【作者】 张春艳；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 繁殖和生长是决定规模化养殖产出的两大主要经济性状,在少胎动物(羊)中,母羊繁殖效益在总效益中占据更大的比例。然而,繁殖性状遗传力低、周期长、选育进展慢、且与生长性状之间的复杂关系是制约养羊经济效益的主要原因。因此,研究山羊繁殖性状影响因素、遗传规律、及其与生长的关系和卵泡发育的分子调控机制,对于利用数量遗传和分子遗传方法提高山羊繁殖力有重要作用,开发提高繁殖力的新技术、建立群体选育和分子育种新方法、快速改良和提高山羊繁殖性能具有重要理论和实践意义。本研究首先对母羊繁殖性状的影响因素和遗传规律进行分析,并探讨与生长性状之间的关系,为山羊繁殖性状的群体选育提供理论依据；从繁殖轴和生长轴的主要激素和细胞因子着手,研究这些基因在卵泡不同发育时期的表达差异；再从基因多态性出发,研究差异表达基因突变后对山羊产羔数和超数排卵性状的影响,通过分析超排母羊血清激素浓度变化趋势,对主效基因进行初步验证,探讨山羊卵泡发育的分子网络调控机制。1、山羊繁殖性状的影响因素、遗传规律及其与生长性状之间的关系以波尔山羊种羊场最近10年(2000-2010)的数据资料,分析年份、季节、母羊年龄和胎次、羔羊性别和同胞数对母羊产羔数、断奶数、妊娠天数和羔羊出生窝重的影响；利用MTGSAM软件,分别用阈性状和线性性状模型估计母羊繁殖性状遗传参数,用DFREML软件估计羔羊生长性状的遗传参数,并分析母体遗传和环境效应对羔羊生长的影响,同时研究母羊产羔数与羔羊生长之间的遗传相关,解析山羊繁殖和生长之间的关系。(1)波尔山羊母羊繁殖性状受年龄、胎次和分娩年份的显著影响(P<0.05),出生窝重还受产羔数和季节的影响；产羔数对母羊妊娠天数无显著影响,表明波尔山羊的多产性和妊娠期与属于多胎动物的猪有类似的规律。(2)母羊繁殖性状遗传力较低(0.09-0.12),重复力较高(0.27-0.57),产羔数、断奶数和窝重之间呈正的遗传相关,而且数值较高(0.66-0.88),表明在生产实际中,只需对其中一种性状(如产羔数)进行选育,便可同时提高三种性状的生产性能。(3)羔羊出生和早期生长性状受母体遗传和环境效应影响很大。直接遗传力和母体遗传力较高,直接遗传和母体遗传之间呈高度的负相关关系,随着羔羊生长发育,这种负相关关系逐渐减弱。(4)母羊产羔数与羔羊早期生长之间呈负相关关系,随着羔羊的生长和发育,相关程度逐渐减弱；出生重和90日龄重在不同同胞数羔羊之间差异显著,达300日龄时,不同同胞数羔羊之间体重已无显著差异。表明双羔和多羔在后期有较强的补偿生长效应,提高母羊产羔数对羔羊后期生长无不利影响。2、不同发育时期卵泡发育相关基因的表达差异利用荧光定量RT-PCR技术,分析繁殖轴(GnRHR, FSH, FSHR, LH, LHR, PRL, PRLR)和生长轴主要激素(GH)及细胞生长因子(IGF-Ⅰ,IGF-Ⅱ, IGFBP-3)在山羊卵泡不同发育时期的表达差异,根据卵泡直径将其分为以下五个不同发育时期：腔前卵泡(解剖卵巢获得)、小卵泡(<2.0 mm)、中等卵泡(2.0-4.0 mm)、大卵泡(4.0-8.0mm)和排出的卵子,探讨卵泡动态发育的调控机制,发现：(1)FSH、LH及其受体基因相对表达量随卵泡直径增大而逐渐增高,在腔前卵泡中表达最低，在大卵泡和卵子中大量表达(P<0.05)。(2)PRL及其受体基因相对表达量在卵泡优势化以前极低,在大卵泡和卵子中较高(P<0.05)。(3)GH基因相对表达量在卵泡发育各时期均较高,在腔前卵泡中的表达量最高(P<0.05)。(4) IGF-Ⅰ、IGF-Ⅱ和IGFBP-3基因相对表达量在不同发育时期的卵泡中均较高；卵子中IGFBP-3基因相对表达量显著高于各发育时期的卵泡。3、山羊产羔和超数排卵性状标记基因的鉴定以繁殖轴(GnRHR, LHR, FSH, FSHR, PRL, PRLR)和生长轴(GH, IGFBP-3)主要激素和细胞因子为候选基因,采用PCR-SSCP、PCR-RFLP、基因克隆和测序等技术,分析了候选基因在4个山羊品种(波尔、马头、波-马杂交羊、麻城黑山羊)共计780个样本中的多态性及其与产羔数的关系；同时,超数排卵处理57只母羊,分析基因多态性与超数排卵效果的关系,以及不同基因型母羊在超排过程中血清雌二醇和孕酮浓度的变化差异,筛选并初步验证影响山羊繁殖性状的标记基因,发现：(1) GnRHR基因外显子1发生G 137 A突变,引起MspⅠ酶切多态性,但在四个山羊品种中只检测到AA和AB两种基因型；AA型波尔山羊和马头山羊平均产羔数和第三胎产羔数显著大于AB型(P＜0.05)，但是,不同基因型的杂交羊产羔数无显著差异。超排后,与AB型相比,AA型母羊卵巢上的小卵泡数显著增高,血清雌激素水平上升较快,而且峰值水平较高,孕激素下降和上升变化较明显。表明,GnRHR突变是影响母羊繁殖性状的主效基因。(2) FSHB基因外显子3发生A278 G突变,引起MnlⅠ酶切多态性,在四个山羊品种中存在AA、AB和BB三种基因型。AA型波尔山羊和杂交羊第三胎和各胎平均产羔数显著大于AB型和BB型,而AB型和BB型之间无显著差异。不同基因型的马头山羊各胎产羔数虽无显著差异,但变化趋势与其他山羊品种一致,而且超排后,AA型母羊卵巢上的大卵泡数显著高于AB和BB型,血清雌激素和孕酮浓度变化更明显。表明,FSHB基因是影响山羊繁殖性状的主效基因。(3) FSHR基因外显子1发生C272T突变,引起HaeⅢ酶切多态性,但在四个山羊品种中只检测到AA和AB两种基因型,AA型母羊产羔数显著大于AB型。超数排卵处理后,AA型母羊卵巢上小卵泡、大卵泡和黄体数均显著多于AB型,囊肿率较低,雌激素峰值显著升高,且变化趋势更明显。表明,FSHR基因是影响山羊产羔数和超数排卵性状的主效基因。(4)LHR基因外显子11发生G 94 A突变,引起MspⅠ酶切多态性,在波尔和杂交羊中检测到三种基因型(AA、AB和BB型),但AA型频率极低。在马头山羊和黑山羊中只检测到AB和BB两种基因型；在波尔山羊中,BB型个体第三胎产羔数和各胎平均产羔数显著高于AB型。马头山羊超数排卵后,BB型个体卵巢上的小、中、大卵泡数和黄体数高于其他基因型,但差异均不显著,BB型母羊在超排期间雌激素峰值显著高于其他基因型,孕激素浓度变化无显著差异。表明,LHR基因突对波尔山羊产羔数产生显著的影响。(5)GH基因分别在外显子2和5发生A 781 G和A 1575 G突变,引起HaeⅢ酶切多态性,两个突变位点在四个山羊品种中均只检测到两种基因型(AA和AB,CC和CD型)。马头山羊AB和CC型母羊产羔数显著大于AA和CD型,聚合两个突变位点,波尔和马头山羊ABCD型母羊各胎次的产羔数显著高于AACD型；AB和CC型母羊超排后卵巢上的黄体数显著增多,雌激素变化明显,峰值显著升高。表明,GH基因突变对母羊产羔和超数排卵性状产生显著的影响。(6) IGFBP-3基因在转录区发生A 137 G突变,引起BfaⅠ酶切多态性,在四个山羊品种中存在AB和BB三种基因型,但AA型频率极低；AB型马头山羊各胎次产羔数均显著高于AA和BB型；超排后,AB型母羊卵巢上中等大小的卵泡数显著多于BB型,血清雌激素上升迅速,峰值显著升高,孕激素下降和上升明显。表明,IGFBP-3是影响马头山羊产羔和超数排卵性状的主效基因。(7)PRL基因在转录区发生C 407 A突变,引起SduⅠ酶切多态性,在四个山羊品种中存在AA、AB和BB三种基因型,基因型频率均表现为BB＞AB＞AA。关联分析表明,AB杂合型母羊各胎次产羔数比纯合型高、超数排卵效果较好,但无显著差异,血清雌激素和孕激素浓度变化与其他基因型相比差异不显著。表明,PRL基因突变对母羊产羔和超数排卵性状无显著影响。(8) PRLR基因外显子10发生C 53 G突变,引起MspⅠ酶切多态性,在四个山羊品种中存在AB和BB两种基因型,BB型母羊平均产羔数显著高于AB型；不同基因型母羊超排性状和血清激素水平变化无显著差异。表明,PRL基因突变对母羊产羔和超数排卵性状无显著影响。更多还原

