter increased (Table 3-5). The greatest change in palmitic acid content occurred before dry matter deposition had reached 50%. In line F435, 50% dry matter corresponded to a maturity rating of less than 5 and in the F78114 line less than 10. These maturity ratings correspond to very immature stages of development. The seeds sampled were not only high in moisture but small in size. The oil sample extracted was estimated to be near the minimum limit of the analytical methodology. However, the greatest changes in oil composition appear to be in these earliest developmental stages. A more accurate description of the rate of specific fatty acid deposition may be obtained from sampling seed only in maturity classes under rating ten. Linoleic acid percentages declined slightly from 5.9% to 3.8% during the accumulation of dry matter in the F435 line (Table 3-6 and Figure 3-11). In the F519-9, there may have been a slight increase in linoleic acid content, but no change was indicated in the F78114 line (Figures 3-12 and 3-13). A previous study using 'Florunner' indicated no change in linoleic acid percentage during development (Sanders, 1980b).











% Palmitic Acid
12






11 .tx. a .



a~~ a~O
a2
. a .


17 1 - 1110 11


R2- 0.01 Y - 2.71 - O.006X


% Dry Matter
X Palmitic Acdd
a2-


Figure 3-10. Regression plot of the percent palmitic acid versus the percent dry matter of peanut seed sampled from all four plants of line F519-9.















Table 3-5. The percent palmitic acid and standard errors for high, moderate, and low oleic acid peanut genotypes sampled at various stages of dry matter deposition.


Percent Palmitic Acid and Standard Error Genotypes
Percent F78114 F519-9 F435
Dry Matter Mean *SE Mean SE Mean SE

10-29 15.7 -18.9 - -30-39 14.2 0.56 10.8 1.59 9.0

40-49 11.7 0.35 9.8 0.18 7.8 0.22

50-59 12.2 0.13 9.3 0.21 7.9 0.17

60-69 - - 9.2 0.13 7.6 0.16

70-79 --- - 7.7 0.19


*Standard error of the mean. Insufficient data.
















Table 3-6. The percent linoleic acid and standard errors for high, moderate, and low oleic acid peanut genotypes sampled at various stages of dry matter deposition.


Percent Linoleic Acid and Standard Error Genotype s
Percent F78114 F519-9 F435
Dry Matter Mean *SE Mean SE Mean SE

10-29 37.8 -110.7 - -30-39 37.1 2.07 30.3 5.65 5.9

40-49 34.2 0.94 26.7 1.39 4.5 0.80

50-59 36.2 0.40 25.5 0.55 4.4 0.31

60-69 - - 25.1 0.46 3.3 0.21

70-79 --- - 3.8 0.42


*Standard error of the mean. 1Insufficient data.











% Linoleic Acid




7 . .
Ela





r 03
4 . . -3. . . .
13
3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . .


R2. 0.23 Y - 7.9 - 0.07X


% Dry Matter
X Linale Acid
-2-


Figure 3-11. Regression plot of percent linoleic acid versus percent dry matter of peanut seed sampled from all four plants of line F435.











% Li
40

35 30

25 20

15 10

5
11


noleic Acid


0 20


30 40 50 60 70 1
% Dry Matter
X Linoloic Acid
-5-


R 2. 0.1Y - 7IXI

Figure 3-12. Regression plot of percent linoleic acid versus percent dry matter of peanut seed sampled from all four plants of line F519-9.


.-.g.



. . . . . . . .





S. I .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I I. . I . . . . . . . . . .












% Linoleic Acid 42



383

36

3 .
34 . 13. . J .C, . S . .

323

303

2811
20 30 40 50 60 70
% Dry Matter X Linoloic Acid a6
R2 - 0.0008
Y - 35.32 + 0.009X

Figure 3-13. Regression plot of percent linoleic acid versus percent dry matter of peanut seed sampled from all four plants of line F78114.








68

one objective of this study was to identify a stage of development where a proportionate increase in the percentage of oleic acid, relative to other fatty acids, may indicate variation in enzyme activity. A change in the relative percentage of fatty acids during seed development may also indicate separate systems operating at different times. Based on dry matter, the difference in oleic acid content from the most immature to most mature was from 73.3% to 77.9%. This amount of variation is minimal and is within the range of normal variation for the line (Table 3-1). The amount of variation in linoleic acid due to maturity was also found to be minimal over the stages of development sampled. These results indicate that there is limited, if any, differential enzyme activity during the development period after 30% dry matter deposition. Since the greatest variation in the fatty acid composition occurs in the earliest stages of development, less than 30% dry matter deposition, it may be valuable to sample more of these immature seeds and more closely examine these developmental stages. If, however, the high oleic acid character is a result of reduced activity of the enzymes in the oleic acid desaturase system, and the fatty acid composition of the oil is relatively stable over development, then the stage of development that would yield the most active transcription of the desaturase enzymes would simply be the stage at which the total oil content is most rapidly increasing. In








69

'Florunner', total lipid content has been shown to increase most rapidly in the earliest stages of development, stages 1-5 (Sanders, 1980a). However, since rate of dry matter deposition has also been shown to be genotype specific, further investigation would be appropriate to examine the rate of oil deposition in F435 to find the developmental stage where genes for fat synthesis are most active.














CHAPTER IV

RESTRICTION FRAGMENT LENGTH POLYMORPHISM IN THE GENUS ARACHIS


Introduction

In the development of improved cultivars, genetic markers can be valuable tools for the plant breeder (Helentjaris, 1989). Markers can be used for determining incidence of natural outcrossing, identifying genetic linkages, construction of genome maps, determining degree of relatedness, and differentiating selfs and crosses. In recent years, a new class of genetic markers has been revealed with the use of restriction endonucleases. These markers are referred to as restriction fragment length polymorphisms (RFLP).

RFLPs have several advantages over morphological markers. In many crops they are more numerous than morphological markers (Beckmann, 1983). There is also a lack of dominance in RFLP markers. There are no multiallelic forms in RFLP markers nor pleiotropic affects on economically important traits. RFLP markers also have potential in mapping quantitative traits, screening genetic resources for important quantitative traits, determining the relationship between quantitative trait loci and "Mendelian"










genetic loci, isolation of causative genes, varietal identification, and determining ancestry and taxonomic relationships (Helentjaris, 1989).

RFLP clone sets and linkage maps are currently

available in maize (Zea mays L.), tomatoes (Lycopersicon esculentum Mill.), Brassica spp., wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), lettuce (Lactuca sativa L.), soybeans [Glycine max (L.) Merr.], and rice (Oryza sativa L.). The establishment of protocol specific to peanut is necessary to begin the development of the RFLP clone sets and linkage maps. It has been noted that there are substantial RFLP marker differences between species and genera. Considerable information has been obtained on maize and Brassica. However, self-pollinated species, such as tomato, wheat, and soybean, have not yielded much polymorphism among cultivars and mapping has been difficult (Apuya et al., 1988; Keim et al., 1989; Helentjaris et al., 1986). Peanut is also self-pollinated, and like soybean, it is very uniform with few morphological markers. There are also few isozyme markers in peanut (Cherry and Ory, 1972; Thomas and Neucere, 1974). To date, no work has been published on RFLPs in peanut, (Arachis spp.).

With an economically important trait, such as the high oleic acid character in peanut, there can be significant advantages to the development of RFLP markers. Varietal identification can be important, especially since a U. S.










patent application has been filed for the the high oleic acid trait. The isolation of DNA fragments unique to the high oleic acid line may lead to the molecular characterization of the gene. The identification of linkage between the simply inherited high oleic acid character and quantitatively inherited characters could also benefit breeders in the development of improved cultivars.

Initial steps in development of RFLP in peanut include: the isolation of purified nuclear DNA; the construction of a genomic DNA library; the isolation of suitable random genomic DNA clones for use as labeled probes; and the hybridization of labeled probes to Southern blots to identify polymorphism. These initial steps were the objectives of this study. All of these steps are described herein, with the exception of the genomic DNA library which was constructed by Dr. M. K. U. Chowdhury. This DNA library was constructed from genomic DNA extracted from the high oleic acid peanut line, F435. The F435 DNA was restriction digested with PstI and cloned with vector pUCl9. Clones derived from this library were used as probes in screening for RFLP among the Arachis species and genotypes.

Materials and Methods

DNA was isolated from four lines of A. hvpocraea: line F435, the high oleic acid line; line F519-9, a 'Sunrunner' component line; line F78114, a high yielding Virginia botanical type; and PI 262090, a Virginia botanical type








73

plant introduction. In addition, DNA was isolated from four species of perennial peanut: Arachis spp. (not speciated); A. glabrata; A. pintoi; and A. repens.

Two methods of DNA isolation were employed and

compared. One was a modification of the method used by Saghai-Mahoof et al. (1984) using a buffer of alkyltrimethyl-ammonium bromide (CTAB extraction). The other was a modification of the potassium acetate precipitation method of Dellaporta et al. (1983). DNA Extraction

Extraction 1 (CTAB)

Young mature leaf tissue was collected from the

selected peanut lines and species. After collection it was immediately frozen in liquid nitrogen and stored at -700C. The tissue was removed from the -70�C storage as needed. Prior to extraction the tissue was ground under liquid nitrogen to a fine powder and again stored at -70�C until needed.

Total cellular DNA was isolated from the powdered leaf tissue by a modified version of Saghai-Maroof et al. (1984). To approximately 500 mg of each ground tissue was added seven ml of CTAB extraction buffer [50 mM Tris (pH 8.0), 0.7 M NaCl, 10 mM EDTA, 1% hexadecyltrimethylammonium bromide (CTAB), 0.1% 2-mercaptoethanol (BME)] . The tissue and buffer was mixed vigorously to a homogenous suspension, then incubated at 65�C for 90 minutes. During incubation, tubes










were mixed by inversion every fifteen minutes. After incubation, tubes were air cooled for five minutes. Then 4.5 ml of chloroform/isoamyl alcohol (24:1) was added and the tubes mixed by inversion for five minutes. Tubes were then centrifuged at room temperature for 10 minutes at 2500 rpm. After spinning, the upper aqueous layer was pipetted off into new tubes, and extracted with equal volume of the chloroform/isoamyl alcohol (24:1). Again, tubes were mixed by inversion for five minutes then centrifuged for 10 minutes, and the supernatant pipetted off into new tubes. To these tubes were added 50 Al of 10 mg/ml RNase A (Promega Inc.) in 10 mM Tris-HCl (pH 7.5) and 15 mM NaCl. The tubes were then mixed by inversion for five minutes and incubated at room temperature for 30 minutes. After incubation, DNA was precipitated by adding equal volume of isopropanol to the tube and mixing gently by inversion. Precipitated DNA was removed with a wire hook and rinsed first with 3-4 ml of

0.2 M NaOAc in 76% ethanol. The precipitate remained suspended in the ethanol for 20 minutes. The precipitate was then rinsed with 10 mM NH4OAc in 76% ethanol and transferred to a 5 ml Eppendorf tube containing 1.0 ml TE [10 mM Tris and 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0]. Quantity and quality was determined by measuring absorbance at 260 nm and 280 nm. DNA quality was further confirmed by agarose gel electrophoresis. DNA samples from each of the lines and species were diluted to uniform











concentrations. Restriction endonuclease digestions were made using DNA and 30 units of EcoRI restriction enzyme according to the manufacturer's recommendations. Three A~l of RNase mix were also added and the tubes incubated at 37*C for five hours. Two A~l of loading buffer were added and each digestion loaded onto an agarose gel. A lambda HindIII-digested marker was also included in each gel as a reference for measuring fragment sizes. The gel was run for fifteen hours at 22 volts and 65 milliamps. It was then rinsed with distilled water, stained with 500 Al of ethidium bromide, and photographed under 300 nm wavelength ultraviolet light. The photographs were then examined to evaluate the quality and the concentration of the DNA extracts.

Extraction 2 (potassium acetate)

The eight ground frozen tissue samples used in the extraction 1 method were weighed to the same approximate quantities (500 mg) and placed into eight separate 30 ml Oak Ridge tubes. To each tube was added 15 ml of extraction buffer [50 mM EDTA (pH 8.0), 100 mM Tris (pH 8.0), 500 mM NaCl, 10 mM BME] and 1.0 ml of 20% sodium dodecyl sulfate (SDS). The contents of the tubes were then mixed thoroughly and incubated in water bath at 650C for ten min. The tubes were then cooled and 5.0 ml of 5 M potassium acetate were added to each. The contents of the tubes were then mixed vigorously followed by incubation at 00C for 20 minutes.










After incubation, the tubes were centrifuged in a refrigerated centrifuge at 25,000 X g for 20 min. The supernatant from each tube was poured through Miracloth filter (Calbiochem) into clean 30 ml tubes, and the DNA precipitated by adding equal volume of isopropanol. The contents of the tubes were mixed gently, incubating at -20�C for 30 min. The precipitated DNA was pelleted by centrifugation at 20,000 X g for 15 minutes. The supernatant was gently poured off and the pellet dried by inverting the tube over a paper towel for 30 min. The DNA pellets were then removed from the Oak Ridge tubes and placed into Eppendorf tubes with 700 Al of DNA buffer, TE. After the pellets were dissolved, the tubes were spun in a microfuge for 10 minutes to remove insoluble debris. The supernatants were then transferred to new Eppendorf tubes and purified with a phenol/chloroform extraction. An equal volume of phenol was added to each tube and mixed gently by inversion. The upper buffer phase was pipetted off into clean Eppendorf tubes and equal volumes of phenol/chloroform (1:1) added and gently mixed. The upper buffer phase was pipetted off and mixed with an equal volume of chloroform. These upper buffer phases were then pipetted off into clean Eppendorf tubes to each of which was also added 75 gl of 3 M sodium acetate and 500 gl of isopropanol. After mixing, the DNA was pelleted by microfuge spinning for 10 seconds. The supernatant was discarded and the pellets washed with 80%










ethanol. The pellets were redissolved in 400 A~l of DNA buffer TE. Quality and quantities of the DNA extracts were determined as in the extraction 1 procedure.

The extraction method 1 was repeated using tissue

samples collected from the same species and genotypes. For this extraction, the samples collected were immature not fully expanded leaf tissue plucked from stem apices. For the first extraction, leaves were young but fully mature. Southern Blotting

Samples of DNA extracted from the four genotypes of A. hvpogaea and the four Arachis species that had been determined to be of acceptable quality were digested with EcoRl and run out on a gel as previously described. A lambda marker was also included on the gel. Gels were stained and photographed, also as previously described.

The blotting procedure used was adapted from E. M. Southern (1975). After staining and photographing, gels were treated for 10 min. with a 0.25 M solution of HCl. The gels were then rinsed twice and soaked with constant stirring for 45 minutes in a denaturing solution of 1.5 M NaCl and 0.5 M NaOH. The gels were then neutralized by soaking 45 minutes in 1 M Tris with 1.5 M NaCl (pH 8.0), also with constant stirring. DNA was transferred from the gel to Hybond N blotting membrane (Amersham Corp.) using the capillary method described by Maniatis et al. (1982) with 3X SSC (1X SSC = 0.15 M NaCl and 0.015 M sodium citrate). This










blotting structure was then allowed to stand for 12-24 hours. The filter was then removed from the stack and soaked in 3X SSC for 5 minutes at room temperature. Next, the filter was wrapped in a clear plastic wrap and the DNA side of the filter exposed to ultraviolet light for 6 minutes. The blot was then air dried and stored in a plastic bag at 4�C until needed. Probe Preparation

Plasmid DNA was isolated by a method adapted from

Birnboim and Doly (1979). Inocula from individual library colonies were placed into tubes containing 5 mls of LB broth [10 g Bacto-typtone (Difco), 5 g yeast extract (Difco), and 5 g NaCl in 1 liter of water with pH adjusted to 7.2]. The LB broth also contained 0.2% maltose and 0.1 g ampicillin. The genomic DNA used to construct the peanut library was isolated from the high oleic acid peanut line F435. It was digested and cloned into the PstI site of pUCI9.

The cultures in broth were then transferred to 10 ml centrifuge tubes and centrifuged in a table-top centrifuge for seven minutes. The supernatants were discarded and the pellets resuspended in 800 il of cold STET buffer (80 g sucrose, Triton X-100, 200 ml of 0.25 M EDTA, 50 ml of 1 M Tris-HCl, and deionized H20 to a total of 1 L with pH adjusted to 8.0) in 1.5 ml Eppendorf tubes. Each tube then received 60 Al of 10 mg/ml lysozyme and was placed in boiling water for two minutes. The tubes were then spun in










a microfuge for 20 minutes. The gelatinous pellets were removed with toothpicks and discarded. Supernatants were purified with phenol/chloroform extraction and digested with RNase A. The plasmid DNA was then precipitated, dissolved in 100 Al TE, and stored at -200C as described in the plant DNA isolation above. Clones were evaluated for insert size by digesting 2 Ag plasmid DNA with 8 units PsI, then electrophoresing and staining as described above.

Clones that could be isolated from the vector were selected to be used as probes. Isolation of inserts was done by PstI-digesting 40 A~l of plasmid DNA as previously described. The separation was by electrophoresis and gels were stained and examined under ultraviolet light to determine the location of the inserts on the gel. Incisions were made in the gel 1-2 mm to the advance of the insert. Pieces of NA45 membrane (Schleicher and Schuell, Inc., Keene, NH), approximately 5 X 25 mm in size, were soaked in gel buffer. one piece of membrane was inserted into each incision. The gel was then returned to the electrophoresis apparatus and run for an additional 30 minutes at the same voltage.

The membranes containing bound DNA inserts were rinsed with 400 Al of low salt buffer, then eluted with high salt buffer (1M NaCl, 0.1 mM EDTA, 20mM Tris, and 0.5 M Arginine). To elute the DNA, tubes were heated to 700C and after 20 minutes the membranes were turned in the tubes.








