Abstract
The Chinese green tree viper (Trimeresurus stejnegeri stejnegeri), one of the most common snakes in Southeast Asia, can be a good model species for evolutionary and behavioral research. However, there is no high polymorphic co-dominant marker that can be used for individual-based genetic analyses available for this species. Therefore, we developed 20 polymorphic microsatellite loci for T. s. stejnegeri in Taiwan by screening a microsatellite-enriched DNA library. The allele numbers of these loci ranged from 3 to 22, and the observed heterozygosity were 0.042-1.000. The probability of false parent non-exclusion was 2 × 10−5 for those loci that conformed to the Hardy–Weinberg equilibrium. These highly polymorphic microsatellite markers are useful for both individual-based and population-level analysis. Furthermore, this is the first set of microsatellite markers developed for Trimeresurus, and could be applied to closely related species to address various research questions on evolution and animal behavior.
Snakes, characterized by numerous types of morphological and behavioral adaptations, are an ideal system for studying questions on sexual selection, such as mating system and strategies. However, most field and experimental studies about these topics in snakes are limited to a few taxonomic groups (e.g. natricines, rattlesnakes or slatey-grey snake) (reviewed in Shine, 2003; Rivas and Burghardt, 2005; Uller and Olsson, 2008; Dubey et al., 2009; Clark et al., 2014). The Chinese green tree viper (Trimeresurus stejnegeri stejnegeri), arboreal sit-wait predators, is one of the most common venomous snakes in Southeast Asia. They are a good model species for studying sexual selection and behavioral research because they are philopatric, easily captured, and often have high recaptured rate (Wang, Lin and Tu, 2003; Lin, Li and Tu, 2006). However, in previous studies only one nuclear locus and a few dominant markers (e.g. mtDNA gene sequences and amplified fragment length polymorphism (AFLP) fragments) were reported (Creer, 2003; Creer et al., 2004). There is no co-dominant marker available for individual-based genetic analyses such as parentage and individual recognition in this species. Microsatellites are ideal markers to address issues on mating systems and population structure because they have high degree of polymorphism and can be easily scored (Zane, Bargelloni and Patarnello, 2002). Therefore, we developed 20 polymorphic microsatellite markers from the Chinese green tree viper.
Characteristics of 20 Trimeresurus stejnegeri stejnegeri microsatellite loci.
A microsatellite-enriched library was constructed using genomic DNA extracted from the tissue (obtained by clipping a small tail segment) of a T. s. stejnegeri viper from Tsaochiao, Taiwan. DNA was extracted using the Gentra Puregene Tissue Kit (Qiagen). The hybridisation capture procedure reported by Glenn and Schable (2005) was followed to construct a microsatellite-enriched library using the 5′-biotinylated tetramer (GATA)10 probes, and more details also descripted by Chang et al. (2014); then 96 clones were randomly selected to sequence. Seventy-four clones contained microsatellite repeats, including di-, tri-, tetra-, penta-, and hexa-nucleotide microsatellite motifs, 32 of which contained more than one repeat motif after one round of enrichment. The sequences were trimmed using Sequencher v4.1.4 (Gene Codes Corporation), and the PCR primers were designed using Primer3 (Rozen and Skaletsky, 2000) and Msatcommander v1.0.8 (Faircloth, 2008). The forward primers of each pair were modified on the 5′ end with a CAG-tag (5′-CAGTCGGGCGTCATCA), and a GTTT pig-tail sequence was optionally added to the 5′ end of the reverse primers. In addition, a fluorescently labelled CAG-tag primer was used as the third primer for the PCRs. Each reaction was performed in 10 μl volumes containing approximately 30 ng of DNA template, 0.05 μM forward primer, 0.5 μM reverse primer, 0.5 μM fluorescently labelled CAG-tag primer, 0.2 mM dNTPs, 1.5 mM MgCl2, and 0.25 U Taq DNA polymerase with 1× PCR buffer (Ampliqon, Denmark). Two touchdown PCR thermoprofiles (Don et al., 1991) were adopted with a 10°C decrease in the annealing temperature from 55°C to 45°C (TD55) or from 65°C to 55°C (TD65). The TD55 thermoprofile comprised an initial denaturation at 95°C for 3 min, followed by 8 cycles of denaturation at 95°C for 20 s, the highest annealing temperature of 55°C (decreased 0.5°C per cycle) for 20 s, extension at 72°C for 30 s, followed by 8 cycles of denaturation at 95°C for 20 s, 50°C for 20 s, and 72°C for 30 s, followed by 24 cycles of denaturation at 95°C for 20 s, 45°C for 20 s, and 72°C for 30 s, and a finally extension at 72°C for 7 min. The thermoprofile of TD65 was the same as that of TD55 except that the annealing temperature at each stage was higher by 10°C. Amplicons were mixed with the LIZ600 standard and electrophoresed using ABI3730XL DNA Analyzer (Applied Biosystems). Raw data of allele sizes were scored using Peak Scanner v1.0 (Applied Biosystems).
Twenty microsatellite loci were successfully amplified, and the polymorphisms were distinguished on the basis of 24 T. s. stejnegeri adult individuals collected from the same population in field at Tsaochiao in the northern Taiwan (24°37′N, 120°51′E). Cervus v3.0.3 (Kalinowski, Taper and Marshall, 2007) was used to calculate the number of alleles (k), the observed heterozygosity () and expected heterozygosity () of each locus, and the parental exclusion probabilities. We tested deviation from the Hardy-Weinberg equilibrium (HWE) by using GenePop v4.2.1 (Rousset, 2008) and detected null alleles by using Cervus v3.0.3 and Micro-Checker v2.2.3 (Van Oosterhout et al., 2004).
The microsatellite loci were polymorphic from 3 to 22 alleles and and values from 0.042 to 1.000 and from 0.196 to 0.963, respectively (table 1). Nine loci significantly deviated from the HWE (), possibly because of the null alleles. The probability of false parent non-exclusion was 2 × 10−8 and 2 × 10−5 for all loci and for those loci that conformed to the HWE, respectively. In addition, compared to previous studies, we reported more loci that contained simple (i.e., non-compound) microsatellite tetra-repeat for T. s. stejnegeri than the other vipers in the same subfamily such as Crotalus species (Munguia-Vega et al., 2009; Oyler-McCance and Parker, 2010; Pozarowski et al., 2012). Loci with simple repeats are less biased by homoplasy than compound repeats and thus are better in size scoring and estimating population parameters (Garza and Freimer, 1996).
These highly polymorphic microsatellite markers are not only useful for individual-based but also for population-level or subspecies genetic studies of T. s. stejnegeri. Furthermore, this is the first set of microsatellite markers developed so far for Trimeresurus species. These markers could potentially be used in other Trimeresurus species to address a various array of population genetics issues.
Acknowledgements
We are grateful to Dr. C.M. Hung for improving the manuscript. We thank C.Y. Lai, C.F. Lin, Y.J. Liang, and colleagues at the Wild Animal Rescue Centre for supporting the fieldwork in the past decade. We thank the Animal Adoption Program of Taipei Zoo and the Wildlife Rescue Project supported by the Forestry Bureau, Council of Agriculture, Executive Yuan, Taiwan.
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Footnotes
Associate Editor: Judit Vörös.
