半滑舌鳎微卫星标记遗传连锁图谱的构建

利用全基因组测序方法筛选出微卫星标记,以渤海近海野生个体和人工养殖的半滑舌鳎(Cynoglossus semi-laevis)为亲本交配产生的F1全同胞家系为作图群体,构建了半滑舌鳎雌、雄微卫星标记遗传连锁图谱。用320对引物对父母本和92个F1个体进行遗传分析,共得到288个分离标记,其中包含112个偏分离标记(P<0.05)。其中雌性框架图包含242个标记,分布在21个连锁群上,总长度1 311.9 cM,标记间平均距离为4.9 cM,图谱覆盖率为83.3%;雄性框架图定位标记218个,21个连锁群,总长度1 316.2 cM,标记间平均距离为5.5 cM,覆盖率为82%。半滑舌鳎遗传连锁图谱的构建为半滑舌鳎重要经济性状QTL定位、分子标记辅助育种和性别控制奠定了重要基础。     

卤淡水工厂化养殖半滑舌鳎探析

半滑舌鳎(Cynoglossus semilaevis Günther)属鲽形目、舌鳎科,俗称鳎米、牛舌头等。半滑舌鳎为底栖大型鱼类,以肉质细嫩、味道鲜美、经济价值高著称,是我国南北方市场广泛认可的优质名贵鱼类。由于种群繁殖能力弱,近海捕捞强度大,自然界中半滑舌鳎数量日渐稀     

半滑舌鰨养殖群体雌雄比例与自然性逆转率

利用半滑舌鳎（Cynoglossus semilaevis Günther）性别连锁微卫星标记scaffold1128₃43和性腺组织切片的方法,研究半滑舌鳎8个普通家系和养殖场雄性亲鱼遗传性别与性逆转情况。首先,8个普通家系（28、30、38、39、40、44、57、69）遗传性别检测结果显示,遗传雌性比例最低为39家系（37.93%）,最高为38家系和40家系（55.00%）。对其中4个家系（28、39、44、57）的两次遗传性别比例检测结果对比发现,家系间遗传雌性比例存在差异。其次,对4个家系（28、39、44、57）和养殖场雄性亲鱼的遗传与表型性别鉴定结果表明,不同家系表型雌鱼比例为18.75%⁴6.88%,低于各家系的遗传雌性比例,不同家系间自然性逆转率存在显著性差异（P<0.05）。研究还发现,繁育场所用的普通雄性亲鱼中平均有28.42%的伪雄鱼。本研究结果表明,自然性逆转现象在半滑舌鳎养殖群体中普遍存在,半滑舌鳎不同家系的表型雌鱼比例存在显著差异,这为培育半滑舌鳎高雌性苗种提供了理论依据。     

半滑舌鳎微卫星标记的开发及其在F1家系中分离方式分析

利用富集文库-菌落原位杂交的方法筛选半滑舌鳎( Cynoglossus semilaevis)微卫星标记,构建富含(AC)和(AG)序列重复的半滑舌鳎微卫星富集文库,随机从文库中挑选1060个克隆,筛选得到883个(83.3％)阳性信号,菌落PCR检测742个阳性克隆片段的长度在500～1 200 bp范围内,随机挑选其中50个克隆进行测序,共设计引物33对,进行退火温度优化、多态性检测后,共得到20个多态的半滑舌鳎微卫星标记.对其进行分离方式分析,其中有17个位点符合孟德尔分离定律,共表现5种分离方式,可用于半滑舌鳎遗传连锁图谱的构建；另外有3个位点偏离孟德尔分离定律(P＜0.05).结果同时证明富集文库-菌落原位杂交是半滑舌鳎微卫星标记大量筛选和高密度遗传连锁图谱构建的一种行之有效的方法.     

