The polymorphism in the β4-defensin gene and its association with production and somatic cell count in Holstein-Friesian cows

The aim of this study was to find a polymorphism of the bovine β4‐defensin gene and search for its association with milk yield and composition and with the somatic cell count in milk. The data were from the years 1999 to 2004 on 212 Holstein‐Friesian (HF) dairy cows, descended from 70 sires. Based on the sequence of the bovine β4‐defensin gene (GenBank no. AF008307 ) the primers were designed for the amplification of the 924‐bp or 393‐bp long fragments. The 924‐bp long fragment was sequenced and the sequence was compared with that available in the GenBank. Ten putative nucleotide sequence polymorphisms were found in the intron of the bovine β4‐defensin gene. One of them, a C→T transition at position 2239, that creates a new NlaIII (Hin1II) restriction site, was genotyped with polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) in a cohort of 212 HF cows. The CC genotype was the most common (72%). The heterozygous CT genotype was found in 26% of the genotyped cows and four cows (2%) were TT homozygotes. In order to determine the relationship between the polymorphism of the β4‐defensin gene and milk production traits a multi‐trait repeatability test‐day animal model was used. The Derivative‐free Multivariate analysis program was used for computation. The differences between estimates for genotypes were checked using Student's t‐test. The model included the animal genotype, year‐season of calving and parity as fixed effects and the animal additive genetic effect and permanent environmental effect of individual cows as well as dates of the tests as random effects. Significant associations were found between the RFLP‐NlaIII and milk fat, protein and lactose contents. Also, a significant effect was shown of the defensin genotype on the somatic cell count in the milk.     

Polymorphism of insulin‐like growth factor 1 gene is associated with breast muscle yields in chickens

Insulin‐like growth factor‐1 (IGF1) plays an important role in muscle development in chickens. In this study, an F2 chicken population of 362 individuals, obtained from an intercross between high breast muscle yield line males and low breast muscle yield (LB) line females, was constructed for investigating the associations between IGF1 gene and breast muscle yields. The IGF1 sequence was investigated in the grandparents. There were no differences in the exon sequences. However, sequence analysis of the IGF1 promoter revealed a known single nucleotide polymorphism (g.570C > A) in LB line grandparents. PCR – restriction fragment length polymorphism was used for screening the F2 population, which was evaluated for body weight (BW), carcass weight (CW), breast muscle weight (BMW), and breast fillet weight (BFW). Significant associations with the polymorphism were detected for BMW, BFW, BMW% and BFW%, although there were no associations between the polymorphism and BW or CW. The allelic effect on BMW, BFW, BMW% and BFW% acted in additive and dominance modes. We confirmed that the g.570C > A polymorphism is significantly associated with breast muscle yields in the F2 population. Therefore, this polymorphism in the IGF1 gene may help improve breast muscle yields by marker‐assisted selection.     

An independent confirmation of a quantitative trait locus for milk yield and composition traits on bovine chromosome 26

Several reports have demonstrated that bovine chromosome 26 (BTA26) harbours significant or suggestive quantitative trait loci (QTL) for milk production and composition traits in dairy cattle. Our previous study showed that a C/T substitution in the bovine TCF7L2 gene on BTA26 was significantly linked to QTL for protein yield (PY) in a Canadian dairy cattle population. Actually, this polymorphism was one of the markers derived from a genome‐wide screening of QTL for milk PY using an amplified fragment length polymorphism technique combined with a DNA pooling strategy. In the present study, 990 Holstein bulls with complete genotype and phenotype data from 14 sire families were analysed to confirm, if the QTL effects exist in other populations. Statistical analysis revealed that this marker was significantly associated with PY, protein percentage, milk yield and fat yield (FY) (p < 0.001) in the US Holstein population. These results indicate that this QTL region has a pleiotrophic effect on different milk traits and is portable in different populations.     

