几种DNA分子标记技术及其在水产动物中的应用

本文介绍TRFLP、RAPD和AFLP分子标记技术的原理及其特点，并论述了三种DNA分子标记技术在水产动物种质资源和遗传育种研究中的应用。     

AFLP分子标记技术及其在水产动物中的应用

AFLP分子标记技术是DNA指纹技术的重大突破，由于其快速、简便、有效，因而在医学、农业、林业、畜牧业、渔业等领域中得到了广泛的应用。近年来人们不断地将这一技术完善和发展，使得AFLP成为迄今为止最有效的分子标记。本文简述了AFLP技术的基本原理、技术流程以及在水产动物中的应用情况。     

DNA分子标记技术

本文概述了DNA分子标记技术的特点、种类,以及常用分子标记技术:限制性片段长度多态性(RFLP)、随机扩增多态性(RAPD)、扩增长度多态性(AFLP)的原理、特点和操作方法。     

鱼类遗传研究中AFLP标记技术的应用

扩增片段长度多态性(Amp lified Fragm ent Length Polymorph ism,AFLP)是一种新的DNA指纹技术,具有多态性丰富、结果可靠、所需DNA量少等优点。AFLP标记技术在鱼类遗传研究上已得到广泛的应用,并取得了一定成果。     

分子标记技术在水产育种与种质鉴定中的应用

分子标记技术有限制性酶切片段长度多态性、DNA指纹图谱、扩增片段长度多态性、随机扩增多态性、序列特异性扩增区、DNA和RNA测序以及SSLP技术。综述了分子标记各种技术的研究方法和研究成果，介绍了分子标记技术在物种(品种)鉴定与系统发育研究、辅助育种、监测遗传渐渗等方面的应用成果。     

扩增片断长度多态性（Amplified fragment length polymorphism，AFLP）在水产动物研究中应用前景

扩增片断长度多态性（Amplified fragment lengthpolymorphism，AFLP）标记技术是一种新的DNA指纹技术主要用于检测DNA多态性。AFLP技术是根据基因组DNA水平的差异进行检测，不受组织和器官、发育阶段、生理条件等诸多因素影响，因此在水产动物中应用AFLP技术具有广阔的前景，可作为水产动物种质鉴定、亲缘关系及种质分类研究的理想标记。     

SSR和ISSR分子标记技术及其在遗传多态性方面的应用

SSR(Simple SequenceRepeat)和：ISSR(Inter-Simple Sequence Repeat)分子标记技术是近几年发展起来的、以PCR为基础的DNA多态性检测技术。它们通常表现为共显性、呈孟德尔式遗传、具有较好的稳定性和多态性、遗传信息量大，目前已广泛应用于基因组研究的各个领域。本文主要论述了SSR和ISSR分子标记技术的原理及实验中的技术要点。并总结了其在物种亲缘关系和遗传多态性研究、DNA指纹库的建立、遗传图谱的构建和基因定位及分子标记辅助育种等方面的应用。     

微卫星标记技术及其在鸡遗传多样性研究中的应用

微卫星标记技术是目前在鸡遗传标记研究中运用较广泛的一种分子标记技术。微卫星DNA作为一种分子标记,具有数量大、分布广且均匀、多态信息含量高等特点。本文就微卫星标记的结构特点及其在鸡遗传中的应用作一综述。     

水产种苗种质检测的好方法——RAPD技术

RAPD技术是以PCR为基础的一种分子标记技术，具有简单快捷、DNA用量少等优点。本文主要介绍该技术测定水产种苗遗传多样性的方法。探讨了RAPD在水产经济动物的群体遗传多样性方面的应用。     

微卫星标记在海洋经济贝类中的应用

微卫星标记是一种DNA分析手段,是种群遗传学研究中广泛应用的分子遗传标记。本文探讨了海洋贝类微卫星DNA的开发现状,介绍了微卫星的特点。阐述了微卫星标记技术应用于海洋贝类遗传多样性研究、遗传连锁图谱的构建、杂交子代的鉴定及杂种优势和繁殖成功率分析以及物种鉴定等方面的情况,并说明了微卫星标记技术存在的问题和发展趋势。     

西伯利亚鲟（Acipenser baeri）养殖群体遗传结构的AFLP分析

本研究构建了西伯利亚鲟（Acipenser baeri）扩增片段长度多态性（AFLP）分析体系,分析了DNA提取、双酶切反应、连接反应、预扩增反应、选择性扩增反应和银染等步骤。用7对引物对24尾西伯利亚鲟进行了AFLP分析,每对引物扩增的位点数在24～39之间,共扩增出了234个位点,多态性位点为116个,多态性比例为48.83%,平均Nei氏基因多样性指数H为0.4297,平均Shannon氏指数I为0.1233,个体间平均遗传距离为0.1872,表明西伯利亚鲟养殖群体的遗传多样性程度比较低,同时还构建了西伯利亚鲟的聚类图。     

