Title: Genetic, developmental, and molecular characterization of a high oleic acid peanut (Arachis hypogaea L.)
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Title: Genetic, developmental, and molecular characterization of a high oleic acid peanut (Arachis hypogaea L.)
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Language: English
Creator: Moore, Kim M., 1951-
Copyright Date: 1990
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GENETIC, DEVELOPMENTAL, AND MOLECULAR
CHARACTERIZATION OF A HIGH OLEIC ACID
PEANUT (Arachis hypoqaea 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













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.



















TABLE OF CONTENTS


Paae


ACKNOWLEDGEMENTS .. .. .. .. * * * * 11


LIST OF TABLES .. .. .. . ... * * * * v


LIST OF FIGURES. .. ... .. * * * * * vlll


ABSTRACT ... .. .. .. * * * * . xl


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 .....


. .17


* * *
. . *
* * *


. * *
. *
. * *
. . *


Summary . . . . * * *

VARIATION IN FATTY ACID COMPOSITION
IN DEVELOPING SEED OF Arachis hypoqaea 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


III











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

V SUMMARY ................... 109

REFERENCES .. .. .. . ... . . . . . 112

BIOGRAPHICAL SKETCH .. .. .. .. . . . . 120














LIST OF TABLES


Table Face

1-1 01eic and linoleic acid content of
vegetable oils. .. ... .. .. * * 9

2-1 01eic 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 tcate . ... .. 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 F1 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 BC, 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 Page

2-8 Phenotypic segregation of oleic acid content
for F progeny from the cross between high
(F4355 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 01eic and linoleic acid content of three
peanut breeding lines .. ... ... .. 44

3-2 R2 Values of individual plants and for a
composite of all plants for each genotype
for maturity versus dry matter .. .. .. 47

3-3 R2 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. hypoqaea 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
8. hypoqaea lines and four perennial Arachis
species at two wavelength. Extraction was
method 2 (potassium acetate) using young
mature leaf tissue. .. .. .. .. .. .. 84









Table


Pagg


4-3 Absorbances of DNA extracts from four
8. hypoqaea 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. hypoqaea 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















LIST OF FIGURES


Fiqrure 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


Pase


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








Pase


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 atga on peanut
genotypes illustrating an unacceptable
autoradiograph based on poor fragment


definition.


. . . . . . . 94


HPIl6 on peanut
sizes in kilobases. .

HPI6 on peanut
size in kilobases ..

atgg on peanut
sizes in kilobases. .

coxl on peanut
sizes in kilobases. .

HPI67 on peanut
sizes in kilobases. .

HPI72 on peanut
sizes in kilobases. .

HPI58 on peanut
sizes in kilobases. .

HPI52 on peanut
sizes in kilobases. .

rrn5-rrl8 on peanut
sizes in kilobases. .

HPI54 on peanut
sizes in kilobases. .


Autoradiograph of probe


4-6


95


96


98


99


100


101


102


103


104


105


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














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 hypoqaea 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. 01eic 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 Fl, F3, and backcross








generations. Among the four 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. h pogaea 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 hypoqaea L., is a native lequme 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_. hypoqaea, 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 hyp~oqaea can be

divided into two subspecies, based on morphological

differences. These subspecies are hypoqaea and fastigiata.

The subspecies hypogaea is subdivided into variety hypoqaea,

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,

C3 5(0H),, to which organic acids (fatty acids) are bound,
substituting for one, two, or all three of the hydroxyl









15.9 %C


14.9 % Candy


d 9Snah7 Butter
0.97 %


i Crushed for 011

IIOther
0 %


24.


37.0 %


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










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 18-

carbon 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











SPalmitic

52.0 % 11.0 Stearic
9~Oleic

1.1X % Linollec
2.5 %2
1.4 % O Arachidic

SBehenic

Lignocaeri

28.0 %Z
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%. 01eic 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%. 01eic 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 8. hypoqaea botanical

types and two other Arachis species, A. monticola and A.

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.] 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 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










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 1-

3. 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).










