Study On Molecular Mechanisms Of Cytoplasmic Male Sterile In Alfalfa (Medicago Sativa L.)

Alfalfa(Medicago sativa L.)is a superior leguminous herbage.Cytoplasmic male sterility(CMS),a common biological phenomenon in higher plants,is often used in cross breeding of crops.With the discovery of cytoplasmic male sterility lines and maintainer lines of alfalfa,the research on its mechanism is deepening.Transcriptome and small RNA sequencing technology was used in this study to the molecular mechanism of cytoplasmic male sterile lines in alfalfa.The results revealed the molecular mechanism of alfalfa male sterile lines,and provided a theoretical basis for cross breeding of alfalfa male sterile lines.The main research results are as follows:1?A total of 54.43 G clean reads were obtained by transcriptome sequencing.High quality reads were assembled into 95 679 Unigenes,with an average length of 768 bp.The base numbers with a mass value(Q)greater than or equal to 30 accounted for more than 90.87% of the total base numbers.The anthers of sterile lines MSGN1 A and maintainer lines MSGN1 B of alfalfa were analyzed for gene expression,and the differentially expressed genes(DEGs)were screened out.Compared with maintainer line MSGN1 B,sterile line MSGN1 A had a total of 187 differentially expressed genes,including 169 down-regulated genes and 18 up-regulated genes.The GO and COG functional classification of 187 DEGs revealed that differentially expressed genes with catalytic activity,metabolic process,signal transduction mechanism,carbohydrate transport and metabolism,cell process,synthesis and other functions may be closely related to alfalfa cytoplasmic male sterility.KEGG functional classification and enrichment analysis of 187 DEGs showed that the main metabolic pathways related to cytoplasmic male sterility of alfalfa included "carbohydrate metabolism" pathway,"pentose and glucuronic acid conversion" pathway and "oxidative phosphorylation" pathway.Ten differentially expressed genes were randomly selected and the expression pattern was verified by real-time quantitative PCR.The results showed that the realtime quantitative PCR analysis results of 9 DEGs were consistent with the RNA-seq sequencing results.2?Through identification,1 478 miRNAs were detected in this study,including 1 351 known miRNAs and 127 novel miRNAs.Of these,1 351 known miRNAs belonging to 136 miRNA families.|log2(FC)|?1;FDR?0.01 was used as the screening criteria to detect the differentially expressed miRNAs.A total of 130 differentially expressed microRNAs were screened,including 90 up-regulated miRNAs and 40 down-regulated microRNAs.GO annotation results of differentially expressed microRNA target genes show that microRNA target genes are mainly involved in the following functions: “metabolic process”,“signal biological process” and “catalytic activity”.KEGG pathway analysis of differentially expressed miRNA target genes results show that the major metabolic pathways including “amino acid biosynthesis”,“carbon metabolism”,“plant hormone signaling”,“oxidative phosphorylation”,“starch and sucrose metabolism” etc.Among the differentially expressed miRNAs,miR159,miR172,miR396,miR169 and other microRNA target gene functions may lead to pollen abortion.Quantitative PCR was performed on 8 randomly selected differentially expressed microRNA,and the results of 6 microRNAs were consistent with the expression patterns of small RNA sequencing.