Establishment of paternity testing system using microsatellite markers in Chinese Holstein

To estimate the efficiency of microsatellite markers in paternity testing among Chinese Holstein, 30 microsatellite loci were used to differentiate 330 Chinese Holstein genotypes, according to the calculation of the allele frequency, number of alleles, effective number of alleles, genetic heterozygosity, polymorphic information content （PIC）, and the exclusion probability in this cattle population. The results demonstrated that the exclusion probability ranged from 0.620 in locus BM1818 to 0.265 in locus INRA005 with the average of 0.472 and 11 microsatellite markers exceeding 0.5. The combined exclusion probability of nine microsatellite markers was over 0.99. The result showed that paternity testing of Chinese Holstein was basically resolved using the nine microsatellite markers selected.

Association of a missense mutation in the luteinizing hormone/choriogonadotropin receptor gene (LHCGR) with superovulation traits in Chinese Holstein heifers

Background： Upon binding luteinizing hormone in the ovary, the luteinizing hormone/choriogonadotropin receptor （LHCGR） is necessary for follicular maturation and ovulation, as well as luteal function. We detected mutations in the LHCGR gene and evaluated their association with superovulation. Methods： Using polymerase chain reaction-single strand sequencing, we examined polymorphisms in LHCGR and Chinese Holstein heifers. conformation polymorphism （PCR-SSCP） and DNA the genotypes associated with superovulation traits in 127 Results： A G/T polymorphism （ss52050737） in exon 11 was significantly associated with the total number of ova and the number of transferable embryos. Conclusions： LHCGR may be a new predictor for superovulation in Chinese Holstein heifers.

Evaluation of Breeding Programs Combining Genomic Information in Chinese Holstein

Current study adopted gene flow theory and selection index method to compare the breeding efficiency of three breeding plans in the Chinese Holstein cattle using ZPLAN software. Simulated conventional progeny-testing program （PT） and young sire program （YS） were compared with breeding program using genomic selection （GS） taking parameters derived from Chinese Holstein breeding system. The results showed that, GS shortened generation interval by 1.5-2.2 years, and increased the genetic progress by 30-50%, comparing to PT and YS, respectively. Economic analysis showed that GS could obtain a higher breeding efficiency, being 119 and 97% higher than that of PT and YS, respectively; and GS was also powerful in improving functional traits with a low heritability. Main factors affecting breeding efficiency in GS were further discussed, including selection intensity, accuracy and the cost of SNP genotyping. Our finding provided references for future designing and implementing GS in Chinese dairy population.

Mapping QTLs Affecting Economic Traits on BTA3 in Chinese Holstein with Microsatellite Markers

It had been demonstrated that the strong and highly significant quantitative trait locus（QTL） can affect protein percentage on Bos Taurus Autosome 3（BTA3） at the position 52 cM, near the microsatellite DIK4353, with the 95% confidence interval spanning from 25 to 57 cM in Chinese Holstein population using QTL-express, MQREML, and GRIDQTL softwares. This study herein focused on such region of fine mapping QTLs for milk production and functional traits with 16 microsatellite markers with coverage of 33 cM between the markers BMS2904 and MB099 on BTA3 in a daughter-designed Chinese Holstein population. A total of 1 298 Holstein cows and 7 sires were genotyped for 16 microsatellites with ABI 3700 DNA sequencer. The variance components QTL linkage analysis（LA） and linkage-disequilibrium（LD） analysis（LA/LD） was performed to map QTLs for 7 traits, i.e., 305-d milk yield, fat yield, protein yield, fat percentage, protein percentage, somatic cell score and persistency of milk yield. Four strong and highly significant QTLs were detected for fat yield, fat percentage, protein percentage and somatic cell score at the position 40, 30, 27 and 26 cM, respectively. Two minor QTLs for milk yield and persistency of milk yield were identified at 42 and 46 cM, respectively. These findings provided a general idea for the fine mapping of the causal mutation for milk production and functional traits on BTA3 in the future.

