Paternal genetic diversity, differentiation and phylogeny of three white yak breeds/populations in China

The white yak, a type of unique and valuable farm animals on the Qinghai-Tibet Plateau, are mainly distributed in Tianzhu (County of Gansu Province), Menyuan, Huzhu and Ledu (three Counties of Qinghai Province) in China. In the present study, the Y-chromosomal genetic diversity, differentiation and phylogeny of three Chinese white yak breeds/populations (Tianzhu, Huzhu and Menyuan) were comprehensively explored using five Y-SNPs (SRY4, USP9Y, UTY19, AMELY3 and OFD1Y10) and one Y-STR (INRA189) markers. The results showed that six Y-haplotypes (H1Y1, H9Y1, H10Y1, H11Y2, H12Y2 and H13Y2) were identified in 97 male yak from three white yak breeds/populations. Among these haplotypes, H1Y1, H10Y1 and H11Y2 were shared by all of breeds/populations and H12Y2 was shared by Tianzhu and Huzhu populations. However, H9Y1 and H13Y2 haplotypes were only detected in Menyuan and Tianzhu white yak populations, respectively. The Y-haplotype diversity was maximum in Huzhu white yak (0.7500 ± 0.0349), the medium in Tianzhu white yak (0.6881 ± 0.0614) and the lowest in Menyuan white yak (0.5720 ± 0.0657). The total Y-haplotype diversity of three white yak breeds/populations was 0.7567 ± 0.0233, indicating rich paternal genetic diversity in white yak. The FST values showed a moderate differentiation between Tianzhu and Menyuan (FST = 0.0763, P < 0.05) populations, but a weak differentiation between Huzhu and Tianzhu white yak breeds/populations (FST = 0.0186, P > 0.05) and Huzhu and Menyuan (FST = − 0.005, P > 0.05) populations. The clustering analysis revealed a close genetic relationship between Huzhu and Menyuan white yak, both were far from Tianzhu white yak breed. The phylogenetic analyses showed that white yak had two Y-haplogroups/lineages (Y1 and Y2) with two potential paternal origins. The findings of present study provide new insight into the basic information for the formulation of molecular breeding programs of white yak. Moreover, it also contributes to the conservation and utilization of this special animal genetic resource.

The mammalian Y chromosome is considered as a symbol of maleness with the characteristics of paternal inheritance, lower mutation rate, and less susceptibility to recombination and reverse mutation 4,5 . It has been used to explore the paternal genetic diversity, origination and population genetic structure of domestic animals. In recent years, two kinds of molecular markers, including Y chromosome microsatellites (Y-STRs) and single nucleotide polymorphisms (Y-SNPs), have been used to investigate the yak paternal population genetics, which showed that Chinese yak breeds/populations owned rich paternal genetic diversity with two different haplogroups/lineages [6][7][8][9][10] . In our recent maternal genetic study on white yak, we found that each of the three Chinese white yak breeds/populations (Menyuan, Huzhu and Tianzhu) carried special maternal genetic information with relatively rich genetic diversity 11 . However, to date, there is no comprehensive paternal genetic analysis of these three Chinese white yak breeds/populations in literature. In this context, the present study explored the Y chromosomal genetic diversity, differentiation, and phylogeny of three Chinese white yak breeds/populations. The Y-SNPs and Y-STR markers were used to clarify the status of white yak genetic resources and to provide theoretical basis for the protection and utilization of white yak populations for future breeding plans and policies.

Materials and methods
Ethical approval. Based on the recommendations of the Regulations for the Administration of Affairs Concerning Experimental Animals of China, the Institutional Animal Care and Use Committee of the Academy of Animal Science and Veterinary Medicine, Qinghai University approved all the animal experiments used in this study. Specific consent procedures were not required for this study following the recommendation of the Regulations for the Administration of Affairs Concerning Experimental Animals of China.
Sample collection and genomic DNA extraction. The blood samples from 34, 32, and 31 male white yak were randomly collected by jugular venipuncture from the core breeding tracts of Tianzhu County of Gansu Province, Huzhu and Menyuan Counties of Qinghai Province in China, respectively (Table 1, Fig. 1). The genomic DNA was extracted using blood DNA extraction kit (Aidlab Biotechnologies Co., Ltd, China) and stored at -20 ℃ for future use. PCR amplification, sequencing and data analysis. By referring to the reported information on five Y-SNPs (SRY4, USP9Y, UTY19, AMELY3, and OFD1Y10) and one Y-STR (INRA189) 7,9,10,12 , the six pairs of primers were synthesized and then used to amplify these markers as previously stated. The purified PCR products were sequenced and typed commercially (Shanghai Sangon Biotech Co., Ltd). The CHROMAS 2.6.6 (Technelysium Pty Ltd, South Brisbane, Australia) was used for sequencing output, while, BIOEDIT 7.2.5 was used for multiple sequence alignment 13,14 . Meanwhile, the typing results of Y-STR INRA189 were analyzed with GENEMARKER 1.91 to determine its allele size 15 . The Y chromosome haplotype/haplogroup (Y-haplotype/ haplogroup) of each male white yak was determined by combined analysis of the above two kinds of molecular markers. The number of Y chromosome haplotypes of white yak breeds/populations was determined by DNASP 5.10.01 and ARLEQUIN 3.11 software 16,17 . Sampling sites and haplotype distributions maps were created using ArcGIS 10.7 to show geographic relationship. In addition, the Y chromosome haplotype diversity (Hd ± SD) was calculated to evaluate the paternal genetic diversity of three white yak breeds/populations. The pairwise fixation index (F ST ) values were calculated using ARLEQUIN 3.11 software to indicate the differentiation degree amongst the breeds/populations. The UPGMA (unweighted pair-group method with arithmetic means, UPGMA) tree was constructed using MEGA 7.0 based on F ST to reveal genetic relationships among white yak breeds/populations 18 . Based on nucleotide variations between different haplotypes, the median-joining (MJ) network was drawn using NETWORK 10.1 to reveal the phylogenetic relationship among haplotypes/haplogroups 19 .

