数量分类学方法在曼陀罗属种子形态分类上的应用

用数量分类学的方法对35个来源的曼陀罗属和木曼陀罗属种子形态进行了聚类分析。结果表明,重瓣曼陀罗(Datura fastuosa)、毛曼陀罗(D.innoxia)、洋金花(D.Metel)和神圣曼陀罗(D.meteloides)在所选取的形态特征上表现出一致性;多刺曼陀罗(Datura ferox)和栎叶曼陀罗(Datura quercifolia)也表现出较高的形态一致性;无刺曼陀罗(Datura inermis)、勃哈蒂曼陀罗(Datura bernhadtii)、湿地曼陀罗(Daturaceratocaula)和曼陀罗(Datura Stramonium)等在形态上较相近。     

准噶尔盆地南缘和阿拉善左旗9个梭梭居群种间系统发育和亲缘关系研究

对荒漠植物梭梭进行ITS序列测定,研究不同梭梭居群系统发育和亲缘关系.材料源于准噶尔盆地南缘及内蒙古阿拉善左旗.结果表明:所测序列经排序后全长732bp.遗传距离矩阵表明:阜康与阿左旗的梭梭居群间遗传距离最小;石河子150与富蕴的居群遗传距离最大.系统发育树结果显示:石河子121、克拉玛依、杜热和150的居群聚为一支;阜康与阿左旗居群以92％的自展支持率相聚;戈壁藜等三种植物作为外类群.     

基于ITS和trnL-F序列鉴定边陲黄耆为独立的物种

乌拉特黄耆(Astragalus hoantchy Franch.)、边陲黄耆(A.dshimensis Gontsh.)和A.coluteocarpus Bioss.为形态上相似的3个近缘物种。为了明确它们之间的分子亲缘关系,探讨分子证据是否支持边陲黄耆为一个独立的物种,测定了这3个物种共33个样本的ITS和trn L-F序列。基于ITS+trn L-F联合序列,运用Mega 6.06软件计算了种内和种间的遗传距离,并利用Mega 6.06和Mr Bayes 3.2软件分别构建了最大似然(ML)树和贝叶斯(BI)树。结果显示:乌拉特黄耆、边陲黄耆和A.coluteocarpus Bioss.的种内平均遗传距离分别为0.000、0.001和0.003;乌拉特黄耆和边陲黄耆之间平均遗传距离为0.010,而这2个物种与A.coluteocarpus Bioss.之间的平均遗传距离分别为0.015和0.016;乌拉特黄耆和边陲黄耆种间的平均遗传距离远大于其物种内的遗传距离;ML树和BI树具有相似的拓扑结构,均分成了3个分支,其中每个物种的所有个体都聚集在一个单独的分支上且不存在种间交叉现象。本研究揭示了这3个近缘物种的分子亲缘关系,分子证据说明边陲黄耆应该是一个独立的种。     

基于DNA条形码技术对新疆罂粟科部分植物系统发生

以新疆地区4属5种罂粟科植物为研究对象,利用DNA测序技术测定植物叶绿体rbcL基因、核基因ITS,并研究将这两种特定基因联合作为DNA条形码在识别新疆地区罂粟科植物的可行性和有效性。对15条新疆罂粟科植物的ITS-rbcL序列进行信息分析,发现其基因片段多态性位点较多,种内平均遗传距离小于种间。另外,以4种毛茛属植物做外类群对31种罂粟科植物进行聚类分析。结果显示：同属植物基本聚在一起,说明这一联合片段用来鉴定罂粟科植物中属的分类阶元是可行的。所选择的ITS-rbcL片段对罂粟科植物的分类鉴定有一定的参考价值。通过研究还发现,新疆鳞果海罂粟与伊犁秃疮花的亲缘关系较该属其他植物更为接近;新疆重点保护植物红花疆罂粟与黑环罂粟为近缘种;rbcL基因由于其保守度较高,并不适合作罂粟科植物的进化标记基因。     

