Genetic diversity estimation of Yunnan indigenous goat breeds using microsatellite markers

Abstract Background To assess the genetic diversity of seven Yunnan indigenous goat populations (Fengqing hornless goat, Mile red‐bone goat, Longling goat, Ninglang black goat, Black‐bone goat, Yunling black goat, and Zhaotong goat), their population structures were investigated using 20 microsatellite markers. Results The results indicated that the genetic diversity of these goats was rich. The observed heterozygosity ranged from 0.4667 ± 0.0243 to 0.5793 ± 0.0230, and the mean number of alleles ranged from 4.80 ± 1.61 and 4.80 ± 1.64 to 6.20 ± 2.93. The population structure analysis showed that these seven goat populations were separated into two clusters, consistent with the results from phylogenetic networks, pairwise differences, and STRUCTURE analyses. We speculate that this may have been caused by natural geographical isolation, human migration and economic and cultural exchanges. We suggest removing CSRD247 and ILSTS005, two loci identified to be under positive selection in the present study, from the microsatellite evaluation system of goats. Conclusions The present study may provide a scientific basis for the conservation and utilization of Yunnan indigenous goats.

Yunnan is located in a low-latitude inland area at the southwestern border of China with a subtropical plateau monsoon type climate (Liu, Wang, & Zhang, 2016). In addition, Yunnan Province contains the largest ethnic group in China, including 25 ethnic minorities, with populations over 6,000. Among these individuals, 15 ethnic groups are unique to Yunnan, and this population accounts for more than 80% of the total ethnic population of China (Pu, 2010). Furthermore, Yunnan is one of the important birthplaces of human civilization in China. The Yuanmou people, who lived 1.7 million years ago, were the earliest human beings in China and Asia (Gao, 2015;Yao, Deng, & Zhu, 2005). During the Warring States Period, the Yi tribe lived in Yunnan (Pu, 2010). Moreover, the number of plant and animal species in Yunnan is the highest in China.
Goats are the most important source of meat in this region and play an important role in economic development and social stability.
Additionally, goats are also the objects of totem worship of many local minorities, affecting the spiritual worlds of the local indigenous peoples (Lai, 2012).
According to historical records, the domestication of local goats occurred 3,000 years ago (China National Commission of Animal Genetic Resources, 2011). Through long-term natural selection and artificial selection, a large number of indigenous goat breeds with environmental adaptations and special genetic phenotypes have been created (Wang et al., 2017). For example, Ninglang black goat (NLG) and Yunling black goat (YLG) have black coats, Zhaotong goat (ZTG) and Longling goat (LHG) have extremely high slaughter rates, Mile red-bone goat (HGG) and Black-bone goat (WGG) have significant bone pigmentation, and Fengqing hornless goat (FQG) are naturally hornless or have horn dysplasia (China National Commission of Animal Genetic Resources, 2011).
Therefore, an assessment of the diversity and population structure of these goats not only helps to understand their domestic history and conservation status but also plays an important supporting role for future breeding improvement and industrial development of local goat populations.

| Animals, DNA extraction, and genotyping
The experimental conditions in the present study were approved by the Committee on the Ethics of Animal Experiments of Southwest University (No. [2007] 3) and the Animal Protection Law of China (this study did not directly carry out any relevant experiments on animals). All samples were collected from venous blood. The sample supply department guaranteed that after blood collection, the blood collection site was disinfected with alcohol to ensure no infection. At the same time, the animals were observed to survive normally after 1 week without any adverse effects. The genomic DNA from 175 individual blood samples of seven Yunnan indigenous goat populations (sample information see Table 1), which was supported by the Yunnan Animal Science and Veterinary Institute, was extracted by a standard phenol-chloroform protocol (Sambrook & Russell, 2001) and quantified with a DTX microplate reader (Beckman Coulter).
Approximately 2 µl of PCR product was diluted with 10 µl of auto-
A phylogenetic neighbor-joining network was reconstructed with Reynolds genetic distance (Reynolds, Weir, & Cockerham, 1983) by using the PHYLIP software package (Felsenstein, 2005) and visualized by SPLITSTREE4 (Kloepper & Huson, 2008). STRUCTURE 2.3.3 was utilized to assess the population structure with Bayesian clustering and an admixture model from K = 2 to K = 11 in 100 runs. Assignment clusters were made with a burn-in of 50,000, and 100,000 Markov Chain Monte Carlo iterations. CLUMPP software (Jakobsson & Rosenberg, 2007) was used to merge all runs for each K, and the results were visualized by DISTRUCT 1.1 (Rosenberg, 2004). Finally, the best K value was estimated using the STRUCTURE Harvester (Earl & vonHoldt, 2012) online tool.
The F IS within the population ranged from 0.024 (p = 0.2311) in FQG to 0.214 in WGG (p = 0.0004). No population generated a p-value for inbreeding coefficients that was significantly different from zero, indicating an adjusted nominal level (5%) of 0.00036 based on 2,800 randomizations of the p-value for F IS (Table 4).
In addition, according to the chi-square test of F ST , all populations showed significant divergence (p < 0.05) from each other (Table 5

