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Association of bovine CD4 and STAT5b single nucleotide polymorphisms with somatic cell scores and milk production traits in Chinese Holsteins

Published online by Cambridge University Press:  25 March 2011

Yanghua He ,
Qin Chu ,
Peipei Ma ,
Yachun Wang ,
Qin Zhang ,
Dongxiao Sun ,
Yi Zhang ,
Ying Yu  and
Yuan Zhang
Yanghua He
Affiliation:
Key Laboratory of Agricultural Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China
Qin Chu
Affiliation:
Key Laboratory of Agricultural Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, 100097, Beijing, P.R. China
Peipei Ma
Affiliation:
Key Laboratory of Agricultural Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China
Yachun Wang
Affiliation:
Key Laboratory of Agricultural Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China
Qin Zhang
Affiliation:
Key Laboratory of Agricultural Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China
Dongxiao Sun
Affiliation:
Key Laboratory of Agricultural Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China
Yi Zhang
Affiliation:
Key Laboratory of Agricultural Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China
Ying Yu*
Affiliation:
Key Laboratory of Agricultural Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China
Yuan Zhang*
Affiliation:
Key Laboratory of Agricultural Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193, Beijing, P.R. China
*
*For correspondence: yuying@cau.edu.cn; changy@cau.edu.cn
*For correspondence: yuying@cau.edu.cn; changy@cau.edu.cn
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Abstract

CD4+ T cells play a key role in the immune response of pathogen-induced mastitis in dairy cattle. Mammary gland factor STAT5b is involved in the regulation of CD4+T cell differentiation during inflammatory response and milk production. Little is known about the genetic variation effects of bovine CD4 and STAT5b genes on somatic cell score (SCS) and milk production traits in dairy cattle. The aim of the study was to investigate the single nucleotide polymorphisms (SNPs) of bovine CD4 and STAT5b in Chinese Holsteins and to analyse their association with estimated breeding values (EBVs) for SCS and milk production traits. In the present study, SNPs of CD4 (NC_007303 g.13598C>T) and STAT5b (NC_007317 g.31562 T>C) were identified and genotyped in Chinese Holstein population. The results showed that both SNPs were significantly associated with the EBVs for milk yield and protein yield in Chinese Holstein cows, and the SNP in CD4 was associated with the EBV for SCS (P<0·01). The additive effect of CD4 SNP on protein yield was significant (P<0·05), and the dominant effect of STAT5b SNP was significant on milk yield and protein yield (P<0·01). Cows with combination genotype C7 (CCTT: CD4 g.13598C>T and STAT5b g.31562 T>C) had the highest SCS EBV but lower milk yield, while cows with C2 (TTTC) produced more milk, fat and protein than the other eight combination genotypes. These results suggested that the SNPs in CD4 and STAT5b may be potential genetic markers for SCS and milk/protein yields selecting and warrant further functional research.

Keywords

CD4STAT5bsingle nucleotide polymorphismssomatic cell scoremilk production traitsChinese Holstein

Information

Type
Research Article
Information
Journal of Dairy Research , Volume 78 , Issue 2 , May 2011 , pp. 242 - 249
Copyright
Copyright © Proprietors of Journal of Dairy Research 2011

Bovine mastitis is the most significant disease affecting dairy herds worldwide (Wang et al. Reference Wang, Xu, Ma, Gao, Ren and Chen2006; Viguier et al. Reference Viguier, Arora, Gilmartin, Welbeck and O'Kennedy2009). Mastitis results in increased somatic cell count (SCC) which diminishes milk quality and causes a higher milk loss in dairy cows (Hagnestam-Nielsen et al. Reference Hagnestam-Nielsen, Emanuelson, Berglund and Strandberg2009). Because of the positive genetic correlation (0·4–0·8) between mastitis and somatic cell score (SCS) (Rupp & Boichard, Reference Rupp and Boichard1999; Hu et al. Reference Hu, Wang, Li, Wang, Lai, Li and Zhong2009) a widely used strategy to control mastitis in dairy cattle has been to select against cows with increased SCS (Mark et al. Reference Mark, Fikse, Banos, Emanuelson and Philipsson2000: 2002; Rupp et al. Reference Rupp, Bergonier, Dion, Hygonenq, Aurel, Foulon and Foucras2006).

