Hoya R. Br. (Apocynaceae) is composed of 200–300 species worldwide and is an important epiphyte component of tropical and subtropical forests (Liede and Albers, 1994). It is mainly distributed in the tropical rainforests of Southeast Asia, Australia, and islands of the Indian and Pacific oceans (Wanntorp, 2009). China is one of the main distribution areas of Hoya, with 39 species mainly occurring in Yunnan, Guangxi, Guangdong, and Hainan provinces (Li and Jiang, 1977; He et al., 2009a, b, c, 2011a, b, 2012).
In China, many species of this genus occur in a very narrow distribution range. For example, H. ledongensis Shao Y. He & P. T. Li is restricted to the central mountain areas of Hainan Province and H. jianfenglingensis Shao Y. He & P. T. Li is only known from Jianfengling Nature Reserve, Hainan (He et al., 2011a, b). For the latter species, it is estimated that there are fewer than 60 individuals in its whole range (He et al., 2011a). Therefore, conservation of these lands that provide narrow distribution ranges for these species should be prioritized. Knowledge of genetic diversity and population structure of Hoya species can provide important information for their conservation. Furthermore, both of these species occur at high elevations (ca. 1000 m) and are sympatric in the area of the Jianfengling Nature Reserve. Hoya ledongensis flowers from May to June, and H. jianfenglingensis flowers from May to July (He et al., 2011a, b). The overlap in flowering time between the two species and the insect-pollination mating system for Hoya provide an opportunity for interspecific hybridization. Molecular markers can be used to address these evolutionary questions on conservation genetics and natural hybridization in this genus. With advances in high-throughput sequencing technologies, transcriptome-derived simple sequence repeat (SSR) markers can be easily obtained and are also increasingly used in conservation genetic studies (Yu et al., 2004; Chen et al., 2010; Wu et al., 2012). However, to date, there has been no report of SSR markers in Hoya. In the current study, we developed and characterized 12 transcriptome-derived SSR markers for H. ledongensis and tested their transferability to its congeneric species H. jianfenglingensis.
METHODS AND RESULTS
Fifteen individuals were sampled from each of two natural populations of H. ledongensis in Bawangling (CJL: 19°08′13.6″N, 109°10′37.7″E) and Baisha (BSL: 18°45′50.3″N, 108°57′48.8″E), and six individuals were sampled from the population in Jiangfengling (LDL: 18°46′27.3″N, 108°52′29.6″E) in Hainan Province. An additional six individuals of the congeneric species H. jianfenglingensis were collected from Bawangling (CJJ: 19°13′12.0″N, 109°08′07.6″E), Hainan Province (Appendix 1). Genomic DNA was extracted from silica-dried leaves using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). The leaf transcriptome of H. ledongensis was sequenced as described by Liu (2012); based on these data, we selected 46 pairs of transcriptome-derived primers with five or more repeats of dinucleotide or trinucleotide motifs.
To screen the expressed sequence tag (EST)-SSR primers, PCR amplification was conducted using two individuals from each of the two H. ledongensis populations (CJL and BSL) in a final reaction volume of 20 µL containing: 1.2 µL of DNA (15 ng/µL), 10 µL of 10× PCR KOD buffer, 4 µL of dNTPs (2.5 mM, Mg2+), 0.6 µL (10 µM) of each primer, and 0.6 µL KOD polymerase (TOYOBO Ideas & Chemistry, Osaka, Japan). PCR was performed under standard conditions for all primers using the following cycling conditions: 3 min of denaturation at 94°C; followed by 30 cycles of 40 s at 94°C, 45 s at the annealing temperature of each primer set, and 40 s at 72°C; with a final extension of 10 min at 72°C. The PCR products were first resolved with electrophoresis in a 1.5% agarose gel to assess if the amplification was successful for the expected sized products of each primer pair. These experiments produced PCR products with expected sizes that were successfully amplified from 28 primer pairs designed for H. ledongensis.
To test the polymorphism level of these 28 primer pairs, PCR was conducted using all of the H. ledongensis and H. jianfenglingensis samples in a final reaction volume of 20 µL, using the conditions detailed above. Amplified products were resolved in an 8% polyacrylamide gel electrophoresis (PAGE), and gels were stained with 0.1% silver nitrate. The band size was calculated by comparison with a 50-bp DNA ladder (TaKaRa Biotechnology Co., Dalian, China). Our results showed that 12 transcriptome-derived SSR markers were polymorphic in H. ledongensis (Table 1).
POPGENE version 1.31 (Yeh et al., 1999) was used to calculate the population genetics parameters for H. ledongensis and H. jianfenglingensis, respectively. Two to 11 alleles were detected for these loci (Table 2). These polymorphic loci had observed heterozygosity from 0.133 to 0.867 and expected heterozygosity from 0.128 to 0.894, respectively.
Out of the 12 polymorphic microsatellite loci, six, seven, and one in the CJL, BSL, and LDL populations of H. ledongensis, respectively, exhibited significant deviations from Hardy–Weinberg equilibrium (HWE; Table 2). The deviations from HWE might be an effect of null alleles at these loci, despite the fact that null homozygous individuals were absent in these populations. To test this, we used MICRO-CHECKER (version 2.2.3; van Oosterhout et al., 2004) to check if there were null alleles. We found that null alleles were present at five markers (SSR-8, SSR-41, SSR-45, SSR-46, and SSR-31) in the population BSL, at three markers (SSR-8, SSR-31, and SSR-23) in the population CJL, and at one marker (SSR-14) in the population CJJ. Because HWE was also observed in populations with null alleles at some loci, homozygote excess of populations should be another factor for the deviations from HWE. No significant linkage disequilibrium was observed between these markers; therefore, they can be considered independent across the genome. Furthermore, cross-species amplification of these 12 markers was successful in H. jianfenglingensis and only one locus in the CJJ population exhibited a significant deviation from HWE.
To the best of our knowledge, this is the first study to report SSR markers in a species of Hoya. We have identified and verified 12 markers for H. ledongensis that can also be used for the investigation of its congener species H. jianfenglingensis. The primers designed in this study can be applied to the investigation of genetic diversity and population structure of Hoya and other related species. Furthermore, natural hybridization between Hoya species can be tested with these markers. This work provides an important tool for the development of scientific conservation strategies and testing natural hybridization hypotheses in Hoya.
Genetic diversity of 12 polymorphic markers developed in three populations of Hoya ledongensis and one population of H. jianfenglingensis.a
This study was financially supported by the Science and Technology Program of Guangdong Province (2013B020302006).