Carrier (oxid)~ 2-Linoleoyl PC/PE



Carrier (red) 2-Oleoyl PC/PE


(oxid)
Desotur ase
I2-Lyso PC/PE


C oA

Oleoyl transferose


Oleoyl CoA


NAD H



NAD

NAD H:Corrier
Redu cta se


+A =rdcdnctnmieoeiedncetd
NADH = reiduced nicotinamide adenine dinucleotide

CoA = coenzyme A
red = reduced
oxid = oxidized
PC/PE = p hosphotidylcholine/p hosphotidy lethonolamine
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. 01eic 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 8. hypoqaea.

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 (E. hypoqaea

ssp. fastigiata var. vulgaris). This botanical type

accounts for only 10.8% of the total U.S. peanut acreage.

The Virginia botanical type (E. hyp~oqaea ssp. hypoqaea var.










hypoqaea) 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 Fl generation

were planted in the field at the University of Florida

Agronomy Farm, near Gainesville, in July 1986. These F1

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 H2SO,

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. 01eic 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.







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,) 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 100"C for 3 minutes.

After heating, the reaction vials were cooled and 1 ml of

deionized H20 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

pl 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

250*C. The oven temperature was programed for an initial

temperature setting of 190"C for 3 minutes, then increasing

at the rate of 3"C per minute until reaching a final

temperature of 220*C. 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 Fl 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 F1 progeny from the F435

X F78114 cross and each parent, and backcrosses between Fl

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 BFz,
some fatty acids may be dissociated from glycerol. Total


















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
_22991 J*t-el

11.5% 1.70

1.7% 0.30

47.4% 0.04

33.4% 0.03

0.9% 0.80

1.0% 0.04


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 t .lc


F435
non
Fatty Acid sapon sapon

Palmitic 6.7% 9.2%

Stearic 2.0% 3.2%

01eic 80.8% 79.%

Linoleic 2.5% 2.9%

Arachidic 1.1% 2.5%

Behenic 1.4% 2.5%

* tcate compares the means of each










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

BF3 is not strong enough for complete saponification.
However, the degree of saponification by BF3 aprnl

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 F1 seed had oleic acid levels similar to the

F78114 parent (Table 2-4), regardless of the genotype on

which the seed were borne. F2 seed from 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 % 01eic % 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

vl 25
S25 324

.5~ 20
S20
c: 17 tXI~~~R ~l17
~c 15
15-
O 13 gI i XI I II t 13
I 11
o 10 10 0

3 6
Z 5 5 5




/6 30 b 3Z 3 6/ 60 6b 6 62' 66 1 16

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 Fz progeny from the cross
between high (F435) and normal (F78114) oleic acid lines.

01eic 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 01eic Oleic Low:High 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 .2-.5
F435 X F519-9
BC (F, X F435) 7 4 1:1 .2-.5
BC (F, X F435) 6 5 1:1 .5-.9
















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


Oleic Acid Composition
Fz Observed F3 Expected
Cross F, Normal- High Ratio
F7811 X 45 ih 4 l h
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













S30 t- 29
o 26
25 25
p 25- 24

c 20 -
m0
u~15 15
O 15 -14 1
I LR~ 12 ~Z ~~~iR\ 1P~12 12 1
.0 10
E 10


3 3 436S k 6616 9S
Percet Olec Aci
Fiur 2-.Feunydsrbto fnme fF fsrn npeoyi
clse bae noecai otn.Dt a enpoe o l aiisfo
the rossof F19-9and 435

























01eic 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 for F2 progeny from 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 F1 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 2-

3).

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 Fz progeny from the
cross between high (F435) and normal (PI 262090) oleic
acid lines.


01eic Acid
Composition
Family Cross Normal High X2
(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

cr 16
S 15-



` 10 g







the crs ofP PI 2609 and F435.04








40

(Brunklaus-Jung and Robbelen, 1987). Results presented

here indicated major genes control the oleic and linoleic

acid content in peanut. Together, the F F2' 3, and BC1

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 hypoqaea 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 non-

destructive 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 P.

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. 01eic 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 80.1 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 temperature-

controlled oven at 110*C. 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 80*C for 90

minutes. The vials were then cooled and 1 ml of 1.8 M H2SO,

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










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 for 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. R2 values of individual plants and for a
composite of all plants for each genotype for maturity
versus percent dry matter.



Genotyp~e Plant R2
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



T o ) - - -- - - - - -- - - - - - - ----------O ---- ) ~ ~ . . . . .. . .~. .. .

