羊肚菌栽培菌株遗传多样性分析及种特异性RAPD-SCAR标记开发

近年来中国的羊肚菌Morchella spp.栽培技术取得了长足进步,但基础研究薄弱影响其稳产和高产,国内外尚无羊肚菌栽培菌株种质资源遗传多样性的研究报道。本文对来自全国12省份的36个羊肚菌栽培菌株进行了ITS系统发育分析,并采用RAPD进行了遗传多样性评价。结果表明,结合有效的参考菌株序列,通过ITS序列分析可以将供试菌株进行区分和鉴定,在36个菌株中,26个菌株属于梯棱羊肚菌Morchella importuna,其他10个菌株属于六妹羊肚菌M. sextelata;将自40条RAPD引物中筛选出的14条用于供试菌株遗传多样性分析,共扩增出124条多态性条带;UPGMA聚类可将供试菌株分为两大类群,分别对应于ITS系统发育分析中的梯棱羊肚菌和六妹羊肚菌两个物种,梯棱羊肚菌种内菌株多态性高于六妹羊肚菌。OPA17引物和OPA18引物分别在AA02和AA15菌株中扩增出具有唯一性的特征条带,对两个特征条带进行回收测序后,设计出两个特异性SCAR的引物,它们能有效地从36个供试菌株群体中将菌株AA02和AA15鉴别出来。本文首次全面系统地采用ITS分析鉴别了我国羊肚菌栽培菌株的种性,采用RAPD分子标记系统地评价了羊肚菌栽培菌株的遗传多样性,并验证了RAPD分子标记转化为菌株特征性SCAR标记的可行性。     

Identification and Assessing the Cultivars of Laminaria Lamx. （Phaeophyceae） with Molecular Markers

Molecular markers were used to identify and assess cultivars ofLaminaria Lamx. and to delineate their phylogenetic relationships. Random amplified polymorphic DNA (RAPD) analysis was used for detection. After screening, 11 primers were selected and they yielded 133 bands in all, of which approximately 99.2% were polymorphic. The genetic distances between gametophytes ranged from 0.412 to 0.956.Two clusters were formed with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on the simple matching coefficient. All cultivars ofLaminariajaponica Aresch. used for breeding in China fell into one cluster. L.japonica from Japan, L. saccharina (L.) Lam., and L. angustata Kjellm.formed the other cluster and showed higher genetic variation than L. japonica from China. Nuclear ribosomal DNA (rDNA) sequences, including internal transcribed spacers (ITS1 and ITS2) were studied and aligned. The nucleotides of the sequences ranged from 634 to 668, with a total of 692 positions including ITS1, ITS2, and the 5.8S coding region. The phylogenetic tree obtained by the neighbor-joining method favored, to some extent, the results revealed by RAPD analysis. The present study indicates that RAPD and ITS analyses could be used to identify and assess Laminaria germplasm and to distinguish some species and, even intraspecies, in Laminaria.     

Identification and Assessing the Cultivars of Laminaria Lamx.(Phaeophyceae) with Molecular Markers

Molecular markers were used to identify and assess cultivars ofLaminaria Lamx. and to delineate their phylogenetic relationships. Random amplified polymorphic DNA (RAPD) analysis was used for detection. After screening, 11 primers were selected and they yielded 133 bands in all, of which approximately 99.2% were polymorphic. The genetic distances between gametophytes ranged from 0.412 to 0.956.Two clusters were formed with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on the simple matching coefficient. All cultivars of Laminaria japonica Aresch. used for breed ing in China fell into one cluster. L. japonica from Japan, L. saccharina (L.) Lam., and L. angustata Kjellm.formed the other cluster and showed higher genetic variation than L. japonica from China. Nuclear ribosomal DNA (rDNA) sequences, including internal transcribed spacers (ITS1 and ITS2) were studied and aligned. The nucleotides of the sequences ranged from 634 to 668, with a total of 692 positions including ITS1, ITS2, and the 5.8S coding region. The phylogenetic tree obtained by the neighbor-joining method favored, to some extent, the results revealed by RAPD analysis. The present study indicates that RAPD and ITS analyses could be used to identify and assess Laminaria germplasm and to distinguish some species and, even intraspecies, in Laminaria.     

