波尔山羊及其杂交改良后代群体遗传结构与亲缘关系研究

利用RAPD标记和POPGENE、PHYLIP及SPSS等统计分析软件,研究了波尔山羊及其杂交改良后代群体的遗传结构及遗传亲缘关系。结果表明,波尔山羊与其杂交改良后代间特别是高代杂种间具有相似的多态基因座百分率、等位基因数和基因多样性。随着级进杂交改良代数的递增,高代杂种之间以及与改良亲本之间的群体遗传结构的差异逐渐缩小,群体间的基因分化降低,基因流动和遗传相似度增加,遗传距离缩小,高代杂种与波尔山羊间具有更近的遗传亲缘关系。 Abstract:The genetic structure and relationship of Boer goat and its upgrading offspring to Tangshan Diary goat were studied using the RAPD marker and some statistical program,such as POPGENE,PHYLIP and SPSS.The results indicated that there were the similar percentage of polymorphic loci,observed and effective number of alleles,gene diversity between Boer goat and its upgrading offspring,especially higher upgrading offspring.With the increasing of upgrading,the difference of population structure decreased as well as the genetic distance and differentiation among higher upgrading offspring and their improving parental,but gene flow and genetic identity increased.There was a close genetic relationship between higher upgrading offspring and Boer goat.     

3个山羊群体中4个微卫星DNA多态性及其与杂种优势的关系

利用4个微卫星标记（OarFCB11,OarAE101,McM218,McM38）对波尔山羊、太行山羊和河北奶山羊的等位基因频率、群体多态信息含量、有效等位基因数、杂合度和遗传距离进行了遗传检测,并测定了波尔山羊与河北奶山羊及太行山羊的杂交效果。结果表明：4个微卫星标记在波尔山羊、太行山羊和河北奶山羊3个品种中存在多态性,可以用于山羊遗传多样性的评估;从不同品种来看,太行山羊的遗传变异程度最大,而波尔山羊的遗传变异程度相对较小;波尔山羊与河北奶山羊的遗传距离大于与太行山羊,波尔山羊与河北奶山羊的杂种优势高于与太行山羊,与实际杂种优势测定结果相符。 Abstract: Gene frequency, polymorphism information contents, number of effective alleles, heterozygosity and genetic distances were studied in Boer goat, Taihang goat and Hebei dairy goat using four microsatellite markers（OarFCB11,OarAE101,McM218,McM38）. The crossing effects on Hebei dairy goat and Taihang goat with Boer goat were tested. The results indicated that there are genetic polymorphisms at four microsatellite markers in three goat breeds. Four microsatellite markers can be used for genetic diversity evaluation in goat breeds. The genetic variability of Taihang goat is the highest, and Boer goat is the lowest in three goat breeds. Genetic distances between Boer goat and Hebei dairy goat is bigger than that between Boer goat and Taihang goat. The heterosis between Boer goat and Hebei dairy goat is higher than that between Boer goat and Taihang goat. It accords with testing results on actual heterosis.     

波尔山羊杂交后代及其亲本随机扩增多态DNA研究

利用随机扩增多态DNA技术研究了波尔山羊、唐山奶山羊、青龙本地山羊以及波尔山羊与这两个山羊群体杂交后代共计128个山羊个体的随机扩增多态DNA。结果表明：（1）总群体平均遗传多样性指数（Hsp）为0.6974,群体遗传分化指数为0.9706,山羊群体间平均遗传距离指数（0.1314～0.2052）明显大于群体内的相应值（0.0582～0.1440）,上述结果说明,所研究山羊群体不仅具有较为丰富的遗传多样性,而且其核基因组遗传变异主要存在于群体间。（2）山羊群体间的分子聚类关系与各群体间的亲缘关系基本一致。 Abstract:The random amplified polymorphic DNA（RAPD）of 128 individuals was studied,which were from Boer goat, Tangshan dairy goat,Qinglong native goat and their hybrids crossbred with Boer goat.The average index of genetic polymorphism for whole population（Hsp）and the index of genetic differentiation were 0.6974 and 0.9706,respectively.The average index of genetic distance between populations（0.1314～0.2052）was significantly higher than that within populations（0.0582～0.1440）.All of these indicated that the genetic polymorphism was not only abundant,but also the genetic variation was mainly existed between goat populations.The molecular dendrogram among goat populations was in accord with their genetic relationship.     

