CLUSTERING OF GENES FOR 20 kd ZEIN SUBUNITS IN THE SHORT ARM OF MAIZE CHROMOSOME 7

CARLO SOAVE, REM0 REGGIANI, NATALE DI FONZO, FRANCESCO SALAMINI

Istituto Biosintesi Vegetali C. N . R., I20133 Milano, Italy

Istituto sperimentale per la Cerealicoltura, Sezione di Bergamo P . 0. Box 164, I 24100 Bergamo, Italy

AND

Manuscript received June 16, 1980 Revised copy received January 5, 1981

ABSTRACT

Zein is the major storage protein of the endosperm of maize kernels. When this alcohol-soluble protein is subjected to SDS polyacrylamide gel electropho- resis, it is resolved into four fractions of different molecular weight: 10, 14,20 and 22 kilodaltons (kd). Each fraction is heterogeneous with respect to isoelec- tric pH. For example, the 20 kd fraction contains a t least seven subfractions as revealed by isoelectric focusing in polyacrylamide gels. In this report, we pre- sent evidence that the structural genes coding for the 20 kd proteins are clus- tered on the short arm of chromosome 7 , a region that also bears loci regulating endosperm zein biosynthesis [opaque-2 (02) and defective endosperm-B30 (De*- B30)] . The organization of these zein genes suggests that the evolution of at least some of the maize genome has occurred as the result of repeated dupli- cation and divergence of chromosome segments.

EIN is an alcohol-soluble protein that accumulates in the protein bodies of maize endosperm (BURR and BURR 1976). When analyzed by polyacrylamide

gel electrophoresis in the presence of SDS or urea, zein is fractionated into several molecular components (MOSSE 1966; MISRA, MERTZ and GLOW 1975; ALEX- ANDRESCU, CHIHAIA and COSMIN 1976). Subjected to isoelectric focusing (IEF) in polyacrylamide gels, zein produces a more complex banding pattern (SOAVE et al. 1975; RIGHETTI et al. 1977). Two-dimensional analysis (IEF-SDS) shows that some of the IEF zein bands can be further resolved in two major components with molecular weights of 20 and 22 kilodaltons (kd) (VALENTINI, SOAVE and OTTAVIANO 1979). This extensive heterogeneity is not an artifact since (1) the various zein polypeptides differ slightly in their primary structure ( BIETZ, PAULIS and WALL 1979; VITALE, SOAVE and GALANTE 1980); (2) the products synthe- sized in vitro by zein mRNA exhibit the same SDS or IEF heterogeneity as native zein (VIOTTI et ai. 1978) ; ( 3 ) hybridization studies between cDNA copies of zein mRNA and maize DNA demonstrate the existence of multiple zein genes (VIOTTI et al. 1979) ; and (4) cloned cDNA copies of zein mRNA are able to hybridize with mRNA specific for particular zein IEF bands (PARK, LEWIS and RUBEN- STEIN 1980). GENTINETTA et al. (1975) have shown that the zein IEF banding Genetics 97: 363-377 February, 1981.

364 c. SOAVE et al.

pattern is genotype specific. Moreover, the association of zein IEF variants with chromosomes 4 , 7 and 10 (VALENTINI, SOAVE and OTTAVIANO 1979; SOAVE et al. 1978a) has been confirmed by VIOTTI et al. (1980), using in situ hybridization. Of particular interest was the finding that three 20 kd zein band loci map on chromosome 7 (SOAVE et al. 1978a). This suggested the possibility of finding other 20 kd genes on this chromosome, a test of the hypothesis that zein genes belong to clustered, multigene families.

