细胞核rDNA序列用于罗非鱼及其杂交种鉴别的初步研究

罗非鱼种间,尤其是尼罗罗非鱼与杂种尼奥罗非鱼之间,很难区分。本文对尼罗罗非鱼、奥利亚罗非鱼、莫桑比克罗非鱼和杂交种尼奥罗非鱼（尼罗罗非鱼♀×奥利亚罗非鱼♂）和红罗非鱼的核糖体DNA内部转录间隔子1（ITS1）序列及其两侧的18S和5．8s部分序列特征进行分析,以筛选种间鉴别标记。PCR扩增产物大小约700bp,测序结果表明,（去除引物后）18S长146bp,5．8S长66bp,不同种类之间无差异;ITS1长383-483bp,因种类不同而异,其GC含量大于AT含量,达到67．1％。序列比对分析结果表明,18S和5．8s片段高度保守,但各有3个变异位点可以把上述几个种和杂种相互区分开;18S序列上有一个UnbI限制性酶切位点,可作为尼罗和尼奥罗非鱼的鉴别标记。ITS1序列种间变异大,系统发育分析表明,所研究的5个种聚成2个类群,尼罗与尼奥罗非鱼为一组,莫桑比克-红罗非鱼-奥利亚罗非鱼为另一组。组内种间遗传距离较小,尼罗和尼奥罗非鱼的种间遗传距离为0．006;莫桑比克、奥利亚和红罗非鱼的种间遗传距离在0．007-0．009之间;两组罗非鱼之间的遗传距离较大,在0．030-0．035之间,表明罗非鱼ITS1序列多态性较高,适合于种类区分。结合部分18S和5．8S序列,细胞核rDNA具有鉴别罗非鱼及其杂种的潜力。     

罗非鱼雌、雄群体遗传差异的RAPD分析

从40个随机引物中分别筛选出15和18个,对奥利亚罗非鱼和尼罗罗非鱼雌、雄群体进行RAPD分析,结果显示:奥利亚罗非鱼雌性群体的多态位点比例为56.25%,基因多样性指数为0.2358,Shannon氏指数为0.3417,群体内的遗传相似指数为0.8625;雄性群体的多态位点比例为57.50%,基因多样性指数为0.2356,Shannon氏指数为0.3418,群体内的遗传相似指数为0.8375。尼罗罗非鱼雌性群体的多态位点比例为47.44%,基因多样性指数为0.1788,Shannon氏指数为0.2637,群体内的遗传相似指数为0.7667;雄性群体的多态位点比例为64.10%,基因多样性指数为0.2347,Shannon氏指数为0.3486,群体内的遗传相似指数为0.6769。试验结果表明,奥利亚罗非鱼雌、雄性群体的遗传多样性程度接近,而尼罗罗非鱼雄性群体的遗传多样性要比雌性群体丰富,遗传变异比雌性群体大。     

Comparative time-course study on pituitary and branchial response to salinity challenge in Mozambique tilapia (<Emphasis Type="Italic">Oreochromis mossambicus</Emphasis>) and Nile tilapia (<Emphasis Type="Italic">O</Emphasis>. <Emphasis Type="Italic">niloticus</Emphasis>)

The physiological response of Mozambique and Nile tilapia transferred from fresh to brackish (15 ppt) water was compared during a one-week time course. Response in the pituitary was measured by the gene expression pattern of prolactin (PRL I), growth hormone (GH), and calcium-sensing receptor (CaSR), while the response in the gills was measured by the gene expression pattern of the prolactin receptor (PRL-R), Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) and Na⁺/Cl⁻ cotransporter (NCC), and by activity and expression of Na⁺/K⁺-ATPase (NKA). The time-course curves of plasma osmolality levels indicate a rapid elevation 24 h after transfer, which later decreased and maintained at stable level. PRL I expression decreased in both species, but with stronger response in the Nile tilapia, while no differences were found in the slightly elevated levels of GH mRNA. The branchial response demonstrated a faster up-regulation of NKA and NKCC in the Mozambique tilapia, but similar levels after a week, while Nile tilapia had stronger and constant down-regulation of NCC. The time-course response of the measured osmoregulatory parameters indicate that 24 h after transfer is a critical time point for brackish-water adaptation. The differences in responses to saltwater challenge between Mozambique and Nile tilapia shown in this study may be associated with the differences in saltwater tolerance between these two tilapiine species.     

