Fine Mapping And Preliminary Characterization Of A QTL GW5 For Grain Width And Weight In Rice (Oryza Sativa L.)

Rice is the most important staple food crop and a primary food source for about half of the world's population. As farmland is decreasing and the global population increasing, there is an urgent need to secure grain production. The grain shape, including grain length, grain width, length/width ratio and grain thickness, is one of important apperence qualities in rice, which may have an effect on both grain yield and milling quality. The grain shape is a complex quantative trait controlled by multiple genes, and most of the grain-based researches are limited to the use of the primary populations targeted QTL at present. In this study, we have gotten a chromosome segement substitution line with narrow grain, CSSL28, derived from Asominori (wide cultivar)/IR24 (narrow cultivar) with Asominori as the recurrent parent. The mapping population was constructed from the cross between CSSL28 and parental line, Asominori. Traits of grain length, grain width and length/width ratio were used for characterization, genetic anaylsis, primary mapping and high resolution mapping QTL anaylsis. We identied and preliminarily characterized the QTL GW5 underlying grain width, length/width ratio and grain wight, and also investigated the differences of yield components and cell morphological between CSSL28 and Asominori. The main conclusions are as follows:The significant differences existed in grain shape between Asominori and CSSL28 (P<0.01). CSSL28 shows 83.6%and 126.6%in width and length/width ratio of paddy rice, 91.6%and 123.7%in brown rice grain, which were compared with these of Asominori respectively. But, none of other agronomic traits show significant difference, such as plant height, plant and panical shape, etc. For factors influencing rice yeild, there are also no significant differences between the substitution lines and background parent in tiller number, seed setting rate up, but in grain weight per plant, grain weight per panicle and 1,000-grain weight(P<0.01). Cytological analysis showed that the number of external parenchyma cells in rice hull caused the grain width differences between Asominori and CSSL28, and the vascular cross-sectional area in carpopodium of CSSL28 was significantly lower than that of Asominori. Meanwhile, the grain filling rate of Asominori was higher than the CSSL28 in 5-20 days after flowering, and the former has a higher percentage of grains with chalkiness. Moreover, the brown rice rate (P<0.01) and milled rice rate (P<0.05) in Asominori were significantly higher than that in CSSL28.Based on secondary F2 populations derived from the backcross of CSSL28 and Asominori, the QTL GW5 controlling both the grain width and length/width ratio was dissected into a single gene despite the nontraditional Mendelian segregation ratio between the plant with slender grains and wide ones in the F2 population. Meanwhile, the ratios of genotypes of SSR marker and frequency distribution of grain width in BC4F2 indicated that the indica-japonica hybrid partial sterility gene S31(t), being linked with GW5, was the reason of the observed distortion of segregation in the BC4F2 population. For genetic anaylsis, the phenotype of narrow grain in CSSL28 was controlled by a dominant nuclear gene. Then, the GW5 was further mapped between CAPS makers Cw5 and Cw6 on chromosome 5, within a 21kb recombination hotspot region of BAC clone (OJ1097_A12), using the 2,180 segregants showing the wide grain phenotype in BC4F2 population and markers developed in our laboratory. The result of sequencing indicated that the genome of Asominori habored a 1.2kb deletion compared with that in CSSL28. Within targeted region, two opening reading frams(ORFI and ORF3) was predicted in Asominori, but three ones (ORF1, ORF2 and ORF3) in CSSL28. In another words, ORF2 was located in the deleted region. Also, there is no difference exsited in the coding region of ORFI and ORF3. Thus, we teneatively designated the ORF2 as the GW5 gene. Genotyping analyses in 46 rice cultivars and 12 wild rices showed that the deletion prevailed in wide grain rice cultivars and defined a domestication related rice gene.The expression of GW5 in narrow cultivar was confirmed using the RT-PCR analysis. The protein was predicted to contain a nuclear localization signal (NLS) and an arginine rich domain. Transient expression in onion epidermal cells showed the GW5-GFP fusion protein is exclusively localized to nuclear. The yeast two-hybrid showed that the GW5 may physically interact with polyubiquitin and played an important role in the ubiquitin proteasome pathway. However, the 2.5kb fragement covering the ORF2 and the over-expression of ORF2 could not restore the narrow phenotype in Asominori. These results suggensted the ORF2 was incompleted for GW5, and the partial cDNA was isolated using RACE, which was detected with Southern blot.