Comparative Transcriptome Studies On The Dimorphic Style Of Primula Sikkimensis

Primula sikkimensis is a perennial herb of Sect.Sikkimensis Balf.f.in Primula genus,mainly distributed in the Eastern Himalayas.This species is mainly distributed in western Sichuan,northwestern Yunnan and southeastern Tibet in China.Primula sikkimensis is a typical dimorphic style plant with self-incompatibility breeding system,which is an ideal research model species for studying the formation and evolution mechanism of dimorphic style.In this study,a total of 12 individuals（6long-styled morph and 6 short-styled morph）from 6 populations of P.sikkimensis were sampled.High-throughput transcriptome sequencing was performed on the Illumina Nova Seq 6000 platform to compare the transcriptome and expression patterns of genes related to dimorphic styles between the two style types in P.sikkimensis.The aim of this study is to preliminarily explore the molecular mechanism of the formation of the dimorphic style in P.sikkimensis.The main results are as follows:1.RNA-seq sequencing and assemblyRNA-seq sequencing and de novo assembly without reference genome were performed on 12 P.sikkimensis samples.After quality control,a total of 552,982,058 clean reads were obtained,including 73.7Gb of total bases and 92.7%–93.58% of Q30 bases,indicating that the sequencing quality of each sample was high.After splicing and assembling,we got transcriptome data of P.sikkimensis with 254,333 transcripts and 228,285 Unigenes.2.Functional annotation for transcriptomeP.sikkimensis was annotated based on the egg NOG database,and the protein sequences of 40,369 genes were annotated to at least one database,including 23,366 sequences that obtained GO functional annotation,38,300 sequences that obtained KOG database,23,559 sequences that obtained KEGG functional annotation,and1,152 genes that obtained CAZy annotation.In GO annotations,the top annotations are cells,cell parts,and cell processes.After being annotated by KOG,they were divided into 24 functional annotation categories,with the common ones including posttranslational modification,protein turnover,chaperones;signal transduction mechanisms;transcription.The KEGG annotation identified 47 metabolic pathways,and the first three metabolic pathways were carbohydrate metabolism,amino acid metabolism,and signal transduction.3.Identification of differentially expressed genes and gene ontology and pathways mapping of DEGsBased on the transcriptome data of P.sikkimensis,through differential gene analysis,1,528 differentially expressed genes were screened out between pin flower and thrum flower,among them,1338 DEGs were up-regulated and 190 DEG were down-regulated.Among these differential genes,1,102 genes were annotated,including 983 up-regulated genes and 119 down-regulated genes.Through the GO enrichment analysis of differential genes,we found that these genes are mainly involved in developmental growth involved in morphogenesis,cell growth and pollination,etc;Through KEGG enrichment analysis,we found that these genes are mainly involved in Toll and Imd signaling pathway,Neurotrophin signaling pathway,NF-κB signaling pathway and MAPK signaling pathway,etc.4.Expression pattern of S-locus related gene in the dimorphic style of P.sikkimensisThe dimorphic style development of Primula was controlled by the S-locus.We analyzed the expression pattern of three genes related to S-locus: GLO1,GLO2,CYP734A50,and three linkage markers Pv SLL1,Pv SLL2,and Pv Glo in P.sikkimensis.By using the maximum expected algorithm to analyze the expression levels,we found that Pv SLL1,Pv SLL2,and GLO1 were expressed in the long-styled morph and short-styled morph,but the difference between morphs was not significant.Pv Glo gene was not expressed in both long and short morph.CYP734A50 was only weakly expressed in one sample of thrum flower.GLO2 was absent in pin flower and only expressed in thrum flower.The above results indicated that differentially expressed genes associated with long-styled morph and short-styled morph in P.sikkimensis were mainly involved in such pathways as morphological development and growth of pollen tubes.There were differences in expression patterns of six genes of S-locus related to the formation of dimorphic style in long-styled morph and short-styled morph,and the gene GLO2 was short-styled morph specific,which control the style-length dimorphism and promote the rise of the anther position.This study provided some evidences for studying the process and mechanism of the formation of the dimorphic style,and helped to understand the significance of the adaptive evolution of the heterostyly.