The inheritance of protein and lysine in pearlmillet (Pennisetum typhoides (Burn.) Stapf and E. C. Hubbard) grain

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The inheritance of protein and lysine in pearlmillet (Pennisetum typhoides (Burn.) Stapf and E. C. Hubbard) grain
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Barnett, James Bynum, 1931-
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THii I i:RITIANCiE OF PROUTIN AND LYSINE IN
PIARLMI, LET (Pennisetum typhoides
(H rn.) Stapf and E. C. iHubbard) GRAIN












By

JAMES BYNUM BARNETT












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










UNIVERSITY OF FLORIDA 1975

























To

my wife, Martha, our children, Charles, James,

George, and Virginia, and my wife's mother, Mrs. A. L. Hill,

each of whom has sacrificed and willingly given

of what they had toward the completion of this goal.
















AC KNOWLEDGMEINTS


I acknowledge the guidance, encouragement, and helpful criticism received from the members of my Supervisory Committee. Each member has contributed not only to this study but to my total education. Dr. Rex L. Smith, Conitteo Chairman, has always been available for discussions and lending aid. Dr. James Soule gave me a better understanding of fruit crops, Dr. V. E. Green, Jr., imparted to me some knowledge and appreciation of tropical agriculture and agronomy. Dr. R. C. Robbins' assistance and guidanice in the analytical phase of this study was invaluable. Dr. R. C. Littel's critique of the statistical analysis gave me a better understanding of that discipline.

I consider each a close personal friend and am honored thaLt they served on this committee.

















iii
















TABLE OF CONTENTS



AC i W I,*I) 1 I V ,-; . . . . . . . . i i



A 'i'RACT . . . . . . . . . . . vii

I N R Dt )UCT I ON . . . . . . . . . . 1
The Protein Problem . . . . . . 1
Possible Solutions to the Problem. . . . 6

SCOPE AND PURPOSE OF THIS STUDY . . . . . 14

:MATERIALS AND METHODS . . . . . . . 17
The Diallel Cross. . . . . . . . 17
Analytical Procedures . . . . . . 19
Protein Analysis . . . . . . 19
Amino Acid Analysis . . . . . 20
Lysine Analysis . . . . . . 20
Statistical Analysis ........... 21

RESULTS . . . . . . . . . . . 22
Protein Inheritance. . . . . . . 22
Combining Abilities . .......... 22
Heritability Estimates. . . . . 33 Genetic Parameters. . . . . . 34
Lysine Inheritance . . . . . . . 37
Quantitative Analytical Methods. . . . 37

D C, I I LON... ............ ..................... 43

CO C I S iONS . . . . ........ . 49

LLTLEATURE CITED .............. ... 50

BIOGRAPHICAL SKETCH ............ ..... 55










iv
















LIST OF TABLES

TA I P 1'Paje

I COMPARISON OF ESSENTIAL AMINO ACIDS IN CEREAL
GRAINS EXPRESSED IN g/16g N .......... 3

2 011, AND MINERAL COMPOSITION OF SOME CEREAL
(GRA I N S . . . . . . . . . . 16

3 PROTEIN AND LYSINE CONTENT OF INBRED LINES USED
IN THE DIALLEL CROSS . . . . . . 17

4 PROTEIN CONTENT OF F GENERATION HYBRIDS AND SELFS RESULTING FROM A DIALLEL CROSS OF FIVE
INBRED PARENTS. . . . . . . . . 23

5 MEAN PROTEIN CONTENT OF ALL PROGENY OF A DIALLEL CROSS, GAINESVILLE ........ . 25

6 MEAN PROTEIN CONTENT OF ALL PROGENY OF A DIALLEL CROSS, DELRAY BEACH ....... . 26

7 ANALYSIS OF VARIANCE FOR PROTEIN OF PROGENY RESULTING FROM A DIALLEL CROSS WITH FIVE INBRED
PARENTS ............ .... .. 27

8 VAITANCEI ANALYSIS FOR COMBINING ABILITY FOP, PR)TEi:IN, GAINESVILLE. . . . . . . 28

9 VARIANCE ANALYSIS FOR COMBINING ABILITY FOR PROTEIN, DELRAY BEACH .... ........ 29

10 ESTIMATES OF GCA, SCA, AND RECIPROCAL VARIANCES
ASSOCIATED WITH EACH PARENT, AND ENVIRONMENTAL
VA RIANCE MEANS FOR PROTEIN, GAINESVILLE .... 30

11 EST'i MlATES OF GCA, SCA, AND RECIPROCAL VARIANCES
Ai;SCOCIATED W[TI EACH PARENT, AND ENVIRONMENTAL
VARIANCE MEANS FOR PROTEIN, DELRAY BEACH. . 31

12 MIA' SQUARES AND ESTIMATES OF VARIANCE FOR
COMBINING ABILITIES AND RECIPROCALS OF PROTEIN. 35

13 ANALYSIS OF VARIANCE FOR GENETIC PARAMETERS OF
PROTEIN INHERITANCE ...... .. . . 36


v










Li ST OF TAl3LES (continued)

TA I I.E Page 14 ANALYSIS FOR ESSENTIAL AMINO ACIDS. . . . 38 15 ,r; Y;IN CONTENT OF PNiOGEINY FROM A DIALTI,L CRO:;S, 39 I 6 'VAIATV I 'ON (I' EXTRACT I ON FOR TIlE CONCON RAPID I,Y;INI TECHNIiNUii; AND COMPARISON OF LYSINE
VAI,XliS BY Till11 CON4CON TECHNIQUE TO TIIOSE OF THE
AMI&, O ACID ANALYZER . . . . . . . 40

17 COMP'IARISON BETWEEN DBC AND MICRO-KJELDAHL ANALYSIS FOR PROTEIN, REPLICATION I,
GATNESVILLE . . . . . . . . . 41

18 COMPARISON BETWEEN MICRO- AND MACRO-KJELDAHL ANALYSES FOR PROTEIN, REPLICATION II,
GAINESVILLE .............. .. . . 42





































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i( I 1)i i ;is:~ tL i. n I rc( ;ntcd to thi (Gr;dllat: (Cunci l
()I ti I iii vn i :;it y of F'lorida in Partial Fulfil m nt
kl t i,, Iti ili tn-cmin L:; for the Deqree of Doctor of PIhil osop '

TI'Hii IiUI';ITAN'iJCL OF PROTEIN AND LYSINE IN
PIARLMIIL.LT (Pennisetum typhoides
(Burn.) Stapf and E. C. lubbard) GRAIN By

James Bynum Barnett

June, 1975

C a i roman: Rex L. Smith
Major Depairtment: Agronomy

A diallel cross was made using inbred lines of

pearlmillet selected on the basis of variation in protein ana lysine content. The F1 generation was planted in a randomized block design at two locations with two replicaL ions at each location. Each replication contained the six inbred lines produced by selfing and the 30 progeny produced by crossing the five lines in all possible combinations.

Total protein (N x 6.25) was determined by rapid micro;,jeoldahl technique. Statistical analysis indicated protein in pearlmillet is influenced by the presence of dominant

,* t: ion for Lower Iprotein with evidence of maternal.

ins hi 'l ar influenced by environment. Rej ressi on

': Ailysis indicated no linear relationship between total essential amino acids and total protein.

Analysis of lysine inheritance was based upon a comp-Aete :nino acid analysis of samples representing a complete



vi i











;I i cr(f:;:;I:; f riin a repl ication including inbred lIarents il not rcirol:i lack of replication made comrlrjete

it i, tical analysis isp):;!:;ible. Repression analyses indi(c,id li near relationship exists between lysine and total :;et.itial amino acids with a very low correlation coefficient with a negative correlation between lysine and total i 'ltein. A positive correlation exists between lysine aind the total of methionine and threonine.

Dye-binding techniques for protein and a rapid quantitative analysis technique proved unreliable when used in pearlmillot. The cause of this unreliability was undetermined.






























viii

















INTRODUCTION


The Protein Problem


Internationally, cereal grains are the major source of protein in the human diet while legumes and animal protoins act as supplemental sources. Nicol (1971) reports sorqhum and millets contribute 63% of the total dietary protein in West African diets while rice contributes up to 76' of the total dietary protein in the Far East. Estimation of the contribution of cereal grains to the dietary

prtote'in in Latin America is complicated by the fact that in sore, areas rice is the staple grain while in others corn is the basic grain in the diet. Altschul (1965) estimates cereal grains produced more protein for human consumption than all animal and legume sources combined. The importance of protein quality and quantity in cereal grains is secondary in developed countries where animal products are the major source of protein.

The prominence of cereal grains as a major protein

source in human diets and problems which are inherent have only recently begun to be elucidated. Howe et al. (1965) ;, igests that where sufficient cereals to satisfy the caloric


1






2



r l uiremn('lIs wr' i njsted, there would( be an adequate Iprolwin balance only if the amounts and kinds of the ce real proteins were comparable to animal proteins. All cereal tIIiins when analyzed on a whole-grain basis are, however, below F'AO recommended levels in one or more essential amino acids. Lysine, for example, is the primary limiting amino acid in all cereal grains. Threonine is second limiting except in maize where tryptophane and lysine are colimiting (Table 1).

A concomitant problem is the loss of protein during prccessing. The principal source of this loss is through milling where the germ, which is proportionally higher in protein than the endosperm,or the seed coat, including the aleurone layer, may be removed. Loss of either or both of these seed fractions lowers both the quality and the quantity of protein in the finished product.

Varying degrees of protein malnutrition often results in developing areas of the world where the average diet consist of cereal grains of low nutritive value supplemented with small amounts of animal or legume protein. This form of malnutrition is debilitating in adults. In pre-school children whose protein needs are high in relation to caloric requirements, severe cases of protein malnutrition result in the permanent impairment of mental capacity. The caloric requirement may be met with a cereal-based diet after weaning but a protein deficiency will result unless sufficient






3







TABLE 1

COMPARISON OF ESSENTIAL AMINO
ACIDS IN CEREAL GRAINS
EXPRESSED IN g/16g N

+ + + ++ ++Ani~o acid Pearlmillet+ Sorghum Wheat+ Rice FAO++ les inr 2.6 2.5 2.7 3.26 4.2 2.1. 2.1 2.1 3.22 2.4 IT I onine 4.9 4.1 3.3 4.03 2.8 thlionine 2.5 2.7 2.5 2.2 2.2 isoleucine 4.3 4.9 3.6 4.0 4.2 Tryptophane 2.3 1.1 1.2 1.68 1.4 Vl Iino 5.7 4.8 4.5 5.75 4.2 I ine 17.4 24.2 6.8 8.53 4.8 1'lrlny l a;lanine 4.9 4.9 5.7 4.4 2.8 Tyrosine --- --- --- 1.89 --Source: +Burton et al., 1972.

++Juliano et al., 1968.
+4+Suggested FAO requirements for good amino acid
balance (Food and Agr. Org., 1957).






4



.1,,qlimis and/or animal protein sources are supplied. These i icjct :; make Lit imperative that good quality protein be m ido av il dale as cheaply as possible. Improving the protein quality and quantity in cereal grains appears to be a

1 ,:,iblc approach to this problem.

Population pressures and cultural mores also contribute to the protein problem. Available arable land is decreasing and the population is increasing and the competition between man and animals for the available cereal grains is becoming more pronounced. Per capita consumption of animal products increases as people become more affluent. This also creates a pressure on available grain supplies forcing the price of both grain and animal products upward. This trend mkes both grain and animal products less attainable by the majority of the population in underdeveloped countries where unemployment is high and wages are low.

Cultural patterns and mores often limit the progress w:ich might be made in improving protein quality and quantity in local diets. Improved high protein rices are considered less palatable in some areas of Asia (Juliano et al., 1965). High lysine corn has not found acceptance in many ,> a; of Latin America. Local taboos and prohibiti ons often i liiate potential sources of animal protein. Dietary i, d cultural patterns are difficult to change and progress will oe slow where change is required for improved protein

Jw t r it ion.










Adequate proLein balance from plant sources can be Chi vl i dd by selection of plant foods that complement one another in essential amino acids. A cereal-legume mixture, for exancipie, when eaten in proper balance will often fulfill inu:;in protein requirements (Johnson et al., 196.8) However, economic limitations and lack of understanding of nutritional requirements frequently rule out food selection as a realistic approach to improved diets. Moreover, children are restricted during their most critical stage of development to what is available in the home.

Improved protein balance can also be accomplished by

the supplemental amino acids in processed foods. The difficulty of this approach is implementation of the program. 'ie people in greatest need of such a program normally consume very little commercially processed food. It is either unavailable or too expensive.

Another alternative, which also has limitations, is the improvement of protein quality and quantity in cereal grains. Several approaches have been suggested by Kamra (1971) most of which are based upon selection and breeding. !'wo precautions in a program of quality improvement are to

(1) maintain the proper balance between essential and non:;: ntial amino acids in the grain and (2) avoid selection for a single nutrient without regard to total composition of h! grain (Munck, 1964). A third precaution concerning ijl)lt,bility and acceptability would also be appropriat, in





6



vi ew of the experiences with high protein rice and high l y; i Ino corn.


Possible Solutions to the Problem


Advanccf; have been made over tne last half century in breedingj crops for increased production with little cmpilasis on increased nutritional quality. Kamra (1971) attributes this lack of progress to a general lack of awareneos of the nutritional requirements of monogastric animals andi the non-availability of simple, quantitative, inexpensive and non-destructive mass screening methods to aid in the selection of breeding material. A general lack of awareness of the importance of cereal grains and imbalance among their amino acids in the diets of developing cultures may also have attributed to this situation.

Interest in the improvement of protein in some cereal

crops began early in the twentieth centruy. Woodworth et al. (1952) reported on the results of 50 generations of selection for protein and oil content in corn and shortly afterward Frey (1949, 1951) published on the inheritance of protein inii corn. These studies indicate crude protein could be doiolied in corn but no serious work was begun since the increase was largely in the form of nutritionally poor zein. Di:,'o'lry of the opaque-2 and thoe floury-2 genes which vlin;i ficantly alter the amino acid patterns in maize (:Ir tz and Bates, 1964, 1965) created renewed interest in < i, inheritance in cereal grains.






7



Increai; ing the quality and the quantity of protein

in c lil1 grains has received increasing attention over the last decade. The International Rice Research Instit ut (i i
The inferior quality of cereal proteins is attributable to the ratio among the protein fractions in the grain. Prolamines low in lysine content constitute the major percontaqe (40-60%) of seed proteins in most cereals, with iluLelins of intermediate lysine content constituting most o)f I:hf' remainder. The smallest protein fraction in cereals










i:; t 1 (jIobu I in; which are characteristically high r in I 1 i i;x x pt ion:; to this are rice, with 8% prolamin( s uand oats wLth 12'% (Nelson, 1969). These differer:es in alino alcid content among various protein fractions of the aj ldin sucjgest thio possibility of improvement through miiiftions which would suppress the synthesis of protein fractions low in the desired amino acids with a compensatinq synthesis of other fractions with higher levels of the u si red amino acids.

