Two mutations in the 5′‐flanking region of the KITLG gene are associated with litter size of dairy goats

In this study, Xinong Saanen (SN) and Guanzhong (GZ) dairy goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the 5′‐flanking region of the KITLG gene by DNA sequencing and primer‐introduced restriction analysis–polymerase chain reaction. Two novel SNPs (g.13090G>T and g.13664C>A) were identified (GenBank Accession no. KM658964). Furthermore, g.13090G>T and g.13664C>A loci were closely linked in SN and GZ breeds (r² > 0.33). Association analysis results showed that g.13090G>T and g.13664C>A SNPs significantly affected litter size (P < 0.05). The litter size of individuals with the combined genotype GG/CC from both dairy goat breeds was greater than that of individuals with TT/AA in average parity (P < 0.05). Known biochemical and physiological functions, along with our results, indicated that GG/CC could be used in marker‐assisted selection to choose individuals with greater litter size from both breeds. These results extend the spectrum of genetic variation in the caprine KITLG gene and may contribute to genetic resources and breeding of goats.     

Novel SNPs of butyrophilin (<Emphasis Type="Italic">BTN1A1</Emphasis>) and milk fat globule epidermal growth factor (EGF) 8 (<Emphasis Type="Italic">MFG</Emphasis>-<Emphasis Type="Italic">E8</Emphasis>) are associated with milk traits in dairy goat

Butyrophilin (BTN1A1) and milk fat globule epidermal growth factor (EGF) 8 (MFG-E8) genes are both milk fat globule membrane proteins. BTN1A1 plays a key role in the secretion of milk lipid and production which has effects on performance traits, while the MFG-E8 is vital for the development of the mammary gland and phagocytic clearance of apoptotic cells. Therefore, BTN1A1 and MFG-E8 gene are candidate genes for quantitative traits in mammalian animals with respect to milk performance traits. The objective of this study is to investigate variations in goat BTN1A1 and MFG-E8 gene and analyze their associations with growth trait and milk performance. In this study, the goat BTN1A1 gene showed a novel single-nucleotide polymorphism (SNP): XM_001494179:g.8659C>T, resulting in a missense mutation: CTT (Leu)>TTT (Phe) at position 377 aa of the BTN1A1 (526 aa); the goat MFG-E8 gene showed four novel SNPs: NC_007319: g.843delA, 6417delC, 14892T>C and 14996A>C, only the 14892T>C result in a synonymous mutation. The associations between genotypes and production traits were analyzed. Significant statistical results implied that HinfI locus of BTN1A1 gene is associated with milk fat yield (P = 0.004), total solid (P = 0.002), solid-non fat (P = 0.018) and first milk yield (P = 0.030). The DA and EcoRV loci of MFG-E8 gene are associated with milk fat yield (DA locus: P = 0.000; EcoRV locus: P = 0.033) and total solid (DA locus: P = 0.002; EcoRV locus: P = 0.015) in the Xinong Saanen dairy goat.     

Analysis of genetic variability at codon 42 within caprine <Emphasis Type="Italic">prion protein</Emphasis> gene in relation to production traits in Chinese domestic breeds

In this study, genetic variability at codon 42 within prion protein (PRNP) gene and its associations with production traits were investigated in 2002 goats from four Chinese domestic breeds. The frequencies of allele “A” ranged from 0.353 to 0.562 in analyzed goat breeds with Hardy–Weinberg equilibrium (P > 0.05) except Xinong Sannen (XNSN) dairy breed. The establishment of relationships between different genotypes and growth traits was performed in Inner Mongolia white Cashmere (IMWC) breed and revealed an association of the polymorphism with body weight at 7-year-old goats (P = 0.033). The individuals with genotype GG showed heavier body weight than those with genotype AA. Moreover, association analysis detected two significant associations between different genotypes and cashmere yield and fiber length in IMWC breed (P = 0.009, P = 0.048, respectively). In addition, three significant associations of different genotypes with density of milk (a.m. and p.m.), solids-not-fat of milk (P = 0.013, P = 0.009 and P = 0.002), respectively, were found in XNSN breed. Genotype GG had better milk quality than others. These findings suggested that the polymorphism of codon 42 within PRNP was a useful DNA marker for eliminating or selecting excellent individuals in relation to production traits in marker-assist selection breeding of goat.     

