RAPD方法对中华绒螯蟹长江、辽河、瓯江三群体的遗传多样性分析

用RAPD技术检测了中华绒螯蟹（E．sinensis）长江、辽河、瓯江三个群体各10个个体的基因组DNA的多态性。经20个随机引物扩增得到每一个体的多态片段，通过对群体内及群体间共享片段的分析，得到三群体间的相似率数据，为中华绒螯蟹的种群关系的研究提供了重要的资料。

五种鲤科鱼类的RAPD分析兼论稀有鮈鲫的系统位置

本文作者采用20个随机引物，对五种鲤科鱼类，即稀有鲫、中华细鲫、麦穗鱼、唐鱼和草鱼的基因组进行了DNA随机扩增分析（RandomAmplifiedPolymorphicDNA，即RAPD）。其目的是为了探讨稀有鲫的系统分类位置。研究结果表明在所研究的物种中，稀有鲫与中华细鲫和唐鱼有更近的亲缘关系。

Increasing the genetic uniformity of bighead carp [Aristichthys nobilis (Richardson)] by means of spontaneous diploidization of gynogenetically activated eggs

Three groups of gynogenetic diploid bighead carp were successfully obtained by means of artificial gynogenesis. The activation rates of gynogenesis varied from 75.9% to 98.8%, and the frequency of spontaneous diploidization was around 0.4%. Over 2000 normally gynogenetic diploid fry were obtained in three gynogenetic groups. The haploid karyotype consisted of nine metacentric, 12 submetacentric, three subtelocentric chromosomes and 45 arms. The chromosome number was 48 from gynogenetic diploid. The results showed that the genetic material of offspring was maternal. The aneuploid hybrid embryos of bighead carp and Xingguo red common carp with chromosome numbers ranging from 28 to 73 did not survive post hatch, likely the result of incompatibility between the nucleus and the cytoplasm of two parents. Sixty RAPD primers from three groups were used for total DNA amplification of gynogenetic offspring, maternal and 'paternal' fish. A total of 451 bands were amplified from three kinds of samples above. From maternal bighead carp, 256 bands were amplified; however, there were 251 shared bands between maternal and gynogenetic bighead carp. From artificial gynogenetic offspring, two 'paternal' DNA segments without an expression function were found. An UPGMA tree showed that gynogenetic offspring were closely clustered and the genetic identity among them was very high (0.956).

Genetic structure and low-genetic diversity suggesting the necessity for conservation of the Chinese Longsnout catfish, Leiocassis longirostris (Pisces : Bagriidae)

Genetic variation and phylogenetic relationship of Leiocassis longirostris populations from the Yangtze River were investigated at mitochondrial DNA level. The samples were collected from the upstream and mid-downstream of the Yangtze River. Three mitochondrial DNA fragments, ND5/6, cytochrome b (Cyt b) and control region (D-loop), were amplified and then digested by 10 restriction endonucleases. Twenty-three D-loop fragments randomly selected were sequenced. Digestion patterns of ND5/6 by AluI and HaeIII, D-loop by HinfI and RsaI, and Cyt b by HaeIII were polymorphic. Ten and eighteen haplotypes were obtained from RFLP data and sequence data, respectively. The individuals from upstream and mid-downstream of the Yangtze River were apparently divided into two groups. The average genetic distance was 0.008 and 0.010 according to the two data. Low diversities and decreasing abundance indicated that Leiocassis longirostris may be in severe danger and reasonable measures of fishery management should be taken.

Amplified fragment length polymorphism analysis to identify the genetic structure of the Gymnocypris przewalskii (Kessler, 1876) population from the Qinghai Basin, China

Amplified fragment length polymorphism (AFLP) was used to analyse the genetic structure of 45 individuals of Gymnocypris przewalskii (Kessler, 1876), an endangered and state-protected rare fish species, from three areas [the Heima (HM), Buha (BH) and Shaliu rivers (SL), all draining into Qinghai Lake]. A total of 563 polymorphic loci were detected. The HM, BH and SL populations have 435, 433 and 391 loci, respectively (Zhu and Wu, 1975), which account for 77.26%, 76.91% and 69.45% of the total number of polymorphic loci of each population, respectively. The Nei indices of genetic diversities (H) of the three populations were calculated to be 0.2869 (HM), 0.2884 (BH) and 0.2663 (SL), respectively. Their Shannon informative indices are 0.4244, 0.4251 and 0.3915, respectively. Research results show that the mean genetic distance between HM and BH is the smallest (0.0511), between BH and SL is the second shortest (0.0608), and between HM and SL is the largest (0.0713), with the mean genetic distance among the three populations being over 0.05. Data mentioned above indicate that the three populations have a certain genetic differentiation. The total genetic diversity (H-t = 0.3045) and the mean value of genetic diversity within the population (H-s = 0.2786) indicate that the variations have mainly come from within the population.

