坛紫菜种质材料DNA指纹图谱的构建

采用20对简单重复序列(SSR)引物对福建省坛紫菜种质资源库中保存的44个坛紫菜种质材料进行了遗传分析,共检测到了81个多态性位点,每对引物检测到的等位基因介于2~6个之间,平均为4.05个,引物的PIC值介于0.15~0.79之间,平均为0.57。然后根据3对引物(Phes08,Phes03和PC13)扩增出的16个多态性位点构建了44个坛紫菜种质材料的指纹图谱库,使得每一种质材料均具有其特异的DNA指纹图谱。并根据指纹图谱中各扩增位点条带的有无,将指纹图谱转化成了计算机可以识别的数码指纹,开发了相应的数码指纹识别软件,为坛紫菜种质的自动化鉴定及坛紫菜种质资源库的信息化管理奠定了基础。     

5个紫菜属物种丝状体的微卫星初步遗传分析

文章利用68对微卫星引物对坛紫菜（Porphyra haitanensis）、半叶紫菜（P.katadai）、条斑紫菜（P.yezoensis）、少精紫菜（P.oligospermatangia）和皱紫菜（P.crispata）5个紫菜属物种的丝状体进行初步的遗传分析,共筛选出20对高多态性适用引物,在10份紫菜丝状体样品扩增位点的多态性比例为95.24%,每对引物可扩增的等位基因位点数为1.22～4.05,平均为2.37;各样品之间的遗传多样性指数为0.254 6～0.899 4;不同物种的遗传距离为0.106 0～1.406 6,同一物种的丝状体材料优先聚类为一支,表明筛选出的微卫星引物具有良好的遗传分辨能力。     

微卫星标记在坛紫菜丝状体品系DNA指纹构建中的应用

用从条斑紫菜EST数据库中筛选合成的微卫星引物对8个坛紫菜丝状体品系进行微卫星DNA指纹扩增。5个微卫星引物共扩增出32条带，其中3对引物所扩增出的5个条带(AU192094—127、AU187410—335、AU187410—190、AU194267—203和AU194267—328)被用来构建8个坛紫菜丝状体品系的DNA指纹。在这个图谱中，每个丝状体品系都有独一的指纹模式，彼此很容易被区分开。所获得的DNA指纹图谱，可用来进行孟德尔分离研究，及为坛紫菜纯系鉴定提供分子基础。     

4种紫菜叶状体的ISSR分子标记分析

4种紫菜样品分别为采自青岛海区的野生条斑紫菜（Porphyra yezoensis）、华北半叶紫菜（P.katadai var.hemiphylla）、少精紫菜（P.oligospermatangia）和浙江象山海区人工栽培的坛紫菜（P.haitanensis）.取其10～15 cm叶状体为材料,采用ISSR（Inter-simple sequence repeats）分子标记进行分析.从100个ISSR引物中筛选出22个引物,扩增出清晰、可重复的条带共计247条,其中多态性条带比例达95.5%.根据扩增结果进行的聚类分析显示,条斑紫菜和少精紫菜的亲缘关系最为接近,在聚类图中二者首先聚在一起,接着与坛紫菜相聚,最后与华北半叶紫菜汇聚.本研究还讨论了紫菜属海藻的遗传多样性以及种间的特异性ISSR分子标记.本研究目的在于为紫菜遗传变异、亲缘关系以及分类研究、探索新的分析手段.[中国水产科学,2006,13（3）：371-377]     

ISSR标记在坛紫菜不同色泽丝状体种质鉴定中的应用

ISSR标记在坛紫菜不同色泽丝状体种质鉴定中的应用=Application of ISSR markers in germplasm identification of different color’s Porphyra haitanensis filament strains［刊，中］/谢潮添（集美大学水产学院，厦门361021）,纪德华,陈昌生,徐燕,张元//水产学报，—2007，31(1).-105～111 用ISSR分子标记技术对4个不同色泽坛紫菜（Porphyra haitanensis）丝状体品系（分别为桔黄色，褐绿色，棕红色和紫色）及1个野生型对照丝状体品系（褐红色）进行了遗传分析，共筛选出11条引物，PCR反应得到了129个扩增位点，多态位点比率达88.4％，有效等位基因数为1.6369，期望杂合度为0.3609，Shannon多样性指数为0.5265；根据Nei方法计算了这5个坛紫菜丝状体品系间的遗传相似性系数和遗传距离，结果发现4个不同色泽丝状体间的平均遗传距离为0.5727，它们同野生型对照的平均遗传距离为0.6564，同时应用UPMGA法对它们进行了聚类分析。最后应用引物812扩增的4个多态性位点构建了这5个不同坛紫菜丝状体的DNA指纹图谱，使每一品系都具有独特的DNA指纹，并将其转化成了计算机可以识别的数码指纹，可以方便地应用于坛紫菜丝状体的种质鉴定中。结果表明ISSR分子标记技术可以作为坛紫菜丝状体种质鉴定的有效技术手段，提供快捷、准确的鉴定结果。图3表5参24 关键词：坛紫菜; ISSR标记; 遗传分析; 种质鉴定 E-mail: cschen@jmu.edu.cn     

