【作者】 张润锋；

【导师】 陈宏；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2007， 博士

【摘要】 本研究采用PCR-RFLP和PCR-SSCP技术,检测了南阳牛、秦川牛、郏县红牛、中国荷斯坦牛和牦牛5个群体共521个个体的MRF家族、MSTN、GHRH和GHR基因的遗传变异,分析了其遗传结构和遗传多样性,并对南阳牛、秦川牛和郏县红牛3个中国黄牛群体在上述位点的多态性与其生长性状进行了相关分析,以检验这些基因的多态对南阳牛、秦川牛和郏县红牛3个黄牛群体生长发育的遗传效应,以期发现对重要经济性状具有显著效应的遗传标记,为中国黄牛的高效选育和分子标记数据库的建立、种质资源保护与利用提供遗传学依据。本研究获得了以下结果:1.牛MSTN基因变异及其与生长性状的相关分析1.1 PCR-SSCP检测结果表明,MSTN基因外显子1和3在5个群体中均未发现多态。利用限制性内切酶TaqI对5个群体MSTN 5’调控区约1346 bp片段进行PCR-RFLP分析,发现该片段不存在限制性内切酶TaqI的多态位点,证明已经公布的牛MSTN序列(GenBank No. AF348479)存在测序错误,-360位(相对于ATG转录起始点)碱基应该为A。1.2利用限制性内切酶DraI对5个群体MSTN 5’调控区约1346 bp片段进行PCR-RFLP分析,结果表明牛MSTN 5’调控区PCR-RFLP-DraI多态位点存在2个等位基因T和A。南阳牛、秦川牛、郏县红牛、中国荷斯坦牛和牦牛等位基因T/A的基因频率分别为0.955/0.045、0.973/0.027、0.972/0.028、0.918/0.082和1.000/0.000。这个多态表现为TT、TA和AA 3种基因型。牦牛个体全部为TT型,即在牦牛群体中未出现T>A突变;南阳牛、秦川牛和中国荷斯坦牛群中出现了TT和TA两种基因型,无AA纯合体;而在108头郏县红牛群体中,除了TT和TA型个体,还出现了1个AA纯合个体。在5个群体中,TT基因型为优势基因型,T为优势等位基因。群体卡方适合性检验表明,南阳牛、秦川牛和中国荷斯坦牛3个群体处于Hardy-Weinberg平衡状态;而郏县红牛群体偏离Hardy-Weinberg平衡状态。中国荷斯坦牛、郏县红牛、南阳牛、秦川牛和牦牛5个群体遗传多态指数h/Ne/S/PIC分别为0.1505/1.1772/0.2835/0.1345、0.0864/1.0946/0.1842/0.0826、0.0545/1.0576/0.1279/0.0530、0.0523/1.0552/0.1237/0.0501和0.0000/1.0000/0.0000/0.0000,因此其群体遗传多态性丰富程度从大到小依次为中国荷斯坦牛、郏县红牛、南阳牛、秦川牛和牦牛。1.3利用最小二乘法对MSTN-DraI多态位点与3个黄牛群体(南阳牛、秦川牛、郏县红牛)生长性状进行相关分析,结果表明年龄对南阳牛体重、体高、体斜长、胸围和坐骨端宽影响极显著(P<0.01),说明不同年龄阶段南阳牛的生长发育存在明显差异,生长快慢与年龄有直接关系。多重比较表明,18月龄南阳牛TT型个体的体高显著高于TA型个体(P<0.05),6月龄两种基因型个体之间胸围差异显著(P<0.05)。2.牛MRF基因家族多态性及其与生长性状的相关分析2.1利用PCR-RFLP和PCR-SSCP技术在牛MRF基因家族4个基因(Myf5、MyoD、Myf6、MyoG)检测到4个多态位点。PCR-RFLP-TaqI检测到牛Myf5-1位点存在三种基因型,该位点多态是由该基因内含子2的1948 bp (Accession No. M95684)处的SNP (CAA/CGA)引起的。在南阳牛、秦川牛、郏县红牛、中国荷斯坦牛和牦牛5个群体中等位基因A/B的频率分别为0.173/0.827、0.242/0.758、0.248/0.752、0.262/0.738和0.031/0.969,等位基因B为优势等位基因。牦牛群体在该位点未发现AA型纯合体。卡方检验表明5个群体均处于Hardy-Weinberg平衡状态。PCR-SSCP检测到牛Myf5-2位点有1个SNP(A>T),位于Myf5基因外显子1的609 bp (Accession No. M95684),未引起氨基酸的改变。在南阳牛、秦川牛、郏县红牛和牦牛4个群体中,该多态表现为AA、AB和BB三种基因型,等位基因A/B的频率依次为0.443/0.557、0.475/0.525、0.467/0.533和0.199/0.801。在南阳牛、秦川牛和郏县红牛3个黄牛群体中,杂合个体所占比例较小,基因频率分布接近相等;在牦牛群体中,BB型为优势基因型,B为优势等位基因。南阳牛、秦川牛、郏县红牛和牦牛4个群体在该位点均偏离Hardy-Weinberg平衡状态。利用PCR-SSCP技术检测到牛MyoD基因内含子2 (MyoD-intron2)和牛Myf6基因编码区分别存在1个SNP (C>T),Myf6位点的SNP位于Myf6基因(Accession No. AB110601)的338 bp,未引起氨基酸的改变。南阳牛、秦川牛、郏县红牛和中国荷斯坦牛在MyoD-intron2位点等位基因A/B的频率分别为0.904/0.096、0.882/0.118、0.926/0.074和0.980/0.020。南阳牛、秦川牛和郏县红牛在Myf6位点等位基因A/B的频率分别为0.983/0.017、0.995/0.005和0.997/0.023。在这2个位点上,发现AA和AB两种基因型,牦牛全部为AA型个体,说明牦牛群体的这两个位点可能已经达到纯合;其它所研究群体均未发现BB型纯合体,AA型纯合体占绝大多数,AA基因型为优势基因型,A为优势等位基因,且群体处于Hardy-Weinberg平衡状态。南阳牛、秦川牛和郏县红牛3个黄牛群体的Myf5-1和Myf5-2位点属于(或接近)中度多态位点,MyoD-intron2和Myf6位点属于低度多态位点。南阳牛、秦川牛和郏县红牛3个黄牛群体平均PIC分别为0.2023、0.2175和0.2123,因此其遗传多态性丰富程度从高到低依次为秦川牛、郏县红牛和南阳牛。2.2利用最小二乘法对这4个位点多态与南阳牛、秦川牛和郏县红牛3个黄牛群体生长性状进行相关分析,结果表明:在Myf5-1-TaqI位点,18月龄南阳牛AA型个体的体高极显著高于AB和BB型个体(P<0.01);郏县红牛AA型个体的坐骨端宽显著高于其它两种基因型个体(P<0.05) ;而秦川牛AA型个体的体高和十字部高显著低于AB和BB型个体(P<0.05); Myf5-2位点3种基因型对南阳牛体高和体斜长影响显著,6月龄和12月龄BB型个体体高均显著高于AA型个体(P<0.05);6月龄的BB型个体的体斜长也显著高于AA型个体(P<0.05)。在秦川牛群体中,杂合个体的腰角宽显著高于纯合个体(P<0.05);在MyoD-intron2位点,秦川牛除了体高和十字部高外,AB型个体的其它性状均显著高于AA型个体(P<0.05)。在Myf6位点,18月龄南阳牛AA型个体的坐骨端宽显著高于AB型个体(P<0.05)。3.牛GHRH基因外显子3多态与生长性状的相关分析3.1利用PCR-RFLP检测了5个群体GHRH基因外显子3限制性内切酶HaeⅢ的酶切多态性。