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

Abstract

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 (Pi) 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.

Keywords

• chloroplast genome
• paphiopedilum parishii
• sequence divergence
• polymorphic dna markers
• indels
• ssrs