As shown in the previous report (Tian et al., 2001), we have identified seven 57H mutants genes, esp2 and Glup1 to glup6. However, the detail genetic behavior of Glup5 remained to be described. In this report, we describe the inheritance mode of Glup5 mutant character and the genetic relationship between Glup5 gene and other 5 genes involving in 57H mutation.

The three independent Glup5 mutants, EM675, EM 677 and PMT 19, were so far isolated by the screening of mutant lines derived from the mutation treatment of fertilized egg cells of a rice cultivar Taichung65 (TC65) with MNU. SDS-PAGE and western blot analyses of seed storage proteins in three kinds of 57H mutants showed that Glup5 mutations increased significantly the amount of glutelin precursor protein with M.M 60kD and decreased the accumulation of both of 40kD acidic and 20kD basic mature glutelins (Fig. 1). In addition, Glup5 mutations decreased markedly the accumulation level of 26kD globulin and 13kD prolamin. All of Glup5 mutations exhibited the opaque phenotype in grain in which the central potion was transparent as same as the normal grain while the outer potion was translucent and floury. F₁ seeds derived from crosses between the wild type cultivar TC65 as a female parent and a Glup5 mutant EM675 as a male parent was normal in the SDS-PAGE profile of seed storage protein as well as in grain appearance. However, F₁ seeds from crosses between Glup5 mutants as a female parent and TC65 as a male parent showed 57H in the seed storage proteins and opaque in the grain phenotype. In F₂ seeds derived from the self-pollinated F₁ plants, the segregation

modes of normal and 57H phenotyps fitted well to the expected ratio of co-dominance inheritance, 1:1 (Table 1). In addition, the complete linkage relationship was observed between 57H character in seed storage proteins and opaque phenotype in grain, suggesting that Glup5 gene result in not only the abnormal accumulation of proglutelin but also the anomalous grain filling.

Recombinants showing the normal phenotype segregated in all of F₂s derived from crosses between Glup5 mutant lines and each of marker lines for 5 kinds of 57H mutant genes (Table 1), indicating that Glup5 gene is independent from other six 57H mutant genes. F₂ seeds from the cross of Glup5 with esp2 segregated into a ratio of 3 (Glup5 type): 2 (esp2 type): 3 (normal) without double mutant type, suggesting that esp2 gene is epistatic to Glup5 gene. Double mutant types having extremely high amount of proglutelin were detected in F₂ seeds derived from crosses between Glup5 and glup4, indicating that they have the additive effect on the proglutelin accumulation each other. Double mutant types were not observed in opaque F₂

seeds from cross combinations of Glup5 with Glup1, glup2 and glup3 while opaque and normal seeds segregated into the expected ratio of 1:1, suggesting that Glup5 are epistatic to these three genes.

Preliminary trisomic analysis showed that trisomic segregation for Glup5 was found in F₂ derived from a cross combination of EM675 with Triplo 12 while disomic segregation was observed in F₂s of crosses between EM675 and other eight trisomic lines (Data not shown). This results suggest that glup5 gene locates on chromosome 12.

The details including F₃ analysis and RFLP mapping are under investigation.

Acknowledgement

This work was supported by the Ministry of Education, Science and Culture of Japan (Grant no. 12138506) and BRAIN.

Reference

Tian, H.D., T. Kumamaru, Y. Takemoto, M. Ogawa and H. Satoh, 2001. Gene analysis of a new 57H mutant gene, glup6, in rice. RGN 18: 48-49.