群体遗传不平衡条件下的结构基因遗传共适应特性

本研究以柴达木山羊、柴达木绒山羊和辽宁绒山羊三个群体共147只山羊为材料,利用聚丙烯酰胺凝胶电泳（PAGE）技术检测了5种血液蛋白质（酶）基因座的遗传多态性,并进行了结构基因遗传共适应的研究,结果发现：45个基因座组合中有10个基因座组合处于遗传不平衡状态,并且这些遗传不平衡皆单纯由遗传共适应差异造成;除辽宁绒山羊Tf-PA-3组合的遗传不平衡包含非等位基因间的遗传共适应差异外,其他基因座组合的遗传不平衡皆由等位基因间的共适应差异,即单基因座的遗传不平衡造成;LAP-EsD组合的共适应差异在群体间有遗传传递现象。 Abstract:With the technology of PAGE,the genetic polymorphism of blood protein and enzyme was investigated,and genetic co-adaptability among structural genes was studied in three goat populations(147 goats) including Chaidamu goat(CS),Chaidamu Cashmere goat(CRS) and Liaoning Cashmere goat(LRS) in Qinghai Province,China.The results were showed that the genetic disequilibrium of 10 locus combinations was found among 45 locus combinations in the three goat populations,and these genetic disequilibria were caused only by the difference of genetic co-adaptability among genes,because there didn′t exist the linkage disequilibrium among non-allelic genes.The genetic disequilibrium including the difference of genetic co-adaptability between non-allelic genes was only found at Tf-PA-3 locus combinations in LRS population,the other ones were all caused by the genetic disequilibrium at a single locus.The difference of genetic co-adaptability of LAP-EsD locus combinations could be messaged among different populations.     

乌羊和小香羊的RAPD分析

用40条多态引物对乌羊、小香羊、南江黄羊、黑山羊、川东白山羊、波尔山羊和马头山羊7个品种(或群体)进行RAPD分析,其中28条引物扩增出多态性谱带,并用于进一步对12只乌羊个体和12只小香羊个体基因组进行扩增.扩增产物以1.5%琼脂糖凝胶(含0.5μg/ml溴化乙锭)电泳分离.Nei氏公式计算品种间的遗传距离指数和品种内的遗传相似指数,NJ法构建系统聚类图.结果表明:乌羊和川东白山羊间的遗传距离最小,亲缘关系较近,而小香羊与各品种间的遗传距离都较大,亲缘关系较远.乌羊群体及小香羊品种都具有一定的遗传稳定性。 Abstract:A total of 40 primers generated polymorphic loci were used in random amplified polymorphism DNA(RAPD)analysis in seven goat breeds(or populations)including Wu goat,Small-Xiang goat,Nanjiang Brown goat,Black goat,Chuandong white goat,Boer goat,Matou goat.28 of them were selected for further amplification in individuals of 12 Wu goats and 12 Small-Xiang goats because of their different amplified patterns among seven goat breeds.Amplification products were separated by 1.5% agarose gel(contain 0.5μg/ml ethidium bromide)electrophoresis.Genetic distance indexes among breeds and genetic similarity indexes within breed were calculated by Nei,and the phylogenetic tree was constructed by NJ method.The results indicate that the genetic distance index between Wu goat population and Chuandong white goat breed is smallest,therefore Wu goat population is closely related to Chuandong white goat breed.While the genetic distance indexes between Small-Xiang goat breed and any other goat breeds are all big,so Small-Xiang goat breed is far related to any other goat breeds.The results also indicate that the genetic stability either among the Wu goat population or the Small-Xiang goat breed reaches some extent.     

