中华倒刺鲃微卫星标记筛选及特征分析

以乌江流域中华倒刺鲃(Spinibarbus sinensis)为材料,构建了中华倒刺鲃微卫星富集文库。以乌江彭水电站所采集的32个个体的基因组DNA为模板,进行PCR扩增,结果筛选出8个多态性微卫星位点,其等位基因数4~10,多态信息含量0.6072~0.8515;观测杂合度0.5625~1.0000,期望杂合度0.6791~0.8795,Hardy中华倒刺鲃微卫星标记筛选及特征分析Weinberg平衡偏离指数-0.2009~0.3805;卡方检验结果表明,3个位点不同程度地偏离了Hardy中华倒刺鲃微卫星标记筛选及特征分析Weinberg平衡(P<0.05);各位点两两之间不存在显著的连锁不平衡现象。     

中间球海胆野生和养殖群体遗传结构的微卫星分析

利用28对微卫星DNA分子标记对中间球海胆的1个野生和1个养殖群体进行了遗传多样性分析。结果表明：在28个基因座中，共检测到91个等位基因，每个基因座位检测到的等位基因数为2～6个，2个群体的平均等位基因数均为3.071 4，平均有效等位基因数分别为2.231 9、2.227 1，平均观察杂合度分别为0.523 4、0.536 5，平均期望杂合度为0.486 8、0.499 3，平均多态信息含量为0.447 7、0.439 6，群体间的多态性差异不显著。2个群体间的遗传相似系数为0.758 7，遗传距离为0.276 2。经HardyWeinberg平衡的卡方检验，有50％的位点显著偏离HardyWeinberg平衡。通过F检验发现，两个群体均处于不同程度的杂合子过剩状态。群体间发生分化程度很弱，遗传变异主要来自群体内个体之间。     

斑节对虾养殖群体遗传多样性的同工酶和RAPD分析

采用聚丙烯酰胺凝胶电泳（PAGE）和RAPD方法对厦门养殖斑节对虾（Penaeus monodon Fabricius）群体的遗传多样性进行分析。9种同工酶共检测到21个座位，其中多态座位13个，多态座位比例为61．90％，预期杂合度0．151，观察杂合度0．120，Hardy—Weinberg遗传偏离指数（d）为-0．208，存在杂合子缺失。经x^2拟合度检验，多数座位偏离Hardy—Weinberg平衡，表明群体未达到随机交配。14个10bp引物共获得了83个标记，单个引物获得的标记数为2～11个，平均每个引物扩增出5．93个座位，其中多态标记数68个，多态位点比例为81．93％，杂合度为0．246，基因多样性为0．260，Shannon’S信息指数为0．397。两种方法均表明该斑节对虾养殖群体的遗传多样性水平较高，但可能已有近交衰退发生。     

倒刺鲃属3种鱼类种间杂交的初步研究

采用人工催产和干法授精技术,进行了以中华倒刺鲃、倒刺鲃为母本与其父本及黑脊倒刺鲃雄鱼的种间杂交实验,获得4个杂交组合和2个自交组合,在(27±0.5)℃温度条件下,对其F1卵膜径、受精率、孵化率、孵化时间、畸形率、子代早期形态特征与成活率进行了比较,同时,观察了胚胎发育及其异常现象,结果表明:以中华倒刺鲃为母本的F1卵膜径差异不显著;以倒刺鲃为母本的F1卵膜径,在多细胞期时差异不显著,在耳石期时,倒刺鲃♀×中华倒刺鲃♂F1卵膜径显著大于母本(P<0.05)。杂种F1的受精率、孵化时间与母本差异不显著,畸形率显著高于母本(P<0.05)。中华倒刺鲃杂种F1的孵化率较高,与母本差异不显著(P<0.05)。倒刺鲃♀×中华倒刺鲃♂F1的孵化率与母本之间差异不显著,倒刺鲃♀×黑脊倒刺鲃♂F1的孵化率显著低于母本(P<0.05)。中华倒刺鲃杂种F1仔鱼早期发育阶段存在死亡高峰期,成活率显著低于母本(P<0.05)。中华倒刺鲃种间杂交和倒刺鲃♀×黑脊倒刺鲃♂杂种F1既具有父本特征,同时又具有母本特征,初步证明倒刺鲃属种间杂交是可行的。     

