SNP variation in ADRB3 gene reflects the breed difference of sheep populations

The β3-adrenergic receptor (ADRB3), a G-protein coupled receptor, plays a major role in energy metabolism and regulation of lipolysis and homeostasis. We detect the single nucleotide polymorphism (SNP) variation in full-length sequence of ovine ADRB3 gene in 12 domestic sheep populations within four types by polymerase chain reaction-single strand conformation polymorphism and sequencing to reveal the breed difference. Twenty-two SNPs, 12 of which in the exon 1 and ten in the intron, were detected, and 12 new exonic and four new intronic SNPs were found. Most SNPs presented in Shanxi Dam Line and least ones in Dorset. The average SNP number in both meat and dual purpose for meat and wool breeds was significantly higher than general and dual purpose breeds for wool and meat. Frequency of each SNP in studied breeds or types was different. The 18C Del and 1617T Ins majorly existed in dual purpose breeds for wool and meat. The 25A Del, 119C>G and 130C>T were mostly found in the meat and dual purpose for meat and wool breeds. The 1764C>A more frequently presented in meat than in other types. The majority of variations came from within the populations as suggested by analysis of molecular variance. Close relationship presented among the Chinese and western breeds, respectively. In conclusion, SNPs of ovine ADRB3 gene can reflect the breed difference and within- and between-population variations, and to a great extent, the breed relationship.     

Novel single nucleotide polymorphism identification in interleukin-6 gene of Pakistani sheep

The present study aimed to identify single-nucleotide polymorphism (SNP) in coding and non-coding regions of interleukin-6 (IL-6) gene of Pakistani sheep. The IL-6 gene of 205 animals from nine sheep breeds were sequenced for screening of SNP. Characterizing the IL-6 gene revealed thirteen SNP sites within the intronic region of IL-6 gene. The novel SNPs found in the present study can serve as genetic marker for association studies with susceptibility/resistance to parasite infection in sheep. This is first report of SNP polymorphism of IL-6 gene of Pakistani sheep.     

Computational and artificial neural network based study of functional SNPs of human LEPR protein associated with reproductive function

Genetic polymorphisms are mostly associated with inherited diseases, detecting and analyzing the biological significance of functional single-nucleotide polymorphisms (SNPs) using wet laboratory experiments is an arduous task hence the computational analysis of putative SNPs is essential before conducting a study on a large population. SNP in the leptin receptor (LEPR) could result in the retention of intracellular signalling due to the structural and functional instability of the receptor causing abnormal reproductive function in human. In this first comprehensive computational analysis of LEPR gene mutation, we have identified and analyzed the functional consequence and structural significance of the SNPs in LEPR using recently developed several computational algorithms. Thirteen deleterious mutations such as W13C, S93G, I232R, Q307H, Y354C, E497A, Q571H, R612H, K656N, T690A, T699M V741M, and L760R were identified in the LEPR gene coding region. Backpropagation algorithm has been developed to forestall the deleterious nature of SNP and to validate the outcome of the tested computational tools. From ConSurf prediction three SNPs (Q571H, R612H, and T699M) were highly conserved on LEPR protein and the most deleterious variant R612H had one hydrogen bond abolished and severely reduced protein stability. Molecular docking suggested that the mutant (R612H) LEPR had lowest binding energy than native LEPR with the ligand molecule. Thus the energetically destructive changeover of ARG to HIS in R612H could possibly affect the LEPR protein structural stability and functional constancy due to interruption in the amino acid interactions and could result in reproductive disorders in human and increases the complication in obstetric and pregnancy outcome.     

Identification of single nucleotide polymorphisms in the ovine acetyl-CoA carboxylase-alpha gene