柴达木山羊群体遗传结构及系统地位的研究

采用简单随机抽样法对柴达木山羊群体进行了遗传检测,从毛色、外型特征、体尺、血液蛋白与DNA多态性等5个方面进行了群体遗传结构分析,并探索了其系统地位。结果表明：(1)毛色和形态特征的平均表型异质度分别为0.3419、0.5207;(2)血液蛋白在6个基因座上具有多态性,平均基因座杂合度为0.2584;(3)DNA-RAPD标记表现丰富的多样性,基因多样度为0.4085～0.5318。通过对柴达木山羊与其他国内山羊的系统聚类,初步探索了该山羊群体的形成及所属,这些都表明柴达木山羊是一个比较古老的未经较高强度选育的地方山羊品种,其基因资源是我国山羊遗传资源中宝贵的一部分。Abstract: Genetic structure and character of Chaidamu Goats were studied through simple random sampling. Genetic structure was analysed from five aspects, and phylogeny status was also investigated. The results indicated that: (1) the average phenotypic heterogeneity degree of coat color and morphological character were 0.3419 and 0.5207,respectively; (2) polymorphous blood albumen existed in 6 loci and the average loci heterozygosity was 0.2584; and (3) polymorphism existed in marked genes by DND-RAPD with diversity of 0.4085～0.5318. Phylogeny status was investigated through clustering by Ward’s method on Chaidamu Goats and other domestic goats. All these indicated that Chaidamu Goats was a domestic goat with less intensively selected breed.     

蟋蟀精子表面LCA及ConA结合糖复合物的分布变化

LCA and ConA-binding glycoconjugates on cricket (Teleogryllus emma) sperm surface were detected with fluorescence microscope after FITC labelling for better understanding of the distribution of glycoconjugates during spermatogenesis and spermiogenesis. FITC-LCA and FITC-ConA were bound on the spermatocytes, and their distribution changes in the process of spermiogenesis were observed .In the testis sperm, FITC-LCA and FITC-ConA were mainly bound on the head and neck region. That is different from the mark pattern of spermatophore sperm, in which the nucleus, neck region and front of the tail showed obvious fluorescence mark, especially the acrosome complex and neck region exhibited stronger mark. The mark patterns of FITC-LCA and FITC-ConA were similar,though the former was distinctly clearer than the latter. But a little difference still exists in both of them. For example in the ninth stage of spermatid, FITC-LCA mark is located on the spermatid head and neck region, and FITC-ConA mark on the spermatid head, neck and front of the tail region. When fixed germ cells were treated with PBS instead of lectin solution, or fixed cells were incubated with lectin solution, which have been treated with 0.1 mol/L specific sugar inhibitor, i.e.α-D-mannose for FITC-LCA and FITC-ConA, and α-D-glucose for FITC-ConA, no mark was observed on the cells. Those results indicate that FITC-LCA conjugated glycoconjugates has the α-D- mannose residue, and FITC-ConA conjugated glycoconjugates has the α-D-mannose and α-D-glucose residue. The investigations show that the changes in glycoconjugates distribution of cricket sperm is similar to those of other insects and mammals. The evidence exhibit that a common rule of the glycoconjugates distribution on the sperm surface is followed by most of animal sperm which may relate to the function of sperm physiology.     

桑树对花叶病抗性遗传规律的研究

用7个母本、5个父本品种,按NCII杂交设计,进行桑树对花叶病抗性的遗传分析。首次证明：桑树对花叶病的抗性具有数量遗传特点,符合加性－显性模型,主要受加性基因控制,广义遗传力和狭义遗传力中等,一般配合力显示绝对优势,具有超显性现象。母本品种含有比父本品种更多的显性基因。 Abstract　Seven maternal and five paternal varieties of mulberry were used in inheritance experiment by NCII mating design. Genetic study of mulberry resistance to the mosaic disease was carried out. The results showed for the first time that mulberry resistance to the mosaic disease showed charateristics of quantitative inheritance, it tallied with the model of additive-dominance, it was mainly controlled by additive genes. Broad heritability and narrow heritality were moderate. The general combining ability showed absolute superiority. The resistance showed overdominant phenomenon. The maternal variety had more the dominance gene than paternal.     

