利用微卫星DNA标记分析7个绵羊群体遗传多样性与系统发育

摘要:实验室检测了甘肃藏羊、甘南藏羊、湖羊、青海藏羊、小尾寒羊、滩羊、岷县黑裘皮7个绵羊群体268只个体15个微卫星座位等位基因,进而开展了等位基因变异分析、杂合度分析、哈代—温伯格比率检验、遗传距离估算、系统发生树构建和遗传结构推导。结果表明：7个绵羊群体在15个微卫星座位上共发现187个等位基因。哈代—温伯格比率偏差检验中共发现43个“群体-座位”偏离了哈代—温伯格比率,其中湖羊偏离哈代-温伯格比率的“群体-座位”数最多。群体杂合度分析表明青海藏羊群体的遗传多样性较丰富,而湖羊和岷县黑裘皮羊的遗传多样性较低。遗传距离和系统发生树的分析表明,滩羊、小尾寒羊和湖羊亲缘关系较近,类群起源上享有共同的祖先,岷县黑裘皮羊与其它6个绵羊群体遗传关系较远,7个绵羊群体的遗传结构推演为三类。     

应用线粒体DNA序列探讨羚牛分类地位

为探讨羚牛分类学地位,应用聚合酶链式反应（PCR）扩增了羚牛、绵羊、山羊和斑羚（青羊）线粒体DNA（mtDNA）细胞色素b基因（Cyt b gene）,并测序,结合GenBank检索序列,对9种偶蹄类动物、1种奇蹄类动物Cyt b gene序列差异进行分析,构建了分子系统树（最优NJ树和唯一MP树）。通过本研究分析表明羚牛与羊亚科动物亲缘关系最近,将羚牛归入羊亚科较为合理。     

甘肃境内6个牦牛群体微卫星标记遗传多样性及遗传结构分析

为了研究甘肃境内牦牛(Bos grunniens)群体的遗传多样性和遗传结构,促进甘肃境内牦牛群体遗传资源的合理利用和保护,研究采用微卫星标记技术检测了甘肃境内6个牦牛群体(240头个体)15个微卫星座位的等位基因。结果共检测到了146个等位基因,其中在玛曲牦牛群体检测到的等位基因数最多(106个),天祝白牦牛群体最少(74个)。期望杂合度(He)、观察杂合度(Ho)和多态信息含量(PIC)的分析结果表明,6个牦牛群体中来自于合作市的3个牦牛群体的遗传多样性较丰富,而天祝白牦牛、肃南牦牛和天祝牦牛群体的遗传多样性相对较低。DA遗传距离和Nei氏标准遗传距离(DS)的分析结果以及DA和DS遗传距离构建的NJ系统树的分析结构均表明,玛曲牦牛、碌曲牦牛和夏河牦牛群体具有较近的亲缘关系,说明这3个群体可能具有相同的原始祖先。天祝白牦牛、肃南牦牛和天祝牦牛群体的遗传距离较近,另为一类,可能来源于相同的原始祖先。主成分分析和遗传结构推导分析的结果与系统树的分类结果一致,研究将甘肃境内的6个牦牛群体分为两个类群,分类结果与其地理分布具有一致性。     

甘肃高山细毛羊微卫星遗传多样性及亲子鉴定研究

利用多重PCR和DNA测序技术对116只甘肃高山细毛羊(Ovis aries)13个微卫星座位的多态性进行检测,并结合父母本记录,开展亲子鉴定研究,以期为绵羊种质资源评价和亲子鉴定研究提供合理有效的微卫星座位.结果表明,甘肃高山细毛羊13个微卫星座位共检测到111个等位基因,在DRB 1-INTRO2座位检测到的等位基因数最多(14个),SRCRCP5座位最少(5个).ILSTS011座位观察杂合度最高(0.888),MAF214座位最低(0.500),除MAF214座位为中度多态外,其余12个座位均为高度多态,13个微卫星座位平均观察杂合度(Ho)为0.743,期望杂合度(He)为0.727,平均多态信息含量(PIC)为0.689.表明甘肃高山细毛羊遗传多样性较丰富.通过Cervus 2.0计算,13个微卫星座位累计父权排除概率(CPE)达到了0.999 94,11个具有高度多态的座位(除MAF214和OARJMP29外)CPE达到0.999 85,12个座位达到0.999 91,其中MAF214座位(中度多态)对CPE的影响较小,DRB1-INTRO2座位(高度多态)对CPE的影响较大,对亲子鉴定的贡献较大.本研究所分析的13个微卫星座位能够有效的提高亲子鉴定要求的精确度,可使实验操作更简便,成本降低,更适用于生产实际.     

