Ilex kaushue S. Y. Hu (Aquifoliaceae) is an evergreen tree growing in dense forests at elevations between 400 and 1000 m in southern China (Chen et al., 2008). The species is one of the primary sources of ku-ding-cha (Hao et al., 2013), a tea used in traditional medicine that has been consumed for thousands of years in China. Modern medicinal research has demonstrated that ku-ding-cha has significant pharmacological effects, including as an antidiabetes and antiobesity drug, as well as an antioxidant (Hao et al., 2013). Ilex kaushue lives in a fragmented habitat with extremely small population sizes, and was included in a conservation program carried out in 2011 by the State Forestry Administration of China (Chen et al., 2014). Furthermore, I. kaushue is an economically important crop due to its wide-spread use for tea, and there has been rapid development in its cultivation in southern China (Guo et al., 2005).
Evidence supports that small natural populations and modern plant breeding can lead to a reduction in overall genetic diversity (Tanksley and McCouch, 1997; Leimu et al., 2006). Low levels of genetic diversity put wild populations at risk and jeopardize the continued ability to improve crops (Reif et al., 2005). Therefore, assessment and preservation of genetic diversity of I. kaushue are important concerns. Although Zhang et al. (2003) developed molecular markers (RAPD) for use in the study of I. kaushue, further genetic diversity research is necessary at the population and species levels to assess and protect its germplasm resources. Assessment and conservation of genetic diversity of a species requires development of efficient codominant microsatellite markers. In this study, 16 microsatellite loci for I. kaushue were isolated and characterized, which will be useful for assessment and conservation of genetic diversity of I. kaushue.
METHODS AND RESULTS
We sampled 12 I. kaushue trees in a natural population (Baisha County/ Hainan Province, China [QS]: 19°08′50.38″N, 109°16′14.9″E) and 10 trees in a cultivated population (Dapu County/Guangdong Province, China [DM]: 24°16′40.31″N, 116°28′02.83″E). Voucher specimens of each population were deposited in the Guangxi Institute of Traditional Medical and Pharmaceutical Sciences herbarium (GXMI; accession numbers Ik-012-ZQW and Ik-008-HYF, respectively; Appendix 1). Genomic DNA (gDNA) was extracted from silica gel–dried leaves using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). We mixed gDNA of all wild-collected individuals to be shotgun sequenced by Sangon Biotech (Shanghai, China) using 454 GS FLX Titanium (Roche Applied Science, Branford, Connecticut, USA). The 454 sequencing technique is described in detail in Margulies et al. (2005).
We obtained 29,247 reads ranging from 32 to 691 bp with an average read length of 401 bp, for a total of 11,736,223 bases. All reads were further screened for microsatellite motifs implemented in the program SSRHunter 1.3 with the default parameters (Li and Wan, 2005). A total of 1109 sequences containing 1104 dinucleotide, 259 trinucleotide, and nine tetranucleotide repeats were obtained. Of these sequences, those containing at least six dinucleotide or trinucleotide repeats and sufficient lengths at either end of the repeat motif were chosen for primer design using Primer Premier 5.0 (Clarke and Gorley, 2001); a total of 631 sequences, containing 691 dinucleotide and 82 trinucleotide repeats, were subjected to primer design. The settings for Primer Premier were as follows: (i) each search range of sense primer and antisense primer was at each end of the repeat motif; (ii) the primer length was between 17 and 25 bp; (iii) the PCR product size was between 100 and 400 bp long; (iv) the annealing temperature of primers was between 50°C and 64°C, and the difference in annealing temperature between the forward and reverse primers was <4°C; (v) the GC content was between 40% and 60%; (vi) there was not obvious hairpin structure within the primer; and (vii) other parameters followed the default settings of “High” stringency in the search criteria. A total of 78 primer pairs were successfully designed for a total of 99 repeats including 65 dinucleotide, 18 trinucleotide, and 16 compound repeats. These primers were tested for polymorphism in 22 individuals from the two populations.
Characteristics of 16 microsatellite markers in Ilex kaushue.
PCR reactions were performed in a 20-µL reaction volume containing 50–100 ng of gDNA, 0.5 µM of each primer, and 10 µL of 2× Taq PCR Master Mix (0.1 U/µL Taq polymerase, 0.5 mM dNTP each, 20 mM Tris-HCl [pH 8.3], 100 mM KCl, and 3 mM MgCl2 [Tiangen Biotech, Beijing, China]). PCR amplifications were conducted under the following conditions: 95°C for 5 min; followed by 35 cycles at 94°C for 45 s, at the annealing temperature for each specific primer (optimized for each locus, Table 1) for 45 s, 72°C for 45 s; and a final extension step at 72°C for 5 min. PCR products were resolved on 6% polyacrylamide denaturing gel using a 10-bp or 25-bp DNA ladder (Invitrogen, Carlsbad, California, USA) as a reference and were visualized by silver staining.
Results of initial primer screening in two populations of Ilex kaushue.
Sixteen primer pairs were successfully amplified; these products exhibited the expected sizes and showed clearly defined banding patterns with a maximum of two alleles in each locus per individual. The number of alleles per locus (A) and the observed and expected heterozygosity (Ho and He) of the two populations were estimated by GenAlEx version 6 (Peakall and Smouse, 2006). Linkage disequilibrium (LD) and deviation from Hardy–Weinberg equilibrium (HWE) were calculated by GENEPOP version 4.2 (Raymond and Rousset, 1995).
Across the cultivated and wild populations, A varied from one to nine, and a total of 73 alleles were scored in 22 individuals (Table 2). Ho and He in the natural and cultivated populations ranged from 0.000 to 1.000 and from 0.000 to 0.785, respectively. No pairs of loci showed significant LD. The P value of tests for HWE ranged from 0.024 to 1.000 (Table 2). Only locus KDC66 in population DM significantly deviated from HWE (P < 0.05), which may be due to overdominant selection or admixture from different resources given the high level of heterozygosity for this locus.