glup4 controlling a 57H character was located on choromosome 12 in rice

VI. Gene and genome structure 41. glup4 controlling a 57H character was located on choromosome 12 in rice H. Satoh1, W. X. Li1, Y. TAKEMOTO�, T. Fm�, T. KUMASMARU1, L.Q. Qu1 and M. OGAwA2 1) Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan 2) Home Economy, Yamaguchi Prefecture University, Yamaguchi, 753-8502 Japan Glutelin, a major storage protein in rice, is synthesized as 57kD glutelin precursor on the ER, transported to the vacuole via the Golgi appartus, and formed by cleavaging the precursor into a subunit (4OkD) and b subunit (2OkD) glutelins in the vacuole (Yamagata et al.1982; Takaiwa et al. 1986; Masumura et aLl989). Kumamaru eta!. (1988) reported a high deposition of 57kD polypeptides (57H) mutant, CM1787, which is from Japonica rice cultivar �Kinmaze� induced by MNU treatment, and revealed that this character was controlled by a recessive gene, esp2, located on chromosome 11. Takemoto et a!. (1996) demonstrated that esp2 mutation was caused by the inhibitation of post-translational modifycation of glutelin precursor. 57H mutations are valuable materials for the study on genetic regulation of post-translational modification of glutelin precursor. So far five genes, esp2, Glupi, glup2, glup3 and g!up4 have been identified as 57H mutation in rice (Kumamaru et al.1988, Satoh et a!. 1994, Satoh et a!. 1995). The chromosome location of g!up4 was not known. In this report, we showed that the glup4 is non allelic to other four 57H genes and is located on choromosome 12. EM956 and EM960 were characterized by the increased content of 57kD polypeptides with remarkably decreased 26kD globulin in addition to the decreased amount of both 4OkD and 2OkD glutelins in SDS-PAGE. Both the mutants were crossed with their normal parent cultivar T65. The phenotypes of F1s were normal. The segregation of normal and mutant types in F2 showed a good fit to a 3:1 ratio. These results indicated that the 57H character of EM956 and EM960 are due to a single recessive gene. All mutant types in F2 population had decreased 26kD globulin together with increased 57kD polypeptide accumulation. These results suggested that the reduction of 26kD globulin was the pleiotropic effect of 57H gene in EM956 and EM960. These mutants were crossed with each other. The phenotype of F1 was 57H type and all of the F2 seeds derived from this cross combination were the mutant type, showing that the 57H character of EM956 and EM960 was controlled by the same gene. Table 1 shows the segregation pattern in F2 seeds derived from crosses between EM956 and marker lines of esp2, Glupl, glup2 and glup3. F1 seeds of the cross between esp2 and EM956 showed the normal phenotype, and F2 seeds were classified into normal, esp2 and EM956 types. Since the double mutant was sterile, segregation of these three types fitted a 9:3:3 ratio. In the cross between Glupi and EM956, the F1 seeds were 57H phenotype, and F2 seeds were classified into Glupi, normal, EM956 and double mutant types, of which the segregation ratio in F2 fitted to 9:3:3:1. F1 seeds of the cross between glup2 and EM956, were normal for the 57H character, and F2 seeds were classified into normal, 57H and double mutant type. As g!up2 and EM956 types were not distinguishable from each other, the F2 segregation ratio fitted 9:6:1. F1 seeds of the cross between glup3 and EM956 showed the normal phenotype, and F2 seeds were classified into normal, glup3, EM956 and double mutant types, with the segregation ratio of 9:3:3:1. These results indicate that the 57H gene of EM956, designated as glup4 , is independent of esp2, Glupi, glup2 and glup3, and that glup4 shows the additive effect with these four genes. Previously, we thought that glup4 was located on chromosome 5 (Unpublished). However, in the results of detailed linkage analyses no linkage was found with marker genes located on chromosome 5. Furthermore, trisomic analysis of EM960 revealed that the segregation in the F2 seeds from the cross between Tnplo 12 and the mutant fitted well to the expected ratio of trisomic segregation (Table2). From these results, we concluded that glup4 is located on chromosome 12. .

