贵州山羊品种DQA1基因第3外显子遗传变异分析

目的:通过筛选3个贵州地方山羊种DQA1基因Exon3的SNPs位点并做相应的遗传变异分析,为今后山羊抗病育种及地方山羊品种的选育提供理论依据。方法:采用构建品种DNA池与直接测序的方法,对三个贵州地方山羊种群共514只个体的DQA1基因外显子3进行遗传变异分析。结果:在3个山羊品种中共筛选得到4个SNPs,G71A(同义突变)、T100C(Asn→Ser)、G202A(Pro→Leu)、A223G(Met→Thr)。生物信息学分析发现,G202A位点变异虽未引起mRNA二级结构的变化,但最小自由能降低,结构稳定性增强;DQA1基因Exon3的不同基因型的蛋白质二级结构中均不含有α螺旋。结论:三个贵州地方山羊种DQA1基因外显子3中含有较丰富的多态性,G202A位点变异在mRNA二级结构及蛋白质二级结构中与其他基因型具有较明显的差异。     

三种绵羊BMP15基因第二外显子多态性及与产羔数的相关性分析

绵羊存在影响多胎性状的不同主效基因,选择影响Romney Hanna绵羊和Cambridge绵羊高繁殖力的骨形态发生蛋白15 (bone morphogenetic protein 15, BMP15)为候选基因,采用PCR-SSCP的方法检测BMP15基因外显子Ⅱ第747位点(T747→C)和755位点(T755→C)在蒙古羊、甘肃高山细毛羊、小尾寒羊三种绵羊母羊中的多态性,同时还研究了上述两处突变对三种绵羊产羔数的影响。表明:(1)一共检测到野生纯合型AA、突变杂合型AB (T747→C)、AC (T755→C)三种不同的基因型,AA为优势基因型,A为优势等位基因;(2)三种基因型在甘肃高山细毛羊中均被检测到,而蒙古羊和小尾寒羊中未检测出AB基因型;(3)突变杂合型蒙古羊(AC)比野生纯合型(AA)的平均产羔数多0.27只(p<0.05)。(4)AC的基因型频率,双羔母羊和多羔母羊均高于单羔母羊。根据以上实验推测,BMP15第755位点发生的T→C突变(AC型)对蒙古羊一胎产双羔影响十分显著,甘肃高山细毛羊中AC基因型的绵羊其产羔数有比AA基因型和AB基因型多的趋势,因此该位点可能是一个影响绵羊高繁殖力潜在的DNA标记。     

贵州白山羊GOLA-DQA1基因多态性及生物信息学分析

目的:通过筛选贵州白山羊GOLA-DQA1基因SNPs位点,为进一步研究MHC基因多态性与山羊免疫性状的相关性提供依据。方法:选取同一生长环境中的贵州白山羊母羊157只构建品种DNA池并进行测序,结合SNPs位点测序峰高比值估算等位基因频率,利用软件对不同基因型的RNA和蛋白质二级结构进行预测。结果:在贵州白山羊DQA1基因发现6个SNPs位点G+2930A(同义突变)、T+2959C(Asn→Ser)、G+3061A(Pro→Leu)、A+3082G(Me→Thr)、C+3463A(Trp→Cys)、C+3525T(Arg→His)。生物信息学分析发现,C+3463A变异位点使RNA二级结构更加稳定;A+3082G变异导致自由能增加,稳定性降低。蛋白质结构预测发现除A+3082G基因型与原序列具有相同的二级结构外,其他基因型均引起蛋白质二级结构的改变。结论:贵州白山羊GOLA-DQA1基因具有丰富的多态性,但SNPs位点等位基因频率差异较大。     