三角帆蚌微卫星位点筛选及多态性分析

采用磁珠富集法,以生物素标记的(CA)10寡核苷酸为探针,构建了三角帆蚌(Hyriopsis cumingii)基因组微卫星富集文库。根据微卫星位点的侧翼序列设计引物,随机挑选扩增出与预期大小相符的32对引物,引物荧光标记后对24个三角帆蚌个体分别进行PCR扩增,共筛选出20对多态性较好的引物。结果表明,20个微卫星位点的等位基因数为5～20,有效等位基因数2.321 3～13.260 3。观察杂合度和期望杂合度分别为0.318 2～1.000 0和0.582 5～0.946 1;PIC值0.547 2～0.919 9;其中14个位点符合Hardy-Weinberg平衡(P>0.05)。本研究筛选的20个微卫星标记可作为三角帆蚌遗传多样性、种群遗传结构等研究的理想分子标记。     

秦皇岛海域野生牙鲆群体遗传多样性分析

用18对多态性微卫星标记对采自秦皇岛海域的90尾野生牙鲆(Paralichthys olivaceus)进行遗传分析。18个微卫星位点共检测出161个等位基因;各位点等位基因数为7~11个,平均为8.9;有效等位基因为3.7~8.0,平均有效等位基因为5.9;各个位点多态信息含量为0.69~0.86,平均值为0.80;Shannon多样性指数平均值为1.9;观测杂合度(Ho)值为0.33~0.87,平均值为0.64;期望杂合度为0.74~0.89,平均期望杂合度(He)为0.84。χ2检验表明,18个位点中有9个的等位基因分布偏离了哈迪温伯格平衡(P<0.05)。研究结果表明,秦皇岛海域野生牙鲆群体遗传信息含量丰富、等位基因分布均匀,遗传多样性较高,但是,存在Hardy-Weinberg不平衡现象。     

黑脊倒刺鲃的人工繁育技术

<正>倒刺鲃属鱼类属于鲤科鲃亚科,属内有5种,分别为黑脊倒刺鲃、中华倒刺鲃、倒刺鲃、云南倒刺鲃、多鳞倒刺鲃,其中黑脊倒刺鲃也叫光倒刺鲃,在福建俗称光鱼、粗鳞。此属鱼类易给人的错觉:被认为是草鱼。目前,有关倒刺鲃属鱼类的研究报道主要是前3种(黑脊倒刺鲃、中华倒刺鲃、倒刺鲃),分布于我国南方乃至西南地区诸水系。3个种各有特点:中华倒刺鲃生长速度中等,绝对怀卵量大,雌鱼性成熟年龄4龄~5龄;倒刺     

倒刺鲃属三个种之间种间杂交的初步研究

采用人工催产和干法授精技术,进行了以中华倒刺鲃、倒刺鲃为母本与其父本及黑脊倒刺鲃雄鱼的种间杂交实验,获得4个杂交组合和2个自交组合,在(27±0.5)℃温度条件下,对其F₁卵膜径、受精率、孵化率、孵化时间、畸形率、子代早期形态特征与成活率进行了比较,同时,观察了胚胎发育及其异常现象,结果表明:以中华倒刺鲃为母本的F₁卵膜径差异不显著;以倒刺鲃为母本的F₁卵膜径,在多细胞期时差异不显著,在耳石期时,倒刺鲃♀×中华倒刺鲃♂F₁卵膜径显著大于母本(P<0.05)。杂种F₁的受精率、孵化时间与母本差异不显著,畸形率显著高于母本(P<0.05)。中华倒刺鲃杂种F₁的孵化率较高,与母本差异不显著(P<0.05)。倒刺鲃♀×中华倒刺鲃♂F₁的孵化率与母本之间差异不显著,倒刺鲃♀×黑脊倒刺鲃♂F₁的孵化率显著低于母本(P<0.05)。中华倒刺鲃杂种F₁仔鱼早期发育阶段存在死亡高峰期,成活率显著低于母本(P<0.05)。中华倒刺鲃种间杂交和倒刺鲃♀×黑脊倒刺鲃♂杂种F₁既具有父本特征,同时又具有母本特征,初步证明倒刺鲃属种间杂交是可行的。     