Glutelin, a major storage protein in rice, is synthesized as 57kD glutelin precursor on the ER, transported to the vacuole via the Golgi appartus, and formed by cleavaging the precursor into a subunit (4OkD) and b subunit (2OkD) glutelins in the vacuole (Yamagata et al.1982; Takaiwa et al. 1986; Masumura et aLl989). Kumamaru eta!. (1988) reported a high deposition of 57kD polypeptides (57H) mutant, CM1787, which is from Japonica rice cultivar �Kinmaze� induced by MNU treatment, and revealed that this character was controlled by a recessive gene, esp2, located on chromosome 11. Takemoto et a!. (1996) demonstrated that esp2 mutation was caused by the inhibitation of post-translational modifycation of glutelin precursor. 57H mutations are valuable materials for the study on genetic regulation of post-translational modification of glutelin precursor.

So far five genes, esp2, Glupi, glup2, glup3 and g!up4 have been identified as 57H mutation in rice (Kumamaru et al.1988, Satoh et a!. 1994, Satoh et a!. 1995). The chromosome location of g!up4 was not known. In this report, we showed that the glup4 is non allelic to other four 57H genes and is located on choromosome 12.

EM956 and EM960 were characterized by the increased content of 57kD polypeptides with remarkably decreased 26kD globulin in addition to the decreased amount of both 4OkD and 2OkD glutelins in SDS-PAGE. Both the mutants were crossed with their normal parent cultivar T65. The phenotypes of F1s were normal. The segregation of normal and mutant types in F2 showed a good fit to a 3:1 ratio. These results indicated that the 57H character of EM956 and EM960 are due to a single recessive gene. All mutant types in F2 population had decreased 26kD globulin together with increased 57kD polypeptide accumulation. These results suggested that the reduction of 26kD globulin was the pleiotropic effect of 57H gene in EM956 and EM960. These mutants were crossed with each other. The phenotype of F1 was 57H type and all of the F2 seeds derived from this cross combination were the mutant type, showing that the 57H character of EM956 and EM960 was controlled by the same gene.

Table 1 shows the segregation pattern in F2 seeds derived from crosses between EM956 and marker lines of esp2, Glupl, glup2 and glup3. F1 seeds of the cross between esp2 and EM956 showed the normal phenotype, and F2 seeds were classified into normal, esp2 and EM956 types. Since the double mutant was sterile, segregation of these three types fitted a 9:3:3 ratio.

In the cross between Glupi and EM956, the F1 seeds were 57H phenotype, and F2 seeds were classified into Glupi, normal, EM956 and double mutant types, of which the segregation ratio in F2 fitted to 9:3:3:1.

F1 seeds of the cross between glup2 and EM956, were normal for the 57H character, and F2 seeds were classified into normal, 57H and double mutant type. As g!up2 and EM956 types were not distinguishable from each other, the F2 segregation ratio fitted

9:6:1.

F1 seeds of the cross between glup3 and EM956 showed the normal phenotype, and F2 seeds were classified into normal, glup3, EM956 and double mutant types, with the segregation ratio of 9:3:3:1.

These results indicate that the 57H gene of EM956, designated as glup4 , is independent of esp2, Glupi, glup2 and glup3, and that glup4 shows the additive effect with these four genes.

Previously, we thought that glup4 was located on chromosome 5 (Unpublished). However, in the results of detailed linkage analyses no linkage was found with marker genes located on chromosome 5. Furthermore, trisomic analysis of EM960 revealed that the segregation in the F2 seeds from the cross between Tnplo 12 and the mutant fitted well to the expected ratio of trisomic segregation (Table2). From these results, we concluded that glup4 is located on chromosome 12.