Synthesis of amino acids is regulated to correspond closely to demand for incorporation into proteins by the plant, therefore, cereal grains contain very low quantities of free amino acids (Nelson, 1969). An induced or spontaneons mutation causing a loss of sensitivity in control of amino acid synthesis could cause an over-synthesis with a resulting increase in free amino acids. No mutation of this type has been identified to date (Nelson, 1969).

The more promising types of mutations are those which cdjus .e a gross change in the structural components of the endosperm or seed structures. The high lysine mutants in maize are of this gross structural change type. Theoretically, opaque-2, the high lysine gene, inhibits the synthesis of zein, the prolamine fraction of the protein, in maize.
'i'l- offec:ts of mutations in which one amino acid is sub; itittd for another in protein synthesis have not been i);,(;rt-ant in changing protein quality and quantity.






9



It bcam e increasingly apparent as the components of s;eeid i(ro( ein w('er' ucidated that significant changes in tile amino acid composition of cereal protein could occur onrly ilhrough mutations. More specifically these changes occur through mutations which reduce synthesis of the ae c hol-soluble prolarnine fraction and increase synthesis ciI other protein fractions (Johnson et al., 1968; Munck, 1964). iertz and Bates (1964, 1965) found a significant reduction in zein, the prolamine fraction of corn protein, in mutant strains homozygous for either the opaque-2 or the floury-2 gene with a corresponding increase in lysine. iTello et al. (1965) found the same effect when the opaque-2

gocne was incorporated into various races of corn. More recently Nelson and Chang (1974) studied the effect of enhancing protein and lysine content in corn by incorporating a gene conditioning multiple aleurone cell layers,

which are high in lysine.

Significant improvement in protein quality or quantity in cereal grains can be accomplished through selection and breeding and by screening mutations, either spontaneous or induced. Manipulation of major genes offers the most ciriaatic and the most rapid method of increasing protein 2ality or quantity but generally major genes are not

I ilI,. Selection and breeding hold the most |promise ior protein improvement when sufficient variability is preient. The re-,earcher can attempt to induce mutations










iich will, achieve the desired improvements as a; last reP. olygenes are less dramatic in changing protein

quaili y nid quantity but can be concentrated through sound i aIsl a procedures. They are the most limiting but also thi most predictable of the genetic procedures.

Factors which may influence the total protein or the levels of certain amino acids in the grain should be considered as well as the form in which the grain is consumed. Differences in structural components of the seed have a marked influence on their ability to transmit genetically increased protein and amino acid potentials. Milling procedures and cooking methods may remove portions of the seed which are highest in good quality protein. Cultural practic9es may also significantly alter both the quality and the quantity of protein in the endosperm (Sawhney and Naik, 1970).

Proteins in the germ are much superior in protein

quality to those of the endosperm (Nelson, 1969), therefore, increasing the relative size of the embryo for quality and quantity of protein in the diet would be of importance where tAn whole seed is consumed. No studies have been made to

-xpilore the possibilities of significantly changing the amino acid composition of the embryo but it has been noted that both the opaque-2 and the floury-2 genes of maize increase embryo size when incorporated into new lines










(7ra, 7 L) h''ie l ffect of this increase has nrt bI en



T''1 aleurone layer, usually one cell layer in thick::i:, iis hi (jh in protein and contains significant amounts of Lthc globulin fraction. Increasing the thickness of this layer may significantly increase both the quality and the quantity of protein in the grain. The improved protein (c intent by the amylose extender gene in maize is believed to be due to the increased thickness of the aleurone layer (Kamra, 1971).

Inferior quality of cereal proteins may be attributed to the small fraction of globulins which are high in lysine and the large fraction of prolamines of low lysine content. (liobulins are abundant in the aleurone layer and the embryo while prolamines are found in the endosperm. Any factor which significantly alters the ratio of these protein fractions will have significant effects on the protein quality and quantity of the grain.

One aspect of the problem unrelated to the breeding a;n~d genetics is the lack of a simple, quantitative, nondc;structive method of analysis for total protein and amino acids.

Dye-binding techniques have been developed which

estimate protein in some cereal grains. Udy (1971) developed this technique originally for wheat and later expanded it to





12



Stra i :;. tiu ; al. (1970) reported the t,'-l iiol w,l:; wt'l ,t ltt c to) ric(? ajld other grains. Kamnra (1972) nt ii'ioned th le effectiveness of dye-binding for the quail itit ivo estimation of lysine in grains. The method was use'':!

In:; iltghl and Axteoll (1973) to isolate the high lysine irrnt

in sorghum. Kaul et al. (1969) described a method of microscopic screening of rice grains for protein content using a dye-binding technique. Johnson and Craney (1971) reported a rapid biuret method for estimation of protein in grains. Parial and Rooney (1970) found a high correlation among results from dye-binding, biuret, and micro-Kjeldahl analyses. Concon and Soltess (1973) described a rapid micro-Kjoldahl process in which digestion is completed within 10 minutes.

The major problem confronted in the search for a rapid quantitative analytical technique for lysine or total amino ils is hydrolysis. Standard methods require special equipment and a great deal of time. Palter and Kohler (1969) developed a survey hydrolysis procedure in which up to 300 samples per week may be processed, but even this method is t(oo slow to meet the requirements of a breeding program in u.iici thousands of segregating genotypes must be analyzed.

Quantitative analytical methods for lysine include g as 'hromat:ographic (Zscheile and Brannaman, 1972), c(lo9Irl tr:ic (Concon, 1972; Ghosh and Bose, 1973), and thiniaiyel chromatographic techniques (Heathcote and Haworth,











) 211 it th ;:: methods require hydr)liysis pt i er to .:i ()r thie waino acids.

















SCOPE AND PURPOSE OF TIIIS STUDY


Pearlmillet (Pennisetum typhoides (Burn.) Stapf and E. V. Hublard) is a robust annual bunchgrass grown on 20 million hectares as a grain crop for human consumption (Burton and Powell, 1968; Burton et al., 1972). It is best adapted to the tropics and will grow and mature seed on soils too infertile and too dry for other grain crops. Y'he ability to produce in the dry, infertile areas of the tropics has made it a major food source. Good nutritional qualities of the grain justify the research in production and improvement of this crop presently being advocated (Dewit, 1969).

Pearlmillet has a protein content ranging from 8.8% to 20.91, with lysine varying from 1.9% to 3.4% of the protoin (Burton et al., 1972; Wallace, unpublished data). These data are in agreement with those of Swaminathan et al.,

(1971) whii found 8 to 20% protein, with lysine ranqli nl from . o o t rht lr tcill, 11 ry ) ptophlan ranI inq I iom

0.7 to L.7 of the protein. Losses occur during milling, i t approximately 85% of the proteins in the whole grain normally remain after processing (Dewit, 1969).



14










Millet has a content and balance of essential amino

acids equal to or better than most other cereals (Table 1) and is higher than rice, wheat, or maize in fat and mineral

content (Table 2).

The grain yield potential has not been established, out there is evidence to suggest pearlmillet yields may compare favorably with corn and sorghum where good manaqement including use of adopted varieties, irrigation, and fertilizers was practiced (Burton and Powell, 1968; Burton

et al., 1972).

Purposes of this study were (1) to determine the

ihoritability of protein and lysine, the primary limiting amino acid, in the grain of pearlmillet and (2) to investijtLe the qualities and adaptability of pearlmillet of -



















TABLE 2

O11 AND) MINERAL COMPOSITION OF SOME CEREAL GRAINS


Oil Ca P al (ra in % mg mg 'cailI mil let 4.5 46 314 Winter wheat 1.8 46 354 Corn 3.9 22 268 P ice--brown 1.9 32 221 3.3 28 287



S()urcr,: Composition of foods. USDA Agriculture llandbiok
No. 8.

















MATERIALS AND METHODS


The Diallel Cross


A diallel cross was made during the summer of 1973

u;in.g six inbred lines received from Tifton, Georgia. Line were( selected on the basis of variation in protein and lysine content as measured by Wallace and Block (unpublished) as shown in Table 3.



TABLE 3

PROTEIN AND LYSINE CONTENT OF INBRED LINES
USED IN THE DIALLEL CROSS

Protein Lysine
Line % (as % protein)

1-23 11.24 2.'5

T-27 15.31 2.58 T-140 10.12 2.53 T-4 10.31 3.70 1- 8 16.12 2.15 T-98 19.12 2.00








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Progeny from the diallel cross were planted in a

split plot design over two locations with two replications at each location. Replications contained the six inbred lines produced by selfing the parental lines and the 30 F1s (including reciprocals) derived by crossing the six lines in all possible combinations.

Location I was planted on the Agronomy Farm at the University of Florida, Gainesville. Each plot consisted of three rows 5.5 m in length, with 1 m between rows. Plants were spaced approximately 0.5 m apart in the rows. One application of 403 kg/ha or 10-10-10 fertilizer was broadcast before planting. Heads from the center row of each plot were randomly selected and bagged with a kraft bag and an aluminum screen protector bag to minimize bird damage. Heads remained on the plants until they were fully matured.

Location II was planted the same season (summer, 1974) on the Morikami Farm, University of'Florida Experiment Station, Delray Beach, Florida. Plots were bedded due to the high water table in this area and covered with black plastic to minimize weed competition. The beds were approximately 30 cm highand 1.5 m wide. The plots were fertilized with 403 kg/ha of 10-10-10 fertilizer broadcast prior to bedding. Each plot consisted of two rows 90 cm apart and 5.5 m long placed upon the bed. Plants were spaced approximately 0.5 m apart in the rows. Bedding and covering the beds with plastic minimized interplot competition. Heads from both











)tw; witiuin each plot were randomly selected and ba((gged w\iii 1h 1h a kra ftL )ag( and an aluminum screen protect !. iils rma lined on Lthe plants until they were fully mat Jr .

Al] baqged heads were harvested and three heads were

I lid(,omIly selected from each plot for use in the quantitative analysis for protein and lysine. Line I-18 and its progeny welo. eliminated due to poor germination and poor seed set in several plots.


Analytical Procedures


Equal measures of seed from each of the three heads selected were bulked for estimation of the plot mean for protein and lysine. Samples were ground in a Norris hammer mill through a 1 mm screen and oven dried for 24 hours at 1000 C before analysis. Samples used in the analysis for lysine and the dye-binding capacity (DBC) technique for estimation of protein were further ground in a mortar and pestle using approximately 5 ml acetone as a wetting agent.


I>rotein Analysis

Protein (N x 6.25) was determined by the rapid microj ,ifJn]1 procedure described by Concon and Soltess (1973).

() hii,:itely 2.3 g of K2S04/MgO mixture and 2.3 ml I12 4 C;:er aIdded to a ground sample of 50 mg. The mixture was cated until frothing occurred then approximately 1 ml 30% ii202 was added. Heating at a lower temperature, approximately






20



iO" C, was' contiinu(i until clearing. Distillation was by

tnda d uicro-ij 'lda l procedure (AOAC, 1965). Ar-tnracy ol I;)- pileeodure was verified by correlating result!-s of r,ii(dom ly selected samples with those from the macro-jrldah1 prn c dure (AOAC, 1965). Data generated by the microKlidahiit analysis were used to determine the accuracy of dye-binding techniques for protein estimation in pearlmil I t.


Amino Acid Analysis

Ground, oven-dried samples of 100 g each were hydrolyzed in 100 ml 6N HCI in a nitrogen-saturated atmosphere at 400 C for 24 hours. The solute was brought to dryness in a rotary evacuator at 400 C and added with 10 ml 0.01N HCl. A 1:10 (v/v) dilution was used for the complete amino acid determinat ion.

Fifteen samples representing a complete set of crosses from a replication, including inbreds but not reciprocals, w, analyzed on a Jeol Model JLC-6AfJ Amino Acid Analyzer. 'Si: Ce Analysis

Lysine was estimated as grams of lysine/16 g N using ti( r-apid lysine determination technique described by Cone ,n (1972). A protein extraction was made from a 500 mg s'7;,,ie of ground seed using 1.5 ml 70% ethanol followed by

5.5 ml 0.5, NaOH. Nitrogen content of the extract was it(,iinined by rapid micro-Kjeldahl procedures (Concon and






21



<; ::;, 1972) u:;in a 0.5 ml. aliiquot. Din itrobr ,nz ie

:u ll a (i)N ;) wa:; used to determine lys.i ne cont(:nt col 1 imtl rica lly. A Bausch and Lomb spectrophotometer (i ,l1 20) was used to measure absorbance at 460 nm rather tAin al 300 nm as indicated in the literature. The spectoi inol ticter tacked sensitivity at 360 nm to distingui sh among samples whose differences were visually apparent. Accuracy of this procedure was verified by correlating results with data on 15 samples analyzed with the amino acid analyzer.


Statistical Analysis


Protein data were analyzed using Griffing's (1954)

method 3 (in which all Fls are used), model I (all effects except error are considered fixed) for estimates of general combining ability (GCA) and specific combining ability (,CA) in protein inheritance. Method 3, model II, in which A1ll ff(ects are considered random variables, was used to cim; i atc heritability with the formula for the genetic variance estimate (2 G) modified to include the reciprocal varilace estimate (82). Estimates of heritability (h2) in :so: iired sense were calculated.

The same data were analyzed by the Hayman (1954)

M:n hod of diallel cross analysis for estimation of genetic eli-cots. Gene action parameters estimated by this method are additive, dominance, and maternal effects.
















RESULTS


Protein Inheritance


A highly significant difference was found between

crosses. All other parameters were non-significant (Table 7). Location differences were non-significant but observation of mean values of crosses compared across locations (Table 4) suggests the environmental effect upon crosses was variable in both the positive and the negative directions masking possible location effects.

Table 5 and 6 contain the means of each cross (by location) arranged with the inbred parents forming the diagonal. In this format reciprocals are also located diagonally, from one another. Reciprocal differences in some lines are apparent.


Combining Abilities

Data at both locations were subjected to analysis of variance for combining ability (Tables 8 and 9). Estimates of GCA and SCA differed with the reciprocals showing no significant difference between locations. Parental reciprocal estimates (8 ) (Tables 10 and 11) indicate the major reciprocal differences of approximately the same magnitude



22













-- x r 'H cm cn 0 a r m 0 L n m G N v o 00 T 0 C) J H N N ,- Cl A N








S, < -4 0 -4 L r-4 rQ.. '. * * 0 (- LA 0 L A 0 0 C 4 N






1 o -4 r- H 0-1 (N N H -- r- N -4












-- 4
0 0A
1- "N N N N r- '- m N n 4N














it





E-4 I I I I I I i t -1 H a)








24




CN
(N '2r CO in o Mn a) C) 'O 0 N -1
C, Cyt o a o m a m k in I b 1-4 )- c) in (7) r- 10 r- C2 "T CN S . . ,- -4










+ OD ( N L
O . . . . * *







0 C) ,-.



(,N 0 C ) 0 C- 4 0 m 0 D m f L) U


(Ym 0a r. rrO
r-4 ,-4 ,-l r-A i i r-4 ,-4 r-4 r-4 -4 r- 4

cl)


-O O


0U)1 m oQo a i 0 D c r C- I I )

O O C0 0 -r 0 (Y) r- C 0r N 41


A N N

4J



C)


o) E IE


I I I I I I I I I I I I

4.)




