​Dominant-genotype Frequency Analysis of Economic Traits Related to SNP Candidate Markers in Three Yak Populations

(El-Komy et al., 2021) Domestic yak (Bos grunniens) has perfect plateau adaptability and is one of the mammals living in the Qinghai-Tibet Plateau, which has the highest altitude (>3000 m) in the world. This animal provides milk, meat, wool, labor, fuel and other daily necessities for local herdsmen and is thus an important life and economic resource (Nie et al., 2020).
       
The molecular markers (MMs) of economic traits in cattle and buffalo have been largely identified to be related to their growth traits and milk production (Ujan et al., 2011; Lyu et al., 2020; El-Komy et al., 2021). Conversely, only a few candidate genes related to the economic traits of yak have been reported. For example, a single nucleotide polymorphism (SNP; G1069C) from MC4R is significantly (P<0.01) correlated with the body weight of 18-month-old Maiwa yak (Cai et al., 2015). A new SNP (C19913T) in exon 7 of the LPL gene is reportedly responsible for the Phe-to-Ser substitution of amino acids, which have significant effects on the body weight (P<0.01), carcass body weight (P<0.05), eye area and visceral fat weight of yak (Ding et al., 2012).
       
At present, the yak population in the Tibet Plateau of China subsists mostly on extensive grazing and breeding methods. Considering the slow technological development in the region, the production level of yak breeding is low. Therefore, screening the frequency of dominant genotypes of economic-trait-related markers in Tibetan yak populations is needed to assess the breeding potential of these populations for future Tibetan yak breeding. In the present study, we aimed to identify the dominant-genotype distribution of 12 previously known MMs in three Tibetan yak populations and to evaluate the breeding potential for genetic improvement.

Animals and samples
 
A total of 238 yak individuals from three populations were collected, including 34 Sibu (SB), 104 Jiali (JL) and 100 Cawula (CWL) yak individuals (Table 1). Venous blood (5 mL) was collected from each animal and genomic DNA was extracted using a PureLink Genomic DNA Mini Kit (Shanghai, China). Nano Drop 2000 was used to determine DNA concentration and quality.
 

 
Primers and PCR amplification
 
PCR amplification primers were designed using Primer3Plus (http://www.primer3plus.com/) according to the sequence information and previous reports of 12 SNP markers (Table 2).
 

       
The 10 µL PCR amplification system included 5 µL of 2×Taq PCR Mix, 1 µL of Primer Mix, 1 µL of DNA (20 ng/μL) and 3 µL of ddH₂O. The PCR amplification-reaction program was as follows: 95°C pre-denaturation for 5 min; 40 cycles each of 94°C denaturation for 30 s, 52°C annealing for 30 s and 72°C extension for 80 s and 72°C extension for 10 min. The PCR product genotyped using SNaPshot technology was referenced as conducted in previous studies (Wang et al., 2021; Cheng et al., 2020).
 
Data analysis
 
A Microsatellite Toolkit (Park, 2012) was used to analyze genotype frequency and genetic-diversity parameters, such as observed heterozygosity (H_O), expected heterozygosity (H_E) and polymorphism information content (PIC). Arlequin 3.5 software (Excoffier and Lischer, 2010) was used for Hardy-Weinberg equilibrium (HWE) analysis.

Results of genetic-diversity parameter analysis (Table 3) showed that the distribution of H_E in the CWL population was 0.000 (Hesx1-G618C and OXGR1-A347G) to 0.4984 (MyoD1-C1710T) and that of H_O ranged from 0.0000 (Hesx1-G618C and OXGR1-A347G) to 0.5400 (CAPN_G-1222A). The PIC of the MyoD1_C1710T locus was the highest (0.373). In the JL population, CAPN_G-1222A had the highest H_E (0.5023) and H_O (0.5098) and CAPN_G-1222A (0.3749) and GHSR_T1387C (0.3731) had the highest PICs. In the SB population, the highest H_E was found in GHSR_T1387C (0.5035) and MyoD1_C1710T (0.4965). MyoD1_C1710T and CAPN_G-1222A had the highest H_O and five sites, namely, ACSL1_A2079T, GHSR-T1387C, MyoD1_C1710T, CAPN_G-1222A and TMEM-18_C1267T, were medium polymorphic (0.25<PIC<0.5). The HWE results further revealed that no loci considerably deviated from the HWE within the population. This finding implied that the genetic diversity of the three yak populations was well maintained and that the three populations had not undergone artificial selection for economic traits.
 

       
Table 4 shows the genotype frequencies of 12 SNPs in the three yak populations. The HesxI gene is known to indirectly affect the growth and development of animals (Kaminski et al., 2008). A previous report has found that HesxI_G-618C and HesxI_T226C are significantly correlated (P<0.05) with the weight and height of 12-month-old hornless yak (Zhang et al., 2019). The present study showed that the HesxI_G-618C locus was a homozygous G/G type in the three populations and more than 90% of individuals carried the T/T type of the HesxI_T226C locus. Thus, whether these two markers can be used for the molecular breeding of these three Tibet yaks requires further research.
 

       
Myogenic differentiation 1 (MyoD1) from the muscle regulatory factor (MyoD) family is an important transcription factor that regulates skeletal-muscle production (Talbot et al., 2016). It can activate muscle-gene transcription, transform non-muscle cells into muscle cells, regulate muscle-cell fusion and promote the differentiation of myoblasts into myotubes, subsequently fusing them into muscle fibers and thus regulating embryonic muscle development (Hodge et al., 2019). A large number of genetic variations distributed in the MyoD1 gene region are remarkably related to the growth and meat quality of domestic animals (Liu et al., 2008; Ujan et al., 2011). Specifically, a SNP (MyoD1_C1710T) on exon 3 of the MyoD1 gene has been identified to be significantly correlated with body-size traits (P<0.05) in Shenzha yak and C/T is the dominant genotype (Huang et al., 2019). Herein, we found that the C/T genotype of MyoD1_C1710T had the highest frequency (51.00%-67.64%) within all three yak populations.
       