AFLP共显性标记的分离、应用与AFLP技术的改良

AFLP作为新一代的分子标记,其应用越来越广泛,技术也越来越成熟。文章综述了AFLP多态带转化为SNPs、SSR等共显性标记的研究现状及AFLP改进技术的应用发展前景。     

二倍体和四倍体泥鳅线粒体ND-5/6基因多态性的PCR-RFLP分析

采用PCR-RFLP分析方法,对洞庭湖、武汉两地的二倍体和四倍体泥鳅线粒体DNAND-5/6基因多态性及4个群体遗传变异和遗传关系进行了研究。结果表明,120尾泥鳅mtDNAND-5/6扩增片段长度均为2.2kb;从13种限制性内切核酸酶中筛选出6种多态性内切酶(HaeⅢ、DraⅠ、RsaⅠ、TaqⅠ、HinfⅠ、MspⅠ),对扩增产物进行酶切,共检测到66种单倍型。泥鳅群体内单倍型多样性和核苷酸多样性分别为0.7679～0.9385和0.01041～0.03212;其中,洞庭湖二倍体核苷酸多样性最高(0.03212),武汉二倍体其次(0.02452),二倍体群体内核苷酸多样性高于四倍体。群体间的核苷酸多样性(π)大小为0.02588～0.04144,平均值为(0.031737±0.000005)。群体间的核苷酸歧化距离(δ)大小为0.00462～0.01617,平均值为(0.010659±0.000003);其中,武汉二倍体和四倍体之间的核苷酸歧化距离最大(0.01617),武汉四倍体和洞庭湖二倍体之间的核苷酸歧化距离最小(0.00462)。MonteCarlo模拟x2检验表明,4个群体间的单倍型频率存在极...     

No AFLP sex-specific markers detected in Pangasianodon gigas and P. hypophthalmus

Sex-specific DNA markers are useful for hatchery management. Sex identification at early ages can reduce broodstock rearing costs. This study employed the Amplified Fragment Length Polymorphism (AFLP) approach for the identification of sex-linked markers in Pangasianodon gigas and P. hypophthalmus. Eight DNA pools (4 females, 4 males) each from P. gigas and P. hypophthalmus were screened using a total of 570 and 102 different primer combinations, respectively. None of the 570 primer combinations gave sex-associated amplification for P. gigas while 31 of the 102 primer combinations gave apparent sex-associated amplification across the pooled DNA samples of P. hypophthalmus. However, none of the 45 SCAR markers derived from the presumed sex-specific fragments showed sex specificity when tested using DNA of individual P. hypophthalmus males and females.     

Comparison of conventional and molecular techniques to investigate the intestinal microflora of rainbow trout (Oncorhynchus mykiss)

The concept of the use of probiotic organisms or prebiotic compounds to modify the fish gut microflora is becoming a popular topic for investigation. A major flaw in many such studies is a failure to consider fully the nature of the established microflora, which is to be modified pre-, or probiotically. Since it is widely accepted that a large proportion of bacteria are non-culturable, the use of conventional bacteriological (culture) techniques alone to investigate fish intestinal microflora may be expected to bias results. We report a study designed to investigate the normal intestinal microflora of rainbow trout (Oncorhynchus mykiss) using both conventional bacteriological and molecular methods.Over an 18 month period, the intestinal microflora of a single population of laboratory-raised rainbow trout was investigated. Bacteria isolated using bacteriological techniques were identified using the BiOLOG system and 16S rRNA gene sequencing. Dominant bacteria consistently were Aeromonas sp. and Carnobacterium piscicola, demonstrating that the microflora is stable in fish kept in defined conditions. Restriction Fragment Length Polymorphism (RFLP) and 16S rRNA gene sequence analysis was used to investigate anaerobic and non-culturable bacteria. An obligate anaerobe, Clostridium gasigenes, was shown to be among the dominating intestinal microflora.     

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.     

