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

Date Published: May 6, 2019

Publisher: Public Library of Science

Author(s): Yunyan Zhang, Mengyuan Zhang, Yimin Hu, Xin Zhuang, Wuqin Xu, Pengfu Li, Zhongsheng Wang, Branislav T. Šiler.


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.

Partial Text

Parrotia subaequalis (H.T. Chang) R.M. Hao & H.T. Wei, the focal species of our study, is a member of the genus Parrotia C. A. Mey. in the Hamamelidaceae family. 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]. Therefore, P. subaequalis is widely cultivated as a horticultural and ornamental tree in North America, Europe and East Asia [3, 4]. However, the natural population size of the wild P. subaequalis has sharply declined due to its narrow geographic distributions in disjunct montane ecosystems of eastern China, serious habitat destruction and the species’ alternate-year fruit production [5, 6]. Additionally, 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]. Thus, 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.

This study is the first to assemble and characterize the transcriptomes of two individuals of P. subaequalis using RNA-sequencing technologies on the Illumina HiSeq 2500 platform. This large set of annotated unigenes and pathways will remarkably enlarge the transcriptomic resources and putative gene functions of P. subaequalis. In addition, we successfully and efficiently developed the first set of 27 novel polymorphic EST-SSR markers for P. subaequalis from the two transcriptomic datasets. These polymorphic EST-SSR markers displayed a relatively high genetic diversity in P. subaequalis and high transferability in five related Hamamelidaceae species, suggesting that they are useful and powerful molecular tools to facilitate future studies on population genetics, molecular breeding and germplasm identification of P. subaequalis and other Hamamelidaceae species. Taken together, these results produced by our study indicated that high-throughput next-generation sequencing technology is a cost-effective and convenient approach to mining abundant novel molecular resources for non-model organisms.