Mining and characterization of novel EST-SSR markers of Parrotia subaequalis (Hamamelidaceae) from the first Illumina-based transcriptome datasets.

Yunyan Zhang,Pengfu Li,Yimin Hu,Xin Zhuang,Wuqin Xu,Mengyuan Zhang,Zhongsheng Wang,Branislav T Šiler

doi:10.1371/journal.pone.0215874

Abstract

Parrotia subaequalis is an endangered Tertiary relict tree from eastern China. Despite its important ecological and horticultural value, no transcriptomic data and limited molecular markers are currently available in this species. In this study, we first performed high-throughput transcriptome sequencing of two individuals representing the northernmost (TX) and southernmost (SJD) population of P. subaequalis on the Illumina HiSeq 2500 platform. We gathered a total of 69,135 unigenes for P. subaequalis (TX) and 84,009 unigenes for P. subaequalis (SJD). From two unigenes datasets, 497 candidate polymorphic novel expressed sequence tag-simple sequence repeats (EST-SSRs) were identified using CandiSSR. Among these repeats, di-nucleotide repeats were the most abundant repeat type (62.78%) followed by tri-, tetra- and hexa-nucleotide repeats. We then randomly selected 54 primer pairs for polymorphism validation, of which 27 (50%) were successfully amplified and showed polymorphisms in 96 individuals from six natural populations of P. subaequalis. The average number of alleles per locus and the polymorphism information content values were 3.70 and 0.343; the average observed and expected heterozygosity were 0.378 and 0.394. A relatively high level of genetic diversity (HT = 0.393) and genetic differentiation level (FST = 0.171) were surveyed, indicating P. subaequalis maintained high levels of species diversity in the long-term evolutionary history. Additionally, a high level of cross-transferability (92.59%) was displayed in five congeneric Hamamelidaceae species. Therefore, these new transcriptomic data and novel polymorphic EST-SSR markers will pinpoint genetic resources and facilitate future studies on population genetics and molecular breeding of P. subaequalis and other Hamamelidaceae species.

Highlights

IntroductionThis species is a diploid (2n = 2x = 24) deciduous tree characterized by unique exfoliating bark, obovate leaves in green, yellow, red or purple, and distinct apetalous bisexual flowers [1, 2]
The two raw sequencing datasets were uploaded to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/Traces/sra; Biosample accession SAMN10502180 for P. subaequalis (TX) and SAMN10509852 for P. subaequalis (SJD))
With the help of Trinity version 2.5, these above clean reads were de novo assembled into 117,794 transcripts with an average length of 674 bp and an N50 length of 1268 bp for P. subaequalis (TX) and 145,619 transcripts with an average length of 672 bp and an N50 length of 1245 bp for P. subaequalis (SJD) (Table 1)

Summary

Introduction

This species is a diploid (2n = 2x = 24) deciduous tree characterized by unique exfoliating bark, obovate leaves in green, yellow, red or purple, and distinct apetalous bisexual flowers [1, 2]. As an endangered Tertiary relict tree, P. subaequalis is categorized as ‘extremely endangered’ by the International Union for Conservation of Nature (IUCN) [7] and the Chinese Plant Red Book (Grade I Key Protected Wild Plants) [8]. Collection of the wild germplasm resources, plant breeding, and improvement of genetic variability of P. subaequalis has been attracting increasing amounts of attention from cultivators and researchers because of its high value in gardening applications and extant endangered survival

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