乌江思林水电站库区鱼类增殖放流效果初步研究

为探究乌江思林水电站鱼类增殖放流效果,2013年和2014年对思林(沙沱)鱼类增殖放流站放流的中华倒刺鲃(Barbodes sinensis)和白甲鱼(Varicorhinus simus)部分个体进行了荧光标记,共标记放流1.98万尾中华倒刺鲃和4.56万尾白甲鱼,并在2014年与2015年进行了2个周期的回捕调查。结果显示,回捕调查捕获鱼类12种,共计2687尾;其中,中华倒刺鲃和白甲鱼的渔获物占比分别为1.60%和0.71%,未见荧光标记个体。通过走访调查,收集到标记中华倒刺鲃7尾(2014年6尾,2015年1尾)、白甲鱼7尾(2014年4尾,2015年3尾),回捕率分别为0.013%~0.049%和0.009%~0.030%,回捕地点共7个,距离放流地点226~24 105 m。研究表明,思林(沙沱)鱼类增殖放流站的增殖放流活动可对中华倒刺鲃、白甲鱼等起到一定增殖效果。为取得更好的增殖放流效果,建议调整放流方法、放流地点、加强渔政管理和宣传教育。     

吉富品系尼罗罗非鱼抗病性状分子标记筛选及遗传多样性分析

文章采用微卫星标记技术,对吉富品系尼罗罗非鱼(GIFT strain Oreochromis niloticus)抗病群体和易感病群体进行了遗传差异分析。结果显示:在30个微卫星位点中20个能扩增出多态性高、重复性好和扩增条带清晰;抗病群体和易感病群体的平均等位基因数分别为2.5和2.4个;观测杂合度和期望杂合度分别为0.476和0.453,0.44和0.457;多态信息含量PIC分别为0.359和0.379;UNH845和UNH189位点在抗病群体中偏离Hardy-Weinberg平衡(P0.05),GM180位点在易感病群体偏离Hardy-Weinberg平衡,两个群体间的遗传距离和遗传分化指数0.055和0.054。此外GM251和GM462位点的差异等位基因片段(135bp和198bp)与抗病性状存在一定的连锁关系。以上结果表明,吉富品系尼罗罗非鱼抗病群体与易感病群体之间的遗传多样性差异不明显,筛选出2个与抗病性状相关的微卫星标记,为开展吉富品系尼罗罗非鱼抗病品种分子标记辅助选育奠定了基础。     

基于AFLP分析的乌江四川裂腹鱼种群遗传结构及多样性研究

本研究采用扩增片段长度多态性(AFLP)分子标记技术,以筛选出的7对引物,对采自贵州乌江的30尾四川裂腹鱼(Schizothorax kolzovi)的遗传结构和多样性进行分析。结果显示,用7对引物获得的扩增片段数为5~73,扩增片段长度范围为69~500 bp,共检出扩增位点856个。其中,多态位点796个,占扩增总位点数的92.99%。该种群含30个基因型,其观测等位基因数(Na)、有效等位基因数(Ne)、Nei多样性指数(H)、Shannon信息指数(I)平均值分别为1.933 6、1.338 8、0.212 2和0.338 7。个体之间遗传距离为0.169 1~0.531 9,平均为0.306 8。根据遗传距离,采用UPGMA法和NJ法得到了拓扑结构相似的30个个体的系统树,该系统树由2个分支组成。其中,个体1为一个分支,而其余29个个体聚为另一个分支。乌江四川裂腹鱼种群具有较丰富的遗传多样性。然而,受水电站建坝、河流污染和酷渔滥捕的影响,该种群分布区已缩小,种群数量减少,处于生态受威胁状态,需要开展对乌江四川裂腹鱼种群及种质资源的保护。     

