Pangenome and multi-tissue gene atlas provide new insights into the domestication and highland adaptation of yaks

Background The genetic diversity of yak,a key domestic animal on the Qinghai-Tibetan Plateau(QTP),is a vital resource for domestication and breeding efforts.This study presents the first yak pangenome obtained through the de novo assembly of 16 yak genomes.Results We discovered 290 Mb of nonreference sequences and 504 new genes.Our pangenome-wide presence and absence variation(PAV)analysis revealed 5,120 PAV-related genes,highlighting a wide range of variety-specific genes and genes with varying frequencies across yak populations.Principal component analysis(PCA)based on binary gene PAV data classified yaks into three new groups:wild,domestic,and Jinchuan.Moreover,we pro-posed a‘two-haplotype genomic hybridization model'for understanding the hybridization patterns among breeds by integrating gene frequency,heterozygosity,and gene PAV data.A gene PAV-GWAS identified a novel gene(Bos-Gru3G009179)that may be associated with the multirib trait in Jinchuan yaks.Furthermore,an integrated transcrip-tome and pangenome analysis highlighted the significant differences in the expression of core genes and the muta-tional burden of differentially expressed genes between yaks from high and low altitudes.Transcriptome analysis across multiple species revealed that yaks have the most unique differentially expressed m RNAs and lnc RNAs(between high-and low-altitude regions),especially in the heart and lungs,when comparing high-and low-altitude adaptations.Conclusions The yak pangenome offers a comprehensive resource and new insights for functional genomic studies,supporting future biological research and breeding strategies.

Levels of Germinable Seed in Topsoil and Yak Dung on an Alpine Meadow on the North-East Qinghai-Tibetan Plateau

In order to clarify the interactive mechanism between grazing yak and alpine meadow on the Qinghai-Tibetan Plateau,our study assessed seed density（by species） in the topsoil of alpine meadow with different grazing intensities in the Tianzhu area,north-eastern margins of the Qinghai-Tibetan Plateau and their rates of occurrence in yak dung.Seed density in the topsoil of the lightly grazed,moderately grazed,heavily grazed and extremely grazed alpine meadows in November,2010 were 1 551,1 692,2 660 and 1 830 grains m-2,while in the same meadows in April,2011 densities were 1 530,2 404,2 530 and 2 692 grains m-2,respectively.In the cold season pasture,mean seed density in yak dung from November to April in the lightly grazed,moderately grazed,heavily grazed and extremely grazed sites were 121,127,187,and 120 grains kg-1of dry yak dung.The proportion of total seed numbers in yak dung to soil seed bank in lightly grazed,moderately grazed,heavily grazed and extremely grazed alpine meadow was 1.40,2.62,0.69,and 0.90%.12 species out of the 47 were not found in topsoil but were found in yak dung,10 species out of 45 were not found in yak dung but were found in the topsoil.Endozoochorous dispersal by yaks is therefore very important for soil seed bank and plant biodiversity and population dynamics in alpine meadows.

Supplementation with yak (Bos grunniens) bone collagen hydrolysate altered the structure of gut microbiota and elevated short-chain fatty acid production in mice

In this study, yak bone collagen hydrolysate(YBCH)was produced by mixed proteases and provided to standard-diet mice at a different dose(low dose(LD), medium dose(MD), and high dose(HD))to investigate its effects on the composition of gut microbiota and short-chain fatty acids(SCFA)production. It was found that YBCH was mainly composed of small molecular peptides whose molecular weight below 2 000 Da. Notably, supplementation with different doses of YBCH could significantly downregulate the ratio of Firmicutes to Bacteroidetes in the fecal microbiota. At the family level, the Lachnospiraceae abundance was significantly reduced in the YBCH gavage groups(mean reduction ratio 41.7 %, 35.2%, and 36.4% for LD, MD, and HD group, respectively). The predicted functions of gut microbes in the MD group were significantly increased at “lipid metabolism” and “glycan biosynthesis and metabolism”. Moreover, the SCFA production in the YBCH groups was elevated. Especially, the concentration of acetic acid, propionic acid, and butyric acid in the MD group was separately increased 79.7%, 89.2%, and 78.8% than that in the NC group. These results indicated that YBCH might be applied in the development of functional food for intestinal microecological regulation.