Fine Mapping QTLs Affecting Milk Production Traits on BTA6 in Chinese Holstein with SNP Markers

Our previous studies demonstrated that the region around markers BMS470 and BMS1242 on BTA6 showed a linkage to 305-d milk yield and composition traits in the Chinese Holstein population. We herein focused on such narrow region to fine map milk production QTLs with 15 SNPs across 25 Mb with each SNP in 1 Mb within most regions in a Chinese Holstein population with daughter design. 1 449 Holstein cows and 11 sires were genotyped for such SNPs by using TaqMan probe and RFLP assays. Multipoint linkage analysis across family revealed a QTL affecting milk yield between PPARGC1A C4075T and SLC34A2 T1713C. Meanwhile, within family analysis found three milk yield QTLs （two in CR T60984131G-CEP135 C501T and one in PDLIM5 A106C-OPN T3907, a fat yield QTLin UGDH T1670C-CR T60984131G region, and two protein yield QTLs in TBC1D1 G501C-UGDH T1670C and PPARGC1A C4075T-SLC34A2 T1713C, respectively. Associations between aforementioned significant SNP markers and milk production traits were further implemented. We found significant associations of PPARGC1A C4075T, SLC34A2 T1713C with milk yield （P0.05, P0.01, P0.01）, UGDH T1670C, and CR T60984131G with fat yield （P0.01, P0.01）, and PPARGC1A C4075T, SLC34A2 T1713C, UGDH T1670C and OPN T3907 with protein yield （P0.01, P0.01, P0.01, P0.01）. Our findings implied that QTLs affecting milk production traits on BTA6 were pleictropism or multigenic effect and PPARGC1A and OPN may be the causal mutations behind milk production QTLs on BTA6 in the Chinese Holstein population.

Novel SNPs in IL-17F and IL-17A genes associated with somatic cell count in Chinese Holstein and Inner-Mongolia Sanhe cattle

Background： Bovine mastitis is the most common and costly disease of lactating cattle worldwide. Apart from milk somatic cell count（SCC） and somatic cell score（SCS）, serum cytokines such as interleukin-17（IL-17） and interleukin-4（IL-4） may also be potential indicators for bovine mastitis. The present study was designed to investigate the effects of single nucleotide polymorphisms（SNPs） in bovine IL-17 F and IL-17 A genes on SCC, SCS and serum cytokines in Chinese Holstein and Inner-Mongolia Sanhe cattle, and to compare the m RNA expression variations of the cows with different genotypes.Results： A total of 464 lactating cows（337 Holstein and 127 Inner-Mongolia Sanhe cattle） were screened for SNPs identification and the data were analyzed using fixed effects of herd, parity, season and year of calving by general linear model procedure. The results revealed that SNP g.24392436 C ＆gt; T in IL-17 F and SNP g.24345410 A ＆gt; G in IL-17 A showed significant effects on SCC and IL-4 in Holstein（n = 337） and on IL-17 and IL-4 in Sanhe cattle（n = 127）. The homozygous GG genotype of SNP g.24345410 A ＆gt; G had significantly higher m RNA expression compared with the heterozygous AG genotype.Conclusions： The results indicate that IL-17 F and IL-17 A could be powerful candidate genes of mastitis resistance and the significant SNPs might be useful genetic markers against mastitis in both dairy and dual purpose cattle.

Genetic parameters for somatic cell score and production traits in the fi rst three lactations of Chinese Holstein cows

The objectives of this study were to estimate genetic parameters of lactation average somatic cell scores （LSCS） and examine genetic associations between LSCS and production traits in the first three lactations of Chinese Holstein cows using single-parity multi-trait animal model and multi-trait repeatability animal model. There were totally 273605 lactation records of Chinese Holstein cows with first calving from 2001 to 2012. Heritability estimates for LSCS ranged from 0.144 to 0.187. Genetic correlations between LSCS and 305 days milk, protein percentage and fat percentage were -0.079, -0.082 and -0.135, respectively. Phenotypic correlation between LSCS and 305 days milk yield was negative （-0.103 to -0.190）. Genetic correlation between 305 days milk and fat percentage or protein percentage was highly negative. Genetic correlation between milk fat percentage and milk protein percentage was highly favorable. Heritabilities of production traits decreased with increase of parity, whereas heritability of LSCS increased with increase of parity.