Uo C 4 cv
'-4 ,-)






25












TABLE 5

MEAN PROTEIN CONTENT OF ALL PROGENY
OF A DIALLEL CROSS, GAINESVILLE



Fmeale nMale parents
ijare nts 1-23 T-27 T-140 T-4 T-98 1-23 16.42 13.92 15.09 15.45 13.99 T-27 20.79 19.66 14.67 15.15 17.c6 T- 140 13.79 16.03 23.12 17.68 14.86 i 1 15.40 18.56 17.37 16.90 17.49 T-98 16.08 15.06 17.98 15.26 17.68




















TABLE 6

MEAN PROTEIN CONTENT OF ALL PROGENY
OF A DIALLEL CROSS, DELRAY BEACH


Femla le Male parents parents 1-23 T-27 T-140 T-4 T-98
(%)

1-23 14.96 14.77 15.94 13.02 13.75 T-27 21.51 23.48+ 14.79 10.11 11.37 T- 140 17.22 12.70 21.46 lr,.23 17.40 T-4 16.32 14.34 17.01 19.43 15.76 T-98 15.55 11.07 14.15 16.52 19.05



,Missinq value estimated by Yates method (Cochran and Cox,






27









TABLE 7

ANALYSIS OF VARIANCE FOR PROTEIN OF PROGENY
RESULTING FROM A DIALLEL CROSS WITH FIVE INBRED PARENTS


Source df MS

Locations 1 12.945

Error (a) 2 1.164



Crosses 24 25.123** Locations x Crosses 24 0.572

Error (b) 48 4.49



**Significant at .01.






















TABLE 9

VARIANCE ANALYSIS FOR COMBINING ABILITY FOR
PROTEIN, GAINESVILLE

:Source of Mean va: i dance df squares C, dining ability;

General 4 1.751 Specific 5 2. A06 Reci procl 10 4. 359 Error 48 2.24







29














TABLE 9

VARIANCE ANALYSIS FOR COMBINING ABILITY FOR
PROTEIIN, DELRAY BEACH

S:ureo of Mean v.1 in e df squares Combining ability:

General 4 7.991* Specific 5 9.875** Reo, i pi c-a1 10 4. 765* )l ,or 48 2.24



*Significant at .05.

**S;iinificant at .01.



















TABLE 10

ET;'I' IA; OF GCA, SCA, AND RECIPROCAL VARIANCES ASSOC IATID W1TIH EACH PARENT, AND ENVIRONMENTAL L
VARIANCE MEANS FOR t'o)TEIN, GAINESVILLE

2 a2 2 2 Cen t g s r e 1-23 3.5570 0.0025 3.4238 0.1269 T-27 -0.2719 0.5983 4.5627 0.1269 T-140 -0.4476 1.8200 0.0487 0.1269 T-4 -0.1614 0.9769 0.3773 1269 1'-98 -0.4953 -0.5026 1.0450 0.1209


















T'A IllE 11

1:I;'Tl;IATi'S OF GCA, SCA, AND RECIPROCAL VARIANCE:S ASSO()CIATED WI'YHl EACH PARENT, AND ENVIRONMENTAL
VARIANCE MEANS FOR PROTEIN, DELRAY BEACH

2 2 2 .2
Parent g s r e 1-23 1.4735 3.9958 4.0854 0.12(9 T-27 1.9195 4.0026 4.6339 0.1269 'i-140 0.4737 0.5017 0.4172 0.1269 T-4 -0.3991 0.5833 1.4831 0.1269 T-98 0.0937 1.4675 0.1915 0.1269






32



occui ,l in thi, s ini crosses at both locations. These ,iata S,,'I:;t tIc cnv i runmlicnt had little effect upon rec i proca l etl tv't ill prop'ny from lines 1-23 and T-27. Environirnt dl ci ict:; appeared to have an influence on progeny from li:i:; T-4 and T-98, however. Differences in combining abilities between locations are an indication of the effect of environment on protein content.

The GCA of each of the five inbred lines in this study was non-significant at Gainesville. Estimates associated with each parent show line 1-23 superior to all other lines in that location, this being evidenced by the consistency of the progeny in approaching the parental value in protein content (Table 5).

The GCA variance estimate ( 2) associated with each
g
parent indicates lines T-27 and 1-23 are superior to all other lines tested at that location (Table 11). The most striking difference in the parental GCA variance estimates between locations is that line T-4 has a negative value while four lines have negative values at the Gainesville location.

Analysis of SCA shows non-significant effect at Gainesvil.e (Table 8) and a highly significant effect at Delray BE;ach (Tables 8 and 9). Parental estimates for SCA (2) si)ow; line T-140 to be superior to other lines at Gainesville (Table 8) and lines T-27 and 1-23 superior at Delray Beach (Taoles 10 and 11).











i 1 ,i i I it y :;timates

lir Lrtabi I it ies in both the broad sense an( the

n, it rtow sernso were estimated assumi n g ;id it i ve v,ri a r

2) to e(ll twice the GCA variance (02 ) (Griffinq,

1954). Total genetic variance was estimated by the formula

2 22 2 2
2 = 282 + + 8
G g s r where

8G = total genetic variance,

2
= estimate of GCA
g 2
( = estimate -f SCA, and
s


a = estimate of reciprocal effects.
r


The phenotypic variance (d 2) was estimated by the forinula

2 2 2





.2
P e




error.


iiarit,hbility is then estimated by the formula

2 262
2
P

ia the narrow sense and











.2
2


in the broad sense.

Nairow-sense heritability estimates for both locations were negative due to the negative values in the GCA variance estimate (Table 12). Broad-sense heritability was extremely variable between locations with a value of

0.008 at Gainesville and 0.54 at Delray Beach. Genetic Parameters

The Hayman (1954) method of diallel analysis indicated dominance for protein content. The negative values

ill aean squares were interpreted as indications of dominInce for lower protein content (Table 13). The consistency with which the progeny deviated in the negative direction from the mid-parent value (the mean of the pl)rents) for protein content confirms this interpretation.

Analysis of data also indicates no significant

q: netic difference among parental lines. The Yates (1947) m-thod of interpretation of data reveals a significant

mt,rnal effect which is evidenced by significant reciprocal differences involving some lines.












C) 'o (N 3




0>






f1



H4 Lf) Ll)

0





U






O >
0 o


Coo




-4
Q) C) 14 04 mm U)(5 -4 c: m c in









04 44 p








-4-O




0 15 U 0 0 W>1 0 4: m OrO Ui U U)> (9n U)






















TABLE 13

ANALYSIS OF VARIANCE FOR GENETIC PARAMETERS
OF PROTEIN INHERITANCE


::ou 11ce ot Mean var fiance df squares Piarental 4 5.93 DoaM i nance 10 -883.32 MLtu'rnal effect 4 17.13

r r 6 5.58






37



Lysine Inheritance


Analysis of inheritance for lysine was limited to

h(:;e samples analyzed on the amino acid analyzer, di,, to the unrealiable results of the rapid analytical technique for lysine (Table 16). There was no replication, hence statistically valid inferences cannot be made. Hypotheses, however, may be formulated for verification in further work.

Regression analysis of the data (Table 14) indicates a linear relationship exists between lysine and total essential amino acids but with a very low correlation

coefficient. A negative correlation (r = -0.44) exists between lysin' and protein content with a strong positive correlat in);i between lysine and the sum of methionine and threonine (1 0.76). A linear relationship was not indicated between LoLta essential amino acids and total protein although the data indicate a slight negative correlation may exist.


Quantitative Analytical Methods


TiiE DBC method for screening samples for protein

content as reported by Kaul et al. (1970) was unreliable wh ao used with pearlmillet (Table 17). The rapid, quantitatiVve analytical technique for lysine reported by Concon (1972) also proved unreliable (Table 16). The rapid microjeldahl technique reported by Concon and Soltess (1973) proved very accurate and consistent and correlated well (r 0.79) with the macro-Kjeldahl results (Table 18).


















0 H n r) 1 oo I oA (





C) L 0 L 4 L( ) O 0 0

(NW r'1 rn i n Lr r- rn (N4


-4 q C :T r- ON 0 r--4 (n O Lr) r- k.0
ND 0 n LM M -I M --4
N W N T N 0 H0 ON M) 0 rM
C (N H N C (N r He ( N 0 0 N H m N ON (Nm NL -4 CmNl N



o 0 (N ON a i N OW 0 Lo MCMN (N H ,44 H (,




0 N IV A NON tN~ N H f N (M HML


U O i (N (N (N ( H (N > r (N '0 CI H C

--I O O -I q C C O- O O


-- 44
01 0


C > ar r-4 r4 N m O r4m 4 L 4 u lhO 4i: Lon

Ko ON m m r- m r- L CN C mC) lJ rSm m (co or (N a N n o O (Y 1 coa O




l m m 'l o -i mU m t m C m tD
)- O H O I I I H I H N LA LA ( +0












o o T m
T N ON 0 COm





L (N H I N H (N O I -(-4 O-4 ( H I
a) O O I 0
4) N 0 0 H C 0 ( N



0 0 0 w

Ol (N H H I4 O N 14 1) H f ON -4 4 It f-4 CO -























TABLE 15

LYS INE CONTENT OF PROGENY
FROM A DIALLEL CROSS


i t ',ll e Male parents Sen~ I s 1-23 T-27 T-140 T-4 T-98 1-23 1.54 1.64 1.14 1.56 2.27 '-27 1.04 0.68 1.24 L.78 T'-140 0.79 1.21 1.79 T-4 1.35 0.73

'-98 t1.62






40



TABLE 16

V\'ALUATMI; O1' TRACTIONN FOR THE COHCON RAP !
I;1.1NE;; ''il;CillJlQTl', AND COMPAP I';ON OF' LY:;INL VALUES
BY TiEll CONC(,,'J TI;ClN IQUE TO TIIObIH' OF THE AMINO ACID ANALYZER

NJit roj'n (q/100g Lysilie
cr-ould smp le) (grams/lysine/16q N
Ground I N Amino acid Concon I::s ,'d Extracted* Extracted analyzer method 1 2.78 1.35 48 1.43 2.63 2.95 0.83 28 1.32 1.32 2.55 L.13 44 1.13 2.41 4 2.95 2.48 84 1.56 3.74 6 2.11 1.31 62 2.27 3.34 9 2.08 1.92 92 0.75 1.33

10 2.55 1.79 70 1.16 1.94 12 3.17 1.04 33 1.61 1.45 13 3.67 1.36 37 0.76 1.08 16 2.80 2.05 72 1.20 2.13 18 2.39 2.00 84 1.79 1.36 19 2.74 1.35 49 1.32 1.45 24 2.80 0.87 31 0.73 1.57 31 2.82 1.31 46 1.63 2.00



*:tracted as described by Concon (1972).






41



TABLE 17

CO()MPARI;()N 131:TWI":EN DBC AND MICRO-KJE ,DAHiL
ANAIE,,i S FOR PROTEII N, REPLICATION I, (GAINESVILLE


Cross DBC values Micro-Yjr 1dahl absorbance % protein I-i ; I f ed .29 16.71 1-23 x T-27 .28 13.86 1-23 x T-140 .27 14.38 1- 3 x T-4 .29 12.44 1-23 x T-98 .26 14.77 T-27 selfed .31 19.63 i'-27 x 1-23 .32 20.99
x T-140 .32 14.96 x T-4 .30 14.96 x T-98 .26 17.25 T-H40 selfed .31 23.32 ''-140 x 1-23 .31 13.99
x T-27 .26 15.94 X T-4 .30 17.88 x T-98 .26 14.77 T-4 selfed .29 16.71 T-4 x 1-23 .30 17.59
x T-140 .29 17.36 x T-98 .29 17.49 1-98 oIlfcd .31 17.64 i-98 x 1-23 .30 15.68
x T-27 .32 14.38 x T-140 .29 17.49 x T-4 .31 14.97






42



TABLE 18

,'! Im ( )n N ITWEJ IN MICRO- AND MAC 0'- (. JELDA ,
ANA LY tOR PROTiI REPLICATE 0( l 11, GAINE;VIILLL


C ros Macro-K j e dah 1 M i c ro-K h % Protein % Protein 1-23 16.12 16.13 1-213 x T-27 14.15 13.99
x T-140 15.47 15.80 T-4 18.84 18.46 x T-98 13.59 13.22
T-27 selfed 19.53 19.70 T-27 x 1-23 19.78 20.60
X T-140 14.25 14.38 x T-4 15.56 15.35 x T-98 17.91 17.88 T-140 selfed 22.78 22.93 '-140 x 1-23 13.31 13.69
x T-27 16.50 16.13 x T-4 17.62 17.49 T-98 14.91 14.96 'I- slfed 17.81 17.10 T- :: -23 13.31 13.21
: T-27 18.00 18.08 T- 140 17.42 17.38 T-I8 17.44 17.49
8 :; i ,I 18.38 17.68 T'-'8 x 1-2 16.22 16.48
.: T-27 15.09 15.74 x T-140 17.72 18.27 x T-4 15.00 15.55

















DISCUSSIOi


The geneticc components of protein inheritance are

comle:x. ihlen one considers that production of all aminr ,cihis are polygenically controlled and amino acid sequence of each protein is genetically determined the complexity oit protein inheritance becomes apparent. The problem is further complicated by evidence of maternal influence (,Helson, 1969) and divergence in modes of inheritance in which the same character may follow different patterns of inheritance in various lines within the same species (Balint, 1970).

Another force influencing protein content is the

environment. Cultural practices such as the rate and time of fertilizer application influence protein content of many cereal grains (Swaminathan et al., 1971). Increased protein ro fertilizer application increases the prolamine fraction wit i ,nen ;ing the more (desirable globulin and albminitl

I t uII: I ul i ln in p)rote in (f lowe I b iolo ic ( l va I ti .

(I at I( L (1968) reported high protein genetic tra its whf at are associated with a more efficient and complete tr;nslocation of nitrogen from the plant to the grain rath .r t.a, with differential nitrogen uptake or accumulation by it"* p lant.4

43





44




Thidly, tLhe array of genes which control prot in

i;i rlance arc le i ec-td by environmental factors. ijqi ic: cnt gjenoLyp -on-environment interactions have behe n i1irtd in sorglhum (Crook and Casady, 1974; Deyoe and :e llnbierer 1965; Liang et al., 1968), rice (Sampath ct. Al., 1968; [ii]lerislambers et al., 1973), wheat (Johnson

Sl., 1968), and pearlmillet (Mlahadevappa, 1967). It

i)ccomes evident the phenotypic expression of protein content in an individual plant is a balance between the genetic potential, the environmental conditions, and the interaction between the two.

The results of this study are further evidence of the complex nature of protein inheritance. The significarmCe of environmental differences due to location appears to be masked by other interactions.