Random amplified polymorphic DNA markers for three Japanese laminarian species

ABSTRACT: Random amplified polymorphic DNA (RAPD) was studied to detect genetic markers for three economically important Japanese laminarian species, Laminaria japonica Areschoug, L. religiosa Miyabe and L. ochotensis Miyabe, which were sampled from their representative localities on the coasts from Tsugaru Straits to the Sea of Japan. DNA templates for RAPD were extracted from lamina using a DNA extraction kit and were purified with glassmilk. Reproducible RAPD markers for these three species were detected using three random primers from a total of 15 tested. In L. japonica and L. religiosa , these RAPD markers were confirmed to be useful for populations from other localities. The three species studied showed high intraspecific and interspecific band sharing index (BSI) values. Like annual typical L. religiosa from other localities, biennial individuals identified as putative L. religiosa from Atsuta could be discriminated from L. japonica but not from L. ochotensis by any of the RAPD genetic markers studied so far.     

Investigation into the mode of inheritance of allozyme and random amplified polymorphic DNA markers in tilapia Oreochromis mossambicus (Peters)

Feral Australian Oreochromis mossambicus (Peters) (Pisces: Cichlidae) and an interspecific hybrid population (most probably originally derived from crosses of O. mossambicus and O. niloticus stocks) were used as model organisms to study the inheritance patterns of 24 allozyme loci and 31 random amplified polymorphic DNA (RAPD) loci in tilapia. Single‐paired matings of parents of known genotype were used to generate families, and 10–15 full‐sib offspring from each mating were used to test for mode of inheritance. The majority of allozyme and RAPD loci tested segregated in a Mendelian fashion. Allozyme markers in general showed co‐dominant inheritance patterns, while RAPD markers conformed to expectations for band presence/absence under a dominant allele model. Although only a small number of families and offspring were used, the results highlight the suitability of allozymes and RAPDs as genetic markers for population analysis in tilapia.     

Evaluation of genetic relationship among six Labeo species using randomly amplified polymorphic DNA (RAPD)

Labeo species constitute an important group of fish with intense diversity and potential for commercial aquaculture in many Southeast Asian nations including the Indian subcontinent. The present investigation involves the comparative analysis of randomly amplified polymorphic DNA (RAPD) profiles of six Labeo species viz., L. bata (bata), L. calbasu (calbasu), L. dyocheilus (dyocheilus), L. fimbriatus (fimbriatus), L. gonius (gonius) and L. rohita (rohu) at the nuclear DNA variation level. Fifteen decamer random primers were chosen from 40, which amplified a total of 449 DNA fragments ranging in size from 400 to 3000 bp. Both monomorphic and polymorphic DNA bands were identified based on their presence or absence that could be used for specieswise differentiation. Similarity coefficients were calculated to quantify the genetic variation within and between species. On an average, the highest intra‐species genetic similarity value was found in calbasu (0.93) followed by rohu and fimbriatus (0.91), bata (0.87), gonius (0.86) and dyocheilus (0.77). The interspecies genetic similarity estimates among the species of Labeo were used to deduce their phylogenetic relationships. The cluster analysis showed two main clusters, one with calbasu, rohu, fimbriatus and gonius and another with bata and dyocheilus. The study provides evidence that RAPD could be used for genetic differentiation of closely related species.     

