Cloning And Function Identification Of GL4 Controlling Grain Length And Seed Shattering In African Wild Rice(Oryza Barthii)

It is critical for agricultural scientists to increase crop productivity to meet the rapidly growing demands of food in Africa and the world.Grain size is a major determinant of 1,000-grain weight which is composed of grain length,width,thickness and grain filling in rice and many other cereals.Among all the determinants of the rice productivity,the heritability of 1,000-grain weight is the highest O.barthii,which is the wild progenitor of African cultivated rice(O.glaberrima),is an important germplasm for improving African cultivated rice and Asian cultivated rice(O.sativa).The seed of O.barthii is typically longer and larger than that of O.glaberrima.Therefore,revealing the molecular genetic basis of selecting small seed during the domestication of O.glaberrima will contribute to improving the grain yield of rice.In this study,W1411 and IRGC102305 were used to develop introgression line.One introgression line(GIL25)displays longer and larger grain.A locus controlling grain length on chromosome 4(Grain Length 4;GL4)was detected through an F2 population derived from a cross between GIL25 and IRGC102305.The main results are as fellows:1.Compared with IRGC1023 05,the grain length of GIL25 was increased by 13.6%,and the 1,000-grain weightwas increased 16.8%,respectively,ultimately resulting in grain yield increased by 14.3%.Larger grain of GIL25 is caused by cell elongation along the longitudinal axis in glumes.Besides,it was much more easier for the seeds form GIL25 to fall off than IRGC102305.An F2 population containing 186 individuals derived from a cross between GIL25 and IRGC102305 was developed.A locus controlling grain length was detected on the chromosome 4 named GL4.GL4 was then narrowed down to a 5.9-kb region between markers M3 and M4 by another 6500 plants.there was only one annotated gene ORGLA04G0254300 in the mapping region.The gene ORGLA04G0254300 from GIL25 was used to construct complementary vector.All the 12 independent transgenic lines showed longer and larger grains than the controls.It demonstrats that ORGLA04G0254300 was the GL4 gene.2.Five mutations in the GL4 coding sequences between GIL25 and IRGC 102305 were found.These includes four SNPs and one indel.An association test by 67 O.glaberrima and 16 O.barthii with grain length and nucleotide polymorphism revealed that the strongest signal was present at the SNP2 site.A site-specific mutation vector at SNP2 was constructed and introduced to IRGC 102305.The function of SNP2 on grain size and seed shattering was verified by transgenic plant in African rice.3.Based on resequencing data of 93 O.glaberrima and 94 O.barthii,African cultivated rice can be separated into two categories nucleotide diversity analysis showed that African cultivated rice carrying T at SNP2 was strongly selected.4.GL4 protein has a high identity with SH4/SHA1,its orthologous gene in Asian wild rice.By comparing near-isogenic lines NIL-GL4or,NIL-GL40S and NIL.GL40g,we found that Oggl4 reducedseed shattering and grain length at the same time,but OsGL4 only reduced seed shattering but could not affect grain length.These results show that selection of Oggl4 was an imperfect solution.In conclusion,the selection on a SNP in GL4 resulted in the decrease of grain length and the loss of seed shattering during domestication of African rice.The cloning of GL4 provides new insights into the diverse domestication practices in African rice,and also paves the way for enhancing rice yield.