| Pepper(Capsicum annuum L.)is a very important economic crop around the world.Utilization of male sterile lines can reduce the cost of pepper seed production and improve the seed purity.At present,for pepper male sterilities,researches on cytoplasmic male sterility(CMS)are relatively deep.Yet although two candidates of the CMS gene have been cloned,the restorer-of-fertility(Rf)gene has not been found,reports on fertility related genes are relatively few,the molecular mechanism of pepper CMS is not yet clear.In present study,the RNA-seq was utilized to analyze the anther transcriptomes of a pepper cytoplasmic male sterile line 121A and the corresponding near-isogenic restorer line 121C.85,144 unigenes were assembled de novo,among which 4,326 unigenes were found with higher expression in 121A,while 7,061 unigenes were found with higher expression in 121C.The reliabilities of the results were verified by semiquantitative RT-PCR and quantitative real-time PCR analyses of 20 unigenes which were chosen according to the expression difference.60 unigenes were selected as male fertility related candidate genes for further analyses according to the expression difference and gene annotation.Reanalysis of the transcriptome data shows that 30,643 genes(86.72%)of the Zunla-1 genome were mapped.738 genes exhibit higher expression in 121 A,while 1,426 genes exhibit higher expression in 121C.Comparative analyses between the de novo assembled transcriptome and the mapping result reveal that the genes with higher expression in 121C are significantly more than that in 121 A,and the genes with the highest expressions difference show higher expressions in 121C;the GO items with the largest numbers of differential expression genes are "Cell" and "Cellular process",and the metabolic processes with the largest numbers of differently expressed genes are "Starch and sucrose metabolism" of the carbohydrate metabolism process and "Oxidative phosphorylation" of the energy metabolism process.The expressions of 43 top scored genes from differential expression analysis of the de novo assembled transcriptome were verified by semiquantitative RT-PCR.Among the 17 genes that specifically expressed in the restorer line,8 were chosen for expression profile analysis during anther development.The result shows that all the genes are "late" genes.Combining the result of semiquantitative RT-PCR analysis,28 genes were chosen for VIGS analysis among the male fertility related candidate genes.Function analysis shows that one gene named CaAMS is a fertility related gene.Silencing of the gene can lead part of the anthers to abortive.Sequence analysis shows that there are two alternative splicing forms in CaAMS,while the two splice variants exhibit no tissue specificity.Moreover,no sequence difference was found in the two splice variants and the promoters between 121A and 121C.Subcellular localization analysis shows CaAMS is a nuclear locating protein.Expression analysis shows that the expression of CaAMS in 121C is obviously higher than that in 121A,and the gene expresses in both leaf and flower organs,while it prefers to express in anther of early development.The two splicing variants exhibit different expression profiles.The stage with highest expression level of CaAMS-1 in 121C is the early-uninucleate stage;while for CaAMS-2,it is the tetrad stage.This research analyzed pepper anther transcriptome comprehensively,screened and analyzed the fertility related candidate genes,characterized the sequence and expression profile of the fertility related CaAMS gene,provides important data support and reference information for future studies on pepper male fertility regulation network and creation of new male sterile lines. |
