Chloroplast DNA restriction fragment length polymorphism in Sequoia sempervirens D. Don Endl., Pseudotsuga menziesii (Mirb.) Franco,Calocedrus decurrens (Torr.), and Pinus taeda L.

Summary The extent and type of chloroplast DNA restriction fragment length polymorphism was determined among individual tree samples of coast redwood, Douglas fir, incense-cedar, and loblolly pine. A total of 107 trees was surveyed for three restriction enzymes (BamHI, EcoRI, HindIII) and six chloroplast DNA probes from petunia (P3, P4, P6, P8, P10, S8). The probes comprise 64% of the petunia chloroplast genome. Polymorphisms were detected in all species but loblolly pine. Coast redwood and incense-cedar had a small number of rare variants, whereas Douglas fir had one highly polymorphic region of insertions/deletions in sequences revealed by the P6 probe from petunia. The mutation hotspot is currently being studied by DNA sequence analysis.     

Phylogenetic relationships among cultivated Allium species from restriction enzyme analysis of the chloroplast genome

Summary The genus Allium contains many economically important species, including the bulb onion, chive, garlic, Japanese bunching onion, and leek. Phylogenetic relationships among the cultivated alliums are not well understood, and taxonomic classifications are based on relatively few morphological characters. Chloroplast DNA is highly conserved and useful in determining phylogenetic relationships. The size of the chloroplast genome of Allium cepa was estimated at 140 kb and restriction enzyme sites were mapped for KpnI, PstI, PvuII, SalI, XbaI, and XhoI. Variability at restriction enzyme sites in the chloroplast DNA was studied for at least three accessions of each of six cultivated, old-world Allium species. Of 189 restriction enzyme sites detected with 12 enzymes, 15 mutations were identified and used to estimate phylogenetic relationships. Cladistic analysis based on Wagner and Dollo parsimony resulted in a single, most-parsimonious tree of 16 steps and supported division of the species into sections. Allium species in section Porrum were distinguished from species in sections Cepa and Phyllodolon. Two species in section Rhiziridium, A. schoenoprasum and A. tuberosum, differed by five mutations and were placed in separate lineages. Allium cepa and A. fistulosum shared the loss of a restriction enzyme site and were phylogenetically closer to each other than to A. schoenoprasum. This study demonstrates the usefulness of restriction enzyme site analysis of the chloroplast genome in the elucidation of phylogenetic relationships in Allium.     

绣球属(Hydrangea L.)植物分子系统学及系统发育分析

该研究基于对绣球属(Hydrangea L.)的大尺度取样,选取国内外61种绣球属和近缘属植物,分别基于核基因片段(ITS)和叶绿体基因片段(rbcL,trnL-F,atpB)重建了绣球属及其近缘种属的系统发育关系。结果表明:(1)核基因与叶绿体基因树之间在树形上没有明显的冲突,进而基于核基因和叶绿体基因联合数据重建了绣球属及其近缘种属的系统发育关系。(2)基于联合数据构建的系统树确认了2个大分支,并得到了果实顶端截平与否这一形态学证据的强力支持;每个大分枝又分为4个类群,共确定了8个类群。部分类群也得到了广义宏观形态性状的支持,如第1类群得到了叶形、花粉以及种子形态的支持。因此,该系统发育关系的重建对于全面理解绣球属及其近缘种属的演化关系具有重要的启发。     

