Development of microsatellite markers for a Chinese endemic plant, Torreya yunnanensis (Taxaceae)

Premise of the Study Microsatellite markers were developed in Torreya yunnanensis (Taxaceae) to investigate the genetic diversity, phylogeography, and population structure of the species. Methods and Results Sixteen primer sets were identified using Illumina 2 × 100‐bp paired‐end sequencing and bioinformatic screening. Most primers also amplified in T. fargesii, T. grandis, T. jackii, and T. nucifera. Conclusions These results indicate the utility of these microsatellite markers in T. yunnanensis for future studies of molecular ecology as well as their applicability across the genus.


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Torreya yunnanensis C. Y. Cheng & L. K. Fu (Taxaceae) is a Chinese endemic evergreen tree that is highly valued for its timber, as well as for its ornamental and ecological benefits. Wild resources of T. yunnanensis have declined sharply as a result of overexploitation and deforestation. It was listed as endangered in the China Species Red List (Wang and Xie, 2004) and is now rare in the field. Very little research has been done on this species, except for recent studies on chemical composition, cultivation techniques, and community structure (e.g., Hou et al., 2015;Fu et al., 2016;Li et al., 2016). Although conservation of this species is urgent, little is known about its genetic diversity, which is important for planning conservation strategy (Avise and Hamrick, 1996;Jiang et al., 1997).
Microsatellites, also called simple sequence repeat (SSR) markers, are a neutral molecular marker widely distributed in the nuclear genome of eukaryotes. Because of advantages such as high polymorphism, ability to facilitate genotyping, and low demand of DNA quality, microsatellites have been widely used as DNA markers in population genetic studies and parentage analysis. In this study, 16 polymorphic microsatellite markers were developed using Illumina 2 × 100-bp paired-end sequencing and bioinformatics screening. We believe these markers are useful for investigating genetic diversity and population structure for T. yunnanensis and other Torreya species.

METHODS AND RESULTS
A total of 64 T. yunnanensis individuals were sampled from five wild populations in this study (Appendix 1). The sample sizes were small for each population (ranged from 7-18) because T. yunnanensis is an endangered species and is rare in the field. Young leaves were collected and dry preserved with silica gel. Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method (Doyle, 1987). A normalized DNA library was constructed using the TruSeq Stranded DNA Sample Preparation kit (Illumina Inc., San Diego, California, USA) for one sample from the Misha population. The normalized DNA library was sequenced using the Illumina HiSeq 2500 (Illumina Inc.). A total of 5,362,980,012-bp paired-end raw sequences were trimmed to remove adapter sequences and low-quality sequences using Trim Galore (http://www. bioinformatics.babraham.ac.uk/projects/trim_galore/). De novo assembly based on trim reads was performed with Velvet 1.2.10 (Zerbino and Birney, 2008). A total of 50,381,156 trim reads with an average length of 98.7 bp (4,975,006,766 bp in total) were produced and de novo assembled into 283,057 contigs with an N50 length of 1823 bp. SSR detection was performed with MISA (Thiel et al., 2003) using the following criteria: ≥6 repeat units for dinucleotides, ≥5 for tri-, and ≥4 for tetra-, penta-, and hexanucleotides. A total of 4146 SSRs were identified. Of them, trinucleotide repeat motifs (50.19%) were the most common, followed by di-(44.19%), tetra-(3.14%), penta-(0.58%), and hexanucleotide (1.91%) repeat motifs. Primer pairs were designed using Primer3 (Koressaar and Remm, 2007;Untergasser et al., 2012) with the default conditions. Fifty primer pairs amplifying SSRs containing dinucleotide or trinucleotide motifs were randomly selected and tested in four individuals. Sixteen primer sets displaying consistent amplification (Table 1) were selected for further polymorphism tests on 64 individuals. All PCRs were performed in 20-μL volume (50-100 ng of genomic DNA, 10 μL of 2× EasyTaq PCR SuperMix polymerase [TransGen Biotech Co., Beijing, China], 0.5 μM of each primer pair [the forward primer was fluorescently labeled with FAM, HEX, or TAMRA]) under the following conditions: 5 min denaturation at 95°C; 32 cycles of 30 s at 95°C, 1 min at specific annealing temperatures, and 1 min at 72°C (Table 1); and a final extension of 72°C for 10 min. PCR products were separated on an ABI PRISM 3730 Genetic Analyzer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with a GeneScan 500 LIZ Size Standard. Allele peaks were scored using GeneMarker Hardy-Weinberg equilibrium was tested with GENEPOP version 3.4 (Rousset, 2008), and the P values were then tested using Bonferroni correction. The exact tests for genotypic linkage disequilibrium for each pair of loci in each population were performed with GENEPOP version 3.4.
According to MICRO-CHECKER analysis, there is no evidence for scoring error due to stuttering, large allele dropout, or the existence of null alleles. Fourteen loci were polymorphic in five wild populations (Table 2) and two loci (Ty49984 and Ty61395) were monomorphic. Values for A, H o , and H e of 14 loci ranged from 2-12, 0.000-1.000, and 0.000-0.869 (Table 2) . Significant deviation from Hardy-Weinberg equilibrium was found in loci Ty13986, Ty53899, Ty60665, and Ty72305 (P < 0.05) after Bonferroni correction. Most of the F values were negative, indicating low levels of inbreeding in these populations. There was no evidence of linkage disequilibrium among pairs of loci in the sample. The sequences of these microsatellite loci have been deposited in the GenBank database (Table 1), and the raw sequences from highthroughput sequencing have been deposited in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI; accession no. SRR6428886). Cross-amplification of these 16 primer sets was tested in five individuals in each of the following species: T. fargesii Franch., T. grandis Fortune ex Lindl., T. jackii, and T. nucifera (L.) Siebold & Zucc. Most primers also amplified in these species (Table 3).

CONCLUSIONS
Genetic diversity in wild T. yunnanensis populations was shown to be high. The SSR markers that we developed were polymorphic and will be a useful tool to investigate the genetic diversity, population structure, and levels of gene flow, as well as to optimize breeding for T. yunnanensis and related species. Significant deviations from Hardy-Weinberg equilibrium at *P < 0.05, **P < 0.01, ***P < 0.001 after Bonferroni correction.