基于DNA条形码技术对新疆罂粟科部分植物系统发生分析

以新疆地区4属5种罂粟科植物为研究对象,利用DNA测序技术测定植物叶绿体rbcL基因、核基因ITS,并研究将这两种特定基因联合作为DNA条形码在识别新疆地区罂粟科植物的可行性和有效性。对15条新疆罂粟科植物的ITS-rbcL序列进行信息分析,发现其基因片段多态性位点较多,种内平均遗传距离小于种间。另外,以4种毛茛属植物做外类群对31种罂粟科植物进行聚类分析。结果显示:同属植物基本聚在一起,说明这一联合片段用来鉴定罂粟科植物中属的分类阶元是可行的。所选择的ITS-rbcL片段对罂粟科植物的分类鉴定有一定的参考价值。通过研究还发现,新疆鳞果海罂粟与伊犁秃疮花的亲缘关系较该属其他植物更为接近;新疆重点保护植物红花疆罂粟与黑环罂粟为近缘种;rbcL基因由于其保守度较高,并不适合作罂粟科植物的进化标记基因。     

利用DNA条形码对10种苍耳属杂草的鉴定

使用ITS、ITS2、psbA-trnH、rbcL和matK等序列对截获的10种苍耳属(Xanthium)杂草进行扩增测序,比较各序列的扩增和测序效率,采用最近距离法和相似性搜索算法评价不同序列的鉴定效率。采用最佳鉴定序列计算种间K2P距离并构建系统进化树。结果表明,ITS、psbA-trnH2个序列的扩增效率和鉴定效率最高;ITS序列的种间变异最大,其次是psbA-trnH序列,matK序列的种间变异最小;ITS序列的NeighborJoining(NJ)树可明显地将不同苍耳属杂草种分开;psbA-trnH序列可明显地将国内苍耳种和检疫性苍耳种分开。说明利用DNA条形码能够准确地鉴别苍耳属杂草,ITS和psbA-trnH序列是鉴别苍耳属杂草较理想的条形码组合。该研究结果为苍耳属杂草的分子鉴别提供了科学依据与新的思路。     

忍冬科的系统发育核糖体DNA ITS区序列证据

以败酱草科Valerianaceae的Centranthu rubber为外类群,使用Phylip 3.65软件对忍冬科Caprifoliaceae 12个属12个代表种的ITS区序列进行了系统发育分析.采用最大简约法分析获得了5个最简约树,步长为580,一致性指数(CI)和维持性指数(RI)值分别为0.783 2和0.741 5.利用5个最简约树获取严格一致树.结果表明:①荚蒾属Viburnum与接骨木属Sambucus是姐妹群,自展支持率100%;并与忍冬科内七子花属Heptacodium、锦带花属Weigela构成姐妹群,虽然自展支持率为60%,从遗传距离上可看出荚蒾属与忍冬科内其他属之间仍有较近的亲缘关系,所以支持荚蒾属和接骨木属置于忍冬科内.②忍冬属Lonicera与鬼吹萧属Leycesteria聚成一小支,并构成姐妹群,自展支持率为100%;并与毛核木属Symphoricarpos、莛子镳属Triosteum构成姐妹群,自展支持率为100%,故不支持将毛核木属置于北极花族内Linnaceae;北极花属Linnaea、六道木属Abelia、猬实属Kolkwitzia、双盾木属Dipelta聚成姐妹群,自展支持率为100%,说明四者亲缘关系较近,仍置于北极花族内.     

新疆野扁桃与其近缘种的亲缘关系

野扁桃（Amygdalus ledebouriana Schlecht）为野生珍稀濒危植物资源,我国仅分布在新疆。为研究野扁桃与栽培扁桃及其近缘种的亲缘关系,对新疆野扁桃、栽培扁桃、栽培桃和桃属部分物种的叶绿体matK基因进行测序,基于Kimura 双参数模型计算种间遗传距离,并以邻接法（NJ）构建系统发育树。结果表明：野扁桃与蒙古扁桃和榆叶梅的遗传距离较小,与栽培扁桃和桃属物种（桃、山桃和蟠桃）的遗传距离较大;栽培扁桃与山桃、蟠桃和桃的亲缘关系较其与野扁桃、榆叶梅和蒙古扁桃的亲缘关系更近。     