在南阳牛、秦川牛、郏县红牛、中国荷斯坦牛和牦牛5个群体中,等位基因A/B的频率分别为0.314/0.686、0.259/0.741、0.243/0.757、0.265/0.735和1.000/0.000。在牦牛群体中未发现GHRH exon3-HaeⅢ多态,全部个体为AA型。在南阳牛、秦川牛、郏县红牛和中国荷斯坦牛群体中主要为BB型和AB型个体,等位基因B为优势等位基因。南阳牛、秦川牛、郏县红牛和中国荷斯坦牛4个群体处于Hardy-Weinberg平衡状态。南阳牛、秦川牛、郏县红牛和中国荷斯坦牛群体的遗传多态指数h/Ne/S/PIC分别为0.431/1.757/0.474/0.338、0.384/1.623/0.559/0.310、0.368/1.582/0.590/0.300和0.390/1.638/0.548/0.314。该位点的0.25<PIC<0.50,属于中度多态位点。5个群体的遗传多态性丰富程度从大到小依次为南阳牛、中国荷斯坦牛、秦川牛、郏县红牛和牦牛。3.2利用最小二乘法对GHRH exon3-HaeⅢ位点多态与南阳牛、秦川牛和郏县红牛3个黄牛群体生长性状进行相关分析和多重比较发现,对于6月龄南阳牛,除了胸围和坐骨端宽外,体重、体高、体斜长和日增重在杂合个体与纯合的BB型个体之间差异显著(P<0.05);24月龄时,AA型和AB型个体的体重和日增重显著高于BB型个体(P<0.05)。GHRH exon3-HaeⅢ多态对郏县红牛的体斜长和胸围的效应达到显著水平,表现为BB型个体显著低于杂合个体(P<0.05)。4.牛GHR基因外显子10多态与生长性状的相关分析4.1利用PCR-SSCP研究了GHR基因外显子10的SNP (A>G)在5个群体中的遗传特性。在南阳牛、秦川牛、郏县红牛、中国荷斯坦牛和牦牛5个群体中,等位基因G/A的频率分别为0.443/0.557、0.444/0.556、0.495/0.505、0.164/0.836和0.114/0.886。牦牛群体未发现GG型个体;在牦牛和中国荷斯坦牛群体中主要为AA型,等位基因A为优势等位基因。在南阳牛、秦川牛和郏县红牛群体中,3种基因型分布比较均衡。南阳牛、秦川牛、牦牛3个群体处于Hardy-Weinberg平衡状态,而郏县红牛和中国荷斯坦牛群体则偏离Hardy-Weinberg平衡状态。南阳牛、秦川牛和郏县红牛3个群体在该位点0.25<PIC<0.50,属于中度多态位点,中国荷斯坦牛和牦牛群体PIC<0.25,属于低度多态位点。依据遗传多态指数,南阳牛、秦川牛和郏县红牛3个黄牛群体在该位点的群体多态丰富程度比较接近一致。4.2对牛GHR基因外显子10的SNP基因型对南阳牛、秦川牛和郏县红牛生长性状的遗传效应进行了分析。在南阳牛生长过程中,GHR基因外显子10的SNP (A>G)对坐骨端宽性状的效应显著(P<0.05);6月龄南阳牛纯合GG型个体的体高显著高于杂合个体和纯合AA型个体(P<0.05)。12月龄南阳牛GG型个体的胸围显著高于杂合个体(P<0.05)。更多还原

【Abstract】 Genetic variations of MRF family, MSTN, GHRH and GHR genes were detected by PCR-RFLP and PCR-SSCP DNA marker techniques in 521 individuals of five bovine populations (Nanyang cattle, Qinchuan cattle, Jiaxian Red cattle, Chinese Holstein, and Yak), and association analysis were carried out to evaluate the effects of genotypes of polymorphic loci on growth traits of three Chinese cattle populations (Nanyang, Qinchuan and Jiaxian Red ). The objective was to detect the hereditary characteristics and to explore molecular markers with significant effects on economic important traits for efficient selection and improvement of Chinese cattle, and to provide genetic information for construction of molecular marker database, protection and utilization of breed resource of Chinese cattle. The results were as follows:1. Relationship between genetic variation of MSTN gene and growth traits in Nanyang, Qinchuan and Jiaxian Red cattle populations1.1 No polymorphism in MSTN exon 1 and 3 was detected using PCR-SSCP in the five populations. PCR-RFLP tests using restriction endonuclease TaqI were carried out in the 1346 bp fragment of bovine MSTN 5’regulatory region in the five populations. The results showed that no TaqI polymorphism in the five populations. Therefore, this study identified an occurrence of sequencing error in the publishing sequence (Accession No. AF348479) and confirmed the presence of nucleotide A at position -360 (relative to ATG start codon).1.2 PCR-RFLP tests using restriction endonuclease DraI were carried out in the 1346 bp fragment of bovine MSTN 5’regulatory region in the five populations. The results showed the polymorphic PCR-RFLP-DraI in MSTN 5’regulatory region was a bi-allelic loci with allele T and A. The allele frequencies of T/A of Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak populations were 0.955/0.045, 0.973/0.027, 0.972/0.028, 0.918/0.082, 1.000/0.000 respectively. There were three genotypes (TT, TA and AA) in this locus. Yak individuals were all genotype TT, that is, no mutation (T>A) was detected in Yak population. In Nanyang and Qinchuan cattle, individuals with genotype TT and TA were found and no individuals with genotype AA was detected, whereas in Jiaxian Red cattle only one homozygous AA individual was detected. Genotype TT and allele T were dominant in the five populations. The Chi-Square tests showed Nanyang, Qinchuan cattle and Chinese Holstein populations were at Hardy-Weinberg equilibrium, but Jiaxian Red cattle population was at Hardy-Weinberg disequilibrium. Genetic diversity indexes of h/Ne/S/PIC in Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak populations were 0.1505/1.1772/0.2835/0.1345, 0.0864/1.0946/0.1842/0.0826, 0.0545/1.0576/0.1279/0.0530, 0.0523/1.0552/0.1237/0.0501 and 0.0000/1.0000/0.0000/0.0000,so the population genetic polymorphisms from higher to lower were Chinese Holstein, Jiaxina Red, Nanyang, Qinchuan cattle and Yak.1.3 Associations between MSTN-DraI polymorphism and growth traits in Nanyang, Qinchuan and Jiaxian Red cattle were analyzed by least square means (LSM) method. The results showed that age had significant effects on body weight, withers height, body length, heart girth and hucklebone width in the Nanyang cattle (P<0.01), which indicated growth of Nanyang cattle differed significantly at different growth stages and directly correlated with age. Multiple comparison results showed MSTN-DraI genotypes affected hucklebone width of six-month Nanyang cattle and individuals with genotype TT had higher withers height than those with genotype TA (P<0.05).2. Genetic variations of MRF gene family and their associations with growth traits in Nanyang, Qinchuan and Jiaxian Red cattle populations2.1 Four polymorphic loci were detected in four genes (Myf5, MyoD, Myf6, MyoG) of bovine MRF gene family by PCR-RFLP and PCR-SSCP techniques.Three genotypes were detected by PCR-RFLP-TaqI technique in bovine Myf5-1 locus. This polymorphism was caused by a SNP (CAA/CGA) located in intron 2 at position 1948 bp of bovine Myf5 sequence (Accession No. M95684). The allele frequencies of A/B in Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak were 0.173/0.827, 0.242/0.758, 0.248/0.752, 0.262/0.738 and 0.031/0.969. Homozygous AA individual was not detected in Yak population. Allele B was dominant in five populations which were all at Hardy-Weinberg equilibrium. A SNP (A>T) located in bovine Myf5 exon 1 at position 609 bp (Accession No. M95684) was detected by PCR-SSCP and did not change the amino acid sequence. In Nanyang, Qinchuan, Jiaxian Red cattle and Yak population, there were three genotypes and allele A/B frequencies were 0.443/0.557, 0.475/0.525, 0.467/0.533 and 0.199/0.801. The heterozygous genotype frequency was low and allele frequencies of A and B were almost equal in Nanyang, Qinchuan and Jiaxian Red cattle populations. Genotype BB and allele B were dominant in Yak population. Nanyang, Qinchuan, Jiaxian