吉富罗非鱼不同选育群体的遗传多样性

用微卫星和扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分子标记对海南、广州和青岛吉富品系尼罗罗非鱼的遗传多样性和遗传分化进行了研究。9对微卫星引物和4对AFLP引物的分析结果一致。微卫星分析表明,青岛吉富鱼的平均等位基因数(4.8)、平均观测杂合度(0.528)、平均多态信息含量(0.605)最高,海南吉富鱼的平均等位基因数(4.4)、平均观测杂合度(0.479)、平均多态信息含量(0.549)最低。AFLP分析显示,青岛吉富鱼的多态位点比例(48.4%)和基因多样性(0.245)最高,海南吉富鱼的多态位点比例(36.3%)和基因多样性(0.147)最低。这些表明,青岛吉富鱼的遗传多样性最高,海南吉富鱼最低。遗传分化分析表明,两两群体间遗传分化显著(微卫星FST为0.07~0.11,P<0.01;AFLPFST为0.24~0.29,P<0.01)。AMOVA分析显示,大部分遗传变异(微卫星的分析结果为91.26%;AFLP的为67.6%)来源于群体内个体间,表明吉富鱼选育品系尚具有进一步选育的潜力。     

A preliminary genetic map of Zhikong scallop (Chlamys farreri Jones et Preston 1904)

Zhikong scallop (Chlamys farreri Jones et Preston 1904) is one of the most important aquaculture species in China. The development of a genetic linkage map would provide a powerful tool for the genetic improvement of this species. Amplified fragment length polymorphism (AFLP) is a PCR‐based technique that has proven to be powerful in genome fingerprinting and mapping, and population analysis. Genetic maps of C. farreri were constructed using AFLP markers and a full‐sib family with 60 progeny. A total of 503 segregating AFLP markers were obtained, with 472 following the Mendelian segregation ratio of 1:1 and 31 markers showing significant (P<0.05) segregation distortion. The male map contained 166 informative AFLP markers in 23 linkage groups covering 2468 cM. The average distance between markers was 14.9 cM. The female genetic map consisted of 198 markers in 25 linkage groups spanning 3130 cM with an average inter‐marker spacing of 15.8 cM. DNA polymorphisms that segregated in a 3:1 ratio as well as the AFLP markers that were heterozygous in both parents were included to construct combined linkage genetic map. Five shared linkage groups, ranging from 61.1 to 162.5 cM, were identified between the male and female maps, covering 431 cM. Amplified fragment length polymorphism markers appeared to be evenly distributed within the linkage groups. Although preliminary, these maps provide a starting point for the mapping of the functional genes and quantitative trait loci in C. farreri.     

An assessment of genetic diversity in wild and captive populations of endangered Japanese bitterling <Emphasis Type="Italic">Tanakia tanago</Emphasis> (Cyprinidae) using amplified fragment length polymorphism (AFLP) markers

ABSTRACT:   The Japanese bitterling Tanakia tanago (Cyprinidae) is on the verge of extinction in the wild, placing great importance on captive breeding programs for current conservation of the species. However, the loss of genetic diversity during captive breeding is an ongoing matter of concern. Since some captive populations have been almost monomorphic in mitochondrial DNA (mtDNA), this hampers assessments of their genetic diversity during captive breeding. To more accurately assess their genetic diversity, one wild and three captive populations were examined using amplified fragment length polymorphism (AFLP) markers. Estimates of average heterozygosity and nucleotide diversity ranged 0.0479–0.1920 and 0.0023–0.0088, respectively, enabling comparison of genetic diversity among the wild and captive populations, and among year-classes of captive populations. Significant differences in numbers of amplified fragments and proportions of polymorphic fragments were observed among year-classes of all populations. The indices of genetic diversity calculated from AFLP seemed to be, however, less sensitive to weak bottlenecks. No continuous decrease in genetic diversity in nuclear DNA was detected in presently captive populations. This supports the possibility of re-introduction of the captive populations into the original habitats, although survival and reproductive ability in the wild must be taken into consideration.     

4种沼虾的SRFA指纹研究↑（*）

本文用沼虾属（Ｍａｃｒｏｂｒａｃｈｉｕｍ）的４个主要种：罗氏沼虾（Ｍ．ｒｏｓｅｎｂｅｒｇｉ）、海南沼虾（Ｍ．ｈａｉｎａｎｅｎｓｅ）、日本沼虾（Ｍ．ｎｉｐｐｏｎｅｎｓｅ）、粗糙沼虾（Ｍ．ａｓｐｅｒｕｌｕｍ）作材料，构建了它们的ＳＲＦＡ－ＤＮＡ指纹，经比较得到这４个种的ＳＲＦＡ分子标记，并构建了它们之间的谱系关系图，发现日本沼虾和海南沼虾在遗传上比较接近，属于同一“支序”，粗糙沼虾与另外３者在遗传上都相距较远。