中国大陆绒螯蟹线粒体16SrDNA序列变异与分子鉴定标记

选取了中国大陆东部6个水系合计110个绒螯蟹个体,通过DNA序列测定和PCR/RFLP分析,对线粒体16S rDNA部分片段的序列变异进行研究.结果表明,在401bp的16S rDNA片段中,合浦绒螯蟹(Eriocheir japonica hepuensis)与中华绒螯蟹(E.j.sinensis)之间存在3～4个固定的碱基替代;合浦绒螯蟹的闽江、九龙江和南流江种群之间未发现碱基变异,表现为1种单元型(C型);中华绒螯蟹长江和辽河的种群中,有1个固定的碱基替代,表现为A,B两种单元型.单元型之间的碱基变异反映了绒螯蟹地理种群之间的遗传歧异.经Dra I酶切形成的16S rDNA酶切片段差异,为2个亚种的鉴定提供了一种准确、简捷的DNA分子标记.对长江水系部分水域绒螯蟹的分子鉴定提示,长江下游至长江口以中华绒螯蟹的2个单元型为主,但已混有合浦绒螯蟹的单元型.在江苏、安徽渔场中的饲养种群分别属于单元型A型和B型.16S rDNA的PCR/RFLP差异可作为正确鉴定中华绒螯蟹和合浦绒螯蟹的分子鉴定标记;16S rDNA片段中1个固定位点的碱基替代可作为区分中华绒螯蟹两种单元型的分子鉴定标记.     

基于mtDNA COI基因序列的盐肤木种群遗传变异

基于mtDNA COI基因序列对贵州、湖北和四川的盐肤木(Rhus chinensis Mill.)种群进行遗传变异分析.研究共测得盐肤木9个种群50个个体的mtDNA COI基因长度为1.37kb的序列,在测得的序列中有3个核苷酸位点存在变异(占所测核苷酸序列的0.22%),T、C、A、G 4种核苷酸的组成分别为34.1%、21.8%、22.5%、21.6%,其中A+T含量(56.6%)高于G+C含量(43.4%).共检测到3个单倍型(HapA、HapB和HapC),HapA出现在所有种群中,是出现频率最高的单倍型,占所测序列的90%,该单倍型可能是祖先单倍型;HapB由安县的1个个体和竹山的3个个体共享,而HapC只存在于榕江的1个个体.分析表明盐肤木种群具低水平单倍型多样性(0.000 2)和核苷酸多样性(0.187 0),基于该基因序列的盐肤木种群间遗传多样性和遗传分化均较小.     

基于rDNA部分序列的亚洲小车蝗遗传多样性分析

以核糖体DNA序列中一段基因为目的片段,对内蒙古地区15个地点的75个亚洲小车蝗样本进行了DNA序列的遗传多样性分析,通过与GenBank中已知蝗虫的核糖体DNA序列的对比、剪切得到288bp的目的片段序列.分析结果表明:在目的片段序列中共检测到19个变异位点,其中包括16个置换(11个颠换和5个转换)和3个碱基插入,占碱基总数的6.60%;目的序列显示出一定的GC偏好性.15个种群的FST在0.050 6~0.402 8之间,均值为0.149 5;基因流(Nm)在0.231 2~5.933 6之间,均值为1.988 0,表明15个小车蝗种群间存在遗传分化,基因交流处于中等水平.正镶白旗小车蝗种群的核苷酸多样性指数(Pi)和平均核苷酸差异数(K)在15个种群中最大,分别为0.028 0和8.000 0,表明正镶白旗种群相对于其他14个地理种群的种内变异度最高.在所测的75条序列中共有8种单倍型,其中正镶白旗和四子王旗小车蝗独享的两个单倍型与其他单倍型差异较大,表明正镶白旗和四子王旗的部分小车蝗个体的遗传变异度较大.Mantel检验表明,15个小车蝗种群的遗传距离与地理距离无显著的相关关系.     