Red cattle and Yak populations were all at Hardy-Weinberg disequilibrium.Two SNP (C>T) were detected in intron 2 of bovine MyoD gene and coding sequence of bovine Myf6 gene respectively. In Myf6 gene the SNP did not cause any change in amino acid sequence. The frequencies of allele A/B at MyoD-intron2 locus in Nanyang, Qinchuan, Jiaxian Red cattle and Chinese Holstein were 0.904/0.096, 0.882/0.118, 0.926/0.074 and 0.980/0.020. The frequencies of allele A/B at Myf6 locus in Nanyang, Qinchuan and Jiaxian Red cattle were 0.983/0.017, 0.995/0.005 and 0.997/0.023. There were two genotypes at the two loci respectively. Individuals of Yak population were all genotype AA, which indicated Yak population was homozygous at the MyoD-intron2 and Myf6 loci. No homozygous BB individual was found and homozygous AA individual was the majority in the others which were all at Hardy-Weinberg equilibrium. Genotype AA and allele A were dominant in the studied populations.Myf5-1-TaqI polymorphism and Myf5-2 SNP were (or closer to) moderate polymorphic and MyoD-intron2 and Myf6 loci were low-polymorphic in Nanyang, Qinchuan and Jiaxian Red cattle. The means of PIC of population at the four loci of Nanyang, Qinchuan and Jiaxian Red cattle were 0.2023, 0.2175 and 0.2123, therefore the genetic polymorphisms from higher to lower were Qinchuan, Jiaxina Red and Nanyang cattle.2.2 Association analysis between the polymorphisms and growth traits of Nanyang, Qinchuan and Jiaxian Red cattle populations were carried out. The results were as follows:For Myf5-1-TaqI polymorphism, eighteen-month Nanyang individuals with genotype AA had higher withers height compared to individuals with genotype AB and BB, and Jiaxian Red cattle with genotype AA had higher hucklebone width than those with genotype AB and BB, whereas Qinchuan cattle with genotype AA had lower withers height and height at hip cross than the other two kinds of individuals (P<0.05).The Myf5-2 SNP had significant effects on weithers height and body length of Nanyang cattle (P<0.05). Homozygous individuals of six-month and twelve-month Nanyang cattle with genotype BB had higher withers height than the other homozygous animals, and homozygous BB animals of six-month Nanyang cattle had higher body length than the other homozygous animals (P<0.05). Compared with homozygous animals in Qinchuan cattle, heterozygous individuals had higher hip width (P<0.05).For MyoD-intron2 SNP, except withers height and height at hip cross, body length, hucklebone width, rump length, heart girth and hip width of heterozygous Qinchuan animals were higher than homozygous animals (P<0.05). For Myf6 SNP, eighteen-month Nanyang cattle with genotype AA had higher hucklebone width when compared with heterozygous animals (P<0.05).3. Association of GHRH exon3-HaeⅢpolymorphism with growth traits in Nanyang, Qinchuan and Jiaxian Red cattle populations3.1 The polymorphism in exon 1 of bovine GHRH gene was investigated by PCR-RFLP- HaeⅢin the five populations. The frequencies of allele A/B in Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak populations were 0.314/0.686, 