Results on the linkage map location of the 20 kd zein structural genes are re- ported; moreover, the linkage relationships among these genes and Opaque-2 (02) and Defective endosperm-B30 (De*-B30) are discussed. The latter two genes regulate the level of synthesis of zein. In the presence of the mutant alleles of both loci, the synthesis of 22 kd family polypeptides is depressed more than those of other families (SOAVE et al. 1976; SOAVE et ut. 1978b; DI FONZO et ul. 1979; DI FONZO et al. 1980). The finding in the same chromosome region of regu- latory and structural elements for zein synthesis may have some important evolutionary implications.

MATERIALS A N D METHODS

Stocks: Stocks summarized in Table 1 were from the collection of inbreds at the Institute of Cereal Crops, Bergamo, or at the Institute of Botany and Genetics, Piacenza; De*-B30, a spontaneous dominant opaque mutant, was reported by SALAMINI e t aZ. (1978). The EMS- induced recessive viviparous mutant of Table 1 was identified by allele testing with a standard up9 mutant obtained from the Maize Genetics Cooperative Stock Center, Urbana, Illinois. At least two 02 alleles of independent origin were used, one from an Italian open-pollinated variety (PIVA, SALAMINI and SANTI 1967) and the second from the Maize Genetics Cooperative Stock Center. The translocation stock T7-9 (4363) was received from the Maize Genetics Cooperative Stock Center. The translocation breakpoints in both chromosomes are listed as being at the

TABLE 1

Genotype and inbred line background of the stocks utilized in the experiments described in the text

Experiment number Genotype Background

1 T7,9(4363), wx, zp l , zp2, zp3 - 1 wx, Z p l , z p 2 , z p 3 MI4 2 02, z p l , z p 2 , zp3, zp5, zp6, zpi2 W64A 2 02, Z p l , ZpZ, Zp3 ,Zp5 ,Zp6 ,Zp l2 A69Y 3 De*-B30, Z p l , Zp2,Zp3 B37

3 ,f , z p l , ~ 2 , Z P ~ B3 7 4 Z p l , Zp2: Zp3, ZplS A652 4 z p l , zp2, zp3, zp16 H96 5 0 2 , vp9 , ZPI, ZP2, z p 3 , Zp2l A69Y 5 02, vp9, z p l , zp2, zp3, zp21 A69Y 6 02, Z p l , Zp2,Zp3, zpS,ZpI&, zp21, zp29 Oh45 6 0 2 , z p l , zp2, zp3,Zp6, zpl6,Zp21,Zp29 H96 7 ZPf, ZP2,ZP3,ZP6 ZP21, A69Y

Det-B30

7 z p l , zp2, zp3, zp6, zp21 38-1 1

ZEIN GENE CLUSTERS 365 centromeres (LONGLEY 1961). The recessive wx allele is closely linked to the centromere in chromosome 9.

Zein po1ypept:de nomenclature: About 30 zein polypeptides, each having a distinct isoelectric pH, have been described (GENTINETTA et al. 1975). These zein proteins ( Z p ) were numbered consecutively from most basic isoelectric pH ( Z p i ) to least basic. Several inbred lines have been compared to a hypothetical, generalized IEF pattern containing all known Z p bands. The absence of a particular band at a particular pH in an inbred line is designated by the symbol zp followed by the band number. All of the IEF bands analyzed in this report were shown, by two dimen- sional IEF-SDS electrophoresis, to belong to the 20 kd fraction of zeins.

ZEF e2ectrophoresis: Individual kernels from segregating ears were classified as to endosperm phenotype. The endosperm was powdered with a mortar and pestle and the meal (about 200 mg) extracted by shaking for 2 hr with 3 ml of 70% (v/v) ethanol containing 1% (v/v) 2-mercapto- ethanol. The meal was re-extracted twice. Only the zein protein fraction is alcohol-soluble. The extract was dried in vacuo and the zein redisssolved at a concentration of about 7 mg/ml in 0.01 M Trisglycine, pH 8.2,6 M urea and 1 % 2-mercaptoethanol.

