Transcriptome And Small RNA Sequencing Analysis Of Multi-pistil Mutant (mp1) In Alfalfa

Alfalfa(Medicago sativa)is an autotetraploid cross-pollination crop and a perennial forage legume,it is known as the “King of the forages” owing to its high yield,good quality and fine palatability.Alfalfa multi-pistil mutant(mp1)exhibited more than one to two pistils compared with its wild-type plant.Our previous research revealed that mp1 was partial-female-sterile,significantly decreasing the production yield.Thus,we conducted high-throughput sequencing of the samll RNAs and transcriptomes of the alfalfa mutant florets(mp1)and its wild-type florets(WT),to excavate the miRNA and its target genes that may be involved in multi-pistil development,illuminating the molecular mechanism of pistil mutation,boosting the development and utilization of innovative germplasm resources in alfalfa.By sequencing and correlation analysis,we obtained the main results:1)Using the Illumina HiSeqTM2500 platform,we conducted the transcriptomes sequencing analysis of alfalfa mutant florets(mp1)and its wild-type florets(WT).A total of 199,546,986 paired-end clean reads were screened from the WT and mp1 transcriptomes.After de novo assembly,a total of 151,359 unigenes were obtained and then were annotated in NT,KO,Swiss-Prot,PFAM,GO and KOG databases.In the level of P-value adjusted < 0.05,2306 differentially expressed genes were identified in mp1 vs.WT,including 1100 up-regulated unigenes and 1206 down-regulated unigenes.Through the GO function and KEGG enrichment analysis of differentially expressed genes,we found that differentially expressed genes were significantly enriched in the DNA replication and synthesis pathways and cell metabolic pathway.Among them,the up-regulated genes may related to multi-pistil development were mainly enriched in DNA metabolic process,DNA polymerase activity,DNA integration,DNA replication,as well as in response to biotic stress concentration.By the transcription factors analysis of differentially expressed genes,we found that a total of 41 unigenes belonging to 14 different transcription factors' families,among them MADS-box genes(c68049_g1,c60978_g2)may participate in development of multi-pistils.2)We conducted small RNA sequencing analysis of alfalfa mutant florets and its wild-type florets.A total of 1,819,505 and 2,028,422 sRNA(length range of 18-30nt)were obtained from that two libraries respectively,and all sRNA were classified and annotated.Through the miRNA family analysis,a total of 150 known miRNA were identified,which belonged to 72 families.Through the new miRNA prediction,a total of 80 new miRNA were obtained and these new miRNA may be unique in alfalfa,among them,9 new miRNA were mutant-specific miRNA,which may be associated with multi-pistil mutation.By the analysis of miRNA differential expression,110 differentially expressed miRNA were screened out,including 47 up-regulated miRNA and 63 down-regulated miRNA.Through the prediction of miRNA target genes,there are 160 miRNA targeting 2,319 genes,indicating that one miRNA can simultaneously target multiple target genes.Through the analysis of GO function enrichment of genes targeted by differentially expressed miRNA,35 GO terms were obtained and most target genes were significantly enriched in the defense response,response to stress,regulation of cellular process.Through the KEGG enrichment analysis of genes targeted by differentially expressed miRNA,5 KEGG metabolic pathways were significant enriched,incloduing protein export,homologous recombination,flavone and flavonol biosynthesis,spliceosome and SNARE interactions in vesicular transport.3)Through the correlation analysis of small RNA and transcriptome data,a total of 18 differentially expressed miRNAs and its 27 corresponding target genes were identified,as well as the network diagram of regulation relationship between them.The expression patterns between 18 differentially expressed miRNA and corresponding 27 target genes are not completely consistent.Among them,a total of 6 unigenes(c80545_g1 c11129_g1 c80206_g2,c86423_g2,c76129_g6,c110910_g1)may be a key to pistil development,which were significant enriched in DNA replication and catabolic process.And their corresponding miRNAs(mtr-miR393b-3p,mtr-miR5244,mtr-miR156 j,mtr-miR2673a)may be also involved in the formation of multi-pistils.All in all,the development of alfalfa multi-pistil would be more likely regulated by multiple genes and multiple miRNAs at the same time.Up-regulated differentially expressed genes which were significant enriched in DNA replication and synthesis function may provide the required materials for multi-pistil development,while the down-regulated differentially expressed genes which were significant enriched in organic substance metabolism process may delay the pistil metabolism process for the accumulation of substances and signals of multi-pistil development;Differentially expressed genes may also act as transcription factors opening the gene transcription associated with multi-pistil mutation.Meanwhile,these differentially expressed genes would be regulated by the corresponding miRNAs,and through the complex interaction between miRNA and its target genes may lead to the multi-pistil mutation happen in alfalfa.