Development and validation of EST-SSR markers for Fokienia hodginsii (Cupressaceae)1

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY-NC-SA 4.0), which permits unrestricted noncommercial use and redistribution provided that the original author and source are credited and the new work is distributed under the same license as the original. is a Tertiary relict evergreen conifer of the monotypic genus Fokienia A. Henry & H. H. Thomas (Fu et al., 1999). It is relatively common in China and Vietnam, but many subpopulations have been decimated by legal and illegal logging, as the wood of this cupressaceous tree is much valued for construction (Thomas and Yang, 2013). Currently, F. hodginsii is a protected species (second degree) in China, is on the official List of Rare and Precious Flora and Fauna in Vietnam, and is listed as a Vulnerable species in the IUCN Red List of Threatened Species (Thomas and Yang, 2013). Applying eight inter-simple sequence repeat markers, Tam et al. (2011) investigated the genetic variation of F. hodginsii in Vietnam and found low genetic variability at both the population and the species level. Simple sequence repeat (SSR) markers have the advantage of producing mostly codominant markers, and high-throughput sequencing technologies, such as the Illu-mina platform, allow for cost-effective development of these markers. In this study, 129 novel expressed sequence tag (EST)– SSR markers were developed from transcriptome data of F. hodginsii. Eleven of the 129 EST-SSRs demonstrated clear polymorphism among 45 individuals from three populations of F. hodginsii and were transferable in Thuja occidentalis L. (Cupressaceae), T. standishii (Gordon) Carrière (Cupressa-ceae), Platycladus orientalis (L.) Franco (Cupressaceae), and Chamaecyparis obtusa (Siebold & Zucc.) Endl. (Cupressaceae). The other 118 markers were monomorphic in nine randomly selected individuals from three populations of F. hodginsii. The development of these novel EST-SSR markers is valuable for conservation of F. hodginsii and other related conifer species. METHODS AND RESULTS One seedling of F. hodginsii was collected from Taoyuandong, Hunan Province , China (Appendix 1), and planted in a greenhouse at Sun Yat-sen University. Two months later, its fresh leaves were harvested and total RNAs were extracted immediately using a modified cetyltrimethylammonium bromide (CTAB) method (Fu et al., 2004). Then, a normalized cDNA library was constructed with the Illumina protocol and subsequently sequenced using the HiSeq 2500 Sequencing System (Illumina, San Diego, California, USA). Reads containing primers or adapters, unknown " N " bases, or more than 10% bases with a …