An investigation of the possible causes for the loss of productivity in genetically improved farmed tilapia strain in Fiji: inbreeding versus wild stock introgression

Four microsatellite markers and a mitochondrial DNA (mtDNA) fragment were used to investigate two possible explanations for a reported decline in productivity of genetically improved farmed tilapia (GIFT) in Fiji: (i) a decline in genetic diversity (GD) and (ii) genetic introgression from feral tilapia populations. Genetic diversity was estimated using θ and allelic richness, while Bayesian clustering was used to assign individuals to genetic groups (K=2 or 3) to test for introgression. Differentiation among groups was estimated using F_ST analysis. Results indicate that genetic diversity had declined compared with a GIFT reference stock from WorldFish Centre, while there was little evidence for introgression from feral tilapia populations. Loss of genetic diversity most probably resulted from practices that have not actively managed genetic resources in the hatchery. While GIFT is considered to be an improved line of Nile tilapia (Oreochromis niloticus), mtDNA analysis here revealed haplotypes assigned previously to three discrete Oreochromis species (O. niloticus, Oreochromis mossambicus and Oreochromis aureus) in both the Fijian strain and the WorldFish Centre strain. Possible sources for the three divergent lineages are discussed. Results have implications for the management and future expansion of the tilapia culture industry in Fiji as well as in other Pacific island nations.     

RAPD fingerprinting of the eel-loaches Pangio filinaris and Pangio piperata: preliminary evaluation

Random amplified polymorphic DNA (RAPD) analysis was applied to two morphologically similar species of the increasingly popular ornamental eel‐loaches, Pangio filinaris and P. piperata, collected from Kemat River, Terengganu and Padang Sanai River, Kedah, West Malaysia. Forty primers were tested in a preliminary screening. Of these, five (OPA‐03, OPA‐09, OPA‐11, OPA‐13 and OPA‐18) were chosen for their ability to provide consistent amplification. RAPD analysis differentiated the two species, with each showing different RAPD patterns for each primer used. The five primers generated a total of 82 scorable loci with up to 83% and 60% polymorphism in P. piperata and P. filinaris respectively. The RAPD banding patterns of these two species ranged from 2 to 14 fragments, with a size range of 250–2000 bp. Genetic similarity within species ranged from 0.610 to 0.985 (mean 0.799±0.14) for P. piperata and from 0.824 to 0.934 (mean 0.885±0.04) for P. filinaris respectively. The interspecies genetic similarity ranged from 0.386 to 0.486 with an average value of 0.434±0.040. Our study revealed RAPDs as useful genetic markers for the taxonomic clarification and assessment of genetic variability of the eel‐loaches.     

Growth comparison of Asian Nile and red tilapia strains in controlled and uncontrolled farm conditions

Growth of several genetically improved Nile tilapia (GIFT or Genetically Improved Farmed Tilapia, FaST or Freshwater Aquaculture Center Selected Tilapia, SEAFDEC-selected) and domesticated red tilapia (BFS or Binangonan Freshwater Station, NIFI-red or National Inland Fisheries Institute red, HL or Hacienda Luisita) stocks were compared in controlled (tank) and uncontrolled farm conditions (lake-based cages) with unselected NIFI or Chitralada Nile tilapia as control. Specific growth rate differed significantly (P = 0.009) in tank-reared Nile tilapia stocks where GIFT grew best at 1.358%/day followed by FaST (1.307%/day), control stock NIFI (1.257%/day) and SEAFDEC-selected (1.202%/day). Genetic effect explained 84.4% of the variance in growth of Nile tilapia in tanks. Although Nile tilapia growth in cages followed the same trend where GIFT grew best at 1.570%/day, no significant stock differences (P = 0.479) were noted. Meanwhile, red tilapia reared in either tanks or cages showed no significant stock differences in terms of growth. However, survival of the red tilapia stocks in cages differed significantly with HL having the highest percentage survival at 93.3%. The different growth responses of the Nile tilapia stocks especially under controlled (tank) farm conditions were largely due to genetic factors (stock differences).Under uncontrolled farm conditions, environmental factors were generally observed to have also affected the survival and to some extent, the growth of Asian Nile and red tilapia stocks.     

Simulating the economic viability of Nile tilapia and Australian redclaw crayfish polyculture in Yucatan, Mexico

Tilapia culture in rural communities in the state of Yucatan, Mexico, has been increasing in recent decades. Polyculture of tilapia with other more commercially valuable species provides an opportunity to substantially improve the economic yields of rural producers. The economic viability of implementing Nile tilapia with Australian redclaw crayfish in polyculture was analyzed using profitability indicators such as internal rate of return (IRR) and net present value (NPV). A bioeconomic model was developed to simulate three production densities, accounting for investment recuperation in time horizons of 5, 10 and 15 years. Simulations showed a notable improvement in profitability when farms adopt the polyculture strategy, particularly over the 5-year horizon. Nile tilapia with Australian redclaw crayfish polyculture shortens investment return time and buffers risk related to changes in tilapia sale price. Of the three studied polyculture combinations the optimum outcome was achieved with medium redclaw crayfish density.     

