Fine Mapping And Functional Analysis Of Candidate Gene Rf2 In Cotton CMS-D8

Cytoplasmic male sterility（CMS）is a maternally inherited inability to produce functional pollen that derives from the mitochondrial and nucleus chimeric gene interactions in higher plants.Cotton is an important economic crop,grown worldwide,and has obvious heterosis in yield components.The CMS system,seed production,based on cotton sterile line,maintainer line and restorer line is an ideal tool for utilizing cotton heterosis.At present,there are a variety of sterile cytoplasmic male sterile lines in cotton that have achieved three-lines matching.The G.Trilobum（D8）cytoplasmic male sterile（CMS-D8）lines has stable inheritance,complete abortion,and is easy to restore,and it is gametophytic sterility,offsprings of hybrid are fertile.For the few reports on the research of the restorer gene Rf2.So,the fine mapping and cloning of the restorer gene Rf2 is of great significance to broaden the source of restorer lines,improve the efficiency of restorer line transfer and create new restorer lines.In this study,the genome of the CMS-D8 restorer line was used as the reference genome for Rf2 fine mapping,integration with transcriptome analysis determined the potential candidate genes,and then the candidate genes were initially verified by VIGS.The main results of this study are:（1）CMS-D8 restorer line（D8R）high-quality genome assembly.Pacbio Hi Fi sequencing generated 96 Gb of high-quality Pacbio Hi Fi reads data in this study.In addition,a Hi-C library was constructed,yielded 315.77 Gb of clean data.The genome of the CMS-D8 restorer line was initially assembled by Pacbio Hi Fi,assisted by Hi-C assembly.The genome size was 2.38 Gb,the N50 was 98.3 Mb,and a total of 77,674protein-coding genes were annotated in the genome.BUSCO software was used to evaluate the assembled CMS-D8 restorer line genome and annotations,and the complete alignment of conserved genes was 99.5%and 99.4%,respectively.The D05chromosome where the CMS-D8 restorer gene Rf2 was located and the Ghir＿D05chromosome of the TM＿1 genome were analyzed,and it was found that there were abundant variations between the two chromosomes,and the genetic backgrounds were quite different.Further analysis of chromosome D05 of D8R and chromosome 9 of 13wild cottons showed that D05 of D8R had good collinearity with chromosome 9 of G.Trilobum.（2）The Rf2 fine mapping.A BC₁F₁ population of 5293 individual plants was constructed.In accordance with the previous preliminary mapping,developed new In Del markers which were utilized to further narrow down the candidate interval of Rf2.The fine mapping revealed a candidate interval of 268 kb on chromosome D05 between55453752-55722097.（3）RNA-seq analysis to identify genes involved in fertility restoration.A total of1721 genes exhibited significant differential expression in the anthers of D8R,D8A,and D8B lines.In particular,three anthers development encoding genes such as GhD8R＿D05G3745,GhD8R＿D05G4197,and GhD8R＿A04G0023 had shown up-regulation in the restorer line.While GhD8R＿D05G4197 and GhD8R＿A04G0023related to the tapetum development,showed no expression in the anthers of the sterile line.In the candidate interval of Rf2,q RT-PCR verified the expression patterns of GhD8R＿D05G3836 and GhD8R＿D05G3837 were significantly higher in the flower buds of the restorer line compared to the maintainer and sterile lines.（4）Functional verification of candidate genes.According to the q RT-PCR analysis of the expression patterns of GhD8R＿D05G3836 and GhD8R＿D05G3837 in the three lines,the GhD8R＿D05G3837（GhD8R＿PPR2）gene was selected for VIGS verification.Using CMS-D8R as the experimental material,the VIGS experiment preliminary confirmed a significant decrease in the expression of the GhD8R＿D05G3837 and would lead to inactivity of pollen benzidine staining.It was preliminarily verified that the gene was related to the anther development and pollination,but the fertility restoration function of the gene needs to be further verified.Yeast two-hybrid screening library point-to-point verification that the protein interacting with GhD8R＿PPR2 is the ATP-dependent RNA helicase,GhD8R＿DBP6.