利用mtDNA D-loop区研究中国10个绵羊品种的遗传多样性与起源

中国家绵羊的起源目前还不清楚,存在双起源说和三起源说,争论较大。为了进一步研究绵羊的起源和遗传多样性,根据已知家养绵羊(Ovis aries)线粒体基因组序列,利用 Primier premier5.0 设计引物,对我国 10 个家养绵羊品种 133 只个体的 mtDNA D-loop 区进行测序并利用 Laser Gene、MEGA4、Clustalx1.83 等软件对结果进行分析 ,结果标明,整个 D-loop 区为 1 106~1 182 bp,共检测到 103 种单倍型,155 个多态位点,表明我国家养绵羊品种遗传多样性丰富。通过构建 NJ 网络进化树,得出中国家养绵羊分为两个分支,羱羊(O. ammon)(AJ238300)与盘羊(O. vignei)(AY091490)聚为一类,而摩佛伦羊(O.musmon)(AY091487)与一个分支聚为另一类。说明,摩佛伦羊对中国家养绵羊起源与进化的贡献更大。本研究所测定的中国家养 10 个品种分为 A、B 两大支系表明家养绵羊至少有两大母系起源,且单倍型以亚洲 A 型为主。本研究从物种水平上评价了我国家养绵羊品种的遗传多样性,指出了中国家养绵羊分为两个分支,为确立中国家养绵羊种群关系和保护种质资源提供了理论依据。     

藏绵羊DQA座位基因的适应性进化研究

主要组织相容性复合体(major histocompatibility complex,MHC)是脊椎动物重要的功能基因家族之一,其所编码的MHC分子具有维持机体世代适应环境变化的能力和功能,是研究动物适应性进化研究的最佳遗传标记.本研究选择青藏高原的主要畜种资源——藏绵羊(Ovis aries)为研究对象,利用聚合酶链式反应-单链构象多态(polymerase chain reaction-single strand conformation polymorphism,PCR-SSCP)技术对甘肃(欧拉,乔科和甘加型)和青海(青海欧拉型和青海高原型)的5个藏绵羊生态群体的DQA座位基因遗传特征进行研究,以期揭示其与藏绵羊高原适应性进化的机制;结果在藏绵羊DQA座位的2个基因中发现了33个等位基因和125个核苷酸变异位点,表明5个生态群体藏绵羊的DQA座位基因均具有丰富的多态性;分析证明平衡选择是维持藏绵羊DQA座位基因多态性的主要机制之一,甘肃和青海5个藏绵羊生态群体DQA座位基因区域之间的变化(among groups)小于同一区域不同群体间的变化(among population with groups),进一步说明藏绵羊DQA座位基因发生了正选择作用和适应性进化.研究结果将为藏绵羊的遗传改良和种质创新提供基础数据.     

鸭微卫星标记的筛选及其遗传多样性分析

采用磁珠富集法从AFLP片段中筛选微卫星标记,并对5个福建地方鸭品种的遗传多样性进行检测。基因组DNA用EcoRⅠ/MseⅠ内切酶酶切的同时与接头连接,酶切连接产物与用生物素标记的探针杂交,应用磁珠捕获100~2000bp含有微卫星序列的DNA片段并通过pGEM-T载体转化到大肠杆菌DH5a感受态细胞中,构建富集微卫星序列的基因组文库。随机挑选46个阳性克隆,测序获得14条含有微卫星的DNA序列并递交到GenBank（登录号：FJ599499~FJ599512）。设计合成14对微卫星引物,通过PCR优化从中选择5对引物用于5个福建地方鸭品种的遗传多样性分析。结果显示,5对微卫星引物共检测到31个等位基因,各微卫星基因座的有效等位基因数为1.969 7~2.834 4,多态信息含量和杂合度的平均值分别为0.5133和0.7480,遗传多样性丰富,说明磁珠富集法适合用于鸭微卫星标记的分离与筛选,筛选得到的5个微卫星位点可作为有效的遗传标记用于福建省地方鸭品种遗传多样性的研究。     