Acetyl-CoA carboxylase (ACACA) is the rate-limiting enzyme in the biosynthesis of palmitic acid and long-chain fatty acids. The dietary intake of palmitic acid, which represents approximately 22% of sheep milk fatty acids, increases low-density lipoprotein (LDL) levels and the risk of developing human cardiovascular diseases. Following the candidate gene approach for improving sheep milk composition, and as a first step in assessing the possible influence of the ovine ACACA gene on milk fatty acid composition and its potential use as an animal genetic model of human atherosclerosis disease, we present here an investigation into the genetic variability of the ovine ACACA gene. We sequenced approximately 6.6 kb of ovine ACACA cDNA, including most of the coding sequence of the protein (except 348 bp), in Spanish Churra sheep. A total of 22 synonymous single nucleotide polymorphisms (SNPs) were identified in the analysed sequence, which were genotyped in a set of eight sheep breeds with different productive aptitudes (dairy, meat and double aptitudes). Two of the SNPs identified, SNP03 (c.1450T>C) and SNP15 (c.5134T>C), which appeared to be breed-specific variations, were situated in the gene sequence coding for the biotin-carboxylase (BC) and acetyl-CoA carboxyltransferase (ACCT) domains of the protein, respectively. Particularly interesting is SNP12 (c.4579G>A), which displayed higher frequencies in the dairy-specialised breeds relative to the meat-producing breeds. Moreover, in the dairy breeds studied, the frequency of this SNP showed a positive correlation with the degree of dairy specialisation. A previously described alternative splicing site (Ser-1200) affecting an important regulatory region of the enzyme was observed in one of the Churra animals. Despite the high genetic variability observed in this gene, none of the identified SNPs caused an amino acid change. However, these polymorphisms could be in linkage disequilibrium with other mutations showing a functional effect on the ACACA enzyme. Hence, the characterisations of the allelic variants reported herein lay the groundwork for evaluation of the potential use of these SNPs as genetic markers of fat content and fatty acid composition in sheep dairy products.     

SNP detection and haplotype analysis in partial sequence of MSTN gene in sheep

Polymerase chain reaction (PCR) products of the MSTN gene amplified from sixty sheep of nine Chinese indigenous sheep breeds and one imported sheep breed were sequenced to identify the single-nucleotide polymorphisms (SNPs) in a 378-bp fragment including intron 2 and exon 3 of the MSTN gene. A total of fifteen SNPs (A1937C, T1942G, C1956T, A1972C, A1990G, A2008C, A2011G, C2019T, A2025C, A2027C, T2085G, T2173C, C2198T, C2210T and C2213T) were detected among the sixty sequenced individuals and they were all located in intron 2. Twelve haplotypes were identified from these fifteen SNPs, of which haplotype I (CGTCGCGTCCGCTTT) and VIII (ATCAAAACAATTCCC) were the two major and basic ones with frequencies of 12.25% and 77.80%, respectively. Haplotype VIII was distributed in all sheep breeds and all individuals of the meat or meat-wool type sheep breeds were homozygous with respect to this haplotype. This suggests that haplotype VIII might be related to meat production traits in sheep. Haplotype I was only distributed in the fur, lambskin type and fur-meat type sheep breeds. This suggests that haplotype I may have some relationship with fur traits in sheep.     

五指山猪IGF2基因5′调控区单核苷酸多态性分析

利用PCR产物直接测序法, 对五指山猪、滇南小耳猪、香猪、梅山猪和大白猪共60个样本的IGF2基因5＇调控区部分片段的单核苷酸多态性进行了研究。找到13个SNP, 分别是: C5872T、C5888T、A5976G、C6010T、T6029A、C6037T、C6043T、C6063T、C6112T、C6164T、G13520A、G13563A和G13669A。T6029A为T←→A碱基颠换, A5976G、G13520A、G13563A和G13669A为A←→G转换, 其他均为C←→T转换。针对13个SNP位点得到23种组合基因型。统计各位点等位基因和基因型以及各组合基因型在总群体与各品种内的分布频率, 发现3个小型猪在A5976G、C6164T和G13669A位点上的优势等位基因均分别为G、T和A, 而梅山猪和大白猪的优势等位基因均分别为A、C和G; H19型为3个小型猪的特征组合基因型, 而另两个猪品种为H15型。同时对123头五指山猪IGF2基因C5888T位点进行了PCR-RFLP分析, 研究表明该位点C为优势等位基因(0.8536), CC为优势基因型(0.7235)。卡方检验表明该位点处于Hardy-Weinberg平衡状态。这些结果可为五指山猪等小型猪的生长发育规律、矮小机制等方面的研究提供遗传学依据。     

Single nucleotide polymorphisms in the open reading frame of the stearoyl-CoA desaturase gene and resulting genetic variants in Canadian Holstein and Jersey cows.