云斑尖塘鳢微卫星分子标记的筛选与检测

用磁珠富集法分离云斑尖塘鳢的微卫星序列,由所获得的1032个克隆中筛选出146个阳性克隆,经测序81个含有微卫星序列,52个为完美型,27个为非完美型,2个为复合型,其中43个微卫星序列重复次数在10以上。所获得的云斑尖塘鳢微卫星序列中除探针中使用的CA重复单元和GA重复单元外,还有TAC等其他类型的重复单元。设计合成38对微卫星引物,其中29对引物可稳定扩增出条带,使用这些引物对云斑尖塘鳢48个个体进行检测显示:观测杂合度平均值为0.63,期望杂合度平均值为0.43。29对引物中1对引物表现为单态,7对表现为高度多态,14对表现为中度多态,7对表现为低度多态,多态性较为丰富,说明本研究开发的绝大部分微卫星分子标记较为理想。     

Microsatellite–centromere mapping in walking catfish Clarias macrocephalus (Günther, 1864) using gynogenetic diploids

Thirty new microsatellite loci were isolated from a microsatellite‐enriched genomic library of walking catfish Clarias macrocephalus. The CA motif was the most abundant, although several other motifs were also isolated. Two gynogenetic diploid families, each consisting of a female and 50 offspring, were genotyped at 56 microsatellite loci, including 30 loci developed in the present study and 26 loci reported in the previous study. Overall, 33 anonymous microsatellites derived from genomic library and 11 EST‐linked microsatellites were mapped to their centromeres. High levels of chiasma interference were apparent in the walking catfish genome as indicated by an average frequency of second‐division segregation (y) of 0.643 ± 0.248. Twenty‐six loci (59%) showed a high microsatellite–centromere recombination, with a frequency >0.67, and three loci had recombination frequencies >0.9. This study demonstrated that gene–centromere mapping provided a rapid method for the expansion of the initial linkage map of walking catfish.     

鲇细胞 b基因序列变异及遗传多样性分析

从４个种群的鲇（Ｓｉｌｕｒｕｓａｓｏｔｕｓ）线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ，ｍｔＤＮＡ）扩增出约５００ｂｐ的细胞色素ｂ基因，经ＣｌｕｓｔａｌＸ同源排序后得４００ｂｐ序列。用ＰｏｐＧｅｎ３２软件计算遗传距离和遗传一致度。１７个个体间的遗传 距离的变化范围为０～０．０２１１，遗传一致度的变化范围为１～０．９７９１。结果显示，在长江上游的支流中，雅砻江种群在该基因片段上基本不存在变异，在被分析的４个个体中，其细胞色素ｂ基因的序列完全一致；岷江种群和乌江种群在该基因片段上有一定的变异。沅江上游的贘阳河种群在该基因片段上基本不存在变异，在被分析的５个个体中，其细胞色素ｂ基因的序列完全一致。但各个种群间在该基因片段上差异较大。在４个种群的鲇Ｃｙｔｂ基因比较分析的基础上，构建了遗传关系聚类图，雅砻江、岷江、乌江、贘阳河４个种群各聚为一支后，雅砻江和岷江２个种群再聚为一支，乌江和贘阳河２个种群再聚为另一支。     

中华绒螯蟹37个多态性微卫星标记在亲子遗传中的分离方式分析

为了评估基因组扫描方法获得的中华绒螯蟹微卫星标记的遗传方式,随机选择60个候选三核苷酸微卫星标记,首先利用中华绒螯蟹F1家系双亲及其6个F1子代共8个样品进行PCR扩增验证和多态性检测,随后对多态性标记位点在80子代个体中的亲子遗传分离类型及连锁关系进行了分析。结果显示,42个（70.00%）位点得到清晰扩增产物,其中有5个单态微卫星位点和37个多态微卫星位点。多态位点中23个位点子代基因基因型为1：1：1：1分离类型,11个位点属1：1分离类型,余下3个位点为1：2：1分离类型。37个多态位点中35个位点（94.59%）的分离符合孟德尔分离比（P>0.05）,scaffold430598_213690和scaffold21303_16865位点显著偏离1：1：1：1分离比。35个分离比符合孟德尔分离比的位点中,scaffold240262_150253、scaffold216209_138892、scaffold293154_172768三个标记发生连锁关系,scaffold285640_169721和scaffold427534_212914发生连锁关系,scaffold507500_231891和scaffold92860_68250标记发生连锁关系。以上结果表明开发的候选微卫星标记适用于中华绒螯蟹亲子鉴定和遗传图谱构建。     