黄姑鱼异质雌核发育子代的遗传分析

为了解黄姑鱼（Nibea albiflora）异质雌核发育子代的基因纯合情况，利用微卫星标记（SSR）和扩增片段长度多态性标记（AFLP）对黄姑鱼异质雌核发育家系进行遗传鉴定和分析。结果显示：（1）雌核发育家系在4个SSR位点和5对AFLP引物组合扩增出的位点均未发现父本特异性等位条带，表明雌核发育体比率为100%。（2）用于遗传分析的7个SSR位点在雌核发育家系和正常交配家系中均未见完全纯合的情况，雌核发育家系7个SSR位点的平均纯合度为0.382，是正常交配家系（0.161）的2.37倍。雌核发育家系各个体的纯合位点数为0~6个，纯合位点所占比例为0~85.7%。（3）5对AFLP引物共扩增出182条清晰的扩增条带，其中有21条父本特异性条带和16条母本特异性条带。16条母本特异性条带中有7个条带在雌核发育家系中显著偏分离（P<0.05）。雌核发育家系和正常交配家系多态性条带比例分别为14.7%和20.3%。（4）雌核发育家系与母本的遗传相似度高于与正常交配家系的遗传相似度，正常交配家系同父本和母本的遗传距离大致相同。研究结果表明，黄姑鱼异质雌核发育二倍体家系的遗传纯合度显著高于正常交配家系，人工诱导雌核发育是促进基因纯合的一个有效途径，它不仅可以加速有利基因的纯合固定，还可以加速有害基因的淘汰，从而有效提高育种效率。     

条斑紫菜品系评价方法的探讨

总结并分析了“国家级紫菜种质库”中有关紫菜种质材料的原始数据和资料,调查研究了条斑紫菜不同品系的形态结构等生物学特性及环境适应性表现;通过对条斑紫菜不同品系经济性状的定量测评、丝状体生长发育特征的测评、品系产品生化成分的分析评价以及分子标记技术在条斑紫菜种质评价分析中的应用等研究,初步得到了条斑紫菜品系的评价方法;筛选...     

草鱼野生群体和人工繁殖群体遗传结构的比较研究

采用新型分子标记SRAP(Sequence-related amplified polymorphism)对草鱼(Ctenopharyngodon idella)1个野生群体(来自邗江草鱼国家级原种场)和2个人工繁殖群体(分别来自淡水中心良种场和无锡前洲水产良种场)进行遗传多样性分析,从不同引物组合中筛选出8组条带清晰、多态性丰富的引物组合,每个引物组检测到的位点数为12～21个,在3个草鱼群体中共检测到120个位点,其中多态性位点有92个,多态位点比例为76.67%,显示了较高的多态性。野生群体与2个人工繁殖群体多态位点比例分别为67.62%、59.81%、53.33%,平均杂合度分别为0.214 3、0.211 0、0.172 2;邗江野生群体与2个人工繁殖群体间的遗传距离分别为0.098 0、0.115 9,两个人工繁殖群体间遗传距离为0.095 9。结果表明,草鱼人工繁殖群体遗传多样性有所下降。比较各扩增位点显性基因型频率在不同区间的分布发现,人工繁殖群体低频位点明显减少而隐性纯合基因位点显著增加。     

中国对虾SRAP分子标记体系正交优化及在遗传多样性分析中的应用

利用正交设计L16(45)对影响中国对虾SRAP分子标记分析的5个因素(Taq酶,Mg2+,模板DNA,dNTP和引物浓度)在4个水平上进行了优化,筛选出各反应因素的最佳水平为:20μl反应体系中包含1.0UTaq酶,2.0mmol/L的Mg2+,40.0ng的模板DNA,0.125mmol/L的dNTP以及0.4μmol/L的引物,退火温度为53.5℃。研究表明,各因素的不同水平对扩增结果有显著影响,其中Mg2+影响最大,影响大小顺序依次为Mg2+,Taq酶,引物,dNTP和模板DNA。利用优化的SRAP分子标记体系对中国对虾"黄海1号"第12世代选育群体进行了遗传多样性分析,实验结果表明,此标记技术可作为中国对虾遗传分析的良好技术工具。遗传多样性分析共筛选出8对可以扩增出清晰稳定条带的引物组合,共获得171个位点,其中多态性位点154个,占90.08%;有效等位基因数为1.7741,期望杂合度均值为0.4219,Shannon多样性指数为0.6055。上述参数值均高于应用AFLP技术对前几代选育群体的遗传多样性分析结果。     