中华鳖MyoD1基因SNP鉴定及其与生长性状的关联分析

为了研究生肌决定因子1(MyoD1)基因多态性与中华鳖生长性状的相关性,采用直接测序法在MyoD1基因上共检测到6个SNP位点(T-49G、A-38G、C91T、A187T、C880T和T1522A),其中C880T位于外显子上,属于错义突变。对从同批繁殖、同块稻田养殖的2冬龄中华鳖群体中随机选取的178只个体中各位点的基因型进行检测。结果显示,所有位点在中华鳖群体中的平均有效等位基因数、平均观测杂合度和平均期望杂合度分别为1.636 5、0.349 3和0.375 4,除A187T位点外,其余5个位点的基因型频率分布均符合Hardy-Weinberg定律。采用一般线性模型分析各位点与中华鳖生长性状之间的相关性,研究发现,T-49G位点GG基因型个体的背甲宽显著大于TT基因型,A-38G位点AG基因型个体的背甲宽显著大于AA基因型,A187T位点TT基因型个体的体高显著大于AA基因型,T1522A位点AA基因型个体的体质量显著大于TT、TA基因型,其余位点不同基因型个体间的生长性状均不存在显著差异。T-49G、A-38G、A187T和T1522A位点与生长性状显著相关,可作为中华鳖分子标记辅助育种的候选标记。     

湖羊、东湖杂种羊POMC基因外显子3单核苷酸多态性及其与生长性状的关联分析

阿黑皮素原(Pro-opiomelanocortin, POMC)在动物采食和能量平衡调控中发挥重要作用, 文章对绵羊POMC基因外显子3进行扩增和测序, 筛选多态性位点, 并分析多态位点与湖羊和东弗里生×湖羊杂种羊生长性状的相关性。测序后发现湖羊POMC基因外显子3有2个单碱基突变(g.273 T/C和g.456 G/A), 根据273位点处发生的T/C突变, 建立PCR-RFLP分析方法, 并对162只湖羊和130只东湖杂种羊进行检测分析。结果发现, 在湖羊群体中检测到TT(0.469)、TC(0.438)和CC(0.093)3种基因型, 而在东湖杂种羊群体中仅检测到TT(0.754)和TC(0.246)两种基因型。POMC基因外显子3的273位点多态性与生长性状的相关性研究结果显示：湖羊群体中CC基因型个体的2月龄断奶重、4月龄尻高及TC基因型个体4月龄体长和管围均显著高于TT型个体(P<0.05); CC基因型个体的4月龄重、6月龄重极显著高于TT和TC基因型个体(P<0.01); CC基因型个体的4月龄体高和体长极显著高于TT型个体(P<0.01), 且显著高于TC基因型个体(P<0.05)。此外, CC型个体的管围极显著高于TT基因型个体(P<0.01)。东湖杂种羊群体中TC基因型个体的2月龄断奶重、4月龄重及4月龄体高、体长、胸深和管围都显著高于TT型个体(P<0.05), TC型个体的6月龄重极显著高于TT型个体(P<0.01)。研究结果表明, POMC基因外显子3与绵羊生长性状相关, C等位基因对体重及体尺性状的增加更有利。该结果为进一步探讨POMC基因作为绵羊生长性状的辅助选育标记奠定了基础。     

贵州宗地花猪的GDF9基因多态性及生物信息学分析

以贵州宗地花猪和贵州从江香猪2个地方猪种为研究对象,构建基因组DNA池,扩增GDF9基因第1外显子和第2外显子序列,采用直接测序法对2个品种的GDF9基因进行单核苷酸多态性进行检测,利用生物信息学软件预测不同多态性位点对GDF9基因m RNA二级结构、蛋白质二级结构的影响。结果表明,在2个品种中共检测到T~(997)A、C~(1009)T、T~(1014)C、G~(1137)T和A~(1196)T 5个SNPs位点,其全位于第二外显子上。T~(997)A、C~(1009)T和A~(1196)T均为错义突变。T~(997)A导致编码的苯丙氨酸(Phe)变为异亮氨酸(Ile)、C~(1009)T导致编码的亮氨酸(Leu)变为苯丙氨酸(Phe)、A~(1196)T导致编码的赖氨酸(Lys)变为甲硫氨酸(Met)。SNPs位点对于GDF9基因的m RNA二级结构、蛋白质二级结构有一定影响。     

