Assessment of association between variants and haplotypes of the IGF2 gene in beef cattle

Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation. The aim of this study was to examine the association of the IGF2 polymorphism with growth traits in beef cattle breed. Four single nucleotide polymorphisms (SNPs: 1–4) were identified in the bovine IGF2 by sequencing pooled DNA samples (Pool-Seq) and forced polymerase chain reaction–restriction fragment length polymorphism (Forced PCR–RFLP) methods. The result of haplotype analysis of four SNPs showed that eight haplotypes and eighteen combined genotypes were revealed, and the linkage disequilibrium and evolutionary relationship were assessed in 1522 individuals representing four purebred cattle breeds from China. The statistical analyses indicated that the 4 SNPs and 18 combined genotypes or haplotypes are associated with the body weight at 18 and 24 months in Jiaxian cattle population (P < 0.05 or P < 0.01). Our results provide evidence that polymorphisms in the IGF2 gene are associated with growth traits, and may be used for marker-assisted selection in beef cattle breeding program.     

Novel polymorphisms of SIX4 gene and their association with body measurement traits in Qinchuan cattle

Sine oculis homeobox homolog 4 (SIX4) gene belongs to the sine oculis/SIX gene family, which includes six members in vertebrates. SIX4 gene plays a crucial role in skeletal myogenesis, and its genetic variations or deficiency may cause hypopituitarism, suggesting that SIX4 gene is a potential candidate gene affecting body measurement traits (BMTs) in animals. Herein, the objectives of this study were to identify genetic polymorphisms of bovine SIX4 gene and to analyze potential association between single nucleotide polymorphisms (SNPs) and body measurement traits in Qinchuan cattle. In the present study, we investigated polymorphisms of SIX4 gene in 426 Qinchuan cattle using DNA sequencing and polymerase chain reaction–restriction fragment length polymorphisms. Three novel SNPs were identified within bovine SIX4 gene. Associations between body measurement traits and SIX4 gene polymorphisms were investigated, and significant statistical associations were found between polymorphisms of these three SNPs and body measurement traits (P < 0.05). Hence, based on results obtained from this study, we conjectured that SIX4 gene may have potential effects on body measurement traits in Qinchuan cattle population and could be used for marker-assisted selection.     

Identification and genetic effect of haplotype in the bovine BMP7 gene

Bone morphogenetic proteins (BMPs) are peptide growth factors belonging to the transforming growth factor-beta (TGF-β) superfamily, and some members of the BMP family support white adipocyte differentiation. In this study, we focused on the BMP7 which singularly promotes the differentiation of brown preadipocytes. Haplotypes involving 5 single nucleotide polymorphism (SNP) sites in the bovine BMP7 gene were identified and their effect on body weight was analyzed. 16 haplotypes and 18 combined haplotypes were revealed and the linkage disequilibrium was assessed in the cattle population with 602 individuals representing three main cattle breeds from China. The results showed that haplotypes 3, 10 and 14 were predominant and accounted for 75.64%, 69.85%, and 83.36% in Nanyang, Qinchuan and Jiaxian cattle breeds, respectively. The statistical analyses indicated that the SNP 1, 4, and 5 are associated with the body weight, body length, and heart girth at 12 and 24 months in Nanyang cattle population (P < 0.05), whereas there is no significant association between their 16 haplotypes and 18 combined haplotypes. Our results provide evidence that some SNPs and haplotypes in BMP7 are associated with growth traits, and may be utilized as a genetic marker in marker-assisted selection for beef cattle breeding programs.     

Haplotype distribution in the GLI3 gene and their associations with growth traits in cattle

The glioma-associated oncogene family zinc finger 3 gene (GLI3) mediates in all vertebrates hedgehog (Hh) signaling that plays an essential role in the induction and patterning of numerous cell types during invertebrate and vertebrate development. In this study, a total of 6 single nucleotide polymorphisms (SNPs: 1–6) were identified by polymerase chain reaction–single stranded conformational polymorphism (PCR–SSCP) and DNA pool sequencing, including all 13 exons and 12 exon–intron boundaries within the bovine GLI3 gene. 16 haplotypes and 13 combined genotypes were revealed and the linkage disequilibrium was assessed in 708 individuals representing three main cattle breeds from China. The statistical analyses indicated that the SNP2, 3 and 4 are associated with the body weight at birth and 6 months in Nanyang cattle population (P < 0.05). No significant association was detected between 11 combined genotypes and body weight at five different ages. Our results provide evidence that polymorphisms in the GLI3 gene are associated with growth traits, and may be used for marker-assisted selection in beef cattle breeding program.     