Thin-layer polyacrylamide gel isoelectric focusing was carried out as described by MOTTO et al. (1979). The gel slab contained 4.815 g acrylamide and 0.185 g bisacrylamide, 2% carrier ampholytes prepared with equal volumes of pH 6-8 and pH 7-9 range Ampholine (LKB Fro- ducts) and was 6 M urea. Protein samples (about 150 p g in 30 pl) were applied to slots precast near one end of the gel and the gradient established by electrophoresis for four h r at IO" with a constant wattage of 13 W. Staining and destaining were as described by MOTTO et al. (1979).

Linkage calculations: Linkage values were calculated according to the product method (IMMER 1930). When fewer than 5 individuals were observed in any particular class of a segre- gating F,, the 3-class maximum likelihood method was used (KRAMER and BURNHAM 1947). Linkage data reported in the lower part of Figure 6, which gives the results of several linkage experiments, were calculated by the weighted-average method (IMMER and HENDERSON 1943 ; KRAMER and BURNHAM 1947).

RESULTS

Expression of zp uariation: The zein protein variation described here is limited to the presence or absence of IEF bands. For example, in Figure 1, one line (Ea 2121) is null for zp5, 6, 7,12 and 26, which are expressed in the other line (Lo 38). The F, hybrid between these two parents exhibits the Lo 38-specific bands, although in an apparently dosage-dependent way. If the maternal parent is Lo 38, the variant bands are expressed more intensely than if Lo 38 is the paternal parent (the endosperm is triploid, with two of the genomes being contributed by the maternal parent). The mapping of the 20 kd family of polypeptides is based on a similar variation that we have found for the z p l , 2, 3, 6, 16, 21 and 29 poly- peptides.

Preliminary evidence for the association of 20 kd zein genes and the short arm of chromosome 7: The results of two preliminary experiments indicated a strong association between the short arm of chromosome 7 and the 20 kd zein family. In the first of these experiments, a genetic stock bearing the translocation T 7-9 (4363) homozygous for the closely linked wx allele (about 4 crossover units from the translocation breakpoint) and lacking zein bands Zpl , Zp2 and Zp3, was crossed with the inbred line NI14 in which these bands are expressed. The selfed F, plants (F, kernels) segregated normal and wx seeds. The IEF profiles of the zein extracted from indixidual F, seeds are shown in Figure 2. The following could be seen: (1) co-inheritance of the Zpl , 22 and Zp3 bands in F, kernels,

366 c. SOAVE et at.

4 5 4 6

4 7

4 1 2

A A A B ABB B

FIGURE 1.-The zein IEF patterns of the inbreds Ea 2121 (A) and Lo38 (B) and of their reciprocal F,s. In the Fls, the intensity of the staining of bands 5, 6, 7, 12 and 26 (which are present in Lo38 and absent in Ee2121) appears to be related to the dose of the Lo38 parental genome. The staining intensity of band 22 (which is present in Ea2121 and absent in Lo38) is related to the dose of the Ea2121 parental genome (the endosperm tissue is triploid and the female parent contributes two doses to the F,).

and (2) an apparent linkage between the wx trait and the zp2, zp2 and zp3 null alleles. Among the 33 wz seeds, only two recombinants were found. The re- combinant seeds had all three M14-specific IEF bands. These gave a linkage value between wx and the loci of genes coding for Zp2, Zp2 and Zp3 as 5.1 C.U.

(crossover units; Table 2). Since the wx to centromere distance in T 7-9 (4363) is 4.0 c.u., these three Z p genes are presumably close to the centromere of chro- mosome 7.