The First Internal Transcribed Spacer (ITS-1) of Ribosomal DNA as a Molecular Marker for Phylogenetic and Population Analyses in Crustacea

The objective of the present study is to explore the feasibility of using the first internal transcribed spacer (ITS-1) of ribosomal DNA as a molecular marker for studying the interspecific and intraspecific genetic variations among crustaceans. We designed primers that could amplify ITS-1 from a majority of taxonomic groups of crustaceans. The gene was found to exhibit a high degree of length polymorphism among different groups, ranging from 182 bp in the barnacle Balanus amphitrite to approximately 820 bp in the spiny lobster Panulirus japonicus. With respect to differences between congeneric species, it was found that the ITS-1 sequences of 3 mitten crabs, Eriocheir sinensis, Eriocheir leptognathus, and Eriocheir formosa, exhibit 5.4% to 16.3% nucleotide divergence, suggesting that ITS-1 is informative for phylogenetic analysis at the species level. Yet there are extensive (0.9%–2.3%) variations within individual E. formosa, so that phylogenetic analyses could be obscured. ITS-1 was found to vary between 2 geographical populations of the shrimp Penaeus japonicus. The variations involved substitutions as well as insertions/deletions between shrimp from Australia and South China Sea. These results show that ITS-1 is highly divergent among different crustaceans and could be an appropriate marker for molecular systematic studies at the species and population levels, although the presence of intragenomic variation needs to be taken into consideration. Received August 15, 2000; accepted February 9, 2001     

Molecular phylogeny of Trichomonadidae family inferred from ITS-1, 5.8S rRNA and ITS-2 sequences

The Trichomonads have been the subject of several molecular studies that reported some discrepancies both at the lower and higher taxonomic levels. The purpose of this study was to make an extensive phylogenetic analysis of the Trichomonadidae using ITS-1/5.8S/ITS-2 sequences, to better understand its phylogeny and the usefulness of this marker. ITS-1/5.8S/ITS-2 sequences of 36 strains from 14 species belonging to Trichomonadidae and Monocercomonadidae were analysed, in which 20 were newly determined. Maximum likelihood, maximum parsimony, neighbour joining, and Bayesian phylogenetic methods were employed in order to reconstruct and compare the evolutionary history of this group. Tetratrichomonas gallinarum and four strains of Tetratrichomonas sp. isolated from bull genital organs were found closely related, confirming the classification of the latter, probably as a new species. The monophyly of Tritrichomonadinae and Trichomonadinae subfamilies were corroborated, with the exclusion of Trichomitus batrachorum from the latter since it grouped consistently with Hypotrichomonas acosta. Tritrichomonas foetus, Tritrichomonas suis and potentially also Tritrichomonas mobilensis seemed to correspond to the same species. Monocercomonas sp. and Ditrichomonas honigbergii emerged as independent lineages, with their phylogenetic positions undetermined. Neither Trichomonadidae nor Monocercomonadidae were supported as monophyletic groups. The ITS-1/5.8S/ITS-2 seems to be a reliable locus for phylogenetic studies in the Trichomonadida, mainly at lower taxonomic levels, and at least up to the family level.     