80

After another 25 minutes at 70�C, the membranes were removed from the tubes and the eluate extracted with 400 Al of phenol/chloroform (1:1) and again with chloroform. The aqueous phase was then pipetted into another clean Eppendorf tube to which was added 100 gi of 7.5 M ammonium acetate and 800 Al of absolute alcohol. The tubes were incubated overnight at -20�C. DNA pellets were recovered by microfuging for 15 minutes then washing with 500 Al of 70% ethanol. The DNA was air dried and redissolved in 20 Al of TE buffer. Two Al of each isolated DNA was mixed with 18 Al of H20 and run on an agarose gel. The lambda marker was also included on the gel. The relative concentrations and fragment sizes were estimated from this gel. The tubes with the remaining inserts in DNA buffer were stored at -20�C until needed for oligolabeling. Radio-labelinQ Probes

Probes were labeled with 32P by the primer extension method of Feinberg and Vogelstein (1983). The method consisted of denaturing the DNA by heating to 95�C for 10 min. then rapid cooling on ice to prevent renaturing. The appearance of the gel from the insert isolation procedure was used to estimate the amount of insert DNA used. Two to four microliters of the insert DNA were placed in a 500 Al Eppendorf tube. Also into the tube were placed 10 Al of OLB (Finberg and Vogelstein, 1983), 6 Al of bovine serum albumen 3mg/ml (BSA), 2 Al of Klenow enzyme (lU/gl), 4 Al of 32p-










labeled deoxycytidine 5'-triphosphate (dCT32P) (3000ci/mM), and enough double-distilled H20 to bring the total volume to 50 Al. The tubes were then incubated at 37�C for 30 minutes. Then to each tube 50 Al of OLB stop mix [2.0 ml of 1 M TrisHCI (pH 7.0), 400 ml 5 M NaCl, 0.5 M EDTA (pH 8.0), and 12.5 Al 20% SDS] were added. Unincorporated nucleotides were removed by liquid chromatography with G50 Sephadex in 1X NTE buffer (100 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA). Probes were then denatured by boiling for 5 minutes and immediately placed in ice for 5 min. The probes were then ready for immediate use.

Prehybridization and Hybridization of Blots

Southern blots were pre-hybridized to prepare for probe hybridization. Blots were first soaked for a few seconds in deionized water, then in 3X SSC. Next blots were placed into a heat sealed plastic bag along with 30 mls of prehybridization solution (7% SDS in 3X SSC with 30 Al of denatured salmon sperm DNA). Prehybridization was conducted for 3-4 hours at 65�C.

Denatured probes were then injected into the sealed bag containing the blot and the pre-hybridization solution. Hybridization was conducted for 16 hrs. at 65�C. Blots were washed for two times for 15 min. each in 3.5 liters of 3X SSC at 65�C. A third rinsing was done at the same temperature and for the same time but in 0.3X SSC. Blots were then removed and allowed to air dry on paper towels.










Using a Geiger counter, the radioactivity was checked to estimate the exposure time necessary for autoradiography. Blots were then place into a film case with a piece of Kodak XAR-5 x-ray film and Cronex Hi-Plus intensifying screens (E. I. Dupont de Nemours and Co.). The film was exposed at

-70*C for 3 days to 2 weeks depending on the radioactivity of the blot. After appropriate exposure, the film was developed and the hybridization patterns examined.

Results and Discussion

The quality and quantity of DNA can be estimated from

calculations using the absorbance measured at 260 nanometers

(nm) and 280 nm. The absorbance at 260 nm divided by the absorbance at 280 nm indicates the purity of DNA with respect to protein contamination (Berger, 1987). The closer the ratio is to 1.8 the more pure the DNA. Concentrations can be estimated by multiplying the absorbance measured at 260 nm by a factor of 50 and then times the dilution rate of the sample analyzed. Absorbances measured on the DNA extracted using extraction method 1 with young but mature leaf tissue is shown in Table 4-1. Two genotypes, F435 and A. repens, showed ratios close to 1.8 indicating good quality DNA. The absorbance ratios of the other genotypes were less than the 1.8 optimum. None of the genotypes extracted using extraction method 2 were of optimum quality (Table 4-2). Photographs of stained gels and hybridization patterns of mature leaf DNA extracted by both methods were















Table 4-1. Absorbances of DNA extracts from four A. hypogaea lines and four perennial Arachis species at two wavelengths. Extraction was method 1 (CTAB) using young mature leaf tissue.

Absorbances

Genotype Wave length Concentration

260nm 280nm 260/280 ag/Ui* F435 0.202 0.114 1.77 1.01

F519-9 0.085 0.052 1.63 0.43

F78114 0.052 0.037 1.41 0.26

PI 262090 0.197 0.137 1.43 0.99

A. sPP- 0.101 0.066 1.53 0.51

A. clabrata 0.033 0.027 1.22 0.17

A. pintoi 0.092 0.024 1.56 0.46

A. repens 0.044 0.024 1.83 0.22


* Calculated by A260 X 50 X dilution rate of 100.















Table 4-2. Absorbances of DNA extracts from four A. hypogaea lines and four perennial Arachis species at two wavelength. Extraction was method 2 (potassium acetate) using young mature leaf tissue.


Absorbances


Genotype


F435

F519-9 F78114 PI 262090 A. sPD.

A. glabrata A. pintoi A. repens


Wave LenQth
260nm 280nm 260/280

0.053 0.039 1.36 0.037 0.027 1.37 0.066 0.047 1.40 0.037 0.033 1.23 0.058 0.043 1.35 0.052 0.040 1.30 0.045 0.032 1.41 0.072 0.051 1.41


Concentration
acr/al*

0.27 0.19 0.33 0.19 0.29 0.26 0.23 0.38


* Calculated by A260 X 50 X dilution rate of 100.








85

compared to determine DNA quality (Figure 4-1). The overall quality of the DNA, as indicated by the gel, was not acceptable because of lane to lane variation in DNA concentration and fragment distribution. There was also narrowing of the lanes which appeared to affect the running rates of the fragments. Narrow lanes ran slower, probably due to compounds binding to the DNA and restricting its migration through the gel. Because extraction method 1 was slightly faster, less complex, and at least equal to or better than, in the quality of DNA extracted by method 2 (Figure 4-2), all subsequent DNA extractions were performed using extraction method 1.

To further improve the quality of DNA extracted,

another tissue source was examined using extraction method

1. Immature leaves that were not fully unfolded and had not entirely emerged from the apical bud were used as the tissue source. Absorbances were measured and are shown in Table 43. There was improvement of extracted DNA quality from every genotype, as seen by the 260/280 absorbance ratios (Table 4-3). Gels of the endonuclease digested DNA showed a good quality by the even, non-narrowed band width along the length of the lanes (Figure 4-3). Because of the improvement in DNA quality associated with the immature leaf tissue, all subsequent isolations were made from that tissue source.














1 2 3 4 5 6 7 8


1 F435
2 F519-9 3 F78114
4 PI 262090
5 A. sp.
6 A. glabrata
7 A. pintoi 8 A. repens




Figure 4-1. DNA extracts from eight peanut genotypes using extraction method 1 on mature leaf tissue.















2 3 4 5 6 7 8


1 F435
2 F519-9 3 F78114
4 PI 262090
5 A. sp.
6 A. glabrata
7 A. vintoi 8 A. repens




Figure 4-2. DNA extracts from eight peanut genotypes using extraction method 2 on mature leaf tissue.
















Table 4-3. Absorbances of DNA extracts from four A. hypogaea lines and four perennial Arachis species at two wavelengths. Extraction was method 1 (CTAB) using immature leaf tissue.


Genotype F435

F519-9 F78114 PI 262090 A. spp. A. glabrata A. pintoi A. repens


Absorbances

Wave Length
260nm 280nm 260/280

0.179 0.099 1.81

0.148 0.079 1.87


0.086 0.093 0.180 0.088 0.053 0.022


0.047 0.053 0.098 0.053 0.029 0.012


1.83 1.75

1.84 1.66 1.82 1.83


Concentration


Concentration
uA/Al*

0.90 0.74 0.43 0.46 0.90 0.44 0.27 0.11


* Calculated by A260 X 50 X dilution rate of 100.




Full Text

PAGE 1

GENETIC, DEVELOPMENTAL, AND MOLECULAR CHARACTERIZATION OF A HIGH OLEIC ACID PEANUT (Arachis hypogaea L.) By KIMM. MOORE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1990

PAGE 2

ACKNOWLEDGEMENTS The author expresses his most sincere appreciation to his major advisor, Dr. David Knauft, for his advice, encouragement, and expert editorial assistance in completing this manuscript. Appreciation is also extended to Dr. Al Norden for his initial encouragement to pursue this particular research project. A special thanks is extended to Dr. Sherlie West for his support, advice, and understanding that led to the completion of this project. Appreciation is also extended to the other members of his advisory committee, Dr. Ken Buhr, Dr. Paul Lyrene, and Dr. Ken Quesenberry, for their thoughtful suggestions in this dissertation. A very special thanks is extended to Dr. Rex Smith who, although not a member of the advisory committee, provided excellent support and invaluable input. Thanks is also extended to Dr. Kamal Chowdhury for his assistance on the analysis of RFLPs. Thanks is also extended for the technical assistance of Karen Bedigian, Harry Wood, Jeff Seib, and Natalie McGill. The author also wishes to acknowledge both the Florida Foundation Seed Association and Proctor and Gamble, Incorporated, for their funding of portions of this research. ii

PAGE 3

TABLE OF CONTENTS ACKNOWLEDGEMENTS LIST OF TABLES .. LIST OF FIGURES . . ABSTRACT CHAPTERS I II III IV INTRODUCTION .. THE INHERITANCE OF HIGH OLEIC ACID IN PEANUT. . . . . . . . . . . . . . . Introduction ..••...•.•.. Materials and Methods .•.•.. Results and Discussion Saponification Versus Direct Esterification •.. Cross with F78114 .•. Cross with F519-9 ..... . Cross with PI 262090. Summary . . . . . . . . . . . . . . VARIATION IN FATTY ACID COMPOSITION IN DEVELOPING SEED OF Arachis hypogaea L. Introduction •..... Materials and Methods .. Results and Discussion RESTRICTION FRAGMENT LENGTH POLYMORPHISM IN THE GENUS Arachis. . . . . .. Introduction ...•...•.•..•• Materials and Methods. DNA Extraction ..•.. Extraction 1 (CTAB) ..•.•. iii Page ii V . . viii xi 1 17 17 19 24 24 26 34 37 37 41 41 43 46 70 70 72 73 73

PAGE 4

V REFERENCES Extraction 1 (CTAB) ...••.•. 73 Extraction 2 (potassium acetate) 75 Southern Blotting. . . . . . . •. 77 Probe Preparation 78 Radio-labeling Probes 80 Prehybridization and Hybridization of Blots. . . . . 81 Results and Discussion. . 82 SUMMARY. 109 BIOGRAPHICAL SKETCH. 112 120 iv

PAGE 5

LIST OF TABLES Table Page 1-1 Oleic and linoleic acid content of vegetable oils ........ . 9 2-1 Oleic and linoleic acid content of the four peanut breeding lines used in crossing .... 21 2-2 A comparison of two methods of fatty acid analysis, saponified versus nonsaponified. Palmitic, stearic, oleic, linoleic, arachidic, and benhenic acid contents are shown from four different oil sources. Also shown are values of tcalc . . . . . . 25 2-3 A comparison of total areas integrated on chromatographs of saponified oils versus nonsaponified oils. Four different oil sources are included. Also included is the percent of the total fatty acids measured when saponification is performed that are measured when saponification is not performed . . . . . . . . . . . . . . . 2-4 F 1 progeny from the cross between high (F435) and normal (F519-9), (F78114), and (PI 27 262090) oleic acid phenotypes ........ 28 2-5 Segregation data for F 2 progeny from the cross between high (F435) and normal (F78114) oleic acid lines .•.....• 2-6 BC 1 oleic acid phenotypic segregation ratios for two peanut lines (F78114 and F519-9) crossed to a high-oleic-acid peanut line 31 (F435). . . . . . . . . . . . . . . . . . 32 2-7 Phenotypic segregation of F 3 families from crosses between high (F435) and normal (78114 or F519-9) oleic acid peanut lines .. 33 V

PAGE 6

Table 2-8 2-9 3-1 Phenotypic segregation of oleic acid content for F progeny from the cross between high (F435j and normal (F519-9) oleic acid peanut 1 ines . . . . . . . . . . . . . . . . . . Segregation data for F 2 progeny from the cross between high (F435) and normal (PI 262090) oleic acid lines ...•... Oleic and linoleic acid content of three peanut breeding lines .....•..•. 36 38 44 3-2 R 2 values of individual plants and for a composite of all plants for each genotype for maturity versus dry matter . . . 47 3-3 R 2 values for regressions of the percent oleic acid with maturity rating as the independent variable compared with dry matter as the independent variable for three genotypes, F519-9, F435, and F78114 51 3-4 The percent oleic acid and standard errors for high, moderate, and low oleic acid peanut genotypes sampled at various stages of dry matter deposition ..•..•........ 60 3-5 The percent palmitic acid and standard errors for high, moderate, and low oleic acid peanut genotypes sampled at various stages of dry matter deposition ..•..•.•...•.• 63 3-6 The percent linoleic acid and standard errors for high, moderate, and low oleic acid peanut genotypes sampled at various stages of dry matter deposition ......•....... 64 4-1 Absorbances of DNA extracts from four Ahypogaea lines and four perennial Arachis species at two wavelengths. Extraction was method 1 (CTAB) using young mature leaf tissue. . . . . . . . . . . . . . . . . . . . 83 4-2 Absorbances of DNA extracts from four Ahypogaea lines and four perennial Arachis species at two wavelength. Extraction was method 2 (potassium acetate) using young mature leaf tissue .........•.... 84 vi

PAGE 7

Table Page 4-3 Absorbances of DNA extracts from four Ahypogaea lines and four perennial Arachis species at two wavelengths. Extraction was method 1 (CTAB) using immature leaf tissue .. 88 4-4 Genomic DNA clones, library cell locations, and approximate sizes of inserts isolated for production of radio-labeled probes 92 4-5 Gene clones used as radio-labeled probes. 93 4-6 Pair-wise indices of genetic similarity of four Ahypogaea lines and four Arachis species. The similarity index was calculated by dividing the total number of DNA fragments common between two genotypes by the total number of unique fragment sizes represented by the paired genotypes ......•.•.• 107 vii

PAGE 8

Figure 1-1 1-2 LIST OF FIGURES End use of peanuts as percent of the total 1984 u. s. production .•. The fatty acid percentages of the total fatty acid composition of peanut oil .• 1-3 The currently proposed biochemical pathway for desaturation of oleic acid to linoleic Page 4 6 acid in higher plants .........•.. 14 2-1 Frequency distribution of number of F 2 offspring in phenotypic classes based on oleic acid content. Data has been pooled for all families from the cross of F78114 and F435 •. 30 2-2 Frequency distribution of number of F 2 offspring in phenotypic classes based on oleic acid content. Data has been pooled for all families from the cross of F519-9 and F4 3 5. . . . . . . . . . . 3 5 2-3 Frequency distribution of number of F 2 offspring in phenotypic classes based on oleic acid content. Data has been pooled for all families from the cross of PI 262090 and F435. . . . . . . . . . . . . . . . 39 3-1 Regression plot of the percent dry matter versus maturity classification of peanut seed sampled from all four plants of line F519-9 . 48 3-2 Regression plot of the percent dry matter versus maturity classification of peanut seed sampled from all four plants of line F435 .. 52 3-3 Regression plot of the percent dry matter versus maturity classification of peanut seed sampled from all four plants of line F78114 . 53 viii

PAGE 9

Figure 3-4 3-5 3-6 3-7 3-8 3-9 3-10 3-11 3-12 3-13 4-1 4-2 Regression plot of the percent oleic acid versus maturity classification of peanut seed sampled from all four plants of line F519-9 54 Regression plot of the percent oleic acid versus maturity classification of peanut seed sampled from all four plants of line F435 .. 55 Regression plot of the percent oleic acid versus maturity classification of peanut seed sampled from all four plants of line F78114 . 56 Regression plot of the percent oleic acid versus the percent dry matter of peanut seed sampled from all four plants of line F519-9. 57 Regression plot of the percent oleic acid versus the percent dry matter of peanut seed sampled from all four plants of line F435 .. 58 Regression plot of the percent oleic acid versus the percent dry matter of peanut seed sampled from all four plants of line F78114 . 59 Regression plot of the percent palmitic acid versus the percent dry matter of peanut seed sampled from all four plants of line F519-9 . 62 Regression plot of percent linoleic acid versus percent dry matter of peanut seed sampled from all four plants of line F435 65 Regression plot of percent linoleic acid versus percent dry matter of peanut seed sampled from all four plants of line F519-9 . 66 Regression plot of percent linoleic acid versus percent dry matter of peanut seed sampled from all four plants of line F78114 . 67 DNA extracts from eight peanut genotypes using extraction method 1 on mature leaf tissue. . . . . . . . . . . . . . . . . . DNA extracts from eight peanut genotypes using extraction method 2 on mature leaf tissue. . . . . . . . . . . . . . . . . . ix 86 87

PAGE 10

Figure 4-3 4-4 4-5 DNA extracts from eight peanut genotypes using extraction method 1 on immature leaf tissue . ....... . Clones separated from pUC .. Autoradiograph of probe atpa on peanut genotypes illustrating an unacceptable autoradiograph based on poor fragment definition •.•.......••... 89 91 94 4-6 Autoradiograph of probe HPI16 on peanut genotypes with fragment sizes in kilobases. 95 4-7 Autoradiograph of probe HPI6 on peanut genotypes with fragment size in kilobases 96 4-8 Autoradiograph of probe atp6 on peanut genotypes with fragment sizes in kilobases .• 98 4-9 Autoradiograph of probe cox! on peanut 4-10 4-11 4-12 4-13 4-14 4-15 genotypes with fragment sizes in kilobases. 99 Autoradiograph of probe HPI67 on peanut genotypes with fragment sizes in kilobases .. 100 Autoradiograph of probe HPI72 on peanut genotypes with fragment sizes in kilobases .. 101 Autoradiograph of probe HPI58 on peanut genotypes with fragment sizes in kilobases .. 102 Autoradiograph of probe HPI52 on peanut genotypes with fragment sizes in kilobases .. 103 Autoradiograph of probe rrn5-rrn18 on peanut genotypes with fragment sizes in kilobases .• 104 Autoradiograph of probe HPI54 on peanut genotypes with fragment sizes in kilobases .. 105 X

PAGE 11

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy GENETIC, DEVELOPMENTAL, AND MOLECULAR CHARACTERIZATION OF A HIGH OLEIC ACID PEANUT (Arachis hypogaea L.) By Kim M. Moore May 1990 Chairman: David A. Knauft Major Department: Agronomy Shelf life and nutritional value are important factors affecting the quality of peanuts and peanut products. These factors are directly related to the chemical composition of the oil. Peanut oil is composed of 95% triacylglycerides which vary in their composition by the relative proportions of component fatty acids. Typically, 90% of the total fatty acid composition is made up of three fatty acids, palmitic, oleic, and linoleic acids. Oleic acid has been shown to be more desirable for both stability and nutrition than palmitic or linoleic acids. In 1987, a peanut line high in oleic acid was identified. This peanut line was crossed with four other peanut lines and cultivars to determine the mode of inheritance of the oil character. Segregating F 2 populations were analyzed along with F 1 , F 3 , and backcross xi