Composition Analysis on Intermuscular Fatty Acid of Qinghai Datong Yak

[ Objective] The study aimed to provide a theoretical basis for rational utilization of Datong yak resource. [ Method] By qualitative and quantitative determination with gas chromatograph, the composition and content of intermuscular fatty acid of Datong yak were compared with those of Datong yellow cattle. [ Result] Comparing with Datong yellow cattle, Datong yak had lower saturated fatty acid （SFA） content and higher polyunsaturated fatty acid （PUFA） content at extremely significant level （P 〈0.01 ）, while the difference of monounsaturated fatty acid （MUFA） contents between them was not significant （P〉0.05）. The PUFA/SFA and n-6/n-3 PUFA of Datong yak were in accordance with the recommended proportions. [ Conclusion] The intermuscular fatty acid composition of Datong yak is superior to that of local yellow cattle, which indicates that Datong yak meat has high nutritive value and wide development prospect.

Cloning and Bioinformatics Analysis of Lfcin Gene of Datong Yak

[Objective] This study was to clone Lfcin gene from Datong yak, so as to provide reference for applying this gene in feed industry and breeding industry. [Method] Using PCR technology, the lactoferricin(Lfcin)-encoding gene was obtained from genome of Datong yak; then it was cloned into pGEM-T easy vector, and then sequenced; the sequencing results were subsequently aligned with the sequences of dairy cow accessed in GenBank. Moreover, amino acid sequences of Lfcin gene from various species including yak, dairy cow, human and mouse were used for sequence alignment and phylogenesis analysis. [Result] The second exon of lactoferrin(LF) from Datong yak, which is 778 bp in length, was obtained, within which the coding region of Lfcin gene is 75 bp (25 amino acid residues); sequence analysis showed that there is discrepancy of eleven bases between Datong yak and dairy cow; Lfcin proteins from various species shared high homeology, of which that from Datong yak and dairy cow were completely identical; phylogenesis analysis showed that cladogram based on Lfcin was consistent with species evolutionary law. [Conclusion] This study laid a foundation for the prokaryotic or eukaryotic expression of Lfcin gene and further understanding the activity of Lfcin protein.

Study on Carcass Traits of Changtai Yak

For studying the germplasm charactedsUcs and carcass traits of Changtai yak, 8 adult yaks （5 male, 3 female） were selected and tested. The results showed that the pre-slaughter weight of the adult male and female Changtai yak was 364.32 and 266.83 kg, respectively, which presented extremely significant difference （P〈0.01）. The carcass weight, net bone weight net meat weight, and the ratio of bone to meat also showed extremely significant difference （P〈O.01）, which were 186.60 and 125.67 kg, 39.74 and 25.00 kg, 147.84 and 100.83 kg, 1：3.73 and 1： 4.03, respectively. The dressing percentage had significant difference which was 51.15% and 49.34%, respectively （P〈0.05）. The neat meat percentage and carcass meat production rate showed no significant difference which were 40.54% and 37.66%, 79.29% and 80.24%, respectively （P〉0.05）. It indicated that Changtai yak has better growth performance and meat performance potential, and worth of further studying and breeding.

Study on the Correlation between Blood Enzyme Activity and Production Performance in Tibetan Yaks