利用RAPD技术对葱属品种遗传关系的分析

利用随机扩增多态性DNA(RAPD)技术分析了我国栽培的几个主要葱属品种。所用的20个引物,有11个能扩增出重现性好且稳定的谱带,10个品种共扩增出102条带,其中68条具多态性。根据DNA谱带计算的10个品种相似系数范围为0.571～0.947。对10个品种的UPGMA聚类分析表明,章丘大葱(Allium fistulosum var. gigantum cv. Zhangqiu Welsh onion)、天津五叶齐（A. fistulosum var.gigantum cv. Tianjin Wuyeqi Welsh onion）、上海分葱（A. fistulosum var. caepitosum cv.Shanghai Spring onion）、南京冬葱（A. fistulosum var. caepitosum cv. Nanjing Winter onion）和韩国大葱（A. fistulosum var .gigantum cv. Korea Welsh onion）关系较近。根据聚类结果,将章丘大葱、天津五叶齐、上海分葱、南京冬葱、韩国大葱、细香葱（A. schoenoprasum）以及楼葱（A. fistulosum var. viviparum）归为葱组;而将胡葱（A. ascalonicum）、洋葱（A. cepa）及洋葱和大葱的杂交种归为洋葱组。最后根据RAPD标记,讨论了各品种之间的亲缘关系。     

二十八份玉米自交系的RAPD亲缘关系分析

采用RAPD技术,对28份玉米自交系的亲缘关系进行分析。旨在DNA水平上揭示玉米自交系之间的亲缘关系,为进一步提高玉米杂种优势利用水平提供有益的信息从100个10bp随机引物中筛选出24个多态性较好的引物,对28份玉米自交系DNA进行扩增,扩增出24张DNA指纹图谱,其中多态性DNA谱带106条,占总扩增带数的64％。利用DNA扩增结果进行聚类分析,建立了28个玉米自交系的亲缘天系树状图,将供试材料划分为五个类群,RAPD分析结果与已知系谱的亲缘关系基本一致。     

Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.

Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.     

The use of ISSR and RAPD markers for detecting DNA polymorphism,genotype identification and genetic diversity among barley cultivars with known origin

The potential of bulk analyses of RAPD and ISSR-PCR markers for fingerprinting purposes was evaluated using ten RAPD and ten ISSR primers. The phylogenetic relationships of 16 barley cultivars from different countries, and all having a known pedigree, were analysed using 353 PCR markers (125 RAPDs and 228 ISSRs). The band profiles generated were reproducible in spite of the different DNA extractions, PCR techniques, electrophoretic methods and gel scorings used. The RAPD primer S10 and four ISSR primers (811, 820, 835 and 881) were both able to distinguish all cultivars. A strong and quite linear relationship was observed between Resolving Power (Rp) of a primer and its ability to distinguish genotypes. The dendrograms obtained using these two molecular markers are in agreement with their known origin, showing clusters that separate very well the spring/winter and six-rows/two-rows cultivars. Thus, bulk analyses of RAPD and ISSR PCR markers provides a quick, reliable and highly informative system for DNA fingerprinting and also permit to establish genetic relationships which agree with, by other means, known origin of the cultivars.     

甘蔗种质间亲缘关系及特异标记的RAPD分析

采用RAPD技术分析甘蔗种质问的亲缘关系及特异标记。筛选出25个扩增多态性较强的随机引物,构建了41份甘蔗种质的RAPD指纹图谱,并对RAPD数据进行UPGMA聚类分析。甘蔗栽培品种之间、栽培品种与近缘种之间以及近缘种相互间的遗传相似系数变异范围为0．56～0．92,表明所研究的甘蔗种质之间的亲缘关系较近。此外,发现某些甘蔗亲本种质具有特异RAPD标记带。     

The phylogeographic importance of the Strait of Gibraltar as a gene flow barrier in terrestrial arthropods: a case study with the scorpion Buthus occitanus as model organism

The phylogenetic relationship between Buthus occitanus populations across the Strait of Gibraltar was investigated using nuclear 18S/ITS-1 DNA sequences and mitochondrial 16S and COI DNA sequences. All analyses showed that the European samples are highly separated from North African samples, and also suggest the existence of three main groups within this species complex, i.e., an European, an Atlas (=Moroccan samples) and a Tell-Atlas group (=Tunisian samples). The European clade was subdivided into three distinct subclades. The application of a previous calibration of the molecular clock of another buthid species suggested that most of the detected mitochondrial DNA lineages including the European lineages are about three times older than the re-opening of the Gibraltar Strait, and consequently, that other and older vicariant events are responsible for the observed phylogeographic structure of this species complex. Concerning the Moroccan samples, a discordance between nuclear and mitochondrial gene markers was observed. The 18S/ITS-1 gene tree could not resolve the phylogenetic relationships among the Moroccan B. occitanus subspecies and the closely related species B. atlantis, whereas mitochondrial genes suggested the co-existence of several old phylogenetic lineages in Morocco. We hypothesized that this difference may be explained by male-biased gene flow and gene conversion at the tandemly repeated 18S/ITS-1 gene regions.     