DNA序列在悬钩子属植物分子系统学研究中的应用进展

悬钩子属植物种类繁多,类群复杂,而且多为多倍体和杂种。该文就近年来国内外有关DNA序列在悬钩子属植物分子系统学研究中的应用现状和进展进行了综述,并对中国悬钩子属植物系统发育研究进行了展望。研究认为:叶绿体DNA序列多应用非编码区,且多与ITS序列联合分析;核基因组中ITS序列应用最为广泛,主要用于研究悬钩子属空心莓组与木莓组的进化关系、栽培品种间亲缘关系及部分杂种和多倍体的起源等;在该属植物中发现了ITS个体内多态性,但未进行ITS假基因检测,其系统学应用价值需重新评价;低拷贝核基因只有GBSSI和LEAFY有相关应用。同时认为,悬钩子属植物系统学研究中应用的DNA序列及研究类群均较少,缺乏对整个悬钩子属全面而系统的研究。指出应进一步选择具有代表性的样本、筛选合适的DNA片段,并结合形态学、孢粉学和细胞学等手段对中国悬钩子属植物系统关系进行深入研究。     

Phylogenetic analysis of Asian<Emphasis Type="Italic"> Symplocos</Emphasis> (Symplocaceae) based on nuclear and chloroplast DNA sequences

Thirty species and one variety of Symplocos (Symplocaceae), including all taxa distributed in Japan, were phylogenetically analyzed with DNA sequence data. The evolution of morphological characters is discussed on the basis of the phylogenetic relationships obtained. All species were Asian, except one, S. austromexicana, from Mexico. The nuclear ribosomal internal transcribed spacer (ITS) region and two intergenic spacer regions, between trnL and trnF, and between trnH and psbA of chloroplast DNA were used. The topologies of trees obtained from ITS and the chloroplast intergenic spacers are largely congruent. S. sonoharae, the representative of the subgenus Symplocos in Asia, was sister to all other species. The position of the American species, S. austromexicana, which also belongs to the subgenus Symplocos, was not well resolved. The phylogenetic tree based on combined sequence data largely supports the monophyletic origin of the infrageneric sections proposed earlier. However, the phylogenetic relationship between them is not well resolved, probably due to rapid diversification. The section Palura, a deciduous group, is well defined in the DNA analysis, suggesting its independent status in the genus Symplocos. In spite of their morphological divergence, the three endemic species of the Bonin Islands are monophyletic. The occurrence of curved seeds seems to be homoplastic, scattered over the phylogenetic tree without showing a particular infrageneric relationship.     

Chloroplast DNA inheritance and variation in <Emphasis Type="Italic">Leucadendron</Emphasis> species (Proteaceae) as revealed by PCR-RFLP

The inheritance of chloroplast DNA (cpDNA) in Leucadendron species was studied by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. A total of 100 progeny from five interspecific crosses involving seven parental species were tested, and all progeny exhibited the cpDNA restriction fragment pattern of the female parent, indicating that cpDNA in Leucadendron is maternally inherited. PCR-RFLP was also employed to study cpDNA variation among 21 Leucadendron species. Parsimony analysis using a heuristic search resulted in a phylogenetic tree that showed limited agreement to the taxonomic classification of Leucadendron species, based on morphological characteristics. The incongruence between cpDNA phylogenetic and taxonomic groupings in Leucadendron may be due to reticulate evolution involving a combination of hybridization and introgression, convergent evolution and/or lineage sorting at the interspecific, intersubsectional and intersectional levels.     

Phytogeny of Epidermophyton floccosum and other dermatophytes

Eleven strains of Epidermophyton floccosum were compared with 5 Microsporum and 5 Trichophyton species with respect to the restriction fragment length polymorphism (RFLP) of the mitochondrial DNA to reveal their phylogenetic relationships. The phylogeny of 11 species showed that the three dermatophyte genera could not be separated from each other and could be considered to be congeneric. This result is not inconsistent with the results from ribosomal RNA sequences.     

