华北地区小丛红景天种群的AFLP遗传多样性

利用扩增片段长度多态性(AFLP)标记, 对分布于华北地区5个山脉的25个小丛红景天(Rhodiola dumulosa)自然种群的776个样品进行了遗传多样性和遗传结构的研究。结果表明: 华北地区小丛红景天种群具有较高的遗传多样性, 4对选择扩增引物共扩增出398条清晰的条带, 其中多态带312条, 种群的平均多态位点百分率为78.46%, 种群总的Nei’s基因多样性为0.364 9, 总Shannon多态性信息指数为0.542 2。华北地区小丛红景天种群间的遗传分化系数G_st = 0.150 7, 基因流Nm = 2.817 9, 表明种群间遗传分化较低, 有一定的基因交流。AMOVA分析结果也表明: 华北地区小丛红景天的遗传变异主要存在于种群内, 地理单元间有一定的遗传分化, 而种群间的遗传分化较低。STRUCTURE的分析和UPGMA聚类分析结果一致, 结果显示地理分布距离相近的种群优先聚在一起。Mantel检验也进一步证实, 华北地区小丛红景天种群的遗传距离与地理距离间呈显著的正相关关系(r = 0.512 9, p < 0.001)。种群的遗传多样性与海拔呈显著的负相关关系(p < 0.05), 而与坡向没有显著相关性。用Dfdist软件分析海拔对遗传多样性的影响, 结果表明没有显著的受选择位点。     

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.     

Genotypic Diversity among Brazilian Isolates of Sclerotium rolfsii

Thirty isolates of Sclerotium rolfsii Sacc. from different hosts and regions of Brazil were studied in relation to morphology, mycelial compatibility, analysis of genomic DNA through random amplified polymorphic DNA (RAPD), variation within the nuclear rDNA [internal transcribed spacers (ITS)] and sequencing of ITS fragments. There was considerable variability among isolates in relation to the number, size and location of sclerotia on the medium surface. Thirteen mycelial compatibility groups (MCG) were identified among 23 isolates. Seven isolates were only self‐compatible. With the exception of group 3, where all the isolates came from soybean, there was no apparent correlation between group and isolate origin. On the basis of RAPD profiles, 11 haplotypes (A to K) were identified. There was an association between the RAPD groups and MCG. Haplotypes A, B, D, G, I and K belonged to MCG groups 1, 2, 3, 4, 5 and 6, respectively. All other RAPD haplotypes contained incompatible isolates. Polymerase chain reaction (PCR) amplification with primers 4R and 5F amplified two fragments containing ITS1, ITS2 and 5.8 S rDNA sequences, that were present in all isolates, with molecular sizes of 739 and 715 bp. Restriction analysis of PCR products showed that the two fragments had sequence divergency which is referred to as ‘ITS types’. Four arbitrarily chosen soybean isolates (2, 6, 7 and 23) and two non‐soybean isolates (11 and 22) were used to investigate the variation within the ITS sequence and its role in the phylogeny. The strict consensus of nine most‐parsimonious trees inferred from the data set which included six isolates of S. rolfsii, four of which have two different ‘ITS types’, showed three well‐supported groupings. The neighbour‐joining tree inferred from the data set also showed three major clades as did the parsimony tree. The major difference was that in the neighbour‐joining tree the ‘ITS type’ 11 was resolved and grouped in one clade. These results show that the ‘ITS types’ within isolates are almost always phylogenetically distinct. There was no clear correlation between ITS‐based phylogeny and isolate origin.     

用ITS序列研究杨属各组之间的系统发育关系

杨树是重要的工业用材树种。我国杨树遗传资源丰富 ,分布范围广泛 ,不少种为我国特有。开展杨属系统发育和分子进化研究 ,对丰富的杨树遗传资源保存和利用有着重大意义。杨属 (Ｐｏｐｕｌｕｓ)全世界约 10 0余种 ,属下通常分 5个组[1] 。胡志昂等[2 ] 对杨属不同组间的过氧化物酶同工酶进行了研究 ;李宽钰等[3] 利用ＲＡＰＤ标记技术对白杨组、青杨组、黑杨组 2 0个种作了遗传分析。但是在杨属系统分类上还存在着许多混乱 ,同物异名、同名异物现象相当普遍。本文以杨属 5个派主要代表种为材料 ,用ＰＣＲ产物直接测序法测定杨树ＩＴＳ序列 ,…     

A modified AFLP with fluorescence-labelled primers and automated DNA sequencer detection for efficient fingerprinting analysis in plants

A modified AFLP (amplified fragment length polymorphism) technique is described. Fluorescence-labelled primers were used in the selective amplifications. The amplified fragments were detected on denaturing polyacrylamide gels using an automated ALF DNA sequencer with the fragment option. The modified AFLP technique avoids the use of isotopes or silver staining, but gives a much higher resolution than other AFLP detection systems.     

