两种珍稀白蝶兰属(兰科)叶绿体基因组比较分析

Comparative analysis of chloroplast genomes of two rare Pecteilis species(Orchidaceae)

关键词:  龙头兰, 景洪白蝶兰, 叶绿体基因组, 分子标记, 系统发育

Key words:  Pecteilis susannae, P. hawkesiana, chloroplast genome, molecular markers, phylogeny

摘要:

为理解珍稀濒危兰科植物龙头兰(Pecteilis susannae)和景洪白蝶兰(P. hawkesiana)的叶绿体基因组的基本特征,开发用于物种鉴定、保护遗传学和系统发育分析的分子标记,该研究利用二代测序技术对龙头兰和景洪白蝶兰进行浅层基因组测序,采用生物信息学分析方法进行叶绿体基因组的拼接、组装和注释,并与其他近缘物种进行比较基因组分析和系统发育分析。结果表明:(1)龙头兰和景洪白蝶兰的叶绿体基因组大小分别为154 407 bp和153 891 bp,由一对26 550 bp和26 523 bp的反向重复序列(IR)、84 204 bp和83 756 bp的大单拷贝区(LSC)、17 103 bp和17 089 bp的小单拷贝区(SSC)组成; 均注释了111个唯一基因,包括77个蛋白质编码基因、30个tRNA基因和4个rRNA基因。(2)在叶绿体基因组中分别鉴定出94个和92个简单重复序列(SSRs)。(3)二者之间存在706个单核苷酸多态性(SNPs)位点和152个插入缺失(InDels)位点,其中cpInDel 067等可以区分2个物种。(4)观察到1个差异较大的基因(accD)和9个高变区(rps19-psbA、matK-trnQ-UUG、psbM-psbD、trnT-UGU-ndhJ、accD-psaI、ycf4-cemA、clpP-psbB、ndhF-trnL-UAG、rps15-ycf1)。(5)系统发育分析结果显示,龙头兰、景洪白蝶兰和鹅毛玉凤花(Habenaria dentata)的亲缘关系较近。在白蝶兰属2种叶绿体基因组研究中获得的SSR位点、InDels和高变区序列可为物种鉴定、开发利用及其资源保护提供有价值的遗传信息。

Abstract:

Pecteilis susannae and P. hawkesiana are rare and endangered species with important medicine and ornament value. However, little is known about the genetic information of these two species. In order to understand the basic characteristics of the chloroplast genome of these two Pecteilis species, and to develop molecular markers for species identification, conservation genetic and phylogenetic analysis, the genome skimming approach using next-generation sequencing methods was used to generate chloroplast DNA sequences in this study. The chloroplast genomes were assembled and annotated by bioinformatics analysis. Simple sequence repeats(SSRs), single nucleotide polymorphisms(SNPs), and insertions and deletions(InDels)were identified. Furthermore, comparative chloroplast genomic and phylogenetic analyses were conducted with closely related species. The results were as follows:(1)The newly sequenced chloroplast genomes of P. susannae and P. hawkesiana were 154 407 bp and 153 891 bp in size. They comprised a pair of 26 550 bp and 26 523 bp inverted repeats(IR)that separated a large 84 204 bp and 83 756 bp single copy region(LSC)and a small 17 103 bp and 17 089 bp single copy region(SSC), respectively. Both chloroplast genomes contained 111 unique genes, including 77 protein-coding genes, 30 tRNA and 4 rRNA genes.(2)Ninety-four simple sequence repeats(SSRs)were identified in the P. susannae chloroplast genome and 92 in that of P. hawkesiana.(3)Comparisons of two chloroplast genomes revealed that there were nucleotide variations including 706 single-nucleotide polymorphism sites and 152 InDels between the two Pecteilis species, of which several markers(cpInDel 067)could discriminate the two Pecteilis species.(4)The one most divergent gene(accD)and the nine most divergent intergenic regions(rps19-psbA, matK-trnQ-UUG, psbM-psbD, trnT-UGU-ndhJ, accD-psaI, ycf4-cemA, clpP-psbB, ndhF-trnL-UAG, rps15-ycf1)among genomes were detected.(5)The phylogenetic analysis based on the chloroplast genome sequences revealed that P. susannae, P. hawkesiana and Habenaria dentata are closely related. The molecular markers(SSRs, InDels and hotspots)developed from the chloroplast genomes of two Pecteilis species in the present study can be used to identify related species and provide valuable genetic resources in utilizing and conserving natural resources.