Somatic cells in the milk of mastitis cows are primarily lymphocytes, neutrophils and macrophages. A typical characteristic of mastitis is an influx of activated CD4+T lymphocytes to the mammary gland. A cluster of differentiation 4 (CD4) proteins is expressed on the surface of CD4+T cells. CD4 protein participates in cell-mediated immunity through combining with MHC class II and mediating signals for bacteria recognition. Rivas et al. (Reference Rivas, Schwager, Gonzále, Quimby and Anderson2007) found that dairy cows infected with Staphylococcus aureus showed a distinct increase of CD4+T cells in mammary gland at the early stage of the infection. Soltys & Quinn (Reference Soltys and Quinn1999) showed that the increased αβ T cells observed in milk from cows with Staph. aureus mastitis were primarily due to increased numbers of CD4+T cells. Thus, CD4 protein and CD4+T cells play key roles in host resistance during the development of mastitis.

Inflammatory disease and immune responses are polygenic traits by nature. Numerous studies have demonstrated that variation in the inflammatory and immune response to bacteria is partially mediated by single nucleotide mutation of immune-related genes (Shelley & Hill, Reference Shelley and Hill2003; Leyva-Baca et al. Reference Leyva-Baca, Schenkel, Martin and Karrow2008). Oyugi et al. (Reference Oyugi, Vouriot, Alimonti, Wayne, Luo, Land, Ao, Yao, Sekaly, Elliott, Simonsen, Ball, Jaoko, Kimani, Plummer and Fowke2009) showed that the SNP in CD4 gene was associated with an increased risk of HIV-1 infection in man. Khatib et al. (Reference Khatib, Monson, Schutzkus, Kohl, Rosa and Rutledge2008) reported that allele G of SNP12195 in STAT5a was associated with a significant decrease in fat and protein percentages as well as SCS in dairy cattle. Selvaggi et al. (Reference Selvaggi, Dario, Normanno, Celano and Dario2009) found that SNP C6853 T in exon 7 of STAT5a appears to improve milk production in Italian Brown cattle with CC cows produced more milk than CT cows. Our previous study reported that the haplotype of two SNPs in STAT5a was associated with milk yield, protein yield and fat yield in Chinese Holsteins (He et al. Reference He, Sun, Yu, Wang and Zhang2007). STAT5 (STAT5a and STAT5b), an important member of the Jak/STAT inflammatory signalling pathway, is known as a prolactin-induced mammary gland factor (Wakao et al. Reference Wakao, Gouilleux and Groner1994; Li & Rosen, Reference Li and Rosen1995; Darnell, Reference Darnell1997). Activation of STAT5a and STAT5b has a permissive role in the initial stages of CD4+T lymphocyte differentiation during inflammatory response (Wilson et al. Reference Wilson, Rowell and Sekimata2009).

Bovine CD4 gene (GenBank accession number: NC_007303) is located on chromosome 5, including seven exons and six introns. STAT5b gene (NC_007317) is located on chromosome 19 and consists of nineteen exons and eighteen introns. Several SNPs in bovine CD4 and STAT5b genes were annotated in the NCBI database. In the present study, we hypothesized that polymorphisms in the two genes may contribute to variation in EBVs for SCS and milk production traits. Thus, two SNPs in CD4 and STAT5b genes in Chinese Holsteins were characterized and their genetic effects on the EBVs for SCS and milk production traits were investigated.

Materials and Methods

Resource population

A total of 1013 Chinese Holstein cows from eight sire families (78–211 daughters per sire) were collected randomly from 20 dairy herds (8–179 cows per herd) in Beijing, China. All of the cows were fed on the same lactation diet according to energy recommendations for lactating Chinese Holstein cows. They were of different parities ranging from 1 to 3 and were milked three times a day. All of the blood samples were collected from the jugular vein.

