FIASCO法筛选鳜鱼微卫星标记

通过FIASCO(Fast Isolation by AFLP Sequences Containing repeats)法构建鳜鱼(Siniperca chuatsi)基因组微卫星富集文库,分离微卫星DNA序列并对其特征进行分析。鳜鱼基因组DNA经MseⅠ限制性内切酶消化后,选取200~800 bp的片段与MseⅠ接头连接,用生物素标记的寡核苷酸探针(AC)8、(CT)8、(AT)7、(GATA)8、(GATT)7与其杂交,杂交复合物结合到包被有链霉亲和素的磁珠上,变性洗脱获得单链目的片段,经PCR扩增形成双链,然后克隆到pGEM-T载体上,转化至DH5α中,首次成功构建鳜鱼基因组微卫星富集文库。对其中100个阳性克隆进行测序,60个(60%)含有微卫星序列(GenBank Accession Number:DQ789247~DQ789306)。成功设计了47对鳜鱼微卫星引物,并合成21对引物进行PCR扩增,结果筛选出18个多态性微卫星标记。结果表明:FIASCO法能有效提高筛选微卫星标记的效率。本研究筛选的微卫星标记可以用于鳜鱼遗传背景分析和遗传连锁图谱构建,并将为鳜鱼基因组结构分析、标记辅助育种以及数量性状位点(QTL)基因的定位等研究提供候选微卫星标记。     

鲫血清转铁蛋白cDNA克隆及系统发育进化序列分析

鱼类血清转铁蛋白是鱼类血清中一种非血红素结合铁的β—球蛋白。从GenBank数据库查询发表的鱼类转铁蛋白cDNA或基因序列,根据铁离子结合及转运功能位点,设计并合成了两对引物P1、P4以及P2、P3,克隆出鲫血清转铁蛋白cDNA中的核心片段,长度为866bp。再根据克隆出的核心片段分别设计上游及下游两对引物P5、P6以及P7、P8,随后用RACE方法分别克隆出鲫血清转铁蛋白cDNA的5’端(787bp)和3’端(1081bp)以及全长cDNA,最后在计算机上排列出鲫血清转铁蛋白全长cDNA,长度为2444bp。比较了14种鱼血清转铁蛋白cDNA序列的同源性,其同源性在30％～80％之间,结果显示鲤科鱼类(鲫、银鲫、鲤及斑马鱼等)具有很近的亲缘关系;同时进行了系统发育进化分析,证实了鱼类血清转铁蛋白进化的保守性和氨基酸序列的高度同源性。     

鳙鱼微卫星分子标记的筛选

采用常规方法从鳙鱼(Aristichthys nobilis)血液中提取基因组DNA,经限制性内切酶Sau3AI酶切后,选取250～750bp大小的片段构建鳙鱼基因组文库。采用人工合成的重复序列(CA)15、(AG)12、(AAG)8用同位素标记作为探针,通过原位杂交筛选基因组文库,获得鳙鱼的微卫星序列。进一步用引物设计软件Primer Premier 5．0设计引物。通过杂交获得99个阳性克隆,经测序筛选出82个微卫星序列,设计引物65对。     

胭脂鱼微卫星富集文库的构建及其应用前景展望

以湖北武汉和四川宜宾人工增殖放流的胭脂鱼子一代酒精浸泡鳍条样本为材料,提取完整基因组DNA,选用人工合成的生物素标记(AAAG)7探针,采用FJASCO(fast isolation by AFLP of sequences containing repeats)磁珠富集法构建胭脂鱼(Myxocyprirnus asiaticus)微卫星富集文库.以Msel-N引物组和设计合成的微卫星核心序列引物(AAAG)5,用双引物PCR法筛选含有微卫星的阳性克隆,从54个阳性克隆中共获得22个微卫星序列,其中完美型(perfect)共15个(占68.2%),非完美型(imperfect)6个(占27.3%),混合型(compound)1个(占4.5%);(AAAG/ITYC)n序列在胭脂鱼的基因组DNA中含量非常丰富.根据微卫星侧翼序列最终设计并合成了18对胭脂鱼微卫星引物,经其有效性检验后,可应用于胭脂鱼遗传多样性、种群遗传结构等进一步研究及人工增殖放流效果评估.     