~e! DD









20 *-- -**** -- -- -- -- --- -- -


Maturity Rating
% Dry Matter
R2 = 0.64
Y *34.1 + 10.51nX

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.








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 RZ

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 3-

4). 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. R2 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.




R2

Genotype Maturity Dry Matter

F519-9 0.30 0.62

F435 0.12 0.13

F78114 0.21 0.24










% Dry Matter
80~--1"-111--l



500



O D



30

3nI II


Maturity Rating
%f Dry Matter
--E)-


R11 0.43
Y = 39.74 + 9.71nX


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



















m O

---- ---- ------- --------.-- ----.------------- --------****.-- --- ------------------------------ ----***

o


% Dry
70 F-


60


40

30

20~


Maturity Rating
% Dry Martter
--t-


R 1, 0.4
Y r 21.74 + 12.43In


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

















0[ag a a a=1







.O
1 I I I I


% Oleic Acid


r


60 1


50


40


30


Maturity Rating
% Oldec Acid
D-E~-


It a 0.30
Y r 42.0 + 0.111nX


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.











% Oleic Acid
82


8 0 -- ---- -- ------------------ ------- -- -- .- -- -. .. .. .. .... .. ..... ...... .. .................


76


74


O D
I3 I


Maturity Rating
% Ol~ec Acid


R s* 0.12
Y 1 714.95 + 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.











% Oleic Acid
50 -----------


D a


D

OO




I I I I _


40



35 1



30


Maturity Rating
% Olekc Acid
--e~--


Ra = 0.21


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












_ __ _I _~


DDD
- - -- - - -- - -- - - -- - -- - - -- g - -- - - - - - - - - - - - - - -
D7









I I I I _s I
D 20 30 40 50 60 70 8
% Dry Matter


% Oleic Acid
70 ------


50 j


40 j


30


% Oleic Acid


R =0.62
Y 8b.32 + 0.461nX


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 1-- -


ao a
I I I I


74


% Dry Matter
% Ol~ec Acid
D-f-


R' = 0.13
Y r 6 1.3 + 3.91nX


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.



































Y r 18.3 + 0.22X m


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.


40 50
% Dry Matter
% Oklec Acid


__


)leic Acid


% O




45





40


O

0 0

O t3
-- -- -- -- -- -- -- -- -- -- - -- -- - -- ID g "g " " "'" "
00r~....r~~.....~~~.~~


- - - - - - -- - - - - - - - -- - - - - -- - - - - -- - - - - -
o0


30 a
2(


0















Table 3-4. The percent oleic acid and standard errors for
high, moderate, and low 01eic acid peanut genotypes sampled
at various stages of dry matter deposition.


Percent 01eic Acid and Standard Error
Genotypes
Percent F78114 F519-9 F435
Dry Matter Mean *SE Mean SE Mean SE

10-29 36.2 -' 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.
SInsufficient data.










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).











% Palmitic Acid
12


11 ~-~.

to D

10 --******--*******- ---*------***--**-***I *********r *****************
9 c......~...,.....t .. (~jr00,r


% Dry Matter
% Palmitic Acid


Rs I 0.0 1
Y = 9.71 -~ 0.006X


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 -' 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.
i 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
Genotypes
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.
i Insufficient data.











% Linoleic Acid


8o




10



DO

3~~ D e)'0~~l
n dl ~0


% Dry Matter
% Linollec Acid


Rs = 0.23
Y = 7.9 0.07X


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


































Rs = 0.1 _
Y 749X~

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.


30 40 50 60 70
% Dry Matter
% Linollec Acid
D-t-


inoleic Acid


0 20


% Li
40

35

30

25

20

15

10

g
1I


m..~

-*** *-********* -- -- ------ -- ----- ------*** -- -- -- -- -- ** ***-**-------------------- -----
m B
-*********************-------************************---****-a -* *****D **************-----******------ -------------------***---
L 13








I I1 I I I I











% Linoleic Acid
42


-O O






30




20
20 30 40 50 60 70
% Dry Matter
% Linoldec Acid
---8-
R' =, 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 mavs 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 (Orvza

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 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 A. hypoqaea: 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 -70*C.