A novel SNP of α-lactalbumin gene in Chinese dairy goats

The α-lactalbumin (α-LA) plays a key role in lactose synthesis in mammary glands of domestic animals. Mutations in the α-LA gene are associated with the milk traits in dairy cattle. In our study, a novel SNP: NO_X06366: g.875 C > T was detected in 708 dairy goat individuals—268 of the Xinong Saanen breed and 440 of Guanzhong breed, which revealed a synonymous mutation in the exon 1 of α-LA gene. The Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) and sequencing techniques showed that there were three genotypes: CC, CT and TT. Moreover, the χ²-test showed that the genotype frequencies of the two breeds were in good agreement with the Hardy-Weinberg equilibrium (P > 0.05). The relationship of the polymorphism of dairy goat α-LA gene with the milk trait and the body size trait was revealed. Individuals with the CC genotype were significantly smaller at chest circumference than those with CT (P < 0.05) in both breeds. But the milk trait and other body size traits of the two dairy goat breeds had no significant association with genotypes studied.     

Identification of a functional SNP in the 3′‐UTR of caprine MTHFR gene that is associated with milk protein levels

Xinong Saanen (n = 305) and Guanzhong (n = 317) dairy goats were used to detect SNPs in the caprine MTHFR 3′‐UTR by DNA sequencing. One novel SNP (c.*2494G>A) was identified in the said region. Individuals with the AA genotype had greater milk protein levels than did those with the GG genotype at the c.*2494 G>A locus in both dairy goat breeds (P < 0.05). Functional assays indicated that the MTHFR:c.2494G>A substitution could increase the binding activity of bta‐miR‐370 with the MTHFR 3′‐UTR. In addition, we observed a significant increase in the MTHFR protein level of AA carriers relative to that of GG carriers. These altered levels of MTHFR protein may account for the association of the SNP with milk protein level.     

Molecular characterization and polymorphisms of the caprine Somatostatin (<Emphasis Type="Italic">SST</Emphasis>) and SST Receptor 1 (<Emphasis Type="Italic">SSTR1</Emphasis>) genes that are linked with growth traits

Somatostatin (SST) and its receptors (SSTR1-5) appear to be important in central regulation of many metabolic systems that affect growth, adiposity and nutrient absorption. In this study, we investigated polymorphisms within the caprine SST and SSTR1 genes and determined their relationship with growth traits. As there were no sequence information of the caprine SST and SSTR1 genes, we explored their DNA sequence and genomic organizations. The caprine SST gene is organized in two exons and is transcribed into an mRNA containing 351 bp of sequence coding for a protein of 116 amino acids. Its protein sequences showed substantial similarity (97–99%) to its respective orthologs from cattle, human and mouse. We also cloned and sequenced a 1.2 kb DNA fragment which contained the major part of the coding region and 3′ UTR of the caprine SSTR1 gene. We then detected the polymorphisms in these determined sequences by PCR-SSCP and DNA sequencing methods in 459 goats from four breeds. Four SNPs (GU014693:g.647T>C, GU014693:g.844A>C, GU014693:g.970T>C, GU014693:g.1039T>A), segregating as two haplotypes (T-A-T-T and C-C-C-A), were identified in intron 1 of the caprine SST gene and showed the associations to body length and body height (P < 0.05). Two SNPs (GU014695:g.801 C>T, GU014695:g.948 C>T) were identified in the caprine SSTR1 gene. Significant associations between the three genotypes of GU014695:801 C>T and body length, body height, and chest circumference was observed (P < 0.05). These results suggest that the caprine SST and SSTR1 genes are strong candidate genes that influence growth traits in goat.     