| D ISCUSS I ON
In the present study, we assessed the genetic diversity and population structure of seven Yunnan indigenous goat populations using 20 microsatellites. The results showed high diversity (heterozygosis levels, PIC, and N A ) of all microsatellite loci across the seven Yunnan goat populations, suggesting that these markers were adequate for assessing the diversity of these populations. In addition, several markers showed a deviation from HWE, which could have been due to the small sample size or the potential occurrence of recent population genetic events (E et al., 2018).
In a previous study, the diversity of three populations (YLG, ZTG, and FQG) analyzed in the present study was estimated, showing that the H o was 0.5517, 0.5299, and 0.5178 in YLG, ZTG, and FQG, respectively (Wei et al., 2014), consistent with the results obtained in the present study, and indicating that the 20 highly polymorphic microsatellite loci utilized were sufficient to estimate the diversity of these seven populations.
In addition, the H o value of every population was smaller than the H E values, and 1-8 markers deviated from HWE within a population. Deviations from HWE occur when individual populations are substructured into smaller flocks within populations that are isolated from each other or improperly managed by humans due to inbreeding Granevitze et al., 2007). In addition, the F IS values for YLG (F IS = 0.160) and ZTG (F IS = 0.174) were higher than the values reported in a previous study (Wei et al., 2014), except for FQG.
Although the p values for F IS in the seven populations were not significantly different from zero after adjusting according to the nominal level (p = 0.00036), the p values for F IS in five populations (LHG, NLG, WGG, YLG, and ZTG) were nearly significant (p = 0.0004).
These findings further suggest that these populations might be more likely to experience inbreeding.  (Wang, Hao, & Yang, 2016). Therefore, the present study provides supporting evidence indicating that a gene flow existed recently or during multicomplex ancient domestication of these four goats.
Moreover, Wei et al. (2014)  China (Yuan, Cheng, & Zhou, 2012 increasing evidence has supported a common genetic pattern in domestic animals (Jin et al., 2012;Lai, Chen, Liu, & Yao, 2007;Liu et al., 2006;Wu et al., 2007) due to the restricted gene flow of livestock between goats as a result of the Hengduan Mountains (Wei et al., 2014). F I G U R E 1 Population average pairwise differences among the seven goat populations. The area above the diagonal shows the average number of pairwise differences between populations, the diagonal elements represent the average number of pairwise differences within population, and the area below the diagonal shows the corrected average pairwise difference Last, but not least important, we identified two markers (CSRD247 and ILSTS005) under positive selection. The domestic and wild animals were clustered according to their geographic locations and breeding and management histories by using microsatellites (Granevitze et al., 2007;Gvozdanović et al., 2018;Su et al., 2017). Those widely used microsatellite markers were codominant and assumed to be neutral drifting. However, positive selection by gradual fixation of dominant genotypes leads to a decline in genetic diversity in the population at this locus and an increase in population disagreement (Gordo et al., 2018;Rasigade, Hollandt, & Wirth, 2018). Therefore, we suggest that these two sites (CSRD247 and ILSTS005) under positive selection should be removed from the microsatellite evaluation system of goats to avoid interference from the positive selection sites on the population structure and genetic diversity evaluation.
However, further studies are needed to determine whether this finding is appropriate.

| CON CLUS ION
In the present study, we estimated the diversity and population structure of 175 Yunnan goats from seven indigenous populations using 20 microsatellite markers. The results indicated that although these goats showed high diversity within the populations, the risk of inbreeding still existed. Second, the two-population F I G U R E 2 Neighbor-joining network of the seven goat populations derived by Reynolds genetic distance by using five of the 20 genomic microsatellite markers F I G U R E 3 Clustering diagrams for the seven goat populations using STRUCTURE. The best K value is 3 according to ΔK = m| L″(K)|/s|L(K)| | 5923 E Et al.
structures clustering from seven goats could be caused by geographical, economic, and cultural characteristics. Finally, we recommend removing CSRD247 and ILSTS005 loci from future studies on microsatellite evaluation system of goats, due to possible positive selection.

ACK N OWLED G M ENT
We thank the Yunnan Animal Science and Veterinary Institute for their contribution of the entire animal DNA and blood samples.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N S
YFH, GXE, and QHH, participated in the experimental design and wrote the manuscript; YJZ, MXC, YHM, LZ performed the laboratory experiment and carried out the bioinformatics data analysis; GXE have read and approved the final manuscript.

DATA ACCE SS I B I LIT Y
We agree to deposit your data to a public repository, and the microsatellite genotypes: Dryad https://doi.org/10.5061/dryad.dk16pr0.