【Abstract】 Both fertility and growth traits are the main two important determinants of economic profitability in large goat producing enterprise. The reproductive efficiency holds a high percentage of the total profitability, especial for the low prolificacy animal, such as goat breed. Nevertheless, the development of goat fertility is restricted by various reasons, such as many influencing factors, low heritability, long breeding cycle and the complex relationship with the growth traits. Therefore, it is necessary to evaluate a thorough knowledge of its reproduction traits and incorporate the reproduction complex into the breeding objective.This study was designed to analyze the effect factors and hereditary rules of goat fertility, as well as its relationship with growth traits and the molecular regulation mechanisms of follicular development, which play an important role in increasing the goat fertility by methods of both quantitative inheritance and molecular breeding. We selected the main genes both from reproductive and growth axles which have significant effect on dam reproduction, and studied the gene differential expression in different follicular developmental stages. And then detected the polymorphisms of these candidate genes and analyzed the relationship with litter size and superovulation performances of dam. The variation trends of serumβ-estradiol (E2) and progesterone (P4) were determined to validate the function and mechanism of these major genes in ovary and follicular development, which could provide the basic theory to explore the new techniques for improving the goat fertility. The main results are as follows:1. The effect factors and hereditary rules of goat fertility, as well as its relationship with growth traits.The hereditary rules for goat reproduction traits including litter size at birth (LSB) and at weaning (LSW), gestation period (GP) and litter weight at birth (LWB) were analyzed by use of the MTGSAM procedure fitting both threshold and linear animal models (y= Xb+Zaa+ZPP+e) in Boer dams from Boer Goat Breeding Station in Yidu, China, during 2000-2010. Influencing factors such as parity and age of dam, kidding year and season, number of sibs and sex of kids were investigated as the fixed effects. The genetic parameters of growth traits of kids were also analyzed by DFREML procedure. By compared with 4 animal models, the effect of maternal genetic and permanent environmental of dam on kids growth, as well as the relationship between reproduction and growth traits were analyzed. The major results showed as following: (1) Parity and age of dam as well as kidding year had significant effect on dam reproduction traits (P＜0.05). The trait of LWB was also significantly affected by the number of sibs and kidding season. The number of sibs had no significant effect on gestation period, which indicates that the prolificacy and gestation of Boer goat breed is more likely to the multiple birth species just for pig.(2) The reproduction traits of Boer dam had low heritability (0.09-0.12) and high repeatability (0.27-0.57). High and Positive genetic correlation estimates were obtained between LSB, LSW and LWB, suggesting that these three traits have the similar hereditary basis, and improve any trait (such as LSB) can synchronously increasing other two traits.