A core set of microsatellite markers for conservation genetics studies of Korean goral (Naemorhedus caudatus) and its cross-species amplification in Caprinae species.

In order to screen microsatellites for conservation genetics studies of the species, a total of 23 microsatellite loci from Korean goral (Naemorhedus caudatus), including 15 previously developed loci and 8 new loci in this study, were tested. Eleven microsatellites were screened and subjected to cross-species amplification using a test panel of four Caprinae species, Japanese serows (Capricornis crispus), Chinese gorals (Naemorhedus goral), Northern chamois (Rupicapra rupicapra) and domestic goats (Capra hircus). In addition, all eleven microsatellites (SY3A, SY12A, SY12B, SY48, SY58, SY71, SY76, SY84, SY84B, SY112, and SY129) satisfied the criteria to be a core set of microsatellites. This core set of microsatellites and cross-species amplification of Korean goral microsatellites were found to be helpful for high-resolution studies for conservation and management of Korean goral and other endangered Caprinae species.

The long-tailed goral (Naemorhedus caudatus) is the only mountain ungulate of the tribe Caprini found in Korea [4]. Since it is known that Korean goral populations have been reduced and fragmented by habitat reduction and road construction [11], understanding the current status of genetic diversity within and between populations is an essential component for their long-term survival. Few molecular studies measuring the genetic diversity of Korean goral have been carried out. Recently, 15 microsatellite loci were isolated and characterized for 20 Korean gorals [1]. Data from that study revealed that nine of 15 loci were in Hardy-Weinberg equilibrium (HWE) and only five of the 9 HWE loci exhibited more than 0.5 in polymorphic information content (PIC), an index of polymorphic level. Therefore, there is need for a sufficient number of polymorphic loci for genetic variability and population structure studies.

Recent studies have demonstrated that the cross-species amplification approach is advantageous as it allows population studies on species for which microsatellites have not yet been developed. Korean goral loci which had successful cross amplification in related endangered species such as Japanese serow (Capricornis crispus), Chinese goral (Naemorhedus goral), and Northern chamois (Rupicapra rupicapra), are thought to be helpful for the conservation genetics study of such species. Here, we report eight novel polymorphic microsatellite loci derived from a Korean goral genomic library and a core set of microsatellite markers that can be used across laboratories for future population genetics studies and conservation management of Korean goral. We also present cross-species amplification of Korean goral loci in other Caprinae species.

The enrichment protocols described by An et al. [1] were used to develop Korean goral specific microsatellites, with a slight modification by An et al. [1]. PCR amplification and genotyping were carried out as described by An et al. [1]. Genomic DNA from 38 Korean gorals, 10 Japanese serows, 7 Chinese gorals, 10 Northern chamois, and 5 domestic goats (Capra hircus) were tested in this study. Deviations from HWE and linkage disequilibrium between loci were tested by the Markov chain method implemented in GENEPOP version 4.0 [8]. Significant levels were adjusted using Bonferroni correction for multiple testing. Expected (He) and observed (Ho) heterozygosities, and PIC were calculated using the CERVUS version 3.0 [5].

A core set of microsatellites for population genetics studies of Korean goral was screened by following criteria described by Kim et al. [6]. We investigated 1) readability of each marker, 2) level of polymorphisms, 3) occurrence of null alleles, 4) level of selective neutrality [2], and 5) linkage equilibrium between loci.

A total of 8 microsatellite loci were successfully optimized for 38 Korean gorals (Table 1). The observed number of alleles per locus ranged from 2 to 13 averaging 6.5 per locus. Expected (He) and observed (Ho) heterozygosity were in the range of 0.417~0.836 (mean = 0.669) and 0~0.711 (mean = 0.437) respectively. Genetic diversity, as indicated by mean He estimates in Korean goral, was not significantly higher than certain ungulates (0.842 in musk deer [10], 0.76 in Chital deer [3]).

Table 1

Characteristics of the 8 newly developed and optimized loci for Korean goral (Naemorhedus caudatus)

*Significant deviation from Hardy-Weinberg equilibrium at α = 0.05. T: annealing temperature, K: number of allele, size range: size range of PCR product, Ho and He: observed and expected heterozygosities, PIC : polymorphic information content.

After correction for multiple comparisons by applying a sequential Bonferroni correction (α = 0.05) [7], Fisher's exact tests revealed four loci that were deviated from HWE. Although these deviations could be caused by null alleles, these results are more likely an artifact of biased sampling from captive specimens exhibiting a deficiency of heterozygotes (i.e. Wahlund's effect) [9].

A total of 23 loci, 8 new loci from this study and 15 loci developed by An et al. [1], were tested to determine whether they meet the criteria to be defined core sets of microsatellite markers as suggested by Kim et al. [6]. Eleven microsatellites satisfied all criteria (i.e. moderate to high polymorphism, no evidence of null alleles, apparent selective neutrality, and no linkage with other loci) and are recommended for future population genetics studies of Korean goral (Table 2). Of the core set of 11 loci tested, all loci were successfully amplified for Japanese serow and Chinese goral, whereas SY12A for domestic goat and SY48 for Northern chamois showed no amplification, and SY71, SY76, SY112, and SY129 have not been investigated for Northern chamois (Table 2).

Table 2

Summary of loci characteristics for inclusion in the core set of Naemorhedus (N.) caudatus microsatellites and cross-species amplification of the N. caudatus loci in four caprine species

*The core set of microsatellites of N. caudatus. +indicates the successful cross species amplification for 11 core set of microsatellites. -implies no amplification. ND: not determined yet (size range is available upon requested). †Based on the expected heterozygosity (He); 0< low (L) <0.4, 0.4 ≤ moderate (M) <0.8, 0.8 ≤ high (H) <0.9. ‡Presence of null allele based on MICRO-CHECKER analysis. §Yes denotes the null hypothesis of selective neutrality against the presence of selection was not rejected for that locus at p = 0.05, and No denotes the null hypothesis of neutrality was rejected for that locus. ∥Linkage disequilibrium were present at p = 0.05 after Bonferroni correction for multiple testing.

This core set of microsatellites can be applied to better understand the genetic diversity and population structure of Korean gorals as well as other endangered Caprinae species. In addition, the applicability of cross-species amplification of Korean goral microsatellites could facilitate high-resolution studies for the conservation and management of other Caprinae species. This could lead to better decision-making in regards to the improvement of conservation management plans.