PAGE 12

generations. Among the four crosses, three F 2 crosses produced segregating ratios of 3 normal to 1 high oleic acid phenotype. One of the crosses produced a segregating ratio of 15 normal to 1 high oleic acid phenotype. Analysis of the F 3 and backcross data further supported a hypothesis of inheritance by two genes, each with a dominant and recessive allele. Further investigations were conducted to determine if variation in gene action occurred during development. Seeds of three peanut genotypes that were low (F78114), moderate (F519-9), and high (F435) in oleic acid were analyzed for fatty acids at different developmental stages. Oleic acid content increased in the early stages of development of all three genotypes. Linoleic acid content was relatively unchanged during development of F519-9 and F78114 but declined during development of the high oleic acid line. Early stages of development may be the best time to isolate mRNA to produce cDNA for screening to characterize the genes. The high oleic acid genotype along with F519-9, F78114, PI 262090, and four species of perennial peanut were compared by variation in DNA fragment length polymorphism. The four Ahypogaea lines were uniform and showed few polymorphic fragments. Polymorphisms were more readily detected among the four perennial peanut species. xii

PAGE 13

CHAPTER I INTRODUCTION The peanut, Arachis hypogaea L., is a native legume of South America. It is a member of the family Leguminosae, tribe Aeschynomeneae, subtribe Stylosanthyenae. The genus, Arachis, is divided into seven sections based on morphology and cross-compatibility. The sectional arrangement of the genus follows the ecological and geographic features of its continent of origin, South America. The currently accepted center of origin for the genus is the Mato Grosso area of Brazil, located just north and east of Paraguay (Wynne and Halward, 1989). The cultivated peanut, A hypogaea, is a member of the section Arachis. This section shows considerable diversity in the area west of the Paraguay River through northern Bolivia to the Andes mountains. Arachis hypogaea can be divided into two subspecies, based on morphological differences. These subspecies are hypogaea and fastigiata. The subspecies hypogaea is subdivided into variety hypogaea, also known as the Virginia type, and variety hirsuta, also known as the Peruvian runner type. The subspecies fastigiata is subdivided into variety fastigiata, commonly 1

PAGE 14

2 known as Valencia type, and variety vulgaris, also known as Spanish type (Stalker and Moss, 1987). Peanut was cultivated before the European exploration of the Americas but has been limited in range to warmer regions of the western hemisphere (Hammons, 1982). The earliest dated peanuts found in association with human activities were estimated at 3800 years old by radiocarbon dating (Hammons, 1982). These samples originated from an archeological site near Las Haldas, Peru. By the time of the European exploration and colonization of the Americas, peanut was grown throughout the warmer regions of the western hemisphere, including the islands of the Caribbean. It was probably on the island of Hispaniola where Europeans first encountered peanut culture. Peanut was then spread by European explorers and travelers to Asia, the Pacific Islands, Europe, southeastern U.S., and both coasts of Africa (Hammons, 1982). Currently, there are twenty-four countries that each produce more than three million kg of peanuts annually. China leads the world in total production with 6,400,000 metric tons produced in 1987. India is second with 4,350,000 metric tons and the United States third with 1,620,000 metric tons. World production in 1987 was over nineteen million metric tons (Commodity Research Bureau, 1988). These production numbers are elevated considerably, relative to twenty years ago, due to the increasing value of

PAGE 15

3 peanut as a source of high-quality edible oil (McGill, 1973). Unlike most countries, where peanut is grown for oil, only 24% of the 1984 U. s. production of shelled peanuts were crushed for oil, as shown in Figure 1-1 (Commodity Research Bureau, 1984). The typical oil content of peanut is approximately 52% (Cobb and Johnson, 1973). With this high level of oil, any factors affecting the quality of the oil will in turn affect the total product quality. The quality of the oil is of particular interest not only to the oil producer, but also to the processors and roasters of peanuts. A number of methods have been developed to measure oil quality quantitatively. Peroxide value, iodine number, and fatty acid content have all been used to quantify oil quality, and all are directly related to the chemical composition of the oil (Cobb and Johnson, 1973). The two principal quality characteristics most affected by the chemical composition are the storage stability of the oil and the nutritional value or liability. Both of these factors are directly related to the degree of unsaturation of the oil and more specifically related to the fatty acid composition. Peanut oil, like other vegetable oils, is composed of monoacylglycerides, diacylglycerides, triacylglycerides, and free fatty acids. An acylglyceride is a glycerol molecule, C 3 H 5 (0H) 3 , to which organic acids (fatty acids) are bound, substituting for one, two, or all three of the hydroxyl

PAGE 16

15.9 " 37.0 " 0.97 " 24.0 " 7.0 " Candy Salted Butter 0 Roasted Iii Crushed for OIi Other Figure 1-1. End use of peanuts as percent of the total 1984 U.S. production.

PAGE 17

5 groups, e.g., mono-, di-, and triacylglycerides. In peanut oil, triaclyglcerides account for more than 95% of the total lipids (Sanders, 1980a). The remainder is made up of approximately 1.7% diacylglycerides, 0.3% monoacylglycerides, 0.7% free fatty acids, and the remaining 2.3% is made up of polar lipids, sterols, and hydrocarbon sterol esters. The fatty acid composition of the triacylglycerides is variable. Typically, however, 90% of the fatty acid composition of peanut oil is made up of three fatty acids: palmitic, oleic, and linoleic (Cobb and Johnson, 1973). Palmitic acid is a 16-carbon, completely saturated, fatty acid. Oleic acid and linoleic are both 18carbon chains with one and two double bonds, respectively. A generalized breakdown of peanut oil fatty acid composition is given in Figure 1-2. Of the oil quality factors dependent on fatty acid composition, storage-stability is most directly related to the degree of fatty acid saturation. The most common cause of oil degradation in storage is oxidation, and the result is termed oxidative rancidity. Although some fatty acids are more prone to degradation than others, regardless of the degree of saturation, the loss of wholesomeness is most commonly caused by oxidative rancidity. This type of rancidity is directly related to the degree of unsaturation of an oil. Oxidation of the double bond in the triacylglycerides and free fatty acids results in the

PAGE 18

11.0 " 1. 1 " 2.5 " 1.4 " Palmitic Stearic ~Oleic fL:J Linoleic D Arachidic fil Behenic Lignoc•ic Figure 1-2. The fatty acid percentages of the total fatty acid composition of peanut oil.

PAGE 19

formation of peroxide groups at or near the double bond. 7 The peroxides then decompose to form acids, alcohols, aldehydes, ketones, and other hydrocarbons that result in the odors commonly associated with rancidity (St. Angelo and Ory, 1973). Therefore, the advantage of more saturation is more stability. The second quality factor, nutritional composition, is also important in the establishment of edible oil quality. Fat chemical composition and level of dietary fat intake have been found to affect level and composition of serum cholesterol (Gustafsson et al., 1985; Bronsgeest-Schoute et al., 1979; Kuusi et al., 1985; Schonfeld et al., 1982). Cholesterol and cholesterol fatty esters are components of the atherosclerotic plaques that restrict arterial blood flow and contribute to heart disease. By altering dietary fat intake both in level and/or composition, serum cholesterol levels can be reduced. A compositional change in dietary fat that has recently been shown to reduce serum cholesterol is a high monounsaturated diet. In a study conducted in 1986, it was concluded that diets high in monounsaturates, i.e., oleic acid, were as effective in reducing serum cholesterol levels as low-fat diets (Grundy, 1986). Since the degree of unsaturation is critical to both nutritional quality and storage-stability, relative proportions of fatty acids in an oil are important in determining total oil quality. Oils higher in

PAGE 20

8 monounsaturated fatty acids would be desirable for both nutrition quality and storage stability. In Table 1-1, a comparison of seven different vegetable oils is shown with their respective oleic acid (monounsaturate) and linoleic acid (polyunsaturate) content (USDA, 1975). As shown in the table, peanut is second only to olive oil in oleic acid content. Variation of fatty acid composition in peanut oil has been shown to be influenced by several factors. It may vary according to variety, location, year-to-year variation, environmental variation, and physiological maturity (Bovi, 1982; Jamieson et al., 1921; Knauft et al., 1986; Worthington et al., 1972; Hartzook, 1969; Norden et al., 1987; Rachmeler, 1988; Worthington and Hammons, 1971; Worthington, 1969; Young et al., 1974). In a comparison of peanut genotypes grouped as Virginia Runner, Virginia Bunch, and Spanish Bunch with respect to growth habit, it was found that oleic and linoleic acid contents varied significantly among groups (Raheja et al., 1987). Total oil content of the three genotypes was very uniform, ranging only from 48.9% to 49.8%. Oleic acid content was much more varied, ranging from 37.6% as a low in the Virginia Bunch to 54.7% as a high in the Virginia Runner type. Linoleic acid was also varied both within and among

PAGE 21

Table 1-1. Oleic and linoleic acid content of vegetable oils. Oil Source % Oleic Acid % Linoleic Corn 28 53 Cottonseed 21 50 Olive 76 7 Peanut 47 29 Safflower 15 72 Sesame 38 42 Soybean 20 52 (USDA, 1975) 9 Acid

PAGE 22

10 peanut types, ranging as low as 29.7 in the Virginia Runner to 46.7% in the Virginia Bunch. In a survey of 110 genotypes, some assayed over more than one growing season, wide variation in fatty acid composition was observed (Worthington and Hammons, 1971). The genotypes consisted of Virginia, Spanish, and runner market types and plant introductions. Palmitic, oleic, and linoleic were the fatty acids with the most variability. Palmitic acid ranged from 6.7% to 13.7%, and the average of all 110 genotypes was 10%. Oleic acid ranged from 35.8% to 71.4%, and with an average of 45.0%. Linoleic acid ranged from 11.1% to 40.1% with an average of 29.9%. The oleic and linoleic acid contents had a strong negative correlation and the genotype with the highest oleic acid level also had the lowest linoleic content. In the Worthington and Hammons study and the Raheja et al. study, the Spanish varieties consistently showed lower oleic acid and higher linoleic content than the Virginia and runner varieties. Higher peroxide values, indicating less stability, have been previously reported for Spanish varieties compared with runner and Virginia varieties (Picket and Holley, 1951) (Fore et al., 1953). In another study, 82 peanut genotypes were tested for their fatty acid composition (Worthington et al., 1972). The genotypes represented a wide variation in genetic background. The three major fatty acids varied as follows:

PAGE 23

11 palmitic 7.4-12.9; oleic 35.7-68.5; and linoleic 14.1-40.3. Another study of 40 peanut cultivars also included 12 plant introductions representing all four A. hypogaea botanical types and two other Arachis species, Amonticola and nambyguarae (Treadwell et al., 1983). Palmitic acid ranged from 7.5-11.8%; oleic acid ranged from 39.3-56.6%; and linoleic acid ranged from 26.0-38.9%. These values are well within the range recorded throughout the literature. The highest oleic and lowest linoleic values recorded for peanut are 80% oleic and 2% linoleic (Norden et al., 1987). The variant fatty acid levels were found in an experimental breeding line, F435, of Spanish botanical type. The highest oleic acid level previously published was 71.4% and the lowest linoleic acid level was 11.1%. Earlier evaluations of F435 were found to have typical Spanish botanical type values for oleic and linoleic, 50% oleic and 26% linoleic. The oleic and linoleic acid contents of F435 deviated sufficiently from previously established ranges within Arachis to justify a genetic study of the character. Variant fatty acid phenotypes have been identified in other oil seed crops and some have been found to be controlled by major genes. In a mutant soybean [Glycine max (L.) Merr.J line, high linoleic acid was found to be controlled by two alleles at one locus (Wilcox and Cavins, 1985). In flax (Linum usitatissimum L.) two mutant lines were found to have increased linoleic and reduced linolenic

PAGE 24

12 acid in the seed oil. Analysis of progeny from crosses between the mutants and normal parental lines showed that the variation in linoleic and linolenic was controlled by two unlinked genes with additive gene action (Green, 1986). In a high oleic acid sunflower (Helianthus annuus L.) line the phenotype was found to be controlled by two genes, one with partial dominance (Miller et al., 1987). For the high oleic acid character to be expressed one gene must have at least one dominant allele present. When inheritance is simple it is sometimes possible to trace the character to a single protein or enzyme in a biochemical pathway. In the production of plant storage fats and oils, many enzymes are involved. Fat synthesis begins in the plastids, using translocated sugars as the carbon source. The synthesis of fats from carbohydrates proceeds by the esterification of fatty acids onto a glycerol backbone. The glycerol is derived from glycolysis and is formed by the reduction of dihydroxyacetone phosphate (Salisbury and Ross, 1985). The fatty acids are synthesized from molecules of acetyl CoA adding two carbons at a time to the chain. More than 99% of the fatty acids in peanut oil are made of even-numbered carbon chains. These even numbered fatty acid chains are formed through the fatty acid synthase system. Fatty acid synthase catalyzes a series of reactions where one molecule of acetyl-CoA and seven molecules of a three-carbon compound, malonic acid in the

PAGE 25

13 form of its CoA thioester malonyl-CoA, are linked to form palmitic acid (16:0). The reaction evolves seven molecules of carbon dioxide and requires the reducing power of 14 NADPH (nicotinamide adenine dinucleotide phosphate) (Lehninger, 1982). Palmitic acid is formed in plastids, where it is also lengthened by two carbons forming stearic acid (18:0). Stearic acid, still in the plastid, is then desaturated to oleic acid (18:1) with the enzyme stearoyl ACP desaturase. This system is well understood and established up to the formation of oleic acid. The mode of introduction of the second and third double bonds are not so clearly defined. The substrates are not firmly established and the enzyme system appears to be membrane bound (Stumpf, 1989). The most probable mechanism is outlined in Figure 13. One carrier protein and at least three important enzymes that have not been characterized may affect the protein structure of the enzymes or carrier protein, which will also affect the activity of these species. Changes in the activities of the enzymes will affect the formation of the fatty acids and ultimately alter the final ratios of the various fatty acids. Experimental data have shown that the system is also affected by the type of tissue examined, the temperature at which the tissue is grown, the light regime to which the tissue was exposed, and the age of the tissue (Stumpf, 1989).

PAGE 26

NADH Carrier ( oxid) 2-Linoleoyl PC/PE t Carrier (red) + 2-0leoyl PC/PE NADH:Carrier {oxid) Reductase a12 Desaturase 2-Lyso PC/PE + NAO = reduced nicotinamide adenine dinucleotide NADH = oxidized nicotinamide adenine dinucleotide CoA = coenzyme A red = reduced oxid = oxidized CoA Oleoyl transferase Oleoyl CoA PC/PE = phosphotidylcholine/phosphotidylethanolamine Figure 1-3. The currently proposed biochemical pathway for desaturation of oleic acid to linoleic acid in higher plants.

PAGE 27

15 Variation in fatty acid composition has been shown to be related to the maturity of the peanut seeds tested, which corroborates in vitro experimental data. In 1969, Worthington reported varying fatty acid contents of peanuts of four different maturity classes. The maturity classes were measured in weeks from gynophore penetration of the soil. Oleic acid content ranged from 41.2% in the earliest maturity class to 52.1% in the most mature class. A reduction in linoleic acid content also occurred over maturity, ranging from 32.3% in the most immature to 28.9% in the most mature. In another study, a comparison was made between the fatty acid composition of the triacylglycerides versus the free fatty acids over varying maturity classes of 'Florunner' peanut oil (Sanders, 1980b). Some fatty acids, palmitic and oleic, varied over maturity in both triacylglyceride and free fatty acid forms. Linoleic acid was relatively stable over maturity in both triacylglyceride and free fatty acid forms. If the high oleic acid character in peanut is controlled by major genes, the mutant line may be helpful in determining the pathways of fatty acid synthesis. Since it is known that the rate of synthesis of both oleic and linoleic acid varies during seed maturation in peanut, a comparison of rates between a normal peanut line and the mutant (F435 line) may be valuable in determining the

PAGE 28

16 biochemical pathway of oleic acid desaturation to linoleic acid. To date, simply inherited characters of economic value have not been identified in peanut. If the high oleic acid character is controlled by only a few major genes, it could be an important subject for gene isolation and transformation into other peanut cultivars and possibly other oil seed species as well. Preliminary work necessary for molecular transformation would require the development or adaptation of protocol compatible with genus Arachis and ultimately A hypogaea. The objectives of this dissertation research were threefold. The initial objective was to determine the mode of inheritance of the high oleic acid character. The second objective was to determine if the rate of formation of oleic acid or linoleic acid varied in the high oleic acid line in relation to a particular stage of seed development. The third objective was to develop or adapt molecular genetic protocol that could lay the ground work for the gene isolation and molecular transformation of the high oleic acid character.

PAGE 29

CHAPTER II THE INHERITANCE OF HIGH OLEIC ACID IN PEANUT Introduction Fatty acid composition is an important determinant of quality in edible oils. Oil stability and nutritional quality are both dependent on the relative proportions of the saturated and unsaturated fatty acids that constitute the oil. Oxidative rancidity increases with increased levels of polyunsaturated fatty acids. Oxidation of the carbon double bonds of fatty acids produces acids, aldehydes, ketones, and other hydrocarbons that cause odors and flavors commonly associated with rancidity (St. Angelo and Ory, 1973). Therefore, the total amount of unsaturation is inversely proportional to the keeping quality of the oil. Fats with more saturation are less prone to oxidation during storage and processing than polyunsaturates. From a nutritional standpoint, polyunsaturates have been desirable for their role in lowering plasma cholesterol levels. However, a recent study showed that human diets containing oils high in monounsaturates were as effective in lowering serum cholesterol levels as were low-fat diets (Grundy, 1986). It was also demonstrated that beef cattle and swine fed diets high in monounsaturates produced meats with 17

PAGE 30

18 significantly higher levels of unsaturation (St. John et al., 1987). Peanut oil varies in both quantity and relative proportion of fatty acids. Although there are eight fatty acids in peanut found in quantities greater than 1.0%, palmitic (16:0), oleic (18:1), and linoleic (18:2) constitute approximately 90% of the total fatty acid composition (Cobb and Johnson, 1973). Generally, palmitic acid constitutes nearly 10%, and the oleic and linoleic acid proportions together make up 80% of the fatty acid composition in peanut oil (Ahmed and Young, 1982). The variation in composition has been related to maturity, temperature, planting date, location, market grade, and peanut genotype (Bovi, 1982; Harris and James, 1969; Holaday and Pearson, 1974; Knauft et al., 1986; Mozingo et al., 1988; Norden et al., 1987; Young et al., 1972; Young et al., 1974). In 1987, Norden et al. reported a peanut line, F435, with 80% oleic acid and 2% linoleic acid. This line extended the known variability of these two fatty acids, which had been reported to range from 36% to 71% for oleic acid and from 11% to 43% for linoleic acid (Bovi, 1982; Norden et al., 1987; Treadwell et al., 1983). The F435 line is a Spanish botanical type (Ahypogaea ssp. fastigiata var. vulgaris). This botanical type accounts for only 10.8% of the total U.S. peanut acreage. The Virginia botanical type (A. hypogaea ssp. hypogaea var.