A second preliminary experiment utilized deficiencies for the short arm of chromosome 7. Ears of W64A 02, which is null for Z p l , 2 , 3 , 5 , 6 and 22, were pollinated with A69Y 02 ,Zp l , Zp2, Zp3, Zp5, Zp6 and Zp12 pollen that had

ZEIN GENE CLUSTERS 367

FIGURE P.--Zein IEF patterns of normal and w r endosperms from an F, obtained by crossing Ml4 Wx (P,) with T7-9(4363) wr (P,). = recombinant F, endosperm phenotypes.

been X irradiated (1,000r). Forty-seven of 7,467 seeds from this cross were opaque-2. Analysis of the putative chromosome 7 hemizygotes indicated fre- quent (25 of 47) and simultaneous loss of the wild-type allele a t the 0 2 locus with loss of some or all of the 20 kd polypeptide genes by which the two lines contrasted (see Table 3). The frequency of loss appears to be higher for Zp2, Z p 2 , Z p 3 and Zp6 than for Zp5 and Zp22; apparently the former four genes are more closely linked to 0 2 than the latter two.

Results of additional linkage tests: The Z p l , 2 and 3 gene loci were found to be closely linked (0.5 c.u.) to De*-B3O (Table 4). De*-B30 is about 6 C.U. from 02 on the short arm of chromosome 7 (see APPENDIX); it is fairly certain that the Z p l , 2 and 3 gene loci are on the short arm of chromosome 7 and do not re- combine at an observable frequency. The other 20 kd family genes for which variants have been found ( Z p 6 , 2 6 , 2 2 and 29) were mapped in relation to this seemingly stable gene complex and the endosperm markers 02 and V p9.

The gene controlling Zp26 appears closely linked to the Zp2, 2 , 3 complex (see Table 5 and Figure 3 and 6). Zp22 is closely linked to 0 2 (see Table 6 and

TABLE 2

Linkage between ihe endosperm marker wx iranslomted io the shori arm of chromosome 7 and near the zein 20 kd genes Zpl, Zp2, and Zp3 from 137 seeds of the cross

T 7-9 (4363) wx zpl Z P ~ zp3 (Erperimeni I ) + + + + + -

Segrcgnting genes Number of observed phenotypes Re"hination

A R AR Ab aR ab p e n t S.E.

W r ZPIY ZP2Y ZP3 97 5 2 33 5.1 1.9

368 c. SOAVE et al. TABLE 3

Loss (*) of 20 kd bands in selected opaque seeds from crosses of W64Ao2, zpl, zp2,zp3, zp5, zp6, zp12 females with A69Y, 02, Zpl, Zp2,Zp3,Zp5,

Zp6, Zpl2 X-irradiated pollen (Experiment 2 )

Seed phenotype IEF zein band number

No. 1,2,3 5 6 12

Total 7420 Normal Analyzed 20

Frequency of loss (%) 0 0 0 0

Opaque-Plump

Opaque-Small osr wrinkled

I2 5 3 2 2 1

10 3 3 2 1 1 1 1

* * * *

* * * * * *

* * * * * * * *

* * *

* * * * *

Total 47 18 8 17 11 Frequency of loss among 02 kernels (%) 38.3 17.0 36.1 23.4

Figures 4 and 6). The data presented in Table 7 and Figure 5 show the map position of two additional 20 kd genes, Zp6 and Zp29, with respect to the other mapped zein genes on the short arm of chromosome 7. Table 8 adds information on the map location of Z p l 6 with respect to Zpl , 2 , 3 and Zp21. The linear order of those genes, inferred from all these experiments, is summarized in Figure 6. The genes encoding Zp5 and 12, although suspected of being on chromosome 7

TABLE 4

Linkage relationships between the locus carrying genes Zpl, Zp2 and Zp3 and the De*-30 locus. The progeny kernels were from the backcross f f ,+ De*-B30 zpl zp2 zp3 f

(Experiment 3 ) zpl zp2 zp3 --I + x zpl zp2 zp3f

Expected in case Genotype Observed of independence

+ + + De*-B30 98 49 + + + + 0 49 zp i , zp2, zp3 De*-B30 1 49 zpl , zP2, z p 3 I+ 98 49