Cytochrome P450 4A11 (CYP4A11) can be used to diagnose renal cell carcinoma (Kim et al., 2020) and is related to the occurrence of non-alcoholic fatty liver and coronary artery disease (Gao et al., 2020). In particular, a recently discovered SNP marker of the CYP4A11 gene (CYP4A11_G4806A) is significantly correlated with weight and tube circumference in Maiwa yak and G/A genotype is significantly higher in these phenotypes than that of individuals with G/G (P<0.05) (Guan et al., 2019). In the current study, we found that the CYP4A11_G4806A locus had a high frequency of the G/G genotype (73.53%-85.58%) in the three yak populations, the G/A genotype had a certain proportion of distribution, ranging from 14.42% (JL) to 26.47% (SB). Whether the two SNPs of CYP4A11 can be extensively used as MMs of yak growth traits requires further clarification.
       
Transmembrane protein 18 (TMEM18) participates in the migration and regulation of neural stem cells in vivo and in vitro (Jurvansuu et al., 2008) and regulates adipocyte differentiation through the nervous system to affect obesity (Gutierrez-Aguilar et al.,  2011). In previous studies, some mutations from TMEM18 gene have been confirmed to be related to appetite in humans (Larder et al., 2017) and growth traits in cattle (Crispim et al., 2015). Another study has suggested that the TMEM18_C1267T and TMEM18_C44 47T loci are significantly associated with body height and slaughter rate (P<0.05) in Tianzhu yak (Zhang et al., 2017) and that T/T and C/T are the dominant genotype of these loci, respectively. However, the current study found that the two loci from the TMEM-18 gene (C1267T and C4447T) carried the highest frequency of the C/C genotype in the three populations, whereas T/T in TMEM18_C1267T was rare.
       
Calpain 4 (CAPN4) is extensively known to be associated with beef quality traits (Colle et al., 2017). An SNP (CAPN_G-1222A) in the CAPN4 gene’s promoter region is significantly related (P<0.05) to multiple meat quality and growth traits of Gannan yak, including cooking rate and water-loss rate (Niu et al., 2015). This study found that the CAPN_G-1222A locus had abundant polymorphisms in the three populations. Thus, this locus had potential in the molecular breeding of meat-quality traits in the three yak populations.
       
Growth hormone (GH), growth hormone receptor (GHR) and growth hormone secretagogue receptor (GHSR) are important regulatory genes for body growth and development (Wegmann et al., 2017; Wang et al., 2020). GHSR can stimulate the release and secretion of GH and thus participate in the body’s growth and energy metabolism (Zhang et al., 2017; Lv et al., 2018). Particularly, an SNP (T1387C) at the 52  UTR of the GHRS gene is confirmed as significantly related to the body weight in Maiwa yak (P<0.05) (Hai et al., 2017) and the dominant gene has a C/C genotype. In the present study, the frequency of the C/C genotype of GHRS_SNP (T1387C) in the three Tibetan yak populations was 32.69%-45.00%. This finding indicated that the three yak populations carried abundant dominant genotypes in this locus.
       
Uncoupling proteins (UCPs), as an anion carrier member of the inner mitochondrial membrane, maintain energy metabolism and homeostasis in vivo (Krauss et al., 2005). Some studies have shown that UCP2 is associated with body weight in humans and domestic animals (Oliveira et al., 2017; Oguzkan-Balci et al.,  2013). Coincidentally, an SNP (UCP2_T1499C) of UCP2 gene is related to the weight of Maiwa yak and individuals with the C/T genotype have significantly higher weight (P<0.05) than those with the T/T genotype (Hao et al., 2019). In the current work, we found that the dominant genotype C/T was a rare genotype in the three Tibetan yak populations.
       
A series of studies has shown that long-chain acyl-CoA synthetase 1 (ACSL1) is the most important quantitative-trait locus and functional candidate gene that affects fatty acid composition in pork. Moreover, the polymorphism of the ACSL1 gene is significantly related (P<0.05) to the composition and ratio of fatty acid in the skeletal muscle of cattle (Widmann et al., 2011). Some genetic variations in ACSL1 are also substantially related to the milk-production traits of Holstein cows (Liang et al., 2020). In Gannan yaks, the two SNPs (ACSL1_A2079T and ACSL1_G2409A) in the promoter region of the ACSL1 gene are significantly related (P<0.05) to the protein rate and milk-fat rate (Zhao et al., 2019). The present study found that these two loci were rich in polymorphisms in three Tibetan yak populations.

This study analyzed the genotype distribution of 12 candidate SNP genetic markers related to the economic traits of yak in three Tibetan yak populations. Some of them may have value in the breeding selection of Tibet yaks in the future, although extensive and in-depth research is required.

Healthy breeding of Tibetan characteristic livestock (XZ201901NA02), differential expression analysis of several factors related to fat metabolism in Yak (XZNKY-2020-C-007Z03), National Meat cattle and Yak Industrial Technology System (CARS-37), Research on the impact of oregano oil in yak productive performance (XZNKY-2020-C-007Z05), New breed cultivation of Ali yak (XZ202001ZY0021N).