山羊抑制素α亚基基因HaeⅡ酶切多态性及其与产羔数性状的关联分析

以323 只马头山羊、努比山羊、波尔山羊和海门山羊为试验材料, 利用PCR-SSCP、PCR-RFLP和克隆测序等方法对山羊抑制素α亚基基因(INHA)编码区一个片段进行多态性分析, 发现在该基因第284位(登录号: L28815)存在G→A突变, 该突变引起HaeⅡ酶切位点改变; HaeⅡ-RFLP分型结果显示G等位基因为优势等位基因, INHA不同基因型平均产羔数表现为GG>AG>AA。结果显示: INHA可能是影响山羊产羔数性状的一个主效基因, G等位基因可能与高产性状呈正相关。     

乐至黑山羊PRLR基因外显子10多态性与产羔数的关系研究

设计2对特异性引物对乐至黑山羊PRLR基因第10外显子进行了PCR-SSCP检测,并研究该基因与产子性能的相关性。结果表明,P1引物扩增片段不存在多态性;P2引物扩增片段存在多态性,表现为AA,AB,AD和CD 4种基因型,测序结果表明,4种基因型都在该片段第89、94、146和157位存在C→T、A→C、C→G、G→C的突变;此外AA型还在61位发生C→T的突变;AD型还在175位发生A→G的突变;CD型还在24位发生T→C的突变,96位发生C→T的突变,通过统计分析发现AD型平均产羔数优于其他3种基因型,并且与AB型差异达到显著水平(P<0.05)。因此认为PRLR基因对于乐至黑山羊产子性能有一定的影响。     

甘肃肉羊新品种选育群GDF9基因外显子2多态性及其与产羔性状关联分析

根据GenBank发布的绵羊GDF9基因外显子2的序列设计4对引物,采用PCR-SSCP技术分析GDF9基因外显子2在甘肃内羊新品种选育群羊中的单核苷酸多态性,并与产羔性状进行关联分析.结果表明,GDF9基因的扩增片段在所检测的新品种群羊中存在PCR-SSCP多态性,检测到3种基因型(AA、AB和BB),而在32只无角陶赛特母羊群中只检测到AA和AB基因型.测序结果显示,GDF9基因编码区第978位碱基发生A→G突变,但没有导致氨基酸的改变;第994位碱基发生G→A突变,导致Ⅴ变成Ⅰ(缬氨酸→异亮氨酸).新品种选育群羊产羔数的最小二乘均值关系为AB＞ AA＞ BB,统计分析结果初步表明3种基因型之间差异不显著(P＞0.05).故该区域可能不是影响新品种群羊繁殖力的功能结构区城.     

中国美利奴羊H-FABP基因多态性及其与部分肉质性状相关性的研究

利用聚合酶链式反应-单链构象多态（PCR-SSCP）技术检测中国美利奴羊(Chinese Merino)心脏型脂肪酸结合蛋白基因（H-FABP）外显子2的单核苷酸多态性（SNPs）和遗传多态性,分析其与肌内脂肪（IMF）含量、肌纤维直径和肌纤维密度的相互关系,为该品种绵羊的分子标记辅助选择提供理论依据。结果显示,H-FABP基因外显子2有AA、AB和BB 3种基因型,AA型和BB型在778位均发生了C缺失,939位均发生了A→G转换,BB型还在789位发生了T→C转换,该突变导致所编码氨基酸发生了缬氨酸→丙氨酸的替换;BB型为IMF的优势基因型,与AB型相比差异显著(P<0.05),与AA型相比差异极显著（P<0.01);BB型对肌纤维直径存在负相关。结果提示,中国美利奴羊H-FABP基因外显子2具有多态性,该基因可能是中国美利奴羊肉质性状的主效基因,或者与控制这些性状的主效基因相连锁。     

