Screening And Functional Analysis Of Candidate Genes For CMS-D2 Fertility-restoring Gene Rf₁ In Upland Cotton

Cotton is an important fibre and oil crop.Heterosis is an important way exploited to increase yield in cotton production.In production practices,the ideal way of cotton hybrid seed production is three-line hybrid.The key to cultivating strong dominant three-line hybrids is to create excellent restorer lines.Therefore,it is very important to carry out basic research on restoration genes.CMS-D2 is one of the major cytoplasmic male sterility in cotton.However,the restorer gene Rf₁ has not been isolated and cloned yet,and the global gene expression patterns of CMS-D2 and its interaction with the restorer gene Rf₁ remain unclear.In this study,the full-length transcript sequencing was performed in anthers of the CMS-D2 restorer line using Pac Bio single-molecule real-time sequencing technology,combined Illumina RNA-seq data of"three line"materials and hybrid line（F₁）,and restorer gene Rf₁ is located on Chr＿D05 in the previous study of the project.Rf₁candidate genes were further screened for structural analysis and preliminary functional verification.The main results were as follows:1.107,066 isoforms from 44,338 loci were obtained,including 10,086 novel isoforms of novel genes and 66,419 new isoforms of known genes.A total of 56,572alternative splicing（AS）events,1,146 lnc RNAs and 61 fusion transcripts were identified,and 10,466 genes with selective polyadenylation（APA）loci,and 60,995novel isoforms with predicted open reading frames（ORFs）were further identified.In addition,there are 819 FLNC reads that have not been mapped to the TM-reference genome.These findings provide a basis for genome annotation and transcriptome research in upland cotton.2.Using transcriptome data from A,B and R,the following three comparisons of gene expression levels were performed:A vs B,R vs B and R vs A.The results showed that a total of 1295,1721 and 837 differentially expressed genes（DEGs）were identified respectively.GO and KEGG enrichment analysis were performed for the identified DEDs.The GO categories‘binding’had the highest enrichment ratios in all comparison groups.In comparison groups A vs B,and R vs B,the KEGG pathways of‘Cutin,suberine and wax biosynthesis’were enriched significantly.The KEGG pathway of‘Circadian rhythm-plant’was enriched significantly in R vs A comparison group.3.The DEGs were specifically expressed in R but hardly expressed in A and B,including FLNC reads that were not unmapped to TM-1 reference genome,on Chr＿D05 and its homologous Chr＿A05,and on scaffolds.6 candidate genes that might be restoration genes verified by q RT-PCR.The results showed that the relative expression of Gh772CCS and Gh61CCS in R was significantly higher than that of A and B.Further research found that the relative expression of these two genes in F₁ was significantly higher than A,and the relative expression of R was approximately twice that of F₁.Analysis of the expression levels of the two genes in three-line of anther and different tissues of the restorer line.The results showed that the two genes were specifically highly expressed in different tissues of the restorer line,especially in the anther tissue,indicating that these two candidate genes may be Rf₁.4.Using RACE technology,the full-length transcripts of Gh CCS772 and Gh CCS61 were obtained,and then preliminary predictive analysis was performed.The VIGS technology was used to silence the two genes.Results showed that the expression level of target gene was decreased in all positive strains.Except for a few strains with high silencing efficiency,other strains generally had lower silencing efficiency.It shows that the silencing efficiency of the same gene in different positive strains is different.Pollen viability testing found that most of the positive strains had reduced pollen viability,but overall was insignificant.Anther fertility observed abnormal development of some anthers.It shows that these two genes may be potential fertility restoration genes of CMS-D2 or genes related to anther development.