绿僵菌属4个生防潜力菌株的多基因鉴定

传统的绿僵菌分类鉴定以形态特征为主要依据,其局限性使种内仍包含着复杂的类群。DNA分子鉴定使绿僵菌属、种分类更为细化和准确,对其育种、杀虫机理、生态学等的研究非常重要。本文对绿僵菌4个有生防潜力的菌株IPPM010202、IPPM2029、IMI330189和M200614的5个持家基因即核糖体转录间隔区序列(ITS1-4)、β微管蛋白(beta-tubulin)、RNA聚合酶Ⅱ大亚基(RPB1)、RNA聚合酶Ⅱ第二大亚基(RPB2)和翻译延伸因子(EF-1α)的同源序列进行了克隆和测序,通过构建系统发育树进行了多基因进化分析。结果表明,4个生防菌株分别位于进化树的3个分支上,鉴定菌株IPPM010202和IPPM2029同属于平沙绿僵菌Metarhizium pingshaense,IMI330189为蝗绿僵菌M.acridum,M200614属于低温绿僵菌M.frigidum,它们之间亲缘关系很近,5个基因的遗传分化距离只有0.021~0.036。ITS1-4序列有较高变异频率和变异幅度,分类效率优于其他基因;EF-1α序列在鉴别菌株和分析亲缘性上有优势。多基因的系统进化分析能够弥补单基因分析的局限,取得更为准确的鉴定和更为细化的分类结果。     

吉林省球孢白僵菌遗传多样性与亚洲玉米螟化性相关性分析

利用ITS序列分析和ISSR分子标记对吉林省玉米主产区的49株寄生亚洲玉米螟的球孢白僵菌进行了遗传多样性分析。ITS序列分析表明,各菌株间的亲缘关系与其地理来源无关。分别根据9个采集地,以及寄主化性类型划分供试菌株类群,利用ISSR分子标记技术进一步分析表明,不同地理类群中榆树地区的遗传多样性最高,双辽地区最低;双辽和通化地区间的遗传分化系数最高,梨树和通化地区间的遗传距离最大。根据寄主化性类型划分类群得出一代玉米螟分离菌株遗传多样性指数最高,一代兼二代区最低;一代兼二代区菌株遗传分化系数最高,而一代区和二代区的遗传距离最大。由此可见,吉林省球孢白僵菌遗传多样性水平较高,种群的异质性较强,遗传多样性同地理位置无关,与寄主化性类型存在相关性。     

Phylogenetic analysis of cercospora and mycosphaerella based on the internal transcribed spacer region of ribosomal DNA

ABSTRACT Most of the 3,000 named species in the genus Cercospora have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few. Mycosphaerella is an extremely large and important genus of plant pathogens, with more than 1,800 named species and at least 43 associated anamorph genera. The goal of this research was to perform a large-scale phylogenetic analysis to test hypotheses about the past evolutionary history of Cercospora and Mycosphaerella. Based on the phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), the genus Mycosphaerella is monophyletic. In contrast, many anamorph genera within Mycosphaerella were polyphyletic and were not useful for grouping species. One exception was Cercospora, which formed a highly supported monophyletic group. Most Cercospora species from cereal crops formed a subgroup within the main Cercospora cluster. Only species within the Cercospora cluster produced the toxin cercosporin, suggesting that the ability to produce this compound had a single evolutionary origin. Intraspecific variation for 25 taxa in the Mycosphaerella clade averaged 1.7 nucleotides (nts) in the ITS region. Thus, isolates with ITS sequences that differ by two or more nucleotides may be distinct species. ITS sequences of groups I and II of the gray leaf spot pathogen Cercospora zeae-maydis differed by 7 nts and clearly represent different species. There were 6.5 nt differences on average between the ITS sequences of the sorghum pathogen Cercospora sorghi and the maize pathogen Cercospora sorghi var. maydis, indicating that the latter is a separate species and not simply a variety of Cercospora sorghi. The large monophyletic Mycosphaerella cluster contained a number of anamorph genera with no known teleomorph associations. Therefore, the number of anamorph genera related to Mycosphaerella may be much larger than suspected previously.     

Fish-isolated Naegleria strains and their phylogeny inferred from ITS and SSU rDNA sequences

Effort was made to identify Naegleria strains isolated from organs of fish, using phylogenetic analyses of SSU rDNA and ITS sequences. Eighteen fish-isolated strains studied enlarged substantially the so far available set of Naegleria strains characterized by both molecular markers. The phylogenetic analyses of separate and concatenated SSU rDNA and ITS sequences revealed phylogenetic relationships of strains under study; however, they failed to solve classification of fish-isolated strains into species. The sequence similarity of strain-representatives of Naegleria species as well as data obtained on intragenomic variation of ITS sequences discouraged the authors from the definition of new species. The results of the present study provide evidence of a need to re-evaluate the current practice of setting boundaries between species of the genus Naegleria. Sequences obtained in this study have been deposited in GenBank with accession numbers DQ768714-DQ768743.     

