Cloning And Molecular Evolution Of ObSH3 Controlling Seed Shattering In African Wild Rice(Oryza Barthii A.Chev.)

Crop domestication is a process of reshaping wild species to be adapted for the cultivation environment and to meet human needs.Several morphological characteristics,such as plant architecture,seed size and dispersal,had been changed during domestication.The key step in the evolution during domestication of cereal crops is the elimination of seed dispersal.Isolation and identification of key domesticated genes of seed dispersal and the analysis of their molecular evolution could provide new insights into the crop origin and evolution.In this study,we identificated a key gene of seed shattering during African rice domestication by the method of map-based cloning,main results for this study are as following:1.ObSH3(Oryza barthii Seed Shattering 3)was mapped on the long arm of chromosome 3 with the population derived from the cross between W1411(Oryza barthii A.Chev.)and IRGC104165(Oryza glaberrima Steud.).ObSH3 was finally mapped between the molecular markers SNP29 and SNP31 wtih the secondary segregation population.In the fine mapping region,the genomic interval of IRGC104165 was 17-kb,however,the genomic sequence of the W1411 was 63-kb with a 45.5-kb insertion compared to IRGC104165,which contained six predicted genes(ORF1-ORF6).The quantitative RT-qPCR analysis showed that only ORF3 was highly expressed in the region of abscission layer,so we designated it as a candidate gene for ObSH3.2.We knocked out the ORF3 gene of W1411 using the CRISPR-Cas9 genome editing system and three T1 transgenic plants with homozygous mutations including 1-bp insertion,5-bp deletion and 19-bp deletion exhibited loss of seed shattering.These results indicated that the ORF3 was the ObSH3 gene.Moreover,the consequence of the transgenic lines of the complementation vector,overexpression vector and RNAi vector further confirmed that the ORF3 was the ObSH3 gene.3.We found that ObSH3 was mainly expressed at the abscission layer of seed,leaf,and stem through RT-qPCR analysis.The GUS signal correlated well with the RT-qPCR results.mRNA in situ hybridization results corroborated that ObSH3 was expressed at the abscission layer of spikelet pedicel,the expression pattern of ObSH3 was consistent with its role of controlling abscission layer cellular development.4.ObSH3 is a YABBY transcript factor,the sub-cellular localization results of full-length ObSH3 with GFP fusion protein showed that the GFP signal was detected in the nucleus,which was in accordance with the prediction that ObSH3 was a transcription factor.Amino acid sequence analysis showed that zinc finger domain and YABBY domain of ObSH3 orthologs were highly conserved in monocots.5.The genetype and geographical distribution of SH3 and SH4/GL4(another gene controls seed shattering on chromosome 4)in 91 O.glaberrima landraces were investicated.It was found that 11 landraces only carried the SH3 deletion mutation,51 landraces only carried the SH4 mutation,and the remaining 29 accessions carried both SH3 deletion mutation and SH4 mutation.The 11 accessions only carrying SH3 deletion mutation were mainly distributed in the country of Guinea,Sierra Leone and Liberia of west Africa,suggesting that this area could be the origin center of the SH3 deletion mutation.Identification of ObSH3 not only sheds light on the molecular mechanism underlying abscission layer development but also provides new insights into the African rice domestication.