26. Genetic analyses on glutelin mutants in rice 

L.Q. qu1, T. kumamaru1, H. satoh1 and M. ogawa2

1) Laboratory of Plant Genetic Resources, Faculty of Agriculture. Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka. 812-81 Japan

2) Home Economy,Yamaguchi Prefecture University, Sakurahatake. Yamaguchi, 753 Japan

 Glutelin acidic subunits (a subunits) of rice are less conserved than basic subunits (P subunits) (Wen and Luthe 1985). They might, therefore, be the best targets for improving the amino acid composition of rice glutelin. Eight glutelin mutant lines were found by screening about 3000 mutant lines derived from the treatment of fertilized eggs with MNU (N-methyl-N-nitrosourea) (Satoh et al. 1997). In this study, we report the inheritance of the mutated characters and the gene loci of three of a -2 subunit (38kD) less mutants combined with increased amount of a -1 or of a -3 subunit (39kD/37kD).

Three a -2 subunit less mutant lines used in this study were characterized as follows: EM 278 combining with increased a -1 subunit (a -1 H/a -2 L), CM 1707 with increased a -1 and a -3 subunits (a -l,3H/a -2 L) and EM 659 with increased a -3 subunit (a -3H/a -2 L) (Fig. 1). Extracted glutelins of mutant lines were electrophoresed using the discontinuous buffer system of Laemmli (1970) on a slab gel containing a linear of 15% to 25% acrylamide/0.05% to 0.67% BIS concentration gradient.

The mutant lines were crossed reciprocally with their original cultivar, Kinmaze. The SDS-PAGE band patterns of glutelin subunits in F1 seeds showed intermediate types between mutants and Kinmaze in all of the cross combinations with slight differences between reciprocal cross combinations, indicating that the genes have dosage effects. The phenotypes of F2 seeds from the crosses were classified into 4 types — normal (P1P1P1), semi-normal (P1P1P2), semi-mutant (P1 P2P2) and mutant (P2P2P2) types. The segregation mode of the F2 seeds of the crosses between Kinmaze and mutants are shown in Table 1. Since the segregation of the four types fitted the expected ratio of 1:1:1:1, the three mutant characters could be considered to be controlled by a single incompletely

Research Notes 79

Fig. 1. SDS-PAGE analysis of seed storage proteins in three a -2 less
mutants of rice.

l:Kinmaze 2: CM 1707 3:EM278 4:EM659
Table 1. Segregation of glutelin types in F2 from the crosses between mutants and the original
cultivar
Segregation in F2
Cross combination P1P1P1 P1P1P2 P1P2P2 P2P2P2 c 2(1:1:1:1)
Kinmaze X EM278 24 23 28 25 0.56
Kinniaze X CM1707 21 26 26 26 0.92
Kinmaze X EM659 24 23 26 27 0.40

dominant gene respectively.

The mutant lines were crossed with each other. The phenotypes of the F1 seeds in the cross combinations between EM 278 and CM 1707, CM 1707 and EM 659 showed intermediate types and that of F2 seeds could also be classified into 4 types—mutant I(P1P1P1), semi-mutant I (P1P1P2), semi-mutant II (P1P2P2) and mutant II (P2P2P2) types. The segregation mode of F2 seeds among mutant lines are shown in Table 2. The segregation also fitted the 1:1:1:1 ratio. In the cross combination between EM 278 and

80 Rice Genetics Newsletter Vol. 14

Table 2. Segregation of glutelin types in F2 from the crosses between mutants


 
Segregation in F2
Cross combination P1P1P1 P1P1P2  P1P2P2 P2P2P2 c 2(1:1:1:1 or 1:2:1)
EM278 x CM1707 26 23  26 25 0.24
CM1707 x EM659 24 25  22 23 0.22
EM278 x EM659 26 48 26 0.16

 
 
Table3. Linkage analysis among Glu4-c,spl6 and eg genes
segregation in F2
Cross combination +++ + +Glu4-c + Glu4-cGlu4-c m*++ m+Glu4-c mGlu4-c Glu4-c cv(%)
spl6 X Glu4-c 16 56 37 18 8 1 17.2
eg X Glu4-c 18 55 36 16 9 2 24.2

* m: recessive marker genes,

EM 659 the F1 seed also showed intermediate type, but it was difficult to distinguish the semi-mutant I and semi-mutant II types from each other in F2 segregation. So, these two types were counted together as F1 (intermediate) type. The segregation fitted the ratio of 1 (EM 278 type): 2 (FI type): 1(EM 659 type). Hence, the three mutant lines were considered to be intragenic mutants at the same locus, i.e. they were allelic mutants. The fact that polypeptide patterns differed significantly, though the mutants had the mutation at the same locus, suggested that the amino acid compositions of the same gene might be changed greatly by different inducement, Iida et al. (1994) reported three mutants for glutelin induced by g -ray or EMS treatment and named the relevant genes as glul (t), glu2 (t) and g!u3 (t). The genes we report here are named G!u4-a, Glu4-b and Glu4-c tentatively.

A series of trisomics with extra chromosome from No.4 to No. 12 and marker gene lines concerning of chromosomes 1, 2 and 3 were crossed with EM659 (Glu4-c) to determine the chromosome on which the genes are located. Glu4-c gene was found to be linked with spl6 and eg genes located on chromosome 1 through F2 and F3 analyses. The cross-over values between Glu-4c and both marker genes, spl6 and eg, were estimated to be 17.2% and 24.2%, respectively (Table 3). Since the cross-over value between spl6 and eg genes was 7% (Kinoshita 1995), the order of the genes must be eg—spl6—glu4-c on

Research Notes 81

chromosome 1 (Fig. 2).

Iida et al. (1994) reported a glu3 gene located on chromosome 1. Similarly another Glu1 (t) gene which was found in local rice cultivars was also reported to be located on chromosome 1 (Uemura et al. 1995). Deduced from the chromosomal locations of the two genes and our result, it seems that the three genes e.g. Glul (t), glu3 (t) and Glu4 (t) are either allelic or these genes are closely linked to each other.

References

Iida, S., M. Kusaba and T. Nishio. 1994. RFLP mapping of mutated genes lacking a glutelin

subunit of rice glutelin. Breed. Sci. 44 (Suppl. 2): 193.

Kinoshita, T., 1995. Report of committee on gene symbolization, nomenclature and linkage

groups III. Current linkage maps. RGN 12: 94-96.

Laemmli, U.K., 1970. Cleavage of structural protein during the assembly of the head of 

bacteriophage T4. Nature 227:680-685.

Satoh, H., L.Q. Qu. T. Kumamaru and M. Ogawa. 1997. Glutelin mutants induced by MNU

treatment in rice. RGN 14: 81-84.

Uemeru, Y.J., H. Satoh. M. Ogawa, H. Suehisa.T.I. Katayama and A.Yoshimura, 1995.

Chromosomal location of genes encoding glutelin polypeptides in rice. In Rice Genetics III: 471-476. Wen. T.N and D.S. Luthe. 1985. Biochemical characterization of rice glutelin. Plant Physiol. 78:

172-177.