山羊β乳球蛋白基因的克隆及其在转基因小鼠乳汁中的高效表达 Goat β-lactoglobulin Gene Cloning and High Expression in the Mammary Gland of Transgenic Mice

通过长距离PCR从山羊基因组DNA分两段扩增山羊β乳球蛋白（β-lactoglobulin,BLG）基因,扩增出的两个片段分别克隆到T载体上,利用BLG基因序列自身存在的NarI单酶切位点进行拼接,获得了全长为7.2kb的山羊BLG基因克隆,并构建了它的真核表达载体,经酶切鉴定和序列分析证实了克隆的正确性。用线性化的BLG基因显微注射小鼠受精卵以建立转基因鼠,经PCR和Southern印迹分析证实获得了6只首建者（Founder）转基因小鼠（3♀,3♂）,在泌乳期采集两只F0代转基因雌鼠乳汁并用ELISA测定山羊β乳球蛋白的含量,其表达水平分别为23.49 mg/mL和2.19 mg/mL。 Abstract:To clone goat β-lactoglobulin (BLG) gene,two fragments were amplified from goat genomic DNA by LD-PCR method.The fragments were inserted in T-vectors before being spliced into the whole 7.2 kb BLG gene at a single restriction enzyme site of NarI.Consequently,the eukaryotic expression vector was constructed.All the clones were proved to be correct by restriction enzyme cutting and sequencing analysis.Six Founders (3♀,3♂) of goat BLG transgenic mice were obtained by microinjection and BLG genes integration were confirmed by both PCR and Southern blot analyses.The milk was collected from two lactating female transgenic mice and goat BLG protein contents were measured with ELISA.The results showed that goat BLG protein in milk of the two mice were 23.49 mg/mL and 2.19 mg/mL,respectively.     

Variation of 423G>T in the Agouti Gene Exon 4 in Indigenous Chinese Goat Breeds

The Agouti gene plays an important role in pigment synthesis in domestic animals. A transversion of 423G>T recognized by BanII was found after a fragment (178 bp) of the goat Agouti gene exon 4 was amplified and sequenced. To investigate its genetic effect and diversity, 677 individuals from 12 indigenous Chinese goat breeds and one imported goat breed from South Africa (Boer goat) were analyzed by PCR-RFLP. Two alleles, T and G, and three genotypes, TT, TG, and GG, were detected. Allele T had a higher frequency in most goat breeds and, combined with the coat color phenotype, is believed to be responsible for the black phenotype or to be linked with the causative site in the goat. The results also indicate that the 423G>T transversion showed lower genetic diversity in goat breeds with black coat color in China. Genetic differentiation among the 13 goat populations was 0.2023. The clustering of populations based on the 423G>T site was basically consistent with the variation of coat color.     

贵州地方山羊品种的RAPD分析

用180条引物对黔东南小香羊、贵州白山羊、贵州黑山羊和黔北麻羊4个贵州地方山羊品种(种群),以及南江黄羊和波尔山羊进行RAPD分析,其中27条引物扩增出多态性图谱。这27条引物共扩增出281条带,多态带为115条,平均多态频率为40．92％(范围20％～80％);每条引物平均扩增条带为10．41条(范围4～16条);扩增带分子量在210～2800bp。贵州白山羊与贵州黑山羊之间的遗传距离指数(0．0605)最小,而波尔山羊与其他品种之间的遗传距离指数(0．1059～0．1488)最大。NJ法聚类结果显示,贵州白山羊与贵州黑山羊间的亲缘关系最近,其次为黔北麻羊,而黔东南小香羊与其他3个贵州地方品种的亲缘关系较南江黄羊还远。分析表明,黔东南小香羊在遗传上为一独立的品种;而贵州地方山羊品种间具有较近的亲缘关系,遗传变异较小,具有较高的遗传稳定性。     

Cloning and expression levels of genes relating to the ovulation rate of the Yunling black goat

This study investigated the nucleotide sequences and tissue expression levels of genes relating to the ovulation rate in Yunling black goats, a famous Yunnan province, China, local breed with low fecundity. Five genes, FSHB, FSHR, BMP15, BMPR1B, and ESR2, were investigated; the complete cDNA sequences of these genes were 390, 2088, 1185, 1509, and 1585 bp, respectively, and compared with Boer goats (a more fecund breed), the sequence identities were 99%, 99%, 99%, 100%, and 99%, respectively. There were two base differences in FSHB and BMP15, four in FSHR, and three in ESR2. There were fewer follicles and oocytes in Yunling black goats than in Boer goats. Expression levels of FSHB, FSHR, and BMP15 genes in Yunling black goats were lower, and expression levels of BMPR1B and ESR2 genes were higher. Serum FSH content was lower in Yunling black goats, but serum estrogen content was higher. Protein expression levels of FSHR, BMP15, BMPR1B, and ESR2 in ovary and pituitary correlated positively with gene mRNA expression levels. In Yunling black goats, the mRNA expression levels of FSHB, FSHR, and BMP15 positively correlated with litter size, but those of BMPR1B and ESR2 correlated negatively. Together, base changes and variations in mRNA and protein expression levels of genes relating to the ovulation rate result in low fecundity in the Yunling black goat. Reduced BMP15 and FSHR levels may be related to the observed fewer oocytes and, consequently, fewer follicles.     