Oidium neolycopersici: intraspecific variability inferred from amplified fragment length polymorphism analysis and relationship with closely related powdery mildew fungi infecting various plant species

Previous works indicated a considerable variation in the pathogenicity, virulence, and host range of Oidium neolycopersici isolates causing tomato powdery mildew epidemics in many parts of the world. In this study, rDNA internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) patterns were analyzed in 17 O. neolycopersici samples collected in Europe, North America, and Japan, including those which overcame some of the tomato major resistance genes. The ITS sequences were identical in all 10 samples tested and were also identical to ITS sequences of eight previously studied O. neolycopersici specimens. The AFLP analysis revealed a high genetic diversity in O. neolycopersici and indicated that all 17 samples represented different genotypes. This might suggest the existence of either a yet unrevealed sexual reproduction or other genetic mechanisms that maintain a high genetic variability in O. neolycopersici. No clear correlation was found between the virulence and the AFLP patterns of the O. neolycopersici isolates studied. The relationship between O. neolycopersici and powdery mildew anamorphs infecting Aquilegia vulgaris, Chelidonium majus, Passiflora caerulea, and Sedum alboroseum was also investigated. These anamorphs are morphologically indistinguishable from and phylogenetically closely related to O. neolycopersici. The cross-inoculation tests and the analyses of ITS sequences and AFLP patterns jointly indicated that the powdery mildew anamorphs collected from the above mentioned plant species all represent distinct, but closely related species according to the phylogenetic species recognition. All these species were pathogenic only to their original host plant species, except O. neolycopersici which infected S. alboroseum, tobacco, petunia, and Arabidopsis thaliana, in addition to tomato, in cross-inoculation tests. This is the first genome-wide study that investigates the relationships among powdery mildews that are closely related based on ITS sequences and morphology. The results indicate that morphologically indistinguishable powdery mildews that differed in only one to five single nucleotide positions in their ITS region are to be considered as different taxa with distinct host ranges.     

ITS片段作为轮枝菌DNA条形码的评价研究

以轮枝菌属中的重要植物病原性轮枝菌及其近似种共13种35个菌株为材料,探讨ITS片段作为轮枝菌DNA条形码的可行性。结果表明,ITS 成功区分了研究涉及的10个种,其 PCR 扩增和测序成功率为97.1%,序列鉴定成功率为94.5%,但对个别亲缘关系较近种的物种鉴别能力较弱。     

Identification of Armillaria isolates from Bhutan based on DNA sequence comparisons

Armillaria root rot is a serious disease in fir and mixed conifer forests of Bhutan, Eastern Himalayas. The species causing this disease have, however, never been identified. The aim of this study was to identify field isolates collected at four localities in Bhutan. Identification was based on RFLP analysis of the IGS-1 region, comparisons of ITS and IGS-1 sequence data with those available on GenBank, cladistic analyses and sexual compatibility studies. Isolates were found to reside in two distinct RFLP groups. RFLP group 1 isolates from Pinus wallichiana at Yusipang had RFLP profiles and IGS-1 sequences similar to those of Armillaria mellea ssp. nipponica . Although ITS sequence data are not available for A. mellea ssp. nipponica , sequences from this DNA region were most similar to the closely related A. mellea from Asia. The RFLP profile and IGS-1 sequences for RFLP group 2 isolates from Abies densa at Changaphug, Tsuga dumosa at Chimithanka as well as Picea spinulosa and T. dumosa in the Phobjikha valley were similar to those published for Armillaria borealis , Armillaria cepistipes , Armillaria gemina and Armillaria ostoyae . Distance analysis based on IGS-1 and ITS sequence data indicated that these isolates are closely related to A. cepistipes , Armillaria gallica and Armillaria sinapina . The isolates were, however, sexually incompatible with tester strains of A. cepistipes , A. gallica and A. sinapina . Although closely related to these species, they appear to represent a distinct taxon that will be referred to as Bhutanese phylogenetic species I (BPS I) until basidiocarps are found and the species can be described.     