青海湖裸鲤的种群结构和线粒体DNA变异

对青海湖裸鲤55个个体Cytb基因全序列进行了测定和分析,探讨了种群结构和群体遗传多样性。用MEGA2.1软件分析了碱基组成和序列变异;以黄河花斑裸鲤为外群,构建了单倍型的NJ树;用Arlequin Ver.2 000程序计算了群体内遗传变异值(Fst)。结果显示,青海湖裸鲤群体没有显著的种群结构,提示青海湖裸鲤群体内存在广泛的基因交流;种群的遗传多样性较低(π=0.7828±0.0532),青海湖裸鲤种群很可能在历史上遭受过严重的“瓶颈效应”。     

梭鱼和鲻鱼线粒体16S rRNA和COⅠ基因片段的比较分析

运用PCR技术,扩增了梭鱼和鲻鱼线粒体16S rRNA和COⅠ基因片段,并比较分析了其种间的序列差异。获得16S rRNA基因的540～560 bp碱基序列,出现26个碱基的插入与缺失位点;获得COⅠ基因的602～604 bp碱基序列,出现2个插入缺失位点;16S rRNA和COⅠ基因的序列中G平均含量最低,且(A+T)含量高于(G+C)含量,与其他鱼类中的16S rRNA和COⅠ基因片段研究结果相一致。在16S rRNA基因片段中,梭鱼出现1种单倍型,鲻鱼为2种单倍型;在COⅠ基因片段中,梭鱼样品中检测到3个单倍型,鲻鱼样品中检测到5个单倍型。以Takifugu poecilonotus为外群,构建的NJ系统发育树,基于16S rRNA和COⅠ基因序列获得的NJ系统树基本一致,鲻鱼和梭鱼种内个体分别聚为一支,显示16S rRNA和COⅠ基因适合于梭鱼和鲻鱼的物种鉴定。     

基于COⅠ基因和D-loop序列的东海鳓鱼种质资源遗传变异研究

以东海区鳓鱼背部肌肉的线粒体DNA作为模板,对16个个体的CO Ⅰ基因和D-loop序列进行了扩增,通过对PCR产物进行双向测序,最终得到了691 bp的CO Ⅰ基因片段和718 bp的D-loop序列片断.用DNASP软件分析得知,16个CO Ⅰ基因序列定义了6个单倍型(在GenBank登录号:HM030765-HM030768,HM030769-HM030770),在6个单倍型中,共检测到6个变异位点,其中有5个变异位点发生在第三密码子位置上,有1个变异位点发生在第一密码子位置上.东海区鳓鱼16个个体D-loop序列共定义了14个单倍型(GenBank登录号:HM030781,HM030783-HM030792,HM030794-HM030796).除去插入/缺失位点,14个单倍型共检测到30个多态位点,主要发生在D-loop序列的两端区域;D-loop序列还检测到80个插入或缺失位点,碱基插入主要发生在340～419 bp之间的中央区域,以40 bp重复片断插入的形式进行.每个个体含有2～4个连续重复片断.对鳓鱼进行了遗传多样性分析,CO Ⅰ基因序列的单倍型多样性为0.617,核苷酸多样性指数为0.001 37,平均核苷酸差异数为0.950.D-loop序列的单倍型多样性为0.983,核苷酸多样性指数为0.009 98,平均核苷酸差异数为6.367.综合分析,东海区鳓鱼的遗传多样性并不高,有必要采取综合措施对其进行种质资源保护.     

基于线粒体基因的杂色山雀中国大陆种群遗传多样性研究

杂色山雀(P arus varius)为东亚特有鸟类、分布呈片段化.为了深入了解杂色山雀中国大陆各地理种群遗传水平现状,分析了杂色山雀8个地方种群49个样本的线粒体Cytb基因全序列(1143bp)和部分ND5基因序列(957 bp),所测样品全长共2.1 kb.结果显示:T、C、A、G碱基的平均含量分别为21.5％、37.4％、28.9％、12.2％,A+T含量(50.4％)略高于G+C含量(49.6％);发现17个变异位点,其中单变异位点为7个,简约信息位点数为10个;检测到14个单倍型,8个地方种群共享一个单倍型(Hap-1),说明这个单倍型是个较古老的单倍型,也反映了这几个群体间的分化是不显著的.5个种群的单倍型多样性为0.822 39,核苷酸多样性为0.000 82,平均核苷酸差异数为4.663 83,总体上看,杂色山雀具有较低的单倍型多样性和核苷酸多样性.五个种群的Nm值为5.282 21～17.33645,Fst值为0.002 07～0.086 47,构建的系统发生树和TCS网络图进一步分析表明杂色山雀遗传多样性较低、基因交流非常频繁、种群间的分化不显著.中性检验显示偏离中性期望值,表明种群在进化的过程中出现过群体急速扩张事件.     