Genetic diversity assessment in Portugal accessions of <Emphasis Type="Italic">Olea europaea</Emphasis> by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.     

杨属植物分类的研究进展和展望

自林奈建立杨属（Populus）以来,众多的学者对杨属的分类进行了研究,并建立了较多的分类系统。目前的国际杨属分类,总体上还停留在传统形态分类阶段,存在明显的地域和国别特征。尽管各国在主流上都采用“属—组—种”的分类阶元,但在种、亚种、变种的划分上,却存在很大的不同。中国和美国分别是细分派和聚合派的代表,两国的分类系统存在很大分歧,但各有优点和缺点。即使在国内,杨属分类也还有很多需要解决的问题。新兴的分子系统学研究,为杨属的分类修订提供了一些有益参考,但总体贡献不大。国际杨属分类的趋势和终极目标是建立基于系统发生的统一分类系统。这需要基于种群和综合物种概念,融合形态学、生态学、生物地理学、基因组生物信息学和进化生物学等的综合系统分类来实现。     

木霉属3个中国新记录种

对来自黑龙江的木霉属真菌进行系统分类研究,报道了该属3个中国新记录种：平展橙红木霉Trichoderma auranteffusum,塔梗木霉T. pyramidale 和革菌生木霉T. thelephoricola,对其宏观和微观特征提供了详细描述及图示,系统发育分析为上述种的分类地位提供了佐证。     

西花蓟马检测鉴定技术研究进展

西花蓟马是一种世界性的入侵害虫,2003年首次在北京发现,目前仅在我国局部地区发生。其危害严重,潜在适生区广,且具有进一步扩散蔓延的趋势。快速准确的检测鉴定技术是防止其进一步扩散蔓延,保障我国农业生产健康发展的必要前提。综合相关报道,用于西花蓟马的检测鉴定技术包括传统的形态学特征鉴定法、由此衍生的计算机软件识别法,以及分子检测技术,如RAPD-PCR法、DNA序列分析法、PCR-RFLP法、SCAR分子标记技术、实时荧光定量PCR检测技术以及蛋白质分析技术等。其中,基因芯片和DNA条形编码技术作为新兴的物种鉴定手段,在西花蓟马等入侵物种的检测鉴定中具有广阔的应用前景。     

兔痒螨和水牛痒螨第二转录间隔区（ITS-2）基因序列分析

为了探讨水牛痒螨株和兔痒螨株的分类地位,采用 PCR 技术扩增了四川水牛痒螨株和四川兔痒螨株的第二内部转录间隔区（ITS-2）基因,并与 GenBank 中注册的 5 个国外痒螨株的同源基因进行了比较。序列分析发现：兔痒螨株和水牛痒螨株的序列长度分别为 277 bp 和 281 bp,两序列间存在多处转换、颠换和缺失。四川水牛痒螨株同四川兔痒螨株间及国外痒螨分离株间的 ITS-2 基因同源性较低（87.1%～88.0％）; 而四川兔痒螨株与国外痒螨分离株的同源性较高（95.5％～100.0％）。用痒螨 ITS-2 基因构建的 MP,NJ,ME 及 UPGM 树中,兔痒螨株和水牛痒螨株在不同的系统树中其位置比较固定,且两者相距均较远。根据痒螨 ITS-2 基因同源性分析和系统树构建结果以及其他已报道的相关证据,作者认为：兔痒螨株和水牛痒螨株可能为痒螨属 Psoroptes 中两个不同的种,兔痒螨分离株为马痒螨 P. equi ;而水牛痒螨株与来自兔、山羊、绵羊和黄牛等痒螨株亲缘关系较远,可能为痒螨属中的另一个独立有效种。     

RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species.