5个山羊品种CSN1S2基因的Alw26I酶切

利用PCR-RFLP技术对西农萨能奶山羊、关中奶山羊、陕南白山羊、安哥拉山羊和波尔山羊5个山羊品种的170个个体的αs 2酪蛋白(CSN1S2)基因进行多态性分析,结果表明：扩增大小为310 bp的片段经限制性内切酶Alw26I酶切后表现多态,且5个山羊品种该基因座位均处于Hardy-Weinberg平衡状态。西农萨能奶山羊、关中奶山羊、陕南白山羊、安哥拉山羊和波尔山羊的基因杂合度/有效等位基因数/Shaanon信息熵/PIC值分别为0.1589/1.1889/0.2955/0.1463, 0.4114/1.6981/0.6017/0.5171,0.1653/1.1980/0.3046/0.1516,0.0646/1.0691/0.1463/0.0625,0.0541/1.0572/ 0.1270/ 0.0526。分析结果显示,关中奶山羊的遗传多样性最丰富,表现为高度多态;其次是西农萨能奶山羊和陕南白山羊,而安哥拉山羊和波尔山羊的遗传变异程度最低。     

Novel SNPs in the caprine stearoyl-CoA desaturase (SCD) and decorin (DCN) genes that are associated with growth traits in Chinese goat breeds

Stearoyl-CoA desaturase (SCD) is an iron-containing enzyme involving in the biosynthesis of monounsaturated fatty acids (MUFA) in mammary gland and adipose tissue, while decorin (DCN) consists of a protein core and a single dermatan or chondroitin sulfate glycosaminoglycan chain, contributing multifunctionally to matrix assembly, modulation of the activity of growth factors and cell migration and proliferation. However, few studies have focused on the genetic variability of them in goat. Herein, five Chinese goat breeds (1229 animals) were analyzed. Based on DNA pooling and PCR-RFLP, three nucleotide substitutions, one of which caused a amino acid substitution, were detected in SCD gene and three haploids (A, B, C) were constructed. According to SSCP analysis and DNA sequencing methods, a 2-bp deletion and two other SNPs were found existing in another analyzed gene DCN, and three haploids (X, Y, Z) were built. Associations between the genotypes and the growth traits (body length, body height, chest circumference, cannon circumference) were also analyzed. For SCD gene, genotype CC individuals had significant greater body height in Guanzhong and body length in both Guanzhong and Xinong saanen than genotype BC individuals (P < 0.05). For DCN gene, individuals with genotype XX was obviously higher than that with genotype XY (P < 0.05). These results indicated that genotype CC of SCD gene and genotype XX of DCN gene could be used for the breeding of new breeds of goat in China.     

Effects of genetic variability of the carpine homeobox transcription factor <Emphasis Type="Italic">HESX1</Emphasis> gene on performance traits

HESX1 plays a key role in the development of the forebrain and pituitary gland and produces potential effects on performance traits. The objective of this study was to detect and assess the associations of the possible polymorphisms of six loci within HESX1 gene with performance traits in Chinese 1,119 goats. Only one novel SNP (NM_001494116:g.307049A > G) locating on IVS1 + 348A > G was identified and detected by HaeIII forced-RFLP-PCR. The frequencies of allele “G” varied from 0.025 to 0.245 in analyzed populations with the Hardy–Weinberg equilibrium (P > 0.05). Genotypic and allelic frequencies were found to be significantly different in four breeds (χ² = 147.674, df = 6, P < 0.001; χ² = 157.250, df = 3, P < 0.001, respectively), implying that the distribution of genotypic and allelic frequencies of goat HESX1 gene was significantly associated with different goat utilities (cashmere, meat and dairy). Association analysis results revealed no significant effects of caprine HESX1 gene on body sizes in XNSN population (P > 0.05) and cashmere traits in IMWC population (P > 0.05). Significant statistical of HESX1 gene with body weight was found (*P < 0.05). The genotype AA showed significantly higher body weight than those of AG in 2-year-old age (*P < 0.05), while the AA genotype was senior to AG genotype in 4-year-old body weight trait (*P < 0.05). These suggestions indicated that the HESX1 gene has significant effect on goat body weight depending on ages, which is accordance with the function repressor of the HESX1.     

Associations of <Emphasis Type="Italic">GHSR</Emphasis> gene polymorphisms with chicken growth and carcass traits