江苏省地方鸡品种线粒体DNA遗传多样性分析

江苏省拥有众多优良的地方鸡(Gallus gallus domesticus)遗传资源。这些地方鸡品种经过长期选育,具有肉质好、抗病力强和适应性好等优点,是进行杂交改良的优良素材。为研究江苏省地方鸡遗传资源的群体结构和遗传多样性,本研究对黑狼山、白狼山、鹿苑、溧阳和太湖等5个江苏省地方鸡品种149个个体的线粒体DNA(mitochondrial DNA, mtDNA)控制区(displacement loop, D-loop)全序列进行了PCR扩增和测序。结果表明,5个地方品种mtDNA D-loop区全长为1 231或1 232 bp,共计检测到核苷酸变异位点33个,定义了19个单倍型。总体单倍型多样性和核苷酸多样性分别为0.862±0.017和0.005 91±0.00135。中介网络图分析显示,江苏省地方品种含有A、B、C、D和E共5个分支(单倍型群),其中A和B分支为优势分支。分子方差(analysis of molecular variance, AMOVA)分析表明,群体内变异占66.5%,群体间变异占33.5%,5个群体间的固定指数F统计量(F-statistics, Fst)为0.334 97(P0.01)。上述结果揭示,江苏地方品种具有相对较低的遗传多样性,可能有5个母系来源,品种间分化不明显,少数群体可能受到了外来高产系的侵入。该研究结果为江苏地方鸡遗传资源的保护、开发及利用提供了遗传背景信息。     

中国西南地区主要地方绵羊Cytb基因遗传多样性及系统进化的研究

目前对我国西南地区绵羊(Ovis areis)群体研究的报道较少,为探究我国西南地区绵羊遗传多样性、群体结构和演进历史,本研究利用Oligo 6.0软件设计引物对8个地方绵羊群体和2个引入品种群体183只线粒体细胞色素b (cytochrome,Cytb)基因全长进行测序,并利用MEGA 5.2、DnaSP 5、Phylip 3.695和Network 4.0等软件对其群体进行分析.其研究结果表明,所发现的153个多态位点共确定了62种单倍型;10个绵羊群体的平均单倍型多样度为0.792;平均核苷酸多样度为0.002 43;平均核苷酸差异数为5.054.10个群体的核苷酸多样度(nucleotide diversity,Pi)差异较大,变化范围为0.001 28～0.006 55,核苷酸多样度最大和最小的群体分别为以贵德黑裘皮羊(0.006 55)和昭通绵羊(0.001 28);单倍型多样度(haplotype diversity,Hd)最高和最低的群体分别是西藏羊(0.924)和腾冲绵羊(0.582),结果说明,云南3个绵羊群体的遗传多样性水平较低,其他几个绵羊群体的遗传多样性水平较高.分子方差分析(analysis ofmolecular variance,AMOVA)分析结果说明,10个绵羊群体间的遗传变异为13％,而87％为群体内的遗传变异,两者比例悬殊,说明所选10个绵羊群体间的变异绝大部分来自群体内.通过构建系统进化树和网络系统分析显示本研究中绵羊群体至少可以分为3个支系,但是西南群体以支系A和支系B为主.本研究结果为制定我国西南地区绵羊保种方案以及合理利用等提供理论参考.     

鲮微卫星DNA分子标记的筛选与遗传多样性分析

采用磁珠富集法对已建立的生长较快的西江段野生浅色鲮群体的极大、极小群体各31尾基因组DNA进行了微卫星序列的筛选,128个阳性克隆成功测序93个,其中80个为的微卫星序列,微卫星序列完美型占85％,非完美型占6．25％,混合型占8．75％。利用微卫星序列合成了23对微卫星引物,扩增结果表明,等位基因数为1～10个,扩增片段大小为92～283bp,其中14对引物扩增条带清晰,为中度或高度多态位点,极大、极小群体的平均有效等位基因数和平均多态信息含量分别为3．0784、3．2079和0．5675、0．5974,反映出2个群体遗传多样性均较丰富;高度多态性引物4的位点b,片段大小为119bp,在极大群体中出现频率为61．3％,在极小群体占22．6％,出现频率极大群体高于极小群体近3倍,初步可作为候选差异性分子标记。     

Genetic conservation of South African wattled cranes

The wattled crane (Grus carunculatus), a species highly dependent on wetlands, is the largest and rarest of the six African crane species. The once vast range of the wattled crane now consists of only three disjunct populations. The South African population has shown dramatic declines and supplementation of this population using eggs from south-central Africa has been proposed. The objectives of this study were to compare levels of genetic variation in South African and south-central African populations to determine if such supplementation is needed, and if so, whether the south-central African populations represent a genetically similar source for supplementation. We surveyed genetic variation in samples from South Africa, Zimbabwe, and Botswana using 12 microsatellite DNA loci and a 400-bp fragment of the mitochondrial D-loop. Samples from Zimbabwe and Botswana were deemed genetically similar and pooled to increase sample size. Subsequent analyses indicate that the pooled south-central and South African populations show differentiation in microsatellite DNA genotypes, as well as mitochondrial DNA. As the results from both genetic markers indicate genetic isolation, these populations should be managed as separate entities. As no indication was seen from either microsatellite or mtDNA data that significant loss of genetic diversity has occurred within South African wattled cranes, supplementation from outside populations may not be necessary at this time.     