Estimated breeding values (EBVs) for SCS and five milk production traits (milk yield, fat and protein yields, fat and protein percentages) were used as phenotypes in the study, and were obtained from the official Dairy Data Center of China (Beijing, China). The EBVs for the six traits were estimated using the Genetic Evaluation System adopting the test-day model, which was introduced into China from the Canadian Dairy Network in 2006. Herd-test-day effect, age-parity-season effect and permanent environment effect were included in the test-day model. The average daily heritabilities of the five yield traits ranged from 0·222 to 0·346 and that of SCS ranged from 0·092 to 0·187 in the Chinese Holstein population (Miglior et al. Reference Miglior, Gong, Wang, Kistemaker, Sewalem and Jamrozik2009).

DNA extraction and SNPs investigation

Genomic DNA was extracted from whole blood using DP-318 Blood DNA Kit (Tiangen Biotech Co., China) following the manufacturer's instructions. After genomic DNA isolation, the quantity and quality of DNA were measured using NanoDrop™ ND-2000c Spectrophotometer (Thermo Scientific, Inc.).

Based on the information of the NCBI and Ensembl database, intron 6 of CD4 and exon 16 of STAT5b were annotated as SNPs mutation regions, herein we screened them for identifying SNPs of the two genes by DNA pooling sequencing assays (Bansal et al. Reference Bansal, van den Boom, Kammerer, Honisch, Adam, Cantor, Kleyn and Braun2002). Forty-eight samples out of the 1013 cows were selected randomly to create a DNA pool with equal DNA amounts (50 ng/μl). Two pairs of primers were designed to amplify intron 6 of CD4 and one pair of primer was used for the amplification of STAT5b exon 16:

  • CD4-1F: 5′-CACACTTGGGTCTGGTCCTT-3′,

  • CD4-1R: 5′-GCCGAAGTTGGTGACACAG-3′;

  • CD4-2F: 5′-CCCCCTCCCAGTTCCTTA-3′,

  • CD4-2R: 5′-AGCCTTTCCCTTCCAGTTCT-3′;

  • STAT5b-1F: 5′-CCTAAAACAAAGCCCCGAAT-3′,

  • STAT5b-1R: 5′-CGCTTGGGAGACCTGAGTTA-3′.

Their optimal annealing temperatures were 56°C, 63°C and 62°C, respectively. PCR products were sequenced using the ABI 3730XL (Appliead Biosystems, Foster City CA, USA). Discovery of SNPs was conducted using the software Sequencher 4.2 (demo version, Gene Codes Corporation, Ann Arbor MI, USA).

SNP genotyping

The SNP (NC_007317 g.31562 T>C) in exon 16 of STAT5b was genotyped for 1013 samples using primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) (Ke et al. Reference Ke, Collins and Ye2001). The identified SNP (NC_007303 g.13598C>T) in intron 6 of CD4 was genotyped for 880 samples by PCR-RFLP with the second primer pair (CD4-2F and CD4-2R). Primers for the PIRA-PCR were designed online using PIRA primer design facility (Ke et al. Reference Ke, Collins and Ye2001): forward 5′-CACACACACCTAAAACAAAGCCCCG-3′ and reverse 5′-TTTCCTGACCGGCCCGAAGA-3′.

PCR reactions were performed in a total volume of 20 μl, which included 50–100 ng of genomic DNA, 10 pmol of each primer, 150 μmol of dNTP mix, 1·5 mm-Mg2+, 2·0 μl of 10×PCR buffer and 0·5 unit of Taq DNA polymerase (Takara Biotechnology Co. Ltd.). After an initial denaturation at 95°C for 8 min, PCR was performed by 35 cycles of denaturing at 95°C for 40 s, annealing at 63°C for 45 s, extension at 72°C for 45 s. The final extension was performed at 72°C for 7 min. The PTC-200 PCR machine (Bio-Rad Inc., Hercules CA, USA) was used to carry out PCR reactions. PCR products of CD4 and STAT5b were digested in a total volume of 10 μl using BsiHKAI and BbsI (New England Biolabs, Ipswich MA, USA) at 65°C and 37°C, respectively. Digestion products of CD4 and STAT5b were mixed with 2 μl of loading buffer and subjected to 2% and 5% horizontal agarose gel electrophoresis, respectively. The gels were stained with ethidium bromide for visualization and the genotypes were observed.