长江野生鳡鱼子一代养殖群体的遗传多样性分析

利用ISSR分子标记技术检测了长江野生鳡子一代养殖群体的遗传多样性。在77个引物中, 对筛选出条带清晰、稳定的5个引物扩增结果进行统计, 其中二碱基重复序列2个, 三碱基重复序列1个, 四碱基重复序列2个。共得到24个条带, 其中呈多态性的条带15个, 多态性位点比例达62. 5%。遗传多样性指数为0. 2552, Shannon系数为0. 3707。结果表明, 长江野生鳡子一代养殖群体的多态性位点比例和遗传多样性指数都处于中间水平, 而Shannon系数相对偏低, 个体间遗传差异较大。     

Molecular analysis of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>) by SRAP and SCAR molecular markers

The sequence-related amplified polymorphism (SRAP) technique was used to analyze the gene differentiation between two cultured populations [Freshwater Fisheries Research Center (FFRC) and Qianzhou populations] and one wild population (Hanjian population) of grass carp (Ctenopharyngodon idella). Some loci showed quite different genetic frequencies, attributable to artificial selection, which imply that these fragments are putative markers of germplasm identification. We developed a simple and effective method to further characterize these SRAP fragments. Specific SRAP bands were cut directly from polyacrylamide gels, re-amplified, cloned, and sequenced. Twenty-one putative genetic markers were sequenced, ranging from 137 to 357 bp. The sequences were submitted to the database of the Genome Sequence Survey. A BLAST analysis showed that eight SRAP fragments were highly similar to functional genes, whereas the other 13 had no similarity, indicating that these markers are tightly linked to the germ identification trait although only eight are functional genes. Three primers were designed according to this sequence information and used for PCR amplification of the three populations. A sequence-characterized amplified region (SCAR1) was positively amplified in the artificially cultured populations but not in the wild population. The frequency of the SCAR3 marker in the cultured populations was 87% (26/174), whereas it was only 6% (6/100) in the wild population. A specific band was isolated from all individuals in the wild population with the SCAR3 primers, whereas the specific band was amplified from only seven individuals in the FFRC population and from none of the Qianzhou population. The frequency of SCAR2 in the artificially cultured populations was 96.5%. These results indicate that SCAR1 could be used as a specific molecular marker for population identification. The SCAR markers used in this study offer a powerful, easy, and rapid method for genetic analysis and the discrimination of different populations.     

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     

Analysis on ‘Whole Red’ of patterns Oujiang colour carp (Cyprinus carpio var.color ) by SRAP and SCAR markers

The sequence‐related amplified polymorphism (SRAP) technique was applied to identify the genetic difference between the ‘Whole Red’ (‘WR’) population and the ‘Whole White’ (‘WW’) population. The specific SRAP band, namely SR_2,7^{173 bp} (SR indicates the first two letters of SRAP) from ‘WR’ and ‘WW’, was identified from the amplified bands of 12 primer pairs (PP) screened from 88 SRAP PP. After gel extraction, cloning and sequencing of specific band from the two populations, the sequence was submitted to database of Genome Sequence Survey. blast analysis showed that this SRAP fragment shared high similarity to functional genes of Danio rerio. Four primers (22–26 bases) were designed according to the sequence information. Then, polymerase chain reaction amplification was carried out in the two populations. The experimental results also showed that among four sequence‐characterized amplified region (SCAR) markers, only SC‐3 (154 bp) developing from SR_2,7^{173 bp} (me2‐em7) showed specificity to ‘WR’. Examination with a large sample size showed that SCAR 3 (SC‐3) could be positively amplified with 85% frequency in the ‘WR’ population rather than in the ‘WW’ population. The results indicated that the SC‐3 marker could be used as a specific molecular marker for population identification, providing an effective, easy and rapid method for discriminating different populations and conducting genetic analysis.     

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.     

Species identification of Alaska pollock,Gadus spp., and Micromesistius spp. in cod roe products using a PCR-based method

Fish species identification techniques for authentic food labeling were developed using species-specific PCR primers for cod roe products. A salted, seasoned fish roe product, karashimentaiko (chilli cod roe), is produced from the eggs of Alaska pollock, Theragra chalcogramma, according to the fair trade competition agreement authorized by the Fair Trade Commission of the Japanese government. To examine whether Alaska pollock ovaries or those of other fish species are being used as raw materials for the fish roe products, we developed species identification techniques using PCR amplification of a 255-bp fragment encoding the mitochondrial ATP synthase Fo subunit 6 (ATP6) gene with a species-specific primer set for Alaska pollock mitochondrial DNA. We also designed two species-specific primer sets corresponding to the mitochondrial ATP6 and cytochrome b (cytb) for Gadus spp. and Micromesistius spp. by PCR amplification of 332- and 223-bp fragments, respectively. We examined the species specificity of these PCR-based methods among nine commercially important Gadidae species.     