DGAT1, GH, GHR, PRL and PRLR polymorphism in water buffalo (Bubalus bubalis)

The polymorphism of several genes has been shown to affect the milk composition traits in dairy cattle, including DGAT1‐exon8 K232A, GH‐intron3 MspI, GH‐exon5 AluI, GHR‐exon8 F279Y, PRL‐exon3 RsaI and PRLR‐exon3 S18N. However, the polymorphism and effects of these genes on the milk traits of water buffalo are still unclear. In this study, four DNA pooling samples from Murrah, Nili‐ravi, Murrah‐Nili‐Swamp crossbreed and Chinese swamp buffalo were constructed, respectively, and polymorphism of these sites was investigated using PCR–Single‐strand conformation polymorphism and sequencing. Twenty‐eight inter‐specific single‐nucleotide polymorphism (SNPs) were found in these six assayed gene fragments between buffalo and dairy cattle, including nine intra‐specific SNPs among buffalo groups. All buffalo fixed a K allele genotype in DGAT1‐exon8, MspI⁺ restriction site(c nucleotide) and AluI⁺ site(c nucleotide) at intron3 and exon5 of GH gene, F allele genotype of F279Y mutation in GHR gene, RsaI^? restriction site at PRL‐exon3/exon4 and N allele genotype of S18N mutation at PRLR‐exon3. It provides an indirect evidence that water buffalo have fixed alleles with genotypes reported in dairy cattle, which is thought to be responsible for high milk fat, high protein content and low milk yield. Moreover, three new intra‐specific SNPs were found including 275th bp (c/t) in DGAT1 of Murrah buffalo, 109th bp (t/a) in PRL‐exon3/exon4 and 43rd bp (c/t) in PRLR‐exon3 of Chinese swamp buffalo. Information provided in this study will be useful in further studies to improve buffalo breeding for better lactation performances.     

DNA polymorphism of insulin-like growth factor-binding protein-3 gene and its association with birth weight and body weight in cattle

Insulin‐like growth factor‐binding protein‐3 (IGFBP‐3) is a protein that binds the majority of insulin‐like growth factors in circulation for regulation of its action on growth and metabolism of the animals. Animals belonging to Hariana, Holstein‐Friesian (HF) and their crossbreds (HF × Hariana) were studied using polymerase chain reaction‐restriction fragment length polymorphism and nucleotide sequencing of the IGFBP‐3 gene. A 651‐bp fragment of the IGFBP‐3 gene spanning over a part of exon 2, complete intron 2, exon 3 and a part of intron 3 was amplified and digested with HaeIII restriction enzyme. Three patterns of restriction fragments were observed in HF and crossbred cattle revealing polymorphism in both the populations. The frequency of AA, AB and BB genotypes was 0.65, 0.32 and 0.03 in crossbreds and 0.29, 0.65 and 0.06 in HF respectively. The allelic frequency of the A and B allele was 0.81 and 0.19 in crossbreds and 0.62 and 0.38 in HF cattle respectively. Only one restriction pattern (AA genotype) was observed in all the animals of Hariana breed of Bos indicus showing the absence of polymorphism. Nucleotide sequencing revealed a C → A mutation in the intron 2 region of the IGFBP‐3 gene as the cause of the polymorphism. Least squares analysis revealed a significant effect (p < 0.05) of genotypes on birth weight and body weight (weight at 12, 18 and 24 months of age) of the animals. Animals of AB genotype showed higher birth weight and body weight than the animals possessing AA genotype.     

Analysis of Relationship between Bovine Lymphocyte Antigen DRB3.2 Alleles, Somatic Cell Count and milk Traits in Iranian Holstein Population

The major histocompatibility complex (MHC) is a gene complex closely linked to the vertebrate immune system due to its importance in antigen recognition and immune response to pathogens. To improve our understanding of the MHC and disease resistance in dairy cattle, we gathered 5119 test day records of somatic cell count (SCC) and performance traits of 262 Holstein dairy cows to determine whether the DRB region of the MHC contains alleles that are associated with elevated SCC, milk yield, protein and fat percent of milk. To this purpose, genotyping of animals for DRB3 gene was investigated by polymerase chain reaction‐based restriction fragment length polymorphism (PCR‐RFLP) assay. A two‐step PCR was carried out so as to amplify a 284 base‐pair fragment of exon 2 of the target gene. Second PCR products were treated with three restriction endonuclease enzymes RsaI, BstYI and HaeIII. Twenty‐eight BoLA‐DRB3 alleles were identified including one novel allele (*40). The results in general are in good accordance with allele frequencies of Holstein cattle populations reported by previous studies. Analyses of associations were modeled based on repeated measurement anova and generalized logistic linear methods for production traits and SCC data, respectively. The results of this study showed a significant relationship between the elevated SCC reflecting an increased probability of occurrence to subclinical mastitis and DRB3.2 allele *8 (p < 0.03). The results also revealed significant positive relationships of alleles*22 (p < 0.01) and allele*11 (p < 0.05) with milk fat percent as well as of alleles*24 (p < 0.03) and *22 (p < 0.05) with protein percent. The present study failed to find any association between milk yield and tested alleles. Because of the lack of consistency among results of similar studies, we suggest further investigations to determine the precise nature of these associations with the high polymorphic bovine MHC region to be performed based on haplotypes.     