Restriction enzyme analysis of the nuclear 45s ribosomal DNA of six cultivated Alliums (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of objective molecular characters. Restriction fragment length polymorphisms in the nuclear 45s ribosomal DNA (rDNA) were identified among two of five accessions of each of six cultivated Alliums. Restriction enzyme sites forBamHI,DraI,EcoRI,EcoRV,SacI, andXbaI were mapped. Different lengths of the rDNA repeat unit among the cultivated Alliums were due to sizes of the intergenic spacer. Nineteen polymorphic restriction enzyme sites were discovered and used to estimate phylogenetic relationships. Cladistic analyses based on Wagner parsimony were completed without an outgroup and resulted in two equally most parsimonious trees of 22 steps. A combined analysis of differences at RE sites in the ribosomal (19 characters) and chloroplast (15 characters) DNA generated a single most parsimonious tree of 39 steps. Single trichotomies were observed at 40 and 41 steps. Strict consensus of the three trees of 41 or fewer steps consisted of a lineage forA. tuberosum, a second forA. ampeloprasum andA. sativum, and a third forA. cepa, A. fistulosum, andA. schoenoprasum. Estimates of phylogenetic relationships based on variability at restriction enzyme sites in the rDNA and chloroplast DNA agree with the classification scheme ofTraub. Because of the predominance of autapomorphies, restriction enzyme analysis of the nuclear 45s rDNA is of limited use in estimating phylogenies amongAllium sections. However it is useful in the establishment of interspecific hybridity.     

基于cpDNA和rDNA ITS片段分析西南地区青杨派杨树的系统发育与进化关系

西南地区青杨派杨树种质资源丰富,可为杨树遗传改良提供珍贵的基因资源,但树种之间形态学差异细微,该研究以山杨作为外类群,测定了西南地区及其他地区杨属青杨派17个种(杂种)共36份样本的3个叶绿体片段(atpF-atpH、trnL-F和matK)和核糖体ITS片段,并对其进行系统发育分析,以探讨西南地区青杨派树种的系统进化关系。结果表明:(1)在所有样本中,3个叶绿体片段atpF-atpH、trnL-F、matK的长度分别为605~634bp、957~1 010bp、819bp,3个片段拼接后的联合序列包含29个变异位点和15个信息位点;ITS片段对齐后的长度为646bp,变异位点19个,信息位点17个。(2)基于叶绿体联合序列和ITS片段的杨属青杨派树种的平均遗传距离分别为0.001 3和0.003 6。叶绿体联合片段的MP和Bayes系统树树型基本一致,青杨派树种可以划分为2组,第1组由青杨、三脉青杨、大青杨和辽杨构成;第2组中的小叶杨、小青杨、川杨、德钦杨、昌都杨、乡城杨、康定杨、西南杨、滇杨和藏川杨不能有效区分,且均与缘毛杨的遗传关系较近。(3)基于ITS的系统树与叶绿体联合片段构建的系统树差异不大,仅在于第2组中的小青杨与小叶杨、川杨等差异明显,与第1组中的树种紧密聚拢。     

Genome composition of asymmetric hybrids in relation to the phylogenetic distance between the parents. Nucleus-chloroplast interaction

Summary A series of fusion experiments were performed between protoplasts of a cytoplasmic albino mutant of tomato, Lycopersicon esculentum (ALRC), and gamma-irradiated protoplasts of L. hirsutum and the Solanum species S. commersonii, S. etuberosum and S. nigrum. These species were chosen for their different phylogenetic relationships to tomato. In all fusion combinations except from those between ALRC and S. nigrum, green calli were selected as putative fusion products and shoots regenerated from them. They were subsequently analyzed for their morphology, nuclear DNA composition and chloroplast DNA origin. The hybrids obtained between ALRC and L. hirsutum contained the chloroplasts of L. hirsutum and had the flower and leaf morphology of L. esculentum. After Southern blot analysis, using 13 restriction fragment length polymorphisms (RFLPs) randomly distributed over all chromosomes, all hybrids showed L. esculentum hybridization patterns. No chromosomes of L. hirsutum were found. These results indicate that these hybrids were true cybrids.The putative asymmetric hybrids, obtained with S. commersonii and S. etuberosum, showed phenotypic traits of both parents. After hybridization with species-specific repetitive nuclear DNA probes it was found that nuclear material of both parents was present in all plants. In the case of S. nigrum, which combination has the greatest phylogenetic distance between the fusion parents, no hybrid plants could be obtained. The chloroplast DNA of all hybrid plants was of the donor type suggesting that chloroplast transfer by asymmetric protoplast fusion can overcome problems associated with large phylogenetic distances between parental plants.     

