The Research Of Cross-incompatibility On RNA-seq And Development Of SSR Molecular Markers Between Herbaceous Peony And Tree Peony

Distant hybridization is an important and main way to improve ornamental value of plants and cultivate new horticultural varieties.The cross incompatibility between tree peony and herbaceous peony is still a major obstacle to distant hybridization.In recent years,some related studies involved the distant hybridization incompatibility of tree peony and herbaceous peony,the key reason of that is the obstacle before fertilization.In the previous studies of the research group,the specific situation and the critical time of Paeonia suffruticosa and Paeonia lactiflora before fertilization were identified.In this study,based on the previous work,the key differential genes of cross incompatibility were excavated by transcript sequencing technique,and Paeonia suffruticosa and Paeonia lactiflora were further combined with each other.According to the differential proteomics data of the non-affinity column head,the quantitative analysis was carried out.The results of the study will reveal the molecular mechanism of the non-affinity and the barrier in the distant hybridization of Paeonia suffruticosa and Paeonia lactiflora,and provide the basis for further study of the screening and expression of the non-affinity and important genes in the distant hybridization,so as to provide a theoretical basis for overcoming the distant hybridization disorder and promoting the innovation of the peony germplasm.1.RNA-Seq AnalysisBy using RNA-Seq technique,the changes of the expression of stigma and the expression of stigma in the non-affinity and non-affinity process of the tree peony and herbaceous peony were analyzed,and the total of 1,178.91 Gb Clean Reads sequences were obtained according to the results of the transcriptome sequencing,and the content of GC% was 41.17%.Clean Reads were assembled using Trinity software,then clustered and redundant using TGICL software to obtain 131,265 Unigene sequences with a total length of 98,693,005.Gene structure analysis,as well as CDS prediction and SNP analysis,functional annotation of genes by NR,NT,Swiss-prot,KEGG,KOG and GO databases,a total of 131,265 Unigene annotation results were obtained,which were annotated to the NR database.35,181 were annotated to the NT database of the 45,943 articles.33,186 were annotated to the Swissprot database.33,828 were annotated to the Pfam database,and 34,854 were annotated to the KEGG database,and were annotated to the KOG database.24,594 were annotated into the GO database,most of which were classified into 44 functional entries,25 protein families,and 256 KEGG pathways..2.Differentially Expressed Genes Analysis and Verification5343 differentially expressed genes related to the cross-pollination of Paeonia suffruticosa and Paeonia lactiflora were obtained.Compared with the compatible stigma,the expression of specific genes was down-regulated in the incompatible stigma.These differentially expressed genes are involved in metabolic processes,protein metabolism,transport,transcriptional regulation,redox,defense response and signal transduction.Incompatibility combinations respond to stress caused by cross-incompatibility by increasing the expression of transcription factors and kinases associated with redox and stress response.The data of transcript group combined with Paeonia suffruticosa and Paeonia lactiflora was not parent after distant cross pollination and the RNA-Seq result is verified by the q RT-PCR technology,And the corresponding protein is subjected to PPI analysis.The expression of the differentially expressed genes in each pathway showed that the energy synthesis required for the growth of the pollen tube was blocked,and the signal pathway and the metabolic pathway associated with the polar growth of the pollen tube were blocked.wherein the enolase,the heat shock protein,the calmodulin,the adenoid acid transport enzyme The body exhibits a down-regulation expression.3.SSR Development of Molecular MarkersBased on the data of tree peony transcripts,SSR molecular markers were developed to provide molecular markers for genetic diversity of peony.In this study,Misa software was used to analyze the SSR information and develop the primers.Primer polymorphism analysis was carried out by q RT-PCR amplification.The results showed that a total of 44,181 SSR loci were obtained from 131,265 Unigene obtained by sequencing in Paeonia peony transcript group.The frequency and average distribution distance of SSR were 29.19% and 2.23 kb.Respectively,which were distributed in 38316 Unigene.Single The total number of SSR was 75.19%,15.02%and 6.88%.According to the SSR information sequence in the transcription group,60 pairs of primers are randomly synthesized to carry out polymorphism amplification in16 kinds of peony varieties,15 pairs of primers show the polymorphism.The number of alleles of each site is 4 to 13,the average number of alleles is 8.13,the observed heterozygosity Ho.The expected heterozygosity He.The average values of Shannon’s diversity index I and PIC were 0.7578,0.7903,1.7186 and 0.7308.Further analysis showed that the data of tree peony transcripts could provide abundant SSR loci and provide more abundant and reliable marker selection for genetic diversity analysis and molecular marker-assisted selection of peony.