野猪及杜洛克杂种猪血浆铜蓝蛋白酶多态性的研究

采用聚丙烯酰胺凝胶垂直平板电泳法对12头野猪及20头杜洛克杂种猪血浆铜蓝蛋白酶（Cp）多态性进行了研究。结果发现：①被检野猪及杜洛克二元、三元杂种猪血浆Cp均有CpAB和CpBB型两种基因型,且以CpBB为优势基因型（分别为83．33％,71．43％和53．85％）,Cp^B为优势等位基因（分别为0．9167,0．8571和0．7692）;②被检野猪Cp位点上的基因杂合度为0．1527,而杜洛克二元、三元杂种猪的分别为0．2441和0．3350。     

基于Cytb和COⅠ基因序列的蟒蛇分子鉴定

基于线粒体细胞色素b(Cyt b)和细胞色素c氧化酶亚单元Ⅰ(COⅠ)片段,分析比较了蟒蛇(Python molurus)不同地理种群间的序列差异.通过PCR扩增和序列测定,得到蟒蛇线粒体2个基因片段的总长度分别为1 200 bp(Cyt b)和1 100 bp(COⅠ),其两个基因序列中碱基G含量明显低于其他3种碱基,基因片段的A+T含量都较高.用MEGA4.0软件中的NJ法和UPGMA构建系统树.以红尾筒蛇(Cylindrophis ruffus)作为外群,对福州动物园的5个蟒蛇样品和NCBI上检索的样品进行遗传距离的测算,从遗传距离上来看:有两个群体间的遗传距离很近,一个是Fuzhou1、Fuzhou4和金门蟒蛇种群;另一个是Fuzhou2、Fuzhou3、Fuzhou5、缅甸蟒蛇种群和越南蟒蛇种群;印度蟒蛇种群Python molurus molurus(HM581978)与上述两个群体间遗传距离次之,外群与其他样品距离最远.对5个蟒蛇样品和NCBI上检索的样品物种构建的系统树显示:Cyt b与COⅠ基因所构建的进化树基本一致,Fuzhou2、Fuzhou3、Fuzhou5与缅甸蟒蛇、越南蟒蛇为一支,为东南亚种群,与福建种群Fuzhou4、Fuzhou1、金门蟒蛇种群形成姐妹支,然后与印度种群Python molurus molurus(HM581978)聚到一起,构成大支.实验结果表明福州动物园的蟒蛇是福建本地种群和东南亚种群混养的.     

伴随中国番茄黄化曲叶病毒的卫星DNAβ在本氏烟中的种群变异

采用根癌土壤杆菌介导接种和分子克隆技术,分析中国番茄黄化曲叶病毒(Tomato yellowleaf curl China virus,TYLCCNV)Y10分离物(Y10)的伴随卫星DNAβ(Y10β)在本氏烟中的种群变异.结果显示表明:本氏烟中Y10β种群所有克隆均发生不同程度的变异,在接种60 d及120 d后其种群突变频率分别为2.48×10-3和2.54×10-3,表明该卫星DNAβ在同一寄主体内的侵染时间对其种群变异水平没有明显影响;Y10β种群的碱基突变位点主要分布于DNAβ的富A(A-rich)区,在卫星共同区(SCR)内碱基突变位点最少;其碱基突变类型以A→G,G→A,T→C突变及碱基G缺失等突变类型的比例较高,并且在A-rich区内出现多个克隆在同一核苷酸位点发生相同突变的现象.     