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.     

Systematic implications of chloroplast DNA variation inLepidium sectionsCardamon,Lepiocardamon andLepia (Brassicaceae)

Chloroplast DNA restriction site variation was examined amongLepidium taxa usually classified in sects.Lepia, Lepiocardamon andCardamon. By using 15 restriction endonucleases, filter hybridization experiments, and comparative mapping procedures, a total of 119 variable restriction sites was detected. Of these, 56 were phylogenetically informative and were used in a cladistic analysis. The resulting phylogenetic tree agrees with results derived from morphology, isozyme electrophoresis and the analysis of glucosinolates. The data suggest: 1) The recognition of sect.Lepia, and 2) that sect.Lepiocardamon has close relationships to sect.Cardamon and that both should be amalgamated. Low rates of interspecific chloroplast DNA sequence divergence in sect.Lepia correlated well with morphological data and isoelectric focusing analysis of Rubisco, suggesting that taxa of sect.Lepia are closely related and most likely have diversified recently.     

Intrageneric relationships of maple trees based on the chloroplast DNA restriction fragment length polymorphisms

A maple tree genus,Acer is the largest genus in broad-leaved deciduous trees and contains about 200 species. A delimitation of the genus is clear but the intrageneric classification was controversial because of homoplasies in morphological characters. In this study, a phylogenetic relationship inAcer was inferred based on chloroplast DNA restriction site polymorphisms with 17 restriction endonucleases and previously proposed intrageneric classifications were evaluated. The phylogenetic tree showed that (1) sectionsArguta, Cissifolia, Lithocarpa, Macrantha, Palmata, Spicata, Tataricum, Trifoliata sensu Ogata (1967: Bull. Tokyo Univ. Forests 63: 89–206) were monophyletic groups respectively, (2) sectionsCampestria, Goniocarpa, Platanoidea sensu Ogata (1967) were polyphyletic respectively, and (3) sectionsDistyla andParviflora formed a sister group. An average of estimated nucleotide substitution rates of Acer chloroplast DNA was calculated as 7.9×10⁻¹¹±1.4×10⁻¹¹ nucleotide substitutions par site par year, which coincides well with previously reported rates of perennial plants. Divergence eras of eastern Asia and North American species in both sectionsSpicata andRubra were estimated to be late Miocene. In consideration with previous data, multiple migrations and disjunctions are likely to have formed the eastern Asian and North American disjunct distribution.     

Relationships between species ofLeymus,Psathyrostachys, andHordeum (Poaceae,Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes

We have used total genomic DNA as a probe to size-fractionated restriction enzyme digests of genomic DNA from a range ofTriticeae species from the generaLeymus Hochst.,Psathyrostachys Nevski, andHordeum L., and hybrids betweenHordeum andLeymus to investigate their taxonomic relationships. Genomic Southern hybridization was found to be an effective and simple way to assess the distribution and diversity of essentially species-specific and common, repetitive DNA sequences, and is hence especially useful in evolutionary studies. The DNA sequences ofH. vulgare seem to diverge substantially from those ofH. brachyantherum, H. lechleri, H. procerum, andH. depressum. The genome ofThinopyron bessarabicum shows little homology to those of theLeymus species investigated, confirming thatT. bessarabicum is not an ancestral genome inLeymus. Although the genomes ofLeymus andPsathyrostachys share substantial proportions of DNA sequences, they include divergent repeated sequences as well. Hybridization with a ribosomal DNA probe (pTa 71) showed that the coding regions containing structural genes encoding the 18 S, 5.8 S, and 26 S ribosomal RNA were conserved among the species investigated, whereas the intergenic spacer region was more variable, presenting different sizes of restriction fragments and enabling a classification of the species. The rye heterochromatin probe pSc 119.2 hybridized to DNA fromH. lechleri andT. bessarabicum, but not to DNA from the other species investigated.     

Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia,Asteraceae): Additional information from internal transcribed spacer sequences of nuclear ribosomal DNA

The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.     

Chloroplast DNA variation and the phylogeny ofAsplenium sect.Hymenasplenium (Aspleniaceae) in the New World tropics

Asplenium sect.Hymenasplenium is a well-defined group in Aspleniaceae, distinguished by several morphological and cytological characters. However, interspecific relationships in the section were not clear. In this paper, we report the phylogenetic relationships of 9 Neotropical species of sect.Hymenasplenium determined by chloroplast DNA restriction site variation analyses. From the obtained phylogenetic tree, two major clades: one withA. obtusifolium, A. riparium, A. volubile andA. repandulum and another withA. delitescens, A. ortegae, A. purpurascens, A. laetum andA. hoffmannii were recognized.Asplenium delitescens was shown to have a polyphyletic origin. It was also shown that the epiphytic habit evolved only once in the New World species of sect.Hymenasplenium.     

四种栎属青冈亚属植物叶绿体基因组特征及系统发育研究

栎属青冈亚属植物的系统发育地位长期存在着争议，部分种的种间关系不明确。为揭示宁冈青冈(Quercus ningangensis)、曼青冈(Q.oxyodon)、毛曼青冈(Q.gambleana)、竹叶青冈(Q.neglecta)的叶绿体基因组特征及系统发育关系，该研究选择以上4种栎属青冈亚属植物的成熟叶片进行二代测序，对其叶绿体基因组结构和特征进行分析，并结合相关类群进行系统发育研究。结果表明：(1)宁冈青冈、曼青冈、毛曼青冈、竹叶青冈的叶绿体基因组序列长度分别为160 906、160 883、160 832、160 784 bp,均编码133个基因，包括88个蛋白质编码基因、37个tRNA基因、8个rRNA基因。(2)4种栎属青冈亚属植物偏好以A/T结尾的密码子，质体基因组变异区域主要存在于非编码序列。(3)通过IR边界分析得出，4种栎属青冈亚属植物存在ycf1假基因且在IRb/SSC区域发生扩张。(4)系统发育分析显示，在壳斗科中，水青冈属(Fagus)和轮叶三棱栎属(Trigonobalanus)较早分化出来，栎亚属(subg.Quercus)未形成一个单系群，叶绿体基因组建树结...     

莪术基原植物DNA条形码序列的筛选与鉴定

为寻找适用于中药材莪术基原植物鉴定的DNA条形码序列,探索快速高效的莪术基原植物鉴定的新方法,该文首先利用扩增成功率和测序成功率对中药材莪术三种基原植物,9个样本的7种DNA条形码序列(ITS、ITS2、matK、psbA-trnH、trnL-trnF、rpoB和atpB-rbcL)进行评估,然后利用MEGA6.0软件对获得的高质量的序列通过变异位点分析、遗传距离计算和系统树分析等进一步进行评估,最后将筛选到的DNA条形码序列对未知基原的待测样品进行基原鉴定。结果表明:(1) ITS、ITS2和matK等条形码序列在莪术基原植物中的扩增或测序成功率较低,难以应用于实际鉴定;而psbA-trnH、trnL-trnF和rpoB条形码序列变异位点信息过少,不足于区分莪术的三种不同基原植物;只有atpB-rbcL条形码序列的扩增和测序成功率较高,容易获得高质量的序列,同时序列长度(642～645 bp)理想,变异位点多(11个),可实现莪术的三种不同基原的区分鉴别。(2)待测样品经基于atpB-rbcL序列构建的系统发育树鉴别为温郁金。综上所述,叶绿体atpB-rbcL序列能够准确鉴定莪术不同基原植物,可以作为中药材莪术基原植物鉴定的条形码序列。     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.