Results and discussion
Identification of Y-haplotype/haplogroup in white yak. The (Table S1). The haplotypes/haplogroups were jointly identified based on the alleles of Y-SNPs and Y-STR. According to the previous judgment criterion for yak Y-haplotype 10 , a total of six Y-haplotypes were determined in three white yak breeds/populations, namely H1Y1, H9Y1, H10Y1, H11Y2, H12Y2, and H13Y2 (Table 1, Table S1). The six Y-haplotypes could be further divided into two Y-haplogroups (Y1 and Y2): Y1 haplogroup included three haplotypes (H1Y1, H9Y1, and H10Y1), whereas, the Y2 haplogroup contained H11Y2, H12Y2 and H13Y2 haplotypes (Table S1).
Y-chromosome haplotype diversity of white yak. The frequencies of six Y-haplotypes were different among three white yak breeds/populations (Fig. 1, Table 1). In total, the H11Y2 haplotype was predominant (37.1%) and H13Y2 (1.0%) was scarce in the white yak. Three haplotypes (H1Y1, H10Y1, and H11Y2) were common and shared by all three white yak breeds/populations; however, H12Y2 was just shared by Tianzhu and Huzhu white yak populations. The H9Y1 and H13Y2 haplotypes were exclusively found in Menyuan and Tianzhu white yak, respectively. These results showed that Menyuan and Tianzhu white yak breeds/populations possessed unique paternal genetic information. In our previous study, 5, 3, and 43 specific maternal haplotypes were detected in Menyuan, Huzhu and Tianzhu white yak breeds/populations, respectively 11 . Based on the studies of maternal and paternal genetic markers, it can be concluded that Menyuan and Tianzhu white yak populations possessed specific genetic information; therefore, the conservation and application should be independently carried for these genetic units in the future.
In this study, the total haplotype diversity of three white yak breeds/populations was 0.7567 ± 0.0233. Comparing to the previous report 10 , it showed that the white yak had higher total haplotype diversity (0.7567 ± 0.0233) than 15 other Chinese domestic yak breeds/populations (0.6946 ± 0.0143), but lower than the wild yak population (0.8214 ± 0.1007). This total haplotype diversity revealed that white yak also owned rich paternal genetic diversity. The haplotype diversities of Huzhu, Tianzhu, and Menyuan white yak breeds/populations in this study were 0.7500 ± 0.0349, 0.6881 ± 0.0614 and 0.5720 ± 0.0657, respectively (Table 1). It indicated that the Y-haplotype diversity was maximum in Huzhu white yak but the lowest in Menyuan white yak. Surprisingly, the haplotype diversity of the Huzhu white yak was higher than that of the other previously reported Chinese domestic yak breeds/populations (0.1174-0.7273) but only lower than that of the wild yak population (0.8214 ± 0.1007) 10 . At the same time, the haplotype diversities of Menyuan and Tianzhu white yak breeds/populations were also higher than that of the most Chinese domestic yak breeds/populations (0.1174-0.7273) 10 Table S2). The differentiation among three white yak breeds/populations might have been resulted from the differences in the diversified living environment, geographic isolation and human selection. The UPGMA tree was constructed using R ST values among populations for cluster analysis (Fig. S1). The result showed that Huzhu and Menyuan white yak populations clustered together first and then with Tianzhu white yak. The results of the clustering relationship revealed that there was a close genetic relationship between Huzhu and Menyuan white yak but far genetic relationship with Tianzhu white yak.
Phylogenetic network analysis of white yak. The network analysis showed that six Y-haplotypes were divided into two haplogroups/lineages (Y1 and Y2), suggesting two paternal origins of the white yak. The current observation is consistent with the previous research findings on wild and other domestic yak breeds 10 . In the present study, Y1 and Y2 lineages had three haplotypes in each lineage (Fig. 2) 10 . Therefore, our present findings showed that the population structure composition of Menyuan and Huzhu white yak is different from most of the other Chinese domestic yak breeds but similar to the Pali yak breed. Further exploration at the genome level would be needed to unravel the genomic differences among yak breeds/populations.

Conclusion
In conclusion, the white yak in China showed a rich paternal genetic diversity. The Menyuan and Tianzhu white yak breeds/populations possessed unique haplotypes with a medium differentiation level. The Huzhu white yak and Menyuan white yak had a closer genetic relationship, but they both had far relationships to Tianzhu white yak. The Chinese white yak owned two haplogroups/lineages of Y1 and Y2, an indication of two paternal origins. The population structure composition of Menyuan and Huzhu white yak was different from most of other Chinese domestic yak breeds but similar to Pali yak breed. Given paternal genetic diversity, genetic differentiation and clustering relationship among white yak populations, it is suggested that the conservation of white yak genetic resources should be strengthened to protect their unique and excellent genetic characteristics and to make reasonable utilization. The Huzhu and Menyuan white yak in Qinghai Province and Tianzhu white yak in Gansu Province should be considered as different genetic units.

Data availability
The executable codes and datasets are available from the corresponding author on reasonable request.