山羊MyoD基因家族多态性及与体尺性状的相关性

用PCR-SSCP技术研究了波尔山羊和徐淮山羊2个群体共147个个体MyoD基因家族中3个基因座位的单核苷酸多态性(single nucleotide polymorphism, SNP)。结果表明, 在徐淮白山羊群体中, myf-5基因座发现有3种基因型AA、AB和BB, 波尔山羊均为AA型。在myf-6基因座和myoD 5′侧翼区基因座, 两个山羊群体均检测到了AA和AB型个体。对山羊myf-5、myf-6基因座, myoD 5′侧翼区基因座不同基因型与两品种山羊体尺性状相关分析表明, myf-5基因座对管围和管围指数的效应差异显著(P<0.05)。myf-6基因座对徐淮白山羊体尺性状的效应均不显著(P>0.05), 而对波尔山羊的体高和管围指数效应差异显著(P<0.05)。两个山羊品种myoD 5′侧翼区不同基因型个体的体尺性状差异均不显著。     

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。     

5个山羊品种CSN1S2基因的Alw26I酶切

利用PCR-RFLP技术对西农萨能奶山羊、关中奶山羊、陕南白山羊、安哥拉山羊和波尔山羊5个山羊品种的170个个体的αs 2酪蛋白(CSN1S2)基因进行多态性分析,结果表明：扩增大小为310 bp的片段经限制性内切酶Alw26I酶切后表现多态,且5个山羊品种该基因座位均处于Hardy-Weinberg平衡状态。西农萨能奶山羊、关中奶山羊、陕南白山羊、安哥拉山羊和波尔山羊的基因杂合度/有效等位基因数/Shaanon信息熵/PIC值分别为0.1589/1.1889/0.2955/0.1463, 0.4114/1.6981/0.6017/0.5171,0.1653/1.1980/0.3046/0.1516,0.0646/1.0691/0.1463/0.0625,0.0541/1.0572/ 0.1270/ 0.0526。分析结果显示,关中奶山羊的遗传多样性最丰富,表现为高度多态;其次是西农萨能奶山羊和陕南白山羊,而安哥拉山羊和波尔山羊的遗传变异程度最低。     

Effect of cytoplast on the development of inter-subspecies nuclear transfer reconstructed goat embryo

The aim of this study was to investigate effect of cytoplast on the development competence of reconstructed embryos derived from inter-subspecies somatic cell nucleus transfer (SCNT). First, the development potency of reconstructed embryos produced by transferring Boer goat fibroblast cell nucleus of different ages into enucleated Sannen goat ova was evaluated in order to determine which age of nuclear donor is favorable for the reconstructed embryos development. Secondly, the another component of reconstructed embryos, "cytoplast," was evaluated by comparing the effect of ovum cytoplast derived from Sannen male symbol x Boer female symbol descendant on the reconstructed embryos development to that of Sannen goat ovum cytoplast. The results revealed that the development rate of the reconstructed embryos derived from 2 months old Boer goat somatic cells was the highest, their gestation rate was up to 50%, and one viable male offspring was obtained. The cytoplast derived from the crossbreeding goats improves the development competence of reconstructed embryos, which birth rate was 5.5%. The genetic identification of offspring by using PCR-SSCP analysis confirmed that these cloned kids were derived from the donor. The results above reveal that the cytoplast of Sannen goat ovum could induce the dedifferentiation of somatic cell nuclei derived from Boer goat, but the reprogramming process of these reconstructed embryos seems incomplete, probably due to some incorrect processes happened after implantation. Relatedness components of nucleus donor in cytoplast of the crossbreeding goat may be helpful to induce the dedifferentiation of somatic cell nuclei completely and improve the development competence of the reconstructed embryos.