Development and characterization of EST‐SSR markers for Vitex negundo var. heterophylla (Lamiaceae)

Premise of the Study Vitex negundo var. heterophylla (Lamiaceae) is a dominant shrub in the warm temperate zone of northern China. Expressed sequence tag–simple sequence repeat (EST‐SSR) markers were developed to investigate its genetic diversity and structure. Methods and Results We detected 12,075 SSRs in V. negundo var. heterophylla using transcriptome sequencing. Primer pairs for 100 SSR loci were designed and amplified in three populations of V. negundo var. heterophylla. Sixty loci were amplified, of which 14 were polymorphic. The number of alleles per locus ranged from two to 15, and levels of observed and expected heterozygosity ranged from 0.241 to 0.828 and from 0.426 to 0.873, respectively. All primer pairs amplified PCR products from V. rotundifolia but only four of them amplified products from Leonurus japonicus. Conclusions The identified EST‐SSR markers will be useful for future molecular and reproductive ecology studies of V. negundo var. heterophylla and V. rotundifolia.

http://www.wileyonlinelibrary.com/journal/AppsPlantSci © 2019 Liu et al. cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). Initially, we used 100 primer pairs with high dinucleotide or trinucleotide repeat motifs to amplify products from six individuals belonging to three populations. PCR amplification was performed in a final volume of 20 μL, containing 3 ng of template DNA, 2 μL of 10× buffer (with Mg 2+ ; Tiangen), 1 μL of dNTPs (2.5 mM each), 1 μL of each primer (5 μM), and 1 unit of Taq polymerase (Tiangen). The PCR program consisted of an initial denaturation step at 95°C for 5 min; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at an appropriate temperature for 1 min, and extension at 72°C for 45 s; followed by a final extension step at 72°C for 7 min and 65°C for 30 s. The PCR products were fractionated by electrophoresis using both 2% agarose gels and 6% polyacrylamide gels with a 1-kbp DNA Ladder Marker (Tiangen) as a reference. In total, 52 primer pairs amplified detectable products, of which 14 pairs showed polymorphism among the six tested samples (Table 1; see monomorphic loci in Appendix 2). The putative functions of EST-SSR sequences were determined by BLASTX against the NCBI nr database. The 14 primer pairs were then used with all 83 samples from the three populations to evaluate the overall level of polymorphism. The forward primers were 5′ end-labeled with FAM dye, and final products were fractionated using an ABI 3730XL DNA capillary sequencer (Applied Biosystems, Foster City, California, USA) with a LIZ 500 Internal Size standard (Applied Biosystems). GenAlEx version 6.5 (Peakall and Smouse, 2012) was used to calculate the number of alleles, observed heterozygosity, and expected heterozygosity for each locus. GENEPOP software (version 4.7.0;Rousset, 2008) was used to investigate linkage disequilibrium and to determine deviation from Hardy-Weinberg equilibrium. The number of alleles per locus ranged from two to 15, the levels of  observed heterozygosity ranged from 0.241 to 0.828, and the levels of expected heterozygosity ranged from 0.426 to 0.873 (Table 2). Significant linkage disequilibrium was detected between loci V15 and V30 (P = 0.0109) and loci V25 and V70 (P = 0.0266). Loci V97 and V100 showed significant deviation from Hardy-Weinberg equilibrium in two populations (P < 0.001; Table 2).
To test the transferability of the 14 primers between taxa, they were used with DNA samples from V. rotundifolia and Leonurus japonicus Houtt. (Lamiaceae). All primer pairs successfully amplified products from V. rotundifolia, but only four primer pairs amplified products from some L. japonicus individuals (Table 3).

CONCLUSIONS
We assembled 52,072 unigenes of V. negundo var. heterophylla following transcriptome sequencing and used this data set to develop 14 novel polymorphic EST-SSR primer pairs. All of these primers amplified products in the related species V. rotundifolia. These markers represent a useful resource for reproductive and genetic ecology studies of this species and may provide a valuable tool for revegetation and management in northern China.

ACKNOWLEDGMENTS
The authors thank Dr. Shuping Zhang for field assistance and Prof. Fengning Xiang for technical guidance. This work was supported by the National Natural Science Foundation of China (no. 31470402, 31770361), the Basic Work of the Ministry of Science and Technology of China (no. 2015FY1103003-02), and the Fundamental Research Funds of Shandong University (no. 2017GN0018). The authors also thank PlantScribe (www.plantscribe.com) for editing this manuscript.

DATA ACCESSIBILITY
All sequence information was uploaded to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (accession no. PRJNA491662); primer sequences were uploaded to GenBank (accession no. MH825839-MH825852 and MH892533-MH892570; Table 1 and Appendix 2).