Yak ( Bos grunniens ) is classified as Bovine genus,they live in Qingzang Plateau with elevation more than 3500m and it's neighbor highland,where the weather is high chilly,short of oxygen and large difference of temperature between day and night.But yak have adapted that circumstance after thousands of years' artificial and natural selection and has great anti adversity capacity.Yak is an important sustenance and means of production to Tibetan.China is a country with the largest number of yak,whose amount of livestock on hand accounts for 95% of the world and Tibet region takes the second place next to Qinghai in the number of yak.Tibetan yak has formed some groups for Tibetan's large area,complicated geological ecosystem conditions,geological isolation.The typical better groups are Yadong yak,Jiali yak and Sibu yak. Study about yak fell behind other animals due to the restriction of inconvenient transportation,difficult sampling and poor experimental technology.Study about the relationship between yak blood enzyme activities and milk production was little,only Jing (1992) studied correlation of milk production with serum amylase,esterase in Gansu Jiulong yak,Maiwa yak and no report was found in other groups.This study discussed six Jiali yak blood enzyme activities lactate dehydrogenase (LDH),alkaline phosphatases (AKP),acid phosphatase (ACP),catalase (CAT),amylase (Amy) and superoxide dismutase (SOD),and explored their relationship with milk production in Jiali yak,so as to supply with theoretical evidence by using blood enzyme activity as biochemical assisted selection marker in the breeding of Tibetan yak milk yield. This experiment investigated activity of six blood enzymes from 28 Tibetan yaks and explored its relationship to production performance.The results showed a extremely positive significant difference ( P <0.01) between activity of LDH and milk yield,CAT activity and length of belly hair respectively,while a positive significant difference ( P <0.05) was observed between AKP activity and body weight,Amy activity and body weight and a negative significant difference ( P <0.05) for SOD activity and body weight.Stepwise Regression Analysis showed that activity of LDH could be used for the prediction of milk yield,AKP,CAT,Amy for body weight,CAT,Amy for length of belly hair.So it is expected to use activity of LDH,AKP,CAT,Amy as biochemical genetic marker in the selection of milk yield,body weight and length of belly hair.

Determination and Analysis on Nutritional Components in Placenta of Plateau Yak

Yaks live in Qinghai-Tibet Plateau （4 km above sea level）, and the nutrient components of their placenta were determined, including 17 kinds of amino acids and 11 kinds of trace elements. The results showed that there are abundant amino acids and trace elements in the placenta of yak, equal to that of human and deer, indicating some medical and nutritional value of yak placenta.

Study on the Datong Yak's Skin Development

[Objective] This study was to explore the skin development law of Datong Yak.[Method]Taking Datong yaks at four various growth stages（1 day old,30 days old,180 days old and adult）as experimental materials,we studied histological structure of Datong yak skin by employing the histological methods.[Results]The skins of Datong yak at four various growth stages all showed the characteristics of thinner epidermis and thicker hypodermis,and of less capillary vessels in hypodermis and more pigment cells in epidermis.Datong yak at four various growth stages all showed a fluctuating trend in entire skin thickness,in detail thickening from 1 day old to 30 days old,then thinning to 180 days old,again thickening to adult.[Conclusion]The results provided theoretical basis for further understanding the structural characteristics of Datong yak to adapt to the abominable plateau environment.

Comparative Study on Hydrolase Activities in Tianzhu White Yak Milk and Gannan Yak Milk of Gansu

This study was conducted to compare the hydrolase activities in the milk of Tianzhu white yak and Gannan yak in Gansu. The results showed that the ACP activity in Tianzhu yak milk was significantly higher than that in Gannan yak milk （P〈0.05）; the AKP activity in Tianzhu yak milk was higher than that in Gannan yak milk, but not significant; and the AMC activity in Tianzhu yak milk was significantly lower than that in Gannan yak milk （P〈0.05）. There were no remarkable changes in the activities of the 3 hydrolases with the change of parity, i.e., the effects of parity on enzyme activities in milk were not significant. The comparative study on hydrolase activities in the milk of Tianzhu while yak and Gannan yak helps to understand the characteristics of the milk of the 2 yak breeds, and further provides certain reference basis for the development of yak milk.

