Mapping,Cloning And Function Analysis Of The Restorer Gene Of CMS In Cotton

Cytoplasmic male sterility（CMS）is a trait caused by abnormal mitochondrial genes,which is very common in higher plants.CMS line is an extremely important genetic resource in the utilization of heterosis,and was widely used in cross breeding.Cotton is an important fiber crop in China.With the decline of cotton planting area in China,improving cotton yield is a major goal of cotton breeding.Cotton has obvious heterosis,and the heterosis of yield and quality is widely used in cotton breeding.CMS lines have great advantages in cotton breeding,which can minimize the selfing rate in hybrid breeding,save more time and effort than artificial emasculation and chemical methods.Compared with nuclear male sterility,CMS lines also have certain advantages in the self-reproduction.Therefore,the utilization value of CMS lines in cotton is greater.However,the application of CMS in cotton production is relatively late,and the poor restoring ability of restorer lines is a barrier to the screening of strong heterosis combination.Restorer line transferring through backcrossing is an effective way to screen more prominent heterosis.At present,the restorer gene of CMS in cotton has not been cloned.In order to improve the efficiency of restorer line transferring and to realize the molecular application of restorer gene,it is important to map and clone the restorer gene.In this study,bulked sequencing and genetic mapping were used to locate the restorer gene in 7R13,the restorer line of a cytoplasmic male sterile line 6001A.Through amplicon sequencing and third-generation sequencing,the candidate gene of the restorer gene was cloned,and verified through the callus restoration experiments.Below are the main results:（1）Phenotypic identification.The anther abortion of 6001A was happened at the stage of meiosis,corresponding to the bud length of 5-6 mm;the restorer line 7R13 had a strong restoring effect on the fertility of 6001A.The phenotypic segregation pattern of fertility restoration in F₂ population proved that the fertility restoration was controlled by a single gene,with complete dominance effect.（2）Gene mapping.By s BSA-seq,the Rf gene was initially located in the region of D05:3298333-48126864,an interval of about 15.14 Mb.Two SSR markers,Gh＿4740 and NAU3938,were found in the vicinity of the Rf gene,which were obtained by population genotype analysis and the corresponding physical interval was 2.66 Mb（D05:44749577-47409880）.（3）Evolutionary analysis of region PPR cluster.The mapping region contains a large PPR cluster,which is composed of 20 RFL-PPR homologous sequences.Through genome-wide PPR gene family analysis,it is found that the PPR cluster in the D05 interval has the characteristics of more members,high density,and high homology compared with other chromosomal regions.A total of 54 sequences that were homologous to the region PPR were identified in the whole genome.It was inferred that the PPRs on A04 and D05 were closer to the ancestors of Gossypium,and the importance of D05 homologous PPR cluster in the evolution of cotton was found.The formation of Rf may be the result of site-specific selection,and it is likely to retain the sequence similarity of D05 homologous PPR cluster.The similarity of D05 homologous PPR cluster is higher and its members are more abundant,such characteristics make the D05 cluster have better evolutionary plasticity,and can respond to mitochondrial gene mutation more actively and quickly.（4）Sequence cloning and marker development.Preliminary cloning of regional PPR genes showed that there might be a large number of variations in the region PPR cluster where the Rf located,and the number of PPR genes contained was unknown.TM-1reference genome sequence could not accurately guide the cloning and quantitative analysis of region PPR of restorer lines in this study.Based on the cloned sequence information,two molecular markers were developed,which could be used to test the purity of restorer line 7R13 in production.（5）Homocap-seq was designed to clone the restorer gene.In order to clone restorer genes in a large number of homologous RFL-PPR,Homocap-seq scheme was designed,and a complete set of scripts for amplicon analysis was developed,which has high practicability.By analyzing the restorer line specific amplicons,it was found that there were great differences of the mapping region between the restorer line and the reference sequence.Three PPR genes were cloned according to the high-depth restorer line specific amplicons,and then determined by expression screening,resulting in that two PPR genes were candidates for Rf.Annotated all the PPRs in the Rf gene region through three-generation sequencing,and found that the PPR clusters in the restorer line interval are larger than common upland cotton,and through PCR cloning,the accurate sequences of all the complete and pre-terminated PPRs in the mapping region were obtained.Calculating the relative expression level of PPRs in the Rf region verified the results of amplicon analysis.The analysis of tissue expression patterns showed that n-PPR-1 and n-PPR-2 were specific genes in early anther development.Evolutionary analysis showed that n-PPR-1 might be a new in situ replication product of n-PPR-5 under CMS pressure.Finally,based on the stress sensitive of CMS effect,a rapid verification system of callus was established.Through a transferred CMS genetic transformation receptor,it was verified that n-PPR-1,but not n-PPR-2,could inhibit the salt stress response of the callus of YZ1A,this result proved that n-PPR-1 has the function of restoring CMS-related phenotypes.