Analysis of DNA relationships among Aeromonas species by RAPD (randomly amplified polymorphic DNA) typing

Genetic differences between a collection of aeromonads were studied in two laboratories by analysis of randomly amplified polymorphic DNA (RAPD). A single randomly designed primer, generated reproducible profiles of genomic DNA in both laboratories for Aeromonas salmonicida subspecies salmonicida, although the profiles differed between laboratories. Analysis of atypical strains of A. salmonicida and isolates of the A. hydrophila group produced scattered profiles in both laboratories. The uniform fingerprints produced for A. salmonicida subspecies salmonicida indicate genomic homogeneity. The scattered RAPD profiles of the motile aeromonads demonstrate the genomic diversity of this group. A group of unspeciated motile aeromonads gave uniform fingerprints, suggesting the possibility of a genomically homogeneous species. Although the RAPD technique is susceptible to the effects of minor technical variations, this study has demonstrated that where there is DNA similarity, it can be recognized, and where there is diversity, differentiation can be made. RAPD promises to be useful in epidemiological studies for rapid identification of bacteria where a source of reference DNA is available and may be useful in preliminary investigations of relatedness within groups.     

Inheritance and reliability of random amplified polymorphic DNA-markers in two consecutive generations of common carp (Cyprinus carpio L.)

Random amplified polymorphic DNA (RAPD) markers have been used in a variety of genetic studies in fisheries and aquaculture. Most population studies are performed without preliminary data demonstrating the Mendelian inheritance and reproducibility of RAPD markers. In this study, the inheritance and reproducibility of RAPD markers was examined in two consecutive generations of common carp, Cyprinus carpio L. Variability and segregation of RAPD markers were investigated in one F₁ progeny and three F₂ progenies. Seventy-four RAPD markers were generated by five primers using DNA extracted from the initial ornamental (koi) common carp female and wild-type colour common carp male. Fifty-five of these RAPD markers were transmitted to the F₁ progeny and the inheritance patterns were analysed. Twenty RAPD markers were fully reproducible and demonstrated dominant simple Mendelian inheritance patterns in two consecutive generations. Twenty-four RAPD markers were not reproducible in all progenies. Thirteen markers displayed inheritance ratios in the progenies that did not fit simple Mendelian inheritance patterns. Non-reproducibility of RAPD markers and distorted ratios may be caused by the absence of amplification, poor amplification or by the appearance of artefact bands. Random amplified polymorphic DNA markers with poor reproducibility and non-Mendelian inheritance can lead to misinterpretations of data in population studies, resulting in errors in the estimation of genetic diversity within and between individual populations. Therefore, it is recommended to first identify the set of reproducible RAPD markers that demonstrate Mendelian inheritance before application of the RAPD technique in population studies.     

4种笛鲷属鱼类随机扩增多态性DNA及遗传多样性研究

使用RAPD技术分析了红鳍笛鲷、紫红笛鲷、勒氏笛鲷和约氏笛鲷的种内遗传多样性、种间遗传多样性和种间亲缘关系。在120个随机引物中筛选出扩增效果好的引物36个,平均每个引物在每个个体上产生7．7个条带。种内遗传多样性研究结果显示：遗传距离(D)分别为0．2257,0．2228,0．2679和0．2247;遗传多样性指数(H)分别为0．1854,0．1904,0．2015和0．1865。说明目前湛江近海这4种笛鲷的遗传多样性比较丰富,它们的种质资源仍处于一个良好的水平。种间遗传多样性结果显示：勒氏笛鲷与约氏笛鲷遗传距离(Dpq)最小,亲缘关系最密切,勒氏笛鲷与红鳍笛鲷种间Dpq最大,亲缘关系最远。     

Genetic diversity and species identification of Argulus parasites collected from major aquaculture regions of India using RAPD‐PCR

