FSHβ 基因PCR-SSCP多态性及其与济宁青山羊高繁殖力关系的研究

采用PCR-SSCP技术检测促卵泡素b亚基(follicle-stimulating hormone β, FSHβ)基因5′调控区、外显子1和外显子2在高繁殖力山羊品种(济宁青山羊)和低繁殖力山羊品种(辽宁绒山羊、波尔山羊、安哥拉山羊)中的单核苷酸多态性, 同时研究该基因对济宁青山羊高繁殖力的影响。结果表明: 山羊与绵羊的FSHβ 基因该段核苷酸序列同源性为98%。9对引物中, 只有P9的扩增片段存在多态性。P9的扩增片段在济宁青山羊和辽宁绒山羊中检测到AA、AB和AC 3种基因型; 在波尔山羊中检测到AA、CC和AC 3种基因型; 在安哥拉山羊中检测到AA、BB、CC、AB、AC和BC共6种基因型。测序分析发现BB型与AA型相比在外显子2的第94 bp处有G→A突变, 并引起氨基酸改变(丙氨酸→苏氨酸); CC型与AA型相比在外显子2的第174 bp有一处C→T沉默突变。济宁青山羊AA、AB和AC基因型频率分别为0.686、0.137和0.177。AA基因型济宁青山羊产羔数最小二乘均值比AB基因型的多0.78只(P<0.05), 比AC基因型的多0.64只(P<0.05)。     

山东地方山羊BMP15基因多态性与产羔数性状关联分析

利用PCR、克隆测序、序列拼接获得山羊(Capra hircus)BMP15基因全长。利用F-CSGE技术分析两个外显子,发现山羊BMP15编码序列的第901处发生了A→G单碱基突变,该突变使得第301位氨基酸(成熟蛋白质第32位氨基酸)由丝氨酸变为甘氨酸。利用LDR技术对济宁青山羊、鲁北白山羊和沂蒙黑山羊进行突变检测,并进行其与产羔数的关联分析。结果表明,该突变对济宁青山羊产羔数没有显著影响,但对鲁北白山羊及沂蒙黑山羊产羔数均有显著影响(P0.05)。GG型和AG型的鲁北白山羊产羔数分别比AA型多0.34只(P0.01)和0.31只(P0.01)。AG型沂蒙黑山羊的产羔数比AA型多0.13只(P0.01)。初步表明,BMP15是控制鲁北白山羊和沂蒙黑山羊多胎性状的一个主效基因或是与之存在紧密遗传连锁的分子标记。     

山羊生长激素基因5′调控区的多态性分析

以鲁北白山羊、引进波尔山羊、纯繁波尔山羊以及鲁北白山羊与波尔山羊的杂交一代、回交一代共计274个个体为研究材料,用两对引物分别扩增山羊生长激素(GH)基因5′区的26～239bp以及225～429bp片段,扩增产物经SSCP分析发现均存在多态性.在26～239bp片段上,波尔山羊及杂交后代以AA型个体占多数,而鲁北白山羊则BB型个体较多;在225～429bp片段上,所有种群均以CC型个体较多.对两个片段的纯合型(AA,BB;CC,DD)分别克隆测序发现:(1)26～239bp片段上AA型在第60位发生了C→T的突变,第211位发生碱基C的丢失,(2)225～429bp片段上,DD型存在3处突变,分别为264位由T→C,292位由T→A,372位由C→T.上述结果为首次实验证实山羊生长激素5′调控区存在序列多态性.     

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。     

山羊生长激素基因5调控区的多态性分析

以鲁北白山羊、引进波尔山羊、纯繁波尔山羊以及鲁北白山羊与波尔山羊的杂交一代、回交一代共计274个个体为研究材料,用两对引物分别扩增山羊生长激素(GH)基因5^'区的26－239bp以及225－429bp片段,扩增产物经SSCP分析发现均存在多态性。在26－239bp片段上,波尔山羊及杂交后代以 AA型个体占多数,而鲁北白山羊则BB型个体较多;在225－429bp片段上,所有种群均以 CC型个体较多。对两个片段的纯合型（AA,BB;CC,DD）分别克隆测序发现：（1）26－239bp片段上AA型在第60位发生了C→T的突变,第211位发生碱基C的丢失,（2）225－429bp片段上,DD型存在3处突变,分别为264位由T→C,292位由T→A,372位由C→T。上述结果为首次实验证实山羊生长激素5^'调控区存在序列多态性。     