澳洲鳗鲡微卫星分子标记的筛选与检测

采用小片段克隆法构建了澳洲鳗鲡(Anguilla australis)的部分基因组文库,并用地高辛标记的(CA)15作探针筛选阳性克隆,共获得76条微卫星序列.其中10次以下CA连续重复序列占83.67%,没有检测到30次以上的连续重复序列.根据微卫星序列两端足够长的侧翼序列,设计引物55对,选择合成引物26对,用澳洲鳗鲡3个个体的混合基因组进行引物筛选,其中的18对具有清晰的扩增条带.将筛选出的18对引物对澳洲鳗鲡1个群体的40个个体进行了遗传多样性分析,其中1对引物扩增产物为单态,17对扩增产物呈现多态;17对扩增多态的引物在40个个体中扩增出等位基因数目为5～14,平均为9个.该澳洲鳗鲡群体的PIC、Ho、He的平均值分别为0.715 7、0.677 9、0.7374,所有位点均符合哈迪-温伯格平衡(P=0.05),结果证明这17个微卫星位点适于澳洲鳗鲡群体结构的研究分析.[中国水产科学,2009,16(1):133-138]     

脊尾白虾微卫星富集文库的构建与多态性标记的筛选

采用人工合成的生物素标记(AG)15探针及磁珠富集法构建了脊尾白虾基因组微卫星富集文库.将脊尾白虾基因组DNA经HaeⅢ酶切后,收集400 ～1 200 bp片段,连接特定接头,与生物素标记(AG)15探针杂交并捕获含有微卫星的DNA片段,然后连接到pMD18-T载体中,构建脊尾白虾微卫星富集文库.随机挑取947个克隆,经菌落PCR检验,其中667个为阳性克隆.从阳性克隆中随机选取184个克隆进行测序,有150个克隆含有微卫星序列,共获得199个微卫星序列,其中完美型158个(79.4％),非完美型27个(13.6％),复合型14个(7.0％).根据微卫星序列,设计了119对微卫星引物,64对扩增出稳定的具有特异性的条带,经30个脊尾白虾个体进行多样性评价后,有26个位点表现出多态性.26个微卫星位点获得的等位基因数从3 ～ 16个不等,平均每个位点扩增得到6.5个等位基因.观测杂合度(Ho)、期望杂合度(4)及多态性信息含量值(PIC)的范围分别为0.111 ～ 0.929、0.246 ～0.932、0.231 ～0.909.本研究开发的微卫星位点将为脊尾白虾种群多样性研究、家系识别和种质鉴定提供有效的工具.     

同源雌核发育银鲫子代微卫星杂合位点的分离

使用14对银鲫（Carassius autatus gibelio Bloch）微卫星引物对同源雌核发育的银鲫子代及其亲本进行多态性研究，探讨雌核发育银鲫的母本微卫星杂合位点在同源传代中是否有分离的情况发生。在所选用的14对微卫星引物中，有8对在父母本间检测出不同的多态性，在所检测的第1组银鲫自交的子代中，只有1个个体在所检测的8个微卫星位点均存在变异，有明显的父本特异性条带出现，表现出了大量父本遗传信息，出现率为4．55％；在第2组检测到1个个体只在SCM13位点上出现母本部分特异性条带的缺失、弱化，而在其他位点上没有发现变异，出现率为1．52％。此结果表明，雌核发育银鲫卵子大部分在配子发生过程中没有发生减数分裂，即使用同源精子刺激，也行雌核发育生殖；但是，有极少数卵子在配子发生过程中，可能发生了部分减数分裂，具有部分融合同源精子的能力，从而在子代中检测到父本以及在融合过程中重组来的特异条带，用微卫星分于标记证实了银鲫两种生殖方式的存在，即雌核发育生殖和两性融合生殖。     