Argulus is one of the most important fish parasites that cause heavy economic loss to aquaculture industry. The present investigation was undertaken to study the genetic diversity of the Argulus sp. collected from 13 locations representing major aquaculture zones in India by RAPD analysis and to develop species‐specific markers. Thirteen random decamer primers were used to amplify DNA fragments from three individual parasites of each location. Of the 172 bands scored by the primers, 168 were polymorphic. The per cent polymorphic loci and gene diversity values varied within a range of 8.14–43.02 and 0.0342–0.1727 respectively. Nei's genetic similarity between populations across all the primers ranged from 0.363 to 0.969. The dendrogram based on Nei's genetic distance showed two clusters; Bangalore and Mandi populations forming one cluster, and the rest in another cluster. The clusters also revealed strong correlation with the species identified as A. japonicus and A. siamensis respectively by morphological method. The study thus indicated A. siamensis as the major prevalent species in carp culture farms in India. Species‐specific primers were designed from unique sequences cloned from RAPD fragments that could able to identify A. siamensis and A. japonicus separately.     

Application of the random amplified polymorphic DNA technique in a study of heterosis in common carp, Cyprinus carpio L.

Random amplified polymorphic DNA (RAPD) analysis was applied to three varieties of common carp, Cyprinus carpio L.: Xingguo red carp, German mirror carp and Russian mirror carp. Twenty-seven decamer primers pre-screened from 40 random primers were used to assay polymorphisms within and between the populations. One hundred and ninety-nine fragments were generated in the three populations; 155 of these fragments were polymorphic. The similarity indices and genetic distances within and between the three populations of carp were calculated. The results indicated that the highest value of similarity within populations was obtained for German mirror carp, and the genetic distance between Xingguo red carp and Russian mirror carp was the farthest. It can be presumed that the heterosis between Xingguo red carp and Russian mirror carp is the highest within these three populations.     

Genetic diversity of Nile tilapia populations revealed by randomly amplified polymorphic DNA (RAPD)

Genetic diversity of the Nile tilapia Oreochromis niloticus collected from the river Nile (Cairo, Assuit and Qena) and two Delta lakes (Burullus and Manzalla) in Egypt was examined by the analysis of randomly amplified polymorphic DNA (RAPD). Of 25 primers examined,21 primers produced 230 RAPD bands. The percentage of polymorphic bands in Manzalla (29.4%) and Burullus (24%) populations was low compared with Assuit (30.54%), Cairo (33.5%) and Qena (44.84%) populations. The highest percentage of polymorphic bands was observed in the Qena population,suggesting a greater potential for use in breeding programs. The molecular phylogenetic tree constructed by unweighted pair‐group method of analysis shows Manzalla and Burullus populations strongly linked and separate from the Assuit and Cairo populations, with Qena population as outgroup. The data serve as a baseline analysis of the current genetic diversity found among O. niloticus populations in Egypt.     

尼罗罗非鱼肌肉蛋白质双向电泳体系的建立

为建立1种尼罗罗非鱼肌肉组织蛋白质的双向电泳体系,实验将尼罗罗非鱼肌肉组织蛋白质提取后,用双向电泳(2-DE)分离蛋白质,分别对蛋白质样品的制备方法、IPG胶条长度及p H范围、SDS-PAGE凝胶浓度及染色方法4个关键因素进行了探索和优化。结果显示,采用液氮研磨-丙酮沉淀法制备样品蛋白质,使用24 cm p H 4~7的IPG胶条进行第一向等电聚焦电泳,第二向SDS-PAGE电泳采用浓度为12.5%的凝胶进行,双向电泳后的凝胶采用硝酸银染色法进行染色处理,该条件下扫描所得尼罗罗非鱼肌肉蛋白质二维电泳图谱具有蛋白质分离程度好、斑点清晰、分辨率高及横纹少等优点。研究表明,技术体系适用于尼罗罗非鱼肌肉蛋白质的分离,可用于后续罗非鱼肌肉蛋白质组学研究。     

