基于牡丹涝害胁迫的转录组分析及SSR引物开发

Transcriptomic analysis and simple sequence repeat markers development of Paeonia suffruticosa L.in responses to waterlogging stress

涝害胁迫是限制牡丹种植、生长和高产的最主要的非生物胁迫之一。为了阐明牡丹在涝害胁迫下的作用机制,研究进行了转录组学分析,并开发了SSR标记。利用高通量测序对6个由涝害处理的幼苗和对照的mRNA构建的cDNA文库进行测序。通过组装总共获得73925个基因,创建了牡丹的初始参考转录组数据库。其中780个被鉴定为涝害早期反应基因,其中155个基因上调,625个基因下调。功能分析表明,参与转录因子信号调节、DNA复制、核糖体和嘧啶代谢的基因表达的改变可能在牡丹对涝害胁迫的反应中发挥重要作用。基于这些高通量转录组测序数据,挖掘出5204个SSR标记。在这些标记中,二核苷酸重复占58.13%,三核苷酸重复占27.11%,四核苷酸重复占7.24%。在牡丹耐涝性状靶基因相关的功能注释基因中,发现了110对SSR标记引物,筛选出45对引物,其中12对能扩增出清晰的条带,多态性SSR标记引物占引物总数的26.67%。研究结果不仅有助于阐明牡丹的涝害反应机制,同时也为耐涝牡丹品种的分子标记辅助选择育种提供了宝贵的基因组资源和科学依据。

Waterlogging stress is one of the most severe abiotic stress that limits cultivational expansion,fast growth,and high yield of P.suffruticosa.In order to elucidate the mechanism of P.suffruticosa during waterlogging stress,transcriptomic analysis was performed and SSR markers were developed in this study.Six cDNA libraries constructed from mRNAs of waterlogging-treated seedlings and control were sequenced by using the Illumina sequencing platform.Totally,73925 unigenes were obtained by denovo assembly,creating an initial reference transcriptome dataset of P.suffruticosa.Among them,780 were identified as response genes in early stage of waterlogging including 155 up-regulated genes and 625 down-regulated genes.Functional analysis showed that modifications in the expression of genes involved in signal regulation by transcription factors,DNA replication,ribosome and pyrimidine metabolism might play important roles in responses to waterlogging stress in P.suffruticosa.Based on these high throughput transcriptome sequencing data,5204 SSR markers were excavated.Among these markers,dinucleotide repeats were 58.13%,trinucleotide repeats were 27.11%,and tetranucleotide repeats were 7.24%.110 pairs of SSR marker primers were found in the unigenes of functional annotation related to the target genes of waterlogging stress traits of peony,and 45 pairs of primers were screened,of which 12 pairs could amplify clear target bands,and the polymorphic SSR marker primers accounted for 26.67%of the total primers.This study provides the first large-scale transcriptome annotation of P.suffruticosa which may beneficially elucidate the waterlogging response mechanisms.Besides,it provides a valuable genomic resource and a scientific basis for molecular marker assisted selection breeding in waterlogging-tolerant species of P.suffruticosa.