Genetic structure of Chinese sucker population Myxocyprinus asiaticus in the Yangtze River based on mitochondrial DNA marker

The sequencing analysis of the mitochondrial DNA control region (mtCR DNA) was performed to assess the genetic divergence and population structure of the Chinese sucker Myxocyprinus asiaticus (Cypriniformes Catostomidae) using four sample lots from natural populations of the Yangtze River. The mtCR DNA sequences of approximately 920 base pairs were obtained. A total of 223 nucleotide positions were polymorphic, and these defined 39 haplotypes. Of the 39 haplotypes, 37 (90%) were not shared, and among the populations as a whole there was little sharing of haplotypes. The average haplotype diversity (0.958) and the average nucleotide diversity (0.052) indicated a higher level of genetic diversity of Chinese sucker through the river. Analysis of molecular variation (AMOVA) of data revealed significant partitioning of variance (P<0.001) among populations (60.29%), and within populations (39.71%). The topology according to the neighbor joining and maximum parsimony methods showed mosaic composition of the 39 haplotypes, suggesting that the populations wore not completely divergent. The pairwise F statistic values, however, indicated that the population structuring existed to some extent among the geographic populations. There was a positive relationship between the aquatic distance and the genetic distance (Fst) among the populations (P<0.05). Based on our data, it is suggested that genetic drift, gene flow, and stochastic events are the possible factors influencing the population structure and genetic variation.

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis, with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5-ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5-ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.

PCR-RFLP analysis of mitochondrial DNA ND5/6 region among 3 subspecies of common carp (Cyprinus carpio L.) and its application to genetic discrimination of subspecies

Mitochondrial DNA ND5/6 region was studied by PCR-RFLP analysis among ten representative strains belonging to three subspecies (Cyprinus carpio carpio, Cyprinus carpio haematopterus and Cyprinus carpio rubrofuscus) of common carp (Cyprinus carpio L.). A total of 2.4 kb fragment was amplified and subjected to restriction endonuclease analysis with nine restriction endonucleases subsequently. The results indicated that each subspecies owned one hyplotype and four restriction enzymes (Dde I, HaeIII, Taq I and Mbo I) produced diagnostic restriction sites which could be used for discriminating the three subspecies and as molecular genetic markers for assistant selective breeding of common carp.

Transgenes in F-4 pMThGH-transgenic common carp (Cyprinus carpio L.) are highly polymorphic

To gain information on the integration pattern of pMThGH-transgene, 50 transgenes were recovered from F-4 generation of pMThGH transgenic common carp (Cyprinus carpio L,) and 33 recovered genes were analyzed. The restriction maps of these recovered genes were constructed by digestion with five kinds of enzymes. These transgenes can be classified into 4 types according to their restriction maps. Only one type of transgenes maintains its original molecular form, whereas the other three types are very different from the original one and vary each other on both molecular weight and restriction maps. This implies that the sequences of most transgenes have been deleted and/or rearranged during integration and inheritance. The results of PCR amplification and Southern blot hybridization indicate that MThGH in Type I transgene keeps intact but most of its sequence has been deleted in other three types. All these results suggest that transgenes in F-4 generation of transgenic carp are highly polymorphic. Two DNA fragments concerning integration site of transgenes were cloned from recovered transgenes, and found to be homologous to the 5'UTR of beta -actin gene of common carp and mouse mRNA for receptor tyrosine kinase (RTK), respectively.