Hair and Villus Production Performance of Tianzhu White Yak

[ Objective ] To analyze the hair and villus production performance of Tianzhu white yak and thus provide conditions for the genetic breed ing and product development. [Method] The hair and villus yield as well as villus rate of male and female Tianzhu white yaks from one year to six years old was detected and the villus yield was also determined. And these indexes of hair and villus were analyzed to evaluate the production per formance. [ Result] In Tianzhu white yaks from one year to two years old, the hair and villus yield was lower, but the villus rate and yield were high er. The hair and villus yield, villus rate, and villus yield were all higher in Tianzhu white yaks from three years to six years old but significantly de creased in 7yearold Tianzhu white yaks. In addition, the hair and villus yield was significantly （ P 〈 0.05） or very significantly （ P 〈 0.01 ） higher in male yaks than in female yaks. The villus rate was very significantly （ P 〈0.01 ） higher than in female yaks from five years to seven years old than in male yaks at the same age. [ Conclusion] Tianzhu white yaks from one year to two years old have the best hair and villus production perform ance; Tianzhu white yaks from three years to six years old are in the main phase of hair and villus production; Tianzhu white yaks older than seven years old should not be used for hair and villus production.

Optimization of Parameters of Exogenous Gene Mediated by Liposome to Transfect Yak Mammary Epithelial Cells in Vitro

[Objective] The aim of this study was to optimize conditions of exogenous gene mediated by liposome to transfect yak mammary epithelial cells in Vitro.[Method] Yak mammary epithelial cells were isolated and cultivated in Vitro by the methods of collagenase digestion and tissue adhesion.The expression of cytokeratin in yak mammary epithelial cell was detected by immunocytochemistry technique.With green fluorescence protein as the report gene,yak mammary epithelial cells were transfected with exogenous gene m...

Potential antibacterial activity of berberine against multi drug resistant enterovirulent Escherichia coli isolated from yaks(Poephagus grunniens) with haemorrhagic diarrhoea

Objective:To evaluate the antimicrobial efficacy of berberine,a plant alkaloid.Methods:Five multi-drug resistant（MDR） STEC/EPEC and five MDR ETEC isolates from yaks with haemorrhagic diarrhoea were selected for the study.Antibacterial activity of berberine was evaluated by broth dilution and disc diffusion methods.The binding kinetics of berberine to DNA and protein was also enumerated.Results:For both categories of enterovirulent Escherichia coli（E.roli） isolates, berberine displayed the antibaclerial effect in a dose dependent manner.The MIC50 of berberine chloride for STEC/EPEC isolates varied from 2.07μM to 3.6μM with a mean of（2.95±0.33）μM where as for ETEC strains it varied from 1.75 to 1.96μM with a mean of（1.87±0.03）μM. Berberine bind more tightly with double helix DNA with Bmax and Kd of（24.68±2.62） and（357.8±57.8）,respectively.Berberine reacted with protein in comparatively loose manner with Bmax and Kd of（18.9±3.83） and 【286.2±113.6),respectively.Conclusions:The results indicate clearly that berberine may serve as a good antibacterial against multi drug resistant E.coli.

Comparison of rumen archaeal diversity in adult and elderly yaks(Bos grunniens)using 16S rRNA gene high-throughput sequencing

This study was conducted to investigate the phylogenetic diversity of archaea in the rumen of adult and elderly yaks. Six domesticated female yaks, 3 adult yaks （（5.3±0.6） years old）, and 3 elderly yaks （（10.7±0.6） years old）, were used for the rumen contents collection. Illumina MiSeq high-throughput sequencing technology was applied to examine the archaeal composition of rumen contents. A total of 92 901 high-quality archaeal sequences were analyzed, and these were assigned to 2 033 operational taxonomic units （OTUs）. Among these, 974 OTUs were unique to adult yaks while 846 OTUs were unique to elderly yaks; 213 OTUs were shared by both groups. At the phylum level, more than 99% of the obtained OTUs belonged to the Euryarchaeota phylum. At the genus level, the archaea could be divided into 7 archaeal genera. The 7 genera （i.e., Methanobrevibacter, Methanobacterium, Methanosphaera, Thermogymnomonas, Methanomicrobiu, Meth- animicrococcus and the unclassified genus） were shared by all yaks, and their total abundance accounted for 99% of the rumen archaea. The most abundant archaea in elderly and adult yaks were Methanobrevibacterand Thermogymnomonas, respectively. The abundance of Methanobacteria （class）, Methanobacteriales （order）, Methanobacteriaceae （family）, and Methanobrevibacter （genus） in elderly yaks was significantly higher than in adult yaks. In contrast, the abundance of Ther-mogymnomonas in elderly yaks was 34% lower than in adult yaks, though the difference was not statistically significant. The difference in abundance of other archaea was not significant between the two groups. These results suggested that the structure of archaea in the rumen of yaks changed with age. This is the first study to compare the phytogenetic differences of rumen archaeal structure and composition using the yak model.

