Map-based Cloning And Functional Analysis Of A Major QTL For Grain Width And Grain Weight In Rice.

The grain shape, including grain length, grain width and a length/width ratio, is complex trait controlled by QTL, which may have an effect on both grain yield and quality in rice. We chose an indica cultivar, Fengaizhan-1 (small grain), and a japonica cultivar WY3 (large grain) as the parental lines, because they exhibit great variation in grain weight. The mapping population was constructed from the cross between the above-mentioned parental lines. Traits of grain length and grain width were used for primary-mapping, fine-mapping and high-resolution mapping QTL analysis. We identified and characterized a major QTL GW2 underlying grain width and grain weight in rice. The main results are summarized as follows.First, two major QTL, GW2 and GL3 controlling grain width and grain length in rice were mapped primarily on chromosome 2 and 3, respectively.Second, the candidate region of QTL GW2 was narrowed down to a 8.2kb DNA fragment by fine-mapping and high-resolution mapping analysis, and in this region only one reading frame was predicted, which encoded an functionally unknown RING-type protein.Third, to confirm that GW2 corresponds to the QTL we mapped, we produced transgenic plants expressing different levels of GW2 and examined their grain width and grain weight. Transgenic plants carrying the antisense strands of GW2 that had reduced levels of expression showed wider grain and heavier grain, whereas transgenic plants with sense strands of GW2 that had high levels of expression reduced substantially grain width and grain weight. Therefore, we concluded that we have succeed in isolation of a major QTL GW2 underlying grain width in rice.Fourth, RT-PCR analysis and GW2 promoter-GFP transgenic rice analysis showed that GW2 is expressed constitutively in various tissues or organs. Transient expression in onion epidermal cell showed that GFP-GW2 was localized in the cytoplasm.Fifth, active GW2 protein has expressed in E.coli and biochemical analysis showed that GW2 can ubiquitylated other proteins in vitro, and combined the fact with sequencing of GW2, we can concluded that GW2 is a previously unknown RING-type E3 ubiquitin ligase.Sixth, nearly isogenic lines, NIL (GW2) carring target QTL region (1.4 cM) from WY3 was developed by marker-assisted selection (MAS). Compared with FAZ1, both grain width and grain weight of NIL (GW2) had a substantial increase, consequently, grain production per plant also increased prominently, suggesting that GW2 is a useful locus for increasing grain productivity in crop breeding.Seventh, based on our studies, we proposed a following working mechanism that GW2 functions. As a novel RING-type E3 ubiquitin ligase, GW2 is involved in degradation of the ubiquitin/proteasome pathway, and negatively regulate cell division in rice grain hull. Only 1bp deletion in the coding region in WY3 GW2 allele, which create a premature stop codon, suggesting that it is a null allele. WY3 GW2 null allele results in increase cell division (cell numbers) of grain hull, thereby has larger (wider) grain hull. And, larger grain hull promote milk filling rate of grain, thus plant with WY3 allele will produce larger seeds.