珍稀濒危飘带兜兰叶绿体全基因组种内变异研究(附录)

飘带兜兰(Paphiopedilum parishii)分布范围狭窄,仅在中国、缅甸、泰国以及老挝有少量分布。近年来,因生境破坏和人为滥采而导致飘带兜兰野生种群极度缩减。为开发种内多态性的分子标记用于保护生物学研究,该研究对飘带兜兰4个野生个体经测序、组装、注释获得的叶绿体基因组序列,与已公布的飘带兜兰2个个体的叶绿体全基因组序列进行比对,分析飘带兜兰叶绿体基因组的种内差异。结果表明:(1)飘带兜兰叶绿体基因组具有典型被子植物叶绿体基因组环状四分体结构,基因组长度为154 403～154 809 bp,共编码129个基因,包括78个蛋白质编码基因、39个tRNA基因、8个rRNA基因,以及4个假基因。(2)在飘带兜兰6个个体叶绿体基因组中检测到103～107个SSRs(simple sequence repeats)位点,其中21个SSR位点具有多态性。此外,在6个个体叶绿体基因组中还检测到60个长序列重复,包括17～21个正向重复、18～29个反向重复、9～16个回文重复、4～9个互补重复。(3)通过比较6个个体叶绿体基因组序列的核苷酸多样性,共发现70处变异,包括10个SNPs(single nucleotide polymorphism)、60个插入缺失(InDels)。其中,有3个SNP位点发生了非同义替换,导致编码功能基因的氨基酸发生改变; 19个插入缺失多态性较高,具有开发为分子标记的潜力。(4)通过计算核苷酸多样性值(P_i)共发现8个有变异的区域,P_i值为0～0.006 32,其中变异度较大的是rps3-rpl22、trnL-UAC-rpl32、rpoB-trnC-GCA以及ycf4,这些高变区可开发为分子标记用于评估飘带兜兰遗传多样性。(5)系统发生分析结果表明,飘带兜兰6个个体叶绿体基因组序列聚在一起,与长瓣兜兰互为姐妹群。综上表明,飘带兜兰叶绿体基因组的SSRs、长序列重复、SNPs、InDels以及核苷酸序列呈现了足够的种内多样性,可开发成分子标记用于该种的系统演化及保护生物学研究。

Intraspecific genetic variation within chloroplast genome of a rare and endangered species Paphiopedilum parishii (Orchidaceae)

Paphiopedilum parishii is a rare and endangered species with few localities and fragmented populations found in China, Myanmar, Thailand and Laos. Environmental changes and over-harvesting have led to a decrease in its wild populations. It is important to protect endangered species'' genetic diversity since it provides the species with the ability to adapt and survive. However, little is known about the genetic information of this species. This study aims to detect intraspecific variation and develop polymorphic genetic markers of P. parishii. The complete chloroplast genome of four individuals of wild P. parishii was obtained by sequencing, assembling and annotating, then compared with the existing genomic data of two individuals available from GenBank to detect the intraspecific variation. Further, simple sequence repeats(SSRs), single nucleotide polymorphisms(SNPs), and insertions and deletions(InDels)were identified. The results were as follows:(1)The four new sequencing chloroplast genomes were quadripartite, with a length between 154 403 bp and 154 809 bp, with 129 genes(78 protein coding genes, 39 tRNAs, 8 rRNAs and four pseudogenes).(2)As a result of comparison of six individuals, 103-107 SSR loci were identified in the chloroplast genome of six individuals of P. parishii, and 21 SSRs were polymorphic. And 60 long repeats were found, including 17-21 forward repeats, 18-29 reverse repeats, 9-16 palindromic repeats, and 4-9 complement repeats.(3)In addition, a total of 10 SNPs and 60 InDels were uncovered across the plastome. Three of the non-synonymous mutations caused amino acid changes in functional domains. 19 InDels might be selected for possible chloroplast DNA markers to determine intraspecific variation.(4)The value of nucleotide diversity(P_i)was calculated ranging from 0-0.006 32 suggesting sequences with low variation. Hyper-polymorphic regions, e.g. intergenic spacers rps3-rpl22, trnL-UAC-rpl32, rpoB-trnC-GCA and ycf4 gene were identified as potential barcoding regions.(5)The phylogenetic analyses based on the complete chloroplast genome supported three lineages in Paphiopedilum, and six individuals of P. parishii form a monophyletic group. SSRs, long repeats, InDels, SNPs and nucleotide sequences showed sufficient intraspecific genetic variation in P. parishii. The molecular markers developed here will contribute to further evolutionary studies and conservation of P. parishii.