Combined effects of four SNPs within goat PRLR gene on milk production traits

Xinong Saanen (SN, n = 323) and Guanzhong (GZ, n = 197) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron–exon boundaries of prolactin receptor (PRLR) gene by DNA sequencing, primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Four novel SNPs (g.40452T > C, g.40471G > A, g.61677G > A and g.61865G > A) were identified. The g.61677G > A and g.61865G > A SNPs caused amino acid variations p.Ser485Asn and p.Val548Met, respectively. Both g.40452T>C and g.40471G>A loci were closely linked in SN and GZ goat breeds (r² > 0.33). In addition, there was also a close linkage between g.61677G>A and g.61865G>A loci in both goat breeds. Statistical results indicated that the g.40452T > C, g.61677G > A and g.61865G > A SNPs were significantly associated with milk production traits in SN and GZ breeds. Further analysis revealed that combinative genotype C1 (TTAAGGGG) was better than the others for milk yield in SN and GZ goat breeds. These results are consistent with the regulatory function of PRLR in mammary gland development, milk secretion, and expression of milk protein genes, and extend the spectrum of genetic variation of the caprine PRLR gene, which might contribute to goat genetic resources and breeding.     

Genetic variants in BMP8B gene are associated with growth traits in Chinese native cattle

As a signaling molecule, bone morphogenetic protein 8B (BMP8B) plays an essential role in bone metabolism and is able to regulate thermogenesis and energy balance, which suggests that BMP8B gene may be a new candidate for growth traits. Here, to characterize the effects of BMP8B gene on growth traits, we first used three Chinese indigenous cattle breeds (n = 845) to detect single nucleotide polymorphisms (SNPs). Five novel SNPs of BMP8B gene (g.− 242C>T, g.2164C>T, g.2639T>C, g.2900C>G and g.10817C>T) were identified by DNA pool sequencing and forced PCR–RFLP. And then we associated the five SNPs with four growth traits (body weight, body length, heart girth, and hucklebone width). Results from association analysis showed that the SNPs 1, 2, and 3 affected growth trait(s) markedly (P < 0.05). Further, 6 combined haplotypes were constructed to guarantee the reliability of analysis results. There were also significant differences in body length, heart girth and body weight between the 6 combined haplotypes (P < 0.05), but not in hucklebone width (P > 0.05). Collectively, our results suggest a modulatory role of BMP8B gene in cattle growth and development, and 3 SNPs could be used as molecular markers in early marker assisted selection (MAS) in beef cattle breeding program.     

Single-nucleotide polymorphisms g.151435C>T and g.173057T>C in PRLR gene regulated by bta-miR-302a are associated with litter size in goats

Single-nucleotide polymorphisms (SNPs) located at microRNA-binding sites (miR-SNPs) can affect the expression of genes. This study aimed to identify the miR-SNPs associated with litter size. Guanzhong (n = 321) and Boer (n = 191) goat breeds were used to detect SNPs in the caprine prolactin receptor (PRLR) gene by DNA sequencing, primer-introduced restriction analysis-polymerase chain reaction, and polymerase chain reaction-restriction fragment length polymorphism. Three novel SNPs (g.151435C>T, g.151454A>G, and g.173057T>C) were identified in the caprine PRLR gene. Statistical results indicated that the g.151435C>T and g.173057T>C SNPs were significantly associated with litter size in Guanzhong and Boer goat breeds. Further analysis revealed that combinative genotype C6 (TTAACC) was better than the others for litter size in both goat breeds. Furthermore, the PRLR g.173057T>C polymorphism was predicted to regulate the binding activity of bta-miR-302a. Luciferase reporter gene assay confirmed that 173057C to T substitution disrupted the binding site for bta-miR-302a, resulting in the reduced levels of luciferase. Taken together, these findings suggested that bta-miR-302a can influence the expression of PRLR protein by binding with 3′untranslated region, resulting in that the g.173057T>C SNP had significant effects on litter size.     