Environmental differences were present, but the effect of environment across 25 different genotypes masked this ( ict. Any statistical analysis of an experiment concerni:nmn protein analysis should, therefore, partition the vArianlce into all possible interactions.

Differences between replications in some crosses

(Tail(, 9) cannot be explained. Seed used in growing the

o~,;etirnotion was taken from the same head, hence variations S a;it i',, attributed to possible differences in gqnotype of ta I parents. This is also verified by the analysis of






45



l ia : ilor cjen tic parameters (Table 13). Plots in which eii ernccs occurI11
Closer (examination of the data indicates two significert flcts which are not conclusive but do give some basis tior an hypothesis. When individual plots with replication di; ;erences are counted the Delray Beach location has t;a-e times the number found in Gainesville. Further examination reveals that in every case wither line T-4 or line T-98 or both lines are involved. These same lines also have divergent reciprocal variances across locations (Tables 10 and 11). It may be hypothesized that some tyvi' oi: maternal effect on environment interaction is ,eulrring, when these facts are considered in the light of

-vi Jnnc, of some maternal effect as indicated by the ,raily:;is of genetic parameters (Table 13). Environmental factors which caused the higher incidence of replication diierncess at Delray Beach are unknown.

PIresence of maternal effects as indicated by the data
-fcct. at Delray Beach or line T-98 at Gainesville. These






46



"At indicate maternal effect in pearlmillet may be pr-sent ini ll lin s but is variable clue to interaction with the(



The presence of maternal effects and their interScLon with envir onment complicates the breeding of pearlmillet for high protein in the grain. Any program designed for this purpose should examine not only combining abilities but also reciprocals. These factors influence not only the choice of parents but also whether a chosen line should be used as the male or the female parent.

Plant breeders have placed a great deal of stress upon heritability estimates for specific genetic traits. These estimates, however, are valid only where extensive ra.s
Variability in estimates of heritability for protein content is not unique to millets. Crook and Casady (1974) Yound heritability estimates for this trait in sorghum varied between methods of calculations and with the populaLion. Liang et al. (1968) found components of additive v r-ace to be significantly different between locations. ';"i difference would, in turn, result in significantly

I .tnt her itabil ity est iiat s bet ween locat ions.






4/



11(,rit bi it.y estimates for protein content per e

;ilr p ,i il 1cL wei t, not found in the literature, but the results described by Ahmad et al. (1972) and Mahadevappa (1907) were similar to those in this study. Predominance oKf non-additive gene action and considerable environmental t.: ct both contribute positively to the henominator of the hor i.tability estimate formula. Environmental interaction wti gjcnotype could account for the variability in estimat s,

between locations.

Experience in this work indicates that analysis for only GCA or SCA may prove inadequate when working witn a 1un iLiittively inherited trait. It should be one of a ::le io :: of progrL;s i ve steps lead inq toward a better ulnderstLanding of the gene actions involved in the inheritance of that trait. Only through such analyses can we discover whether protein content per se is controlled by a major gene or genes or if it is the sum of many genes, each of which cont rols the synthesis of a particular amino acid with interactions between control mechanisms.

The limited data on lysine inheritance make detailed analysi: impossible. Certain correlations can be made, how-ever, and may be useful in the formulation of hypothe-es for further study.

Data indicate, when selection is based solely on prot,i content, higher protein is accompanied by lower value;






48



Sihe tl" m iost I limiting amino acids in cereal grains. !his is evidenced by the positive correlation between lysine ,nd the total of methionine and threonine (Green and Phillips, 1974). Other evidence is the negative correlation between lysine and total protein.

Tihe absence of a linear relationship between total essential amino acids and total protein with the very low correlation between lysine and total amino acids indicates progress could be made through selective breeding.

All three analytical techniques, DBC, the Concon rapid lystne, and rapid micro-Kjeldahl, were selected for study becAuse they showed promise of being effective tools in

p ,,irlmillet breeding program designed for protein improvemont. Reasons for the failure of the dye-binding technique and the rapid quantitative lysine technique are unknown. 'J'ire were no correlations which could be attributed to ;enotype (Table 16). One hypothesis is that part of the lysin in piearlmillet is bound in protein complexes which prevent actionin or inhibit dye-binding. Singh et al. (1971) reports evidence for additive and cumulative gene action for lihc values in rice. No other work has been reported on this p ro)] em.

















CONCLUSIONS


The inheritance of protein in pearlmillet is influenced by the presence of dominance for low protein with evidence of maternal effects which are affected by environmental afects. More data must be collected to estimate these eif-octs and their significance in a breeding program.

The inheritance of lysine may be influenced by the presence of dominance in some lines (Table 15). Lack of analysis of replications precluded estimation of environmc.ntal effects. Regression analyses indicated selection a,:ui on total protein may have little effect on total ;su;ntial amino acids but have a negative effect ,n K :re, metii nine, and threonine.

Rapid quantitative analytical techniques for lysine wor' uinreliable when applied to pearlmillet in this work. Pro, ss in breeding for grain with better protein quality will e impeded until this deficiency is corrected.











49
















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ialint, A. 1970. Protein growth by plant breeding. Adademiai Kiado,Budapest.

BPrijgs, F. N. and P. F. Knowles. 1967. Introduction to
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50






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i, L, J. P. 1909. The potential role of pearlmil li as
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.e, Ri. J. 1951. The interrelationships of proteins and
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dCesh, Mira and S. Bose. 1973. Development of rapid tests
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Hillerislambers, D., J. N. Rutger, C. 0. Qualset and W. J.
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,il li iig sLibility of rice vlriettes in A: i,.
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Juliano, Bienvenido O., Juz U. Onate, and Angelita MI. eel
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amra, O. P. 1971. Genetic modification of seed protein
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7: 11-15.

Lianq, G. J. L., E. G. Ileyne, J. II. Chung, and Y. O. Koh.
196E. Tihe analysis of heritability variation for thire agronomic traits in a 6-parent diallel of grain sorghum
(Sorqhum vulgare Pers.). Can. J. Genet. Cytol. 10:
460-469.

Mahadevappa, M. 1967. Investigations of the inheritance of
protein content in pearlmillet. Curr. Sci. 36:
186-188.

Mortz, I. T. and L. S. Bates. 1964. Mutarnt iene that change'
protein composition and increases lysine content of
maize endosperm. Sci. 145: 279.

M ,tez, E. T. anO L. A. Bates. 1965. Second mutant m!ene
affccing the amino acid pattern of maize endosperm
pro tQ-ins. Sci. 150: 1469-1470.










flnkr, 1,. 1964. Plant breeding and nutritional values in
ce ra :;. Iei d i t.as 52: 151-165.

k, .. 1972. Improvement of nutritional values in cereals.
lived i tas 72: 1-128.

.,[eln, O. E. 1969. Genetic modification of prrot'in quality
in plants. In: Advances in Agronomy, Vol. 20, A. G.
rman (ed.) pp. 171-194.

INe;on, O. L. and M. T. Chang. 1974. Effect of multiple
alourone layers on the protein and amino acid content
of maize endosperm. Crop Sci. 14: 374-376.

-icol, B. M. 1971. Protein and calorie concentration.
Nutritional Reviews 29: 83-88.

Pall'er, Rhoda and G. 0. Kiohler. 1969. Survey hydrolysis
procedure for lysine analysis. Cereal Chlem. 46: 22-26.

Sal, Lucila and L. W. Rooney. 1970. Use of dye-binding and biuret techniques for estimating protein in brown
and milled rice. Cereal Chem. 47: 38-43.

Sno;on, P. 1963. Heritability: A second look. In:
Statistical Genetics and Plant Breeding. W. D. Hanson
anid II. F. Robinson (ed.), National Academy of Science
Publication 982, Washington, D.C., pp. 609-614.

Sampath, S., S. Patnaik, and G. N. Mitra. 1968. The breeding
of high protein rices. Current Sci. 9: 248-249.

Sawiuney, D. K. and M. S. Naik. 1970. Amino acid composition
of protein fractions of pearlmillet and effect of nitrogen fertilization on its proteins. Indian J.
Genet. Plant Breeding 29: 395-406.

Singh, C. B., S. Arora, and A. K. Kaul. 1971. Inheritance
of dye-binding capacity value in rice. Current Sci.
40: 28-29.

Sinigh, P. and J. D. Axtell. 1973. High lysine mutant gene
(il) that improves protein quality and biological value
of (rain sorghum. Crop Sci. 13: 178-181,

SwaIrinathan, M. S., M. S. Naik, A. K. Kaul, and A. Austin.
I*7I Genetic upgrading of protein gquli ity and quantily ; in India. Indian J. Genet. Plant Breeding 41:
S() 4 05.






54



'.iello, F., N. A. Alvarez-Tostado, and G. Alvarado. 1965.
A study on tlic improvement of the essential amino
acid 1 bilance of corn protein. I. Correlation between
r-,ci~al and varietal characteristics and lysine
lcvcels of corn. Cereal Chem. 42: 368-384.

<,Ly, D. C. 1971. Improved dye method for estimating protein.
J. AmNir. Oil Chem. Soc. 48: 29A.

Woodwrth, C. M., E. R. Leng, and R. W. Jugenheimer. 1952.
iifty generationss of selection for protein in corn.
A r(n. J. 44: 60-65.

Yates, F. 1947. Analysis of data from all possible reciprocal crosses between a set of parental lines.
Aeledity 1: 287-301.

Escieile, F. P. and Bert L. Brannaman. 1972. A simplified
method for lysine by gas chromatography. J. Agr. Food
Cieim. 22: 537-538.

















BIOGRAPHICAL SKETCH


James Bynum Barnett was born July 30, 1931, in Memphis, T''onlnes :;. lie attended high school at Memphis Technical and anLtred the University of Tennessee Junior College at rL-tin, Tennessee, in 1949.

He entered the U.S. Marine Corps at the outbreak of the )orean Conflict and was sent to Korea after basic training ~ind training as a radio operator. lie rose to the rank of ~staI serqant and in 1953 was recommended for commiss;i :,ing iW the rank of 2nd lieutenant for demonstrating leadership abilityy in combat. He was commissioned in March of 1953. io married Martha Hill of Paris, Tennessee, in December of the same year. lie was released from active duty in 1955 with the rank of ist lieutenant and was honorably discharged in 1962 with the rank of captain.

He returned to Tennessee upon release from active duty ST and attended the University of Tennessee at Knoxville ho, I h it, i ii; I l ) o I ; itqi: c'''ll' l 1 ",' in XAtj n '

in 958. ie assumed management responsibilities of a family ,oo-d farm in Huntsville, Alabama, upon graduation, and was di employeded at Redstone Arsenal with the missile industry.

LIt the missile industry in 1962 and accepted employment










in the iiuntsville Public School System as a biology



Mr. Barnett and his wife felt called to the mission ic iid and were accepted by the Presbyterian Board of World li:; i; ,s ais l(ric uura l/educational missionaries to Mexico illn 1903. They took their post in Teloloapan, Guerrero, :MrNico, after completion of one year of theological training at Columbia Seminary and one year of language training in Costa Rica. Mr. Barnett's duties during that time were similar to those of an agricultural extension agent in an

area comparable in size to the state of Florida.

Mr. Barnett attended Murray State University, Murray,

Ki; niucky, during his furlough year (1969-70), and began work

(n ) f a Master of Science degree in agriculture. lie and his family returned to Mexico in 1970. The Presbyterian Church of: Mexico voted at that time to request that all missionaries be removed from Mexico to allow complete autonomy. Mr. Barnett returned to Murray State University in 1972 and completed work on his Master of Science degree in August,

1972.

Mr. Barnett began work toward a Ph.D. in Genetics in 1972 at the University of Florida. He taught the Genetics

i ty (AY363) six quarters and wrote the manual which is t-e:;.l.y being used in that course. lie taught the genetics S::urs (AY362) during the fall of 1974.






57



i (I Mrs. B3arnett have four children. i1, reci,,ved tia i.l. i e'' in June 1975. Ile belongs t, S iqwa Xi.










c:1 'r if y tIi ,L I have read this study and that in my < ,i i I it c ii ) Irri to acceptable standards of scholar ly
III in and i:; fu I ly adequate, in scojp) and quality, a':; 1 ti :;: ll;: t (t i I or the degree of Doctor of Phi losophy.




Rex L. Smith, Chairman
Associate Professor of Agronony



I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pr(,5entation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.




es Soule
Professor of Fruit Crops



I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, ,as a dissertation for the degree of Doctor of Philosophy.




Victor E. Green, Jr.
Professof of Agronomy



I certify that I have read this study and that in my
in it conforms to acceptable standards of scholarly r:; : _Lionnd is fully adequate, in scope and pqual ity, is dissertation for the degree of Doctor of Phi loo phIy.




a ph e. Robbins
Associate Professor of Food Science










I ('rt.i ly t lht I have read this study and that in my
0.>i;:ion it n' oi mI. : I:o acceptable standards of scholarly
i: < .iI i:; i.ul y adequate, in scope and qua lity, :i li:;t '!ILa iLioil Ior the degree of Doctor of Philosophy.




Ramon C. Littel
Assistant Professor of Statistics



'iji ii lssertation was submitted to the Graduate Faculty of to iCllone of Agriculture and to the Graduate Council, and wais accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

June, 1975




Dean, "ollege of Aqricultu




Dean, Graduate School




Full Text

PAGE 1

THE INHERITANCE OF PROTEIN AND LYSINE IN PEARLMI LLET ( Pennisetum typhoides (Burn.) Stapf and E. C. Hubbard) GRAIN By JAMES BYNUM BARNETT A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1975

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To my wife, Martha, our children, Charles, James, George, and Virginia, and my wife's mother, Mrs. A. L. Hill, each of whom has sacrificed and willingly given of what they had toward the completion of this goal.