Isolation of microsatellite markers by in silico screening implicated for genetic linkage mapping in Japanese pufferfish Takifugu rubripes

ABSTRACT:   The search for dinucleotide repeat microsatellites within scaffolds 1–25 of genome database JGI Fugu v3.0 for the pufferfish Takifugu rubripes revealed that 80% of microsatellite loci consisted of five to 13-fold repeats with locus-specific differences in density. Eleven out of 15 microsatellite loci isolated from the database with which genotyping using wild pufferfish was successfully performed showed polymorphism; that is, the means of the number of alleles and expected and observed heterozygosities at these 11 loci were 21.8, 0.915 and 0.829, respectively. It was confirmed that eight out of the 11 polymorphic loci were inherited through the Mendelian law and one pair of microsatellite loci derived from the same scaffold was linked. These results demonstrated that these loci are useful for constructing a linkage map in the pufferfish as DNA markers.     

Genetic diversity in a Tasmanian hatchery population of Atlantic salmon (Salmo salar L.) compared with its Canadian progenitor population

The level of genetic diversity in a cultured Atlantic salmon (Salmo salar) population from Tasmania, Australia was examined at 11 microsatellite loci and compared with that in its progenitor population from the River Philip in Nova Scotia, Canada. The reference progenitor population consisted of archived scales collected from wild River Philip salmon in 1971 and 1972, not long after salmon from this river were imported into Australia in the mid‐1960s. The Tasmanian hatchery stock had a significant reduction in the mean number of alleles (31–43%) and mean allelic richness (28–39%) across all microsatellite loci compared with the wild Canadian population. Mean heterozygosity levels remained unchanged. Estimates of per‐generation effective population sizes for the Tasmanian population, based on allele frequency temporal variance with the wild progenitor population, ranged from 102–207 individuals and reflected hatchery records.     

Microsatellite loci in tench: isolation and variability in a test population

Because of their high variability and rapid evolution, microsatellites became increasingly important in genetic research, e.g. population structure and differentiation studies, gene mapping and parentage analysis. However, such loci have not been isolated in tench so far. Applying a PCR based method of generating microsatellite enriched DNA fragment libraries we were able to identify nine loci (MTT-1 to MTT-9). The variability of these microsatellite loci was determined in 50 tench individuals originating from a wild population of Lake Döllnsee, Germany. Three loci were found to be monomorphic. The remaining six loci segregated for two to nine alleles. The observed heterozygosities at polymorphic loci were high (0.500–0.959) with only one exception: locus MTT-8 (0.167). These polymorphic microsatellite loci showed a much higher level of genetic variability than the allozyme loci previously studied in the same individuals. Thus, they seem to be more suitable for genetic studies of tench. On the other hand, it remains to be checked in other populations if the three loci that did not show any variation in this population are generally monomorphic in this species.     

Development and evaluation of microsatellite markers for identification of individual Greenshell™ mussels (Perna canaliculus) in a selective breeding programme

A set of 49 microsatellite loci isolated from the endemic New Zealand Greenshell™ mussel, Perna canaliculus, were evaluated for inclusion in a parentage assignment marker suite by assessing their ease of PCR amplification, allele scoring and conformity to Mendelian inheritance in hatchery-produced families. Ten polymorphic loci (mean H_e = 0.78 and polymorphic information content (PIC) = 0.72) were identified as being suitable for parentage assignment. These 10 microsatellite loci gave a combined non-exclusion probability of < 0.001 (probability that an unrelated parent pair will not be excluded from parentage of an arbitrary offspring), based on allele frequencies from 16 broodstock mussels. Simulations predicted an assignment success rate of 99.9% with all 10 loci and > 95% with the best 5 or more loci (mean PIC = 0.84). In actual parentage assignments, 124 offspring from 8 full-sib families were assigned to the correct parent pair with 4 or more loci. We found evidence for null alleles and extensive size homoplasy in many loci, highlighting the importance of thoroughly characterizing and evaluating microsatellite markers prior to parentage assignment and other applications.