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 NaC1, 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 pl of 10 mg/ml RNase A (Promega

Inc.) in 10 mM Tris-HC1 (pH 7.5) and 15 mM NaC1. 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 NHI40Ac 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 pl

of RNase mix were also added and the tubes incubated at 37"C

for five hours. Two pl of loading buffer were added and

each digestion loaded onto an agarose gel. A lambda

Hindlll-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 pl 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 ( H 8.0), 100 mM_ Tris (pH 8.0), 500 mM_

NaC1, 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 65"C 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 0*C 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 pl 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 pl of 3 M
sodium acetate and 500 pl 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 pl 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_.

hypoqaea and the four Arachis species that had been

determined to be of acceptable quality were digested with

EcoR1 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 HC1. The

gels were then rinsed twice and soaked with constant

stirring for 45 minutes in a denaturing solution of 1.5 _M

NaC1 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 NaC1 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 pl of cold STET buffer (80 g

sucrose, Triton X-100, 200 ml of 0.25 _M EDTA, 50 ml of 1 _M

Tris-HC1, and deionized H20 to a total of I L with pH

adjusted to 8.0) in 1.5 ml Eppendorf tubes. Each tube then

received 60 pl 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 pl TE, and stored at -20*C as described in the plant

DNA isolation above. Clones were evaluated for insert size

by digesting 2 yg plasmid DNA with 8 units Pstl, 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 Pstl-digesting 40 pl 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 pl of low salt buffer, then eluted with high salt

buffer (lM NaC1, 0.1 mM EDTA, 20mM Tris, and 0.5 M

Arginine). To elute the DNA, tubes were heated to 70"C 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 pl 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 pl of 7.5 M_ ammonium acetate and

800 pl of absolute alcohol. The tubes were incubated

overnight at -20"C. DNA pellets were recovered by

microfuging for 15 minutes then washing with 500 pl of 70%

ethanol. The DNA was air dried and redissolved in 20 pl of

TE buffer. Two pl of each isolated DNA was mixed with 18 pl

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-labeling 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 pl

Eppendorf tube. Also into the tube were placed 10 pl of OLB

(Finberg and Vogelstein, 1983), 6 pl of bovine serum albumen

3mg/ml (BSA), 2 pl of Klenow enzyme (1U/Crl), 4 pl of 3P-










labeled deoxycytidine 5'-triphosphate (dCT32P) (3000ci/mM),

and enough double-distilled H20 to bring the total volume to

50 pl. The tubes were then incubated at 37"C for 30 minutes.

Then to each tube 50 pl of OLB stop mix [2.0 ml of 1 M Tris-

HCI (pH 7.0), 400 ml 5 M NaC1, 0.5 M EDTA (pH 8.0), and 12.5

pl 20% SDS] were added. Unincorporated nucleotides were

removed by liquid chromatography with G50 Sephadex in 1X NTE

buffer (100 mM_ NaCl, 10 mM Tris-HC1, 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 pl 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

q. re ens, 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 pg/Ul*

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_. glabrata 0.033 0.027 1.22 0.17

A_. Dintoi 0.092 0.024 1.56 0.46

e. re ens 0.044 0.024 1.83 0.22


* Calculated by A26o X 50 X dilution rate of 100.
















































* Calculated by A26 X 50 X dilution rate of 100.


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


Absorbances


Genotype


Wave Length
_260nm 2 80nm 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
Lg/1l*

0.27

0.19

0.33

0.19

0.29

0.26

0.23

0.38


F435

F519-9

F78114

PI 262090

A- spp.

8. qlabrata

_A. pintoi

_A. repens








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 4-

3. 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


2345


678


F435
F519-9
F78114
PI 262090

A. glabrata
A_. Dintoi
B. repens


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
















2 3 4567 8


1 F435
2 F519-9
3 F78114
4 PI 262090
5 A. 292*
6 E. glabrata
7 A. pintoi
8 A. repens




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





Genotype


F435

F519-9

F78114

PI 262090

A. s .

A. glabrata

A. pintoi

e. repens


* Calculated by Ar6 X 50 X dilution rate of 100.


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


Absorbances

Wave Length
260nm 280nm 260/280

0.179 0.099 1.81

0.148 0.079 1.87


Concentration
LIg/pl*

0.90

0.74

0.43

0.46

0.90

0.44

0.27

0.11


0.047

0.053

0.098

0.053

0.029

0.012


0.086

0.093

0.180

0.088

0.053

0.022


1.83

1.75

1.84

1.66

1.82

1.83




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