PAGE 31

19 hypogaea) is the predominant botanical type grown in the U.S., with over 80% of the total U.S. acreage (Holbrook and Kvien, 1989). Spanish lines are generally lower yielding than Virginia types and are not as well adapted to the principal peanut production regions of the U.S., i.e., the warm, humid Southeast. Besides being a Spanish botanical type, F435 is a breeding line with a pod-splitting characteristic that has made it unsuitable for release as a cultivar. To improve the oil quality of the more widely cultivated botanical types, it would be necessary to transfer this high oleic acid characteristic from the Spanish F435 breeding line to Virginia and runner peanut cultivars and adapted lines. An understanding of the mode of inheritance will allow most efficient transfer of this trait to adapted breeding lines and runner and Virginia market type cultivars. The following study was conducted to elucidate the genetic basis of the high oleic/low linoleic acid character. Materials and Methods In the spring of 1986, seed from the high oleic acid line, F435, were planted in a greenhouse along with seed from F78114, a Virginia market type, and F519-9, a component line of the runner market type cultivar, •sunrunner' (Norden et al., 1985). The F78114 had an oleic acid content of 45.4%, which is lower than the midpoint of the range of peanut (Table 2-1). F519-9 had an oleic acid content of

PAGE 32

20 53.6%, which is near the midpoint of the range of oleic in peanut (Table 2-1). Crosses were made between F435 and F78114 and between F435 and F519-9. In both cases reciprocal crosses were made. Seed from the F 1 generation were planted in the field at the University of Florida Agronomy Farm, near Gainesville, in July 1986. These F 1 plants produced F 2 seed that were harvested in December of the same year. These F 2 seed were analyzed for fatty acid composition. Fatty acid analysis was also performed on all parents used in crossing. Fatty acid determinations were made by first extracting oil from the seeds, esterifying the oil, and using gas chromatography to determine the relative proportions of the various fatty acids. Saponification prior to esterification was omitted since the relative proportions of free fatty acids were found to be the same as relative proportions of total fatty acids. This was determined by comparing fatty acid analysis with and without saponification on four different peanut oil sources, F435, F519-9, F78114, and commercial cold press peanut oil. Saponification was performed for comparison by adding 2 ml of 10% KOH in methanol/water (4:1 v/v) to the reaction vials containing the oil extracts and heating the vials to 80C for 90 minutes. The vials were then cooled and 1 ml of 1.8 M H 2 so 4 added to each. One ml of petroleum ether was added to each vial and vigorously shaken. The vials were allowed to stand

PAGE 33

Table 2-1. Oleic and linoleic acid content of the four peanut breeding lines used in crossing. Fatty Acid % Oleic % Linoleic Genotype Mean Range Std. Err. Mean Range Std. Err. F78114 45.4 43.3-46.4 0.42 34.3 32.6-36.8 0.55 F519-9 55.6 50.9-61.5 1.08 25.9 21.1-30.4 0.99 PI 262090 59.8 52.8-63.9 1.56 22.5 19.3-28.3 0.93 F435 80.1 72.6-82.3 1.01 2.2 1. 2-3. 6 0.22 Based on a 10-seed sample from each line.

PAGE 34

22 for a phase separation, but in some cases centrifugation was necessary. The upper petroleum ether phase was pipetted off, and this procedure was repeated. Prior to saponification and/or esterification, the oil was extracted by cutting approximately 0.1 g slices from the end of the cotyledons of an individual seed and soaking these slices in 2 ml of petroleum ether in a 13Xl00 mm culture tube overnight. Embryo ends of the seeds were saved for later planting to produce the subsequent generations. The petroleum ether was then pipetted into a 5 ml reaction vial. For saponification, the petroleum ether was removed by evaporation, leaving the oil extracted from the seed sample. The oil samples in the reaction vials were then ready for saponification. When saponification was not performed, oil was esterified immediately following extraction. Boron-trifluoride in methanol (BF 3 ) was added directly to the vials containing the extracted oil in petroleum ether (Metcalfe and Schmitz, 1961). This mixture was shaken briefly and then heated to 100c for 3 minutes. After heating, the reaction vials were cooled and 1 ml of deionized H 2 0 added to each of the vials. The vials were shaken vigorously and then allowed to stand for clearing and a phase separation. The upper petroleum ether phase containing the fatty acid methylesters was sampled and a 1 l aliquot injected into a gas chromatograph (GC). The first 1000 samples were run on a Varian 3700 with manual

PAGE 35

23 injection and strip chart recorder. All subsequent determinations were performed on a Hewlett-Packard 3690a with automatic sampler, integrator, and flame ionization detector. Both the injector and detector temperatures were 250C. The oven temperature was programed for an initial temperature setting of 190C for 3 minutes, then increasing at the rate of 3C per minute until reaching a final temperature of 220c. The column was a 2 m glass column packed with 10% cyanosilicone (Supelco SP2330) on 100/120 Chromosorb WAW. The detector, column, and settings were the same for both instruments. The relative proportions of the fatty acids were calculated as the percent of the total area under the recorded peaks. Frequency distributions of the fatty acid phenotypes were recorded. Individuals were arranged by percent oleic acid, in classes of 4% increments. Segregating ratios were tested to determine the goodness-of-fit to proposed genetic ratios using the chi-square test. The crosses F435 X F78114 and F435 X F519-9 were repeated in the summer of 1987 and an additional cross of F435 X PI 262090 was also included. The F 1 seed produced from these crosses were analyzed using the same aforementioned procedure. The embryo ends of these seeds were saved and planted to produce subsequent generations. The F 1 , F 2 , F 3 , and backcross generations were produced in a greenhouse at the University of Florida Agronomy Farm near

PAGE 36

24 Gainesville. Seed of the F 2 generation were analyzed for fatty acid composition with same technique, and the embryo ends were planted in the same greenhouse. The F 3 seed produced were also analyzed for fatty acid composition. Backcrosses were also made between F 1 progeny from the F435 X F78114 cross and each parent, and backcrosses between F 1 progeny of the F435 X F519-9 cross and each of those parents. All seed were analyzed for fatty acid composition using the method previously described. Results and Discussion Saponification Versus Direct Esterification Saponification of oil breaks the ester linkages that bind fatty acids to glycerol. Therefore, GC analysis of methyl ester preparations from saponified oil would represent total fatty acid content. However, if the relative proportions of free fatty acids and fatty acids released by the esterification reagent (BF 3 ) were found to be in the same proportions as total fatty acids, saponification would not be necessary. When fatty acid profiles of four different peanut oil sources saponified versus nonsaponified were compared using at-test, there were no differences in palmitic, stearic, oleic, linoleic, arachidic, nor behenic acid contents between the two procedures (Table 2-2). During esterification with BF 3 , some fatty acids may be dissociated from glycerol. Total

PAGE 37

Table 2-2. A comparison of two methods of fatty acid analysis, saponified versus nonsaponified. Palmitic, stearic, oleic, linoleic, arachidic, and benhenic acid contents are shown from four different oil sources. Also shown are values of tea Le. F435 F519-9 F78114 Commercial oil non non non non Fatty Acid sapon sapon sapon sapon sapon sapon sapon sapon *tcalc Palmitic 6.7% 9.2% 9.3% 10.6% 8.2% 10.1% 10.6% 11. 5% 1.70 Stearic 2.0% 3.2% 2.0% 1.8% 3.7% 2.6% 1.9% 1. 7% 0.30 Oleic 80.8% 79.% 54.2% 53.9% 48.2% 47.6% 47.3% 47.4% 0.04 Linoleic 2.5% 2.9% 26.5% 26.7% 35.8% 35.2% 32.1% 33.4% 0.03 Arachidic 1.1% 2.5% 1.2% 1.0% 0.4% 0.5% 1.0% 0.9% 0.80 Behenic 1.4% 2.5% 1.4% 0.9% 0.3% 0.4% 1.5% 1.0% 0.04 * tcalc compares the means of each fatty acid saponified versus nonsaponified. N Ul

PAGE 38

26 area integrated on the chromatograph is an indicator of sample concentration. If samples were measured approximately equal prior to saponification and/or esterification, then the difference in area integrated between the saponified and nonsaponified samples should be equal to the difference between the total fatty acid content and the free fatty acid content of peanut oil. It has been reported that more than 95.0% of peanut oil is made up of triacylglycerides and less than 0.05% free fatty acid (Sanders, 1980a). The difference in integrated areas on the chromatograph between saponified and nonsaponified is not as great as the difference between percent triacylglycerides and percent free fatty acids (Table 2-3). This difference indicates that some saponification is occurring but that the BF 3 is not strong enough for complete saponification. However, the degree of saponification by BF 3 is apparently sufficient to produce a representative sample of the total fatty acid content in peanut oil. Since saponification was found to be unnecessary to determine the content of the fatty acids of interest in this study, the saponification step was omitted. Crosses with F78114 When the high oleic acid line, F435, was crossed with F78114, all F 1 seed had oleic acid levels similar to the F78114 parent (Table 2-4), regardless of the genotype on which the seed were borne. F 2 seed from this family

PAGE 39

27 Table 2-3. A comparison of total areas integrated on chromatographs of saponified oils versus nonsaponified oils. Four different oil sources are included. Also included is the percent of the total fatty acids measured when saponification is performed that are measured when saponification is not performed. Area Integrated Source Saponified Nonsaponified % Nonsaponified* F435 3,048,100 374,215 12.3% 519-9 3,999,500 374,530 7.3% F78114 3,567,899 289,789 8.1% Commercial oil 4,699,600 604,060 12.9% * calculated by area nonsaponified divided by area saponified, times 100.

PAGE 40

Table 2-4. F 1 progeny from the cross between high (F435) and normal (F519-9), (F78114), and (PI 262090) oleic acid phenotypes. Oleic Acid Classification Phenoty12ic Range Cross Normal High % Oleic % Linoleic F78114 X F435 37 0 38.0-65.1 17.2-40.3 F435 X F78114 30 0 36.7-60.4 25.8-36.7 F519-9 X F435 9 0 44.3-70.5 11.4-36. 7 F435 X F519-9 20 0 42.0-71.4 11. 3-39. 3 PI 262090 X F435 13 0 61. 4-67. 9 14.9-20.7 F435 X PI 262090 31 0 61.2-69.8 11.4-19.2 f\J 00

PAGE 41

29 displayed bimodal distributions (Figure 2-1), and were classified as either normal oleic acid (less than 70%) or high oleic acid (70% or greater). This division between high and normal oleic acid content was established from parental phenotypes (Table 2-1) used in the crosses. There was a well defined break in the phenotypic distributions of the F 2 populations which allowed for definitive grouping of individuals into either high oleic acid or normal oleic acid classes. A test of homogeneity was conducted on all F 2 families and there were no differences among families (Table 2-5). In the F 2 segregating population the proportion of seed in the two categories (Table 2-5) was consistent with a 15:1 ratio, indicating that two recessive genes were responsible for the high oleic acid characteristic. F 1 plants used as both male and female parents in crosses with F78114 produced all normal oleic acid seed in the BC 1 generation (Table 2-6), and the backcross to F435 produced a phenotypic distribution consistent with the expected ratio of 3:1 (normal to high oleic acid seed). F 2 embryo ends of sampled seed were used to generate F 3 families. Three F 3 families (Table 2-7), derived from high oleic acid seed, consisted entirely of the high oleic acid phenotype. F 3 families, derived from normal oleic acid seed, consisted of either all normal oleic acid seed or normal to high oleic acid phenotypes in 15:1 or 3:1 ratios.

PAGE 42

30 Cl) 25 0 :l "'C > 20 "'C C \to15 0 'Cl) .!l 10 E :l z 5 5 0 ~u:JiC~..ca..llQI..Da..llOILDCl..110d.J~~~04.1~~61.J&!sa.AZl..21ill_. __ _.fSi~~!RSa..t21J ,,F.... 'l-1!0 .... ~~Ar .... ~'='-;,. .... .... Ar~-,. Ar 1-J. ':J ,-,. ':J'='-,. ':J~-,. E>~-,. o1-,. 1 '-,. 1'='-,. 1q-J. ,,_g .., J .Jg "',,. 1rE>.... c-:,0.... c-:,Ar.... ':J'6.... 0 '}..... 0 E>.... 10.... 1 Ar.... 1'6 .... Percent Oleic Acid Figure 2-1. Frequency distribution of number of F 2 offspring in phenotypic classes based on oleic acid content. Data has been pooled for all families from the cross of F78114 and F435. w 0

PAGE 43

31 Table 2-5. Segregation data for F 2 progeny from the cross between high (F435) and normal (F78114) oleic acid lines. Oleic Acid Classification Family Cross Normal High xz 15:1 1 F78114 X F435 49 3 0.02 .90-.75 2 F78114 X F435 32 2 0.13 .75-.50 3 F435 X F78114 56 5 0.77 .50-.25 4 F435 X F78114 40 0 2.67 .25-.10 5 F435 X F78114 28 3 0.62 .50-.25 6 F435 X F78114 26 3 0.77 .50-.25 7 F435 X F78114 18 1 0.03 .90-.75 Pooled 249 17 0.01 .95-.90 Homogeneity 5.00 .75-.50

PAGE 44

32 Table 2-6. BC 1 oleic acid phenotypic segregation ratios for two peanut lines (F78114 and F519-9) crossed to a high oleic acid peanut line (F435). Observed Ex:gected Low High ratio Cross Oleic Oleic Low:High p F435 X F78114 BC (F 1 X F78114) 17 0 1:0 BC (F 1 X F78114) 37 0 1:0 BC (F 1 X F435) 11 1 3:1 .2-.s F435 X F519-9 BC (F 1 X F435) 7 4 1:1 .2-.s BC (F 1 X F435) 6 5 1:1 .5-.9

PAGE 45

Table 2-7. Phenotypic segregation of F 3 families from crosses between high and normal (F78114 or F519-9) oleic acid peanut lines. Oleic Acid Com2osition F3 Observed F 3 Expected Cross F2 Normal High Ratio F78114 X F435 High 0 54 all high F78114 X F435 High 0 42 all high F78114 X F435 High 0 40 all high F78114 X F435 Normal 47 3 15:1 F519-9 X F435 High 0 45 all high F519-9 X F435 Normal 20 0 all normal F519-9 X F435 Normal 20 0 all normal F519-9 X F435 Normal 16 0 all normal F519-9 X F435 Normal 20 5 3:1 F519-9 X F435 Normal 20 5 3:1 F519-9 X F435 Normal 35 15 3:1 F519-9 X F435 Normal 39 11 3:1 F519-9 X F435 Normal 36 14 3:1 (F435) w w

PAGE 46

34 Cross with F519-9 F 1 seed from the cross of the 'Sunrunner' component line F519-9 with F435 also showed no high oleic acid types, nor were there any reciprocal-cross differences. F 2 seed from this cross also showed a bimodal distribution (Figure 2-2), with seed containing between 45% and 70% oleic acid classified as normal and those containing between 70% and 85% oleic acid classified as high. Classification ambiguities were resolved by considering the proportion of linoleic acid in the seed. All seed classified as high oleic acid had less than 5% linoleic acid, whereas all seed classified as normal oleic acid had more than 10% linoleic acid. F 2 seed from this cross segregated in a 3:1 ratio (Table 2-8) of normal to high oleic acid level, indicating that a single-recessive-gene difference between F435 and F519-9 was responsible for the high oleic acid, with the homozygous recessive condition required for expression of the high oleic acid character. The embryo-ends of the sampled F 2 generation seed were planted to produce F 3 families. In the families analyzed, the F 3 data were consistent with the single-recessive-gene hypothesis (Table 2-7). High oleic acid seed were expected to be homozygous recessive and would breed true in the subsequent generation. The normal oleic acid seed were expected to be either homozygous dominant or heterozygous and either breed true for normal oleic acid or segregated in

PAGE 47

en C "'O > "'O C '+0 L. Cl> ..c E z 35 30 29 25 20 15 10 5 1 0 0 0 1..1&,;1--.........&,;L&.1~.IIUllll~l..&.lu.,a~&.L,1~~~1,.&;lL,l.ll~;,m.J~ca,..aci:L.,CC:LJCa.Jll~:lCI..ICQ.mJILCIC:1J ':,-,. ~~-,_ 6r "!,-,. 6r 1-t':, ,-,. ':,':,-,. ':,~-,. & "!,-,. &1-,. 1 \-,_ 1 ':,-,. 1 ~-,_ tt,"!J-,. "!JAr,... "!J't>,... t, 'i,... t,&,... ':,0,... ':,Ar,... ':,f>,... 0 'i,... 0 &,... 10,... 1 Ar,... 1'6,... tt,'i,... Percent Oleic Acid Figure 2-2. Frequency distribution of number of F 2 offspring in phenotypic classes based on oleic acid content. Data has been pooled for all families from the cross of F519-9 and F435. w U1

PAGE 48

Table 2-8. Phenotypic segregation of oleic acid content for F 2 progeny from the cross between high (F435) and normal (F519-9) oleic acid peanut lines. Oleic Acid Corngosition Family Cross Normal High xz .E (3:1) 1 F519-9 X F435 65 19 0.25 .75-.50 2 F519-9 X F435 32 8 0.53 .50-.25 3 F519-9 X F435 34 13 0.18 .25-.10 4 F519-9 X F435 28 12 0.48 .50-.25 5 F519-9 X F435 9 4 0.23 .75-.50 6 F435 X F519-9 9 5 0.86 .50-.25 7 F435 X F519-9 30 6 1.33 .25-.10 8 F435 X F519-9 25 5 1.11 .50-.25 Pooled 232 72 4.07 .90-.75 Homogeneity 0.90 .99 36

PAGE 49

a ratio of 3:1, normal oleic acid to high oleic acid content. 37 When F 1 plants from the F519-9 X F435 cross were backcrossed to the F435 parent, offspring fit a 1:1 ratio of high to normal oleic acid (Table 2-6). This is again consistent with a single-gene hypothesis, where a homozygous recessive (F435) is crossed to a heterozygote (F519-9) and result in a phenotypic ratio of 1:1. Cross with PI 262090 F 1 seed from the cross of F435 with the PI 262090 were found to have oleic and linoleic acid contents similar to the normal oleic acid parent (PI 262090) (Table 2-4). All F 2 families from this cross segregated in a 3:1 ratio of normal to high oleic acid phenotypes (Table 2-9) (Figures 23) Summary Simple inheritance of fatty acid variants have been reported in other oilseed crop species. In sunflower, (Helianthus annuus L.) it has been reported that a single partially dominant gene is responsible for a high oleic acid phenotype (Urie, 1985). In soybean [Glycine max (L.) Merr.J, two additive alleles at a single locus were found to control linoleic acid content (Wilcox and Cavins, 1985). Induced mutants in rapeseed (Brassica napus L.) produced high linoleic acid and low linolenic acid oil by the effects of two additive alleles at each of two independent loci

PAGE 50

38 Table 2-9. Segregation data for F 2 progeny from the cross between high (F435) and normal (PI 262090) oleic acid lines. Oleic Acid Com12osition Family Cross Normal High xz ( 3: 1) 1 PI 262090 X F435 19 7 0.05 .90-.75 2 PI 262090 X F435 18 6 0.0 1.00 3 F435 X PI 262090 18 6 o.o 1.00 4 F435 X PI 262090 27 6 0.82 .50-.25 5 F435 X PI 262090 23 7 0.04 .90-.75 Pooled 105 32 0.20 .75-.50 Homogeneity 0.71 .50-.25

PAGE 51

Cl) 0 :::, -0 > -0 C 0 L. Q) ..0 E :::, z 20 19 15 10 5 01,.Jg~-ex~..DC101-~~IQQa...1~:lll....ll1001L..M~1..1i0.oa....Dir:~...D0.o...~::.a....ooa..J~~~._.~u ,-,. ~~-,. ~':J-,. ~1-I~~-,. o ,-,. o~-,. o':J-,. 01-Io~-,. 1 '-,. 1~-,. 1':J-,. 11-I1~-,. ,-,. ':Jo" ':J'J:' ':J,_,, ':J 0 " ':Jto" oo" o'l:' 01c" o 0 " o"" 10" 1'J:' 11c" 1" 1to" too" Percent Oleic Acid Figure 2-3. Frequency distribution of number of F 2 offspring in phenotypic classes based on oleic acid content. Data has been pooled for all families from the cross of PI 262090 and F435. l,J \D

PAGE 52

40 (Brunklaus-Jung and Robbelen, 1987). Results presented here indicated major genes control the oleic and linoleic acid content in peanut. Together, the F 1 , F 2 , F 3 , and BC 1 generation data from all three peanut crosses reported, support the hypothesis that the high oleic acid character is controlled by two recessive genes. While the combination of the two genes has not been reported previously, the current study showed one recessive gene to be present in two separate lines (F519-9 and PI 262090). This information about the simple inheritance of high oleic acid in peanut will facilitate genetic improvement of the nutritional quality and storage stability of peanut oil. Transfer of this high oleic acid characteristic to desirable lines and cultivars may be accomplished by traditional backcross breeding. Also, with the development of protocol appropriate to peanut, it may be possible to move this character within Arachis and to other species through molecular genetic methods.