利用AFLP技术筛选锯缘青蟹性别差异DNA片段

采用高盐和酚氯仿异戊醇 (PCI)结合法提取DNA ,利用AFLP技术 ,应用 5 2个引物组合 ,检测了锯缘青蟹 (Scyllaser rata)雌雄基因组DNA的多态性 ,筛选与锯缘青蟹性别相关的分子标记。实验中共扩增出 4 312条带 ,筛选出候选差异DNA片段 74 8条。这些差异DNA片段的获得 ,为研究锯缘青蟹性别的分子标记奠定了基础     

基于遗传连锁图谱筛选虾夷扇贝性别相关AFLP分子标记的方法

基于近年来他人构建的遗传连锁图谱,对2011–2013年2–5月期间采集的雌性、雄性及雌雄同体虾夷扇贝进行性别相关AFLP分子标记筛选。从雄性和雌性遗传连锁图谱上选取合计18对引物组合,经3批次试验,有8对引物扩增出性别相关的条带,其中Eb Mc、Ej Mf和Ei Mk重复性好,有4对引物扩增出雌雄同体特异性条带,5对引物扩增出雌性特异性条带。结果表明,基于高密度遗传连锁图谱筛选性别相关分子标记的方法简单可行,雌雄同体的基因组DNA与雌性和雄性存在显著差异,可能是一个单独的群体。     

半滑舌鳎的性腺分化和温度对性别决定的影响

半滑舌鳎(Cynoglossus semilaevisGünther)是近年来新开发的一种理想的增养殖对象,雌性个体生长速度比雄性快2～3倍。由于温度可影响多种鱼类的性腺分化,因而探索温度对该鱼的性别决定和性腺分化的影响,获得性逆转雄性亲本,用于培育全雌后代,具有重要的应用前景和经济效益。通过石蜡组织切片,对半滑舌鳎早期性腺分化进行组织学观察。发现在24℃饲养条件下,孵化后30 d半滑舌鳎性腺开始分化,其中具有裂隙的性腺原基未来发育为卵巢,而不具裂隙的原基未来发育为精巢。在孵化后25～100 d对半滑舌鳎进行不同温度(16℃、20℃、24℃、28℃、32℃)处理,在9月龄时利用石蜡组织切片鉴定表型性别,观察温度对性腺分化的影响,结果表明,28℃和32℃高温能显著提高群体中的雄性比例,使其分别达到69.2%和66.7%;而低温16℃和20℃对性腺分化影响不大,群体中雄性比例分别为56.5%和57.1%;24℃处理群体中雌雄个体比例接近1∶1。半滑舌鳎雌性特异的遗传性别鉴定技术也检测到高温和低温处理组出现了由基因型雌性向表型雄性逆转的雄性个体。这些结果表明温度影响了半滑舌鳎性腺分化方向。     

半滑舌鳎养殖群体中自然性逆转伪雄鱼的发现

利用雌性特异标记遗传性别鉴定技术,对71尾4龄半滑舌鳎的生理和遗传性别进行鉴定,结果显示32尾生理型雌鱼均能扩增出205bp的雌性特异条带;39尾生理型雄鱼,仅一尾鱼体重显著高于其他雄鱼并扩增出了雌性特异条带,因此这尾鱼遗传上为雌性,是一尾伪雄鱼。对养殖的600尾半滑舌鳎生理雄鱼大规模检测发现,养殖群体自然性逆转伪雄鱼比例为1.66%。对正常雌、雄鱼和1龄自然性逆转伪雄鱼的性腺组织学观察显示,与正常雄鱼相比,伪雄鱼性腺中也有大量的精母细胞,但数量比正常雄鱼的要略少;且未在伪雄鱼的性腺组织中观察到卵母细胞,说明半滑舌鳎性逆转发生在性腺分化期。半滑舌鳎自然性逆转现象的发现有助于解释当前半滑舌鳎养殖群体中雄性率偏高的现象,为半滑舌鳎全雌苗种生产提供了一条新的技术途径,并丰富了鱼类性别分化理论。     

半滑舌鳎(Cynoglossus semilaevis)雌性特异基因CSW3重组蛋白的表达与应用

CSW3基因序列来源于半滑舌鳎(Cynoglossus semilaevis)全基因组测序项目,是经过筛选得到的雌性特异候选基因之一。本研究探索了半滑舌鳎雌性相关基因CSW3的表达与应用,构建了半滑舌鳎雌性特异CSW3基因重组表达载体,通过大肠杆菌进行了体外重组表达,对基因表达产物进行分离纯化,并通过蛋白转染的方法研究了纯化的蛋白对几个性别相关基因的影响。结果显示,雌性特异CSW3重组蛋白注射鱼体后对半滑舌鳎性腺foxl2、sox9a、amh这3种性别相关基因的表达水平具有显著影响,能够引起雌性相关基因foxl2表达上调、雄性相关基因sox9a和amh表达下降。从蛋白水平对雌性相关基因CSW3的基因功能进行初步研究,为半滑舌鳎性别控制及全雌苗育种提供基因资源及技术方法。     

