Isolation and characterization of microsatellite loci from Oxytropis diversifolia (Fabaceae)

Premise of the Study Microsatellite primers were developed for a perennial legume from northern China, Oxytropis diversifolia (Fabaceae), to investigate population genetic structure of this taxon, as well as potential hybridization events with closely related taxa in this genus. Methods and Results One hundred and five primer pairs were designed from Illumina sequence data and screened for suitability. Fifteen of these primer pairs were polymorphic, and these primers amplified tri‐, tetra‐, and pentanucleotide repeats with 10–56 alleles per locus. Cross‐amplification tests in three other Oxytropis species from northern China (O. leptophylla, O. neimonggolica, and O. squammulosa) revealed that all of these loci can be amplified successfully and show polymorphism. Conclusions These primer pairs can be used to assess the genetic diversity and population structure in future studies of O. diversifolia, as well as studies of potential hybridization events with closely related taxa in this genus.

Here, we describe the development of microsatellite markers that will facilitate future research on leaf shape variation in O. diversifolia. In addition, the degree of congeneric cross-transferability of the markers was also assessed in three related Oxytropis species from northern China: O. leptophylla (Pall.) DC., O. neimonggolica C. W. Chang & Y. Z. Zhao, and O. squammulosa DC. We are particularly interested to test for potential hybridization of O. leptophylla with O. diversifolia (H. Wang, Northwest A&F University, Yangling, Shaanxi, China, personal observation).

METHODS AND RESULTS
Total genomic DNA was extracted from a dry leaf sample collected in Urad Zhongqi, Nei Mongol, China (Pop8, Appendix 1; BioSample accession SAMN08408037), using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1990 SRP131738, BioProject ID PRJNA431827) were obtained. The paired-end reads were then processed using Trimmomatic version 0.35 (Bolger et al., 2014) and merged into ~240-bp sequences using FLASH version 1.2.11 (Magoč and Salzberg, 2011). In total, 3,079,710 clean reads assembled into 2,949,319 contigs. SSR_pipeline software (Miller et al., 2013) was used to detect tri-, tetra-, and pentanucleotide repeats on the sequence set, and Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, California, USA) was used to develop primers for 105 loci, prioritizing motif diversity and melting temperature difference ≤1°C. An M13 tag (5′-TGTAAAACGACGGCCAGT-3′) was added to the 5′ end of the shorter primer of each locus. These primer pairs were tested on seven O. diversifolia individuals from different populations (Appendix 1). Each locus was initially amplified individually in 15-μL PCR reactions that contained 1.5 μL of 10× Buffer I, 200 μM of dNTPs, 0.27 μM of M13-tailed primer, 0.07 μM of untailed primer, 0.27 μM of M13 primer (labeled with HEX), 0.1 μL of 1× TaKaRa HS Taq (TaKaRa Biotechnology, Dalian, Liaoning, China), and 1.2 μL of diluted template DNA. PCR thermocycling conditions were an initial denaturation of 95°C for 5 min; 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s; followed by 10 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s; and a final extension at 60°C for 30 min. The PCR products were examined on a 2% agarose gel. Samples that yielded products of expected size were then submitted with a GeneScan 500 LIZ Size Standard (Applied Biosystems, Foster City, California, USA) for genotyping on an ABI 3730xl DNA Analyzer (Applied Biosystems). Resulting chromatograms were scored using GeneMapper version 3.2 (Applied Biosystems).
Of the 105 primer pairs tested, 15 produced repeatable amplicons and showed polymorphism across all seven individuals (Table 1). These 15 pairs were subsequently screened using four populations of O. diversifolia (n = 20, 30, 32, 32, respectively) and additional populations of O. leptophylla (n = 19), O. neimonggolica (n = 20), and O. squammulosa (n = 16) (Appendix 1). Instead of M13-tailed primers, we used primers with a 5′ fluorophore, labeled with 6-FAM, HEX, or ROX (Applied Biosystems, for specific fluorescent dye used for each locus see Table 1). Primer sequences and allele ranges for validated loci were fed into Multiplex Manager (Holleley and Geerts, 2009) to determine the best sets of loci available to include in a multiplex protocol (see Table 1 for pooling groups). The PCR reactions were performed separately for each locus, and the PCR products were then pooled into four groups for genotyping. The 15-μL PCR reactions contained 7.5 μL of 2× TSINGKE Master Mix (Tsingke Biological technology, Xi'an, Shaanxi, China), 0.67 μM of forward primer, 0.67 μM of reverse primer, and 1 μL of diluted template DNA. The PCR thermocycling conditions and genotyping method were the same as above. Resulting chromatograms were scored using Geneious version 9.0.2 (http://www.geneious.com; Kearse et al., 2012).
Genetic diversity parameters were calculated using GenAlEx version 6.503 Smouse, 2006, 2012). Observed and expected heterozygosity levels ranged from 0.048 to 0.897 and 0.567 to 0.968, respectively (Table 2). Alleles per locus ranged from 10 to 56 in O. diversifolia. Tests of pairwise linkage disequilibrium were performed using GENEPOP 4.7 (Rousset, 2008). Only two genotypic disequilibria out of 420 (N876535 and N2720763, N876535 and N350553 in Pop8) were significant at the 5% level after Benjamini-Hochberg correction (Benjamini and Hochberg, 1995). We also used exact tests implemented by GENEPOP software to test for departure from Hardy-Weinberg equilibrium (HWE). A significant departure from HWE was recorded for almost all loci across the four populations ( Table 2) (Table 3).

CONCLUSIONS
The 15 polymorphic microsatellites developed here will be used for population genetic studies on O. diversifolia. Cross-amplification experiments confirmed that these markers should be applicable in O. neimonggolica, O. leptophylla, and O. squammulosa, thus providing a novel population genetic tool in Oxytropis. The lowgenomic-coverage Illumina sequencing reads generated in the present study could potentially be used to assemble high-copynumber gene regions, such as complete or partial chloroplast and mitochondrial genomes, as well as nuclear ribosomal RNA genes. Such gene sequences can be informative in phylogenetic Statistically significant deviation from Hardy-Weinberg equilibrium is indicated as *P < 0.05, **P < 0.01, ***P < 0.001; ns = not statistically significant (P > 0.05). Note: A = number of alleles detected across all individuals; n = number of individuals sampled. a Voucher and locality information are provided in Appendix 1.