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of conjugated linoleic acid (CLA) and mono-unsaturated fatty acids (MUFA) from their saturated counterparts in the mammary gland and adipose tissue of ruminant animals. We hypothesize that single nucleotide polymorphisms (SNPs) in the SCD gene account for some of the differences in SCD activity, and consequently for some of the variations in CLA and MUFA content of milk fat between Holsteins and Jersey cows and within these two breeds. We analyzed the open reading frame of the SCD gene of 44 Holsteins and 48 Jerseys for SNPs by sequencing. Three SNPs: 702A --> G, 762T --> C and 878C --> T were identified in both breeds and a further SNP, 435G --> A, was unique to Holsteins. The SNPs characterized four different genetic variants in Holsteins: A (G(435)A(702)T(762)C(878)), A1 (A(435)A(702)T(762)C(878)), B (G(435)G(702)C(762)T(878)) and B1 (A(435)G(702)C(762)T(878)), with only variants A and B in Jerseys. SNP 878C --> T resulted in a non-synonymous codon change while the rest resulted in synonymous codon changes giving rise to two protein variants, A having alanine and B having valine. Allele A was the most prevalent in the two breeds. These differences may, therefore, contribute to existing variations in CLA and fat content between and within Canadian Holstein and Jersey cows.     

New single nucleotide polymorphisms of androgen receptor gene (<Emphasis Type="Italic">AR</Emphasis>) in the Russian breed of Dzhalginsky Merino sheep

This paper investigates the structure of androgen receptor gene (AR) in the Russian breed of Dzhalginsky Merino sheep. Polymorphisms of the gene were detected using NimbleGen sequencing technology (Roche, United States). Eight single nucleotide polymorphisms (SNPs) and two insertions were detected. Five of these SNPs (c.335T>G, c.339G>A, c.342T>C, c.2491-327T>A, and c.2491-325A>T) and both insertions were identified for the first time. Three SNPs and the insertions are located in the coding part of exon. Insertion c.336_337 is found in most of the animals of this breed and can be used as a genomic marker of the breed. Animals with mutant variant of SNP c.1496+15T>C have significantly lower live weight and body size compared with the wild type genotype. This SNP can be used as a genetic marker of meat production in marker-assisted selection.     

BcSNPdb: bovine coding region single nucleotide polymorphisms located proximal to quantitative trait loci

Bovine coding region single nucleotide polymorphisms located proximal to quantitative trait loci were identified to facilitate bovine QTL fine mapping research. A total of 692,763 bovine SNPs was extracted from 39,432 UniGene clusters, and 53,446 candidate SNPs were found to be a depth >3. In order to validate the in silico SNPs experimentally, 186 animals representing 14 breeds and 100 mixed breeds were analyzed. Genotyping of 40 randomly selected candidate SNPs revealed that 43% of these SNPs ranged in frequency from 0.009 to 0.498. To identify non-synonymous SNPs and to correct for possible frameshift errors in the ESTs at the predicted SNP positions, we designed a program that determines coding regions by protein-sequence referencing, and identified 17,735 nsSNPs. The SNPs and bovine quantitative traits loci informations were integrated into a bovine SNP data: BcSNPdb (http://snugenome.snu.ac.kr/BtcSNP/). Currently there are 43 different kinds of quantitative traits available. Thus, these SNPs would serve as valuable resources for exploiting genomic variation that influence economically and agriculturally important traits in cows.     

Analysis of the non‐recombining Y chromosome defines polymorphisms in domestic pig breeds: ancestral bases identified by comparative sequencing

Sequences from 20 amplicons representing nine different loci and 11369bp from the short arm of the pig Y chromosome were compared using pools of DNA from different European and Chinese breeds. A total of 33 polymorphic sites were identified, including five indels and 28 single nucleotide polymorphisms (SNPs). Three high frequency SNPs within the coding regions of SRY were further analysed across 889 males representing 25 European and 25 Asian breeds or Lines, plus a European Line of Meishan. Two haplotypes seen to be associated with ‘European’ or ‘Chinese’ origin in the initial SNP discovery phase were found to be the most common in their respective groups of breeds in a more detailed genotyping study. Two further SRY haplotypes are relatively rare. One was found exclusively within Tamworth, at low frequency in Retinto, and in three Chinese breeds (Huai, Sahwutou and Xiaomeishan). The other uncommon haplotype is found exclusively in Bamajiang, two further Chinese breeds (Hangjiang Black and Longling) and two European rare breeds (Mangalica and Linderödssvin), but appears based on comparison with other suids to represent an ancestral sequence.     