Phylogenetic analysis and differentiation of Veronica subgenus Stenocarpon in the Balkan Peninsula

The Balkan Peninsula is considered the most important refugium for species during the Pleistocene glaciations and today harbours c. 2000 endemic species, but we know surprisingly little about the evolution of taxa in this region. Veronica saturejoides, V. thessalica and V. erinoides are a group of closely related alpine taxa endemic to the Balkan Peninsula. Here, we analyse four DNA regions [the nuclear chalcone synthase intron (CHSi) and ribosomal internal transcribed spacer (ITS) region and the plastid rpoB‐trnC spacer and trnL‐trnL‐trnF region] and amplified fragment length polymorphism (AFLP) fingerprints to provide a phylogenetic hypothesis for the relationships among these taxa. Additionally, we analyse leaf morphological characters used to distinguish the three subspecies of V. saturejoides. The analyses support the distinction of the three subspecies based on previously intuitively suggested characters. Nuclear chalcone synthase intron data indicate that the southern taxa are genetically much more diverse than the more northern V. saturejoides subsp. saturejoides. Phylogenetic relationships inferred from this region and AFLP fingerprints support the monophyly of V. saturejoides. In contrast, plastid DNA regions suggest a closer relationship of V. saturejoides subsp. saturejoides to V. thessalica. The most likely scenario involves introgression into V. saturejoides subsp. saturejoides from V. thessalica. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 616–636.     

Phylogenetic Relationship ofPopulusSections by ITS Sequence Analysis

ITS sequences of 15 representative species of five sections in the genus Populus L. were determined. By using direct sequencing of PCR product, it was found that the fragments of internal transcribed spacers (ITS) are about 594 bp in length. The length of ITS-1 and ITS-2 is about 220 bp and 210 bp, respectively, while that of 5.8s is 164 bp. Its G+C content is about 69.0%. The number of phylogenetically informative loci is higher in ITS-2 than in ITS-1. Transversion and transition are two main factors that drive the ITS evolution, and more insertions and deletions occurred in ITS-2. Taking Salix matsudana Koidz. and Salix suchowensis Cheng as outgroups, phylogenetic analysis of ITS sequences using PAUP 4.0 software indicated that Populus is monophyletic group and can be divided into two main clades. One is the section Leuce , and the other is the remaining sections.     

广义青篱竹属(Arundinaria)核糖体DNA ITS序列及亲缘关系研究

利用PCR扩增产物直接测序的方法分析广义青篱竹属(Arundinaria)中有关争议类群的代表种或模式种(毛竹为外类群)等18种竹种的核糖体DNA内转录间隔区(Internal Transcribed Spacers,ITS)序列。通过最简约性分析产生的ITS系统发育树表明,供试竹种形成一个自然的单系类群,这说明广义青篱竹属中这些不同的类群归属青篱竹属是合理的。17种竹种可聚为2大分支：其中斑苦竹(A,oleosa)、仙居苦竹(A．hsienchuensis)、茶秆竹(A．amabilis)、长叶苦竹(A．chino)、苦竹(A．amara)、宜兴苦竹(A．yixingensis)、菲白竹(A．fortunei)、翠竹(A．pygmaea)为一个分支;而大明竹(A．graminea)、巴山木竹(A．fargesii)、冷箭竹(A．faberi)、凤竹(A．hupehense)、鼓节矢竹(Pseudosasa japonica cv．Tsutsumiana)、矢竹(Pseudosasa japonica)、短穗竹(Brachystachyum densiflorum)、肿节竹(A．oedogonata)、少穗竹(A．sulcata)组合在另一分支。ITS系统发育树还表明,大明竹与巴山木竹、鼓节矢竹与矢竹、少穗竹与短穗竹和肿节竹关系极为密切,均得到较高的Bootstrap(分别为99％、100％和87％)的支持;茶秆竹与仙居苦竹关系非常密切,茶秆竹可归隶到青篱竹属中;翠竹和菲白竹关系密切,且与苦竹类竹种分为两个分支。     