The bovine annexin 9 gene (ANXA9) is significantly associated with milk-fat yield in a Spanish Holstein–Friesian population

On the basis of QTL studies for milk-fat yield trait on BTA3, annexin 9 protein (ANXA9), fatty acid transport protein type 3 (SLC27A3) and diacylglycerol O-acyltransferase 1 (DGAT1) were selected as candidate genes. Three different single nucleotide polymorphisms (SNPs) of bovine ANXA9, SLC27A3 and DGAT1 genes have been tested in a selective genotyping design for milk-fat yield. Significant allele frequency differences were found for ANXA9 (p = 0.02), in Holstein–Friesian animals with high and low breeding values for milk-fat yield. Regression analysis also showed a significant effect (p = 0.0207) between estimated breeding values (EBVs) for fat milk content and ANXA9 polymorphism. So ANXA9 gene falls into a significant quantitative trait loci interval for milk-fat yield that was previously reported on bovine chromosome 3 in other dairy populations. Our results suggest that the ANXA9 gene polymorphism or a linked segregating QTL contributes to variation in milk-fat yield.     

Differentiating among horse (Equus caballus), donkey (Equus asinus) and their hybrids with combined analysis of nuclear and mitochondrial gene polymorphism

A novel and brief method of differentiating among horse (Equus caballus) and donkey (Equus asinus) and their hybrids (mule, E. asinus × E. caballus and hinny, E. caballus × E. asinus) with combined analysis of nuclear and mitochondrial gene polymorphism (CANMGP) was reported in the present report. A nuclear gene, protamine P1 gene of donkey was sequenced and compared with the known horse sequence from GenBank while a published equid mitochondrial gene, cytochrome b gene of donkey was compared with that of horse. In each of the two genes, a fixed nucleotide substitution within an exon that could be recognized by Dpn II restriction enzyme was found between the two species. Two pairs of primers were designed for amplifying the fragments within the two genes containing the informative nucleotide positions in 65 horses and 41 donkeys and 38 hybrids and conditions of polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) analysis were optimized. Horse, donkey and mule and hinny had their own specific cleavage patterns after the PCR‐RFLP analysis was performed, which made it very easy to identify them from each other. As multiplex PCR can be conducted with the two pairs of primers and only one restriction enzyme is involved in PCR‐RFLP analysis, the method described in the present study is a convenient way to identify horse and donkey and their hybrids. The idea involved in the method of CANMGP can be also used to differentiate other animal species or breeds and their hybrids.     

Genomic organization, localization and polymorphism of porcine PSMB10, a gene encoding the third beta-type proteasome subunit of 26S proteasome complex

The proteasome subunit, beta type 10 (PSMB10) gene regulated by interferon‐gamma is a core part of the 26S proteasome complex, which is an important protein degrading system. Isolation and characterization of swine PSMB10 revealed a conserved structure with other mammalian PSMB10 genes. An A/G nucleotide polymorphism in PSMB10 intron 2 and a C/T single nucleotide polymorphism in exon 5 were detected by polymerase chain reaction restriction fragment length polymorphism. The allele frequencies were significantly different among Tongcheng, Landrace, Large White and Duroc. The porcine PSMB10 was mapped by somatic cell hybrid panel and radiation hybrid mapping on SSC6p14–p15, which is in good agreement with human–pig comparative maps.     

Polymorphism of inhibin βB gene and its relationship with litter size in sheep

The inhibin β_B (INHBB) gene was studied as a candidate gene for the prolificacy of Small Tail Han and Hu sheep. According to the sequence of exon 1 and 2 of bovine INHBB gene, six pairs of primers were designed to detect single nucleotide polymorphisms of exon 1 and 2 of INHBB gene in both high (Small Tail Han and Hu sheep) and low prolificacy breeds (Dorset, Texel and German Mutton Merino sheep) by polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP). Three pairs of primers (primers 1‐1, 1‐2 and 1‐3) were used to amplify the exon 1, and others (primers 2‐1, 2‐2 and 2‐3) to the exon 2. Only the products amplified by primer 2‐3 displayed polymorphism. For primer 2‐3, three genotypes (AA, AB and BB) were detected in Hu sheep and only AA genotype in other breeds. In Hu sheep, frequency of AA, AB and BB genotypes was 0.636, 0.046 and 0.318, respectively. Sequencing revealed 276A > G mutation (based on the amplification region of primer 2‐3) which did not cause any amino acid change because it lay in the 3′ untranslated region. The ewes with genotype BB had 0.58 (P < 0.01) lambs more than those with AA in Hu sheep.     