(3) The maternal genetic and permanent environmental effects were important determinants for kid birth and early growth traits. For which, the additive and maternal genetic heritability were high, and the genetic correlation estimates between direct additive and maternal genetic (ra-m) effect were high and negative. However, the correlation coefficient decreased with the kid growth and development.(4) Negative relationship was observed between litter size and kid early growth, but the correlation coefficient decreased with the kid growth and development. Body weight at birth and 90 days of kids were significant different in various litter sizes, and body weight at 300 days had no significant difference. It implies that the twin and multiple kids have compensatory growth effect in late growth stage, and increasing the litter size has no negative effect on kid growth.2. Gene differential expression in different follicular development stagesDifferential expression of follicular development genes including reproductive hormones (GnRHR、FSH、FSHR、LH、LHR、PRL、PRLR), growth hormone (GH) and cytokines (IGF-Ⅰ、IGF-Ⅱ、IGFBP-3) in different follicular development stages were evaluated by the methods of fluorescent quantization RT-PCR. The follicular development stages were divided into five groups according to the follicular diameter (pre-antral follicle; small:＜2.0 mm; medium:2.0-4.0 mm; large:4.0-8.0 mm; ovum). The aim is to study the regulation mechanism of the follicular development. The main results are as following:(1) The relative expressions of FSH, FSHR, LH and LHR genes were increasing with the follicular development, which was the lowest in pre-antral follicle, and the highest in large and ovum (P＜0.05).(2) The relative expressions of PRL and PRLR genes were low in pre-dominant follicles, and higher in large and ovum(P＜0.05).(3) The relative expression of GH gene was high in all stages, and which was the highest in pre-antral follicle (P＜0.05).(4) The relative expression of IGF-Ⅰ, IGF-Ⅱand IGFBP-3 genes were high in all stages. The relative expression of IGFBP-3 gene in ovum was significant higher than in follicular stages (P＜0.05).3. Identification of major genes associated with goat litter size and superovulation performances.Polymorphisms of candidate genes for goat litter size such as the reproductive hormones (GnRHR、LHR、FSH、FSHR、PRL、PRLR), growth hormone (GH) and cytokine (IGFBP-3) in 780 individuals from 4 goat breeds including Macheng black, Boer, Matou and Boer-Matou crossbred were detected with PCR-SSCP, PCR-RFLP, gene cloning and sequencing. Furthermore,57 dams were superovulated by exogenetic reproductive hormones, and the associations of superovulation performances with above candidate gene polymorphisms were also analyzed. The variation trends of serumβ-estradiol (E2) and progesterone (P4) following the superovulation period in different genotypes were determined and contrasted. Based on the associations between those polymorphisms and litter size as well as superovulation performances, several genes were selected as molecular markers controlling goat reproduction traits. The main results are as follows:(1) A new mutation locating in extron 1 (G 137 A) in goat GnRHR gene was identified, which can be detected by endonuclease Msp I and resulted in two genotypes named AA and AB. Both litter size in the third parity and average litter size in all parity of AA genotype were more than AB genotype in Boer and Matou breed (P＜0.05), which in the crossbred had no significant difference. After the superovulation, contrasting with the AB genotype, dams with AA genotype had significant more numbers of small follicular on ovaries, faster and higher increase of E2 concentration. These results suggested that the mutation in GnRHR gene was major gene controlling dam reproduction traits.