Cloning, Functional Research And Utilization Of Grain Size Gene GL7 In Rice(Oryza Sativa L.)

Grain size,including grain length, width, thickness, and length/width ratio, is one of the most important agronomic trait in rice. It determines not only the appearance quality of rice, but also the grain yield. The grain size is a kind of quantitative trait controlled by nuclear genes that has a high heritability.In the dissertation, we used a slender indica variety P13, crossed with Nipponbare(NPB), Miyang46(MY46), Ⅱ-32 and T461 X. By the genetic analysis of F1 and F2 populations, we found that there was genetic difference in grain size between P13 and the four varieties. In the F1 generation, the grain length was longer than the medium value of the two parents. Using an F2 population of P13 and NPB, we identified five major QTLs for grain size. Among them, a major QTL for grain length has been found in the region between molecular markers RM5508 and RM234 on chromosome 7. To clone GL7, we generated a near isolation line NIL-GL7 by backcrossing with the Japonica cultivar NPB. Using a BC6F2 population consisting of 20160 plants, we finally narrowed down GL7 to an interval of 20.4 kb between the markers CAPS1 and 210 Q. This locus contained two candidate genes, Os07g0603300 and Os07g0603400. Os07g0603400 encodes an uncharacterized protein, and Os07g0603300 encodes a TON1 RECRUIT MOTIF(TRM)-containing protein that shares 20-22% amino acid sequence identity with the Arabidopsis LONGIFOLIA1 and LONGIFOLIA2 proteins, which regulate longitudinal cell elongation in leaves and siliques. The LONGIFOLIA2(also known as TRM1) has been suggested to be involved in cortical microtubule arrays.To understand the function of GL7, we analyzed the expression patterns of Os07g0603300 and Os07g0603400. It showed that NIL-GL7 had a higher expression level of Os07g0603300 and a lower expression level of Os07g0603400 in young panicles than NPB. By using a RACE assay, we found that there were two full-length cDNAs transcribed from Os07g0603300 in P13, named as GL7-S1 and GL7-S2. To further figure out the genomic structure underlying these two transcripts, we sequenced the DNA interval between the markers CAPS1 and 210 Q. We found a 17.1 kb tandem duplication containing GL7 in NIL-GL7. This copy number variation(CNV) at the GL7 locus well explained the unexpected recombinant events observed in the mapping populations.We then transformed NPB with either Ubiquitin promoter driven GL7-S1(pUbi::GL7-S1P13) or GL7-S2(pUbi::GL7-S2P13) and found ~10% longer grain length in these transgenic plants. Furthermore, we found that transforming pUbi::GL7-S1P13 into an indica variety, Zhefu802, resulted in ~21% increase in grain length. The increase in grain length was well correlated with the expression levels of GL7 in transgenic plants. Over-expression of GL7 promoted the longitudinal cell elongation and the formation of densely and regularly packed starch granules. Further genetic analysis indicated that the tandem duplication of 17.1 kb segment at the GL7 locus leads to not only up-regulation of GL7, but also down-regulation of its nearby negative regulator Os07g0603400, which in turn results in an increase in grain length and the improvement of grain appearance quality.We analyzed the polymorphisms of coding sequences of GL7 and Os07g0603400 and the haplotypes of GS3, GW8, GS5, TGW6, GW2 and GL3.1(qGL3) in 96 accessions. Phylogenetic analysis indicated that the beneficial alleles of GL7, GS3, GW8, TGW6 and GS5 contribute to grain length in these accessions and the variations at GS3 and GL7 are major factors affecting grain length diversity. Based on understanding the interaction of different alleles of the grain length-related genes, it is possible to breed varieties with optimal grain size by pyramiding different beneficial alleles of these genes using marker-assisted selection. Sequence analysis indicates that allelic variants of GL7 and its negative regulator are associated with grain size diversity and that the CNV at the GL7 locus was selected and utilized in breeding. Our work demonstrated that pyramiding beneficial alleles of GL7 and other yieldand quality-related genes will contribute to the breeding of elite rice varieties.