黄鳍鲷基因组微卫星的分离

采用磁珠富集法构建黄鳍鲷(Acanthopagrus latus Houttuyn)基因组微卫星富集文库。共挑选60个克隆进行测序,分析发现58个克隆分别含(GA)n或(CA)n两碱基重复单元。进一步通过序列比对,最终获得41个具有特异微卫星序列的阳性克隆。其中,23个克隆含有(GA)n或(CT)n两碱基重复序列,17个克隆含有(GT)n或(CA)n重复序列,另1个含有以上两种重复类型。获得的微卫星序列中,单一型及间断型序列各有20条,另有1条属于复合型序列。序列长度为117~512 bp,平均259 bp。微卫星核心序列两碱基重复5到38次,绝大多数序列重复次数大于10。基于微卫星两端的侧翼序列设计并获得了3对能够在黄鳍鲷基因组有效扩增的微卫星引物。本研究旨为进一步开展黄鳍鲷分子育种及资源评价分析提供基础资料。     

剑尾鱼微卫星DNA的筛选

以剑尾鱼(Xiphophorus helleri)为材料,经MboⅠ限制性内切酶消化基因组DNA后,选取500～2 000 bp的片段连接到经BamHⅠ酶切的pUC18载体上,转化大肠杆菌DH5α构建部分基因组文库.采用设计合成的(AC)7、(GT)7重复序列为引物,PCR筛选部分基因组文库,对其中9个重组阳性克隆进行测序,结果共获得24个微卫星序列,其中Perfect(完美型)13个,占54.2%;Imperfect(非完美型)3个,占12.5%;Compound(混合型)8个,占33.3%.表明(AC/GT)n在剑尾鱼的基因组DNA中含量非常丰富.同时,根据其中3个克隆微卫星的侧翼序列设计引物,PCR扩增剑尾鱼基因组DNA,结果均扩增到目的片段.而且,这3对引物扩增出来的微卫星片段在非选育的剑尾鱼中显示出多态性,而在近交系19代则表现为单态,为剑尾鱼的实验动物化遗传研究提供了理论依据.     

中国对虾微卫星DNA引物的设计及筛选

根据建立的中国对虾(Fenneropenaeus chinensis)部分基因组文库,对筛选的含微卫星DNA序列的克隆设计了28对引物,筛选出5对微卫星多态性引物,并用这5对引物对中国对虾的养殖群体20个个体进行了遗传多样性分析。在这5个微卫星位点中,虽然．RS0871位点是多态位点,但其等位基因数、杂合度和多态信息含量都比较低。其余4个微卫星位点可产生6～8个等位基因,等位基因的大小分布在142～364bp,基本上符合引物设计时理论产物长度。这些微卫星位点的期望杂合度的范围为0．7577～0．8064,表明它们都有较高的杂合度。仅有位点RS0956的观察杂合度与期望值有较大的出入,其他微卫星位点的这两值基本相符。有4个微卫星位点的PIC值较高,在0．7180～0．7709。因此,除RS0871位点以外的4个微卫星位点均可用于中国对虾种群遗传结构分析,这将为中国对虾品种选育、种系评估提供更多的微卫星DNA信息。     

Side-aspect target strength of Atlantic salmon (Salmo salar), brown trout (Salmo trutta), whitefish (Coregonus lavaretus), and pike (Esox lucius)

The side-aspect acoustic target strengths (TS) of 19 Atlantic salmons (Salmo salar), 16 brown trouts (Salmo trutta), 10 whitefish (Coregonus lavaretus) and 9 pikes (Esox lucius) were measured using a 200 kHz split-beam echosounder, in order to study the relationship between TS and fish size indices (length, weight and side area). The effect of side aspect angle on TS was also studied. Linear models between TS and the logarithm of the fish size indices were fitted with length being best for predicting TS. Typically, the standard error of estimate was 1.2–2.9 dB. The side-aspect TS measurements with specimens of known size showed that the linear relationship between full side-aspect TS and the logarithm of fish length for salmonid (Salmo salar + Salmo trutta) was on average 4.7 dB (SE = 0.7), lower than for whitefish and pike combined. The effect of side aspect angle on TS was modelled with cos³ (2α) function. The differences in the TS between full side aspect and head/tail aspect were 17.4, 19.0 and 19.6 dB for salmonid, whitefish, and pike, respectively.     

