蕨类植物芒萁幼孢子体转录组高通量测序及特征分析

De novo Sequencing and Characterization of Juvenile Sporophyte Transcriptome of a Fern,Dicranopteris dichotoma

[目的]采用高通量测序技术Illumina Mi Seq250获得蕨类植物芒萁的孢子体转录组数据,以期为芒萁的生长、发育、代谢调控、微进化机制分析等提供重要的分子信息。[方法]应用生物信息学方法对测序获得的大量单基因簇(Unigene)进行基因功能注释、代谢途径及微卫星分析等。[结果]本研究共获得18 463 296条序列读取片段(reads),总碱基数为4.62Gbp序列信息,经序列组装最终得到63 169个Unigene,平均单条Unigene长度为863 bp,N50为1 587 bp,其中分布在200 500 bp长度区间Unigene占总数的55.4%。数据库中的序列同源性比较表明,26 826个Unigene与其他物种的已知基因具有不同程度的同源性。芒萁转录组中的Unigene根据GO功能大致可分为细胞组成、分子功能和生物学过程3大类47个分支,其中有大量的Unigene与细胞进程、绑定活性、代谢过程和催化活性相关。将Unigene与COG数据库进行比对,根据其功能大致可分为26类。以KEGG数据库作为参考,依据代谢途径可将Unigene定位到257个代谢途径分支。此外,利用MISA软件检索2 6碱基微卫星,共找到13 286个SSR。在不同长度微卫星中,三核苷重复数量最多,占总数的40.41%。在各重复基序类型中出现频率最高的为AG/CT(14.45%)与AAG/CTT(12.39%)。利用重复基序开发的多态性SSR标记,可应用于芒萁不同个体的基因型分型鉴定。[结论]本研究获得了较高质量的芒萁转录组数据库,揭示了芒萁孢子体生长发育过程中表达基因的功能总体特征,可为芒萁进一步的功能基因挖掘和分子标记规模化开发奠定基础。

[Objective]The sporophyte transcriptome of Dicranopteris dichotoma was sequenced by Illumina MiSeq 250 to provide molecular information of its growth,development,metabolism,and the micro evolutionary mecha-nism.[Method]The functional annotations,metabolic pathways and microsatellite analysis of some Unigenes were conducted using bioinformatics methods.[Result]A total of 18 463 296 reads containing 4.62 Gbp of sequence in-formation were generated.A total of 63 169 unigenes were formed by initial sequence splicing,with an average read length of 863 bp and N50 value of 1 587 bp.26 826 unigenes were annotated using BLASTX searches against the Nr,Nt and SwissProt databases.The unigenes of the transcriptome of D.dichotoma were roughly divided into cellu-lar components,molecule function and biological processes categories of 47 branches by gene ontology,of which re-lated with cellular process cell,binding,metabolism processes and catalytic activities.Further annotated based on COG category,Unigenes could be grouped into 26 functional categories.KEGG pathway analysis showed that Uni-genes could be divided into 276 classes based on their metabolic function.Meanwhile,13 286 SSRs （simple se-quence repeats）were mined with repeat motif of 2 to 6 bp by MISA.The trinucleotide repeats were most dominant, accounting for a total of 40.41%.AG/CT （14.45%）and AAG/CTT （12.39%）were the most common repeat mo-tifs.Polymorphic SSR markers were developed from repeat motifs,which could be used for genotyping of different in-dividuals of D.dichotoma.[Conclusion]A higher quality of transcriptome database was obtained in this study, which could reveal the general characteristics of gene expression in the process of growth and development,and lay the foundation for further gene function mining and the large-scale development of molecular markers of D.dichotoma.