Statistical analysis

Associations of the SNPs with EBVs for SCS and five milk production traits were analysed using the mixed model (SAS 9.1):

(model 1)
$$y_{ik} = {\rm \mu} + g_k + a_i + e_{ik} {\rm} $$

where y ik represents each EBV for each trait; μ is overall mean; g k is the effect of the genotypic group k (k=1, 2 or 3. 1 for homozygous genotype TT; 2 for heterozygous genotype TC; 3 for homozygous genotype CC); a i is random residual polygenic effect of the ith cow; e ik is random error. The distribution of a i and e ik is a∼N (0, AσA2) and e∼N (0, $I{\rm \sigma} _e^2 $), respectively. The variance components were executed using AI_REML procedure in the DMU Package (Madsen et al. Reference Madsen, Sørensen and Su2006; Madsen & Jensen, Reference Madsen and Jensen2007).

In model 1, the estimated genotype effects (g k) were further divided into additive effect (A), dominant effect (D) and epistasis effect (I). The additive effect was the mean deviation of two homozygous genotypes (formula 1), and the dominant effect was calculated by the deviation of heterozygous genotype from the mean of two homozygous genotypes (formula 2) (Falconer & Mackay, Reference Falconer and Mackay1996; Rothschild et al. Reference Rothschild, Jacobson, Vaske, Tuggle, Wang, Short, Eckardt, Sasaki, Vincent, McLaren, Southwood, van der Steen, Mileham and Plastow1996).

(formula 1);
$$a = {{TT - CC} \over 2}$$
(formula 2)
$$d = TC - {{TT + CC} \over 2}$$

Where, TT, TC and CC were least squares means of genotype TT, TC and CC, respectively.

The combination effects of the CD4 and STAT5b genes on EBVs for SCS and five milk production traits were analysed with the following model:

(model 2)
$$y_{ik} ={\rm {\mu}} + c_k + a_i + e_{ik} $$

Where y ik, μ, a i and e ik are the same as shown for model 1; and c k is the effect of the combination genotypic group k (k=1, 2, …, 9 as shown in Table 4).

Results

SNPs identification and genotypes

In the present study, six SNPs in CD4 intron 6 and one silent SNP mutation in STAT5b exon 16 were discovered in Chinese Holstein cows. One novel SNP (g.13598C>T) out of six SNPs in CD4 gene and the SNP (g.31562 T>C) in STAT5b were selected for genotyping in the Chinese Holsteins (Fig. 1A, 1B, 1C and 1D). The genotype frequencies of the two SNPs are shown in Table 1.

Fig. 1.

Sequencing and genotyping figures of the two SNPs in bovine CD4 and STAT5b. (A) SNP g.13598C>T in CD4 intron 6 on BTA 5. (B) The length of CD4 g.13598C>T genotypes TT, CT and CC were 735 bp, 735 bp/576 bp/159 bp and 576 bp/159 bp, respectively. M: 200 bp DNA ladder. 1–6 lanes: the genotypes of CD4 gene in six samples. (C) SNP g.31562 T>C in STAT5b exon 16 on BTA 19. The white and grey boxes mean coding and untranslated regions, respectively. (D) The length of STAT5b g.31562 T>C genotypes (TT, TC and CC) were 134 bp, 134 bp/111 bp/23 bp and 111 bp/23 bp, respectively. The 23 bp band has not shown. M: 100 bp DNA ladder. 1–6 lanes: the genotypes of STAT5b gene in six individuals.

Table 1.

Genotypic and allelic frequencies of bovine CD4 and STAT5b genes in Chinese Holsteins

† The number of cows of this genotype

Association and effects of the SNPs

The association of CD4 g.13598C>T and STAT5b g.31562 T>C with EBVs for SCS and five milk production traits in Chinese Holsteins are shown in Table 2. Both SNPs were associated with the EBVs for milk yield and protein yield (P<0·0001), and SNP CD4 g.13598C>T was also associated with the EBV for SCS (P<0·05).