绵羊产羔性状主效基因检测研究

以绵羊BMP15基因和BMPR-IB基因为候选基因,以湖羊、中国美利奴单胎品系、中国美利奴肉用和毛用多胎品系为研究对象,采用PCR-RFLP方法对候选基因进行单核苷酸多态性（SNP）位点检测和基因型分析,同时研究基因对绵羊产羔数的影响。对BMP15基因进行SNP检测,结果未发现多态性位点;对BMPR-IB基因进行多态性检测,结果发现了一个A746 G SNP位点。依据A746 G SNP位点进行基因型分析,结果在各品种（系）羊中发现了3种基因型,即BB、B+和++。等位基因型频率在各品种（系）间差异极显著（P<0.001）,在湖羊中以BB基因型为主,在中国美利奴单胎品系中以++基因型为主, 而在中国美利奴肉用和毛用多胎品系中以B+基因型为主。BMPR-IB A746G位点的变异明显影响绵羊的产羔数,与++基因型母羊相比, BB和B+基因型母羊产羔数明显较多。研究结果同时表明,利用BMPR-IB基因型可以很好的预测母羊的产羔数。研究获得的这些结果强烈表明BMPR-IB为影响绵羊的产羔数的主效基因,可以用于对绵羊产羔数的选择。Abstract: The current study was designed to detect SNPs within BMP15 and BMPR-IB gene and investigate the effect of the genes on sheep litter size. Four sheep lines, HU-Yang, Chinese M erino monotocous, Chinese Merino multiparous for wool production and Chinese Merino multiparous for mutton production, were used in this study. Litter sizes were recorded for each ewe in the four lines. Primers for BMP15 and BMPR-IB gene were designed from database sheep sequence and polymorphisms were detected by PCR-RFLP method. The results showed that there was no polymorphism with BMP15 gene among the four lines, and there was an A / G SNP with BMPR-IB gene at base 746 among the four lines. Three types of genotype (BB, B+ and ++), based on A / G locus, were found within each line. The frequencies of genotypes were significantly different among the lines (P<0.001), with BB genotype primarily existing in HU-Yang, ++ genotype in Chinese Merino monotocous line, and B+ genotype in Chinnese Merino multiparous lines. The A / G mutation influence significantly the sheep litter sizes, and the BB and B+ ewes had significant higher litter sizes than ++ ewes. The results of present study showed simultaneously that the genotype of BMPR-IB was a perfect predictor of the sheep litter sizes. These results intensively indicated that BMPR-IB is a major gene to affect litter size in sheep, and could be used as the molecular genetic marker to select litter size in sheep.     

Polymorphisms of the cocaine-amphetamine-regulated transcript (CART) gene and their association with reproductive traits in Chinese goats

Polymorphisms of the CART gene were investigated by PCR-single-strand conformation polymorphism analysis in 540 samples from 10 goat breeds. Ten novel single-nucleotide polymorphisms as well as three microsatellites were detected; a mutation, 77T → C, led to an amino acid change (Leu → Ser). Associations between polymorphic loci and reproductive traits were analyzed in Chuandong White, Guizhou White and Gulin Ma breeds. Mutation at position 524 had no significant effect on litter size in these three goat breeds. The polymorphism 539C → A differed significantly among the three breeds (P < 0.05); C(7)T(8)/C(9)T(8) at 939 was associated with larger litter size (P < 0.05) than genotypes C(7)T(8)/C(7)T(8) and C(7)T(8)/C(8)T(8). No significant association of birth weight was found with gene variation (524C → T, 539C → A and 939 CnTn). These findings could be valuable for marker-assisted selection for goat breeding.     