Effect of genetic variations within the SH2B2 gene on the growth of Chinese cattle

As an adaptor protein, apart from potentiating Janus kinase 2 (JAK2) activation and promoting the insulin signaling pathway, Src homology 2 B 2 (SH2B2) indirectly takes part in the regulation of glucose uptake through the c-Cb1/CAP/TC10 pathway, which can in turn affect growth. In this study, we identified a 4 bp indel mutation and three single nucleotide polymorphisms (SNPs) within the SH2B2 gene in 959 individuals from five Chinese cattle breeds by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing. Based on the four variations, 12 haplotypes were identified. Additionally, there was a tendency that in every population pair-wise linkages increased progressively from V1–V2 to V3–V4. By association analysis, positive effects of genotypes CC and CT (snp1220 locus), DI (4 bp indel locus), and CC (snp21049 locus) on growth traits were obtained. Furthermore, when combined, individuals with the combination CCDITTCC showed the best performance at an early age. These results were suggestive of an association of snp1220, 4 bp indel and snp12049 with growth performance in Nanyang cattle, indicating possibly the candidate role of the SH2B2 gene in marker assisted selection in a beef cattle breeding program.     

Primers for sequence characterization and polymorphism detection in the Atlantic salmon (Salmo salar) growth hormone 1 (GH1) gene

We report the identification of intraspecific sequence variation in the Atlantic salmon (Salmo salar) growth hormone 1 gene. Rapid and inexpensive assays for polymorphism detection were developed for 10 sites. Five of the assays detected single nucleotide polymorphisms (SNPs) using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses, and five were indel polymorphisms, detected using fragment length analyses. The average within population frequency of the most common allele varied from 0.52 to 0.90, and the average within population heterozygosity varied from 0.02 to 0.37 in seven European salmon populations.     

SNP exploring in the middle and terminal regions of the IGF-1 gene and association with production and reproduction traits in Holstein cattle

Five primer sets were designed in order to identify single nucleotide polymorphisms (SNPs) in middle and terminal exons (2 to 6) and in some flanking intronic regions of the bovine insulin-like growth factor 1 (IGF-1) gene. Sequencing results of PCR products for 10% of animals showed no variant in exons but a SNP at intron 4 was occurred. Both polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and high resolution melting (HRM) methods were developed to genotype samples. The PCR–RFLP results showed the presence of three fragments on agarose gel for the C allele due to two cleavage sites while two fragments for the T allele were observed. Melting curves of 123 bp fragments in HRM analysis showed a difference between temperature melting (T_m) of two homozygous genotypes as the CC genotypes had higher T_m than the TT genotypes. Melting curve of the CT genotype was different and crossed two parallel patterns of homozygous genotypes. The frequencies of the CC, CT and TT genotypes were 0.6, 0.37 and 0.03, respectively. Also, the estimated allele frequencies were 0.785 and 0.215 for the C and T alleles, respectively. Results showed higher accuracy of the HRM analysis compared to the PCR–RFLP method. Least square means (LSMs) comparison of the different genotypes in the SNP showed significant association with milk fat yield trait in the first lactation and open days after the second calving. The polymorphism did not have a significant effect on other milk production or reproduction traits. It seems that other variants or QTLs known in this region underlie genetic variation in the production and reproduction of dairy cattle.     

SNP and haplotype analysis reveal IGF2 variants associated with growth traits in Chinese Qinchuan cattle