Analysis of genetic diversity and population structure of Chinese yak breeds (Bos grunniens) using microsatellite markers

Nine Chinese yak breeds （Maiwa, Tianzhu White, Qinghai Plateau, Sibu, Zhongdian, Pali, Tibetan High Mountain, Jiulong, and Xinjiang） and Gayal were analyzed by means of 16 microsatellite markers to determine the level of genetic variation within populations, genetic relationship between populations, and population structure for each breed. A total of 206 microsatellite alleles were observed. Mean F-statistics （0.056） for 9 yak breeds indicated that 94.4% of the genetic variation was observed within yak breeds and 5.6% of the genetic variation existed amongst breeds. The Neighbor-Joining phylogenetic tree was constructed based on Nei＇s standard genetic distances and two clusters were obtained. The Gayal separated from the yaks far away and formed one cluster and 9 yak breeds were grouped together. The analysis of population structure for 9 yak breeds and the Gayal showed that they resulted in four clusters; one cluster includes yaks from Tibet Autonomous Region and Qinghai Province, one cluster combines Zhongdian, Maiwa, and Tianzhu White, and Jiulong and Xinjiang come into the third cluster. Pali was mainly in the first cluster （90%）, Jiulong was mainly in the second cluster （87.1%）, Zhongdian was primarily in the third cluster （83%）, and the other yak breeds were distributed in two to three clusters. The Gayal was positively left in the fourth cluster （99.3%）.

Novel SNP of EPAS1 gene associated with higher hemoglobin concentration revealed the hypoxia adaptation of yak(Bos grunniens)

Endothelial PAS domain protein 1 gene （EPAS1） is a key transcription factor that activates the expression of oxygen-regu- lated genes. In this study, in order to better understand the effects of EPAS1 gene on hematologic parameters in yak, we firstly quantified the tissue expression patterns for EPASl mRNA of yak, identified polymorphism in this gene and evaluated its association with hematologic parameters. Expression of EPAS1 mRNA was detected in all eight tissues （heart, liver, lung, spleen, pancreas, kidney, muscles and ovary）. The expressions of EPAS1 in lung and pancreas were extremely higher than other tissues examined. Three novel single nucleotide polymorphisms （SNPs） （g.83052 C〉T, g.83065 G〉A and g.83067 C〉A） within the EPAS1 were identified and genotyped in Pali （PL）, Gannan （GN） and Tianzhu White （TZW） yak breeds. Significant higher frequencies of the AA and GA genotypes and A allele of the g.83065 G〉A were observed in the PL and GN breeds than that in the TZW breed （P〈0.01）. Association analysis of the PL breed indicated that the g.83065 G〉A polymorphism was significantly associated with hemoglobin （HGB） concentration in yaks （P〈0.05）. Individuals with genotype AA had significantly higher HGB concentration （P〈0.05） than those with genotype GA and GG. All these results will help our further understanding of biological functional of yak EPAS1 gene in responding to hypoxia and also indicate EPAS1 might contribute to the hypoxia adaptation of the yak.