半滑舌鳎(Cynoglossus semilaevis)雌性特异基因CSW3重组蛋白的表达与应用

CSW3基因序列来源于半滑舌鳎(Cynoglossus semilaevis)全基因组测序项目,是经过筛选得到的雌性特异候选基因之一。本研究探索了半滑舌鳎雌性相关基因CSW3的表达与应用,构建了半滑舌鳎雌性特异CSW3基因重组表达载体,通过大肠杆菌进行了体外重组表达,对基因表达产物进行分离纯化,并通过蛋白转染的方法研究了纯化的蛋白对几个性别相关基因的影响。结果显示,雌性特异CSW3重组蛋白注射鱼体后对半滑舌鳎性腺foxl2、sox9a、amh这3种性别相关基因的表达水平具有显著影响,能够引起雌性相关基因foxl2表达上调、雄性相关基因sox9a和amh表达下降。从蛋白水平对雌性相关基因CSW3的基因功能进行初步研究,为半滑舌鳎性别控制及全雌苗育种提供基因资源及技术方法。     

Application of amplified fragment length polymorphism markers to assess molecular polymorphisms in gynogenetic haploid embryos of turbot (Scophthalmus maximus)

Among the variety of cultured marine species, the turbot Scophthalmus maximus is a fish of growing importance in European aquaculture. In this paper, an advanced application of AFLPs to estimate the genetic diversity of haploid gynogenetic families with the aim of obtaining a preliminary genetic map is presented. Ten EcoRI/TaqI primer combinations were tested in four families comprising diploid mothers and their haploid progenies. The amplified fragment length polymorphism (AFLP) analysis revealed an average of 6.8 polymorphic bands per primer combination and a total number of 88 polymorphisms out of 579 fragments. Among various primer pairs, seven combinations were selected in relation to the quality of profiles and number of polymorphic fragments, to be used in the determination of genetic linkage relationship between AFLP markers within the largest haploid family. Co‐migration of non‐homologous fragments was also investigated in one primer combination adding a fourth selective nucleotide to the three used in the classic TaqI AFLP protocol. Surprisingly, a rate of 38.7% of non‐homologous fragments co‐migrating with monomorphic bands was identified, due to the combined effect of homoplasy and the protocol used. Additional polymorphic markers discovered by this protocol were included in the linkage map. The turbot AFLP linkage map comprises 52 AFLP markers distributed in 12 linkage groups. On the basis of this map, turbot expected total genome length sums up to 1225.6 cM. The results confirm the usefulness of AFLPs in revealing genome segregation in haploid turbot progeny.     

Fish and seafood traceability based on AFLP markers: Elaboration of a species database

Several sociological, health and conservation arguments request a correct labelling of seafood products. Nowadays, molecular genetics is a useful tool for food chain traceability, particularly in regards to species identification. Among the variety of PCR-based molecular markers, AFLPs (Amplified Fragment Length Polymorphisms) have recently been used to investigate genomes of different complexities. This paper assesses the potential use of the AFLP technology to determine fish and seafood species in processed commercial products and domestic stocks. In particular a species database of fish, molluscs and crustaceans has been created with the aim to identify species of origin of seafood products by previously defined AFLP patterns. Different EcoRI and TaqI primer combinations were selected from 20 screened combinations in relation to the total number of detected fragments and polymorphic ones. Most informative combinations were E32/T32, E32/T33, E33/T33, E33/T37, E33/T38, E40/T33, E40/T37, E42/T32, E42/T37. The comparison of informative markers between unknown frozen or fresh products and reference samples has enabled the accurate identification of 32 different species. The taxonomic characterization has been performed either at the species or at the population level depending on the number of available individuals. AFLP variation at the population level is particularly helpful for the stock traceability of domestic strains. Size homoplasy was also investigated in one species to assess the rate of non-homologous comigrating fragments and to detect additional polymorphic markers to be used in stock identification. Results of Band Sharing Index (BSI) and percentage of polymorphic fragments are presented and are discussed in relation to the wide applicability of AFLPs both for fish and seafood safety and authenticity testing in such fields as food traceability and restocking management. The database, available upon request at nonnis@biol.unipr.it, will be continuously updated.     

