Introduction

Yak (Bos grunniens) is a unique bovine specie that inhabits on the Qinghai-Tibet plateau (QTP) and adjacent mountains or subalpine mountainous regions in China. It is known for its better adaptability to the harsh environmental conditions such as high altitude, severe cold, strong ultraviolet radiations, scarce forage and deprived oxygen level1. The white yak, a rare ecological type in domestic yak, produces two types of white fiber (coarse outer hair and a fine down fiber) with relatively high economic value2. Since the 1970s, the down fiber has been used extensively by the textile industry as an alternative to other fine animal fibers in China1. Currently, the white yak is mainly distributed on both sides of the Qilian Mountains on QTP, including Tianzhu County in Gansu Province, and Huzhu, Menyuan and Ledu Counties in Qinghai Province of China3.

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 mutation4,5. 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/lineages6,7,8,9,10. 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 diversity11. 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.

Table 1 Frequencies and diversities of haplotypes in three Chinese white yak breeds/populations.
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Figure 1
figure 1

Sampling sites and haplotype/haplogroup distributions of three Chinese white yak breeds/populations. Maps is created using ArcGIS software. Note: TZ, Tianzhu white yak; MY, Menyuan white yak; HZ, Huzhu white yak. The shaded grey area shows Gansu Province and Qinghai Province of China, and the shaded black area within grey area represents Tianzhu County in Gansu Province, and Huzhu and Menyuan Counties in Qinghai Province. The different colors in each circle represent different haplotypes/haplogroups identified. The size of the circle is proportional to the number of individuals. The distances among breeds/populations were measured from the center of the pie charts.

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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 alignment13,14. Meanwhile, the typing results of Y-STR INRA189 were analyzed with GENEMARKER 1.91 to determine its allele size15. 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 software16,17. 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 (FST) 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 FST to reveal genetic relationships among white yak breeds/populations18. 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/haplogroups19.

Results and discussion

Identification of Y-haplotype/haplogroup in white yak

The amplified fragment lengths of the five Y-SNPs (SRY4, USP9Y, UTY19, AMELY3, and OFD1Y10) were 969 bp, 470 bp, 290 bp, 971 bp and 763 bp, respectively. The corresponding sequences had been submitted to GenBank (Accession No. SRY4: MF683848, USP9Y: MF683849, UTY19: MF683850, AMELY3: MF683851, and OFD1Y10: MF683852). The sequencing results of each of the Y-SNP were analyzed by multiple sequence alignment and 10 previously reported Y-SNPs were also detected (Table S1)9,10. In the typing analysis of the Y-STR INRA189 marker, three alleles of 155 bp, 157 bp and 161 bp were detected. Notably, the allele of 161 bp was only detected in Tianzhu white yak, while, others were found in all three breeds/populations (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-haplotype10, 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, respectively11. 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 report10, 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. The present results indicated a rich paternal genetic diversity in all three Chinese white yak breeds/populations. While, Huzhu white yak showed the highest level of paternal genetic diversity.

Differentiation and clustering relationship among white yak populations

The genetic differentiation index (FST) was used to evaluate the differentiation degree among white yak breeds/populations. The FST values ranged from − 0.0050 to  0.0763, whereas, RST values (linearized FST values) ranged from 0 to 0.0826 (Table S2), indicating the variable differentiation degree amongst the three white yak breeds/populations. Refer to Wright's standard20, the FST values indicated moderate differentiation between Tianzhu and Menyuan white yak breeds/populations (0.0763, P < 0.05), however, the Huzhu white yak population displayed a weaker genetic differentiation from Tianzhu white yak (0.0186, P > 0.05) and Menyuan white yak (-0.0050, P > 0.05) (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 RST 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 breeds10. In the present study, Y1 and Y2 lineages had three haplotypes in each lineage (Fig. 2). The Y1 haplogroup/lineage was observed in 48.45% (47/97) individuals and the Y2 haplogroup/lineage in 51.55% (50/97) individuals (Table 1), indicating that Y2 was a dominant haplogroup/lineage in white yak. Meanwhile, the proportions of Y1 in Menyuan, Huzhu, and Tianzhu white yak were 41.94%, 43.75%, and 58.82%, respectively; however, the proportions of Y2 in Menyuan, Huzhu, and Tianzhu white yak were observed to be 58.06%, 56.25%, and 41.18%, respectively (Table 1). The observed data indicated the dominance of the Y1 haplogroup/lineage in the Tianzhu white yak and Y2 haplogroup/lineage in the Menyuan and Huzhu white yak populations. Previous studies represented that the Y1 haplogroup/lineage was dominant haplogroup/lineage in most of the domestic and wild yak populations except for the Pali yak breed10. 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.

Figure 2
figure 2

Median-joining network of six Y-haplotypes identified in three Chinese white yak breeds/populations based on Y-SNP and Y-STR makers. Colors correspond to different breeds/populations. The size of each haplotype circle denotes the number of observed individuals.

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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.