PAGE 53

CHAPTER III VARIATION IN FATTY ACID COMPOSITION IN DEVELOPING SEED OF Arachis hypogaea L. Introduction A number of studies have been conducted to assess changes in fatty acid composition of developing peanut seed (Holaday and Pearson, 1974; Sanders, 1980a; Sanders, 1980b; Sanders et al., 1982; Worthington, 1969; Young et al., 1968). In these studies, seed from several peanut cultivars commonly cultivated in the southeastern U. s. were assayed for fatty acid and lipid class composition over the course of seed development. The general conclusion of past studies and of principal interest in this study is that the oleic acid content of the oil increased during seed development. It was also noted that palmitic acid decreases as seed matures, and that the linoleic acid composition was relatively stable, though some researchers report a slight decline (Sanders et al., 1982; Young et al., 1968). The high level of oleic acid in peanut line F435 was established in Chapter II to be controlled by two recessive genes. Those two genes are assumed to produce a pronounced alteration of one or more enzymes involved in fatty acid synthesis. Since previous work has shown that oleic acid varies over the course of development in common cultivars, 41

PAGE 54

42 it was thought that there may be additional variation in oleic acid production or rate of production in the developing seeds of line F435. If a unique pattern of oleic and/or linoleic acid production was observed in the F435 line as compared to normal lines, this information could be of considerable value in understanding the biochemical control of linoleic acid synthesis. This information could also be important in the molecular isolation of the gene through further understanding of the fatty acid synthase system. An impediment to developmental studies in peanut has been the lack of a uniform method to determine physiological maturity. Days from pegging is unreliable since the rate of maturation is affected by the location of the peg on the plant. Proposed methods for determination of maturity have included visual examination of the color and structural characteristics of the pod mesocarp (Williams and Drexler, 1981), visual classification of reproductive growth stages (Boote, 1982), dry matter deposition, and level of free arginine in the seed (Tai and Young, 1977). Two of these methods were employed in the current study, the non destructive method by Williams and Drexler and dry matter deposition.

PAGE 55

43 Materials and Methods On February 3, 1989, seeds of three peanut lines were planted in pots in a greenhouse. The lines planted were F78114, F519-9, and F435. F78114 is low in oleic acid; F519-9 is moderate in oleic acid; and F435 is high is oleic acid (Table 3-1). Five seed of each line were planted in each of four pots. One week after emergence, plants were thinned to one plant per pot. After 122 days, plants were removed from the pots and ten pods from each plant were sampled. Due to the indeterminate nature of peanut, a sample of ten pods was considered adequate to represent the greatest variation in maturity possible for each individual plant. Maturity was evaluated using the non-destructive method by Williams and Drexler (1981). This rating system consists of seven developmental stages. Within each stage, there are four distinct subclasses. For the purposes of this study the developmental stages and subclasses were numbered consecutively from 1 to 28 with 1 corresponding to stage 1 subclass g and 28 corresponding to stage 7 subclass g. A total of forty seed per genotype, ten seed per plant, was sampled for analysis. The maturity rating was recorded and the seed immediately sampled for dry matter. In each pod, the seed proximal to the peg was used as the sample

PAGE 56

Table 3-1. Oleic and linoleic acid content of three peanut breeding lines. Fatty Acid % Oleic % Linoleic Genotype Mean Range Std. Err. Mean Range Std. Err. F78114 45.4 43.3-46.4 0.42 34.3 32.6-36.8 0.55 F519-9 55.6 50. 9-61. 5 1.08 25.9 21.1-30.4 0.99 F435 80.l 72.6-82.3 1.01 2.2 3. 6-1. 2 0.22 Based on a 10-seed sample from each line.

PAGE 57

45 seed. These seed were weighed and placed in a temperature controlled oven at 110c. After at least 17 hours the seeds were removed and allowed to cool in a desiccator, then weighed again. The percent dry matter was calculated from these weights. After dry matter was determined, the dried whole seeds were chopped and soaked overnight in approximately 2 ml of petroleum ether. The petroleum ether was then pipetted into a 5 ml reaction vial. The petroleum ether was removed by evaporation, leaving the oil extracted from the seed sample. The oil samples in the reaction vials were then ready for saponification. To the reaction vials containing the oil extracts, 2 ml of 10% KOH in methanol/water (4:1 v/v) was added and the vials heated in a water bath to ao•c for 90 minutes. The vials were then cooled and 1 ml of 1.8 H H 2 So 4 added to each. One ml of petroleum ether was added to each vial and vigorously shaken. The vials were allowed to stand for a phase separation but in some cases centrifugation was necessary. The upper petroleum ether phase was pipetted off and this extraction procedure repeated. The free fatty acids in the 2 ml sample of petroleum ether from each sample were then esterified as previously described (Chapter II) using boron trifluoride in methanol as the esterification reagent. Samples of the fatty acid methyl esters in petroleum ether were then injected into a Hewlett-Packard model 5890a gas chromatograph, also as

PAGE 58

46 previously described (Chapter II). Fatty acid percentages were calculated based on percent area by a Hewlett-Packard 3392 integrator. Dry matter, palmitic acid, oleic acid, and linoleic acid percentages were collected and recorded along with maturity ratings. Linear and non-linear regression analyses were performed on the data and the corresponding correlation coefficients were calculated. Correlation comparisons were made between maturity rating and dry matter accumulation for each genotype. Correlation comparisons for each genotype were also made between dry matter and percent oleic acid, dry matter and percent palmitic acid, and dry matter and percent linoleic acid. Results and Discussion The method of maturity rating by visual examination of pod mesocarp was initially performed to get a quick estimate of maturity prior to chemical analysis. Dry matter deposition was expected to be the preferred method of measuring maturity because of its objectivity over the more subjective visual rating. The two methods had not been previously compared. Non-linear regression analysis showed that a logarithmic function most accurately described the correlation between dry matter and maturity rating for all genotypes. The highest R 2 values for dry matter versus maturity rating were found in the analysis of the F519-9 seeds (Table 3-2) (Figure 3-1). The highest R 2 value for the

PAGE 59

Table 3-2. R 2 values of individual plants and for a composite of all plants for each genotype for maturity versus percent dry matter. Genotype Plant Rz F519-9 1 0.87 2 0.84 3 0.46 4 0.92 1-4 0.64 F435 1 0.47 2 0.47 3 0.69 4 0.37 1-4 0.43 F78114 1 0.59 2 0.54 3 0.59 4 0.55 1-4 0.49 47

PAGE 60

% Dry Matter :: L ... .. ........................... l 60 ........ . D .... .. ~ i:t .... . .. . . . .............. !?. .. ................ . ................. . ............ l 50 .. ..... ............................ .......................................... ] 40 ~.... . ...................................................................... ........................................ JO r .................................................................................. :: ............ : .................... .................... ; ................... ................... .................. 0 5 10 15 20 25 30 Maturity Rating X Dry Matter a . R2 0.64 Y 34.1 + 10.5InX Figure 3-1. Regression plot of the percent dry matter versus maturity classification of peanut seed sampled from all four plants of line F519-9.

PAGE 61

49 F519-9 was 0.92 for plant 4. The overall R 2 for F519-9 was 0.64. This value was the highest of the three genotypes. The original work on the maturity rating system used in this study was performed on 'Florunner'. Since F519-9 is closely related to 'Florunner', a different botanical type from F435, and a different market type from F78114, it may be more likely to conform to the 'Florunner' maturity classification. Therefore, the highest R 2 values might be expected for F519-9. The value of R 2 for dry matter versus maturity rating on the F78114 Virginia market type was lower than for the F519-9 runner line (Table 3-3, Figure 3-2). The F435 R 2 value was slightly lower than the F78114 R 2 values (Figure 3-3). Dry matter has been concluded to indicate level of maturity in peanut (Tai and Young, 1977). However, in the same study, it was also shown that the rate of accumulation and total dry matter at maturity may vary among genotypes. Nevertheless, dry matter appears to be a more definitive measure of maturity than the color and morphology of the mesocarp. Maturity based on color and morphological characteristics can be subjective, besides being genotype dependent. Although there was a correlation between percentage dry matter and maturity rating, dry matter was used as the independent variable in the regression analysis of the fatty acids, palmitic, oleic, and linoleic acids. The maturity rating was used only as the independent

PAGE 62

50 variable in regressions on percentage of oleic acid for the three genotypes. By comparison, the R 2 values associated with maturity rating were found to be lower than the R 2 values of regressions where dry matter was the independent variable (Table 3-3, Figures 3-4 through 3-9). The relative oleic acid percentage increased in all genotypes as dry matter increased from 10% to 50% (Table 34). In line F78114, the oleic acid content increased from 36.2% to 44.1%; in line F519-9, the oleic acid content increased from 25.6% to 53.3%; and in line F435, the oleic acid content increased from 73.7% to 76.7%. The R 2 value for F519-9 was the highest of the three genotypes at 0.62. The R 2 for F78114 was 0.24 and for F435 was only 0.13. Though the R 2 values are low, examination of the figures reveals that there was only one sample of F435 that had less than 40% dry matter and even that sample was well within the range of the oleic acid distribution for that genotype. In all genotypes, however, the samples under 40% dry matter had the lowest oleic acid contents. Previous studies have shown that oleic acid increases with maturity (Sanders, 1980b). If more immature seeds had been sampled, under 40% dry matter, with correspondingly lower oleic acid contents, more of a trend may have been established and R 2 values may have been higher. It may be necessary to sample more seeds to show this in all genotypes.

PAGE 63

51 Table 3-3. R 2 values for regressions of the percent oleic acid with maturity rating as the independent variable compared with percent dry matter as the independent variable for three genotypes, F519-9, F435, and F78114. Genotype F519-9 F435 F78114 Maturity 0.30 0.12 0.21 Dry Matter 0.62 0.13 0.24

PAGE 64

% Dry Matter 80 r _ 70 L ................................... .. .. g-.... ht ............................... .. l I 8 so 0 -~ ... ... -... 0 so .. .. .o .. a ............................................................... ................... . 40 ......... ..... ............................................................................. . Jo 20 r _______ 1 ________________________ _, 0 5 R 2 0.-43 Y 39.74 + 9.71nX 10 15 Maturity Rating X Ory Matter B 20 25 Figure 3-2. Regression plot of the percent dry matter versus maturity classification of peanut seed sampled from all four plants of line F435 U1 IV

PAGE 65

% Dry Matter 70 ,------------------------------60 50 8 .. _, •:.:.:• •;;••~~~••~•...,.._..!ff':': -:':' ':': ':: ::-. B a c c c -~ c a ........... g . . . . . ......... C C C C 40 C 30 ......•. .... Q .. ............. .. .................... .. ...................... ............... .... 20 ............ ...................................................................... ........................................... . 10 ______________________ ......_ _____________ __, 0 5 10 15 20 25 Maturity Rating R 20.41 X Ory Matter a V 21.74 + 12.4.X Figure 3-3. Regression plot of the percent dry matter versus maturity classification of peanut seed sampled from all four plants of line F78114. U1 w

PAGE 66

% Oleic Acid 70 60 .. ...................... . CCC C C 8 C C 50 CB C c 0 .................. l;l ............................................................................................................................................ . C 40 ........................................................................................................................................................................................................................ C 30 .................................... .................................... . C 20 0 5 10 15 20 25 30 Maturity Rating " Olelc Acld a R 1 0.30 Y '42.0 + 0.11lnX Figure 3-4. Regression plot of the percent oleic acid versus maturity classification of peanut seed sampled from all four plants of line F519-9.

PAGE 67

% Oleic Acid 82 80 ........................................................................................... 1:J .............................................................................................................................. . C C C C C a 78 ............ _g .. .. .o .... ........ . C a 76 Cl C ......... .. . ........•.•.. ... . c ................... 5 ....... a ................................................... . C 74 C C 72 ~----+-----.J.-----...J-----....L.----....l 0 5 10 15 20 25 Maturity Rating X Oleic Acid a R 1 0.12 Y 74.95 + 1.0llnX Figure 3-5. Regression plot of the percent oleic acid versus maturity classification of peanut seed sampled from all four plants of line F435. Ul Ul

PAGE 68

% Oleic Acid 50 ,------------------------------C C C C C 45 ...a . . 40 35 C C C C CC C D ....... .. ............................. . Cl C CC C .............................................................................................................................. ,_ ................ . C 30 a......-----'------...J------.L--------1------l 0 5 10 15 20 25 Maturity Rating X Olelc Acid B Figure 3-6. Regression plot of the percent oleic acid versus maturity classification of peanut seed sampled from all four plants of line F78114. 01 CTI

PAGE 69

% Oleic Acid :: ~----------------------------------oo-C C C C C C _____ .. ~r,-C so .. ...................... 0 ...... ....... . ............... . .................... . C 40 ..................................... ' .............................................................. . C 30 ......................................................................................... , ................................... . C 20 .._ ___ .._ ___ ........ ___ _._ ___ --1. ___ -J'--,---J.-----l 10 20 30 40 50 60 70 80 % Dry Matter X Oleic Acid R 1 0.12 B Y 8.32 + 0.411nX Figure 3-7. Regression plot of the percent oleic acid versus the percent dry matter of peanut seed sampled from all four plants of line F519-9. U1 ...J

PAGE 70

% Oleic Acid 82 ------------80 ................................................................................................................................................... ,. ........................ .. a a a 'il 78 a 76 a a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . ... . . .... . ................... a ... '=t:i ........... c .... -EJ a 74 .................................................................................................................................................. a a a a 72 -------------------------------20 30 40 50 60 70 80 % Dry Matter " Olelc Acid B R 2 0.13 Y 61.l + J.9lnX Figure 3-8. Regression plot of the percent oleic acid versus the percent dry matter of peanut seed sampled from all four plants of line F435. Ul (X)

PAGE 71

% Oleic Acid 50 .----------------------------a C a 45 a a ........................ '05 ... Q .. 40 a C C C cC ..................................... :a~ ....................................... . C a C 35 .................................................................................................................................... a 30 '---------'------.L.-------L-----.L-----_J 20 30 R 1 0.24 Y 11.3 + 0.22X 1.11 40 50 % Dry Matter X Olelc Acid B 60 70 Figure 3-9. Regression plot of the percent oleic acid versus the percent dry matter of peanut seed sampled from all four plants of line F78114. lJ1 1..0

PAGE 72

60 Table 3-4. The percent oleic acid and standard errors for high, moderate, and low oleic acid peanut genotypes sampled at various stages of dry matter deposition. Percent Oleic Percent F78114 Dry Matter Mean *SE 10-29 36.2 30-39 36.8 1.50 40-49 44.1 1.04 50-59 42.7 0.49 60-69 70-79 * standard error of the mean. 1 Insufficient data. Acid and Standard Error GenotyQes F519-9 F435 Mean SE Mean SE 25.6 45.9 9.64 73.3 53.3 1.49 76.7 0.90 56.5 2.70 77.5 0.51 55.3 0.37 77.5 0.40 77.0 0.96

PAGE 73

61 The proportion of palmitic acid was found to be unchanged over seed development in the F519-9 line {Figure 3-10). However, there were only three seeds sampled that had less than 40% dry matter. Both the F435 and F78114 showed a decline in percent palmitic acid as dry matter increased (Table 3-5). The greatest change in palmitic acid content occurred before dry matter deposition had reached 50%. In line F435, 50% dry matter corresponded to a maturity rating of less than 5 and in the F78114 line less than 10. These maturity ratings correspond to very immature stages of development. The seeds sampled were not only high in moisture but small in size. The oil sample extracted was estimated to be near the minimum limit of the analytical methodology. However, the greatest changes in oil composition appear to be in these earliest developmental stages. A more accurate description of the rate of specific fatty acid deposition may be obtained from sampling seed only in maturity classes under rating ten. Linoleic acid percentages declined slightly from 5.9% to 3.8% during the accumulation of dry matter in the F435 line {Table 3-6 and Figure 3-11). In the F519-9, there may have been a slight increase in linoleic acid content, but no change was indicated in the F78114 line {Figures 3-12 and 3-13). A previous study using 'Florunner' indicated no change in linoleic acid percentage during development {Sanders, 1980b).

PAGE 74

% Palmitic Acid 12 11 ........................................................................................................................................ ........... Cl .................................................................. . C 10 ....................................................................... .. .. C C 9 C C ........... ...................................................... H ... a C C 8 ............................ c ..................... . 7 10 R 1 0.01 20 Y 1.71 0.00IX 30 C 40 50 60 70 80 % Dry Matter X Palmltlc Acid a Figure 3-10. Regression plot of the percent palmitic acid versus the percent dry matter of peanut seed sampled from all four plants of line F519-9.