Recombination percent Zpi, ZpZ,Zp3-De*-BSO = 0.5 & 0.3

ZEIN GENE CLUSTERS 369

TABLE 5

Linkage relationships among genes Zpl, Zp2,Zp3,Zp16 from 7 6 F, seeds of the cross

$- + + + (Erperiment4) zpl zp2 zp3 zp16

Scgrcgnting gens

A B hD Ab nn ab percent S.E. Number of observed phenotypes

Recombinntion

Z p f , Z p 2 , Z p 3 Z p f 6 56 1 2 17 4.1 1.9

1

+ + + + z p f z p 2 zp3 zp16'

PI = H96, z p f , zp2 , zp3 , z p f 6 ; P, = A652, Z p f , Z p 2 , Z p 3 , Z p 1 6 . In the F, patterns, bands 1 and 2 are not clearly separated, while, in the gel, band 3 was so faint that it does not show in the picture.

FIGURE 3.--Zein IEF pattern of the parents and some F, seeds from the cross

(see Table 3), are not yet mapped with respect to the above 20 kd polypeptide genes.

DISCUSSION

Zein consists of at least 30 different kinds of proteins with similar solubility, molecular weight and amino acid composition, and which differ in net charge (GIANAZZA et al. 1976). A large body of evidence suggests that the charge vari- ability, revealed by IEF, reflects true differences at the level of structural genes coding for zeins (GIANAZZA et al. 1977; VITALE et al. 1980; LARKINS et al. 1979; BIETZ, PAULIS and WALL 1979; FRAU and MELCHER 1978; VIOTTI et al. 1978;

370

1, 2- 3-

21-

c. SOAVE et al.

:: ?!m? ' .

?"?

p1 F1 p 2 02Vp9 02vp9 02Vp9 02Vp9

FIGURE 4.--Zein IEF pattern of the parents, F, and some F2 seeds from the cross

. P, = A69Y, 0 2 , V p 9 , Z p l , Zp2 , Z p 3 , Zp21 . P, = A69Y, 02, vp9 , :p l , + + + + + + zp2 , zp3 , zp21. 02 up9 z p l z p 2 zp3 zp21

4 6

P1 P2

*l, 2 -3 -6

-16

- - - - - -

F 2 Seeds

0 2 + + + + zp1 zpp-7 zp3

FIGURE 5.--Zein IEF pattern of the parents and some F, seeds from the cross

z p 6 + z p 2 1 z p 2 9

4- zp16 4- -I- ----- . PI = OH45 0 2 , Z p l , Z p 2 , Z p 3 , z p 6 , Z p 1 6 . zp21, zp29; P2 = H96$02, :p l ,

zp2 , z p 3 , 2 p 6 , z p 1 6 , Z p 2 1 , Z p 2 9 .

ZEIN GENE CLUSTERS

Zp21 Zp29 CY2 De-830 Vp9 Zp123 Zp16

371

A

EXF! 3

EXF! 4

E X R 5

EXF! 6

E X R 7

APPENDIX

CENTROMERE 9

FIGURE 6.-(A) Results of mapping experiments, the data for which are summarized in Tables 4-8 and in the APPENDIX. (B) Linkage intensities, calculated by the weighted average method, among loci for which estimates were available from more than one experiment. (Map relates to the short arm of chromosome 7.)

PARK, LEWIS and RUBENSTEIN 1980). The data presented here and in previous papers suggest a dosage-dependent expression of the zein genes. This also sup- ports the conclusion that the variability is due to structural gene differences (RIGHETTI et al. 1977; SOAVE et al. 1978a; VALENTINI et al. 1979; MOTTO et al. 1979).

Zein polypeptides can be grouped on the basis of their molecular weight into four classes of about 10,14,20 and 22 kd. Each class is a family of similar poly- peptides, each polypeptide representing one structural gene. The genetic map locations of these structural genes have been determined for seven of the poly- peptides of the 20 kd family.

