A Global Analysis of Y-STR INRA189 Polymorphism in Chinese Domestic Yak Breeds/Populations

Simple Summary Y chromosome-specific markers have been widely used in studying the origin, migration, diversity, and population relationship in several mammalian species. So far, the investigations on yak paternal genetics were mostly based on Y-SNPs information, but very few on Y-STR markers. In this study, we comprehensively investigated the polymorphism of Y-STR INRA189 locus, identified the geographical distribution pattern of its alleles in 15 Chinese domestic yak breeds/populations, and provided basic data for yak paternal genetic analysis. Our results showed that INRA189 is an intermediate polymorphic Y-STR marker with six alleles present among 15 Chinese domestic yak breeds/populations and that the variation among six alleles was due to different numbers of repetitions of a TG dinucleotide motif. Abstract The objective of this study was to probe into the polymorphism of Y-STR INRA189 and identify the geographical distribution pattern of its alleles in the Chinese domestic yak gene pool. We examined the variation at INRA189 locus in 682 male yaks representing 15 breeds/populations in China. The results showed that six alleles, including five reported previously (149, 155, 157, 159, and 161 bp) and a new one identified in this study (139 bp), were detected at INRA189 locus based on genotyping analysis. The frequencies of six alleles varied among the 15 yak breeds/populations with a clear phylogeographical pattern, which revealed the paternal genetic difference among Chinese yak breeds/populations. The average polymorphism information content (PIC) among the 15 yak breeds/populations was 0.32, indicating INRA189 to be an intermediate polymorphic Y-STR marker (0.25 < PIC < 0.5) in yak. Sequence alignment revealed that the variations among six alleles at INRA189 were defined by a TG dinucleotide repeat motif, which repeated for 12, 17, 20, 21, 22, and 23 times, corresponding to the alleles of 139, 149, 155, 157, 159, and 161 bp, respectively. Therefore, we believe that the polymorphic yak Y-STR INRA189 can be used to characterize male-mediated genetic events, including paternal genetic origin, diversity, and evolution.


Introduction
Yak (Bos grunniens) is one of the native mammalian species that lives on the Qinghai-Tibetan Plateau and its adjacent territories. They provide products (milk, meat, hair, hides, and manure for fuel) and services (draught, packing, and riding) to pastoralists and agro-pastoralists living in these areas [1]. China owns rich yak genetic resources with more than 15 million animals represented by 12 officially recognized breeds and several local populations [2].
Y-chromosome-specific polymorphisms (such as single nucleotide polymorphisms (SNPs), insertion-deletion mutations (Indels), microsatellites with short tandem repeats (STRs) and copy number variations (CNVs)) can add significant genetic information to what can be inferred from mtDNA and autosomal variations [3,4]. Thus far, five Y-SNP markers have been used to explore the origin, diversity, and phylogeographical structure of the Chinese domestic yak [5][6][7], whereas a few Y-chromosome-specific microsatellite (Y-STR) markers were identified in yak [8]. Collectively, several previous studies focused on the specificity and polymorphism of INRA189 locus in yak. For example, Han et al. (2000) identified the male specificity and detected three alleles at INRA189 locus in one Bhutanese yak population and three Chinese yak breeds (Gannan, Tianzhu, and Datong) [9]. Nguyen et al. (2005) found two alleles at this locus in one Swiss yak population [10]. In addition, our analysis on INRA189 in three Chinese yak breeds (Gaoyuan, Huanhu, and Datong) indicated that this marker possessed four alleles [11]. It can be seen from the above-mentioned studies that only a few Chinese domestic yak breeds/populations were tested for INRA189 polymorphism, whereas no comprehensive study was reported in all Chinese yak breeds/populations. Under this scenario, the aim of this study was to further evaluate the polymorphism and identify the geographical distribution pattern of different alleles at Y-STR INRA189 locus in 15 domestic yak breeds/populations in China.

Materials and Methods
Blood or tissue samples were collected from 682 male yaks across the distribution ranges of 15 breeds/populations in China, namely Gaoyuan, Datong, Huanhu, Xueduo, Tianzhu, Gannan, Jiulong, Maiwa, Sibu, Niangya, Pali, Leiwuqi, Bazhou, Taxian, and Zhongdian ( Figure 1, Table 1). Five females of the Gaoyuan breed were selected as controls. These yaks were selected randomly based on pedigree information or detailed interviews with owners to minimize the degree of relationship among individuals. The study was approved by the Faculty of Animal Policy and Welfare Committee of Qinghai University.