0.259/0.741, 0.243/0.757, 0.265/0.735 and 1.000/0.000. Yak population was monomorphism in GHRH exon3-HaeⅢlocus, all animals were genotype AA. Individuals with genotype AB and BB were the majority and allele B was dominant in Nanyang, Qinchuan, Jiaxian Red cattle and Chinese Holstein populations which were all at Hardy-Weinberg equilibrium. Genetic diversity indexes of h/Ne/S/PIC in Nanyang, Qinchuan, Jiaxian Red cattle and Chinese Holstein populations were 0.431/1.757/0.474/0.338, 0.384/1.623/0.559/0.310, 0.368/1.582/0.590/0.300 and 0.390/1.638/0.548/0.314. Since the population PICs at GHRH exon3-HaeⅢwere intervenient of 0.25 and 0.50, GHRH exon3-HaeⅢlocus was moderate polymorphic in the five populations. The genetic polymorphisms of five populations from higher to lower were Nanyang, Chinese Holstein, Qinchuan, Jiaxina Red and Yak.3.2 Association analysis and multiple comparisons were carried out between genotypes of GHRH exon3-HaeⅢlocus and growth traits of Nanyang, Qinchuan, Jiaxian Red cattle. For six-month Nanyang cattle, except heart girth and hucklebone width, body weight, withers heights, body length and average daily gain differed significantly between genotype AB and BB animals (P<0.05). Twenty-four-month Nanyang animals with genotype BB had lower body weight and average daily gain compared with animals with other two genotypes (P<0.05). The effects of GHRH exon3-HaeⅢon body length and heart girth reached a significant level in Jiaxain Red cattle, animals with genotype BB were significant lower than those of heterozygous animals (P<0.05).4. Relationship between GHR exon 10 and growth traits in Nanyang, Qinchuan and Jiaxian Red cattle populations4.1 Characterization of the SNP (A>G) located in bovine GHR exon 10 was investigated in the five populations. Allele G/A frequencies of Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak populations were 0.443/0.557, 0.444/0.556, 0.495/0.505, 0.164/0.836 and 0.114/0.886 respectively. Animals with genotype GG were not detected in Yak population. Genotype AA and allele A were dominant in Chinese Holstein and Yak populations. Genotype frequencies were almost equal in Nanyang, Qinchuan and Jiaxian Red cattle populations. Nanyang, Qinchuan cattle and Yak populations were at Hardy-Weinberg equilibrium, whereas Jiaxian Red cattle and Chinese Holstein were at Hardy-Weinberg disequilibrium. In Nanyang, Qinchuan and Jiaxian Red cattle populations, the SNP was moderate polymorphic (0.25<PIC<0.50), but in Chinese Holstein was low polymorphic (PIC<0.25). The genetic polymorphisms of Nanyang, Qinchuan and Jiaxian Red cattle were in close proximity.4.2 The effects of the SNP genotypes of bovine GHR gene on growth traits were evaluated in Nanyang, Qinchuan and Jiaxian Red cattle. The SNP of GHR gene had significant effects on hucklebone width of Nanyang cattle at all growth stages (P<0.05). Six-month Nanyang animals with genotype GG had higher withers heights than heterozygous animals and homozygous AA animals (P<0.05). Homozygous GG animals of twelve-month Nanyang cattle had higher heart girth than heterozygous animals (P<0.05).更多还原