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。     

猪PRLR基因PCR—SSCP多态性与产仔性能的关联分析

采用PCR-SSCP技术分析了杜洛克猪、长白猪、大白猪、马身猪、山西黑猪和山西白猪等6个品种472个个体催乳素受体基因(PRLR)的多态性,检测到A、B、C 3个等位基因和AA、AB、AC、BB、CC 5种基因型.在马身猪中, B等位基因为优势基因,频率为0.55;其他品种中,优势基因为A等位基因,频率分布在0.79～0.89之间;C等位基因除在大白猪频率略高外(0.20),在其他品种中频率都很低,在0～0.09之间.对AA、BB、CC三种纯合子进行克隆测序和同源序列比较,发现在扩增片段内有6处SNP,都发生在PRLR基因的第8内含子,分别是内含子8第26位、54位和99位的C→T突变,47位和68位的A→G突变,63位的G→A突变.利用最小二乘分析研究了PRLR基因型对母猪头胎总产仔数和产活仔数的影响,结果表明PRLR基因不同基因型母猪的头胎总产仔数和产活仔数差异均不显著.     

生长激素基因多态性与山羊体重性状的关系

以鲁北白山羊、波尔山羊以及波尔山羊与鲁北白山羊的杂交一代、回交一代共计224只山羊为材料,根据山羊生长激素基因5′调控区的序列(GenBank登录号:D00476)设计两对引物,用PCRSSCP法进行多态性分析。多态性片段的纯合基因型经克隆测序,发现共有5处突变。对突变位点产生的不同基因型与体重、体尺性状进行分析表明:第一对引物扩增片段波尔山羊AA型个体的初生重、周岁重显著高于BB型和AB型(P<0.05);杂交一代中断奶重、周岁重也以AA型偏高,但不同基因型间的差异均不显著(P>0.05);而鲁北白山羊BB基因型的体重相对于另外两种基因型的偏低,且断奶重的差异达到显著水平(P<0.05)。第二对引物扩增片段不同基因型对体重、体尺没有显著影响。由此初步推断生长激素基因可能是影响山羊体重性状的主基因或与主基因相连锁,可以用该位点对山羊体重性状进行标记辅助选择。     

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.     

7个地方山羊品种遗传多样性及遗传结构分析

检测了7个地方山羊群体在15个微卫星位点的遗传多样性和遗传结构,旨为地方山羊群体的保护利用奠定基础。采集燕山绒山羊、辽宁绒山羊、承德无角山羊、济宁青山羊、太行山羊、武安山羊血液样品及内蒙古绒山羊的耳组织,利用微卫星方法分析遗传多样性及遗传结构。结果表明,7个山羊群体的平均有效等位基因数为4.235 8、平均期望杂合度为0.718 8、平均多态信息含量为0.706 8,均具有高度的遗传多样性;总群体平均近交系数为0.088 3,平均遗传分化系数为0.544 2,平均基因流为0.209 4。武安山羊和承德无角山羊的遗传距离最近,太行山羊和燕山绒山羊的遗传距离最远。系统进化树聚类分析表明,燕山绒山羊与辽宁绒山羊聚为一类,济宁青山羊、承德无角山羊、武安山羊、内蒙古绒山羊和太行山羊聚为一类。综上,7个地方山羊群体遗传多样性丰富,群体间遗传分化程度大,基因交流少,受近交程度影响小,具有较高的利用价值和潜力。     

Polymorphism of bone morphogenetic protein 4 gene and its relationship with litter size of Jining Grey goats

Two pairs of primers (P1 and P2) were designed to detect single nucleotide polymorphisms of exon 2 and intron 2 of bone morphogenetic protein 4 (BMP4) gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Boer, Angora and Inner Mongolia Cashmere goats) by single strand conformation polymorphism. Results showed that no polymorphism was detected for exon 2 (primer P1) of BMP4 gene in four goat breeds. For intron 2 (primer P2), three genotypes (AA, AB and BB) were detected in Jining Grey and Inner Mongolia Cashmere goats, two genotypes (AB and BB) in Angora goats, and only one genotype (AA) in Boer goats. Sequencing revealed one mutation (2203G>A) of BMP4 gene in the genotype BB in comparison to the genotype AA. The differences of litter size between AA, AB and BB genotypes were not significant (P > 0.05) in Jining Grey goats. A pair of primer (P3) was designed to detect polymorphism in the 3' flanking region of BMP4 gene that contained dinucleotide repeated sequence (CA) in the four goat breeds by microsatellite analysis. For primer P3, three genotypes (CC, CD and DD) were detected in four goat breeds. Sequencing revealed one more CA dinucleotide in genotype DD than in genotype CC. The Jining Grey does with genotype CC had 0.55 (P < 0.05) or 0.72 (P < 0.05) kids more than those with genotype CD or DD. These results preliminarily indicated that allele C of BMP4 gene is a potential DNA marker for improving litter size in goats.     