Food and feeding habits of Tilapia aurea (Steindachner) (Cichlidae) in Lake Kinneret (Israel)

An analysis of the food of 274 specimens of Tilapia aurea (Steindachner), which had been collected in Lake Kinneret, showed that this species is mainly a zooplankton consumer.A study of the quantitative dynamics of the food components in the course of a year has proved that the species in question feeds more intensely in spring, this being the time when zooplankton forms are more abundant in the water of the lake. Vegetable detritus, mixed with plankton and benthos forms, served as additional and alternative food.The lack of clearly positive degree of food component selectivity, as well as the low values of the intestine repletion index and of the condition coefficient, account for the slowness in growth of Tilapia aurea in Lake Kinneret.The low values of the growth and feeding indices are due, according to the authors, to the pressure brought to bear by the other local fish species, which are competitors for the same food.     

Food and feeding habits of Tilapia zillii (Gervais) (Cichlidae) in Lake Kinneret (Israel)

Analysis of the gut content of 329 specimens of T. zillii (Cichlidae) collected from Lake Kinneret, has shown great variation in the sorts of food.A study of seasonal dynamics has proved the prevalence in the food of Chironomida pupae (Diptera) in winter and spring and of zooplankton forms in summer and autumn. The additional food consisted of the various groups of algae, the most frequently found being Cyanophyta (100%) and Pyrrophyta (64.16%). An extremely voraceous species, it consumes — while searching for its preferred food — anything that comes its way in the water: algae, scraps of macrophytes, autochthonous and allochthonous insects, and forms of benthic origin, such as Nematoda, Ostracoda, Porifera and Chironomida (larvae).The satiation index is high (between 4.08 and 5.63), in contradiction to the low values of the coefficient of condition (between 3.05 and 3.51), and with the slow rate of growth in Lake Kinneret. The main food of Tilapia zillii, consists of arthropod species with a chitin content (which is eliminated unchanged) of more than 50% of the total weight, and this may account for the poor exploitation of the trophic base.This species of fish may be considered as being detrimental to others (i.e. commercially important species) because of its successful competition for food, and not, as is often incorrectly assumed, because it is an aggressive consumer of their spawn and fry.     

Global conservation status and research needs for tarpons (Megalopidae), ladyfishes (Elopidae) and bonefishes (Albulidae)

We assessed the taxonomic diversity, geographic distributions, life history, ecology and fisheries of tarpons, ladyfishes and bonefishes (members of the subdivision Elopomorpha), which share many life history and habitat use characteristics that make them vulnerable to environmental and anthropogenic stresses in coastal environments. This assessment of Red List status for the International Union for the Conservation of Nature reveals three species considered near threatened or vulnerable, three species of least concern, and 11 data‐deficient species. Although the taxonomy of tarpons appears stable, it is less so for ladyfishes and bonefishes. In aggregate, these species are distributed circumtropically and foray into temperate zones. Although they spawn in marine habitats, larvae of many species disperse into estuarine habitats, which are declining in area or degrading in quality. Several species support high‐value recreational fisheries, or culturally important small‐scale commercial and artisanal fisheries. Nonetheless, no formal stock assessment exists for any species, so improved data collection, information sharing and assessment techniques should facilitate socio‐economic development of individual fisheries. Catch‐and‐release recreational fisheries that promote conservation of tarpon and bonefishes in some regions are promising models to improve the conservation status of these fishes elsewhere, as well as the economic development of these fishing communities. Most tarpons, ladyfishes and bonefishes likely face significant challenges from anthropogenically mediated habitat loss and alteration, and several are vulnerable to both habitat degradation and overfishing. Broader protection and enhancements to fisheries habitat in all regions will benefit these as well as many other coastal fishery species.     