Ghrelin receptor (GHSR), or growth hormone secretagogue receptor, modulates many physiological effects by binding to its ligand and therefore is a candidate gene for chicken production performance. In this study, five polymorphisms (four SNP and a ‘GGTACA’ indel) of GHSR gene were genotyped in a F₂ full sib chicken population to investigate their associations with production traits. Results showed that c.739 + 726T > C (M2) was significantly associated with body weight (BW) at 28 days (BW28), BW90, dressed weight, eviscerated weight, eviscerated weight with giblet, breast muscle weight and leg muscle weight (P < 0.05). Meanwhile, T allele rather than C was positive for chicken body weight gain as individuals with CC had the lowest value of all traits. Otherwise, no significant association of c.264G > A (M1), c.3211-196_3211-181delGGTACA (M3), c.3211 + 75C > T (M4), and c.3211 + 150C > T (M5) with any growth and carcass traits was found. Haplotypes based on five polymorphisms were significantly associated with hatch weight, BW7, BW14, BW21 and breast angle (P < 0.05), as well as BW28 (P < 0.01). Therefore, it was concluded that M2 of the GHSR gene and the analyzed haplotypes were associated with some chicken growth and carcass traits.     

Haplotype combinations of <Emphasis Type="Italic">AdPLA</Emphasis> gene polymorphisms associate with growth traits in Chinese cattle

Ablation of AdPLA in mice could prevent obesity, which implicated AdPLA was a new candidate gene for growth traits. In this study, we screened the polymorphisms of AdPLA gene and revealed three novel SNPs (NC_007330.4:g.43638506 C>T, 43658457 T>C and 43661404 T>C) by DNA sequencing and forced PCR-RFLP methods in 1340 individuals from six breeds. A preliminary single SNP-traits association analysis demonstrated that CC genotype was significantly higher than TT genotype at two growth traits in P6-EcoRII locus (P < 0.05). Moreover, the P8-FbaI locus had a significant association with some growth traits, individuals with TT genotype were higher than CC genotype (P < 0.05). A further analysis confirmed that these two SNPs were in linkage disequilibrium, and haplotype H2, both advantage allelic in P6-EcoRII and P8-FbaI loci, was better than the others for bovine growth traits, which was consistent with single SNP-traits association results. Therefore, this study not only proved that these SNPs were in association with bovine growth traits, but also contributed to evaluate them as genetic markers in bovine genetics and breeding and had potential application in breeding programs.     

Association of IGF-I gene polymorphisms with milk yield and body size in Chinese dairy goats

The association of IGF-I gene polymorphisms with certain traits in 708 individuals of two Chinese dairy-goat breeds (Guanzhong and Xinong Saanen) was investigated. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods were employed in screening for genetic variation. Two novel mutations were detected in the 5'-flanking region and in intron 4 of IGF-I gene, viz., g.1617 G > A and g.5752 G > C (accession D26119.2), respectively. The associations of the g.1617 G > A mutation with milk yield and the body size were not significant (p > 0.05). However, in the case of g.5752 G > C, Xinong Saanen dairy goats with the CG genotype presented longer bodies (p < 0.05). Chest circumference (p < 0.05) was larger in Guanzhong goats with the GG genotype. In Xinong Saanen dairy goats with the CC genotype, milk yields were significantly higher during the first and second lactations (p < 0.05). Hence, the g.5752 G > C mutation could facilitate association analysis and serve as a genetic marker for Chinese dairy-goat breeding and genetics.     

Single nucleotide polymorphisms in bovine <Emphasis Type="Italic">PRL</Emphasis> gene and their associations with milk production traits in Chinese Holsteins

Prolactin (PRL) plays a crucial role in the initiation and maintenance of lactation in mammals. In this study, seven PCR fragments representing most important functional domains of PRL gene were screened for single nucleotide polymorphisms (SNPs) in Chinese Holsteins by single-strand conformation polymorphisms and amplicons sequencing, and their genetic effects on milk production traits were evaluated. A total of four SNPs, including two in the promoter (−1043A>G and −402A>G), one in intron 1 (+2723C>T) and one in exon 4 (+8398G>A) were identified in PRL gene. Statistical results showed significant associations between the promoter genotypes and the milk performance traits in Chinese Holsteins. Cows with genotype P1-GG showed higher milk yields (P < 0.01), while cows with genotype P1-AA showed higher fat contents (P < 0.01). Haplotype analysis of two SNPs in promoter region revealed that the Hap(AG) was significantly associated with increased milk yields and Hap(AA) was associated with increased fat contents (P < 0.01). This is the second study reporting SNPs in the 5′-regulatory region of PRL gene, which interfere with milk production traits.     