Abstract: Twenty-one random and 29 SSR primers were used to assess genetic variation and interrelationships among subspecies and botanical varieties of cultivated peanut, Arachis hypogaea (2n = 4x = 40), and phylogenetic relationships among cultivated peanut and wild species of the genus Arachis. In contrast with the previous generalization that peanut accessions lack genetic variation, both random and SSR primers revealed 42.7 and 54.4% polymorphism, respectively, among 220 and 124 genetic loci amplified from 13 accessions. Moreover, the dendrograms based on RAPD, ISSR, and RAPD + ISSR data precisely organized the five botanical varieties of the two subspecies into five clusters. One SSR primer was identified that could distinguish all the accessions analysed within a variety. Although the polymorphic index content varied from 0.1 to 0.5 for both ISSR and RAPD markers, primer index values were substantially higher for RAPD primers (0.35-4.65) than for SSR primers (0.35-1.73). It was possible to identify accessions, particularly those of divergent origins, by RAPD and (or) ISSR fingerprints. Based on these results, marker-based genetic improvement in A. hypogaea appears possible. None of the 486 RAPD and 330 ISSR amplification products were found to be commonly shared among 13 species of section Arachis and one species each of sections Heteranthae, Rhizomatosae, and Procumbentes. Dendrograms constructed from RAPD, ISSR, and RAPD + ISSR data showed overall similar topologies. They could be resolved into four groups corresponding to the species grouped in four taxonomic sections. The present results strongly support the view that Arachis monticola (2n = 4x = 40) and A. hypogaea are very closely related, and indicate that A. villosa and A. ipaensis are the diploid wild progenitors of these tetraploid species.     

DNA barcodes and microsatellites: How they complement for species identification in the complex genus Tamarix (Tamaricaceae)

DNA barcoding allows the identification of an organism by comparing the sequence of selected DNA regions (barcodes) with a previously compiled database, and it can be useful for taxonomic identification of species in complex genera, such as Tamarix. Many species of this genus show convergent morphology, which leads to frequent errors in their identification. Highly variable genetic markers, such as microsatellites or short sequence repeats (SSR), could be used to differentiate species where DNA barcodes fail. Here, we tested the ability of both, 5 different marker regions (rbcL, matK, ITS, trnH-psbA, and ycf1), and 14 microsatellites, to properly identify Tamarix species, especially those from the Mediterranean Basin, and compared the pros and cons of the different analytical methods for species identification. DNA barcoding allows the genetic identification of certain species in Tamarix. The two-locus barcodes matK + ITS and ITS + ycf1 were the best-performing combinations, allowing up to 69% and 70%, respectively, correct identification. However, DNA barcoding failed in phylogenetically close groups, such as many Mediterranean species. The use of SSR can aid the identification of species, and the combination of both types of data (DNA barcoding and SSR) improved the success. The combination of data was especially relevant in detecting the presence of hybridization processes, which are common in the genus. However, caution must be exercised when choosing the clustering methods for the SSR datasince different methods can lead to very different results.     

尖塘鳢属鱼类线粒体12SrRNA基因序列分析

利用PCR技术扩增和测序了线纹尖塘鳢、云斑尖塘鳢和海丰沙塘鳢线粒体12SrRNA基因,结合从GenBank中下载的部分同源序列,共分析了5种鱼类的系统发育关系。在Kimura2-parameter模型构建的邻接树中,原产泰国的云斑尖塘鳢与原产澳州线纹尖塘鳢均为单系类群,二者为亲缘关系最为密切的姐妹群,海丰沙塘鳢与其它群体的亲缘关系较远,支持将尖塘鳢属从塘鳢属中分出的传统分类处理。尖塘鳢属内云斑尖塘鳢和线纹尖塘鳢鱼类种内DNA序列无差异,而种间差异明显,表明线粒体12SrRNA基因可作为塘鳢科鱼类种类鉴定的良好分子标记。