| Pennisetum purpureum belongs to Pennisetum Rich genus in the family Poaceae.It is widely grown in subtropical and tropical regions as one of the most economically important cereal crops,with high herbage mass,extensive cultivation and long-utilized period.Despite its importance,limited genomic data are available for P.purpureum,which restricts genetic and breeding studies in this species.P.purpureum cv.Purple has purple leaves with high biomass,which is rare in forage grasses.Studies indicated that the synthesis and accumulation of anthocyanins were responsible for the purple leaves,and anthocyanins had positive effects on plants and humans.To enrich the genetic information of Pennisetum,mine genes related to leaf color and develop numerous EST-SSR markers,RNA-Seq were performed between purple-leaved cultivar ‘Purple’ and green-leaved cultivar ‘Mott’.Combined transcriptome and metabolome analysis,the leaf color-related genes were further explored,which provided information for further study on leaf color formation and related genes of monocotyledonous plants.The results of the study are as follows:(1)De novo transcriptome analysis and molecular marker development.In the present study,the transcriptome of P.purpureum was assembled de novo and used to characterize two important P.purpureum cultivars: P.purpureum Schumab cv.Purple and P.purpureum cv.Mott.Based on 6 leaf samples,we obtained a total of 50,756,372high-quality reads.Using Trinity,284,875 de novo assembled transcripts were obtained with an average length of 781 bp and an N50 value of 1,354 bp.Additionally,these transcripts were clustered into 197,466 unigenes with a mean length of 586 bp and an N50 value of 833 bp.Based on functional annotation,a total of 197,466 unigenes were annotated,of which 103,454 were successfully annotated in at least one database and12,195 were annotated in all databases.Among the annotated unigenes,72,485(36.7%),74,148(37.54%),25,683(13%),25,683(13%),54,026(27.35%),55,245(27.97%),28,665(14.51%),56,999(28.86%)had hits in the Nr,Nt,KO,KEGG,Swiss-Prot,Pfam,KOG and GO database,respectively.Plenty of EST-SSR loci were RNA-Seq,and we developed EST-SSR primers by transcriptomic data,and verified them by Pennisetum materials.A total of 21,213 EST-SSRs were identified in 18,587(9.41%)unigenes and these repeats ranged in lengths from 1 to 6 bp.50 of these EST-SSR primer pairs were randomly selected for validation,and 20(40.00%)of them correctly amplified PCR products with rich polymorphisms.For the obtained 128 alleles,121(95.53%)of them were polymorphic,and the numbers of PCR products ranged from four to ten with an average of 6.4.The average polymorphism information content,Nei’s gene diversity and Shannon’s information index of diversity of the EST-SSRs were 0.3347,0.3689,and0.5405,respectively.The genetic diversity analysis of 17 Pennisetum materials showed that they can devided into three clusters,but species can not be devided significantly.(2)The mechanism of anthocyanin accumulation in P.purpureum.based on transcriptome.Scanning by P<0.05,we found 9,074(4,329 up-and 4,745 down-)and10,028(5,548 up-and 4,480 down-)different expressed genes(DEGs)by comparing green leaves with purple leaves and light purple leaves,respectively.In the two comparisons,4,092 and 4,324 DEGs can be annotated in GO database,and 843 and 700 DEGs are enriched in 118 KEGG pathways.A total of 176 genes were found in flavonoid pathway,anthocyanin biosynthesis pathway and flavonoid and flavonol biosynthesis pathway in P.purpureum.Among them,134 genes were annotated into flavonoid biosynthesis pathway,of which 133 were differentially expressed genes.The expression level of 16 genes were significantly different,including upstream genes(CHS,DFR,etc.)and downstream genes(ANR,etc.).These DEGs were annotated as CHS,CYP73 A,CCo AOMT,F3 H,DFR,HCT,ANR,CYP98 A,ANS,F3’H and ANS.To prove the reliability of the transcriptome sequencing data,four DEGs and two differentially expressed factors were verified by q RT-PCR.The results showed that the expression of six genes in quantitative PCR was consistent with the results of transcriptome.(3)The mechanism of anthocyanin accumulation in P.purpureum.based on metabolites.To study the formation mechanism of purple color in P.purpureum,transcriptome and metabolome were performed between purple-leaved cultivar ‘Purple’and green-leaved cultivar ‘Mott’.Metabolites,including luteolin,kaempferol,quercetin,malvidin,apigenin,pelargonidin and cyanidin presented in different quantities when comparing cultivars ‘Purple’ and ‘Mott’.Malvidin probably plays a dominant role in making the color purple in P.purpureum considering its high content and the high connection with color expression.The transcription levels of CHS,CYP73 A,CCo AOMT,F3 H,ANR,DFR and ANS were significantly different between ‘Purple’ and ‘Mott’.These genes were selected as candidate genes possibly involved in purple color formation and accumulation.Moreover,47 differentially expressed transcription factors(TFs)were found in the three differently-colored leaf samples,including 24 MYB and 23 b HLH.Compared with ‘Mott’,15 up-regulated and 12 down-regulated TFs were found.Furthermore,55 SNPs in 11 DEGs and 231 SNPs in 41 differentially expressed TFs were found between‘Purple’ and ‘Mott’,suggesting important roles of SNPs in regulation of gene expression. |
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