“黄海1号”中国明对虾抗逆性状SRAP标记

采用序列相关扩增多态性(sequence-related amplified polymorphism,SRAP)分子标记技术,结合分群分析法(bulk segregate analysis,BSA)对中国明对虾(Fenneropenaeus chinensis)高氨氮和高pH胁迫敏感组和耐受组进行分析研究,筛选出与高氨氮或高pH耐受性相关的遗传标记.应用110对SRAP引物进行筛选研究,根据在BSA基因池产生的差异,筛选出与高氨氮耐受相关引物77对,与高pH耐受相关引物102对,以用于验证分析.根据片段在群体中出现频率和变化规律,筛选出6个可能与高氨氮耐受性相关的分子遗传标记,其中耐受高氨氮负相关标记1个,正相关标记5个；7个可能与高pH耐受性相关的分子遗传标记,其中耐受高pH负相关标记2个,正相关标记5个.对获得的13个序列片段进行回收,连接于pMD-18T载体后转化于大肠杆菌TOP10感受态细胞,进行了克隆和测序.将测序结果进行了BLAST分析比对,发现测得片段序列与数据库中序列同源性较低(一般都低于15％),未找到与之同源性较高的功能基因,推论这些特异性序列片段标记可能与高氨氮或高pH耐受性性状密切相关,后续群体验证工作正在进行,以期筛选出与中国明对虾抗逆性状密切相关的序列特征性片段扩增区域(sequence characterized amplified region,SCAR)分子标记,为分子标记辅助育种提供技术支持.     

条斑紫菜6个品系的SRAP分析

为鉴别条斑紫菜不同品系的种质,使用相关序列扩增多态性(sequence-related amplified polymorphism,SRAP)标记对条斑紫菜的5个选育品系和1个野生品系进行遗传分析,结果从35对引物组合中筛选出可扩增出稳定清晰条带的组合11对,共获得131个扩增位点,其中多态性位点125个,多态性比例高达95.42%。6个品系 间的遗传距离为0.364 3～0.867 9,平均为0.593 0。用UPGMA法进行聚类分析,结果将6个品系分为2个群,所反映的亲缘关系与各品系的来源基本一致,说明SRAP 标记技术可以成为条斑紫菜品系间遗传分析的有效工具。在131个多态性位点中,选择扩增出的4个位点构建了6个品系的指纹图谱。另外,通过ME1/EM6引物组合 扩增得到耐高温品系TM-18的特异性条带,经回收测序和重新设计引物,该条带在其丝状体和叶状体DNA中均能稳定地被扩增出来,可用于该品系的种质鉴别 。     

Rapid identification of Streptococcus iniae by specific PCR assay utilizing genetic markers in ITS rDNA

The 16S-23S intergenic spacers (ITS) of ribosomal DNA from ten independent isolates of Streptococcus iniae and one reference strain ATCC29178 were sequenced, aligned and used to design a polymerase chain reaction (PCR) primer set for rapid and specific detection and identification of S. iniae. This primer set amplified a 377-bp DNA fragment specifically from S. iniae, but not from other common bacterial pathogens of fish or from non-fish pathogens. The PCR conditions were optimized to allow detection of the organism from agar, broth culture or infected fish tissue. The sensitivity of the PCR assay was established by the detection of DNA as low as 0.02 ng or as few as 10 CFU bacterial cells. The establishment of the specific PCR assay provides a useful tool for the identification and diagnosis of fish infection with S. iniae.     

黄姑鱼群体遗传多样性的AFLP分析

对青岛和厦门黄姑鱼群体的遗传多样性进行了AFLP分析，5对选择性引物在两个群体47个个体中，共扩增出461个位点，多态位点265个。青岛和厦门群体的多态位点比例、Nei遗传多样性指数和Shannon遗传多样性指数分别为51.70%、51.99%，0.1022、0.0996，0.1643、0.1622；两个群体遗传多样性在同一水平上。基因分化系数G_st、Shannon遗传多样性指数和AMOVA分析均显示黄姑鱼的遗传变异主要来源于群体内个体间，而群体间无明显的遗传分化。群体的显性基因型频率分布和位点差异数分布显示两个群体有基本相同的群体遗传结构。结果表明，黄姑鱼青岛和厦门群体间无明显的遗传差异，群体间有明显的基因交流。