Comparison of Estimated Genetic Connectedness among Three Herds of Chinese Holstein Cattle

Estimating genetic connectedness among herds is important for the accuracy of dairy cattle genetic evaluation. When selecting between animals raised in different herds, the accuracy of their genetic evaluations can be influenced by the degree of connectedness among these herds. In this study, two methods were used to measure genetic connectedness, CR （genetic connectedness rating ） and GLt （total number of direct genetic links between group）, among herds from Beijing, Shanghai, and Tianjin. Genetic connectedness between the herds from Beijing and Tianjin was 23.95%, between Beijing and Shanghai was 17. 10%, and between Shanghai and Tianjin it was 14.28%. Genetic connectedness between herds from Beijing and Tianjin was the highest and that between Shanghai and Tianjin was the lowest. The correlation coefficient for the two methods was 0. 808. Some suggestions for improved genetic evaluation of dairy cattle were also discussed.

Study on Reproductive Performance of Improved Hybrid from Chinese Holstein Cattle

In order to overcome the problems such as decline in adaptability, low fertility and few utilization years due to continuously increasing coefficient of inbreeding of Chinese Holstein cattle, three dairy-beef dual-purpose cattle varieties （purebred Simmental, Montbeliard and Fleckvieh） were introduced as the male parents to be hybridized with Chinese Holstein cattle, so as to study the improvement effect in the reproduction performance and milk production performance of the hybrid cattle. The results showed that the first-filial generation of Montbeliard had a very significant difference in gestation period from Chinese Holstein cattle （ P 〈0.01）. The parity of Fleckvieh s first-filial generation had a very significant effect on 305 d milk yield （ P 〈0.01）; and for the first-filial generation of Montbeliard, mating times and open days significantly affected 305 d milk yield （ P 〈0.05）, and its total milk yield had a significant difference from Chinese Holstein cattle （ P 〈0.05）. Comprehensively, the reproduction traits of Montbeliard s first-filial generation are better than other two varieties.

A Method for Accuracy of Genetic Evaluation by Utilization of Canadian Genetic Evaluation Information to Improve Heilongjiang Holstein Herds

The objectives of this study were to set up a new genetic evaluation procedure to predict the breeding values of Holstein herds in Heilongjiang Province of China for milk and fat production by utilizing Canadian pedigree and genetic evaluation information and to compare the breeding values of the sires from different countries. The data used for evaluating young sires for the Chinese Holstein population consisted of records selected from 21 herds in Heilongjiang Province. The first lactation records of 2 496 daughters collected in 1989 and 2000 were analyzed. A single-trait animal model including a fixed herd-year effect, random animal and residual effects was used by utilizing Canadian pedigree and genetic evaluation information of 5 126 sires released from the Canadian Dairy Network in August 2000. The BLUP procedure was used to evaluate all cattle in this study and the Estimated Breeding Values (EBV)for milk and fat production of 6 697 cattle (including 673 sires and 6 024 cows) were predicted. The genetic levels of the top 100 sires originated from different countries were compared. Unlike the BLUP procedure that is being used in conjunction with the single-trait sire model in Heilongjiang Province of China now, the genetic evaluation procedure used in this study not only can be used simultaneously to evaluate sires and cows but also increase the accuracy of evaluation due to using the relationships and genetic values of the Canadian evaluated sires with more daughters. The results showed that the new procedure was useful for genetic evaluation of dairy herds and the comparison of the breeding values of these sires imported from different countries showed that a significant genetic improvement has been achieved for milk production of the Heilongjiang Holstein dairy population by importing sires from foreign countries, especially from the United States due to the higher breeding values.