三个不同养殖群体金鱼遗传多样性的RAPD分析

采用随机扩增多态性DNA(RAPD)方法对金鱼3个不同地区养殖群体-天津塘沽(TJ)、江苏苏州(JS)及福建福州(FZ)群体各20ind的DNA多态性进行了分析。在事先优化的反应条件下,所使用的100个随机引物中,有14个引物扩增出清晰稳定的片段,共计103条,大小在200~2500bp,其中多态性片段27条。金鱼总的DNA多态位点百分率为26.21%。数据分析结果显示,3个金鱼群体内遗传多样性偏低,遗传相似度为0.9519~0.9785。金鱼3个群体总的Nei基因多样性指数为0.1061,Shannon信息指数为0.1547。金鱼3个群体间平均遗传距离为0.0375,基因分化系数Gst为0.243,表明3个养殖群体间产生了一定程度的遗传分化。UPGMA法和NJ法的聚类分析显示,两者结果一致。TJ群体和JS群体优先聚类,再与FZ群体聚类。     

尼罗罗非鱼8个养殖群体线粒体控制区遗传多样性和遗传关系分析

 利用线粒体DNA控制区部分序列对中国8个尼罗罗非鱼 (Oreochromis niloticus) 养殖群体(埃及、吉拉达、美国、鹭业、吉诺玛、宝路、广东、新吉富)的遗传多样性和相互间遗传关系进行了分析。在所分析的237个样本中, 可归结为15种单倍型, 其中单倍型BL1为宝路(BL)、埃及(EGY)、吉拉达(GLD)、吉诺玛(GNM)和鹭业(LY) 5个群体所共享, 但没有一个单倍型为所有群体共享。8个群体内的核苷酸多态位点数(S)在4~83, 平均核苷酸差异数(K)的范围为0.50~37.26, 单倍型多样性(h)为0.190 8~0.802 3, 核苷酸多样性(π)为0.000 8~0.056 9。AMOVA分析与群体间两两比较的遗传分化指数(F_ST)表明, 这些罗非鱼群体存在显著的遗传分化与差异(P<0.01)。利用Kimura two-parameter模型分析的系统发育关系表明: 宝路、广东和新吉富3个群体亲缘关系较近, 它们聚为一大支; 埃及、美国、吉拉达、鹭业、吉诺玛这5个群体聚为另一大支。单倍型网络(NETWORK)结果显示, 8个群体并没有明显的谱系分化。本研究结果旨为尼罗罗非鱼种质资源的聚合利用奠定基础。

     

吉富罗非鱼与奥利亚罗非鱼自繁与杂交 F1遗传特性与抗病力分析

以吉富罗非鱼与奥利亚罗非鱼为繁育亲本,采用完全双列杂交繁育4组F1,将初始规格基本一致的4组罗非鱼饲养100 d后,运用“加性-显性”遗传分析模型,分析了4组F1罗非鱼8个生长相关性状杂种优势、遗传效应以及与性状间的相关性.结果表明:(1)F1群体平均优势为0.129 4～0.368 4.除尾柄长超亲优势较大外,其他性状的群体超亲优势较小或表现出负向超亲优势.(2)8个性状的广义遗传率(HB)为0.714 2～0.995 3,表明加性效应和显性效应对性状的遗传变异影响极显著(P＜0.01).除尾柄长外,其他性状的狭义遗传率(HN)介于0.469 4～0.737 9,表明加性遗传方差在表型方差中所占比率较高.(3)体质量、全长、体长、体高、体宽、头长、尾柄长、尾柄高性状之间表型相关在0.776 6～0.999 7范围内,而遗传相关在0.994 1～1.000 0之间,表明这些性状间都存在极显著的正相关.取样结束后,采用3.95×106 CFU/mL的海豚链球菌菌液进行腹腔感染,吉富罗非鱼自繁组F1代12 h后出现死亡,而奥利亚罗非鱼自繁组F1192 h后才出现死亡.384 h后,吉富罗非鱼自繁组F1死亡率为40％,正反交组F1分别为20％和23.3％,奥利亚罗非鱼自繁组F1死亡率最低,为6.67％.研究结果表明,除尾柄长外,杂交F1的其他性状不具备超亲优势,然而杂交可以提高选育后代的抗病力.