Nucleotide variants in prion-related protein (testis-specific) gene (PRNT) and effects on Chinese and Mongolian sheep phenotypes

ABSTRACT

Studies of the ovine prion-related protein (testis-specific) gene (PRNT), including studies of genetic diversity, have highlighted its potential relationship to scrapie infection and economically important ovine traits. PRNT was previously reported to be highly polymorphic in Portuguese sheep. To characterize genetic polymorphisms in this gene in Asian sheep, a direct sequencing method was used to detect polymorphic loci in PRNT in 285 individual sheep from four Chinese and one Mongolian breeds. Seven SNP variants in PRNT were identified, including three novel variants (g.93G>A, g.162G>T, and g.190A>G) and four previously reported variants (g.17 C>T, g.112G>C, g.129C>T, and g.144A>G). In the five breeds that we analyzed, the mutation frequencies of g.190A>G in Lanzhou Fat-tail sheep (LFTS) and g.129C>T in the other four varieties were high (F>0.5). Moreover, thirteen different haplotypes that had a comparable distribution in the tested breeds were also identified; ‘C-G-G-C-A-G-A’ occurred at the highest frequency in the five sheep breeds. Additionally, we previously explored the significance of relationships between polymorphisms in PRNP or PRND and ovine growth performance. Here, we also performed correlation analysis in all tested loci. These loci polymorphisms were significantly associated with ten different growth traits (P<0.05), except for g.93G>A. Meanwhile, in contrast to a previous study, there was no significant association between the seven SNP loci analyzed and our previously reported sheep PRND or PRNP insertion/deletion mutations. Our findings may provide new insights into polymorphic variation in ovine PRNT, which may contribute to genetic improvements in economic traits that are important for sheep breeding.     

牛HTR1B基因的分子遗传特性研究

用PCR-SSCP技术研究了涉及肉牛和奶牛共计7品种HTR1B基因的编码区和3′侧翼区的多态性,以期为牛性情的标记辅助选择积累数据。扩增得到4个片段, 有3个片段存在(SSCP)多态性。对不同的SSCP带型对应片段进行测序, 共发现6个SNP多态位点(G205T、C507T、C546G、C744T、G816A和G942A)。各遗传群体内G205T、C744T、G816A和G942A 位点均处于Hardy-Weinberg平衡, 而C507T和C546G位点只有鲁西牛处于Hardy-Weinberg平衡。奶牛205T等位基因频率显著高于其他肉牛品种(χ2 = 6.87)。奶牛G205T位点多态信息含量为0.25, 其余各位点在不同群体内均小于0.10, 说明牛HTR1B基因较保守。     

Association between expression of reproductive seasonality and alleles of melatonin receptor 1A in goats

To determine whether a link exists between reproductive seasonality and the structure of the melatonin receptor 1A (MTNR1A) gene, the latter was studied in year-round estrous breeds (Jining Grey and Boer goats) and seasonal estrous breeds (Liaoning Cashmere, Inner Mongolia Cashmere, Wendeng milk and Beijing native goats). A large fragment of exon 2 of MTNR1A gene was amplified by PCR using sheep sense and antisense primers in 260 does of six breeds. The uniform 824 bp PCR product was digested with restriction endonucleases MnII and RsaI, and checked for the presence of restriction sites. No polymorphism at the MnII cleavage sites was detected in all six goat breeds and no relationship could be established between the MnII cleavage sites of MTNR1A gene and reproductive seasonality in goats. For polymorphic RsaI cleavage site at base position 53, only genotype RR (267 bp/267 bp) was detected in Jining Grey goats, both genotype RR and genotype Rr (267 bp/320 bp) were found in all other goat breeds, no genotype rr (320 bp/320 bp) was detected in all six goat breeds. Frequency of genotype RR was obviously higher, and frequency of genotype Rr was obviously lower in year-round estrous goat breeds than in seasonal estrous goat breeds. Sequencing revealed one mutation (G52A) in genotype Rr compared with genotype RR. For polymorphic RsaI cleavage site, the differences of genotype distributions were significant (P<0.05) between year-round estrous goat breeds and seasonal estrous goat breeds. These results preliminarily showed an association between genotype RR and year-round estrus in goats, and an association between genotype Rr and seasonal estrus in goats.     