Sequence characterized amplified regions (SCAR) reveal candidate markers linked to growth traits in the Chinese shrimp Fenneropenaeus chinensis

Growth traits in 150 Chinese shrimps Fenneropenaeus chinensis were analyzed by the sequence characterized amplified regions (SCAR) technique in two populations of sixth-generation cultured shrimps with different ranges of body lengths (CP-a and CP-b) and in the wild-type population (WP). First, 240 random primers were used to screen polymorphic fragments by random amplified polymorphic DNA (RAPD). Population analysis revealed nine RAPD markers correlated with growth traits, including seven positively and two negatively correlated ones. The sequences of the RAPD markers were then used to design longer primers for SCAR. Six primer pairs were obtained, and two of these produced polymorphic fragments among the three groups. One amplified 39, 26 and 27 polymorphic fragments, with band frequencies of 78, 52 and 54% in the CP-a, CP-b and WP groups, respectively. The distributions of polymorphic fragments in the three groups were significantly different (P < 0.05) according to a χ ² test, indicating that they may be candidate markers linked to growth traits. Another primer pair amplified three alleles, resulting in six combinations of genotypes among three groups. Since allele A₁ was only found in the population of shorter shrimps, it may be a negative growth marker.     

Formation and growth of free‐living conchosporangia of Porphyra yezoensis: effects of photoperiod,temperature and light intensity

Porphyra are important economic seaweeds in Asia. New Porphyra breeding technology with free‐living conchocelis was being developed to attempt to supplement or replace the primary method. It is crucial to regulate the entire course of conchocelis development exactly for new Porphyra breeding technology. In this research, laboratory culture studies were undertaken on the effects of external factors (photoperiod, temperature and light intensity) on free‐living conchosporangia formation and growth in the Porphyra yezoensis HB line. The results showed that photoperiod, temperature and light intensity were very important factors affecting the formation and growth of free‐living conchosporangia in P. yezoensis. Conchosporangial formation was found at all photoperiods; however, the ratio of conchosporangia to vegetative conchocelis increased with a decrease in the photoperiods. At 57 μmol photons m^?2 s^?1, the optimal light intensity with the highest conchosporangia formation rate was observed, and when the light intensity was 86 μmol photons m^?2 s^?1 or below 42 μmol photons m^?2 s^?1, the conchosporangia formation was inhibited. The optimum temperature for conchosporangia formation was 25 °C, and the free‐living conchosporangia formation rate decreased with the decreased temperatures. Growth experiments showed that a temperature of 25 °C, a light intensity of 86 μmol photons m^?2 s^?1 and a photoperiod of 12 L:12 D were optimum for promoting the growth of conchosporangia. The present results contribute to the understanding of the factors that control the growth and development of free‐living conchosporangia, laying an important foundation for controlling the development of free‐living conchocelis and breeding with free‐living conchosporangia.     

低温对罗非鱼基因组DNA甲基化的影响

以经过连续多代抗寒选育获得的尼罗罗非鱼耐寒品系为实验材料,采用DNA甲基化敏感扩增多态性(methylation sensitive amplified polymorphism,MSAP)分析方法从全基因组水平上探讨了低温适应对罗非鱼DNA序列中CCGG位点的甲基化水平的影响。结果显示,选用的18对选择性扩增引物共检测到849个位点。耐寒品系检测到的位点数为411个,对照组为438个,其中发生甲基化的位点数分别为72和104个,总甲基化率分别为17.52%和23.74%;全甲基化位点分别为37个和65个,全甲基化率分别为9.00%和14.84%;半甲基化位点分别为35个和39个,半甲基化率分别为8.52%和8.90%。结果分析表明,尼罗罗非鱼耐寒品系的总甲基化水平、全甲基化水平和半甲基化水平较对照组均有一定程度的下降。与对照组相比,尼罗罗非鱼耐寒品系基因组DNA甲基化总体水平下降了6.22%,其中以全甲基化位点变异为主,其下降幅度比例明显,为5.84%。由此可见,经过连续多代的低温胁迫可导致尼罗罗非鱼DNA甲基化水平发生改变,发生了去甲基化反应,表现为基因组甲基化程度降低的特征,说明了DNA甲基化与罗非鱼抗寒性反应密切相关。     

“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.     

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.