中国荷斯坦牛和鲁西黄牛mtDNA D-loop序列多态性分析

为了从母系遗传角度深入阐明中国荷斯坦牛和鲁西黄牛的群体遗传多样性以及起源进化,本研究采用PCR测序法测定了9头中国荷斯坦牛和11头鲁西黄牛的线粒体DNAD-loop区的部分序列,并经剪切后进行生物信息学软件分析。结果表明,20个个体D-loop区共711bp,共检测到19种单倍型和50个多态位点。核苷酸多样性（Pi）为0.02133±0.00454,单倍型多样性（Hd）为0.994±0.019,平均核苷酸差异数（k）为15.14620。构建的NJ网络进化树共分为两大支系,其中部分鲁西黄牛与瘤牛聚为一支,而中国荷斯坦牛和部分鲁西黄牛与普通黄牛聚为一支。说明中国荷斯坦牛和鲁西黄牛群体遗传多样性均较高;鲁西黄牛同时含有瘤牛和普通黄牛的血统,而中国荷斯坦牛只含有普通黄牛的血统。     

鲤鱼品系的部分线粒体序列的遗传变异

运用PCR测序法,以9个我国常见鲤鱼（Cyprinus carpio）养殖品种和2个野生群体为研究材料,分析线粒体DNA的16S rRNA、Cyt b和D-loop序列片段,用于分析的序列共有1 457 个位点,其中变异位点32个,简约信息位点21个,共有单倍型16个。分别利用以上3个基因片段以及合并后的序列, 构建NJ和MP进化树。由不同方法获得相似的进化树,由不同基因片段得到的进化树均为两支,其中贝尔湖野鲤（BE）单独成支,其它群体聚为1支,只是由D-loop得到的进化关系更为详细,而由合并后的序列构建的进化树也与利用不同基因序列所得到的进化树并不矛盾。各品系间的分化时间约为3.03×104～1.21×105年前。根据序列的特征,16S rRNA的1个变异位点、Cyt b基因的4个变异位点和D-loop的14个变异位点可以作为鉴定不同的养殖品系和野生群体的SNP,证明mtDNA序列分析可以应用于品系的鉴定。     

绵羊皮肤源EST-SSR标记与羊毛性状的关联性分析

为了筛选与绵羊(Ovis aries)毛性状基因座连锁更强的分子标记,本研究选择9个多态性丰富的绵羊皮肤源EST-SSR位点,检测了德国美利奴羊与中国美利奴羊级进杂交F2代群体的遗传多态性,并分析了标记与毛性状间的关联性.分析结果显示,所选的9个EST-SSR位点在供试群体中共检测到39个等位基因,多态性丰富,其中的6个位点基因型间存在显著差异;其中,与自然长度关联的位点有5个,与伸直长度关联的位点有4个,与纤维直径关联的位点有3个.6个EST-SSR位点基因型间存在显著差异;关联性标记对羊毛性状相关效应的F检验结果表明:33176918位点和33176988位点分别对羊毛自然长度、纤维平均直径具有显著的遗传效应(P＜0.05).研究结果提示,与羊毛性状相关的6个位点中,33176918号位点对羊毛的自然长度有较大的标记效应,该位点的CD基因型是羊毛自然长度的有利基因型;33176988号位点对羊毛的纤维直径有显著地标记效应,该位点的BC基因型是纤维直径的有利基因型.这两个位点可能与控制毛形状的基因座紧密连锁.     

Genetic diversity of a newly established population of golden eagles on the Channel Islands,California

Gene flow can have profound effects on the genetic diversity of a founding population depending on the number and relationship among colonizers and the duration of the colonization event. Here we used data from nuclear microsatellite and mitochondrial DNA control region loci to assess genetic diversity in golden eagles of the recently colonized Channel Islands, California. Genetic diversity in the Channel Island population was low, similar to signatures observed for other recent colonizing island populations. Differences in levels of genetic diversity and structure observed between mainland California and the islands suggests that few individuals were involved in the initial founding event, and may have comprised a family group. The spatial genetic structure observed between Channel Island and mainland California golden eagle populations across marker types, and genetic signature of population decline observed for the Channel Island population, suggest a single or relatively quick colonization event. Polarity in gene flow estimates based on mtDNA confirm an initial colonization of the Channel Islands by mainland golden eagles, but estimates from microsatellite data suggest that golden eagles on the islands were dispersing more recently to the mainland, possibly after reaching the carrying capacity of the island system. These results illustrate the strength of founding events on the genetic diversity of a population, and confirm that changes to genetic diversity can occur within just a few generations.     