绵羊ESR2基因组织表达及其多态性与产羔数关联分析

为了探究雌激素受体2 (Estrogen receptor 2, ESR2)基因在绵羊各组织的表达及其多态性与产羔数之间的关系,本研究利用半定量PCR和实时荧光定量PCR技术检测ESR2基因在不同繁殖力小尾寒羊群体组织中的相对表达量,同时采用Sequenom MassARRAY誖SNP技术对多羔品种绵羊(小尾寒羊,湖羊,策勒黑羊)和单羔品种绵羊(苏尼特羊,草原型藏羊,滩羊) ESR2基因g.73324006C>T位点进行检测,并与小尾寒羊产羔数进行关联分析。半定量PCR表明,ESR2基因在单、多羔小尾寒羊子宫中高表达,在其它组织中等或低丰度表达;单羔群体、多羔群体间荧光定量PCR表明,ESR2基因在单羔小尾寒羊垂体表达量显著高于多羔小尾寒羊(p<0.05);群体遗传学分析表明,g.73324006C>T在小尾寒羊群体中表现为低度多态(PIC<0.25),在滩羊群体中处于中度多态(0.25T在小尾寒羊群体处于哈代温伯格平衡状态(p>0.05);关联分析表明,g.73324006C>T位点多态性与小尾寒羊第一胎、第二胎、第三胎产羔数及平均产羔数均显著关联(p<0.05),CC型各胎产羔数均高于TC型。与FecB (A746G)基因组合后发现,GG-CC和AG-CC基因型母羊产羔数显著高于AA-TC、AA-CC、AG-TC基因型组合(p<0.05)。综上,ESR2与小尾寒羊产羔数密切相关,g.73324006C>T可作为绵羊产羔性状选育的潜在分子标记。     

抑制素基因的研究进展

抑制素是性腺分泌的一种糖蛋白激素,它具有抑制垂体促卵泡素合成和分泌的作用。本文介绍了抑制素α亚基基因（INHA）、抑制素βA亚基基因（INHBA）、抑制素βB亚基基因（INHBB）的克隆、结构、定位、多态性、表达、分子调节及其与繁殖性能和癌症的关系。绵羊INHA、INHBA和INHBB基因分别被定位到2q41→q43、4q26和2q31→q33。INHA、INHBA和INHBB基因对绵羊产羔数都有显著的影响。抑制素βB亚基基因突变的雌性小鼠有明显的发育和繁殖缺陷。INHA 基因与妇女卵巢早衰显著相关。 Abstract：Inhibins are gonadal glycoprotein hormones belonging to the transforming growth factor-βsuperfamily that act to suppress pituitary follicle-stimulating hormone synthesis and secretion. In this paper, we briefly introduced the cloning, structure, localization, polymorphism, expression, molecular regulation of inhibin-α(INHA), -βA (INHBA) and -βB (INHBB) subunit genes and their relationships with reproductive performance and cancer. The inhibin genes (INHA, INHBA and INHBB) had significant effect on litter size in sheep. The ovine INHA, INHBA and INHBB genes had been mapped to chromosomes 2q41→q43, 4q26 and 2q31→q33, respectively. The female mice carrying INHBB mutations suffered from distinct developmental and reproductive defects. The INHA gene was significantly associated with premature ovarian failure in women.     

Polymorphisms of KiSS-1 and GPR54 genes and their relationships with litter size in sheep