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ACKNOWLEDGMENTS I acknowledge the guidance, encouragement, and helpful criticism received from the members of my Supervisory Committee. Each member has contributed not only to this study but to my total education. Dr. Rex L. Smith, Committee Chairman, has always been available for discussions and lending aid. Dr. James Soule gave me a better understanding of fruit crops, Dr. V. E. Green, Jr., imparted to me some knowledge and appreciation of tropical agriculture and agronomy. Dr. R. C. Robbins' assistance and guidance in the analytical phase of this study was invaluable. Dr. R. C. Littel's critique of the statistical analysis gave me a setter understanding of that discipline. I consider each a close personal friend and am honored that they served on this committee. i i i

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS iii LIST OF TABLES V ABSTRACT vii INTRODUCTION 1 The Protein Problem 1 Possible Solutions to the Problem 6 SCOPE AND PURPOSE OF THIS STUDY 14 MATERIALS AND METHODS 17 The Diallel Cross 17 Analytical Procedures 19 Protein Analysis 19 Amino Acid Analysis 20 Lysine Analysis 20 Statistical Analysis 21 RESULTS 22 Protein Inheritance 22 Combining Abilities 22 Heritability Estimates 33 Genetic Parameters 34 Lysine Inheritance 37 Quantitative Analytical Methods 37 DISCUSSION 4 3 CONCLUSIONS 4 9 LITERATURE CITED 50 BIOGRAPHICAL SKETCH 55 iv

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LIST OF TABLES TABLE Page 1 COMPARISON OI' 1 ESSENTIAL AMINO ACIDS IN CEREAL GRAINS EXPRESSED IN g/16g N 3 2 OIL AND MINERAL COMPOSITION OF SOME CEREAL GRAINS 16 3 PROTEIN AND LYSINE CONTENT OF INBRED LINES USED IN THE DIALLEL CROSS 17 4 PROTEIN CONTENT OF F, GENERATION HYBRIDS AND SELFS RESULTING FROM A DIALLEL CROSS OF FIVE INBRED PARENTS 23 5 MEAN PROTEIN CONTENT OF ALL PROGENY OF A DIALLEL CROSS, GAINESVILLE 2 5 6 MEAN PROTEIN CONTENT OF ALL PROGENY OF A DIALLEL CROSS, DELRAY BEACH 26 7 ANALYSIS OF VARIANCE FOR PROTEIN OF PROGENY RESULTING FROM A DIALLEL CROSS WITH FIVE INBRED PARENTS 2 7 8 VARIANCE ANALYSIS FOR COMBINING ABILITY FOR PROTEIN, GAINESVILLE 28 9 VARIANCE ANALYSIS FOR COMBINING ABILITY FOR PROTEIN, DELRAY BEACH 2 9 10 ESTIMATES OF GCA, SCA, AND RECIPROCAL VARIANCES ASSOCIATED WITH EACH PARENT, AND ENVIRONMENTAL VARIANCE MEANS FOR PROTEIN, GAINESVILLE .... 30 11 ESTIMATES OF GCA, SCA, AND RECIPROCAL VARIANCES ASSOCIATED WITH EACH PARENT, AND ENVIRONMENTAL VARIANCE MEANS FOR PROTEIN, DELRAY BEACH. ... 31 1 2 MEAN SQUARES AND ESTIMATES OF VARIANCE FOR COMBINING ABILITIES AND RECIPROCALS OF PROTEIN. 35 13 ANALYSIS OF VARIANCE FOR GENETIC PARAMETERS OF PROTEIN INHERITANCE 36 v

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LTST OF TABLES (continued) TABLE Page 14 ANALYSIS FOR ESSENTIAL AMINO ACIDS 38 15 LYSINE CONTENT OF PROGENY FROM A DIALLEL CROSS 3 9 16 EVALUATION OF EXTRACTION FOR THE CONCON RAPID LYSINE TECHNIQUE AND COMPARISON OF LYSINE VALUES BY THE CONCON TECHNIQUE TO THOSE OF THE AMINO ACID ANALYZER 4 0 17 COMPARISON BETWEEN DBC AND MICROKJELDAHL ANALYSES FOR PROTEIN, REPLICATION I, GAINESVILLE 41 18 COMPARISON BETWEEN MICROAND MACRO-KJELDAHL ANALYSES FOR PROTEIN REPLICATION II, GAINESVILLE 42 vi

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Abstract of Disserta tion Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE INHERITANCE OF PROTEIN AND LYSINE IN PEARLMILLET ( Penn isetum typhoides (Burn.) Stapf and E. C. Hubbard) GRAIN By James Bynum Barnett June, 1975 Chairman: Rex L. Smith Major Department: Agronomy A diallel cross was made using inbred lines of pearlmillet selected on the basis of variation in protein and lysine content. The F^ generation was planted in a randomized block design at two locations with two replications at each location. Each replication contained the six inbred lines produced by selfing and the 30 progeny produced by crossing the five lines in all possible combinations Total protein (N x 6.25) was determined by rapid microKjeldahl technique. Statistical analysis indicated protein in pearlmillet is influenced by the presence of dominant gene action for lower protein with evidence of maternal effects which are influenced by environment. Regression analysis indicated no linear relationship between total essential amino acids and total protein. Analysis of lysine inheritance was based upon a complete amino acid analysis of samples representing a complete vii

PAGE 8

set of crosses from a replication including inbrecl parents but not reciprocals. Lack of replication made complete statistical analysis impossible. Regression analyses indicated a linear relationship exists between lysine and total essential amino acids with a very low correlation coefficient with a negative correlation between lysine and total protein. A positive correlation exists between lysine and the total of methionine and threonine. Dye-binding techniques for protein and a rapid quantitative analysis technique proved unreliable when used in pearlmillet. The cause of this unreliability was undetermined viii

PAGE 9

INTRODUCTION The Protein Problem Internationally, cereal grains are the major source of protein in the human diet while legumes and animal proteins act as supplemental sources. Nicol (1971) reports sorghum and millets contribute 63% of the total dietary protein in West African diets while rice contributes up to 76% of the total dietary protein in the Far East. Estimation of the contribution of cereal grains to the dietary protein in Latin America is complicated by the fact that in soiik> areas rice is the staple grain while in others corn is the basic grain in the diet. Altschul (1965) estimates cereal grains produced more protein for human consumption than all animal and legume sources combined. The importance of protein quality and quantity in cereal grains is secondary in developed countries where animal products are the major source of protein. The prominence of cereal grains as a major protein source in human diets and problems which are inherent have only recently begun to be elucidated. Howe et aj. (1965) suggests that where sufficient cereals to satisfy the caloric 1

PAGE 10

2 requirements were invested, there would be an adequate protein balance only if the amounts and kinds of the cereal proteins were comparable to animal proteins. All cereal grains when analyzed on a whole-grain basis are, however, below FAO recommended levels in one or more essential amino acids. Lysine, for example, is the primary limiting amino acid in all cereal grains. Threonine is second limiting except in maize where tryptophane and lysine are colimiting (Table 1) A concomitant problem is the loss of protein during processing. The principal source of this loss is through milling where the germ, which is proportionally higher in protein than the endosperm, or the seed coat, including the aleurone layer, may be removed. Loss of either or both of these seed fractions lowers both the quality and the quantity of protein in the finished product. Varying degrees of protein malnutrition often results in developing areas of the world where the average diet consist of cereal grains of low nutritive value supplemented with small amounts of animal or legume protein. This form of malnutrition is debilitating in adults. In pre-school children whose protein needs are high in relation to caloric requirements, severe cases of protein malnutrition result in the permanent impairment of mental capacity. The caloric requirement may be met with a cereal-based diet after weaning but a protein deficiency will result unless sufficient

PAGE 11

TABLE 1 COMPARISON OF ESSENTIAL AMINO ACIDS IN CEREAL GRAINS EXPRESSED IN g/16g N Amino acid Pear lmillet + Sorghum Wheat + ++ Rice FAO Lysine 2 6 2 5 2 7 3 26 4 2 llisl i d ino 2.1 2.1 2.1 3.22 2.4 Tin Conine 4.9 4.1 3.3 4.03 2.8 Methionine 2.5 2.7 2.5 2.2 2.2 Isoleucine 4.3 4.9 3.6 4.0 4.2 Tryptophane 2.3 1.1 1.2 1.68 1.4 Va 1 i n e 5.7 4.8 4.5 5.75 4 2 Iiouc i no 17.4 24.2 6.8 8.53 4.8 Pheny 1 a lanine 4.9 4.9 5.7 4.4 2.8 Tyros i no 1.89 +++ Source: Burton et al 1972. + Juliano et al., 1968. Suggested FAO requirements for good amino acid balance (Food and Agr. Org., 1957).

PAGE 12

legumes and/or animal protein sources are supplied. These Factors make it imperative that good quality protein be made available as cheaply as possible. Improving the protein quality and quantity in cereal grains appears to be a feasible approach to this problem. Population pressures and cultural mores also contribute to the protein problem. Available arable land is decreasing and the population is increasing and the competition between man and animals for the available cereal grains is becoming more pronounced. Per capita consumption of animal products increases as people become more affluent. This also creates a pressure on available grain supplies forcing the price of both grain and animal products upward. This trend makes both grain and animal products less attainable by the majority of the population in underdeveloped countries where unemployment is high and wages are low. Cultural patterns and mores often limit the progress which might be made in improving protein quality and quantity in local diets. Improved high protein rices are considered less palatable in some areas of Asia (Juliano e_t a_l. 196 5) High lysine corn has not found acceptance in many areas of Latin America. Local taboos and prohibitions often eliminate potential sources of animal protein. Dietary <-ind cultural patterns are difficult to change and progress will be slow where change is required for improved protein nut rit i on

PAGE 13

5 Adequate protein balance from plant sources can be achieved by selection of plant foods that complement one another in essential amino acids. A cereal-legume mixture, for example, when eaten in proper balance will often fulfill human protein requirements (Johnson e_t al. 1968). However, economic limitations and lack of understanding of nutritional requirements frequently rule out food selection as a realistic approach to improved diets. Moreover, children are restricted during their most critical stage of development to what is available in the home. Improved protein balance can also be accomplished by the supplemental amino acids in processed foods. The difficulty of this approach is implementation of the program. The people in greatest need of such a program normally consume very little commercially processed food. It is either unavailable or too expensive. Another alternative, which also has limitations, is the improvement of protein quality and quantity in cereal grains. Several approaches have been suggested by Kamra (1971) most of which are based upon selection and breeding. Two precautions in a program of quality improvement are to (1) maintain the proper balance between essential and nonessential amino acids in the grain and (2) avoid selection for a sinqle nutrient without regard to total composition of the grain (Munck, 1964). A third precaution concerning palatability and acceptability would also be appropriate i n

PAGE 14

6 view of the experiences with high protein rice and high lysine corn. Possible Solutions to t h e Problem Advances have been made over tne last half century in breeding crops for increased production with little emphasis on increased nutritional quality. Kamra (1971) attributes this lack of progress to a general lack of awareness of the nutritional requirements of monogastric animals and the non-availability of simple, quantitative, inexpensive and non-destructive mass screening methods to aid in the selection of breeding material. A general lack of awareness of the importance of cereal grains and imbalance among their amino acids in the diets of developing cultures may also have attributed to this situation. Interest in the improvement of protein in some cereal crops began early in the twentieth centruy. Woodworth et al (1952) reported on the results of 50 generations of selection for protein and oil content in corn and shortly afterward Frey (1949, 1951) published on the inheritance of protein in corn. These studies indicate crude protein could be doubled in corn but no serious work was begun since the increase was largely in the form of nutritionally poor zein. Discovery of the opaque-2 and the floury-2 genes which significantly alter the amino acid patterns in maize (Mertz and Bates, 1964, 1965) created renewed interest in protein inheritance in cereal grains.

PAGE 15

7 Increasing the quality and the quantity of protein in cereal grains has received increasing attention over the Jast decade. The International Rice Research Institute (IRRI) began studies on the variability of protein content in rice varieties of Asia (Juliano et a_l 1964) and later began screening for high protein rice varieties (Juliano et al 1968). The Nebraska Agricultural Experiment Station systematically screened 16,000 selections of wheat for heritable protein and lysine differences. The data indicate a significant increase in protein content of wheat can be accomplished without sacrificing yield (Johnson e_t a_l. 1968). Lower yield in high protein lines has been the major deterrent in the introduction of high protein commercial varieties. A sorghum program was begun at Purdue University to investigate protein quality and quantity in that crop. One result of tnis program was the discovery of a high lysine mutant gene (Singh and Axtell, 1973). A broad-scaled program designed to upgrade protein quality and quantity in cereal grains was also developed in India (Swaminathan et al. 1971) The inferior quality of cereal proteins is attributable to the ratio among the protein fractions in the grain. Prolamines low in lysine content constitute the major percentage (40-60%) of seed proteins in most cereals, with glutelins of intermediate lysine content constituting most of tht> remainder. The smallest protein fraction in cereals

PAGE 16

8 Is the globulins which are characteristically higher in lysine. Exceptions to this are rice, with 8?; prolamines and oats with 12'i (Nelson, 1969). These differences in amino acid content among various protein fractions of the grain suggest the possibility of improvement through mutations which would suppress the synthesis of protein fractions low in the desired amino acids with a compensating synthesis of other fractions with higher levels of the d"sired amino acids. Synthesis of amino acids is regulated to correspond closely to demand for incorporation into proteins by the plant, therefore, cereal grains contain very low quantities of free amino acids (Nelson, 1969) An induced or spontaneous mutation causing a loss of sensitivity in control of amino acid synthesis could cause an over-synthesis with a resulting increase in free amino acids. No mutation of this type has been identified to date (Nelson, 1969). The more promising types of mutations are those which cause a gross change in the structural components of the endosperm or seed structures. The high lysine mutants in maize are of this gross structural change type. Theoretically, opaque-2, the high lysine gene, inhibits the synthesis of zein, the prolamine fraction of the protein, in maize. The effects of mutations in which one amino acid is substituted for another in protein synthesis have not been important in changing protein quality and quantity.