Insulin-like growth factor 2 (IGF2) is a potent cell growth and differentiation factor and is implicated in mammals’ growth and development. The objective of this study was to evaluate the effects of the mutations in the bovine IGF2 with growth traits in Chinese Qinchuan cattle. Four single nucleotide polymorphisms (SNPs) were detected of the bovine IGF2 by DNA pool sequencing and forced polymerase chain reaction–restriction fragment length polymorphism (forced PCR–RFLP) methods. We also investigated haplotype structure and linkage disequilibrium (LD) coefficients for four SNPs in 817 individuals representing two main cattle breeds from China. The result of haplotype analysis showed eight different haplotypes and 27 combined genotypes within the study population. The statistical analyses indicated that the four SNPs, combined genotypes and haplotypes are associated with the withers height, body length, chest breadth, chest depth and body weight in Qinchuan cattle population (P < 0.05 or <0.01). The mutant-type variants and mutant haplotype (Hap 8: ATGG; likely to be the beneficial QTN allele) was superior for growth traits; the heterozygote diplotype was associated with higher growth traits compared to wild-type homozygote. Our results provide evidence that polymorphisms in the IGF2 gene are associated with growth traits, and may be used for marker-assisted selection in beef cattle breeding program.     

The polymorphisms of bovine <Emphasis Type="Italic">VEGF</Emphasis> gene and their associations with growth traits in Chinese cattle

PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of VEGF gene in 671 individuals belonging to three Chinese indigenous cattle breeds including Nanyang, Jiaxian Red and Qinchuan. Three haplotypes (A, B and C), four observed genotypes (AA, AB, BB and AC) and three new SNPs (6765T>C ss130456744, 6860A>G ss130456745, 6893T>C ss130456746) were detected. The analysis suggested that one SNP (ss130456744) in the bovine VEGF gene had significant effects on birth weight, body weight and heart girth at 6 months old in the Nanyang breed (P < 0.05). The results showed that the SNP (ss130456744) in intron 2 of the VEGF gene is associated with early development and growth of Chinese cattle. These findings raise hope that this polymorphism can be a molecular breeding marker in breeding strategies through marker assisted selection (MAS) in Chinese domestic cattle.     

Characterization of bovine MHC DRB3 diversity in Latin American Creole cattle breeds

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for diseases and immunological traits. However, none of the highly adapted Latin American Creole breeds have been characterized for BoLA gene polymorphism by high resolution typing methods. In this work, we sequenced exon 2 of the BoLA class II DRB3 gene from 179 cattle (113 Bolivian Yacumeño cattle and 66 Colombian Hartón del Valle cattle breeds) using a polymerase chain reaction sequence-based typing (PCR-SBT) method. We identified 36 previously reported alleles and three novel alleles. Thirty-five (32 reported and three new) and 24 alleles (22 reported and two new) were detected in Yacumeño and Hartón del Valle breeds, respectively. Interestingly, Latin American Creole cattle showed a high degree of gene diversity despite their small population sizes, and 10 alleles including three new alleles were found only in these two Creole breeds. We next compared the degree of genetic variability at the population and sequence levels and the genetic distance in the two breeds with those previously reported in five other breeds: Holstein, Japanese Shorthorn, Japanese Black, Jersey, and Hanwoo. Both Creole breeds presented gene diversity higher than 0.90, a nucleotide diversity higher than 0.07, and mean number of pairwise differences higher than 19, indicating that Creole cattle had similar genetic diversity at BoLA-DRB3 to the other breeds. A neutrality test showed that the high degree of genetic variability may be maintained by balancing selection. The F_ST index and the exact G test showed significant differences across all cattle populations (F_ST = 0.0478; p < 0.001). Results from the principal components analysis and the phylogenetic tree showed that Yacumeño and Hartón del Valle breeds were closely related to each other. Collectively, our results suggest that the high level of genetic diversity could be explained by the multiple origins of the Creole germplasm (European, African and Indicus), and this diversity might be maintained by balancing selection.     

Lack of an association of programmed cell death-1 PD1.3 polymorphism with risk of hepatocellular carcinoma susceptibility in Turkish population: A case–control study

Aim

The programmed cell death-1 (PD-1) is a potent immunoregulatory molecule which is responsible for the negative regulation of T-cell activation and peripheral tolerance. Recently, overexpression of PD-1 has been reported to contribute to immune system evasion and poor survival of hepatocellular carcinoma (HCC). A common single nucleotide polymorphism in intron 4 of PD-1 gene called PD-1.3 has been reported to influence PD-1 expression, but its association with HCC has yet to be investigated. The aim of the present study was to investigate whether this polymorphism could be involved in the risk of HCC susceptibility.