The seven genes for the 20 kd polypeptides have properties that characterize a multigene family: multiplicity, sequence homology, overlapping phenotypic function and linkage (HOOD et al. 1975). For example, t hey are located in a seg- ment of chromosome 7 covering 30 crossover units. At least three of these genes are clustered: Z p l , Zp2 and Zp3 are always inherited as a unit, a situation remi- niscent of the organization of chromosome 2 of Bombyx mori, which bears three linked clusters of structural genes for chorion proteins (GOLDSMITH and BASE- HOAR 1978; GOLDSMITH and CLERMONT-RATTNER 1979). A clustered arrange- ment of some chorion protein genes has been found in Drosophila melanogaster (SPRADLING, WARING and MAHOWALD 1979). The histone genes in maize are organized as both clustered and dispersed genes (STOUT and KERMICLE 1979). The observation that in the short arm of chromosome 7 even regulatory loci with similar functions are duplicated (i.e., 0 2 and De*-B30) suggests that duplication of short chromosomal segments has occurred during evolution. Such duplications have given rise to clustered gene families, such as those described here for zein [see REGIER et al. (1978) and SIM et al. (1979) for the evolution of chorion pro- teins in Antheraea polyphemus]. Clustering of genes with overlapping or meta-

372 c. SOAVE et al.

% 0

ZEIN GENE CLUSTERS 373

TABLE 7

Linkage intensities among genes 02, Zpl, Zp2,Zp3,Zp6,Zp21 and Zp29 from 160 F, + zpl zp2 zp3 4- zp16 4- d- 02 + + + zp6 + zp21 zp29

(Experiment 6 ) seeds of the cross --

Segregating genes Number of observed phmotypes Recombination - ~ - _ _

A B AB Ab aB ah percent S.E.

0 2 ZP1, ZP2,ZP3 66 52 42 0 12.6 5.2 02 Z P ~ 101 17 19 23 25.5 2.8 0 2 Zp16 73 46 40 2 19.5 5.1 0 2 zp21 112 6 6 36 7.7 1.4 0 2 Zp29 113 5 2 40 4.3 1.1 Zp1, Zp2,Zp3 Zp6 76 32 44 8 38.5 4.5 Zp1, Zp2,Zp3 Zp16 105 3 8 44 6.6 1.4 zp1, zp2, zp3 zp21 69 391 49 3 22.5 5.0 Zp1, Zp2,Zp3 Zp29 65 43 50 2 17.7 5.1 ZP4 Zp16 82 38 31 9 43.5 4.3 ZP4 zp21 107 13 11 29 16.2 2.2 ZP6 Zp29 104 16 11 29 18.1 2.3 Zp16 zp21 75 38 43 4 27.8 4.8 Zp1 6 zp29 71 42 44 3 23.3 5.0 zp21 Zp29 114 4 1 41 3.1 0.9

bolically related functions is, however, not frequent in eukaryotes: besides the zein o r chorion protein systems, other cases are critically discussed by HOOD, CAMPBELL and ELGIN (1975) , HOOD (1976) , GILES (1978) and COOPER, GORSKI and TUROSCY (1979).

TABLE 8

Linkage intensities among the locus carrying the genes Zpl, Zp2,Zp3 and Zp6 and Zp16 zplzp2zp3zp6zp21

from 150 F, seeds of the cross (Experiment 7 ) + + + + + Phenotype of the seed Expected in

Z p l Zp2 Zp3 Zp6 ZpZ! Obsenred case of independence

+ + + + + 92 63 + + + + ZP21 1 21 ZP1,ZP2,ZP3 '+ ;+ 11 21 zpl,zp2,zp3 + zp21 7 7 f + + Z P ~ + 12 21 + + + Z P ~ zp21 9 7

Z P ~ , ~ P ~ , Z P ~ Z P ~ + 3 7 zpl, zp2, zp3 zp6 zp21 15 2

Seg-egaiing genes Number of observed phenotypes -___-- Recombination -~

A B AB Ab aB ab percent S.E.

