Genetic diversity of Tibetan goats of Plateau type using microsatellite markers

The 10 microsatellite markers (XBM7, XBM11, XBM16, XBM19, XBM24, XBM31, XBM84, TGLA53, SRCRSP-10 and ILS005) were selected to investigate the genetic diversity of Tibetan goat of Plateau type (NM, n=108), and the other 5 goat populations, i.e., Tibetan goat of Valley type (TG, n=36), Baiyu black goat (BY, n=36), Jianchang black goat (JC, n=36), Meigu goat (MG, n=36) and Xinjiang goat (XJ, n=32) were served as control. The mean polymorphism information content, heterozygosity and effective allele number of these 6 populations were 0.660/0.777/4.476, 0.716/0.797/4.9416, 0.631/0.673/3.061, 0.649/0.680/3.125, 0.629/0.680/3.125 and 0.561/0.793/4.840 respectively. The allele frequencies of Tibetan goat of plateau type in 10 microsatellite loci were greatly different with other 5 goat populations. The 6 goat populations were grouped into three distinct clusters: the Tibetan goat cluster (NM goat and TG goat), Sichuan goat cluster (JC goat, MG goat and BY goat), and Xinjiang goat cluster (XJ goat). These 3 distinct clusters were finally clustered together. The genetic differences among populations were in accordance with their geographical and historical origins.


Introduction
The Tibetan goat is mainly distributed in Qinghai-Tibet plateau of China.It is regarded as one of the world's most remarkable domestic animals as it thrives in conditions of extreme harshness and deprivation while providing meat and down for people.The archaeological evidence from Kaluo ruin in Changdu of Tibet suggests that the history of China's Tibetan goat industry is at least 4 000 years old (Wang et al. 1993).At the present time, the total Tibetan goat population is estimated to number around 18 million in China.They were classified as plateau type and valley type (Ouyang et al. 1995).Tibetan goats are famous for their down production and good quality.In fact, what was called Cashmere goat by British businessmen in international cashmere trade during 1770s is Tibetan goat (Wang et al. 1994).There is, however, little study have been undertaken to investigate the genetic characteristics of Tibetan goat using molecular biological techniques.Microsatellites have been commonly utilized for the assessment of genetic diversity, construction of genetic maps, quantitative trait loci mapping and parentage testing etc. (Batendse et al. 1997, Buchanan et al. 1994, Li et al. 2004, Wang et al. 2004, Jin et al. 2005, Agha et al. 2008, Manatrinon et al. 2008, Ślaska et al. 2008, Kusza et al. 2010).Li et al. (2002) analyzed genetic relationships among twelve Chinese indigenous goat populations based on 26 microsatellite markers.Wang et al. (2006) studied microsatellite DNA polymorphism of 9 breeds (populations) of black goats in Sichuan province using 10 microsatellite markers, and the result showed these 9 breeds (populations) have a high genetic diversity.This observation is consistent with population's bodily form, economical purpose and geographical distributions.There is, however, no information is available for genetic diversity of Tibetan goat of plateau type by using microsatellites markers.
Therefore, the objective of this study was to analyze genetic diversity of Tibetan goat by using 10 microsatellite loci.The results would be useful for the protection and utilization of Tibetan goat genetic resources in China.

Sampling and DNA extraction
Genomic DNAs were prepared from whole blood, which were collected from female goats of six populations: plateau-type Tibetan goats in Nima county of Tibetan Autonomous Region (NM, n=108), valley-type Tibetan goats in Mao county of Sichuan province (TG, n=36), Baiyu goats in Baiyu county of Sichuan (BY, n=36), Jianchang black goats in Huili county of Sichuan (JC, n=36), Meigu goats in Meigu county of Sichuan (MG, n=36), and Xinjiang goats in Changji city of Xinjiang Region (XJ, n=32).All breeds were kept at their own origin area.Individuals from each breed were sampled with the proportion of male:female equaling 1:4, according to Barker's (1994) guidelines for sample requirements of genetic diversity evaluation.Owners were questioned in detail to minimize the sampling of closely related individuals.The original and distribution of these six populations were shown in Figure 1.Genomic DNAs from samples were extracted according to procedures described by Wang et al. (2006).

PCR and microsatellite analysis
Ten pairs of microsatellite primers used were synthesized (TakaRa, Dalian, China) and their sequences were shown in Table 1.PCR was accomplished in a total 15 μl of the following mixture: 25 ng/μl of genomic DNA, 20 pmol/l of each primers, 2 U ExTaq DNA polymerase, 100 μmol/l of each dNTPs, standard PCR buffer, 2 mmol/l MgCl 2 and ddH 2 O. PCR amplification was as follows: first step was performed by initial denaturation for 4 min at 94 °C, followed by 33 cycles at 94 °C for 40 s, 54-59 °C for 40 s and 72 °C for 1 min.An extension at 72 °C for 5 min.PCR products were stored at 4 °C.Amplified fragments were analyzed on 9 % polyacrylamide denaturing sequencing gel which was stained in 0.1 % AgNO 3 solution.The PCR product size was calculated according to the pBR322 DNA/mspI marker on the computer.