Introduction
Yak (Bos grunniens) is one of the native mammalian species that lives on the Qinghai-Tibetan Plateau and its adjacent territories. They provide products (milk, meat, hair, hides and manure for fuel) and services (draught, packing and riding) to pastoralists and agro-pastoralists living in these areas [1]. China owns rich yak genetic resources with more than 15 million animals represented by 12 officially recognized breeds and several local populations [2].
Y-chromosome-specific polymorphisms (such as single nucleotide polymorphisms (SNPs), insertion-deletion mutations (Indels), microsatellites with short tandem repeats (STRs) and copy number variations (CNVs)) can add significant genetic information to what can be inferred from mtDNA and autosomal variations [3,4]. Thus far, five Y-SNP markers have been used to explore the origin, diversity, and phylogeographical structure of the Chinese domestic yak [5][6][7], whereas a few Y-chromosome-specific microsatellite (Y-STR) markers were identified in yak [8]. Collectively, several previous studies focused on the specificity and polymorphism of INRA189 locus in yak. For example, Han et al. (2000) identified the male specificity and detected three alleles at INRA189 locus in one Bhutanese yak population and three Chinese yak breeds (Gannan, Tianzhu and Datong) [9]. Nguyen et al. (2005) found two alleles at this locus in one Swiss yak population [10]. In addition, our analysis on INRA189 in three Chinese yak breeds (Gaoyuan, Huanhu and Datong) indicated that this marker possessed four alleles [11]. It can be seen from the above-mentioned studies that only a few Chinese domestic yak breeds/populations were tested for INRA189 polymorphism, whereas no comprehensive study was reported in all Chinese yak breeds/populations. Under this scenario, the aim of this study was to further evaluate the polymorphism and identify the geographical distribution pattern of different alleles at Y-STR INRA189 locus in 15 domestic yak breeds/populations in China.

Materials and Methods
Blood or tissue samples were collected from 682 male yaks across the distribution ranges of 15 breeds/populations in China, namely Gaoyuan, Datong, Huanhu, Xueduo, Tianzhu, Gannan, Jiulong, Maiwa, Sibu, Niangya, Pali, Leiwuqi, Bazhou, Taxian, and Zhongdian ( Figure 1, Table 1). Five females of the Gaoyuan breed were selected as controls. These yaks were selected randomly based on pedigree information or detailed interviews with owners to minimize the degree of relationship among individuals. The study was approved by the Faculty of Animal Policy and Welfare Committee of Qinghai University.  Genomic DNA was extracted using the Aidlab genomic DNA extraction kit (Aidlab Biotechnologies Co., Ltd., China). A pair of primers (PF: 5 -TTTTGTTTCCCGTGCTGAG -3 ; PR: 5 -GAACCTCGTCTCCTTGTAGCC-3 ) described by Kappes et al. (1997) was used to amplify the INRA189 locus [12] following the protocols for PCR amplification and genotyping analysis reported in our previous study [11].
Purified PCR products were scanned together with the GS-500 Liz size standard in ABI 3730XL Sequencer (Applied Biosystems, Foster City, CA, USA). Allele sizes were determined for each individual using GeneMarker v1.97 (http://www.softgenetics.com/GeneMarker.html) and the allele frequencies were estimated using the GENETIX 4.05 software [13]. In order to obtain the informativeness of the INRA 189 marker, the polymorphic information content (PIC) was calculated using PIC_CALC 0.1 software [14]. To confirm the sequence characterization of each allele by DNA sequencing, we cloned PCR amplicons from different alleles into the pUCm-T vector (Sangon Biotech Co., Ltd., Shanghai, China) and carried out sequence alignment using BioEdit v7.2.5 software with Clustal W multiple alignment [15].