RAPD and AFLP techniques for the analysis of genetic relationships in two genera of Decapoda

RAPD was used fur analysing three (sub-)species of mitten crabs (Eriocheir sinensis, E. japonicus, and E. japonicus hepuensis) and three populations of E. sinensis. The results show that their relationships on DNA level are similar to the classical taxonomic hypotheses (Dai, 1991). No diagnostic RAPD marker could be found, but there were statistically significant genetic differences among these taxa (P < 0.001) or populations (P < 0.001). That is, the intraspecific similarities were larger than the interspecific similarities; the intrasubspecific similarities were larger than the intraspecific similarities; and the intrapopulational similarities were larger than the interpopulational similarities. In AFLP analysis, no significant genetic difference has been found between E. sinensis and E. japonicus, but AFLP markers among four species of Macrobrachium (M. rosenbergii. M. nipponense, M. hainanense, and M. asperulum) were found. The DNA similarities among these four species of Macrobrachium are in accordance with morphological similarities.

Frankia的REP-PCR、RAPD-PCR鉴定及遗传多样性研究

本文采用溶菌酶法、CTAB法、微波法及CTAB+微波法等4种方法进行Frankia菌Cp11基因组DNA的提取。结果表明四种方式均可行，但以CTAB法及微波法最为有效。提取方法与REP-PCR带型间关系的研究表明，方法上的差异不会影响到REP-PCR带型的变化。对14株Frankia菌株作REP-PCR分析，并对之进行比较分类，证明REP-PCR方法不但能实现菌株间的差异鉴别，还能有效地进行Frankia菌的分类。这一结果与DNA同源相关性所得的分类结果具有良好的相关性。选用20个随机引物，对分自2个分类接种群的8株Frankia纯培养的总DNA进行随机扩增。其中引物OPW15和OPW16能扩增得到较为稳定的RAPD图谱。扩增产物分子量大都分布在0.5-4Kb之间。从稳定的RAPD扩增图谱看，Frankia菌间存在有丰富的遗传多样性；在选定适当引物情况下，能依据共同带型将Frankia菌化归为同一分类接种群。