Science of Camel and Yak Milks: Human Nutrition and Health Perspectives

Camels and yaks milks are rich in numerous bioactive substances that function beyond their nutritive value. Milk Camel milk is more similar to goat milk and contains less short-chain fatty acids than cow, sheep and buffalo milks, and about 3 times greater vitamin-C than cow milk. One kg of camel milk meets 100% of daily human requirements for calcium and phosphorus, 57.6% for potassium, 40% for iron, copper, zinc and magnesium, and 24% for sodium. Camel milk helps treat liver problems, lowers bilirubin output, lightens vitamin inadequacy and nutrient deficiency, and boosts immunity. Camel milk reduces allergies caused by cow dairy products. Camel milk has low milk fat made mainly from polyunsaturated fatty acids. It lacks ?-lactoglobulin and is rich in immunoglobulins, compatible with human milk. Yak milk has 16.9 - 17.7% solids, 4.9 - 5.3% protein, 5.5 - 7.2% fat, 4.5 - 5.0% lactose, and 0.8 - 0.9% minerals. Yak milk fat is richer in polyunsaturated fatty acids, protein, casein and fat than cow milk. Yak milk casein is used to produce antihypertensive peptides with capacities for producing value-added functional foods and proteins. Continual system-atic education of milk science especially for non-cow species will be an obligation for health implications to be optimally perceived by human populations worldwide.

Physiological and Biochemical Characteristics of Lactation in Half-lactating Yak

Yaks of two lactating types, the half-Iactating yak and the total lactating yak, were investigated in their milk compositions, milk coagulation properties, and contents of three kinds of hormones, glucose, and protein in plasma. The half-lactating Maiwa yak and Jiulong yak contained significantly higher contents of protein, fat, and activities of alkaline phosphatase and Y-glutamyl transpeptidase in milk than that of the corresponding total-lactating yak breeds, with reduced milk yield and similar lactose level and relative percentages of main milk protein components. The half-lactatin yak resembled yaks in a late stage of lactation in their biochemical composition of milk; however, significant differences were also observed, which indicated that the former was in a special stage of lactation quite different from dairy cows. Milk of the total-lactating or half-lactating yaks could be coagulated normally by adding chymosin, with a similar coagulation time. No significant difference was observed between prolactin and progesterone concentrations in plasma of the half-lactating yaks and total-lactating yaks; however, half-lactating yaks had significantly lower level of oestradiol-17 β in plasma than total-lactating yaks.

Effect of Nitrogenous Fertilizer Treatment on Mineral Metabolism in Grazing Yaks

To assess the impact of N fertilization on contents of mineral elements in herbage and the effect of increased forage S on the copper metabolism of grazing yaks, study was conducted during the summer grazing season （2005, 2006, and 2007）. Pasture replicates （20 ha; n=3 per treatment） received the same fertilizer treatment in each growing season, consisting of i） 90 kg N ha^-1 from quickly available nitrogen, ii） 90 kg N ha^-1 from ammonium nitrate, iii） 90 kg N ha^-1 from ammonium sulfate, and iv） control （no fertilizer）. Forage sampling was collected at 60 days intervals following fertilization （10 samples per pasture） for Cu, Mo, Mn, Se, Fe, Zn, Ca, and P. To determine the effect of fertilizer treatment on mineral metabolism in grazing yaks, liver and blood samples were collected at the start and end of the study period in 2005, 2006, and 2007. Ammonium sulfate fertilization increased （P 〈 0.01） forage S concentration. Plant tissue N concentrations were increased by N fertilization, regardless of source in 2005, 2006, and 2007. Yaks grazing S fertilization pastures had lower （P〈0.05） liver and blood Cu concentrations at the end of the study period in 2005, 2006, and 2007, compared with urea, ammonium nitrate, and control. Nominal increases in forage in vitro organic matter digestibility were realized by fertilization, regardless of N source in each year.

Genetic Polymorphism of Milk Protein and Their Relationships with Milking Performances in Chinese Yak

The milk protein polymorphisms were typed by polyacrylamide gel electrophoresis (PAGE)from 109 Maiwa and 100 Jiulong yaks, and the relationships among milk protein polymorphisms,milking traits and milk protein compositions were studied. The results showed thatβ-CN,κ-CN andα-La were monomorphic,αs1-CN andβ-Lg were polymorphic, the dominantgenes were αs1-CN D and β-Lg E,respectively. The frequencies of αs1-CN D were 0.8073and 0.6000 and β-Lg E were 0.9770 and 0.9700 in two populations respectively.The meanheterozygosities were 0.1021 and 0.1867 in two populations. No significant effects onmilking traits and milk protein compositions were observed except for αs1-CN locus onfat percentage in Jiulong yak.