Fine Mapping And Candidate Gene Analysis Of A Rice (Orazy Sativa L.) Grain Weight Gene

As people relying on rice crops to survive, rice is closely related to national food security. Increasing rice yield is an important way to ensure food security. It's the basis of high-yield breeding to mine and research yield-related genes. The aim of this study was that using a large grain of rice material (307R) of the donor parent, three restorer lines IR24, Minghui 63, Shuhui527 as recurrent parents to build backcross populations. Based on previous preliminary position, we gained a controlling grain weight dominant gene, named GLW2, by using recombinant homozygous lines for fine-mapping. And the candidate gene had been validated analysis. The main results were as follows:(1) Three NILs for GLW2 were generated by backcrossing 307R with IR24, M63, and 527R, as the recurrent parents, respectively. By using NILs evaluate the effects of GLW2,we got that GLW2 can make grain length increase by 21%-32% in different contexts, grain width increase by 22%-27%, so that the grain weight increase by 27%-53%, and also increase the number of grain to some extent, so that the yield per plant increase by 15%-26%. These data suggest that gene GLW2 can increase grain weight and ultimately improve the yield per plant mainly by increasing the grain length and width.(2) The observation of grain outer glumes in NILIR24 and IR24 from the paraffin sections and electron microscope show that the total cells number of grain outer glumes in NILIR24 increased by 9.4%, but the number of unit cell area decreased about 41.2%. Compared with IR24, the cell length approximately increased by 59.8% and the width of the cells about increased by 30.4% in NILIR24 of the cell measurements. The all above data show that GLW2 can increase the grain weight by increasing cell size primarily as well as increasing the number of cells.(3) The GLW2 locus was further narrowed down to a 160 Kb interval between the makers H2 and Z4 by using 2500 short-grain individuals generated from a 307R/IR24 BC3F2 population of the same cross. Finally, we limited the GLW2 to a 15 Kb interval between the makers M2 and M8 by fine-genotyping of 8 fixed recombinants.(4) Rice genome annotation displays that the 15Kb interval contains only one gene LOC_Os02g47280. There were four SNP loci between the large grain material 307R and the Nipponbare in the sequence analysis of the gene coding region, however, only one polymorphism (TC ? AA) was conserved between the 307R and IR24, M63 and 527R. We suggest the TC ? AA mutation is the causal one for the large grain size, and the other three variations are between japonica rice and indica rice.(5) We have sequenced the other about twenty materials in the breeding resources nursery of LOC_Os02g47280, and the sequences results show that three large grain materials were consistent with near-isogenic lines, which performed AA genotype, while the small grain materials performed TC genotype. These results further validate the correctness of candidate gene.(6) The expression level of candidate gene by qRT-PCR shows that:the candidate gene was expressed in multiple tissues, but the expression of plant panicle was at higher levels. And the expression of the plant panicle were much more differences between 307R and Nipponbare. It reduced with the development of the panicle in Nipponbare. On the contrary it increased gradually in the 307R, and the expression of third stage in panicle reached the highest level. These results are consistent with the gene as a candidate gene expectations.(7) The subcellular localization analysis shows that GLW2 protein expression was in the cell nucleus by constructing PA7-GLW2-YFP carrier transformed in rice protoplasts instantaneously, and it consistent with its biochemical function as a transcription factor.