粗枝云杉( Picea asperata Mast.)天然群体的遗传变异

西南地区在我国的经济发展和生态环境建设中占重要地位，但也是我国生态环境最脆弱的地区之一，生态系统退化，生态功能减弱，严重制约着西南林业的可持续经营与发展。本项目采用DNA 分子标记SSR 研究不同生境条件下粗枝云杉群体的遗传变异及其时空分布格局，考察遗传变异与复杂的山地生态环境间的潜在联系，系统地揭示粗枝云杉天然群体与环境系统相互作用的生态适应与分子进化机制。粗枝云杉适应性强，生长迅速，在植树造林和工业用材方面占有重要地位，研究成果可为中国西南部亚高山天然林的可持续经营及退化生态系统的恢复与重建提供理论依据和科学指导。主要研究结果如下： 1. SSR 位点变异丰富，等位基因频率的分布格局多样。7 个SSR 标记全是多态位点，每位点的等位基因数变化范围为13~24，平均为19.9 个。SSR 位点的等位基因片段长度范围变化较大。73.1%的等位基因变异遵循逐步突变模型(SSM)而发生1 个重复基元的变化，22.3%和4.6%的变异分别按两阶段突变模型(TMP)发生1 个重复基元以上的变化和在SSR 位点侧翼区发生1 个碱基变化的插入-删除事件。 2. 粗枝云杉拥有中等偏高水平的遗传多样性和相对大的群体间遗传分化。通过分析代表10 个群体的250 个个体在7 个SSR位点的变化，调查了源自中国西南山区的粗枝云杉的微卫星变异。相当高的遗传多样性和强烈的群体分化发生在粗枝云杉中， 其群体平均Nei's 期望杂合度为0.707 ， 群体间遗传距离为0.121~0.224(FST)和0.100~0.537(RST)。然而，群体间遗传距离与地理距离之间无相关性，从而排除了简单的距离分离模式并暗示迁移不是影响粗枝云杉遗传变异格局的主要因素。事实上，使用私有等位基因估算的基因流数量非常低，仅等于0.753。等位基因置换检验(Allele permutation tests)揭示逐步突变及遗传漂变都对群体间分化有贡献。另外，在多数位点检测到显著的群体间遗传差异，这个结果说明自然选择，假设通过环境压力，是引起粗枝云杉微地理分化的主要因素之一。根据SSR基因型，250 个粗枝云杉个体的70%被正确地归类入其各自的来源群体，结果表明微卫星(SSR)对区分来自中国不同生态地理位点的粗枝云杉基因型是有效的。 3. 在SSR、RAPD 和AFLP 位点，显著的群体间遗传结构被发现的，但三种标记间遗传分化程度和群体遗传关系有差异。利用来自10 个群体的247 个个体，我们报告了关于样本粗枝云杉群体间遗传关系的总体看法。根据各自对评价遗传关系的信息能力和适用性，SSR、RAPD 和AFLP 标记被选用，三种技术非常有效地区别这些基因型。使用的SSR、RAPD 和AFLP 标记分别估计平均Dice 相似性系数。Mantel 检验产生显著但相对低的共表型适合度(RAPD = 0.63£AFLP = 0.60和SSR = 0.75)。比较三种标记系统，RAPD 和AFLP 共表型指数相对高地相关(r =0.59)，而RAPD 和SSR 及SSR 和AFLP 之间的相关系数分别是0.53 和0.35。所有系统树，包括不同标记资料结合获得的系统树，反映了多数群体依据它们的地理条件而成某种特定关系。结果暗示单个或结合标记系统能用来深入洞察粗枝云杉遗传研究，并且不同标记系统合并资料能提供更可靠的信息。 Southwestern region plays an important role in economic developmentand ecological construction in China. Yet, it is also one of the weak regionsof ecological environment in China with degraded ecosystem and imperfectfunction, which restricts the sustaining management and development ofsouthwestern forestry. The genetic variation and spatial distribution patternof P. asperata populations originating from different habitats wereinvestigated using SSR molecular markers in this study. The correlationsbetween genetic variation and ecological and environmental conditionswere detected, and the interaction between P. asperata populations andenvironmental system and the mechanism of ecological adaption -molecular evolution were revealed. Given the significant ecological andeconomic roles of the fast-growing and wide-adaptive species in reforestation and production of pulp wood and timber, the study couldprovide a strong theoretical evidence and scientific direction for thesustaining management of subalpine natural forest, and the afforestationand rehabilitation of degraded ecosystem. The results are as follows: 1. The genetic variation at SSR loci was abundant and the distributionof allelic frequencies was uneven. All seven loci were polymorphic, and thenumber of alleles per locus varied from 13 to 24 with a mean valueequaling 19.9. The allele sizes at SSR loci were found to vary widely.73.1% of allelic variation followed stepwise mutation model (SSM) whichresults increase or decrease by one repeat type, and 22.3% and 4.6% wereresulted from two-phase mutation model (TMP) with allele size varying bymore than one repeat type and from insertion-deletion events in theflanking regions at SSR loci with a single basepair changing, respectively. 2. P. asperata possessed a moderate to high level of genetic diversityand considerable genetic differentiation. Microsatellite variation of P.asperata. originating from the mountains of southwestern China wasinvestigated by analyzing variation at seven SSR loci in 250 individualsrepresenting ten populations. A fair degree of genetic diversity and strongpopulation subdivision occurred with the mean gene diversity (H) of 0.707,and genetic distances among populations varying between 0.121 and 0.224(FST) and between 0.100 and 0.537 (RST). However, inter-populationgenetic distances showed no correlation with geographic distances between the population sites. This ruled out a simple isolation by distance modeland suggested that migration does not have a great impact. In fact, theamount of gene flow, detected using private alleles, was very low, equalingonly 0.753. Allele permutation tests revealed that stepwise-like mutations,coupled with genetic drift, could contribute to population differentiation.Moreover, significant genetic differences between populations weredetected at most loci. The results indicate that natural selection, presumablythrough environmental stress, may be one of the main factors causingmicro-geographical differentiation in the genetic structure of P. asperata.Based on SSR genotypes, 70% of the 250 individuals were correctlyclassified into their sites of origin. This suggests that microsatellites (SSRs) are effective in distinguishing genotypes of P. asperata originating fromdiverse eco-geographical sites in China. 3. Using a set of 247 individuals from ten P. asperata populations wereport an overview on the genetic relationship among the sampled P.asperata populations. RAPD, AFLP and SSR were used in terms of theirinformativeness and applicability for evaluate relationship and all threetechniques discriminated the genotypes very effectively. Mean Dicesimilarities coefficient were estimated using RAPD, AFLP and SSR,respectively. The Mantel test resulted in a significant but relatively low fit(RAPD = 0.63, AFLP = 0.60 and SSR = 0.75) of cophenetic values.Comparing the three marker systems to each other, RAPD and AFLP cophenetic indices were highly correlated (r = 0.59), while correlationcoefficient between RAPD and SSR was r = 0.53 and between SSR andAFLP was r = 0.35. For all markers a relatively high similarity indendrogram topologies was obtained although some differences wereobserved. All the dendrograms, including that obtained by the combineduse of all the marker data, reflect some relationships for most of thepopulations according to their geographic conditions. The results indicatethat single or combined marker system could be used to insight into geneticstudy in P. asperata and the combined data of different marker systems canprovide more reliable information.