Number of alleles and allele frequencies
Genotypes were observed according to the size of amplified fragments (a, b, c……): a single band indicated homozygote; two bands indicated heterozygote (Figure 2).If SSR primers gave no amplified product were treated as missing values and they were discarded in the following study.The allele number and frequencies of 10 microsatellite loci in the 6 goat populations were shown in Table 2.The number of alleles per locus ranged from 3 (XBM31) to 8 (XBM16) with an average value of 5.7 in NM plateau type Tibetan goat.According to standard selection of microsatellite loci (Barker 1994), it has been suggested that microsatellite ought to have at least 4 alleles to be useful for the evaluation of genetic diversity, however, 3 alleles per locus were also used to evaluate genetic diversity in some studies (Li et al. 2010), therefore, all 10 microsatellites were used in this study.

Figure 2 Electrophoresis photograph of locus ILS005 in NM Tibetan goat population
There was a great difference in allele frequencies and genotype frequencies among different populations.For example, allele h (XBM16) was only observed in NM goat and TG goat with frequency of 0.017 where not observed in other breeds analyzed (BY goat, JC goat, MG goat and XJ goat).Allele a and b (ILS005 and XBM24) were not observed in XJ goat, but allele g (ILS005 and XBM24) was only observed in this population with frequency of 0.317 and 0.383, respectively.Allele f (XBM7) was only detected in NM goat, TG goat and XJ goat with frequency of 0.250, 0.250 and 0.333, respectively.

Polymorphism information content (PIC), heterozygosity (H) and effective allele number (Ne)
The PIC, H and Ne of 10 microsatellite loci in six goat populations were shown in Table 3.The PIC ranged from 0.832 (TGLA53) to 0.526 (XBM31).The PIC in 10 microsatellite loci was higher than 0.5, indicated that the genetic diversity was abundant.The PIC was higher in The mean effective number of alleles per locus was 4.476 in NM goat, ranged from 3.333 (ILS005, SRCRSP-10, XBM11 to 10.000 (TGLA53).Among the different populations, TG goat had the highest mean effective number of alleles (4.916) ranged from 3.745 (ILS005, XBM 7, SRCRSP-10, XBM11) to 14.925 (TGLA53).BY goat had the lowest mean effective number of alleles (3.061) ranged from 1.876 (XBM11) to 5.988 (XBM24, XBM31).The mean effective number of alleles in 10 microsatellite loci was 3.750 ranged from 3.061 (BY goat) to 4.916 (TG goat), and the mean effective number of alleles per locus ranged from 2.609 (XBM11) to 5.623 (XBM24).These results indicated that the 10 microsatellite loci of these 6 goat populations had abundant genetic diversity.

Genetic distances and population relationship
Estimates of the Da genetic distances among the 6 populations were shown in Table 4.The smallest Da distances were observed between JC goat and MG goat (0.063), and the largest Da distances between BY goat and XJ goat.Using UPGMA cluster method, the dendrogram of relationships among these six goat populations was obtained (Figure 3).The NM goat and TG goat populations were grouped together.JC goat, MG goat and BY goat populations were grouped together, but the XJ goat has separate branch.These three distinct groups were finally clustered together.The UPMG dendrogram showing the genetic relationship among 6 goat populations using Nei's standard genetic distance for 10 microsatellite loci.
populations (Zheng 1989, Sun 1997).TG goat is distributed in Sichuan, but its historical origin is Tibetan goat.In contrast, BY goat is distributed in Baiyu county, Qinghai-Tibet Plateau, but its historical origin is not Tibetan goat (Figure 1 and 3).Although genetic diversity was not studied in these goat populations by molecular biological methods, evidences in chicken and pigs obtained by microsatellite markers (Chen et al. 1991, Wang et al. 2004) are consistent with those by blood protein markers and mtDNA diversity (Chen et al. 1991, Huang et al. 1998, Mo et al. 2003).
In conclusion, Tibetan goat of plateau type has abundant genetic diversity.It is a unique gene pool, developed for a long history by natural selection and artificial selection, with a good adaptation to the extremely cold and low oxygen conditions.The results would be useful for the protection and utilization of Tibetan goat genetic resources in China.

Figure 1
Figure 1The geographic location of each of 6 populations of goats

Table 1
The sequence structure of the 10 microsatellite primer pairs

Table 4
Nei's standard genetic distances (Da) below the diagonal and standard errors above the diagonal between NM and other 5 goat populations