扬子鳄种群的线粒体DNA控制区序列变异性及其对物种保护的启示

测定了来自4个不同地点(包括安徽宣城野生和饲养种群、浙江长兴的饲养种群及在美国的饲养种群)的64个扬子鳄线粒体DNA控制区部分序列.结果共检测出4个单倍型共3个变异位点,与龟类、鸟类、哺乳类及其它鳄类相比较,反央出扬子鳄线粒体区的序列变异性很低.在此基础上,对扬子鳄遗传保护提出了有关建议.     

GENETIC DIVERSITY AMONG CHINESE SIKA DEER (CERVUS NIPPON) POPULATIONS AND RELATIONSHIPS BETWEEN CHINESE AND JAPANESE SIKA DEER

SIKA DEER,CERVUS NIPPON,WAS HISTORICALLY WIDE-SPREAD THROUGHOUT NORTHEASTERN ASIA,FROM THE USSURI REGION TO VIETNAM,INCLUDING THE KOREAN PENINSULA,MAINLAND CHINA AND TAIWAN,AND THE JAPANESE ARCHI-PELAGO,AND UP TO13SUBSPECIES HAVE BEEN DESCRIBED[1].THE FOS…     

Genetic diversity among Chinese sika deer （Cerrus nippon） populations and relationships between Chinese and Japanese sika deer

Sika deer （Cervus nippon） is a cervid endemic to mainland and insular Asia and endangered. We analyzed variation in the mitochondrial DNA （mtDNA） control region for four subspecies to understand the genetic diversity, population structure and evolutionary history in China. 335 bp were sequenced and eight haplotypes were identified based on 25 variable sites among the populations. Sika deer in China showed lower genetic diversity, sug- gesting a small effective population size due to habitat fragmentation, a low number of founder individuals, or the narrow breeding program. AMOVA analysis indicated that there was significant genetic subdivision among the four populations, but no correlation between the genetic and geographic distance. PhyIogenetic analyses also revealed that Chinese sika deer may be divided into three genetic clades, but the genetic structure among Chinese populations was inconsistent with subspecies designations and present geographic distribution. Including the sequence data of Japanese sika deer, the results indicated that Chinese populations were more closely related to Southern Japanese populations than to the Northern Japanese one, and the Taiwan population was closer to populations of Northeastern China and Sichuan than to those of Southern China.     

福建近海蓝圆鲹群体遗传结构分析

测定了福建近海蓝圆鲹(Decapterus maruadsi Temminck&Schlegel)2个群体共60尾个体的线粒体DNA(mtDNA)控制区序列,探讨了闽东和闽南群体遗传结构和遗传多样性.结果显示:获得长度为834～838 bp的控制区部分序列,在所测的60个样本中,共检测到23个变异位点,定义了28个单倍型,平均单倍型多样性(h)为0.948±0.0145,核苷酸多样性(π)为0.004 99±0.002 79,核苷酸差异数(K)为4.173±2.104,提示了福建近海蓝圆鲹具有较高的遗传多样性水平.构建的单倍型邻接关系树没有明显的以地理群体为单位的家系式分支出现,单倍型网络图也未显示出单倍型和地理位置的对应关系.Tajima's D和Fu's Fs中性检验及核苷酸不配对分析暗示了蓝圆鲹闽南群体在63 000年前可能发生过扩张事件,而闽东群体符合中性理论进化,2个群体不同的历史动态可能是由于小冰河期海区间的气候差异和闽东群体的过度利用所致.分子方差分析(AMOVA)表明,遗传变异全部存在于群体内,2个群体间具有紧密的基因流和较低的遗传分化,群体问和群体内的Kimura双参数遗传距离均较小.可知:福建近海蓝圆鲹遗传多样性较高,闽东和闽南群体的遗传结构相似,不存在显著的遗传分化.因此,建议将闽南渔场和闽东渔场作为一个种质资源评估、管理和保护单元.     