Genetic races associated with the genera and sections of host species in the holoparasitic plant Cytinus (Cytinaceae) in the Western Mediterranean basin

* Speciation via race formation is an important evolutionary process in parasites, producing changes that favour their development on particular host species. Here, the holoparasitic plant Cytinus, which has diverse host species in the family Cistaceae, has been used to study the occurrence of such races. * Amplified fragment length polymorphism (AFLP) analyses were performed on 174 individuals of 22 populations parasitizing 10 Cistaceae species in the Western Mediterranean basin. * Neighbour-joining, multivariate ordination analyses, and individual-based Bayesian analyses, clustered Cytinus populations into five well-characterized genetic races that, overall, agreed with the taxonomic sections of their hosts. In the AMOVA, among-races differences accounted for almost 50% of the genetic variation. The isolation-by-distance model was not supported by a Mantel test among Cytinus populations (r = 0.012; P = 0.456). All races showed low within-population genetic diversity, probably as a result of restricted pollen flow aggravated by flowering asynchrony, restricted seed dispersion, or stochastic processes. * The genetic differentiation among the five races of Cytinus is congruent with the view that these races are well-characterized lineages that have evolved independently as a result of selective pressures imposed by their hosts. This pattern, with genetically distinctive groups associated with the infrageneric sections of the host species, has not been reported previously for parasitic angiosperms.     

Genetic Diversity and Genetic Structure of an Endangered Species, Trillium tschonoskii

The genetic diversity and genetic structure of Trillium tschonoskii (Maxim) were investigated using amplified fragment length polymorphism markers. Eight primer combinations were carried out on 105 different individuals sampled from seven populations. Of the 619 discernible DNA fragments generated, 169 (27.3%) were polymorphic. The percentage of polymorphic bands within populations ranged from 4.52 to 10.50. Genetic diversity (H_E) within populations ranged from 0.0130 to 0.0379, averaging 0.0536 at the species level. Genetic differentiation among populations was detected based on Nei's genetic diversity analysis (53.03%) and analysis of molecular variance (AMOVA) (52.43%). AMOVA indicated significant genetic differentiation among populations (52.43% of the variance) and within populations (47.57% of the variance) (p < 0.0002). Gene flow was low (0.4429) among populations. Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. We propose an appropriate strategy for conserving the genetic resources of T. tschonoskii in China.     

AFLP-Based detection of DNA methylation

By using the isoschizomersHpa II andMsp I which display differential sensitivity to cytosine methylation, a modified amplified fragment length polymorphism (AFLP) technique has been developed to investigate DNA methylation profiles in eukaryotic organims. Genomic DNA was digested with a mixture ofEcoR I and one of the isoschizomers, and ligated to oligonucleotide adapters. After two rounds of selective PCR amplification, followed by DNA separation on a Long Ranger gel electrophoresis, a subset of restriction fragments can be displayed on an X-ray film. Comparison of AFLP banding patterns betweenHpa II andMsp I revealed the extent of DNA methylation. The technique has been successfully applied in this study to investigate DNA methylation profiles of apple (Malus domestica cv. Gala) genomic DNA extracted from leaves of field-grown adult trees andin vitro-grown shoot cultures. The results showed that up to 25 percent of AFLP bands were derived from methylated sequences, and among those, a few bands unique to either adult trees orin vitro shoots were observed. These results demonstrated that this protocol is effective in identifying methylated DNA profiles. Both first authors have contributed equally to this work.     

AFLP标记在小香羊遗传多态性检测中的应用

研究了AFLP标记在研究小香羊遗传多态性方面应用的可行性和该山羊个体基因组DNA的AFLP扩增结果。实验应用10条AFLP引物,用PstI酶切,对15只小香羊基因组DNA进行AFLP反应,共获得113个AFLP标记,单引物获得的标记数在2～19之间,小香羊群体相似系数AFLP研究结果为0．913(0．814～0．980)。该研究为评价小香羊的遗传稳定性提供了相关的参数,准确评价尚待和其它品种对比研究后确定。