Genetic mapping of the prion protien gene (PRPN) on bovine chromosome 13

The bovine prion protein gene (PRNP) potentially plays a key role in the development of bovine spongiforme encephalopathy (BSE). In species other than cattle, expression of BSE is clearly dependent on polymorphisms in this gene. PRNP has previously been assigned to the bovine chromosome 13 (BTA13). The present study is an attempt to embed PRNP into a grid of published marker maps. A genetic mapping panel consisting of 266 animals has been genotyped with 19 microsatellites and a polymerase chain reaction‐amplified polymorphism within the PRNP coding region. The linear locus order and the relative distances of these loci are presented. Our linkage map spans 111.6c m of BTA13. The results suggest PRNP to be located telomeric of the microsatellite BMS1580 and centromeric of BM9248 with a log‐likelihood of 2. Our findings further characterize the vicinity of PRNP on BTA13.     

Molecular Typing of a BHV-4 (Bovine herpesvirus 4) Field Isolate

A new BHV-4 (bovine herpesvirus 4) isolated from a case of bovine interdigital dermatitis was characterized by PCR and restriction enzyme analysis. To determine whether the new isolate (PR/1) belonged to BHV-4, DNA from infected cells was specifically amplified by PCR. We used a set of primers spanning a large 2.571 kb conserved region of the BHV-4 genome, including the 3 end of ORF1 (homologous to the EBV BVRF1 gene), ORF2 (homologous to the EBV BXRF1 gene), ORF3 (TK gene) and ORF4 (gH gene) 5 end, respectively. The identity of the amplified product was confirmed by HindIII restriction enzyme digestion and Southern hybridization. No product was observed from the DNA of other bovine herpesviruses tested. The restriction patterns of the PR/1 genome compared to DN 599, MOVAR 33/63 and LVR BHV-4 reference strains showed two kinds of differences, either related or not related to the prDNA (polyrepetitive DNA). Taken together, these data show that PR/1 is a new BHV-4. We would consider that the present report provides a scheme of work for diagnosis and typing of BHV-4 isolates.     

Effects of a novel missense polymorphism within the SIGLEC5 gene on fertility traits in Holstein‐Friesian cattle

The aim of the study was to find functional polymorphism within two exons of the SIGLEC5 (sialic acid‐binding Ig‐like lectin‐5) gene and to examine its effects on the production and fertility traits of cows and bulls. Two hundred seventytwo Holstein‐Friesian cows and 574 bulls were included in the study. Novel missense polymorphism (A > G) within exon 3 causing substitution of amino acid arginine by glutamate in position 260 of SIGLEC5 protein (R260Q) was identified by sequencing and digestion by restriction enzyme Msp I. Basic production and fertility traits of cows and estimated breeding values (EBV) of bulls were analysed. The study demonstrated a significant association of SIGLEC5 R260Q polymorphism with days open and calving interval in cows as well as with breeding value for calving interval in bulls. An opposite effect of SIGLEC5 alleles for production and fertility traits was observed: the allele G increased the breeding value for the protein yield, while the allele A increased the breeding value for the calving interval. The current study suggests the involvement of SIGLEC5 R260Q polymorphism in biological processes related to fertility traits. This finding can be applied as a biomarker for a genetic improvement programme in Holstein‐Friesian cattle.     