PAGE 75

63 Table 3-5. The percent palmitic acid and standard errors for high, moderate, and low oleic acid peanut genotypes sampled at various stages of dry matter deposition. Percent Palmitic Acid and Standard Er;ror Genoty:ges Percent F78114 F519-9 F435 Dry Matter Mean *SE Mean SE Mean SE 10-29 15.7 _, 8.9 30-39 14.2 0.56 10.8 1.59 9.0 40-49 11.7 0.35 9.8 0.18 7.8 0.22 50-59 12.2 0.13 9.3 0.21 7.9 0.17 60-69 9.2 0.13 7.6 0.16 70-79 7.7 0.19 * Standard error of the mean. , Insufficient data.

PAGE 76

64 Table 3-6. The percent linoleic acid and standard errors for high, moderate, and low oleic acid peanut genotypes sampled at various stages of dry matter deposition. Percent Linoleic Acid and Standard Error Genoty12es Percent F78114 F519-9 F435 Dry Matter Mean *SE Mean SE Mean SE 10-29 37.8 _, 10.7 30-39 37.1 2.07 30.3 5.65 5.9 40-49 34.2 0.94 26.7 1.39 4.5 0.80 50-59 36.2 0.40 25.5 0.55 4.4 0.31 60-69 25.1 0.46 3.3 0.21 70-79 3.8 0.42 * Standard error of the mean. 1 Insufficient data.

PAGE 77

% Linoleic Acid 8 C 7 ...................................... ..................... C C 6 .. ...................................................................................................... . a a a 5 a a 4 ................................ .................... a a 3 a O a a ...... .. .. ...... .. .. .... ... .. .... .... .. .... .. .. ...... .... u 9 .. .. 0 C rfl lg 2 20 R 2 0.23 Y 7.1 0.07)( 30 40 50 60 % Dry Matter X Linolelc Acid a Figure 3-11. Regression plot of percent linoleic acid versus of line F435. of peanut seed sampled from all four plants 70 percent 80 dry matter O'I Ul

PAGE 78

% Linoleic Acid 40 35 ...................................... Q ............................................................................................... . 30 .................................................... .. e....... c ............................................................ . C ts C 25 C .................................................................. ...... . C C CD 20 15 ........................... .................................................. . 10 .................. c ................................................................................................................................................................................ , ............... . 5 10 R 2 0.11. Y 7.IX 20 30 40 50 60 % Dry Matter X Llnolelc Acid B Figure 3-12. Regression plot of percent linoleic acid versus of peanut seed sampled from all four plants of line F519-9. 70 80 percent dry matter

PAGE 79

% Linoleic Acid 42 .------------------------------a 40 ....................................................................... Cl .............................................................................................. . 38 ............. c ........................................................ a .......... .. ................................. . a a a a 36 _ ............................................................................................................................................................... -............ .. ,A ,_ a u o .. a c 34 .......................... c ......................... ;; .... : ........... 1:1 ... --i .. ;;i .. a---............................ .. 32 ...... .............. ........... ................ ..... ......... ...... .. ......... .. .. .... .. ......... ..... ........ ..... .. a 30 ...... .. .. .. ......... ..... ......... .. .......... .............. .. .... .............. .............................. . !" a 28 I I I I 20 30 40 50 60 70 % Dry Matter " Lfnoleic Acid a R 2 0.0008 Y 35.32 + 0.009X Figure 3-13. Regression plot of percent linoleic acid versus percent dry matter of peanut seed sampled from all four plants of line F78114. ' -..J

PAGE 80

68 One objective of this study was to identify a stage of development where a proportionate increase in the percentage of oleic acid, relative to other fatty acids, may indicate variation in enzyme activity. A change in the relative percentage of fatty acids during seed development may also indicate separate systems operating at different times. Based on dry matter, the difference in oleic acid content from the most immature to most mature was from 73.3% to 77.9%. This amount of variation is minimal and is within the range of normal variation for the line (Table 3-1). The amount of variation in linoleic acid due to maturity was also found to be minimal over the stages of development sampled. These results indicate that there is limited, if any, differential enzyme activity during the development period after 30% dry matter deposition. Since the greatest variation in the fatty acid composition occurs in the earliest stages of development, less than 30% dry matter deposition, it may be valuable to sample more of these immature seeds and more closely examine these developmental stages. If, however, the high oleic acid character is a result of reduced activity of the enzymes in the oleic acid desaturase system, and the fatty acid composition of the oil is relatively stable over development, then the stage of development that would yield the most active transcription of the desaturase enzymes would simply be the stage at which the total oil content is most rapidly increasing. In

PAGE 81

69 'Florunner', total lipid content has been shown to increase most rapidly in the earliest stages of development, stages 1-5 (Sanders, 1980a). However, since rate of dry matter deposition has also been shown to be genotype specific, further investigation would be appropriate to examine the rate of oil deposition in F435 to find the developmental stage where genes for fat synthesis are most active.

PAGE 82

CHAPTER IV RESTRICTION FRAGMENT LENGTH POLYMORPHISM IN THE GENUS ARACHIS Introduction In the development of improved cultivars, genetic markers can be valuable tools for the plant breeder (Helentjaris, 1989). Markers can be used for determining incidence of natural outcrossing, identifying genetic linkages, construction of genome maps, determining degree of relatedness, and differentiating selfs and crosses. In recent years, a new class of genetic markers has been revealed with the use of restriction endonucleases. These markers are referred to as restriction fragment length polymorphisms (RFLP). RFLPs have several advantages over morphological markers. In many crops they are more numerous than morphological markers (Beckmann, 1983). There is also a lack of dominance in RFLP markers. There are no multiallelic forms in RFLP markers nor pleiotropic affects on economically important traits. RFLP markers also have potential in mapping quantitative traits, screening genetic resources for important quantitative traits, determining the relationship between quantitative trait loci and "Mendelian" 70

PAGE 83

genetic loci, isolation of causative genes, varietal identification, and determining ancestry and taxonomic relationships (Helentjaris, 1989). 71 RFLP clone sets and linkage maps are currently available in maize (Zea mays L.), tomatoes (Lycopersicon esculentum Mill.), Brassica spp., wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), lettuce (Lactuca sativa L.), soybeans [Glycine max (L.) Merr.J, and rice (Oryza sativa L.). The establishment of protocol specific to peanut is necessary to begin the development of the RFLP clone sets and linkage maps. It has been noted that there are substantial RFLP marker differences between species and genera. Considerable information has been obtained on maize and Brassica. However, self-pollinated species, such as tomato, wheat, and soybean, have not yielded much polymorphism among cultivars and mapping has been difficult (Apuya et al., 1988; Keim et al., 1989; Helentjaris et al., 1986). Peanut is also self-pollinated, and like soybean, it is very uniform with few morphological markers. There are also few isozyme markers in peanut (Cherry and Ory, 1972; Thomas and Neucere, 1974). To date, no work has been published on RFLPs in peanut, (Arachis spp.). With an economically important trait, such as the high oleic acid character in peanut, there can be significant advantages to the development of RFLP markers. Varietal identification can be important, especially since a U. s.

PAGE 84

72 patent application has been filed for the the high oleic acid trait. The isolation of DNA fragments unique to the high oleic acid line may lead to the molecular characterization of the gene. The identification of linkage between the simply inherited high oleic acid character and quantitatively inherited characters could also benefit breeders in the development of improved cultivars. Initial steps in development of RFLP in peanut include: the isolation of purified nuclear DNA; the construction of a genomic DNA library; the isolation of suitable random genomic DNA clones for use as labeled probes; and the hybridization of labeled probes to Southern blots to identify polymorphism. These initial steps were the objectives of this study. All of these steps are described herein, with the exception of the genomic DNA library which was constructed by Dr. M. K. U. Chowdhury. This DNA library was constructed from genomic DNA extracted from the high oleic acid peanut line, F435. The F435 DNA was restriction digested with PstI and cloned with vector pUC19. Clones derived from this library were used as probes in screening for RFLP among the Arachis species and genotypes. Materials and Methods DNA was isolated from four lines of Ahypogaea: line F435, the high oleic acid line; line F519-9, a 'Sunrunner' component line; line F78114, a high yielding Virginia botanical type; and PI 262090, a Virginia botanical type

PAGE 85

73 plant introduction. In addition, DNA was isolated from four species of perennial peanut: Arachis (not speciated); Aglabrata; Apintoi; and Arepens. Two methods of DNA isolation were employed and compared. One was a modification of the method used by Saghai-Mahoof et al. (1984) using a buffer of alkyltrimethyl-ammonium bromide (CTAB extraction). The other was a modification of the potassium acetate precipitation method of Dellaporta et al. (1983). DNA Extraction Extraction 1 (CTAB) Young mature leaf tissue was collected from the selected peanut lines and species. After collection it was immediately frozen in liquid nitrogen and stored at -1o•c. The tissue was removed from the -70C storage as needed. Prior to extraction the tissue was ground under liquid nitrogen to a fine powder and again stored at -70C until needed. Total cellular DNA was isolated from the powdered leaf tissue by a modified version of Saghai-Maroof et al. (1984). To approximately 500 mg of each ground tissue was added seven ml of CTAB extraction buffer [50 mM Tris (pH 8.0), 0.7 M NaCl, 10 mM EDTA, 1% hexadecyltrimethylammonium bromide (CTAB), 0.1% 2-mercaptoethanol (BME)] . The tissue and buffer was mixed vigorously to a homogenous suspension, then incubated at 65C for 90 minutes. During incubation, tubes

PAGE 86

74 were mixed by inversion every fifteen minutes. After incubation, tubes were air cooled for five minutes. Then 4.5 ml of chloroform/isoamyl alcohol (24:1) was added and the tubes mixed by inversion for five minutes. Tubes were then centrifuged at room temperature for 10 minutes at 2500 rpm. After spinning, the upper aqueous layer was pipetted off into new tubes, and extracted with equal volume of the chloroform/isoamyl alcohol (24:1). Again, tubes were mixed by inversion for five minutes then centrifuged for 10 minutes, and the supernatant pipetted off into new tubes. To these tubes were added 50 l of 10 mg/ml RNase A (Promega Inc.) in 10 mM Tris-HCl (pH 7.5) and 15 mM NaCl. The tubes were then mixed by inversion for five minutes and incubated at room temperature for 30 minutes. After incubation, DNA was precipitated by adding equal volume of isopropanol to the tube and mixing gently by inversion. Precipitated DNA was removed with a wire hook and rinsed first with 3-4 ml of 0.2 M NaOAc in 76% ethanol. The precipitate remained suspended in the ethanol for 20 minutes. The precipitate was then rinsed with 10 mM NH 4 0Ac in 76% ethanol and transferred to a 5 ml Eppendorf tube containing 1.0 ml TE [10 mM Tris and 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0]. Quantity and quality was determined by measuring absorbance at 260 nm and 280 nm. DNA quality was further confirmed by agarose gel electrophoresis. DNA samples from each of the lines and species were diluted to uniform

PAGE 87

75 concentrations. Restriction endonuclease digestions were made using DNA and 30 units of EcoRI restriction enzyme according to the manufacturer's recommendations. Three l of RNase mix were also added and the tubes incubated at 37C for five hours. Two l of loading buffer were added and each digestion loaded onto an agarose gel. A lambda HindIII-digested marker was also included in each gel as a reference for measuring fragment sizes. The gel was run for fifteen hours at 22 volts and 65 milliamps. It was then rinsed with distilled water, stained with 500 l of ethidium bromide, and photographed under 300 nm wavelength ultraviolet light. The photographs were then examined to evaluate the quality and the concentration of the DNA extracts. Extraction 2 (potassium acetate) The eight ground frozen tissue samples used in the extraction 1 method were weighed to the same approximate quantities (500 mg) and placed into eight separate 30 ml Oak Ridge tubes. To each tube was added 15 ml of extraction buffer [50 mM EDTA (pH 8.0), 100 mM Tris (pH 8.0), 500 mM NaCl, 10 mM BME] and 1.0 ml of 20% sodium dodecyl sulfate (SOS). The contents of the tubes were then mixed thoroughly and incubated in water bath at 65C for ten min. The tubes were then cooled and 5.0 ml of 5 M potassium acetate were added to each. The contents of the tubes were then mixed vigorously followed by incubation at oc for 20 minutes.

PAGE 88

76 After incubation, the tubes were centrifuged in a refrigerated centrifuge at 25,000 X g for 20 min. The supernatant from each tube was poured through Miracloth filter (Calbiochem) into clean 30 ml tubes, and the DNA precipitated by adding equal volume of isopropanol. The contents of the tubes were mixed gently, incubating at -20c for 30 min. The precipitated DNA was pelleted by centrifugation at 20,000 X g for 15 minutes. The supernatant was gently poured off and the pellet dried by inverting the tube over a paper towel for 30 min. The DNA pellets were then removed from the Oak Ridge tubes and placed into Eppendorf tubes with 700 l of DNA buffer, TE. After the pellets were dissolved, the tubes were spun in a microfuge for 10 minutes to remove insoluble debris. The supernatants were then transferred to new Eppendorf tubes and purified with a phenol/chloroform extraction. An equal volume of phenol was added to each tube and mixed gently by inversion. The upper buffer phase was pipetted off into clean Eppendorf tubes and equal volumes of phenol/chloroform (1:1) added and gently mixed. The upper buffer phase was pipetted off and mixed with an equal volume of chloroform. These upper buffer phases were then pipetted off into clean Eppendorf tubes to each of which was also added 75 l of 3M sodium acetate and 500 l of isopropanol. After mixing, the DNA was pelleted by microfuge spinning for 10 seconds. The supernatant was discarded and the pellets washed with 80%

PAGE 89

77 ethanol. The pellets were redissolved in 400 l of DNA buffer TE. Quality and quantities of the DNA extracts were determined as in the extraction 1 procedure. The extraction method 1 was repeated using tissue samples collected from the same species and genotypes. For this extraction, the samples collected were immature not fully expanded leaf tissue plucked from stem apices. For the first extraction, leaves were young but fully mature. Southern Blotting Samples of DNA extracted from the four genotypes of~ hypogaea and the four Arachis species that had been determined to be of acceptable quality were digested with EcoRl and run out on a gel as previously described. A lambda marker was also included on the gel. Gels were stained and photographed, also as previously described. The blotting procedure used was adapted from E. M. Southern (1975). After staining and photographing, gels were treated for 10 min. with a 0.25 M solution of HCl. The gels were then rinsed twice and soaked with constant stirring for 45 minutes in a denaturing solution of 1.5 M NaCl and 0.5 M NaOH. The gels were then neutralized by soaking 45 minutes in 1 M Tris with 1.5 M NaCl (pH 8.0), also with constant stirring. DNA was transferred from the gel to Hybond N blotting membrane (Amersham Corp.) using the capillary method described by Maniatis et al. (1982) with 3X SSC (lX SSC= 0.15 M NaCl and 0.015 M sodium citrate). This

PAGE 90

blotting structure was then allowed to stand for 12-24 hours. The filter was then removed from the stack and soaked in 3X SSC for 5 minutes at room temperature. Next, the filter was wrapped in a clear plastic wrap and the DNA side of the filter exposed to ultraviolet light for 6 minutes. The blot was then air dried and stored in a plastic bag at 4C until needed. Probe Preparation 78 Plasmid DNA was isolated by a method adapted from Birnboim and Doly (1979). Inocula from individual library colonies were placed into tubes containing 5 mls of LB broth (10 g Bacto-typtone (Difeo), 5 g yeast extract (Difeo), and 5 g NaCl in 1 liter of water with pH adjusted to 7.2]. The LB broth also contained 0.2% maltose and 0.1 g ampicillin. The genomic DNA used to construct the peanut library was isolated from the high oleic acid peanut line F435. It was digested and cloned into the PstI site of pUC19. The cultures in broth were then transferred to 10 ml centrifuge tubes and centrifuged in a table-top centrifuge for seven minutes. The supernatants were discarded and the pellets resuspended in 800 l of cold STET buffer (80 g sucrose, Triton x-100, 200 ml of 0.25 M EDTA, 50 ml of 1 M Tris-HCl, and deionized H 2 O to a total of 1 L with pH adjusted to 8.0) in 1.5 ml Eppendorf tubes. Each tube then received 60 l of 10 mg/ml lysozyme and was placed in boiling water for two minutes. The tubes were then spun in

PAGE 91

79 a microfuge for 20 minutes. The gelatinous pellets were removed with toothpicks and discarded. Supernatants were purified with phenol/chloroform extraction and digested with RNase A. The plasmid DNA was then precipitated, dissolved in 100 l TE, and stored at -20c as described in the plant DNA isolation above. Clones were evaluated for insert size by digesting 2 g plasmid DNA with 8 units PstI, then electrophoresing and staining as described above. Clones that could be isolated from the vector were selected to be used as probes. Isolation of inserts was done by PstI-digesting 40 l of plasmid DNA as previously described. The separation was by electrophoresis and gels were stained and examined under ultraviolet light to determine the location of the inserts on the gel. Incisions were made in the gel 1-2 mm to the advance of the insert. Pieces of NA45 membrane (Schleicher and Schuell, Inc., Keene, NH), approximately 5 X 25 mm in size, were soaked in gel buffer. One piece of membrane was inserted into each incision. The gel was then returned to the electrophoresis apparatus and run for an additional 30 minutes at the same voltage. The membranes containing bound DNA inserts were rinsed with 400 l of low salt buffer, then eluted with high salt buffer (lM NaCl, 0.1 mM EDTA, 20mM Tris, and 0.5 M Arginine). To elute the DNA, tubes were heated to 10c and after 20 minutes the membranes were turned in the tubes.