Table 2.

Effects (least squares mean±se)† of CD4 g.13598C>T and STAT5b g.31562 T>C SNPs on five milk production traits and somatic cell score (SCS)

† Bonferroni t test was used for pair comparison in the study. a, b: within a column with no common superscript indicates that means differ at P<0·05. A,B: within a column with no common superscript indicates that means differ at P<0·01. NS: P>0·05

To dissect the genotype effects of the two SNPs, allele additive effect and dominant effect was calculated using formula 1 and formula 2. We found that the additive effect of the CD4 SNP on protein yield was significant (P<0·05), while neither additive effect nor dominant effect of the SNP on SCS was significant (P=0·19 and P=0·11, respectively; Table 3). In addition, the dominant effects of the STAT5b SNP on milk and protein yields were extremely significant (P<0·01) although the additive effects were not significant for both traits (Table 3).

Table 3.

The additive and dominant effects of CD4 g.13598C>T and STAT5b g.31562 T>C on five milk production traits and somatic cell score (SCS)

† The effect of allele T was estimated for CD4 g.13598C>T and STAT5b g.31562 T>C. * P<0·05; ** P<0·01

Association and effects of combination genotypes

Nine combination genotypes of CD4 g.13598C>T and STAT5b g.31562 T>C were detected in 880 Chinese Holstein cows. Their frequencies and combination effects were shown in Table 4. It was noted that the combination effects of the two SNPs were significantly associated with the EBVs for milk yield, protein yield and SCS (P<0·05). Out of the nine combination genotypes, cows with the combination genotype of C2 (TTTC) produced more milk, fat and protein than those of the other eight genotypes. Cows with that of C7 (CCTT) had the highest EBV for SCS and lower EBVs for milk and protein yield, whereas cows with C1 (TTTT) showed the lowest EBV for SCS.

Table 4.

Combination effects of CD4 and STAT5b SNPs on somatic cell score (SCS) and five milk production traits

† Combination genotype: the genotype of CD4 (TT, TC and CC) combining with that of STAT5b (TT, TC and CC) of the same individual. a, b, c within a column with no common superscript indicates that means differ at P<0·05. A, B, C within a column with no common superscript indicates that means differ at P<0·01

Discussion

In this study, we firstly revealed the significant association of CD4 SNP (g.13598C>T) and STAT5b SNP (g.31562 T>C) with the EBVs for milk production traits and SCS in Chinese Holsteins (P<0·05). With the completion of cattle whole-genome sequencing, whole-genome association analysis has been started. In 2010, our research group revealed several significant SNPs related with milk production using Illumina BovineSNP50 BeadChip (Jiang et al. Reference Jiang, Liu, Sun, Ma, Ding, Yu and Zhang2010). However, the two SNPs in CD4 and STAT5b in the present study were not included in the BeadChip. Therefore, the association analysis of the novel SNPs with milk production traits and somatic cell scores is valuable.

Previous studies have demonstrated the importance of CD4+ T cells and STAT5b mammary gland factors in inflammation diseases. During Staph. aureus infection, CD4 knockout mice had poorer bacterial clearance than CD4+ wild-type mice (Mohammed et al. Reference Mohammed, Nasreen, Ward and Antony2000). In STAT5a/STAT5b double knockout mice, the T cells fail to proliferate in response to anti-CD3 stimulation (Moriggl et al. Reference Moriggl, Topham, Teglund, Sexl, McKay and Wang1999). Moreover, the proliferation and differentiation of T lymphocytes and NK cells were impaired in STAT5b-deficient mice (Imada et al. Reference Imada, Bloom, Nakajima, Horvath-Arcidianoco and Udy1998; Igaz et al. Reference Igaz, Tóth and Falus2001). Bovine mastitis is the mammary gland response to pathogenic bacteria, mainly Staph. aureus in China (Gao et al. Reference Gao, Liu, Chen, Shi, Su and Han2010) and is well characterized by increased somatic cells in milk. Studies have found the increased CD4+ T cells during the early stage of bovine mastitis (Taylor et al. Reference Taylor, Keefe, Dellinger, Nakamura and Cullor1997; Soltys & Quinn, Reference Soltys and Quinn1999; Rivas et al. Reference Rivas, Schwager, Gonzále, Quimby and Anderson2007). Here, the significant relationship between CD4/STAT5b combination effects and SCS as well as milk production traits were found in Chinese Holsteins. These results support the pivotal cascade effects of CD4 and STAT5b genes on inflammatory response (Wilson et al. Reference Wilson, Rowell and Sekimata2009).