Methods

The genotype frequency of PD-1.3 polymorphism was determined by using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method in 236 subjects with HCC and 236 cancer-free control subjects matched on age, gender, smoking and alcohol status.

Results

No statistically significant differences were found in the genotype distributions of the PD-1.3 polymorphism among HCC and cancer-free control subjects (P = 0.22).

Conclusion

Our results demonstrate for the first time that the PD-1.3 polymorphism has not been in any major role in genetic susceptibility to hepatocellular carcinogenesis, at least in the population studied here. Independent studies are needed to validate our findings in a larger series, as well as in patients of different ethnic origins.     

Identification of bovine NPC1 gene cSNPs and their effects on body size traits of Qinchuan cattle

NPC1 gene is an important gene closely related to the Niemann–Pick type C (NPC). Mutations in the NPC1 gene tend to cause Niemann–Pick type C, a lysosomal storage disorder. Previous studies have shown that NPC1 protein plays an important role in subcellular lipid transport, homeostasis, platelet function and formation, which are basic metabolic activities in the process of development. In this study, to explore the association between the NPC1 gene variation and body size traits in Qinchuan cattle, we detected four novel coding single nucleotide polymorphisms (cSNPs) in the bovine NPC1 gene, including one missense mutation (SNP1) and three synonymous mutations (SNP2, SNP3 and SNP4). Population genetic analyses of 518 individuals and association correlations between cSNPs and bovine body size traits were conducted in this research. A missense mutation at SNP1 locus was found to be significantly related to the heart girth, hip width and body weight (P < 0.01 or P < 0.05, 3.5-year-old). Two synonymous mutations at SNP2 and SNP3 loci also showed significant effects on hip width (P < 0.05, 3.5-year-old). One synonymous mutation at SNP4 locus showed significant effect on body weight (P < 0.05, 2.0-year-old). Combined haplotypes H₂H₆ and H₆H₆ showed significant effects on body size traits such as heart girth, hip width, and body weight (3.5-year-old, P < 0.01 or P < 0.05). This study provides evidence that the NPC1 gene might be involved in the regulation of bovine growth and body development, and may be considered as a candidate gene for marker assisted selection (MAS) in beef cattle breeding industry.     

Association between two genetic variants of CD226 gene and Cervical Squamous Cell Carcinoma: A case–control study

Cervical carcinoma is a common gynecologic tumor severely influencing the health and life quality of women worldwide. CD226, a costimulatory molecule, is mainly participated in the activation and differentiation of T cells. Recent studies have investigated the association between two genetic variants (rs763361 and rs727088) of CD226 gene and many diseases. In order to evaluate whether these two variants are associated with Cervical Squamous Cell Carcinoma (CSCC), a case–control study including 349 CSCC patients and 380 unrelated healthy controls was carried out to determine the genotypes of these two variants by using the methods of polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and DNA sequencing methods. Significantly increased CSCC risk was observed to be associated with G allele of rs727088 locus (OR = 1.422, 95% CI = 1.129–1.792). We have also observed that increased CSCC risk was statistically associated with rs727088 polymorphism in a dominant model (OR = 1.41, 95% CI = 1.05–1.89). Results of stratified analysis revealed that both rs763361 and rs727088 polymorphisms were not associated with clinical characters. Collectively, this study supports that rs727088 polymorphism may contribute to increased CSCC susceptibility.     

Characterization and mapping of bovine dopamine receptors 1 and 5

A cDNA encoding the bovine dopamine receptor 1 (DRD1) was isolated from a bovine cDNA library, cloned and completely sequenced. The coding region showed 93 and 91% sequence identity on DNA level and 96 and 94% on protein level with its respective porcine and human orthologs. The bovine DRD1 and dopamine receptor 5 (DRD5) were mapped, respectively, to BTA10 and 6 by radiation hybrid mapping. One SNP was found in DRD1 and four in DRD5. Using polymerase chain reaction-restriction fragment length polymorphism, 11 different European cattle breeds were screened for the presence of the DRD1 and DRD5 substitutions. Allele frequencies for DRD1 and DRD5 alleles were very similar across all the breeds examined. Allele frequency discrepancies were found between Belgian Blue beef breed and the other breeds.