(2) A new mutation locating in extron 3 (A 278 G) in goat FSHB gene was identified, which can be detected by endonuclease MnlⅠand resulted in three genotypes named AA, AB and BB in four different goat breeds. Both litter size in the third parity and average litter size in all parity of AA genotype were more than these in AB and BB genotypes of Boer and crossbred (P＜0.05), which in the AB and BB genotypes had no significant difference. The litter sizes in various parity of Matou goat had the same variation trends to the Boer and Crossbred, but showed no significant difference. After the superovulation, dams with AA genotype had significant more numbers of large follicular on ovaries than AB and BB genotypes. The serum E2 and P4 concentration have more significant changes in AA genotype than AB and BB genotypes. These results suggested that the mutation in FSHB gene was major gene controlling dam reproduction traits in Boer and Matou breeds.(3) A new mutation locating in extron 1 (C 272 T) in goat FSHR gene was identified, which can be detected by endonuclease HaeⅢand resulted in two genotypes named AA and AB in four goat breeds. Dams with AA genotype had more litter size than AB genotype (P＜0.05). After the superovulation, dams with AA genotype had significant more numbers of small, large follicular and corpus luteum on ovaries, and lower incidence of ovarian cyst than AB genotype (P＜0.05). The peak value of serum E2 concentration in AA genotype was significant higher than that in AB genotype. These results suggested that the mutation in FSHR gene was high associated with dam litter size and superovulation performances.(4) A new mutation locating in extron 11 (G 94 A) in goat LHR gene was identified, which can be detected by endonuclease MspⅠ. Three genotypes (AA, AB and BB) were detected in Boer and Matou breeds, however, the frequency of genotype AA was too low to be ignored. Only AB and BB genotypes were detected in Matou and Macheng Black breeds. Both litter size in the third parity and average litter size in all parity of BB genotype were more than AB genotype in Boer goat (P＜0.05). After the superovulation, dams with BB genotype had more numbers of small, medium, large follicular and corpus luteum on ovaries than other genotypes, but no significant difference appeared. The peak value of serum E2 concentration in BB genotype was significant higher than that in AA and AB genotypes. These results suggested that the mutation in LHR gene was major gene controlling litter size in Boer dam.(5) Two single nucleotide polymorphisms (SNPs) (A 781 G and A 1575 G) were identified in goat GH gene. Two genotypes (AA and AB, CC and CD) of each mutation were detected in four goat breeds. Neither BB nor DD homozygote genotype was observed. Matou dams with AB or CC genotypes had significant larger litter size than these with AA and CD genotypes (P＜0.05). When combined the two loci, both Matou and Boer dams with ABCD genotype had the largest litter size compared with the other genotypes (P＜0.05). Under the same superovulation treatments, compared with the other genotypes, AB and CC dams had the significant more numbers of corepus luteum, lower更多还原