Mutation in the porcine SERPINA7 gene and its association with boar fertility

The porcine SERPINA7 gene is considered as a positional candidate gene responsible for testis size for its location on X chromosome and its biologically critical role in the development of testis. A nonsynonymous polymorphism (His226Asn or C678A) in the ligand-binding domain of SERPINA7 has been identified, which alters SERPINA7’s affinity to thyroxine and is closely associated with testis size. In this study, a primer mutagenesis strategy was developed to genotype this polymorphism in Chinese indigenous pigs and some western commercial pigs. The C allele existed in all tested Chinese indigenous and wild pigs, while the A allele is specific for western commercial breeds, indicating the occurrence of the mutation is of western origin. The correlation of this polymorphism with different boar fertility traits was assessed using a White Duroc × Erhualian intercross which included 110 F₂ mature boars. The results showed that the C678A polymorphism was closely associated with testis weight and epididymis weight (P＜0.0001 and P=0.0016, respectively) with significant heavier testis weight and epididymis weight in boars carrying the A allele than boars with the C allele. A significant correlation was also observed between this polymorphism and total sperm in the ejaculate (P＜0.01) as well as semen volume (P＜0.05). No statistically significant association of the C678A polymorphism with sperm concentration and sperm motility was found.     

中国西北少数民族东乡族的Y染色体微卫星单倍型分析

运用国际法医学界推荐的"最小单倍型"共7个微卫星位点(DYS19,DYS389Ⅰ,DYS389Ⅱ,DYS390,DYS391,DYS392,DYS393)和1个高多态性微卫星位点DYS385,采用PCR结合银染显色技术,对采自临夏回族自治区东乡县的133个健康男性个体的DNA,进行了Y染色体8个微卫星的等位基因及单倍型分布状况遗传多态性分析.结果显示,东乡族保留着较高的Y-STR遗传多态性,在测出的66个等位基因中共构建了110种单倍型,单倍型多样性为98.9%,表明该系统有较强的个体识别能力.另外,我们的研究可以为东乡族这一穆斯林民族的起源提供有价值的遗传资料.     

曲腿龟甲轮虫和热带龟甲轮虫的mtDNA COI基因和rDNA ITS序列差异以及几种龟甲轮虫的系统发生关系

从芜湖市镜湖和汀棠湖中分别采得曲腿龟甲轮虫(Keratella valga)和热带龟甲轮虫(K.tropica)个体,实验室内对其进行克隆培养.运用常规的分子生物学技术,对各30个曲腿龟甲轮虫克隆和热带龟甲轮虫克隆分别进行了mtDNA COI基因和rDNA ITS的扩增、测序和序列分析.以萼花臂尾轮虫轮虫(Brachionus calyciflorus)为外群,结合Genbank上已登录的几条其它龟甲轮虫COI基因和ITS序列,以及测序所得的60个COI基因和ITS序列构建系统发生树(ML、MP、NJ和贝叶斯树).结果表明,曲腿龟甲轮虫和热带龟甲轮虫间COI基因序列差异百分比为24.9%-26.5%,平均为25.7%.曲腿龟甲轮虫个体间序列差异百分比为0-2%,热带龟甲轮虫个体间序列差异百分比为0-1.5%.两种轮虫间ITS序列差异百分比为6.1%-7.6%,平均为7.25%.曲腿龟甲轮虫个体间序列差异百分比为0-1.1%,热带龟甲轮虫个体间序列差异百分比为0-0.7%.基于COI基因序列和ITS序列构建的四个系统树均支持曲腿龟甲轮虫单倍型和热带龟甲轮虫单倍型各自成支.     