A post-GWAS confirming the genetic effects and functional polymorphisms of AGPAT3 gene on milk fatty acids in dairy cattle

Background: People are paying more attention to the healthy and balanced diet with the improvement of their living standards. Milk fatty acids(FAs) have been reported that they were related to some atherosclerosis and coronary heart diseases in human. In our previous genome-wide association study(GWAS) on milk FAs in dairy cattle, 83 genomewide significant single nucleotide polymorphisms(SNPs) were detected. Among them, two SNPs, ARS-BFGL-NGS-109493 and BTA-56389-no-rs associated with C18 index(P = 0.0459), were located in the upstream of 1-acylglycerol-3-phosphate O-acyltransferase 3(AGPAT3) gene. AGPAT3 is involved in glycerol-lipid, glycerol-phospholipid metabolism and phospholipase D signaling pathways. Hence, it was inferred as a candidate gene for milk FAs. The aim of this study was to further confirm the genetic effects of the AGPAT3 gene on milk FA traits in dairy cattle.Results: Through re-sequencing the complete coding region, and 3000 bp of 5′ and 3′ regulatory regions of the AGPA T3 gene, a total of 17 SNPs were identified, including four in 5′ regulatory region, one in 5′ untranslated region(UTR),three in introns, one in 3′ UTR, and eight in 3′ regulatory region. By the linkage disequilibrium(LD) analysis with Haploview4.1 software, two haplotype blocks were observed that were formed by four and 12 identified SNPs,respectively. Using SAS9.2, we performed single locus-based and haplotype-based association analysis on 24 milk FAs in 1065 Chinese Holstein cows, and discovered that all the SNPs and the haplotype blocks were significantly associated with C6:0, C8:0 and C10:0(P < 0.0001–0.0384). Further, with Genomatix, we predicted that four SNPs in 5′ regulatory region(g.146702957 G > A, g.146704373 A > G, g.146704618 A > G and g.146704699 G > A) changed the transcription factor binding sites(TFBSs) for transcription factors SMARCA3, REX1, VMYB, BRACH, NKX26, ZBED4, SP1, USF1, ARNT and FOXA1. Out of them, two SNPs were validated to impact transcriptional activity by performing luciferase assay that the alleles A of both SNPs, g.146704373 A > G and g.146704618 A > G, increased the transcriptional activities of AGPAT3 promoter compared with alleles G(P = 0.0004).Conclusions: In conclusion, our findings first demonstrated the significant genetic associations of the AGPAT3 gene with milk FAs in dairy cattle, and two potential causal mutations were detected.

A post-GWAS replication study confirming the association of C14H8orf33 gene with milk production traits in dairy cattle

Genome-wide association studies with an Illumina Bovine50K chip have detected 105 SNPs associated with one or multiple milk production traits in the Chinese Holstein population.Of these,38 significant SNPs detected with high confidence by both L1-TDT and MMRA methods were selected to further mine potential key genes affecting milk yield and milk composition.By blasting the flanking sequences of these 38 SNPs with the bovine genome sequence combined with comparative genomics analysis,26 genes were found to contain or be near to such SNPs.Among them,the C14H8orf33 gene is merely 87 bp away from the significant SNP,Hapmap30383-BTC-005848.Hence,we report herein genotype-phenotype associations to further validate the genetic effects of the C14H8orf33 gene.By pooled DNA sequencing of 14 unrelated Holstein sires,a total of 18 with seven novel SNPs were identified.Among them,nine SNPs were in the 5′regulatory region,one in exon 6 and the other in the 3′UTR and 3′regulatory region.A total of nine of these identified SNPs were successfully genotyped and analyzed by mass spectrometry for association with five milk production traits in an independent resource population.The results showed that these SNPs were statistically significant for more than two traits[P<(0.0001–0.0267)].In addition,mRNA expression analyses revealed that C14H8orf33 was ubiquitous in eight different tissues,with a relatively higher expression level in the mammary gland than in other tissues.These findings,therefore,provide strong evidence for association of C14H8orf33 variants with milk yield and milk composition traits and may be applied in Chinese Holstein breeding programs.