大赖草总DNA转化小麦引起的HMW-GS基因变化及大赖草HMW-GS基因的克隆

应用花粉管通道技术将新疆大赖草总DNA导入小麦，用高重序列分析方法，已为大赖草总DNA转入小麦提供了初步的分子证据。在转 化后代中选育出稳定遗传的大穗变异株系，分析表明，这些转化株中蛋白质含量明显增高(13%-17%)。对基因供体新疆大赖草、受 体春麦761、转化株的高分子量谷蛋白亚基(HMW-GS)进行了SDS-PAGE分析，发现这些转化株中HMW-GS发生了很大变化，并在此基础 上，用来自小麦基因组的四对特异引物，以PCR方法扩增供体、受体以及转化株的1Ax、1Bx、1Dx及1Ay、1By、1Dy型HMW-GS全基因 ，比较他们扩增产物的差异，结果表明，受体与转化株在HMW-GS基因1Ax、1Bx位点上的扩增产物差异不大，在HMW-GS基因位点1Dx 和y型基因上的扩增产物有较大差异，说明了受体在基因位点1Dx、1Ay、1By和1Dy上可能发生了多位点插入，可能由于这些基因位 点上的插入引起了转化株的高分子量谷蛋白亚基(HMW-GS)的变化，这就再一次为大赖草总DNA导入提供了直接的分子证据。虽然大 赖草总DNA导入提高了小麦蛋白质的含量，改变了HMW-GS的组成，部分改变了品质评分，但我们感到这些转化株在品质改良方面仍 有很大余地，如何更好地利用新疆大赖草蛋白质的优良特性及避免总DNA导入给转化株带来的不良性状，一个大赖草HMW-GS基因正 被分离及克隆，并准备将其利用于未来的品质育种当中。Total DNA of Leymus racemosus had been transformed into wheat through pollen tube pathway. Analysis of the repeated gene sequence had provided an elementary proof. Some variant cultivars with big ear were screened from their offsprings, and their protein content increased greatly from 13% (receptor)to 17%(transformed). The result from SDS-PAGE analysis of high-molecular-weight glutenin subunits(HMW- GS) respectively in donor(Xinjiang Leymus racemosus), receptor(spring wheat cultivar 761)and transformed wheats, showed the HMW-GS composition changed in the transformed plants. On the basis of the research, Four special pairs primers from wheat(T.aestivum L.) genome were used to amplify complete coding regions of HMW-GS genes on 1Ax、1Bx 、1Dx and 1Ay、1By、1Dy loci of donor、receptor and the big ear transformed cultivars. By comparing amplified PCR products. Faint differences were found among receptor and transformed cultivar's 1Ax、1Bx PCR amplifed products and apparent differences on those of 1Dx、y-typePCR product. We gueseed that there may be some DNA inserts in 1Dx 、1By、1Dy loci resulted in the changes of the HMW-GS among transformed cultivars. This provides second direct molecular witness to the exogenous DNA introduction. Even though the transformed plants have higher protein content, changed HMW-GS composition, partially improved process quality, there still leave much work to improve quality. In order to make full use of the excellent property of Leymus racemosus protein and avoid the disadvantages introducced by total DNA transformation, a HMW-GS gene of Leymus racemosus was being isolated and cloned.

电子束辐照紫花苜蓿愈伤组织后再生苗的RAPD分析

<正>目的:利用RAPD技术研究了电子束辐照紫花苜蓿愈伤组织后引起苜蓿再生苗基因组DNA的变异,为电子束辐照诱变的分子机制研究提供理论依据。

Frankia菌的遗传多样性的RAPD研究

选用20个随机引物，对分自2个分类接种群的8株Frandia纯培养的总DNA进行随机扩增，其中引物OPW15和OPW16能扩增得到较为稳定的RAPD图谱．扩增产物分子量大都分布在0．5～4Kb之间．从稳定的RAPD扩增图谱看，Frankia菌间存在有丰富的遗传多样性；在选定适当引物情况下，能依据共同带将Frankia菌化归为同一分类接种群．