The KiSS-1 and GPR54 genes were studied as candidate genes for the prolificacy in sheep. Four pairs of primers were designed to detect single nucleotide polymorphisms of exon 1 of KiSS-1 gene and exon 1, exon 2 and partial exon 5 of GPR54 gene in high fecundity breeds (Small Tail Han and Hu sheep) and low fecundity breeds (Dorset, Texel and Corriedale sheep) by PCR-SSCP. Polymorphisms in exon 1 of KiSS-1 gene were detected in prolific Small Tail Han sheep (AA, AB and BB genotypes) and Hu sheep (AA and CC genotypes), no polymorphism was found in low fecundity sheep breeds (only AA genotype). Polymorphisms in exon 2 of GPR54 gene were detected in prolific Hu sheep (DD and EE genotypes) and no polymorphism was found in prolific Small Tail Han sheep and low fecundity sheep breeds (only DD genotype). No polymorphism was detected in exon 1 and partial exon 5 of GPR54 gene in five sheep breeds. The polymorphic genotypes were sequenced. While compared the BB genotype with the AA genotype, one nucleotide mutation (G1035A) was detected, which resulted in amino acid change, Val25Met. Five nucleotide mutations were detected from AA to CC genotype (C981T, C996T, T997C, C1034G, C1039T), and among them four caused amino acid changes, that is, Arg7Trp, Phe12Leu, Asn24Lys, Ala26Val. While compared the EE genotype with the DD genotype, two nucleotide mutations (T2360C, A2411C) were detected, which gave rise to amino acid changes, Met90Thr and Asp107Ala, respectively. Genotype frequencies of AA, BB and AB were 0.62, 0.05 and 0.33 in Small Tail Han sheep, respectively. The Small Tail Han sheep ewes with genotype BB or AB had 0.88 (P?<?0.05) or 0.51 (P?<?0.05) lambs more than those with genotype AA; the Small Tail Han sheep ewes with genotype BB had 0.37 (P?>?0.05) lambs more than those with genotype AB. These results preliminarily indicated that the KiSS-1 gene may have some association with prolificacy in sheep.     

检测绵羊BMPR-IB基因多态性寡核苷酸芯片的制备

FecB基因是控制中国美利奴羊排卵率和产羔数的主效基因,由于A746G的点突变而导致绵羊表型的变化。本研究的目的在于根据FecB基因的多态性,制备寡核苷酸芯片检测绵羊FecB基因的单核苷酸多态性（SNP）,设计六条特异性的探针,用基因芯片点样仪将探针点样到醛基修饰的载玻片上,采集绵羊的血液样本,在芯片反应舱中,检测FecB基因A746G点突变,设计对应的软件进行判读,分析检测结果,与PCR-RFLP检测结果完全符合,证明制备的寡核苷酸芯片可以并行、准确而高效地检测FecB基因的多态性,能够作为分子标记辅助选育多胎绵羊的一种合适的检测技术。     

Genome-wide and local pattern of linkage disequilibrium and persistence of phase for 3 Danish pig breeds

Background

A genome wide association study for litter size in Norwegian White Sheep (NWS) was conducted using the recently developed ovine 50K SNP chip from Illumina. After genotyping 378 progeny tested artificial insemination (AI) rams, a GWAS analysis was performed on estimated breeding values (EBVs) for litter size.

Results

A QTL-region was identified on sheep chromosome 5, close to the growth differentiation factor 9 (GDF9), which is known to be a strong candidate gene for increased ovulation rate/litter size. Sequencing of the GDF9 coding region in the most extreme sires (high and low BLUP values) revealed a single nucleotide polymorphism (c.1111G>A), responsible for a Val→Met substitution at position 371 (V371M). This polymorphism has previously been identified in Belclare and Cambridge sheep, but was not found to be associated with fertility. In our NWS-population the c.1111G>A SNP showed stronger association with litter size than any other single SNP on the Illumina 50K ovine SNP chip. Based on the estimated breeding values, daughters of AI rams homozygous for c.1111A will produce minimum 0.46 - 0.57 additional lambs compared to daughters of wild-type rams.

Conclusion

We have identified a missense mutation in the bioactive part of the GDF9 protein that shows strong association with litter size in NWS. Based on the NWS breeding history and the marked increase in the c.1111A allele frequency in the AI ram population since 1983, we hypothesize that c.1111A allele originate from Finnish landrace imported to Norway around 1970. Because of the widespread use of Finnish landrace and the fact that the ewes homozygous for the c.1111A allele are reported to be fertile, we expect the commercial impact of this mutation to be high.