PAGE 17

It became increasingly apparent as the components of seed protein wore elucidated that significant changes in the amino acid composition of cereal protein could occur only through mutations. More specifically these changes occur through mutations which reduce synthesis of the alcohol-soluble prolamine fraction and increase synthesis of other protein fractions (Johnson et a_l 1968; Munck, 1964). Mertz and Bates (1964, 1965) found a significant reduction in zein, the prolamine fraction of corn protein, in mutant strains homozygous for either the opaque-2 or the floury-2 gene with a corresponding increase in lysine. Tello ejt a_l. (1965) found the same effect when the opaque-2 gene was incorporated into various races of corn. More recently Nelson and Chang (1974) studied the effect of enhancing protein and lysine content in corn by incorporating a gene conditioning multiple aleurone cell layers, which are high in lysine. Significant improvement in protein quality or quantity in cereal grains can be accomplished through selection and breeding and by screening mutations, either spontaneous or induced. Manipulation of major genes of fers the most dramatic and the most rapid method of increasing protein quality or quantity but generally major genes are not available. Selection and breeding hold the most promise for protein improvement when sufficient variability is present. The researcher can attempt to induce mutations

PAGE 18

10 which will achieve the desired improvements as a last resort.. Polygenes are less dramatic in changing protein quality and quantity but can be concentrated through sound breeding procedures. They are the most limiting but also the most predictable of the genetic procedures. Factors which may influence the total protein or the levels of certain amino acids in the grain should be considered as well as the form in which the grain is consumed. Differences in structural components of the seed have a marked influence on their ability to transmit genetically increased protein and amino acid potentials. Milling procedures and cooking methods may remove portions of the seed which are highest in good quality protein. Cultural practices may also significantly alter both the quality and the quantity of protein in the endosperm (Sawhney and Naik, 1970) Proteins in the germ are much superior in protein quality to those of the endosperm (Nelson, 1969), therefore, increasing the relative size of the embryo for quality and quantity of protein in the diet would be of importance where the whole seed is consumed. No studies have been made to explore the possibilities of significantly changing the amino acid composition of the embryo but it has been noted that both the opaque-2 and the floury-2 genes of maize increase embryo size when incorporated into new linos

PAGE 19

11 (Kamra, 1971). The effect of this increase has not been quant i f icd The aleuronc layer, usually one cell layer in thickness, is high in protein and contains significant amounts of the globulin fraction. Increasing the thickness of this layer may significantly increase both the quality and the quantity of protein in the grain. The improved protein content by the amylose extender gene in maize is believed to be due to the increased thickness of the aleurone layer (Kamra, 1971). Inferior quality of cereal proteins may be attributed to the small fraction of globulins which are high in lysine and the large fraction of prolamines of low lysine content. Globulins are abundant in the aleurone layer and the embryo while prolamines are found in the endosperm. Any factor which significantly alters the ratio of these protein fractions will have significant effects on the protein quality and quantity of the grain. One aspect of the problem unrelated to the breeding and genetics is the lack of a simple, quantitative, nondestructive method of analysis for total protein and amino acids Dye-binding techniques have been developed which estimate protein in some cereal grains. Udy (1971) developed this technique originally for wheat and later expanded it to

PAGE 20

12 other grains. Kaul oL al. (1970) reported the technique was wc>ll adapted to riec and other grains. Kamra (1972) mentioned the effectiveness of dye-binding for the qualitative estimation of lysine in grains. The method was used by Singh and Axtell (1973) to isolate the high lysine -r-ne in sorghum. Kaul et al. (1969) described a method of microscopic screening of rice grains for protein content using a dye-binding technique. Johnson and Craney (1971) reported a rapid biuret method for estimation of protein in grains. Parial and Rooney (1970) found a high correlation among results from dye-binding, biuret, and micro-Kjeldahl analyses. Concon and Soltess (1973) described a rapid micro-Kjeldahl process in which digestion is completed within 10 minutes. The major problem confronted in the search for a rapid quantitative analytical technique for lysine or total amino -icids is hydrolysis. Standard methods require special equipment and a great deal of time. Palter and Kohler (1969) developed a survey hydrolysis procedure in which up to 300 samples per week may be processed, but even this method is too slow to meet the requirements of a breeding program in wnich thousands of segregating genotypes must be analyzed. Quantitative analytical methods for lysine include gas chromatographic (Zscheile and Brannaman, 1972), coloi im trie (Concon, 1972; Ghosh and Bose 1973), and thinlayer chromatographic techniques (Heathcote and Haworth,

PAGE 21

1 1 \ ')(>'!) All of these methods require hydrolysis pri"r to analysis for the amino acids.

PAGE 22

SCOPE AND PURPOSE OF THIS STUDY Pearlmillet (Pennisetum typhoides (Burn.) Stapf and E. C. Hubbard) is a robust annual bunchgrass grown on 20 million hectares as a grain crop for human consumption (Burton and Powell, 1968; Burton et a_l 1972). It is best adapted to the tropics and will grow and mature seed on soils too infertile and too dry for other grain crops. The ability to produce in the dry, infertile areas of the tropics has made it a major food source. Good nutritional qualities of the grain justify the research in production and improvement of this crop presently being advocated (Dcwit, 1969). Pearlmillet has a protein content ranging from 8.8% to 20.9%, with lysine varying from 1.9% to 3.4% of the protein (Burton et al. 1972; Wallace, unpublished data). These data are in agreement with those of Swaminathan et al (1971) who found 87. to 20% protein, with lysine ranging from J..! i o J. Hv. oi tlif protein, and tryptophan ranging from 0.77; to 1.7% of the protein. Losses occur during milling, but approximately 85% of the proteins in the whole grain normally remain after processing (Dewit, 1969) 14

PAGE 23

Millet has a content and balance of essential amino acids equal to or better than most other cereals (Table 1) and is higher than rice, wheat, or maize in fat and mineral content (Table 2 ) The grain yield potential has not been established, but there is evidence to suggest pearlmillet yields may compare favorably with corn and sorghum where good management including use of adopted varieties, irrigation, and fertilizers was practiced (Burton and Powell, 1968; Burton et al. 1972) Purposes of this study were (1) to determine the heritability of protein and lysine, the primary limiting amino acid, in the grain of pearlmillet and (2) to investigate the qualities and adaptability of pearlmillet of some rapid quantitative methods of analysis reported in the literature for protein and lysine.

PAGE 24

16 TABLE 2 Oil. AND MINERAL COMPOSITION OF SOME CEREAL GRAINS Oil Ca P Cere al grain % mg mg_ Pearlraillet 4.5 46 314 Winter wheat 1.8 46 354 Corn 3.9 22 268 Rice — brown 1.9 32 221 Son (hum 3.3 28 287 Sou rcc Composition of foods. No 8 USDA Agriculture Handbook

PAGE 25

MATERIALS AND METHODS The Diallel Cross A diallel cross was made during the summer of 1973 using six inbred lines received from Tifton, Georgia. Lines were selected on the basis of variation in protein and lysine content as measured by Wallace and Block (unpublished) as shown in Table 3. TABLE 3 PROTEIN AND LYSINE CONTENT OF INBRED LINES USED IN THE DIALLEL CROSS Protein Lysine Line % (as % pr ote in) 1-23 11.24 2.50 T-27 15.31 2.58 T-140 10.12 2.53 T-4 10.31 3.70 1-18 16.12 2.15 T-98 19.12 2.00 17

PAGE 26

18 Progeny from the diallel cross were planted in a split plot design over two locations with two replications at each location. Replications contained the six inbred lines produced by selfing the parental lines and the 30 F^s (including reciprocals) derived by crossing the six lines in all possible combinations. Location I was planted on the Agronomy Farm at the University of Florida, Gainesville. Each plot consisted of three rows 5.5 m in length, with 1 m between rows. Plants were spaced approximately 0.5 m apart in the rows. One application of 403 kg/ha or 10-10-10 fertilizer was broadcast before planting. Heads from the center row of each plot were randomly selected and bagged with a kraft bag and an aluminum screen protector bag to minimize bird damage. Heads remained on the plants until they were fully matured. Location II was planted the same season (summer, 1974) on the Morikami Farm, University of Florida Experiment Station, Delray Beach, Florida. Plots were bedded due to the high water table in this area and covered with black plastic to minimize weed competition. The beds were approximately 30 cm highand 1.5 m wide. The plots were fertilized with 403 kg/ha of 10-10-10 fertilizer broadcast prior to bedding. Each plot consisted of two rows 90 cm apart and 5.5 m long placed upon the bed. Plants were spaced approximately 0.5 m apart in the rows. Bedding and covering the beds with plastic minimized interplot competition. Heads from both

PAGE 27

rows within each plot were randomly selected and bagged with both a kraft bag and an aluminum screen protect'.'bag. he. i It; remained on the plants until they were fully matured. All bagged heads were harvested and three heads were randomly selected from each plot for use in the quantitative analysis for protein and lysine. Line 1-18 and its progeny were eliminated due to poor germination and poor seed set in several plots. Analytical Procedures Equal measures of seed from each of the three heads selected were bulked for estimation of the plot mean for protein and lysine. Samples were ground in a Norris hammer mill through a 1 mm screen and oven dried for 24 hours at 100 C before analysis. Samples used in the analysis for lysine and the dye-binding capacity (DBC) technique for estimation of protein were further ground in a mortar and pestle using approximately 5 ml acetone as a wetting agent. Protein Analysis Protein (N x 6.25) was determined by the rapid microKjeldahl procedure described by Concon and Soltess (1973). Approximately 2.3 g of i^SO^/MgO mixture and 2.3 ml I^SO^ were added to a ground sample of 50 mg The mixture was heated until frothing occurred then approximately 1 ml 30% H~0~ was added. Heating at a lower temperature, approximately

PAGE 28

20 3()0 C, was continued until clearing. Distillation was by the standard micro-Kjeldahl procedure (AOAC, 1960). Accuracy of the procedure was verified by correlatinq results of randomly selected samples with those from the macro-K jo Idahl procedure (AOAC, 1965). Data generated by the microKjeldahl analysis were used to determine the accuracy of dye-binding techniques for protein estimation in pearlmillet. Amino Acid Analysis Ground, oven-dried samples of 100 g each were hydrolyzed in 100 ml 6N HC1 in a nitrogen-saturated atmosphere at 40 C for 24 hours. The solute was brought to dryness in a rotary evacuator at 40 C and added with 10 ml 0.01N HC1. A 1:10 (v/v) dilution was used for the complete amino acid determination Fifteen samples representing a complete set of crosses from a replication, including inbreds but not reciprocals, were analyzed on a Jeol Model JLC-6AH Amino Acid Analyzer. Lysine Analysis Lysine was estimated as grams of lysine/16 g N using tne rapid lysine determination technique described by Cone ^n (1972). A protein extraction was made from a 500 mg sample of ground seed using 1.5 ml 70% ethanol followed by 5.5 ml 0.5% NaOH. Nitrogen content of the extract was determined by rapid micro-Kjeldahl procedures (Concon and

PAGE 29

21 Soltoss, 1972) using a 0.5 ml aliquot. Dinitrobenzene sulfonate (DNBS) was used to determine lysine content colorimetrically. A Bausch and Lomb spectrophotometer (Model 20) was used to measure absorbance at 460 nrn rather than a I 360 nm as indicated in the literature. The spectrophotometer lacked sensitivity at 360 nm to distinguish among samples whose differences were visually apparent. Accuracy of this procedure was verified by correlating results with data on 15 samples analyzed with the amino acid analyzer. Statistical Analysis Protein data were analyzed using Griff ing's (1954) method 3 (in which all F^s are used) model I (all effects except error are considered fixed) for estimates of general combining ability (GCA) and specific combining ability (SCA) in protein inheritance. Method 3, model II, in which all effects are considered random variables, was used to est imate heritability with the formula for the genetic variance estimate (6 ) modified to include the reciprocal ^2 2 variance estimate (6 ) Estimates of heritability (h ) in the broad sense were calculated. The same data were analyzed by the Hayman (1954) mot hod of diallel cross analysis for estimation of genetic effects. Gene action parameters estimated by this method are additive, dominance, and maternal effects.

PAGE 30

RESULTS Protein Inheritance A highly significant difference was found between crosses. All other parameters were non-significant (Table 7) Location differences were non-significant but observation of mean values of crosses compared across locations (Table 4) suggests the environmental effect upon crosses was variable in both the positive and the negative directions masking possible location effects. Table 5 and 6 contain the means of each cross (by location) arranged with the inbred parents forming the diagonal. In this format reciprocals are also located diagonallyfrom one another. Reciprocal differences in some lines are apparent. Combining Abilities Data at both locations were subjected to analysis of variance for combining ability (Tables 8 and 9). Estimates of GCA and SCA differed with the reciprocals showing no significant difference between locations. Parental reciprocal 2 estimates (8 ) (Tables 10 and 11) indicate the major reciprocal differences of approximately the same magnitude

PAGE 31

CO CO H fn 3 W CO < a. a 2 a < w CO CQ a ^ H H >H > H Cm. O Cm M O im < CO CO w o i— IW Cm ^ >-I OP QJ m in ro ro in O m rH I rH rH rH r-H r i rM 1 p— 1 rH ( 1 rH -CI o m a) CQ >i m n rH QJ a rH + rH rH 00 CO 00 rH CO if} rH rH cn rH rH LO —I in Q) — in L0. in O m ro rH >* O r^ rH rH rH rH rH CM rH rH rH r-j H CM n CM o CM O O 00 m CO ro O r-H o\ 00 (N 10 ro ro en 10 0) — CO LO CO co o\ o -r tiO rtN ro rH rH rH rH rH rH CM rH rH rH CM H 01 :-• tn C H a Cm<*> QJ ~ K rH rj> 00 <* CO 0> in CM CT rCO T LO en cn CTl CM CO X) CO tN CT, o * rro ro r-H rH rH rH rH rH CM rH rH CM rH Ql r>* CO CO CO ro rH rs rH O CM rH CTi tN i 1 1 1 1 1 1 1 1 a Eh Eh Eh H H Eh Eh H CO C 0 0 CI) Q) ljh M MH 4H D rH rH CD cu CD CD to CO en CD rH o O (0 ro CO CO CO CO r rr
PAGE 32

24 3 c c u w < [h 0..< CD c U m >< U o Q ChrtP 0) — n 0> P> O 00 CO ca CO CO r IO rH rrH o co 'X) CN LD CJ — VD rr> m CO r '0 m CO m n. rH H H rH H rH rH rH rH rH 01 rH rH •H : • in o c •A fd (U -• c rH ID CO r 10 en Ln in o io n 10 CO 10 CO m oo I I Eh Eh O m (N CN rH l I I H E-t £- CO CTl I E 1 O -r H I o -r rH I Eh t3 o m rH 0) I Eh -4I EH I I Eh I EH -J 0 H in CO OA I Eh CO 00 CM I f l OA o co ^ CN CN i — I I I I M Eh Eh I H CO 0> I Eh do OA I EH

PAGE 33

25 TABLE 5 MEAN PROTEIN CONTENT OF ALL PROGENY OF A DIALLEL CROSS, GAINESVILLE Female parents Male parents 1-23 T-27 T-140 T-4 T-98 (%) 1-23 16. 42 13. 92 15. 09 15.45 13.99 T-27 20.79 19. 66 14. 67 15. 15 17. r -6 T140 13.79 16. 03 23. 12 17.68 14.86 T 4 15.40 13. 56 17. 37 16. 90 17 49 T-98 16.08 15. 06 17. 98 15. 26 17.68

PAGE 34

TABLE 6 MEAN PROTEIN CONTENT OF ALL PROGENY OF A DIALLEL CROSS, DELRAY BEACH Female parents Male parents 1-23 T-27 T-140 T4 T-98 (%) 1-23 14. 96 14.77 15.94 13. 02 13.75 T-27 21. 51 23.48+ 14.79 10. 11 11. 37 T-140 17 22 12 70 21. 46 16, 23 17.40 T-4 16. 32 14. 34 17.01 19. 43 15.76 T-98 15. 55 11. 07 14. 15 16. 52 19. 05 Missing value estimated by Yates method (Cochran and Cox, L'J5^

PAGE 35

27 TABLE 7 ANALYSIS OF VARIANCE FOR PROTEIN OF PROGENY RESULTING FROM A DIALLEL CROSS WITH FIVE INBRED PARENTS Source df MS Locations 1 12.945 Error (a) 2 1.164 Crosses 24 25.123** Locations x Crosses 24 0.572 Error (b) 48 4.49 **Signif icant at .01.

PAGE 36

28 TABLE 3 VARIANCE ANALYSIS FOR COMBINING ABILITY FOR PROTEIN, GAINESVILLE Source of Mean va riance df squares Co 1
PAGE 37

TABLE 9 VARIANCE ANALYSIS PROTEIN FOR COMBINING ABILITY DELRAY BEACH FOR Source of variance df Mean squares Combining ability: General 4 7. 991* Specific 5 9. 875** Rei pr ora 1 10 4. 765* Er i or 48 2.24 *Significant at .05. **S iqni f icant at .01.

