Development and characterization of 33 SNP markers in a critically endangered Hynobius amjiensis using ddRAD sequencing

Hynobius amjiensis was considered as one of the 29 most threatened amphibian in China. To effectively conserve, manage and recover the population of this critically endangered species, 33 single nucleotide polymorphism (SNP) markers were developed using double digest restriction-site associated DNA (ddRAD) sequencing. The minor allele frequency per locus ranged from 0.0161 to 0.5000. The observed heterozygosity and expected heterozygosity varied from 0.0323 to 0.6667 (average 0.3303) and from 0.0317 to 0.5000 (average 0.2772), respectively. The inbreeding coefficient value ranged between − 0.3315 and 0.0000. No significant deviation from Hardy–Weinberg equilibrium (P > 0.05) were found in all loci. These novel SNPs will be helpful for the population genetic assessment and conservation of H. amjiensis.


Introduction
Anji salamander (Hynobius amjiensis) was an endemic species in China. Due to limited distribution and small population size, it was listed as a critically endangered species in the International Union for Conservation of Nature (IUCN) Red List of Threatened Species (Gu and Lau 2004). As one of the 29 most threatened amphibian in China (Zhao 1998), H. amjiensis attracted considerable attention. In recent years, a series of conservation activities which focused on this species were carried out. First, a specific National Natural Reserve was constructed to conserve the species in situ. Additionally, as a key species in national biodiversity survey, it was investigated annually to understand the distribution and population size extensively and deeply by the State Forestry Administration of China from 2012 (Yang et al. 2016). Finally, captive breeding and reintroduction were implemented to try to increase the population size from 2008. However, the basic researches, such as life history, ecology and especially population genetics, were fell far behind the conservation practice. Therefore, to formulate appropriate conservation strategies, it was urgent to perform population genetic studies on this species.
Single nucleotide polymorphism (SNP) had become the most effective molecular marker in conservation genomic researches since its wide genome distribution and high level of polymorphism (Liu et al. 2019). Here, 33 novel SNP loci were developed using double digest restriction-site associated DNA sequencing (ddRAD-seq) in H. amjiensis.

Materials and methods
Thirty-four toe samples of H. amjiensis were collected from Qingliangfeng National Natural Reserve. Genomic DNA was extracted using a standard proteinase K and phenol-chloroform approach (Sambrook et al. 1989   PCR amplifications were performed in a total volume of 20 μL, containing forward and reverse primers at 10 μM of each primer, 10 μL 2 × Taq Plus Master Mix (Vazyme Biotech, China), and 50-100 ng template DNA. The thermal cycling procedure was applied with an initial pre-denaturation step at 95 °C for 3 min, followed by 30 cycles at 95 °C for 15 s, the annealing temperature at 65 °C for 20 s, and elongation at 72 °C for 15 s; a final extension at 72 °C for 10 min. The amplicons for each sample were recycled and purified by 2.0% agarose gel electrophoresis and verified using Sanger sequencing on ABI 3730XL (Thermo Fisher Scientific, US).
Several descriptive statistics were calculated. Specifically, the minor allele frequency (MAF) and P value representing the deviations from Hardy-Weinberg equilibrium (HWE) for each locus were tested using VCFtools (Danecek et al. 2011). The observed heterozygosity (H O ) and expected heterozygosity (H E ) were calculated using PLINK (Chang et al. 2015). The inbreeding coefficient index (F is ) was calculated using Genepop V4.7 (Rousset 2008).

Results
In total, thirty-three SNP loci were identified from 33 primer pairs (Table 1). The minor allele frequency per locus ranged from 0.0161 to 0.5000. The observed heterozygosity and expected heterozygosity varied from 0.0323 to 0.6667 (average 0.3303) and from 0.0317 to 0.5000 (average 0.2772), respectively. The inbreeding coefficient value ranged between -0.3315 and 0.0000. No loci showed significant deviation from Hardy-Weinberg equilibrium (P > 0.05). These novel SNP markers will contribute to the understanding of population genetic structure and be useful for the population conservation of H. amjiensis. Data availability The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval This research was approved by the Ethics Committee of Zhejiang Normal University and it was conducted under Law of the People's Republic of China on the Protection of Wildlife (August 28, 2004).