Differentiation of Salmonella Gallinarum biovar Gallinarum from Salmonella Gallinarum biovar Pullorum by PCR‐RFLP of the fimH gene

In our studies on FimH adhesins expressed by different Salmonella serovars, we cloned and sequenced the fimH genes from Salmonella enterica ssp. Enterica ser. Gallinarum biovar Gallinarum and S. enterica ssp. Enterica ser. Gallinarum biovar Pullorum. Comparison of the nucleotide sequences revealed the presence of a single‐nucleotide polymorphism (SNP) at position 544 bp from the A of the start codon of the fimH open reading frame (ORF). Further analysis of the restriction enzyme sites in fimH gene showed that the SNP at this position is responsible for a sequence specifically recognized by SacI in S. Gallinarum biovar Gallinarum only, making it possible to differentiate both biovars with the use of polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP). Digestion of PCR amplicons of the fimH gene from S. Gallinarum biovar Gallinarum strains with SacI gave two DNA fragments of 554 and 472 bp and only one fragment of 1026 bp for S. Gallinarum biovar Pullorum. This allows a clear differentiation between these two biovars.     

Polymorphism analysis of BMPR1B gene by forced RFLP and PCR-SSCP techniques and expression of the mutation in introgressed sheep

The present study was conducted to screen Kashmir valley sheep with history of prolificacy for the presence of FecB mutation. Forced polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and single strand conformation polymorphism (SSCP) techniques were employed to detect any polymorphism present in bone morphogenetic protein receptor type 1B (BMPR1B) gene. Further, it was aimed at introgressing the FecB mutation into nonprolific noncarrier sheep. A 140-bp fragment of BMPR1B gene was amplified from isolated genomic DNA and subjected to forced RFLP with restriction enzyme AvaII. Three different RFLP patterns were identified. SSCP analysis showed one-to-one correspondence with RFLP patterns. Sequencing of the samples showing different patterns revealed that the wild (+) and mutant (B) alleles were different by a single nucleotide substitution in the form of A109G from wild to mutant allele. It led to change in amino acid from Glutamine (Q) to Arginine (R) from wild to mutant allele. The mutation was only detected in NARI-Suwarna and their crosses; all Kashmir valley sheep with prolific history lacked it. The + allele was abundant in the studied population. The FecB mutation was introgressed in nonprolific noncarrier sheep by crossing ewes with NARI-Suwarna rams possessing the mutation. First generation crossing produced heterozygous (B+) progeny. Some of the F₁ heterozygous ewes gave birth to twins when mated to unrelated NARI-Suwarna rams. It showed that FecB mutation was successfully expressing in those crosses.     

Genetic variation in monoamine oxidase A and analysis of association with behaviour traits in beef cattle

Husbandry of beef cattle requires animals that do not behave aggressively or timidly. The enzyme monoamine oxidase A and the coding gene (MAOA) play an important role in the complex regulation of behaviour. The complete coding region and a part of the non‐coding sequence of the bovine MAOA gene have been analysed in 20 German Angus and 20 German Simmental bulls and cows with the aim of detecting genetic variability. These two cattle breeds are known to differ regarding their behaviour during handling. Five single nucleotide polymorphisms (SNPs) were identified, three of which were found in the coding region of the gene (exons III and XV). One of the SNPs located in exon XV ( NC_007331.3 :g.80340C>T) was found to be a non‐synonymous mutation. The minor allele frequency of this resulting amino acid substitution was significantly different between 543 German Angus and 417 German Simmental calves (0.39 and 0.49, respectively). The potential functional impact of this polymorphism has been tested by in silico analysis, as well as by association analysis using behaviour scores of the genotyped calves for three behaviour tests that assessed the animals’ temperament during tethering, weighing or social separation. In silico analysis did not deliver consistent results arguing for or against a functional impact of the studied amino acid substitution on the function of the biological protein. No significant association was found between this MAOA polymorphism and the behaviour‐related scores analysed in the study.     

Polymorphism of 5'-region of the bovine growth hormone receptor gene

Genes coding for growth hormone (GH) and GH receptor (GHR) are candidates for quantitative trait markers in farm animals. This work describes a search for nucleotide sequence polymorphisms within the 5′‐region of the bovine GHR gene. Two new single nucleotide polymorphisms were found: restriction fragment length polymorphisms (RFLPs) at a Fnu4HI/TseI site (C/T transition at position ?1104), and at a Sau96I site (C/T transition at position ?262). The Fnu4HI/TseI polymorphic site is located within the 1.2‐kbp LINE‐1 retrotransposon upstream of the P1 promoter, while the Sau96I RFLP locates in the P1 promoter for exon 1A. The appearance of the Sau96I RFLP was studied in representatives of two bovine species, Bos taurus and Bos indicus. An absolute correlation was observed between Sau96I genotype and the insertion/deletion of LINE‐1.