Six SNPs were identified in intron 6 of CD4 gene in the Chinese Holstein cows. It is well known that introns are integral elements of genes and perform important functions. Functions of introns include acting as carriers of transcription regulatory elements, as sources of non-coding RNA, as actors in alternative splicing and as activators in gene evolution (Fedorova & Fedorov, Reference Fedorova and Fedorov2003). As the limit of restriction enzymes sites, the novel SNP NC_007303 g.13598C>T out of six SNPs was selected for genotyping in the Chinese Holstein population. Through bioinformatics prediction, we found that the downstream 370–398 bp (Chr5:10617785–10617782, UCSC Genome Browser) of CD4 g.13598C>T (Chr5:10618182) exists as a source of several reported miRNAs (bta -miR-1343, bta-miR-2316, bta-miR-2412, bta-miR-2882, etc. Glazov et al. Reference Glazov, Kongsuwan, Assavalapsakul, Horwood, Mitter and Mahony2009). Its function warrants further study. However, no splicing site (GT or AG) was detected at the SNP (Arenas et al. Reference Arenas, Fairbanks, Vijayakumar, Carr, Escuredo and Marinaki2009). Considering the significant additive effect of the CD4 SNP on the EBVs for protein yield, using new genotyping assays to analyse the effect of other five SNPs in intron 6 of CD4 on SCS and milk production traits in the future is warranted.

STATs are a family of transcription factors that regulate actions of peptide hormones and cytokines. Previous study has revealed STAT5a polymorphism affecting milk production traits and SCS in dairy cattle. Khatib et al. (Reference Khatib, Monson, Schutzkus, Kohl, Rosa and Rutledge2008) reported that allele G of SNP12195 in STAT5a was associated with a significant decrease in fat and protein percentages as well as SCS of cattle. As for the substitution C→T at position 6853 in STAT5a exon 7, CC Italian Brown cows produced more milk than CT cows (Selvaggi et al. Reference Selvaggi, Dario, Normanno, Celano and Dario2009). STAT5b and STAT5a are approximately 95% identical in amino acid sequence (Liu et al. Reference Liu, Robinson, Gouilleux, Groner and Hennighausen1995). Here, we found that the cows with TC genotype of the SNP in STAT5b (g.31562 T>C) showed significantly higher milk and protein yield than cows with TT. The distinct difference among the genotypes was mainly caused by the dominant effect, the marker assistant selection to STAT5b should be further studied in Chinese Holstein population.

Variations of EBVs explained by the SNPs were defined as the proportion of genetic variance of SNPs ($V_{G_{SNP}} $) within genetic variance ($V_{G_{trait}} $) of each trait. A surprisingly narrow range of the genetic variance was contributed by SNPs mutation over many traits and species, which is centered about 0·1% (Keightley & Halligan, Reference Keightley and Halligan2009; Hill, Reference Hill2010). In man, the variation of SNP rs11234027 explained 1·2% of the variance in 25-hydroxy-vitamin D [(OH)D] concentrations across studies, and 0·6% of the variance in 25(OH)D concentrations was due to the variation in SNP rs2060793 (Ahn et al. Reference Ahn, Yu, Stolzenberg-Solomon, Simon, McCullough, Gallicchio, Jacobs, Ascherio, Helzlsouer, Jacobs, Li, Weinstein, Purdue, Virtamo, Horst, Wheeler, Chanock, Hunter, Hayes, Kraft and Albanes2010). After calculation, we found that CD4 g.13598C>T can explain 0·3% genetic variations of milk yield, 0·6% genetic variations of protein yield and 0·3% genetic variations of SCS in Chinese Holsteins. Moreover, STAT5b g.31562 T>C can explain 0·6% genetic variations of milk yield, 0·6% genetic variations of protein yield and 0·06% genetic variations of SCS. Therefore, the two SNPs in our study were considerable in improvement of milk production and prevention of mastitis in Chinese Holsteins. Further testing is needed to investigate their function.