Results and Discussion
In accordance with previous reports [9][10][11], our current results also showed that INRA189 was only present in male yaks, which further confirmed it to be a yak Y-chromosome specific microsatellite marker. In analysis of yak INRA189 polymorphism, previous studies have identified five alleles (149, 155, 157, 159, and 161 bp) at INRA189 locus based on the genotyping analysis of one Swiss population, one Bhutanese population and five Chinese breeds (Gannan, Tianzhu, Gaoyuan, Huanhu, and Datong) [9][10][11]. Herein, we comprehensively examined the polymorphism of INRA189 marker in 15 Chinese yak breeds/populations. Our results showed that six alleles, including those five reported previously [9][10][11] and a new one identified in this study (139 bp), were detected in 15 yak breeds/populations ( Figure 2, Table 1). The frequencies of six alleles varied among 15 yak breeds/populations ( Figure 1, Table 1). The 157 bp allele was predominant and shared among all yak breeds/populations with frequencies ranging between 6.1% in Pali and 100% in Leiwuqi and Zhongdian, followed by the 155 bp allele present in 13 breeds/populations with frequencies varying from 2.9% in Gannan and Maiwa to 93.9% in Pali. The 159 bp allele was only present in five breeds/populations (Datong, Huanhu, Xueduo, Maiwa, and Bazhou) at an overall frequency of 1.6% while the 161 bp allele was only found in Gaoyuan, Tianzhu, and Taxian breeds/populations at 3.1%. Interestingly, private 139 bp and 149 bp alleles were only detected in Gaoyuan breed with frequencies of 0.5% and 1.4%, respectively. The above results showed that 155 and 157 bp alleles were the dominant alleles, but others including 139, 149, 159, and 161 bp alleles were rare alleles in domestic yak. Generally, dominant alleles reflect basic breed/population characteristics while rare alleles display breed/population uniqueness of great value in breeding practice. Therefore, Gaoyuan, Datong, Huanhu, Xueduo, Tianzhu, Maiwa, Bazhou, and Taxian breeds/populations should be paid more attention in their future conservation and breeding programs as they owned rare alleles. Furthermore, Pali breed could be regarded as a unique genetic resource because it had a higher frequency of 155 bp allele, which was different from other yak breeds/populations. It is evident that genetic drift, natural selection, and artificial breeding may have played a role in driving the differences in the number and frequency of alleles at INRA189 locus among the 15 yak breeds/populations. In addition, based on the autosomal microsatellites [16], mtDNA [17], and Y-SNP markers [7], previous studies have explored the genetic differences among some Chinese yak breeds/populations. Here, although the information from INRA189 marker was limited, our results reflected the genetic differences among these yak breeds/populations to some extent ( Figure 1, Table 1). Even though our results are not completely consistent with those of previous studies [7,16,17], INRA189 polymorphism among these domestic yak breeds/populations will add paternal genetic information to what can be inferred from autosomal microsatellites, mtDNA, and Y-SNP markers. Therefore, we believe that the Y-STR INRA189 combining with other molecular markers (e.g., Y-SNPs) can be used to characterize male-mediated genetic events in yak, including paternal origin, evolution, diversity, and population structure. The polymorphism information content (PIC) provides an estimate of the discriminatory power of a locus. The value of PIC reflects the level of polymorphism of microsatellite markers in animal populations. According to the classification standard of Botstein et al. (1980) [18], markers with PIC values greater than 0.5 are considered to be very informative, values between 0.25 and 0.50 are moderately informative while values lower than 0.25 are minimally informative. In our previous study, the results showed that the average PIC for INRA189 marker in three Chinese yak breeds (Gaoyuan, Huanhu, and Datong) was 0.2379, which indicated that this marker was a minimally informative STR marker in yak (PIC < 0.25). However, in this study, the average PIC value among the 15 domestic yak breeds/populations was 0.32, indicating INRA189 to be an intermediately polymorphic Y-STR marker (0.25 < PIC < 0.5). This result changed our previous view on the polymorphism of this marker in yak [11].
The result of sequence alignment in this study revealed the variation among the six alleles defined by a TG dinucleotide repeat motif, which repeated for 12, 17, 20, 21, 22, and 23 times corresponding to the alleles of 139, 149, 155, 157, 159, and 161 bp, respectively (Figure 3). Our result not only identified the sequence of the Y-STR INRA189 locus, but also clarified the phylogeographic pattern among the six alleles in domestic yak.

Conclusions
In conclusion, our results showed that INRA189 locus is an intermediate polymorphic Y-chromosome-specific microsatellite marker in yak. We identified six alleles (139, 149, 155, 157, 159, and 161 bp) with varying frequencies among 15 Chinese yak breeds/populations that followed a clear phylogeographical pattern, indicating paternal genetic differences among these yak breeds/populations. The variations among the six alleles were due to a TG dinucleotide motif.