Zp1, Zp2, Zp3 Zp6 93 21 18 18 30.7 3.1 zp1, zp2, zp3 zp21 104 10 14 22 18.4 2.4 Zp6 zp21 103 8 15 24 17.2 2.3

3 74 c. SOAVE et ai.

Interactions among zein structural genes of maize and their regulatory loci, such as 02, FZ2, 07 and De*-B30 (MERTZ, BATES and NEMON 1964; NELSON, MERTZ and BATES 1965; MCWIRTER 1971; SALAMINI et al. 1978), depend on the molecular weight of the zein polypeptides. For example, the fZ2 allele is known to depress both the 20 and 22 kd family polypeptides. Thus, the 20 and 22 kd families are in some way related, since both are able to respond to a signal from the same regulatory locus. Moreover, fingerprinting experiments ( VITALE, SOAVE and GALANTE 1979) and cross hybridization among cloned cDNA and zein mRNA (PARK et al. 1980) have shown that the 20 and 22 kd families share some sequence homology. Apparently they have diverged early in the evolution of maize since they occupy different chromosomal regions: the 20 kd family is asso- ciated with chromosome 7, while the 22 kd family is located on chromosome 4 (our unpublished results). No information is yet available on the genetic rela- tionship of the two lower molecular weight zein families (9 and 14 kd) to the 20 and 22 kd families.

We are grateful to JOHN T. STOUT for his accurate and critical reading of the manuscript.

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Chromosomal location of zein

Peptide mapping of IEF zein components from

Corresponding editor: R. L. PHILLIPS

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Z E I N G E N E CLUSTERS

APPENDIX

377

Location of a new endospermic trait on the short arm of chromosome 7: In order to locate the 20 kd zein genes on the genetic map of the short arm of chromosome 7, three endosperm markers for this chromosomal region were used, namely opaque-2, viviparous-9 and De*-B30. The first two have been for a long time associated with chromosome 7 (COE and NEUFFER 1976); the last was located only recently by our group and the results reported here. De*-B30 is a dominant mutation that interferes with the synthesis of the 22 kd zein peptides (SALAMINI et al. 1979).

So as to exclude the allelism of De*-B30 with 02, a fact suggested by their common nature as 22 kd zein repressors, a cross between strains homozygous for the two mutant alleles was carried out. I t was then necessary to analyze the F, generation of this cross because De*-B30 is dominant over the normal state. The F, ears clearly segregated opaque and normal seeds: the nonallelic nature of 02 and De*-B30 was then demonstrated. The ratio of opaque to normal seeds, however, deviated strongly from that expected in case of independence of the two markers (13 opaque: 3 normal). Table 1A reports the results from four separate sets of F, ears. From 9883 F, seeds, 313 normal phenotypes (3.17%) were found. The frequency of normal seeds permitted

the recombination frequency ( p ) to be calculated by solving the equation ( )'-I- 2 (9) (%) = 0.0317. The linkage value between the two markers was found to be equal to 6.56 c.u., an estimate that has a standard error of 0.36 C.U. On the basis of this result, the endo- sperm marker De*-B30 was associated with the short arm of chromosome 7 and used in mapping 20 kd zein polypeptides.

TABLE 1A

Linkage intensity between the genes 0 2 and De*-B30 from 26 F , progenies

of the cross -- 02 De*-B30 + ,+

Frequency of normal Progeny number " m a l seeds Opaque seeds (% total)

1978471 (9 ears) 80 321 7 2.42 1979-1581 (5 ears) 59 1564 3.63 1979-1582 (4 ears) 43 1540 2.72

Total (26 ears) 313 95 70 3.17 Recombination percent

197S-1583 (8 ears) 131 3249 3.87

0 2 - De*-B30,= 6.56 f 0.36