Two missense mutations in exon 9 of caprine PRLR gene were associated with litter size

Guanzhong (n = 321) and Boer (n = 191) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions of the prolactin receptor (PRLR) gene by DNA sequencing and PCR‐RFLP. Two SNPs (c.1457G>A and c.1645G>A) were identified that caused amino acid variations p.Ser485Asn and p.Val548Met respectively. Statistical results indicated that the c.1457G>A and c.1645G>A SNPs were significantly associated with litter size in Boer and Guanzhong goat breeds. Further analysis revealed that combined genotype C4 (GGGG) and haplotype G‐G were better than the others for litter size in both goat breeds. These results might contribute to goat genetic resources and breeding.     

Identification of SNPs within the sheep <Emphasis Type="Italic">PROP1</Emphasis> gene and their effects on wool traits

Regarding mutations of PROP1 (Prophet of POU1F1) gene significantly associating with combined pituitary hormone deficiency (CPHD) in human patients and animals, PROP1 gene is a novel important candidate gene for detecting genetic variation and growth, reproduction, metabolism traits selection and breeding. The aim of this study was to detect PROP1 gene mutation of the exon 1–3 and its association with wool traits in 345 Chinese Merino sheep. In this study, on the basis of PCR-SSCP and DNA sequencing methods, ten novel SNPs within the sheep PROP1 gene, namely, AY533708: g.45A > G resulting in Glu15Glu, g.1198A > G, g.1341G > C resulting in Arg63Ser, g.1389G > A resulting in Ala79Ala, g.1402C > T resulting in Leu84Leu, g.1424A > G resulting in Asn91Ser, g.1522C > T, g.1556A > T, g.1574T > C, g.2430C > G were reported. In addition, association analysis showed that three genotypes of P4 fragment were significantly associated with fiber diameter in the analyzed population (P = 0.044). These results strongly suggested that polymorphisms of the PROP1 gene could be a useful molecular marker for sheep breeding and genetics through marker-assisted selection (MAS).     

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.     

A post‐GWAS confirming the SCD gene associated with milk medium‐ and long‐chain unsaturated fatty acids in Chinese Holstein population

The stearoyl‐CoA desaturase (delta‐9‐desaturase) gene encodes a key enzyme in the cellular biosynthesis of monounsaturated fatty acids. In our initial genome‐wide association study (GWAS) of Chinese Holstein cows, 19 SNPs fell in a 1.8‐Mb region (20.3–22.1 Mb) on chromosome 26 underlying the SCD gene and were highly significantly associated with C14:1 or C14 index. The aims of this study were to verify whether the SCD gene has significant genetic effects on milk fatty acid composition in dairy cattle. By resequencing the entire coding region of the bovine SCD gene, a total of six variations were identified, including three coding variations (g.10153G>A, g.10213T>C and g.10329C>T) and three intronic variations (g.6926A>G, g.8646G>A and g.16158G>C). The SNP in exon 3, g.10329C>T, was predicted to result in an amino acid replacement from alanine (GCG) to valine (GTG) in the SCD protein. An association study for 16 milk fatty acids using 346 Chinese Holstein cows with accurate phenotypes and genotypes was performed using the mixed animal model with the proc mixed procedure in sas 9.2. All six detected SNPs were revealed to be associated with six medium‐ and long‐chain unsaturated fatty acids (P = 0.0457 to P < 0.0001), specifically for C14:1 and C14 index (P = 0.0005 to P < 0.0001). Subsequently, strong linkage disequilibrium (D′ = 0.88–1.00) was observed among all six SNPs in SCD and the five SNPs (rs41623887, rs109923480, rs42090224, rs42092174 and rs42091426) within the 1.8‐Mb region identified in our previous GWAS, indicating that the significant association of the SCD gene with milk fatty acid content traits reduced the observed significant 1.8‐Mb chromosome region in GWAS. Haplotype‐based analysis revealed significant associations of the haplotypes encompassing the six SCD SNPs and one SNP (rs109923480) in a GWAS with C14:1, C14 index, C16:1 and C16 index (P = 0.0011 to P < 0.0001). In summary, our findings provide replicate evidence for our previous GWAS and demonstrate that variants in the SCD gene are significantly associated with milk fatty acid composition in dairy cattle, which provides clear evidence for an increased understanding of milk fatty acid synthesis and enhances opportunities to improve milk‐fat composition in dairy cattle.