PAGE 92

80 After another 25 minutes at 70"C, the membranes were removed from the tubes and the eluate extracted with 400 l of phenol/chloroform (1:1) and again with chloroform. The aqueous phase was then pipetted into another clean Eppendorf tube to which was added 100 l of 7.5 M ammonium acetate and 800 l of absolute alcohol. The tubes were incubated overnight at -2oc. DNA pellets were recovered by microfuging for 15 minutes then washing with 500 l of 70% ethanol. The DNA was air dried and redissolved in 20 l of TE buffer. Two l of each isolated DNA was mixed with 18 l of H 2 0 and run on an agarose gel. The lambda marker was also included on the gel. The relative concentrations and fragment sizes were estimated from this gel. The tubes with the remaining inserts in DNA buffer were stored at -2o•c until needed for oligolabeling. Radio-labeling Probes Probes were labeled with 32 P by the primer extension method of Feinberg and Vogelstein (1983). The method consisted of denaturing the DNA by heating to 95"C for 10 min. then rapid cooling on ice to prevent renaturing. The appearance of the gel from the insert isolation procedure was used to estimate the amount of insert DNA used. Two to four microliters of the insert DNA were placed in a 500 l Eppendorf tube. Also into the tube were placed 10 l of OLB (Finberg and Vogelstein, 1983), 6 l of bovine serum albumen 3mg/ml (BSA), 2 l of Klenow enzyme (lU/l), 4 l of 32 P

PAGE 93

81 labeled deoxycytidine 5 1 -triphosphate (dCT 32 P) (3000ci/mM), and enough double-distilled H 2 0 to bring the total volume to 50 l. The tubes were then incubated at 37C for 30 minutes. Then to each tube 50 l of OLB stop mix [2.0 ml of 1 M Tris HCl (pH 7.0), 400 ml 5 M NaCl, 0.5 M EDTA (pH 8.0), and 12.5 l 20% SDS] were added. Unincorporated nucleotides were removed by liquid chromatography with G50 Sephadex in lX NTE buffer (100 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA). Probes were then denatured by boiling for 5 minutes and immediately placed in ice for 5 min. The probes were then ready for immediate use. Prehybridization and Hybridization of Blots Southern blots were pre-hybridized to prepare for probe hybridization. Blots were first soaked for a few seconds in deionized water, then in 3X SSC. Next blots were placed into a heat sealed plastic bag along with 30 mls of prehybridization solution (7% SDS in 3X SSC with 30 l of denatured salmon sperm DNA). Prehybridization was conducted for 3-4 hours at 65C. Denatured probes were then injected into the sealed bag containing the blot and the pre-hybridization solution. Hybridization was conducted for 16 hrs. at 65C. Blots were washed for two times for 15 min. each in 3.5 liters of 3X SSC at 65C. A third rinsing was done at the same temperature and for the same time but in 0.3X SSC. Blots were then removed and allowed to air dry on paper towels.

PAGE 94

82 Using a Geiger counter, the radioactivity was checked to estimate the exposure time necessary for autoradiography. Blots were then place into a film case with a piece of Kodak XAR-5 x-ray film and Cronex Hi-Plus intensifying screens (E. I. Dupont de Nemours and Co.). The film was exposed at -1oc for 3 days to 2 weeks depending on the radioactivity of the blot. After appropriate exposure, the film was developed and the hybridization patterns examined. Results and Discussion The quality and quantity of DNA can be estimated from calculations using the absorbance measured at 260 nanometers (nm) and 280 nm. The absorbance at 260 nm divided by the absorbance at 280 nm indicates the purity of DNA with respect to protein contamination (Berger, 1987). The closer the ratio is to 1.8 the more pure the DNA. Concentrations can be estimated by multiplying the absorbance measured at 260 nm by a factor of 50 and then times the dilution rate of the sample analyzed. Absorbances measured on the DNA extracted using extraction method 1 with young but mature leaf tissue is shown in Table 4-1. Two genotypes, F435 and ~repens, showed ratios close to 1.8 indicating good quality DNA. The absorbance ratios of the other genotypes were less than the 1.8 optimum. None of the genotypes extracted using extraction method 2 were of optimum quality (Table 4-2). Photographs of stained gels and hybridization patterns of mature leaf DNA extracted by both methods were

PAGE 95

83 Table 4-1. Absorbances of DNA extracts from four ft. hypogaea lines and four perennial Arachis species at two wavelengths. Extraction was method 1 (CTAB) using young mature leaf tissue. Absorbances Genotype Wave length Concentration 260nm 280nm 260/280 q/l* F435 0.202 0.114 1.77 1.01 F519-9 0.085 0.052 1.63 0.43 F78114 0.052 0.037 1.41 0.26 PI 262090 0.197 0.137 1.43 0.99 ft. 0.101 0.066 1.53 0.51 ft. glabrata 0.033 0.027 1.22 0.17 ft. pintoi 0.092 0.024 1.56 0.46 ft. repens 0.044 0.024 1.83 0.22 * Calculated by A 2 ~ X 50 X dilution rate of 100.

PAGE 96

84 Table 4-2. Absorbances of DNA extracts from four Ahypogaea lines and four perennial Arachis species at two wavelength. Extraction was method 2 (potassium acetate) using young mature leaf tissue. Absorbances Genotype Wave Length Concentration 260nm 280nm 260/280 uglul* F435 0.053 0.039 1.36 0.27 F519-9 0.037 0.027 1.37 0.19 F78114 0.066 0.047 1.40 0.33 PI 262090 0.037 0.033 1.23 0.19 A.film• 0.058 0.043 1.35 0.29 Aglabrata 0.052 0.040 1.30 0.26 Apintoi 0.045 0.032 1.41 0.23 Arepens 0.072 0.051 1.41 0.38 * Calculated by A 260 X 50 X dilution rate of 100.

PAGE 97

85 compared to determine DNA quality (Figure 4-1). The overall quality of the DNA, as indicated by the gel, was not acceptable because of lane to lane variation in DNA concentration and fragment distribution. There was also narrowing of the lanes which appeared to affect the running rates of the fragments. Narrow lanes ran slower, probably due to compounds binding to the DNA and restricting its migration through the gel. Because extraction method 1 was slightly faster, less complex, and at least equal to or better than, in the quality of DNA extracted by method 2 (Figure 4-2), all subsequent DNA extractions were performed using extraction method 1. To further improve the quality of DNA extracted, another tissue source was examined using extraction method 1. Immature leaves that were not fully unfolded and had not entirely emerged from the apical bud were used as the tissue source. Absorbances were measured and are shown in Table 43. There was improvement of extracted DNA quality from every genotype, as seen by the 260/280 absorbance ratios (Table 4-3). Gels of the endonuclease digested DNA showed a good quality by the even, non-narrowed band width along the length of the lanes (Figure 4-3). Because of the improvement in DNA quality associated with the immature leaf tissue, all subsequent isolations were made from that tissue source.

PAGE 98

86 2 3 4 5 6 7 8 1 F435 2 F519-9 3 F78114 4 PI 262090 5 Afilm• 6 A. glabrata 7 A12intoi 8 Are12ens Figure 4-1. DNA extracts from eight peanut genotypes using extraction method 1 on mature leaf tissue.

PAGE 99

87 2 3 4 5 6 7 8 1 F435 2 F519-9 3 F78114 4 PI 262090 5 Afil2l2. 6 Aglabrata 7 A 12intoi 8 Are12ens Figure 4-2. DNA extracts from eight peanut genotypes using extraction method 2 on mature leaf tissue.

PAGE 100

88 Table 4-3. Absorbances of DNA extracts from four Ahypogaea lines and four perennial Arachis species at two wavelengths. Extraction was method 1 (CTAB) using immature leaf tissue. Absorbances Genotype Wave Length Concentration 260nm 280nm 260/280 g/l* F435 0.179 0.099 1.81 0.90 F519-9 0.148 0.079 1.87 0.74 F78114 0.086 0.047 1.83 0.43 PI 262090 0.093 0.053 1.75 0.46 Afil2R 0.180 0.098 1.84 0.90 Aglabrata 0.088 0.053 1.66 0.44 Apintoi 0.053 0.029 1.82 0.27 Arepens 0.022 0.012 1.83 0.11 * Calculated by A 260 X 50 X dilution rate of 100.

PAGE 101

89 2 3 4 5 6 7 8 1 F435 2 F519-9 3 F78114 4 PI 262090 5 Afilm 6 Aglabrata 7 AQintoi 8 Are12ens Figure 4-3. DNA extracts from eight peanut genotypes using extraction method 1 on immature leaf tissue.

PAGE 102

90 After DNA was isolated, blots were made and probes were isolated from the peanut genomic DNA library (Figure 4-4). Seventy-six library clones were amplified and plasmids were isolated from them. Eighteen of the 76 plasmid inserts were radiolabeled for use as probes (Table 4-4.). In addition to the probes from the genomic peanut DNA library, clones of six maize mitochondrial genes and a rat liver desaturase clone were used as probes (Table 4-5). Five peanut DNA probes and three mitochondrial gene probes produced readable autoradiographs of single or low copy number fragments that could be used in evaluating polymorphism between the genotypes. Unacceptable autoradiographs had poor definition of fragments and in some cases apparent variation in DNA running rates on the original gels that were blotted. Poor definition of fragments is illustrated in Figure 4-5. This type of radiograph can result from a poor quality DNA isolate or from a probe with a complimentary nucleotide sequence occurring at a high copy rate in the DNA on the blot. Variation in DNA running rates on a gel cannot be probe related but only associated with variation in DNA quality. Figures 4-6 and 4-7 illustrate the variation in DNA running rates. DNA from F519-9 (lane 2) on both of the autoradiographs (Figure 4-6 and 4-7) appears to have fragments slightly smaller than DNA from F435 (lane 1) and F78114 (lane 3) on either side. The original gel photograph shows the F519-9 lane wider and with greater accumulation of

PAGE 103

Figure 4-4 Clones separated from pUC. Marker J. 91

PAGE 104

92 Table 4-4. Genomic DNA clones, library cell locations, and approximate sizes of inserts isolated for production of radio-labeled probes. Clones Cell No. Size (basepairs) HPI2 AS 2200 HPI6 All 1000 HPI16 B9 1000 HPI24 D1 5200 HPI33 El 4400 HPI40 Fl 1275 HPI41 F2 600 HPI45 F6 1275 HPI48 F9 850 HPI49 Fll 250 HPI52 G2 1050 HPI54 G4 1050 HPI58 G8 850 HPI60 Gl0 2300 HPI61 Gll 1300 HPI66 HS 600 HPI67 H6 2000 HPI72 Hll 2100

PAGE 105

93 Table 4-5. Gene clones used as radio-labeled probes. Gene Gene Product Source pDs3 Stearyl-CoA desaturase Thiede et al., 1986 atpa ATPase subunit alpha Braun and Levings, 1985 atp6 ATPase subunit 6 Dewey et al., 1985a atp9 ATPase subunit 9 Dewey et al., 1985b coxI Cytochrome c oxidase Isaac et al., 1985 rrn26 26S rRNA Dale et al., 1984 rrn5-rrn18 18S-5S rRNA Chao et al., 1984

PAGE 106

94 3 4 5 6 7 8 --1 F435 2 F519-9 3 F78114 4 PI 262090 5 Afilm• 6 A glabrata 7 A12intoi 8 Are12ens Figure 4-5. Autoradiograph of probe at12a on peanut genotypes illustrating an unacceptable autoradiograph based on poor fragment definition.

PAGE 107

2 3 4 5 lil.Lf. f. '1 1 u~ I I I 1 F435 2 F519-9 3 F78114 4 PI 262090 5 A.film. 6 Aglabrata 7 A. Qintoi 8 Are12ens 6 7 8 .. 4.5 .. 3.0 .. 2.0 Figure 4-6. Autoradiograph of probe HPI16 on peanut genotypes with fragment sizes in kilobases. 95

PAGE 108

2 3 4 5 6 7 8 .. 4.5 1 F435 2 F519-9 3 F78114 4 PI 262090 5 A,. .film• 6 A. glabrata 7 A. :gintoi 8 A. re:gens Figure 4-7. Autoradiograph of probe HPI6 on peanut genotypes with fragment size in kilobases. 96

PAGE 109

97 DNA at the end of the lane than the lanes on either side. This amount of variation visible on the original gel along with the repeating pattern of lane 2 appearing to have slightly smaller fragments, stimulated doubt in the reliability of four autoradiographs of four separate probes. The DNA quality is suspect and the probes should be tested again on blots with better DNA. Acceptable quality radiographs showed that there is DNA fragment length polymorphism within the Arachis genus. Variation in fragments between the species were demonstrated by probes atp6, coxI, HPI72, HPI67, HPI2, HPI58, HPI52, and rrn5-rrnl8 (Figures 4-8 through 4-15). Limited polymorphism was found among the Ahypogaea lines. The most prominent difference found among the four Ahypogaea lines was seen with the hybridization of the coxI probe (Figure 4-9). This autoradiograph showed a fragment absent in F435 that was found in all other lines. Other fragment variations among Ahypogaea lines were produced by probes HPI58, HPI52, and possibly by HPI16 though the quality of the autoradiograph of HPI16 makes it unreliable and the hybridization should be repeated. The relationships between the eight peanut genotypes were analyzed by comparing the number of common fragments between each possible paired combination. Only the best eight autoradiographs were used in this comparison (Figures 4-8 through 4-15). Pair-wise indices of genetic similarity

PAGE 110

2 3 4 1 F435 2 F519-9 3 F78114 4 PI 262090 5 Afilm• 6 Aglabrata 7 A Qintoi 8 AreQens 5 6 7 8 .. 8.0 .. 6.1 .. 4.3 .. 2.0 .. 1.0 Figure 4-8. Autoradiograph of probe atQ6 on peanut genotypes with fragment sizes in kilobases. 98

PAGE 111

2 3 4 5 6 7 8 .. 8.0 +4.3 .. 1.8 .. 1.0 1 F435 2 F519-9 3 F78114 4 PI 262090 5 Afilm• 6 A glabrata 7 A2intoi 8 Are2ens Figure 4-9. Autoradiograph of probe coxI on peanut genotypes with fragment sizes in kilobases. 99

PAGE 112

.23.0 .8.0 .4 . 2 .. 1.1 .0.2 1 F435 2 F519-9 3 F78114 4 PI 262090 5 Afilm 6 Aglabrata 7 A12intoi 8 Are12ens Figure 4-10. Autoradiograph of probe HPI67 on peanut genotypes with fragment sizes in kilobases. 100

PAGE 113

2 3 4 1 F435 2 F519-9 3 F78114 4 PI 262090 5 A gm. 6 Aglabrata 7 A 12intoi 8 Are12ens 5 6 7 8 .. 23.0 .. 9.4 ._ 5.4 .. 4.3 Figure 4-11. Autoradiograph of probe HPI72 on peanut genotypes with fragment sizes in kilobases. 101

PAGE 114

2 3 4 1 F435 2 F519-9 3 F78114 4 PI 262090 5 Afilm• 6 Aglabrata 7 Agintoi 8 Aregens 5 6 7 8 .. 16.0 .. 8.0 .. 3.5 .. 2.2 .. 1.5 Figure 4-12. Autoradiograph of probe HPI58 on peanut genotypes with fragment sizes in kilobases. 102

PAGE 115

2 3 4 1 F435 2 F519-9 3 F78114 4 PI 262090 5 A.film• 6 Aglabrata 7 A. Qintoi 8 AreQens 5 6 7 8 .. 8.0 +3.1 .. 1.9 .. 1.5 Figure 4-13. Autoradiograph of probe HPI52 on peanut genotypes with fragment sizes in kilobases. 103

PAGE 116

1 F435 2 F519-9 3 F78114 4 PI 262090 5 Afilm• 6 Aglabrata 7 A12intoi 8 Are12ens 20.0 6.2 .. 1.9 .. 1.0 104 Figure 4-14. Autoradiograph of probe rrn5-rrn18 on peanut genotypes with fragment sizes in kilobases.

PAGE 117

2 3 4 5 6 7 8 .. 9.8 .. 6.0 .. 1.7 1 F435 2 F519-9 3 F78114 4 PI 262090 5 A. gm. 6 A. glabrata 7 A. 12intoi 8 A,. re12ens Figure 4-15. Autoradiograph of probe HPI54 on peanut genotypes with fragment sizes in kilobases. 105

PAGE 118

106 {Table 4-6) were calculated by dividing the total number of DNA fragments common between two genotypes by the total number of unique fragment size found in the pair of genotypes {Smith et al., 1988). As expected, the highest similarity indices were found between the four Ahypogaea lines. In comparing these four lines the greatest index value was 0.96 found between F78114 and PI 262090. These two lines are both Virginia market-types and are very similar in vegetative growth habit. The other two hypogaea lines {F435 and F519-9) represent two completely different market types. F435 is a Spanish market type, subspecies fastigiata variety vulgaris and the F519-9 line is a runner market type, subspecies hypogaea variety hypogaea. similarity index values calculated between these four lines were as expected based on growth habit and market types. Indices calculated between species showed similar levels of common fragments, whether between one of the Ahypogaea lines and another Arachis species or between two perennial Arachis species. Of the four perennial species, Arepens and Apintoi were most closely related taxonomically. Both have been classified in the same section, Caulorhizae, while Aglabrata and the Afil2P• are in section Rhizomatosae. repens and Apintoi had a similarity index of 0.23, which was not the highest of the species comparisons. The range the indices when two different Arachis species were compared

PAGE 119

Table 4-6. Pair-wise indices of genetic similarity of four Ahypogaea lines and four Arachis species. The similarity index was calculated by dividing the total number of DNA fragments common between two genotypes by the total number of unique fragment sizes represented by the paired genotypes. Genotypes F435 F519-9 F78114 PI 262090 Aglabrata Apintoi 2 0.70 Genotypes* 3 4 5 6 7 8 Similarity Index 0.57 0.58 0.38 0.24 0.24 0.23 0.59 0.61 0.33 0.23 0.27 0.17 0.96 0.24 0.26 0.24 0.26 0.35 0.35 0.28 0.23 0.33 0.38 0.24 0.28 0.32 0.23 *Genotypes: 1-4 Ahypogaea lines l-F435; 2-F519-9; 3-F78114; 4-PI 262090; 5-A . ..PR. line 30b; 6-Aglabrata line 44; 7-Apintoi line 8710; 8-Arepens line 75. I-' 0 -.J

PAGE 120

108 was from 0.17 to 0.38. In comparing two different species, low similarity indices, even between species within the same section of the genus, indicate there are more unique fragments than common fragments between species. These similarity indices then indicate that DNA fragment length polymorphisms are numerous enough to be useful in separating these Arachis species. Further development of RFLPs in peanut may be beneficial as a taxonomic tool where relationships and species demarcations are not clearly defined. The relative lack of polymorphism among hypogaea lines has been observed in other self-pollinated species such as tomato and soybean. Therefore, it would appear that unique DNA fragments are more rare in these genomes. Since only eight probes produced autoradiographs of a quality suitable for evaluation, this is not enough data to suggest fragment length polymorphism cannot be identified in Ahypogaea. Further evaluation using additional unique probes may yield more polymorphic fragments. Other polymorphic fragments may be revealed by different restriction enzymes. By developing more fragment polymorphisms, lines and cultivars of Ahypogaea could be more definitively distinguished. Probes produced from low copy DNA such as mitochondrial genes or probes already known to reveal polymorphism in other self-pollinated species may also be good sources for peanut genome probes.