Polymorphisms of BMPR-IB gene and their relationship with litter size in goats

The bone morphogenetic protein receptor IB (BMPR-IB) gene was studied as a candidate gene for the prolificacy of goats. According to mRNA sequence of ovine BMPR-IB gene, ten pairs of primers were designed to detect single nucleotide polymorphisms (SNPs) of exon 1, exon 2, exon 6 to exon 10 and 3′ untranslated region (UTR) of the BMPR-IB gene in both high prolificacy breed (Jining Grey goat) and low prolificacy breeds (Wendeng Dairy and Inner Mongolia Cashmere goats) by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) method. Only the products amplified by primers P8 and P10 of the 3′UTR displayed polymorphisms. For primer P8, three genotypes (AA, AB and BB) were detected in Jining Grey and Wendeng Dairy goats, two genotypes (AA and AB) were in Inner Mongolia Cashmere goats. Sequencing revealed one mutation (71C→T) of the BMPR-IB gene in genotype BB compared with AA. The differences of least squares mean (LSM) for litter size between genotypes AA, AB and BB were non-significant (P > 0.05) in Jining Grey goats. For primer P10, three genotypes (CC, CD and DD) were detected in Jining Grey and Wendeng Dairy goats and one genotype (CC) in Inner Mongolia Cashmere goats. Sequencing revealed one mutation (130T→C) of the BMPR-IB gene in genotype DD compared with CC. The differences of LSM for litter size between genotypes CC, CD and DD were non-significant (P > 0.05) in Jining Grey goats. These results preliminarily showed that the detected loci of the BMPR-IB gene had no significant effect on prolificacy of Jining Grey goats.     

Association between PCR-SSCP of bone morphogenetic protein 15 gene and prolificacy in Jining Grey goats

The Jining Grey is a prolific local goat breed in P.R. China. Bone morphogenetic protein 15 (BMP15) gene that controls high fecundity of Inverdale, Hanna, Lacaune, Belclare, Cambridge, and Small Tailed Han ewes was studied as a candidate gene for the prolificacy of Jining Grey goats. According to the sequence of ovine BMP15 gene, six pairs of primers were designed to detect single nucleotide polymorphisms in exon 1 and exon 2 of the BMP15 gene in both high fecundity breed (Jining Grey goats) and low fecundity breeds (Boer, Liaoning Cashmere, and Inner Mongolia Cashmere goats) by single strand conformation polymorphism (SSCP). Two pairs of primers (F1/R1 and F2/R2) were used to amplify the exon 1. Four pairs of primers (F3/R3, F4/R4, F5/R5, and F6/R6) were used to amplify the exon 2. Only the products amplified by primer F5/R5 displayed polymorphism. Results indicated that two genotypes (AA and AB) were detected in prolific Jining Grey goats and only one genotype (AA) was detected in low fecundity goat breeds. In Jining Grey goats frequencies of genotypes AA and AB were 0.10 and 0.90, respectively. Sequencing revealed two point mutations (G963A and G1050C) of BMP15 gene in the AB genotype in comparison to the AA genotype. In Jining Grey goats the heterozygous AB does had 1.13 (p < 0.01) kids more than the homozygous AA does. These results preliminarily showed that the BMP15 gene is either a major gene that influences the prolificacy of Jining Grey goats or a molecular genetic marker in close linkage with such a gene.     

Polymorphisms of caprine <Emphasis Type="Italic">GDF</Emphasis>9 gene and their association with litter size in Jining Grey goats

The exons 1, 2 and flanking region of growth differentiation factor 9 (GDF9) gene in five randomly selected does of Jining Grey, Boer and Liaoning Cashmere goats were amplified and analyzed. Thirteen nucleotide differences were identified in GDF9 gene between sheep (AF078545) and goats. Four SNPs (G3288A in intron 1, G423A, A959C [Gln320Pro] and G1189A [Val397Ile] in exon 2) were detected in four goat breeds with different prolificacy, in which G3288A was a new SNP in goats. The results showed that loci 3288, 423 and 1189 in Boer goats, loci 3288 and 423 in Guizhou White goats, loci 423 and 1189 in Liaoning Cashmere goats were all in complete linkage disequilibrium (D′ = 1, r ² = 1), respectively. In moderate (Boer goat) and low prolificacy (Liaoning Cashmere goat) breeds, linkage analysis indicated that there were more fervent linkage disequilibrium among loci 3288, 423 and 1189 than high prolificacy (Jining Grey and Guizhou White goats) breeds. For the 959 locus, the genotype distribution showed obvious difference between high prolificacy breeds and moderate or low prolificacy breeds (P < 0.05 or P < 0.01). The Jining Grey goat does with genotype CC or AC had 0.81 (P < 0.01) or 0.63 (P < 0.01) kids more than those with genotype AA, respectively. The present study preliminarily showed an association between allele C at 959 locus of GDF9 gene and high litter size in Jining Grey goats. These results provide further evidence that the GDF9 gene may be significantly correlated with high prolificacy in goats.     