西藏牦牛mtDNA 16S rRNA基因的克隆及其遗传多样性分析

本研究首次通过克隆、测序获得了11个西藏牦牛类群共111头个体的mtDNA 16S rRNA基因全序列,并分析了西藏牦牛的遗传多样性、分类关系、起源和分化,为进一步保护和合理利用西藏牦牛遗传资源以及探讨牦牛类群的划分提供分子依据。结果表明：西藏牦牛16S rRNA基因全序列长度为1571 bp或1570 bp（LWQ4）;G＋C平均含量37.8%,具有明显的碱基偏倚性;平均核苷酸多样性（Pi）为0.00291,平均单倍型多样性（Hd）为0.8501±0.0009,111个样本共发现48种单倍型并聚为2簇,表明西藏牦牛具有较高的遗传多样性并存在2个母系起源;Kimura双参数遗传距离范围为0.00098-0.00694,11个西藏牦牛类群划分为2大类：嘉黎牦牛、巴青牦牛、丁青牦牛、工布江达牦牛、帕里牦牛、斯布牦牛、康布牦牛、桑桑牦牛、江达牦牛、桑日牦牛为一类,类乌齐牦牛单独为一类。本研究发现类乌齐牦牛具有较丰富的遗传多样性,并且发现了一个序列特异个体LWQ4,需要对其进行更深入的研究。牛种间的聚类结果显示,西藏牦牛与美洲野牛的亲缘关系最近,与普通牛、水牛的亲缘关系相对较远,本研究支持将牦牛从牛属（Bos taurus）中分离出来,作为一个独立的牦牛属（Poephagus）的观点。     

Genetic differences between the two remaining wild populations of the endangered Indian rhinoceros (Rhinoceros unicornis)

The management of rare and endangered species in the wild and in captivity requires an understanding of the characterization of the genetic units within each species and their relationships to each other. The Indian rhinoceros (Rhinoceros unicornis) is an endangered species with a current population size of c. 2800 individuals. We analyzed 26 individuals of known origin kept in captivity and 21 wild ranging individuals of the two remnant large wild populations in Assam (India) and Nepal employing mitochondrial and microsatellite markers to determine whether the two geographically isolated populations show distinct patterns of genetic diversity, and whether the genetic diversity of the populations is influenced by past demographic bottlenecks. We identified 10 different mitochondrial D-loop haplotypes, of which 4 were specific to the Assam population (10 sequences examined) and 6 specific to the Nepal population (19 sequences). Similarly, the microsatellite analysis demonstrated a strong genetic differentiation between the Assam and Nepal populations and allowed to assign each individual to its origin with high confidence. Furthermore, our analyses revealed the occurrence of a bottleneck in the Assam population long before the reported bottleneck in 1908, and it revealed that the Nepal population is a recent (probably post-glacial) colonization. In summary, the extent of genetic divergence between the two remnant R. unicornis populations suggests separate conservation programs (even for captive individuals) as long as the persistence of the entire species is not severely threatened. The microsatellite markers can also be used to determine the origin of confiscated material such as horns.     

Current genetic isolation and fragmentation contrasts with historical connectivity in an alpine lizard (Cyclodomorphus praealtus) threatened by climate change

Assessing levels of genetic diversity, connectivity and historical demography for threatened species provides important information for conservation management. We used a combination of the mitochondrial ND4 gene and seven microsatellite markers to examine both historical and recent population genetic structure and demography of the threatened alpine she-oak skink, Cyclodomorphus praealtus. This species is restricted to the “sky islands” of the Australian alpine region. Based on mtDNA, the New South Wales and Victorian regions are reciprocally monophyletic and highly divergent, with among population variation of 0.9 and net sequence divergence of 4.28%, which suggests that they should be considered separate Evolutionary Significant Units for management purposes. The mtDNA data also indicate historical connectivity between the three Victorian populations. However, a model-based clustering analysis of microsatellite genotypes identified strong population structure in Victoria, with three distinct populations that have no current inter-population gene flow. This suggests that the Victorian populations are effectively isolated from each other, and is indicative of very low dispersal capacity and a high degree of habitat specialisation. This is reinforced by the substantially lower genetic diversity within the lowest elevation population compared to the other higher elevation populations. We found no genetic signature of major changes in effective population size. These data provide a baseline for assessing future impacts of climate change on the genetic structure of this alpine endemic species.