养殖模式及酸和硫化物胁迫对中华乌塘鳢血液生理生化指标的影响

【目的】比较红树林区3种不同生长环境下中华乌塘鳢(Bostrychus sinensis)的血液生理生化指标,并探究其对酸和硫化物胁迫的响应,为中华乌塘鳢红树林生态养殖提供参考。【方法】采集野生、红树林地埋管道生态养殖和池塘养殖的中华乌塘鳢成体,取血测定红细胞(RBC)含量、白细胞(WBC)含量及其组成、总蛋白(TP)含量、超氧化物歧化酶(SOD)活力、谷胱甘肽巯基转移酶(GST)活力和总抗氧化能力(T-AOC),并进行分析比较;采用不同强度的酸或硫化物胁迫中华乌塘鳢72h,每隔24h取血检测。【结果】在所测定的10项指标中,中华乌塘鳢红树林地埋管道生态养殖群体与池塘养殖群体间仅红细胞数量差异显著,而野生群体与养殖群体间有5项存在显著差异。红细胞数量差异表现为池塘养殖群体红树林地埋管道生态养殖群体野生群体。野生群体的中性粒细胞百分比显著高于养殖群体,淋巴细胞正好相反。水体pH值5.5以下或硫化物含量1 225μg/L以上,即能导致中华乌塘鳢产生急性毒理反应,总体表现为红细胞减少,白细胞增加,SOD活力降低,GST活力增强,TP含量升高,T-AOC下降,胁迫强度和持续时间与毒理反应强度呈正相关。【结论】中华乌塘鳢血液生理生化指标对红树林地埋管道生态养殖环境的响应与池塘养殖基本一致,一定浓度硫化物或酸会危害中华乌塘鳢健康状况,硫化物和低pH值不是红树林地埋管道生态养殖的限制因子,但仍是潜在的危害因子。     

Molecular phylogenetic status of Siberian roe deer （Capreolus pygargus） based on mitochondrial cytochrome b from Jeju Island in Korea

In order to elucidate the genetic diversity and taxonomic status of Siberian roe deer （Capreolus pygar- gus） from the Korean peninsula, partial sequences of the mitochondrial cytochrome b gene were analyzed. We col- lected 57 roe deer samples from Mainland and Jeju Island between July 2004 and June 2009. A total of 50 sequences （1,070 bp） were obtained, and seven haplotypes were defined. Five haplotypes were found from Mainland, and two were discovered from Jeju Island. The genetic diver- sity was lower in the Jeju Island population than in the Mainland population. In addition, both the consensus neighbor-joining tree and maximum parsimony tree showed an identical topology between the Mainland and Jeju Island clades. Based on the results, two subspecies of the Korean roe deer were confirmed. Siberian roe deer in Jeju Island showed a distinct difference in the cytochrome b gene compared with other Siberian roe deer, being aunique native species inhabiting only Jeju Island in Korea. Because Siberian roe deer in Jeju Island can be classified at subspecies level, the new subspecies in Jeju was named Capreolus pygargus jejuensis in this study. For this new subspecies, reasonable conservation and management effort is needed in the future.     

Testing reintroduction as a conservation strategy for the critically endangered Chinese alligator: Movements and home range of released captive individuals

The Chinese alligator (Alligator sinensis) is considered the most critically endangered crocodilian as a result of the near total loss of its habitat and its extremely small and fragmented wild populations. Plans for population recovery lie mostly with wetland res- toration and the reintroduction of captive-reared animals. We carried out a first-trial release of 3 adult Chinese alligators (1♂, 2♀) into a pond at the Hongxing conservation site, Xuancheng, southern Anhui Province; the animals were radio-tracked from May to October in 2003. We hypothesized that after a period of adaptation, the alligators would establish definable home ranges. Two (1♂, 1♀) of the 3 alligators were monitored for the whole of the tracking period. The male had an annual home-range size of 7.61 hm2, and the female 4.00 hm2. Water temperature and pond water level were two important factors influencing the alligators’ distributions, and daily movements. The radio-tracked alligators had overlapping home ranges, which notably included the one substantial island in the pond; that island is the only known nesting site of the local native wild alligators. Aggressive interactions between the released alligators and native wild alligators were observed during the breeding season around this island. All the three reintroduced alligators survived the winter of 2003 and were alive in the same pond in 2008. We concluded that the Hongxing conservation site provided a suitable habitat for the reintroduced alligators. However, the low water level in the pond resulting from farmland irrigation in August and September can be a substantial threat to the alligators’ survival. Therefore, regu- lations on irrigation in summer and autumn are needed to balance the water needs of the alligators and agriculture.