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.     

Association Between PCR-SSCP of Bone Morphogenetic Protein 15 Gene and Prolificacy in Jining Grey Goats

The Jining Grey is a prolific local goat breed in P.R. China. Bone morphogenetic protein 15 (BMP15) gene that controls high fecundity of Inverdale, Hanna, Lacaune, Belclare, Cambridge, and Small Tailed Han ewes was studied as a candidate gene for the prolificacy of Jining Grey goats. According to the sequence of ovine BMP15 gene, six pairs of primers were designed to detect single nucleotide polymorphisms in exon 1 and exon 2 of the BMP15 gene in both high fecundity breed (Jining Grey goats) and low fecundity breeds (Boer, Liaoning Cashmere, and Inner Mongolia Cashmere goats) by single strand conformation polymorphism (SSCP). Two pairs of primers (F1/R1 and F2/R2) were used to amplify the exon 1. Four pairs of primers (F3/R3, F4/R4, F5/R5, and F6/R6) were used to amplify the exon 2. Only the products amplified by primer F5/R5 displayed polymorphism. Results indicated that two genotypes (AA and AB) were detected in prolific Jining Grey goats and only one genotype (AA) was detected in low fecundity goat breeds. In Jining Grey goats frequencies of genotypes AA and AB were 0.10 and 0.90, respectively. Sequencing revealed two point mutations (G963A and G1050C) of BMP15 gene in the AB genotype in comparison to the AA genotype. In Jining Grey goats the heterozygous AB does had 1.13 (p < 0.01) kids more than the homozygous AA does. These results preliminarily showed that the BMP15 gene is either a major gene that influences the prolificacy of Jining Grey goats or a molecular genetic marker in close linkage with such a gene.     

Association Between Sexual Precocity and Alleles of KISS-1 and GPR54 Genes in Goats

KISS-1 and GPR54 were regarded as key regulators for the puberty onset and fundamental gatekeepers of sexual maturation in mammals. To explore the possible association between variations in KISS-1 and GPR54 with sexual precocity, mutation screening of exon 1 of KISS-1 and exon 1, exon 3, and partial exon 5 of GPR54 was performed in a sexual precocious breed (Jining Grey goats) and sexual late-maturing breeds (Inner Mongolia Cashmere, Angora, and Boer goats) by PCR-SSCP. The results showed that five novel mutations were identified in exon 1 and partial exon 5 of GPR54 including C96 T, T173C, G176A, G825A, and C981 T. The Jining Grey goats with genotype BB or AB had 1.07 (P < 0.05) or 0.40 (P < 0.05) kids more than those with AA. The Jining Grey goats with genotype DD or CD had 1.80 (P < 0.05) or 0.55 (P < 0.05) kids more than CC, respectively. The present study preliminarily showed an association between alleles B and D of GPR54 with high litter size and sexual precocity in Jining Grey goats.     

Polymorphisms of caprine <Emphasis Type="Italic">POU1F1</Emphasis> gene and their association with litter size in Jining Grey goats

Seven pairs of primers were designed to amplify 5′ promoter region, six exons and partial introns and to detect the polymorphisms of POU1F1 gene in five goat breeds with different prolificacy. The results showed that six mutations were identified in caprine POU1F1 gene including C256T in exon 3, C53T and T123G in intron 3, and G682T (A228S), T723G and C837T in exon 6. The former four mutations were novel SNPs in goat POU1F1 gene. The 53 and 123 loci were in complete linkage disequilibrium in five caprine breeds. Regarding the 256 locus, the Jining Grey goat does with genotype CT had 0.66 kids more than those with genotype CC (P < 0.05), while does with genotype GT had 0.63 (P < 0.05) kids more than those with genotype GG at the 682 locus. The present study preliminarily showed an association between allele T at the 256 and 682 loci of POU1F1 gene and high litter size in Jining Grey goats. Totally 16 haplotypes and 50 genotypes were identified at the above six loci in POU1F1 gene of five goat breeds. Three common haplotypes (hap2, hap3 and hap4) were identified in five goat breeds joined. Four specific haplotypes (hap7, hap9, hap11 and hap13) were detected in Jining Grey goats. The predominant haplotype was hap1 (35.29% and 48.25%) in both Jining Grey and Guizhou White goats, while hap4 (50%) in Boer goats, and hap2 (42.86% and 38.75%) in both Wendeng Dairy and Liaoning Cashmere goats. The most frequent genotypes at six loci in the above five goat breeds were hap1/hap2 (14.38%) and hap1/hap4 (14.38%), hap1/hap2 (38.60%), hap4/hap4 (40.91%), hap2/hap4 (26.53%), hap2/hap5 (20.00%), respectively. The Jining Grey goat does with nine genotypes analyzed of POU1F1 gene showed no obvious difference in litter size.