PAGE 121

CHAPTER V SUMMARY The oil content of peanut is approximately 50%. Since the proportion of oil is so great, the quality of the oil is one of the most important factors in determining the quality of peanut products. Fatty acid composition is an important determinant of oil quality. Therefore, the ability to alter or control fatty acid composition can be a valuable tool to peanut researchers. A number of factors have been shown to affect the fatty acid composition of peanut oil. However, striking differences in fatty acid composition can be achieved by genetic manipulation. With the identification of the high oleic acid peanut line, the known variation of oleic and linoleic acids was sharply increased. Since these two fatty acids are important determinants of peanut oil quality, the increase in phenotypic variation allowed for greater potential of improved oil quality in new peanut cultivars. To use the high oleic acid character in the development of new cultivars it was necessary to determine the mode of inheritance of the character. Crosses were made between the high oleic acid line and three other peanut lines. F 1 , F 2 , F 3 , and backcross generation data showed the character to be 109

PAGE 122

110 controlled by two genes, each with two alleles. The homozygous recessive condition was found to be necessary for the expression of the high oleic acid character. Since the character was found to be simply inherited, further studies were conducted to obtain basic information required in the molecular isolation of the genes. This information included improved understanding of the biochemical synthesis of oleic and linoleic acid and the development of molecular techniques required for molecular genetic manipulations. A study was conducted to determine the developmental changes in the relative proportions of the major fatty acid components of peanut oil. Results corroborate previous research in common peanut cultivars that showed oleic acid increases relative to other fatty acids during development and that linoleic acid remains relatively stable. However, in the high-oleic-acid line it was found that the variation in oleic acid content over development was minimal. Results also indicate that further investigation in changes in fatty acid proportions at more immature stages of development could reveal greater variation in relative proportions of fatty acids. A third investigation was conducted to develop techniques for molecular studies of the Arachis genus. DNA was successfully isolated from five Arachis species and restriction fragment length polymorphisms compared. Molecular variation was found to be limited among the four

PAGE 123

111 A. hypogaea genotypes examined. There was, however, much more variation among the different species examined. It was concluded that for RFLP to be useful in molecular characterization of cultivated peanut additional probes must be tested and DNA from more genotypes evaluated. One fragment difference was found in the high oleic acid line that made it unique from the other Ahypogaea genotypes evaluated. However, further investigation would be necessary to determine if this fragment was a portion of the gene responsible for the high oleic acid character.

PAGE 124

REFERENCES Ahmed, E. M. and c. T. Young. 1982. Composition, quality, and flavor of peanuts. Chap. 17, 655-688. In: Peanut Science and Technology, Editors: H. E. Pattee and c. T. Young. American Peanut Research Education Society, Yoakum, TX. Apuya, N. A., B. Frazier, P. Keim, J.E. Roth, and K. G. Lark. 1988. Restriction fragment length polymorphisms as genetic markers in soybean, Glycine max (L.) merrill. Theoretical and Applied Genetics, 75:889-892. Beckmann, J. S. and M. Soller. 1983. Restriction fragment length polymorphisms in genetic improvement: Methodologies, mapping and costs. Theoretical and Applied Genetics, 67:35-43. Berger, s. L. 1987. Quantifying 32 P-labeled and unlabeled nucleic acids. Chap. 6, 49-54. In: Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques. Editors: S. L. Berger and A. R. Kimmel. Academic Press, Orlando, FL. Birnboim, H. C. and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research, 7(6):1513-1523. Boote, K. J. 1982. hypogaea L.). Growth stages of peanut (Arachis Peanut Science, 9:35-40. Bovi, M. L.A. 1982. Genotypic and environmental effects on fatty acid composition, iodine value, and oil content of peanut (Arachis hypogaea L.). Ph.D. dissertation, University of Florida, Gainesville. Braun, c. J. and c. s. Levings, III. 1985. Nucleotide sequence of the F 1 ATPase alpha subunit gene from maize mitochondria. Plant Physiology, 79:571-577. Bronsgeest-Schoute, D. c., R. J. J. Hermus, G. M. Dallinga Thie, and J. G. A. J. Hautvast. 1979. Dependence of the effects of dietary cholesterol and experimental conditions on serum lipids in man. The American Journal of Clinical Nutrition, 32:2188-2192. 112

PAGE 125

113 Browse, J., P. J. Mccourt, and c. R. Somerville. 1986. Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Analytical Biochemistry, 152:141-145. Brunklaus-Jung, E. and G. Robbelen. 1987. Genetical and physiological investigations on mutants for polyenoic fatty acids in rapeseed (Brassica napus L.). Plant Breeding, 98:9-16. Buchanan, s. ands. Gaylinn (Editors). 1988. 1988 CRB Commodity Year Book. Commodity Research Bureau, New York. Chao, S., R.R. Sederoff, and c. s. Levings, III. 1984. Nucleotide sequence and evolution of the 18S ribosomal RNA gene in maize mitochondria. Nucleic Acids Research, 12:6629-6644. Cherry, J. P. and R. L. Ory. 1973. Electrophoretic characterization of six selected enzymes of peanut cultivars. Phytochemistry, 12:283-289. Cobb, W. Y. and B. R. Johnson. 1973. Physiochemical properties of peanuts. Chap. 6, 209-257. In: Peanuts--Culture and Uses. Editor: c. T. Wilson. American Peanut Research and Education Association Inc., Stillwater, OK. Conn, E. E., P. K. Stumpf, G. Bruening, R.H. Doi. 1987. Outlines of Biochemistry. 5th ed. John Wiley & Sons New York. Crawford, R. V. and T. P. Hilditch. 1950. The component fatty acid and glycerides of groundnut oils. Journal of the Science of Food and Agriculture, 1:372-379. Dale, R. M. K., N. Mendu, and H. Ginsburg. 1984. Sequence analysis of the maize mitochondrial 26S rRNA gene and flanking regions. Plasmid 11:141-150. Dellaporta, S. L., J. Wood, and J. B. Hicks. 1983. A plant miniprepration: Version II. Plant Molecular Biology Reporter, 4:19-21. Dewey, R. E., C. s. Levings, III, and D. H. Timothy. 1985a. Nucleotide sequence of ATPase subunit 6 gene of maize mitochondria. Plant Physiology, 79:914-919.

PAGE 126

114 Dewey, R. E., A. M. Schuster, c. S. Levings, III, and D. H. Timothy. 1985b. Nucleotide sequence of F 0 -ATPase proteolipid (subunit 9) gene of maize mitochondria. Proceedings of the National Academy of Science, 82:1015-1019. Erickson, E. A., J. R. Wilcox, and J. F. Cavins. 1988. Fatty acid composition of the oil in reciprocal crosses among soybean mutants. Crop Science, 28:644-646. Feinberg, A. P. and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry, 132:6-13. Fore, s. P., N. J. Morris, c. H. Mack, A. F. Freeman, and W. G. Bickford. 1953. Factors affecting the stability of crude oils of 16 varieties of peanuts. Journal of the American Oil Chemists Society, 30:298-301. Grande, F., J. T. Anderson, and A. Keys. 1970. Comparison of effect of palmitic and stearic acids in the diet on serum cholesterol in man. American Journal of Clinical Nutrition, 23:1184-1193. Green, A.G. 1986. Genetic control of polyunsaturated fatty acid biosynthesis in flax (Linum usitatissimum) seed oil. Theoretical and Applied Genetics, 72:654661. Gregory, w. c., A. Krapovickas, and M. P. Gregory. 1978. Structure, variation, evolution, and classification in Arachis. Pages 469-481. In: Advances in Legume Science, Editors: R. J. Summerfield and A.H. Bunting, Royal Botanic Garden Surrey, England. Grundy, S. M. 1986. Comparison of monounsaturated fatty acids and carbohydrates for lowering plasma cholesterol. New England Journal of Medicine, 314:745-748. Gustafsson, I., B. Vessby, B. Karlstrom, J. Boberg, M. Boberg, and H. Lithell. 1985. Effects on the serum lipoprotein concentrations by lipid-lowering diets with different fatty acid compositions. Journal of the American College on Nutrition, 4:241-248. Hammons, R. o. 1982. Origin and peanut. Chap 1, 1-20. In: Technology. Editors: H. E. American Peanut Research and Yoakum, TX. early history of the Peanut Science and Pattee and E.T. Young. Education Society,

PAGE 127

Harris, P. and A. T. James. 1969. The effect of low temperature on fatty acid biosynthesis in plants. Biochemistry Journal, 112:325-330. 115 Hartzook, A. 1969. The effect of maturity upon fatty acid composition in the oil groundnut (Arachis hypogaea L.) seeds. Current Science, 38:176. Helentjaris, T. 1989. Future directions for plant RFLP technology and its applications. Pages 268-289. In: Current Communications in Molecular Biology. Editors: T. Helentjaris and B. Burr. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Helentjaris, T., M. Slocum, s. Wright, A. Schaefer, and J. Nienhuis. 1986. Construction of genetic linkage maps in maize and tomato using restriction fragment length polymorphisms. Theoretical and Applied Genetics, 72:761-769. Holaday, c. E. and J. L. Pearson. 1974. Effects of genotype and production area on the fatty acid composition, total oil, and total protein in peanuts. Journal of Food Science, 39:1206-1209. Holbrook, C. c. and c. s. Kvien. 1989. 1988 cultivar census. Peanut Research, 27(1):4. Isaac, P. G., V. P. Jones, and c. J . Leaver. 1985. The maize cytochrome c oxidase subunit I gene: Sequence expression and rearrangement in cytoplasmic male sterile plants. European Molecular Biology Organization Journal, 4:1617-1623. Jamieson, G. S., w. s. Baughman, and D. H. Brauns. 1921. The chemical composition of peanut oil. Journal of the American Oil Chemists Society, 43:1272-1381. Keim, P., B. W. Diers, R. G. Palmer, and R. c. Shoemaker. 1989. Qualitative and quantitative studies of soybean with RFLP markers. Pages 123-141 In: Current Communications in Molecular Biology. Editors: T. Helentjaris and B. Burr. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Knauft, D. A., A. J. Norden, and D. W. Gorbet. 1986. The effect of three digging dates on oil quality, yield, and grade of five peanut genotypes grown without leafspot control. Peanut Science, 13(2):82-85.

PAGE 128

116 Kuusi, T., c. Ehnholm, J. Huttunen, E. Kostiainen, P. Pietinen, u. Leino, U. Uusitalo, T. Nikkari, J.M. Iacono, and P. Puska. 1985. Concentration and composition of serum lipoproteins during a low-fat diet at two levels of polyunsaturated fat. Journal of Lipid Research, 26:360-367. Lehninger, A. L. 1982. Principles of Biochemistry. Worth Publishing, New York. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Metcalfe, L. D. and A. A. Schmitz. 1961. The rapid preparation of fatty acid esters for gas chromatographic analysis. Analytical Chemistry, 33(3) :363-364. McGill, J. F. 1973. Economic importance of peanuts. Chap. 1, pp. 1-15 In: Peanuts-Culture and Uses. Editor: c. T. Wilson. American Peanut Research and Education Society, Stillwater, OK. Miller, J. F., D. C. Zimmerman, and B. A. Vick. 1987. Genetic control of high oleic acid content in sunflower oil. Crop Science, 27:923-926. Mozingo, R. w., T. A. Coffelt, and J. c. Wynne. 1988. Market grade effects on fatty acid composition of five peanut cultivars. Agronomy Journal, 80:73-75. National Peanut Council of America. 1986. USA Peanuts. J. Grimsley. Alexandria, VA. Norden, A. J., o. W. Gorbet, and D. A. Knauft. 1985. Registration of 'Sunrunner' peanut. Crop Science, 25:1126. Norden, A. J., D. W. Gorbet, D. A. Knauft, and C. T. Young. 1987. Variability in oil quality among peanut genotypes in the Florida breeding program. Peanut Science, 14:7-11. Pickett, T. A. and L. T. Holley. 1951. Susceptibility of types of peanuts to rancidity development. Journal of American Oil Chemists Society, 28:478-479. Rachmeler, D. N. 1988. Inheritance of early maturity and fatty acid composition in peanut (Arachis hypogaea L. ) Ph.D. dissertation. North Carolina State University, Raleigh, NC.

PAGE 129

117 Raheja, R. K., s. K. Batta, K. L. Ahuja, K. s. Labana, and M. Singh. 1987. Comparison of oil content and fatty acid composition of peanut genotypes differing in growth habit. Plant Foods for Human Nutrition, 37:103108. Rennie, B. D., J. Zilka, M. M. Cramer, and w. D. Beversdorf. 1988. Genetic analysis of low linoleic acid levels in the soybean line PI 361088B. Crop Science, 28:655-657. Saghai-Maroof, M.A., K. M. Soliman, R. A. Jorgensen, and R. W. Allard. 1984. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proceedings of the National Academy of Sciences, 81:8014-8018. Salisbury, F. B. and c. W. Ross. 1985. Plant Physiology. 3rd. ed. Wadsworth Publishing, Belmont, CA. Sanders, T. H. 1980a. Effects of variety and maturity on lipid class composition of peanut oil. Journal of the America Oil Chemists Society, 57(1):8-11. Sanders, T. H. 1980b. Fatty acid composition of lipid classes in oils from peanuts differing in variety and maturity. Journal of the American Oil Chemists Society, 57(1) :12-15. Sanders, T. H., J. A. Lansden, R. L. Greene, J. s. Drexler, and E. J. Williams. 1982. Oil Characteristics of peanut fruit separated by a nondestructive maturity classification method. Peanut Science, 9:20-23. Schonfeld, G., W. Patsch, L. L. Rudel, C. Nelson, M. Epstein, and R. E. Olson. 1982. Journal of Clinical Investigation, 69:1072-1080. Smith, R. L., M. K. U. Chowdhury, and S. C. Schank. 1988. Use of restriction fragment-length polymorphism (RFLP) markers in genetics and breeding of napiergrass. Soil and Crop Science Society of Florida, Proceedings, 48:13-19. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology, 98:503-517. st. Angelo, A. J. and R. L. Ory. 1973. Investigations of causes and prevention of fatty acid peroxidation in peanut butter. Journal of the American Peanut Research and Education Association, 5:128-133.

PAGE 130

118 St. John, L. c., c. R. Young, D. A. Knabe, L. D. Thompson, S.T. Schelling, S. M. Grundy, and S. B. Smith. 1987. Fatty acid profiles and sensory and carcass traits of tissues from steers and swine fed an elevated monounsaturated fat diet. Journal of Animal Science, 64:1441-1447. Stalker, H. T. and J. P. Moss. 1987. Speciation, cytogenetics, and utilization of Arachis species. Advances in Agronomy, 41:1-40. Stumpf, P. K. 1989. Biosynthesis of fatty acids in higher plants. Chap. 3, 38-67. In: Oil Crops of the World. Editors: G. Robbelen, R. K. Downey, and A. Ashri. McGraw-Hill, New York. Stumpf, P. K., J. B. Ohlrogge, K. C. Oo, and M. R. Pollard, 1977. Biosynthesis of fatty acids and possible chain termination mechanisms in plant tissues. Pages 234-258 In: Regulation of fatty acid and glycerolipid metabolism. Editors: P. K. Stumpf and M. R. Pollard. Pergamon Press, Oxford. Tai, P. Y. P. and c. T. Young. 1977. Inheritance of dry matter deposition and free arginine level in maturing peanuts, Arachis hypogaea L. Peanut Science, 4(1):1-6. Thiede, M.A., J. Ozois, and P. Strittmatter. 1986. Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase. The Journal of Biological Chemistry, 261(28):13230-13235. Thomas, D. L. and N. J. Neucere. 1974. A comparative investigation of peroxidases from germinating peanuts (Arachis hypogaea): Electrophoresis. American Journal of Botany, 61:457-463 Treadwell, K., c. T. Young, and J. c. Wynne. 1983. Evaluation of fatty acid content of forty peanut cultivars. Oleagineux, 38(6):381-385. Urie, A. L. 1985. Inheritance of high oleic acid in Sunflower. Crop Science, 25:986-989. United States Department of Agriculture, (USDA). 1975. Composition of foods. Agricultural Handbook No. 8 Washington, D. c. United States Department of Agriculture, (USDA). 1979. Agricultural Statistics. Washington, D. c.

PAGE 131

119 Wilcox, J. R. and J. F. Cavins. 1985. Inheritance of low linolenic acid content of the seed oil of a mutant in Glycine max. Theoretical and Applied Genetics, 71:7478. Williams, E. J. and J. s. Drexler. 1981. A non-destructive method for determining peanut pod maturity. Peanut Science, 8:134-141. Worthington, R. E. 1969. Developmental changes in peanut lipid Fatty acids. Pages 87-98 In: The Proceedings of the Fifth National Peanut Research Conference. Worthington R. E. and R. o. Hammons. 1971. Genotypic variation in fatty acid composition and stability of Arachis hypogaea L. oil. Oleagineux, 26(11):695-700. Worthington, R. E., R. o. Hammons, and J. R. Allison. 1972. Varietal differences and seasonal effects on fatty acid composition and stability of oil from 82 peanut genotypes. Journal of Agricultural Food Chemistry, 20:727-730. Wynne, J. c. and T. M. Halward. 1989. Cytogenetics and genetics of Arachis. Critical Reviews in Plant Sciences, 8(3):189-220. Young, c. T., M. E. Mason, R. s. Matlock, and G. R. Waller. 1972. Effect of maturity on the fatty acid composition and stability of eight varieties of peanut grown and Perkins, Oklahoma in 1968. Journal of the American Oil Chemists Society, 49:314-317. Young, C. T., R. E. Worthington, R. o. Hammons, R. s. Matlock, G. R. Waller, and R. D. Morrison. 1974. Fatty acid composition of Spanish peanut oils as influenced by planting location, soil moisture conditions, variety, and season. Journal of American Oil Chemists Society, 51:312-315.

PAGE 132

BIOGRAPHICAL SKETCH Kim M. Moore was born on May 23, 1951, in Boulder, Colorado. He lived in Colorado for his primary education but completed his secondary education in Sepulveda, California, where he graduated from James Monroe High School in 1969. He began his higher education at the University of California at Santa Barbara and later transferred to Colorado State University, where he received a B. s. degree in animal science in 1976. After completion of his B. s., he accepted a position as a food technologist for star-Kist Foods, Inc., in their product development division. In 1979, he was promoted to quality control manager of the El Paso, Texas, production facility. In 1984, he enrolled at the University of Florida, Gainesville, Florida, as a graduate student in the Department of Agronomy and received a Master of Science degree in May of 1987 and a Doctor of Philosophy degree in May of 1990. 120

PAGE 133

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. David A. Knauft, Ch rman Associate Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Kenneth L. Buhr Assistant Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Kenneth H. Quesenberry Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I / el l~~ l(uJ~ Sherlie H. West Professor of Agronomy

PAGE 134

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Professor of Horticultural Science This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. May 1990 Dean, of Ag culture Dean, Graduate School

PAGE 135

UNIVERSITY OF FLORIDA II I II IIIIII Ill Ill lllll lllll II IIIIII IIII II llllll 11111111111111111 3 1262 08553 4500