Brown trout (Salmo trutta L.) and Arctic charr (Salvelinus alpinus (L.)) as predators on three sympatric whitefish (Coregonus lavaretus (L.)) forms in the subarctic Lake Muddusjärvi

Abstract  – Brown trout ( Salmo trutta L.) and Arctic charr ( Salvelinus alpinus (L.)) use whitefish ( Coregonus lavaretus (L.)) as their main prey in the subarctic Lake Muddusjärvi. Brown trout dwelled in littoral and pelagic habitat, whereas Arctic charr lived only in epibenthic habitat. Both species shifted to whitefish predation at a length of 20–30 cm. At this size, brown trout fed on larger whitefish than Arctic charr. Whitefish occur in three sympatric forms, differing in their habitat, ecology and morphology. Both the predators preyed primarily upon the small-sized, densely rakered whitefish form (DR), which was the most numerous whitefish form in the lake. DR used both epibenthic and pelagic habitat, whereas two sparsely rakered whitefish forms dwelled (LSR and SSR) only in epibenthic habitat: LSR in littoral and SSR in profundal areas. Sparsely rakered whitefish forms had minor importance in predator diet.     

Habitat use and activity patterns of roach (Rutilus rutilus (L.)), rudd (Scardinius erythrophthalmus (L.)), perch (Perca fluviatilis L.) and pike (Esox lucius L.) in the laboratory: the role of predation threat and structural complexity

Abstract– The role of predation risk and structural complexity in determining the habitat use and activity patterns of roach, rudd and perch was assessed using a series of video-recorded laboratory trials. The time spent in open and structured habitats, vertical swimming heights and activity levels of each species were observed in the presence and absence of a potential, pike, predator. Habitat complexity varied between treatments with artificial stem densities of 200, 400 and 600 stems'm⁻². Predator free trials showed that roach and rudd spent significantly less time in structured habitats than perch. Increasing stem density had no significant effect on the habitat choice of perch but did affect the distribution of roach and rudd. Stem density influenced the vertical swimming height of rudd or perch but not roach, although the effects of habitat complexity on swimming activity were more complicated. Pike were themselves influenced by increases in stem density, only selecting structured habitats when stem densities were less than 600 stems'm⁻². In the presence of a predator, both roach and rudd increased the amount of time spent in structured areas. These observed differences were independent of stem density. Perch, however, decreased the amount of time spent in structural habitats at all stem densities. Predation risk also prompted significant changes in both vertical swimming height and activity levels of potential prey fish. A pike predation success hierarchy of rudd-roach-perch was also observed during the study. The reasons for this pattern are discussed.     

Cu~(2+)、Pb~(2+)、Cd~(2+)、Cr~(6+)对鳙胚胎和仔鱼的急性致毒效应

研究了Cu2+、Pb2+、Cd2+、Cr6+ 4种重金属对鳙胚胎发育及仔鱼存活的影响.试验结果表明,在试验浓度范围内,除0.5、1.0 mg/L的Cd2+可促进胚胎发育速率外,其余3种重金属均不同程度地减慢胚胎孵化速率;受精卵孵化率明显低于对照组;初孵仔鱼出现不同程度的畸形;Cu2+、Pb2+、Cd2+、Cr6+对鳙仔鱼的毒性强度为Cu2+>Cd2+>Pb2+>Cr6+.根据试验数据求得4种重金属对鳙仔鱼的24、48、72、96 h的LC50值及安全质量浓度.     

Resource partitioning between lake-dwelling Atlantic salmon (Salmo salar L.) parr, brown trout (Salmo trutta L.) and Arctic charr (Salvelinus alpinus (L.))

Abstract – Resource partitioning between Atlantic salmon parr, brown trout and Arctic charr was studied throughout the ice-free season in a north Norwegian lake. Juvenile salmon and trout (≤160 mm) utilized the littoral zone and juvenile charr the profundal, while adult trout and charr (>160 mm) were found in both. Juvenile salmon and trout had a similar diet, although trichopteran larvae were more important for the trout and chironomid pupae and three-spined sticklebacks for the salmon parr. Small salmon and trout parr (≤120 mm) had a higher diet overlap than larger parr (121–160 mm). The feeding habits of adult trout were similar to that of juvenile trout, but the former took larger prey items. At the population level, both salmon and trout were generalistic feeders with a broad diet, but at the individual level, both species had specialized on a single or a few prey categories. Juvenile charr were segregated from salmon and trout in both habitat and food utilization; they had a narrow diet consisting of chironomids and zooplankton, possibly reflecting their confinement to the profundal habitat which have a low diversity of potential prey. Larger charr also took zoobenthos and sticklebacks in the littoral zone. ^Note