PAGE 38

30 TABLE 10 ESTIMATES OF CCA, SCA, AND RECIPROCAL VARIANCES ASSOCIATED WITH EACH PARENT, AND ENVIRONMENTAL VARIANCE MEANS FOR PROTEIN, GAINESVILLE Parent a 2 g 3 2 s 8 2 r a 2 e 1-23 3. 5570 0. 0025 3.4238 0. 1269 T-27 -0.2719 0.5983 4.5627 0. 1269 T-140 -0.4476 1. 8200 0. 0487 0. 1269 T-4 -0. 1614 0. 9769 0. 3773 0. 1269 T-98 -0. 4953 -0. 5026 1. 0450 0. I2f-9

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TABLE 11 ESTIMATES OF GCA, SCA, AND RECIPROCAL VARIANCES ASSOCIATED WITH EACH PARENT, AND ENVIRONMENTAL VARIANCE MEANS FOR PROTEIN, DELRAY BEACH Parent 8 2 g 3 2 s 3 2 r a 2 e 1-23 1.4735 3. 9958 4. 0854 0.1269 T-27 1. 9195 4. 0026 4. 6339 0. 1269 T-140 0. 4737 0. 5017 0.4172 0. 1269 T-4 -0. 3991 0. 5833 1.4831 0. 1269 T-98 0. 0937 1. 4675 0. 1915 0. 1269

PAGE 40

32 occurred in the same crosses at both locations. These data suggest the environment had little effect upon reciprocal effect in progeny from lines 1-23 and T-27. Environmental el tocts appeared to have an influence on progeny from lines T-4 and T-98, however. Differences in combining abilities between locations are an indication of the effect of environment on protein content. The GCA of each of the five inbred lines in this study was non-significant at Gainesville. Estimates associated with each parent show line 1-23 superior to all other lines in that location, this being evidenced by the consistency of the progeny in approaching the parental value in protein content (Table 5) 2 The GCA variance estimate (d ) associated with eacn g parent indicates lines T-27 and 1-23 are superior to all other lines tested at that location (Table 11). The most striking difference in the parental GCA variance estimates between locations is that line T-4 has a negative value while four lines have negative values at the Gainesville location Analysis of SCA shows non-significant effect at Gainesville (Table 8) and a highly significant effect at Delrny 2 Beach (Tables 8 and 9) Parental estimates for SCA (0 ) s show line T-140 to be superior to other lines at Gainesville (Table 8) and lines T-27 and 1-23 superior at Delray Beach (Tables 10 and 11)

PAGE 41

Hi v i tabi 1 i t y Estimates IlcritabiliL ies in both the broad sense air! t he narrow sense were estimated assuming additive variance 7 2 ( ~) to equal twice the GCA variance (0 ) (Griffing, i 1954). Total genetic variance was estimated by the form" 7 2 2 2 a: = 2a + a + a G q s r where 2 a = total genetic variance, G 2 8 = estimate of GCA g 2 a = estimate ->f SCA, and s .2 r = estimate of reciprocal effects. 2 The phenotypic variance (3 ) was estimated by the formula 2 2 2 whe re 2 6 error e Heritability is then estimated by the formula .2 26 2 h = __g in the narrow sense and

PAGE 42

34 2 h = G A 2 in the broad sense. Narrow-sense heritability estimates for both locations were negative due to the negative values in the GCA variance estimate (Table 12). Broad-sense heritability was extremely variable between locations with a value of 0.008 at Gainesville and 0.54 at Delray Beach. Genetic Parameters The Hayman (1954) method of diallel analysis indicated dominance for protein content. The negative values in mean squares were interpreted as indications of dominance for lower protein content (Table 13). The consistency with which the progeny deviated in the negative direction from the mid-parent value (the mean of the parents) for protein content confirms this interpretation. Analysis of data also indicates no significant genetic difference among parental lines. The Yates (1947) method of interpretation of data reveals a significant maternal effect which is evidenced by significant reciprocal differences involving some lines.

PAGE 43

2 H w o W u 2 < fa M o (X CO > < fa u O o W W H Eh U w CN a; i — 1 M Eh Q w CO • < Q E2 W << M Eh CO H u H <^ D < a CO 2 2 u H 2 <^ o u PS o .1 <> id i "5 M H Q) Q •H > CO 0) c H 10 o U rsj rC CM o (0 rH n [> •1 1 • (H O o m 1 > 2 QJ O C CO in m O m H • o o o m 1 I > •a rH in U\ &l CO P~ O rin rH vD CTl in O in rro m O H CN in in oo o t H (0 0 o •H oo 0) M u (3 a u u •H 0 3 M < < U M 0 U u <1) > o w IX w

PAGE 44

30 TABLE 13 ANALYSIS OF VARIANCE FOR GENETIC PARAMETERS OF PROTEIN INHERITANCE Source of Mean yorj ance df squares Parental 4 5.9 3 Dominance 10 -883.32 Maternal effect 4 17.13 LIrror 6 5.58

PAGE 45

37 Lysine Inheritance Analysis of inheritance for lysine was limited to those samples analyzed on the amino acid analyzer, due to the unrealiable results of the rapid analytical technique for lysine (Table 16). There was no replication, hence statistically valid inferences cannot be made. Hypotheses, however, may be formulated for verification in further work. Regression analysis of the data (Table 14) indicates a linear relationship exists between lysine and total essential amino acids but with a very low correlation coefficient. A negative correlation (r = -0.44) exists between lysine and protein content with a strong positive correlation between lysine and the sum of methionine and threonine (r 0.7G). A linear relationship was not indicated between total essential amino acids and total protein although the data indicate a slight negative correlation may exist. Quantitative Analytical Methods TiiK DBC method for screening samples for protein content as reported by Kaul et a_l. (1970) was unreliable when used with pearlmillet (Table 17). The rapid, quantitative analytical technique for lysine reported by Concon (1972) also proved unreliable (Table 16). The rapid microKjeldahl technique reported by Concon and Soltess (1973) proved very accurate and consistent and correlated well (r = 0.79) with the macro-K jeldahl results (Table 18).

PAGE 46

u En U x: ta >ii -h ro iH r H e'en CN IN 1— O CN CO 10 o CO CN CI 00 in r o ro cn r— 1 a CTn CN i£> ro ro 00 oa rT ID CO CTl in r CN r0 t" r~rH rH H rH CN H H rH r— 1 H m kO TJ' rH m -v CN in n <* in CO in o ro ro rH no in .-I ro n OA CM CO co CN CO o in cn <£> CN •J' L.O CO io CN ro cn io in CTl oo cn ro ro CN r> 0~> rCN cn r— o ID m o m ^ VD KO (N V.O in o ro cti ro cn cn CO C'l OA CN CO CO CO CN CN O in CN 10 CO CO ro CN CO CO CN Ul C o M fd 10 CN OA o ui rCO m m CA LO rH rH n o r CN rH O Q) m rH m OA in rH CTl lO in ca CO cn CO OA M -H rH y H CN CN o CM CM rH CN H CM CO rH CO C •H o (D *c •5 4-> 00 CD r •^r CN CN r> m CO CN CO ro H 4J O CN -)• r-i 00 in OA CO rH rH r l-> m 0) M i i •* rH rH rH io rH o o rH O rH rH o O 10 rH Ml '.0 m 0 III •rH CN GA CTl CM rO CO CO CM vr r H o CO •+-> rH rfp CO r> 00 in CD CO cn lO o o CM CO 04 c m o : -r rH (N CD co PO rH CO m rH ro m in CN "^r tfl ID r o y 10 CT> cn CO ro rin CN O cn o rH CO ro ro cn cn in 10 rH co LO cn in CO o co lO -r o o 10 rH ro o lO CN rm r> in H -H rH CN rH rH H CN o rH rH H o H lO rCN in r^ rH rH rH CN rH o rH — i o rH rH rH o rH O o T) 00 rrf CO T3 '? CO CJ # CA UJ CN rH OA CU rH rr OA 4h 1 1 CO aj IM 1 1 i 1 CH 1 1 1 rH Eh LH 3) en cw H [-1 Eh Eh E-t rH LH H Eh a> UH i — 1 0) CD to X X rH Eh OJ tfl X X X x n X X X u tfl o o o (0 X CO ro ro ro CO r [-> ~r 5T OA cm 1 r i ci CN IN CM CM CM CN rH rH rH >y* fj M H 1 IH 1 H 1 IH 1 Eh 1 r < 1 EH 1 1 OH 1 Eh 1 Eh l Eh i H 1 Fh

PAGE 47

TABLE 15 LYSINE CONTENT OF PROGENY FROM A DIALLEL CROSS Female Male parents parent a I-2J T-27 T-140 T^A T-98 1-23 1.54 1.64 1.14 1.56 2.27 T-27 1.04 0.68 1.24 1.78 T-140 0.79 1.21 1.79 T-4 1.35 0.73 T-98 1.62

PAGE 48

40 TABLE 16 EVALUATION OF EXTRACTION FOR THE CONCON RAP lb LYSINE TECHNIQUE AND COMPARISON OF LYSINE VALUES BY THE CONCOrJ TECHNIQUE TO THOSE OK THE AMINO ACID ANALYSER Nitrogen (g/100g Lysine ground sample ) (grams/lysine/16g N Ground % N uL 1U Trinrnn Cro: ;s seed Ex tracted* Extracted analyzer method ] 2.78 1.35 48 1.43 2.63 2 2.95 0.83 28 1. 32 1. 32 J 2 • j 5 1.13 44 1.13 2.41 4 2.95 2. 48 84 1. 56 3.74 6 2. 11 1.31 62 2. 27 3. 34 9 2.08 1. 92 92 0.7 5 1. 33 10 2. 55 1.79 70 1. 16 1.94 12 3. 17 1. 04 33 1.61 1.45 13 3. 67 1. 36 37 0.76 1. 08 X. O 2.80 2.05 72 1.20 2.13 18 2.39 2. 00 84 1.79 1. 36 19 2. 74 1.35 49 1.32 1.45 24 2. 80 0. 87 31 0.73 1. 57 31 2.82 1.31 46 1.63 2.00 Extracted as described by Concon (1972).

PAGE 49

41 TABLE 17 COMPARISON BETWEEN DBC AND MICRO-KJELDAHL ANALYSES FOR PROTEIN REPLICATION I, GAINESVILLE Cross DBC values Micro-K j oldahl absorbance % protein 1-23 selfcd .29 16.71 r-2 3 x T-2 7 .28 13. 86 1-2 3 x T-140 .27 14. 38 .1-2 3 x T-4 .29 12. 44 1-23 x T-98 .26 14. 77 T-27 selfed 31 19. 63 T-27 x 1-23 .32 20.99 X T-140 32 14. 96 x T-4 30 14. 96 x T-98 26 17. 25 T-14 0 selfed .31 23. 32 T-140 x 1-23 .31 13. 99 x T-27 26 15. 94 X T-4 .30 17. 88 x T-98 26 14. 77 T4 se 1 fed .29 16.71 T-4 x 1-2 3 30 17. 59 x T-140 .29 17. 36 x T-98 29 17.49 T-9 8 selfed .31 17. 64 T-98 x 1-23 30 15.68 x T-27 32 14. 38 x T-140 .29 17.49 x T-4 .31 14.97

PAGE 50

42 TABLE 18 COMPARISON BETWEEN MICROAND MAC ROKJELDAHL ANALYSES FOR PROTEIN, REPLICAT I ( IN II, GAINESVILLE Cross Macro-Kjeldahl M i cro-K jeldohl % Protein % Protein T-?3 16. 12 16. 13 1-2 3 x T-27 14.15 13.99 x T-140 15. 47 15. 80 T-4 18.84 18.46 x T-98 13. 59 13.22 T-27 selfed 19. 53 19.70 T-27 x 1-23 19.78 20. 60 X T-140 14.25 14. 38 x T-4 15. 56 15.35 x T-98 17. 91 17.88 T140 sol fed 22.78 22. 93 7-140 x 1-23 13. 31 13. 69 x T-27 16. 50 16. 13 x T— 4 17 62 17. 49 T-98 14. 91 14. 96 M'— A qo 1 f nr\ .t T Ol, J 1 "U 17.81 17. 10 T1 x 1-2 3 13.31 x T-2 7 18.00 18. OR x T-140 17.42 17. 38 x T-98 17. 44 17 49 T-98 sol 18.38 17 68 T-98 x 1-23 16.22 16.48 x T-27 15. 09 15.74 x T-140 17.72 18.27 x T-4 15. 00 15.55

PAGE 51

Discussion The genetic components of protein inheritance are complex. When one considers that production of all amino acids are polygenically controlled and amino acid sequence of each protein is genetically determined the complexity of protein inheritance becomes apparent. The problem is further complicated by evidence of maternal influence (Nelson, 1969) and divergence in modes of inheritance in which the same character may follow different patterns of inheritance in various lines within the same species (Balint, 1970). Another force influencing protein content is the environment. Cultural practices such as the rate and time of fertilizer application influence protein content of many cereal grains (Swaminathan et a_l 1971). Increased protein from fertilizer application increases the prolamine fraction without increasing the more desirable globulin and albumin fractions, resulting in protein of a lower biological value Johnson et a_L (1968) reported high protein genetic traits in wheat are associated with a more efficient and complete translocation of nitrogen from the plant to the grain rather than with differential nitrogen uptake or accumulation by the plant. 43

PAGE 52

4 4 Thirdly, the array of genes which control prot in inheritance are affected by environmental factors. Significant genotype-on-environment interactions have been reported in sorghum (Crook and Casady, 1974; Deyoe and Shellenbergor, 1965; Liang et al. 1968), rice (Sampath etal., 1968; Hi 1 lerislambers et al 1973), wheat (Johnson et al., 1968), and pearlmillet (Flahadevappa 1967). It becomes evident the phenotypic expression of protein content in an individual plant is a balance between the genetic potential, the environmental conditions, and the interaction between the two. The results of this study are further evidence of the complex nature of protein inheritance. The significance of environmental differences due to location appears to be masked by other interactions. Environmental differences were present, but the effect of environment across 25 different genotypes masked this effect. Any statistical analysis of an experiment concerning protein analysis should, therefore, partition the variance into all possible interactions. Differences between replications in some crosses (Tabic9) cannot be explained. Seed used in growing the F 1 generation was taken from the same head, hence variations cannot bo attributed to possible differences in genotype of the parents. This is also verified by the analysis of

PAGE 53

45 variance for genetic parameters (Table 13). Plots in which differences occurred were dispersed throughout the field, thus the possibility of an unknown localized effect within the field cannot be the cause. Closer examination of the data indicates two significant f-icts which are not conclusive but do give some basis for an hypothesis. When individual plots with replication differences are counted the Delray Beach location has three times the number found in Gainesville. Further examination reveals that in every case wither line T-4 or line T-98 or both lines are involved. These same lines also have divergent reciprocal variances across locations (Tables 10 and 11) It may be hypothesized that some type of maternal effect on environment interaction is occurring, when these facts arc considered in the light of evidence of some maternal effect as indicated by the analysis of genetic parameters (Table 13). Environmental factors which caused the higher incidence of replication differences at Delray Beach are unknown. Presence of maternal effects as indicated by the data is evidenced in significant reciprocal variances at both locations. Parental variance estimates indicate that lines 1-23 and T-27 are responsible for most of the variance due to this effect with either line T-4 contributing some effect at Delray Beach or line T-98 at Gainesville. These

PAGE 54

46 data indicate maternal effect in pearlmillet may be present in all lines but is variable due to interaction with the environment The presence of maternal effects and their interaction with environment complicates the breeding of pearlmillet for high protein in the grain. Any program designed for this purpose should examine not only combining abilities but also reciprocals. These factors influence not only the choice of parents but also whether a chosen line should be used as the male or the female parent. Plant breeders have placed a great deal of stress upon heritability estimates for specific genetic traits. These estimates, however, are valid only where extensive research has been done with data pooled over genotypes, year and locations or where specific genotypes and locations are involved (Allard, 1966; Briggs and Knowles, 1967; Robinson, 1963). Variability in estimates of heritability for protein content is not unique to millets. Crook and Casady (1974) found heritability estimates for this trait in sorghum varied between methods of calculations and with the population. Liang et a_l. (1968) found components of additive variance to be significantly different between locations. This difference would, in turn, result in significantly (ill !'!'• 1 1 1 heritability estimates between locations.

