Characterization and the broad cross-species applicability of 20 anonymous nuclear loci isolated from the Taiwan Hwamei (Garrulax taewanus)

Variation at the mitochondrial genome has been widely applied to study ecology, conservation, and evolution of animals in the past few decades, but interpretation of the results are somewhat compromised by the nature of uniparental inherence and acting as a nonrecombined super gene. Although nuclear genome is usually considered to evolve much more slowly and is less polymorphic (Li 1997), single nucleotide polymorphic sites (SNPs) are not uncommon in the vertebrate genome (reviewed by Brumfield et al. 2003). Therefore, these SNPs can be used as multiple independent loci to enhance the precision for the genetic estimates of population parameters (e.g. Brumfield et al. 2003) in a nonfemale-biased manner.

Due to the melodic song of males, the Hwamei (Garrulax canosus), commonly found in the central and southeastern China and northern Indo-China, has been introduced into Taiwan in great quantities since the 1970s. Introduction of these birds poses a potential threat to its sister species, the endemic Taiwan Hwamei (Garrulax taewanus) (Li et al. 2006) by means of hybridization (Tu & Severinghaus 2004). Due to the potential detrimental effect of interspecific hybridization (reviewed by Rhymer & Simberloff 1996), details concerning the extant and magnitude of hybridization and genetic introgression between two Hwamei species are critical for formulating proper conservation measures. A set of useful nuclear markers should provide such information. In this study, we identified and characterized 20 anonymous nuclear loci from the Taiwan Hwamei by sequencing random clones from a partial genomic library as suggested by Primmer et al. (2002). We tested the applicability of these loci in Hwamei as well as a wide range of passerines to explore the potential utility of these loci.

We digested total genomic DNA extracted from a Taiwan Hwamei individual with restriction enzyme Sau3A1. Digested DNA fragments were ligated into pBluescript II KS(+) vector (Stratagene) and transformed into JM109 competent cells (Promega). The universal primer T3 and T7 were used to sequence both strands of inserts from 760 random clones by the DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBACE on a MegaBACE 1000 autosequencer (Amersham Biosciences). We designed primers from inserts of 72 clones using program fastpcr 2.3.10 (available at http://www.biocenter.helsinki.fi/bi/Programs/download.htm) to generate amplicons of length between 200 bp and 700 bp.

Polymerase chain reaction (PCR) was set up in a volume of 12.5 µL containing 10–20 ng genomic DNA, 0.2 µm of each primer, 0.5 mm dNTP, 1× PCR buffer and 0.5 U Taq DNA polymerase (Amersham Biosciences). The PCR profile was as follows: 94 °C for 2 min, then 40 cycles of 94 °C for 20 s, annealing at the optimal temperature (Table 1) for 30 s, and 72 °C for 1.5 min, followed by a final extension at 72 °C for 3 min. All the PCRs were performed in an iCycler thermal cycler (Bio-Rad) and sequences were proofread by eyes with the aid of software sequencher (version 4.2, Gene Codes). Only 20 loci (8556 bp in total) with low background noise in sequence chromatograms were subjected to the further characterization.

We characterized these loci by sequencing PCR products of a panel of nine to 15 Taiwan Hwamei individuals (18–30 chromosomes) with unknown relationships. We verified any SNPs by sequencing from both directions and inspecting chromatograms visually. We used nucleotide diversity, θ, to quantify the nucleotide variation of each locus. θ was defined as θ = K/L*[1⁻¹+ 2⁻¹+ 3⁻¹ + … + (n – 1)⁻¹] (Watterson 1975), where K was the number of polymorphic sites, L sequence length (bp), and n the number of chromosomes screened. This equation corrected for the differences in sequence length and number of chromosomes screened among loci.

Results of characterizing 20 anonymous nuclear loci are summarized in Table 1. We identified 46 SNPs from 15 polymorphic loci. SNP frequency was one per 186 bp in average, and comparable to that in two Ficedula flycatchers (Primmer et al. 2002). Nucleotide diversity ranged from 0.00054 to 0.00371 (mean = 0.0078). Using only the most polymorphic SNP site within each locus, we found no evidence of violation to expectations of the Hardy–Weinberg equilibrium and gametic equilibrium (genepop version 3.4, Raymond & Rousset 1995). Consequently, these 15 polymorphic fragments should be regarded as unlinked loci. Our results confirmed that screening of the anonymous nuclear loci is an effective strategy to develop SNP markers for nonmodel organisms.

We attempted to amplify these 20 loci from 17 species of eight passerine families to test their cross-species applicability (Table 2). For all the loci, PCR products of expected sizes could be successfully amplified in some species (ranged from 1 to 16 species; mean = 7.9 species). In the Hwamei, 13 of the loci were applicable, a proportion encouraging further development of interspecific SNP sites for studies of hybridization and genetic introgression between hwameis. Sixteen loci could be amplified from non-Timaliinae passerines (ranged from one to six families). Because the presence of nonspecific bands was a major cause for PCR failures (Table 2), applicability of these loci may be enhanced by optimizing PCR conditions carefully. Nonetheless, the broad cross-species applicability make these anonymous nuclear loci a set of useful molecular tools for studying molecular ecology, conservation genetics and evolutionary biology in a broad range of passerine birds.