Ribosomal DNA Sequence Comparisons of Colletotrichum Graminicola from Turfgrasses and other Hosts

The 5.8S ribosomal gene and the flanking internal transcribed spacers (ITS) 1 and 2 from Colletotrichum graminicola isolates causing anthracnose disease of Agrostis palustris and Poa species were sequenced. Although bootstrap support was not high, two major groups were observed with both UPGMA and parsimony algorithms, one containing isolates from A. palustris and another with isolates from Poa spp. The ITS sequences were also compared with those of isolates of C. graminicola and C. sublineolum from Sorghum spp., Zea mays and Rottboellia cochinchinesis as well as other Colletotrichum species. Except for one isolate from P. annua in Texas, the ITS1 and ITS2 sequences of turfgrass isolates always grouped separately from C. graminicola or C. sublineolum from non-turfgrass hosts with high bootstrap support. ITS sequences of the turfgrass isolates were more similar to those of other species of Colletotrichum, such as C. coccodes and C. dematium, than they were to C. graminicola isolates from other hosts. Turfgrass isolates have ITS sequences which are not identical to those of isolates from Zea mays and Sorghum species demonstrating diversity among fungi conventionally classified as C. graminicola.     

Variation in the nrDNA ITS sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information?

During the past years, nrDNA ITS sequences have supported the identification of many powdery mildew fungi because comprehensive analyses showed that differences in these sequences have always correlated with the delimitation of different species and formae speciales of the Erysiphales. Published data, obtained using direct sequencing of the PCR products, suggested that even one to five nucleotide differences in the ITS sequences delimit different, albeit closely related, species, and/or indicate differences in host range patterns. Here we show that such differences in the ITS sequences can be detected even in a single sample of a powdery mildew fungus. We sequenced the ITS region in 17 samples, representing six powdery mildew species, both directly and after cloning the PCR products. Among these, samples of O. longipes exhibited two or three, samples of O. neolycopersici three or four, those of an Oidium sp. from Chelidonium majus up to seven, and a sample of another Oidium sp. from Passiflora caerulea two different ITS types determined after cloning. No ITS nucleotide polymorphisms were found in samples of O. lycopersici and Erysiphe aquilegiae. This suggests that some powdery mildew taxa are more variable at the ITS level than others. Thus, although the ITS sequences determined by direct sequencing represent robust data useful in delimitation and phylogenetic analysis of distinct species of the Erysiphales, these need to be used with precaution, and preferably determined after cloning, especially when dealing with closely related taxa at species and sub-species levels. With this method a hitherto undetected genetic diversity of powdery mildews can be revealed.     

Characterization of Ribosomal DNA from Venturia inaequalis and Its Phylogenetic Relationship to rDNA from Other Tree-Fruit Venturia Species

ABSTRACT A portion of the 18S ribosomal DNA (rDNA) gene, the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA gene were polymerase chain reaction-amplified from strains and field populations of Venturia inaequalis and assessed for genetic variation. A previously reported optional group I intron in the 18S rDNA gene of V. inaequalis was detected in 75.0% of 92 strains collected worldwide and in 61.1 and 71.2% of 54 and 59 strains from two Michigan orchards, respectively. Sequence and restriction analysis of rDNA revealed four intron alleles, three of which were present both in worldwide strains and in each field population. Two ITS1 alleles were detected and found to be linked to specific intron alleles. The ITS1-5.8S-ITS2 sequences from V. asperata V. carpophila, V. cerasi, V. inaequalis, V. nashicola, V. pyrina, and Cladosporium caryigenum were compared using phylogenetic analysis. Strains of the Venturia species were placed in three distinct monophyletic groups in a phylogenetic tree. The first group comprised V. inaequalis; the second, V. pyrina and V. nashicola; and the third, V. cerasi, V. carpophila, and V. asperata. The described intron and ITS1 alleles in V. inaequalis provide genetic markers for subdividing populations of V. inaequalis, and the ITS1-5.8S-ITS2 sequences are valuable in determining the relationship of the species from tree-fruit crops with other Venturia species.     

DNA条形码技术在入侵植物刺萼龙葵检验检疫中的应用

刺萼龙葵(Solanum rostratum)是危害严重的外来入侵植物,基于种子形态的传统检疫方法有一定的局限性,不依赖于形态学特征的DNA条形码技术是基于DNA序列差异对物种进行鉴定的新方法,是传统形态学鉴定方法的有力补充。以刺萼龙葵及其8种龙葵亚属近缘植物为材料,从Gen Bank数据库中下载rbc L、mat K和内转录间隔区(ITS)序列,进行DNA条形码分析,结果显示ITS鉴定效果优于其他片段,可把刺萼龙葵从其近缘植物中鉴别出来。