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4 / Heritability estimates for protein content per se for pearlmillet were not found in the literature, but the results described by Ahmad et al. (1972) and Mahadevappa (1967) were similar to those in this study. Predominance of non-additive gene action and considerable environmental effect both contribute positively to the henominator of the heritability estimate formula. Environmental interaction with genotype could account for the variability in estimates between locations. Experience in this work indicates that analysis for only GCA or SCA may prove inadequate when working with a quantitatively inherited trait. It should be one of a series of progressive steps leading toward a better understanding of the gene actions involved in the inheritance of that trait. Only through such analyses can we discover whether protein content per se is controlled by a major gene or genes or if it is the sum of many genes, each of which controls the synthesis of a particular amino acid with interactions between control mechanisms. The limited data on lysine inheritance make detailed analysis impossible. Certain correlations can be made, however, and may be useful in the formulation of hypotheses for further study. Data indicate, when selection is based solely on proi content, higher protein is accompanied by lower values

PAGE 56

43 of the throe most limiting amino acids in cereal grains. This is evidenced by the positive correlation between lysine and the total of methionine and threonine (Green and Phillips, 1974). Other evidence is the negative correlation between lysine and total protein. The absence of a linear relationship between total essential amino acids and total protein with the very low correlation between lysine and total amino acids indicates progress could be made through selective breeding. All three analytical techniques, DBC, the Concon rapid lysine, and rapid micro-Kje ldahl were selected for study because they showed promise of being effective tools in a pearlmillet breeding program designed for protein improvement. Reasons for the failure of the dye-binding technique and the rapid quantitative lysine technique are unknown. There wore no correlations which could be attributed to genotype (Table 16). One hypothesis is that part of the lysine in pearlmillet is bound in protein complexes which prevent ext raction or inhibit dye-binding. Singh e_t a^. (1971) reports evidence for additive and cumulative gene action for DBC values in rice. No other work has been reported on this problem.

PAGE 57

CONCLUSIONS The inheritance of protein in pearlmillet is influenced by the presence of dominance for low protein v/ith evidence of maternal effects which are affected by environmental effects. More data must be collected to estimate these effects and their significance in a breeding program. The inheritance of lysine may be influenced by the presence of dominance in some lines (Table 15) Lack of analysis of replications precluded estimation of environmental effects. Regression analyses indicated selection based on total protein may have little effect on total essential amino acids but have a negative effect on ;ine, methionine, and threonine. Rapid quantitative analytical techniques for lysine were unreliable when applied to pearlmillet in this work. Pro'ir ss in breeding for grain with better protein quality will be impeded until this deficiency is corrected. 49

PAGE 58

BIOGRAPHICAL SKETCH James Bynum Barnett was born July 30, 1931, in Memphis, Tennessee. He attended high school at Memphis Technical and entered the University of Tennessee Junior College at Martin, Tennessee, in 1949. He entered the U.S. Marine Corps at the outbreak of the Korean Conflict and was sent to Korea after basic training and training as a radio operator. He rose to the rank of staff sergeant and in 1953 was recommended for commiss ion ing to the rank of 2nd lieutenant for demonstrating leadership ability in combat. He was commissioned in March of 1953. He married Martha Hill of Paris, Tennessee, in December of the same year. He was released from active duty in 1955 with the rank of 1st lieutenant and was honorably discharged in 1962 with the rank of captain. He returned to Tennessee upon release from active duty In i9 r >5 and attended the University of Tennessee at Knoxvilic where li< 1 look his Bachelor of Science degree in Agronomy in 1958. He assumed management responsibilities of a family owned farm in Huntsville, Alabama, upon graduation, and was also employed at Redstone Arsenal with the missile industry. !le Left the missile industry in 1962 and accepted employment

PAGE 59

56 in the Huntsville Public School System as a biology teacher. Mr. Barnett and his wife felt called to the mission field and were accepted by the Presbyterian Board of World Missions as agricul tura 1/educational missionaries to Mexico in 1963. They took their post in Teloloapan, Guerrero, Mexico, after completion of one year of theological training at Columbia Seminary and one year of language training in Costa Rica. Mr. Barnett' s duties during that time were similar to those of an agricultural extension agent in an area comparable in size to the state of Florida. Mr. Barnett attended Murray State University, Murray, Kentucky, during his furlough year (1969-70), and began work on an Master of Science degree in agriculture. He and his family returned to Mexico in 1970. The Presbyterian Church of Mexico voted at that time to request that all missionaries be removed from Mexico to allow complete autonomy. Mr. Barnett returned to Murray State University in 1972 and completed work on his Master of Science degree in August, 1972. Mr. Barnett began work toward a Ph.D. in Genetics in 1972 at the University of Florida. He taught the Genetics Laboratory (AY363) six quarters and wrote the manual which is presently being used in that course. lie taught the genetics lectures (AY362) during the fall of 1974.

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57 Mr. ,1 1 ici Mrs. Barnett have four children. He received the Ph.D. doijrec in June 1975. He belonys tn Sigma Xi.

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LITERATURE CITED Ahmad, Z., B. R. iMurty, and G. Harrinarayana 1972. Protein content in the successive generation of a partial diallel cross in dwarf pearlmillet. Indian J. Genet. Plant Breeding 32: 325-330. Allard, R. W. 1966. Principles of plant breeding. John Wiley and Sons, New York. Altschul, A. M. 1965. Proteins their chemistry and politics. Basic Books, New York. Association of Official Agricultural Chemist, 1965. Official Methods of Analysis. Washington, D.C. Balint, A. 1970. Protein growth by plant breeding. Adademiai Kiado Budapest Priggs, F. H and P. F. Knowles. 1967. Introduction to plant breeding. Reinhold, New York. Burton, G. W. and J. B. Powell. 1968. Pearlmillet breeding and cytogenetics. In: Advances in Agronomy, A. G. Norman (ed.), 20: 49-89. Burton, G. A. T. Wallace, and K. 0. Rachie. 1972. Chemical composition and nutritive value of pearlmiller (po nnisetum typhoides (Burn.) Stapf and E. C. Hubbard) grain. Crop Sci. 12: 187-188. Concon, M. 1972. Determination of lysine in cereal proteins In: Symposium: Seed Proteins. G. E. Inglett (ed.), Av i, Westport Conn., pp. 292-301. Concon, M and Diane Soltess. 1973. Rapid micro-K jeldahl digestion of cereal grains and other biological materials. Analytical Biochem. 53: 35-41. Crook, w. J. and A. J. Casady. 1974. Heritability and inter relationships of grain-protein content with other agronomic traits of sorghum. Crop Sci. 14: 622-624. 50

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51 Ocwit, J. P. 1969. The potential role of pear lmil let as a food in S. Africa. S. Afr. J. i-Jutr. 6: f >4-3f>6. (Plant Breeding Abs. 1970). Doyoe, C. VJ. and J. A. Shellenberger 1965. Amino acids and proteins in sorghum grain. J. Agr. Food Chem. 13: 446-450. Prey, K. J. 1949. The inheritance of protein and certain of its components in maize. Agron. J. 41: 113-117. Frey, K. J. 1951. The interrelationships of proteins and amino acids in corn. Cereal Chem. 28: 123-132. Ghosh, Mira and S. Bose. 1973. Development of rapid tests for the assessment of compounded poultry rations: Separation and estimation of available lysine in compounded poultry rations by TLC. Indian J. Anim. Sci. 43: 38-42. Griffing, B. 1956. Concept of general and specific combining ability in relation to diallel crossing systems. Australian J. Biol. Sci. 9: 463-493. Hayman, B. L. 1954. The analysis of variance of diallel tables. Biometrics 10: 235-244. Heathcote, J. G. and C. Haworth. 1969. An improved technique for the analysis of amino acids and related compounds on thin layers of cellulose. II The quantitative determination of amino acids in protein hydrolysates J. Chromatography 43: 84-92. Hillerislambers, D. J. M. Rutger C. O. Qualset and W. J. Wiser. 1973. Genetic and environmental variation in protein content of rice ( Oryza sativa L. ) Euphytica 22: 264-273. Howe, E. E. G. R. Jansen, and E. W. Gilfillan. 1965. Amino acid supplementation of cereal grain as related t-o the world food supply. Amer. J. Clin. Nutr. 16: 315-320. Johnson, R. M. and C. E. Craney. 1971. Rapid biuret method for protein content of grains. Cereal Chem. 48: 276282. Johnson, V. A., J. W. Schmidt, and R. J. Mattern. 1968. Cereal breeding for better protein impact. Econ. Bot. 22: 16-24.

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52 Juliano, B 0. E. L. Albano, and G. B. Cagampanq. 1964. Variability in protein content, amylose content, and alkali digestibility of rice varieties in Asia. Phil. Ayr. 48: 234-241. Juliano, Bienvenido 0. Juz U. Onate, and Angel ita M. del Mundo. 1965. Relation of starch composit > on protein content, and gelatinization temperature to cooking and eating qualities of milled rice. Food Technology 18: 1006-1010. Juliano, B. O. C. C. Ignacio, V. M Panganiban, md C. M. Perez. 1968. Screening for high protein rice vat ieties. Cereal Sci. Today 13(8): 299-310. Kamra, O. P. 1971. Genetic modification of seed protein quality in cereals and legumes. Pf lanzenziicht 65: 293-306. Kamra, 0. P. 1972. Breeding and screening techniques for seed protein improvement in crop plants. In: Induced Mutations and Plant Improvement IAEA Vienna, pp. 303313. Kaul, A. K. K. D. Dhar, and M. S. Swaminathan. 1969. A rapid dye-binding method of screening single grains for protein characteristics. Current Sci. 14: 330-331. Kaul, A. K. R. D. Dhar, and P. Raghaviah. 1970. The macro and micro dye-binding techniques of estimating the protein quality in food samples. J. Fd. Sci. Tech. 7: 11-15. Liang, G. J. L. E. G. Ileyne, J. H. Chung, and Y. 0. Koh. 1968. The analysis of heritability variation for throe agronomic traits in a 6-parent diallel of grain sorghum ( Sorghum vulgare Pers.). Can. J. Genet. Cytol. 10: 460-469. Mahadevappa, M. 196 7. Investigations of the inheritance of protein content in pearlmillet. Curr. Sci. 36: 186-138. Mertz, E. T. and L. S. Bates. 1964. Mutant qene that changer protein composition and increases lysine content of maize endosperm. Sci. 145: 279. Mertz, E. T. am 1 L A. Bates. 1965. Second mutant qene affecting the amino acid pattern of maize endosperm proteins. Sci. 150: 1469-1470.

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53 Munck, L. 1964. Plant breodinq and nutritional values in cereals. Hereditas 52: 151-165. Munck, L. 1972. Improvement of nutritional values in cereals, ilereditas 72: 1-128. Nelson, 0. E. 1969. Genetic modification of protein quality in plants. In: Advances in Aqronomy, Vol. 20, A. G. Norman (ed. ) pp. 171-194. Nelson, 0. E. and M. T. Chang. 1974. Effect of multiple aleurone layers on the protein and amino acid content of maize endosperm. Crop Sci. 14: 374-376. Nicol, B. M 1971. Protein and calorie concentration. Nutritional Reviews 29: 83-88. Palter, Rhoda and G. G. Kohler. 1969. Survey hydrolysis procedure for lysine analysis. Cereal Chem. 46: 22-26. Pa rial, Lucila and L. W. Rooney. 1970. Use of dye-binding and biuret techniques for estimating protein in brown ana milled rice. Cereal Chem. 47: 38-43. Robinson, P. 1963. Heritability : A second look. In: Statistical Genetics and Plant Breeding. W. D. Hanson and H. F. Robinson (ed.), National Academy of Science Publication 982, Washington, D.C., pp. 609-614. Sampath, S., S. Patnaik, and G. N. Mitra. 1968. The breeding of high protein rices. Current Sci. 9: 248-249. Sawiiney, D. K. and M. S. Naik. 1970. Amino acid composition of protein fractions of pearlmillet and effect of nitrogen fertilization on its proteins. Indian J. Genet. Plant Breeding 29: 395-406. Singh, C. B. S. Arora, and A. K. Kaul. 1971. Inheritance of dye-binding capacity value in rice. Current Sci. 40: 28-29. Singh, R. and J. D. Axtell. 1973. High lysine mutant gene (hi) that improves protein quality and biological value of
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54 Tcllo, F. M. A. Al varcz-Tostado, and G. Alvarado. 1965. A study on the improvement of the essential amino acid balance of corn protein. I. Correlation between racial and varietal characteristics and lysine levels of corn. Cereal Chem. 42: 363-384. Udy, D. C. 1971. Improved dye method for estimating protein. J. Aiiu-r. Oil Chem. Soc 48: 29A. Woodworth, C. M E. R. Leng, and R. W. Jugenheimer. 1952. Fifty generations of selection for protein in corn. Ayron. J. 44: 60-65. Yates, F. 1947. Analysis of data from all possible reciprocal crosses between a set of parental lines. Heredity 1: 287-301. Zscheile, F. P. and Bert L. Brannaman. 1972. A simplified method for lysine by gas chromatography. J. Agr Food Chem. 22: 537-538.

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L cert i fy that I have read this study and that in my opinion itconforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, .is a dissertation tor the degree of Doctor of Philosophy. Rex L. Smith, Chairman Associate Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. es Soule Professor of Fruit Crops I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Victor E. Green, Jr. Professof of Agronomy I certify th.it I have read this study and that in my opinion it conforms to acceptable standards of scholarly preson ta t ion end is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy Associate Professor of Food Science

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I certify that I have read this study and that in my opinion il conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as i dissertation for the degree of Doctor of Philosophy. Thin dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. June, 197 5 Ramon C. Littel Assistant Professor of Statistics Dean, Graduate School