Compared with single SNP analyses, combination genotypes analysis provides more information on gene interactions. Multiple locus analysis used in the study revealed that the combination effects of CD4 g.13598C>T and STAT5b g.31562 T>C SNPs significantly affected milk and protein yields as well as SCS. Cows with combination genotype C2 (TTTC) produced highest milk and protein yields but low SCS, while cows with C7 (CCTT) genotype showed lower milk and protein yields but higher SCS (Table 4). Thus, correct selection of the combination genotypes of the two SNPs may be beneficial for improving milk and protein yields and lowering SCS.

Ron et al. (Reference Ron, Feldmesser, Golik, Tager-Cohen, Kliger, Reiss, Domochovsky, Alus, Seroussi, Ezra and Weller2004) in a daughter design reported QTLs on BTA2, 7 and 27 related with milk production traits and health traits including SCS. With respect to the genomic localization of the bovine CD4 gene, a relevant QTL (6400 Kb∼11 100 Kb) on BTA5 for SCS has been previously mapped, which is close to CD4 gene (BTA5: 10 611 Kb∼10 642 Kb) with 3 608 Kb apart (Holmberg & Andersson-Eklund, Reference Holmberg and Andersson-Eklund2004).

In summary, significant association between the SNPs of CD4 g.13598C>T and STAT5b g.31562 T>C with the EBVs for SCS and protein/milk yield was disclosed in the Chinese Holstein population. Combination genotypic analysis further confirmed the significant effect of the two genes. These results implied that the CD4 and STAT5b genes could be powerful candidate genes or linked to major genes that affect SCS and milk production traits in cattle.

This work was supported by the Earmarked Fund for Modern Agro-industry Technology Research System, the National Key Technologies R & D Program (2008AA101002, 2008BADB2B03-05), the State Major Basic Research Development Program (2006CB102107) and the National “948” project (2010-C14). We are grateful for the help from the China Dairy Data Center in providing blood samples and EBVs data.

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Figure 0

Fig. 1. Sequencing and genotyping figures of the two SNPs in bovine CD4 and STAT5b. (A) SNP g.13598C>T in CD4 intron 6 on BTA 5. (B) The length of CD4 g.13598C>T genotypes TT, CT and CC were 735 bp, 735 bp/576 bp/159 bp and 576 bp/159 bp, respectively. M: 200 bp DNA ladder. 1–6 lanes: the genotypes of CD4 gene in six samples. (C) SNP g.31562 T>C in STAT5b exon 16 on BTA 19. The white and grey boxes mean coding and untranslated regions, respectively. (D) The length of STAT5b g.31562 T>C genotypes (TT, TC and CC) were 134 bp, 134 bp/111 bp/23 bp and 111 bp/23 bp, respectively. The 23 bp band has not shown. M: 100 bp DNA ladder. 1–6 lanes: the genotypes of STAT5b gene in six individuals.

Figure 1

Table 1. Genotypic and allelic frequencies of bovine CD4 and STAT5b genes in Chinese Holsteins

Figure 2

Table 2. Effects (least squares mean±se)† of CD4 g.13598C>T and STAT5b g.31562 T>C SNPs on five milk production traits and somatic cell score (SCS)

Figure 3

Table 3. The additive and dominant effects of CD4 g.13598C>T and STAT5b g.31562 T>C on five milk production traits and somatic cell score (SCS)

Figure 4

Table 4. Combination effects of CD4 and STAT5b SNPs on somatic cell score (SCS) and five milk production traits