Identification and analysis of <Emphasis Type="Italic">MC4R</Emphasis> polymorphisms and their association with economic traits of Korean cattle (Hanwoo)

Signaling by the melanocortin-4 receptor (MC4R) is important for mediation the effect of leptin on food intake and energy homeostasis, and is associated with obesity, energy homeostasis and control of feeding behavior. Presently, the bovine MC4R gene was characterized to detect genetic variation at this locus and to relate it to economic traits in Korean cattle (Hanwoo). Five single nucleotide polymorphisms (SNPs) were identified in the coding region (G709A, C927T, C1069G, C1343A, and C1786T). G709A changed amino acid 166 of the MC4R protein from valine to methionine and C1069G changed amino acid 286 of the MC4R protein from leucine to valine. A SNP at C927T significantly influenced the Marbling score, SNP markers C1069G and C1343A significantly affected the Backfat thickness, and the SNP marker C1786T significantly influenced backfat and Marbling score. The MC4R gene may thus be a candidate gene for carcass traits with MC4R SNPs being potentially valuable as genetic markers for economic traits in Hanwoo.     

猪apoC3基因的遗传变异分析

目的:探究猪apoC3基因多态性,为进一步探讨其对脂肪沉积的影响和表达调控等研究提供依据。方法:选取具有不同脂肪沉积特点的3个猪种(可乐猪、贵州白香猪和大约克猪)构建品种DNA池并进行测序,结合各SNP位点测序峰高比值估算等位基因频率,并利用在线软件对不同基因型的转录结合位点及mRNA二级结构进行预测。结果:在3个猪种的apoC3基因中共发现17个SNPs(2个位于5’侧翼区;1个位于外显子3中,为同义突变;1个位于3’非翻译区,其它13个分别位于3个不同内含子中),其中C813G变异增加了一个转录因子GATA-2结合位点,G2280A变异导致mRNA二级结构最小自由能增加0.4kkal/mol。结论:不同猪种间apoC3基因SNPs位点等位基因频率差异较大,而apoC3基因编码区相对保守。     

Association between melatonin receptor 1A (MTNR1A) gene single-nucleotide polymorphisms and the velvet antler yield of Sika deer

Melatonin, a secretion from pineal gland is ambiguously considered as the key hormone involved in regulation of the antler cycle in Sika deer. To find out more about the roles of melatonin and its receptor gene, we carried out current study to investigate the association between polymorphisms in melatonin receptor 1A (MTNR1A) gene and the antler yield from Sika deer. A total of 251 Sika deer were analyzed in this study, of which consisted of Wusan Sika deer (n = 163) and Dongfeng Sika deer (n = 88). MTNRA gene was amplified by PCR and genotyped by Polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). Three polymorphism loci (C518T, C629G and C635T) were detected in exon2 of MTNR1A gene. The restriction site Ecol881 was used for C518T while a C629G polymorphism locus was digested with Mval restriction endonucleases. In Wusan Sika deer the allele frequencies of C and T were 0.637 and 0.363 for C518T, Also C and G alleles in C629G locus were 0.206 and 0.794. Genotypic frequencies of allele CC, CT and TT were 33.7, 59.9 and 6.4 % respectively, It showed 1.8, 37.4 and 60.7 % for frequencies of genotypes CC, CG and GG. In Dongfeng Sika deer the allele frequencies of C and T were 0.518 and 0.482 for C518T, C and G alleles were 0.375 and 0.625 for C629G. Genotypic frequencies were 10.6, 82.4 and 7.1 % for genotypes CC, CT and TT respectively, and they were 1.1, 72.7 and 26.2 % for genotypes CC, CG and GG. Among three SNPs, only C629G showed significant association (P < 0.05) with average antler yield in Wusan Sika deer, while no SNP was significant in Dongfeng Sika deer. These preliminary results implied that the identified SNPs of MTNR1A gene might influence the antler yield in Wusan Sika deer.