Molecular Diversity of Renibacterium salmoninarum Isolates Determined by Randomly Amplified Polymorphic DNA Analysis

The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture.     

IGS基因及RAPD标记分析在加特隐球酵母基因分类中的应用

根据ITS1-5.8S-ITS2区域的特异核酸序列变化,加特隐球酵母Cryptococcus gattii(≡新型隐球酵母加特变种Cryptococcu neoformans var.gattii)可分为6种基因型。本研究通过检测加特隐球酵母的IGS基因,发现其IGS序列有着更丰富的变异和信息位点。通过结合加特隐球酵母RAPD(随机扩增的多态性DNA)图谱比较研究,与IGS和ITS的序列分析结果大体一致,说明新近发现的加特隐球酵母ITS8型确实有别于以前报道过的其他加特隐球酵母ITS基因型。研究证明IGS1及IGS2基因片段分析可以作为加特隐球酵母基因分类鉴定中有效的辅助鉴别的分子生物学方法,联合多种基因分类鉴定的方法可以更有效地揭示新型隐球酵母加特变种种内不同基因亚型间的遗传进化关系。     

Molecular characterization of some Indian Aloe vera populations through RAPD and ITS markers

The most important and well-known medicinal plant among ~400 species of the genus Aloe is Aloe vera. It is widely used in pharmaceutical, cosmetic, and food industries. Identification and assessment of genetic relationship among the populations and cultivars is needed for conservation and sustainable utilization of this commercially important plant. DNA fingerprinting with random amplified polymorphic DNA (RAPD) marker and Internal Transcribed Spacer (ITS1 and ITS2) of ribosomal DNA sequence analysis were carried out to assess the genetic diversity among populations of Aloe vera collected from geographically different four districts of West Bengal and Jodhpur, Rajasthan. RAPD profiles yielded 158 amplicons showing ~87.34% polymorphism. Analyses of ITS sequences showed that in contrast to ITS2, the length and %GC content (53.6–77.3%) of ITS1 varied within populations. Multiple sequence alignment data reveal that substitutions, insertions, and deletions have arisen at various positions in the ITS regions suggesting polymorphism. A 5′-GGCGCGATGGGCGCCAAGGAA-3′ sequence in ITS1 is conserved in all populations, except AvS4. RAPD dendrogram and topologies of the NJ, Parsimony and ML tree generated from ITS1 sequence revealed that there is a close genetic similarity among AvS1, AvS4, and AvS7 populations. These genetic studies may contribute to plant improvement programs of A. vera.     

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.     

RAPD and internal transcribed spacer sequence analyses reveal Zea nicaraguensis as a section Luxuriantes species close to Zea luxurians

Genetic relationship of a newly discovered teosinte from Nicaragua, Zea nicaraguensis with waterlogging tolerance, was determined based on randomly amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA using 14 accessions from Zea species. RAPD analysis showed that a total of 5,303 fragments were produced by 136 random decamer primers, of which 84.86% bands were polymorphic. RAPD-based UPGMA analysis demonstrated that the genus Zea can be divided into section Luxuriantes including Zea diploperennis, Zea luxurians, Zea perennis and Zea nicaraguensis, and section Zea including Zea mays ssp. mexicana, Zea mays ssp. parviglumis, Zea mays ssp. huehuetenangensis and Zea mays ssp. mays. ITS sequence analysis showed the lengths of the entire ITS region of the 14 taxa in Zea varied from 597 to 605 bp. The average GC content was 67.8%. In addition to the insertion/deletions, 78 variable sites were recorded in the total ITS region with 47 in ITS1, 5 in 5.8S, and 26 in ITS2. Sequences of these taxa were analyzed with neighbor-joining (NJ) and maximum parsimony (MP) methods to construct the phylogenetic trees, selecting Tripsacum dactyloides L. as the outgroup. The phylogenetic relationships of Zea species inferred from the ITS sequences are highly concordant with the RAPD evidence that resolved two major subgenus clades. Both RAPD and ITS sequence analyses indicate that Zea nicaraguensis is more closely related to Zea luxurians than the other teosintes and cultivated maize, which should be regarded as a section Luxuriantes species.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Presence of a short repeat sequence in internal transcribed spacer (ITS) 1 of the rRNA gene of <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae) from geographically different populations in Asia

Nucleotide sequences of the internal transcribed spacer 1 (ITS1)–5.8S–ITS2 region of the nuclear ribosomal RNA gene were determined in the white-backed planthopper (WBPH) Sogatella furcifera (Horváth) to detect molecular variation among regional populations in Asia. We analyzed 932 sequences from 172 individuals (4–9 clones per individual) of 33 populations collected in 1987–2008 from six countries, Japan, China, Taiwan, Vietnam, Philippines, and Papua New Guinea. WBPH showed intra-individual variation in ITS1, which is mainly attributable to the frequency (0–10) of the 66-bp repeat sequence in ITS1. Among the examined clones, the sequences of 5.8S were mostly identical and those of ITS2 were similar. A single planthopper had a maximum of 6 different variants in the number of ITS1 repeats, suggesting highly varied repeat numbers in individual planthoppers. The ITS1 with four repeats was the most frequently (64%) detected. Such a repeat was not observed in two other economically important planthopper species, Nilaparvata lugens (Stål) and Laodelphax striatellus (Fallén). The ITS nucleotide sequences in the WBPH populations in Asia were genetically close and some variations in the sequences were not related to regional populations, indicating that the nucleotide sequences of the ITS region are not useful for geographical discrimination of the WBPH. This closeness seems to be caused by long distance migration and genetic exchange among populations.     

Application of ITS sequence analysis, RAPD and AFLP fingerprinting in characterising the yeast genus Fellomyces

Three molecular techniques, ITS sequence analysis, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to study phylogenetic and genotypic relationships among strains of the genus Fellomyces. In the analyses were included strains isolated predominantly from epiphytic lichens collected in Indonesia, China and Mexico. The polyphasic approach indicated that the Fellomyces isolates are genotypically heterogeneous and that lichens represent a specific environment for selection of large number of the sterigmatoconidia producing species. The phylogenetic and genotypic analysis confirmed the existence of 11 currently accepted Fellomyces species and indicated that several species may be the new representatives of the genus. The RAPD and AFLP analyses demonstrated a higher potential in distinguishing the Fellomyces strains than the ITS regions. Since the sequence analysis showed low or no divergence among several strains, both RAPD and AFLP fingerprinting indicated that the strains may be discriminated at the species level.     

分子标记在鉴别拟船叶藓属和猫尾藓属中的应用

船叶藓科的拟船叶藓属(Dolichom itriopsis)和猫尾藓属植物(Isothecium)非常相似,不易从外部表型上进行属间物种的区分.以尖叶拟船叶藓6个居群、猫尾藓1个居群及疑似尖叶拟船叶藓种1个居群为材料,进行了随机扩增多态性(RAPD)标记分析,结果表明:50个随机引物中有15个引物扩增的产物具有多态性,15个引物共扩增出77条带,其中多态性条带为71条,多态带比率高达92.21%.聚类分析结果表明,疑似种与猫尾藓有较近的亲缘关系,与尖叶拟船叶藓亲缘关系较远.同时测定了疑似尖叶拟船叶藓种的rDNA的ITS全序列,与Genbank已知序列比对结果表明,疑似种与猫尾藓的同源性高达94.74%,而与尖叶拟船叶藓的同源性只有80.64%.进一步支持了RAPD的聚类结果,说明疑似种是猫尾藓而非尖叶拟船叶藓.     

Molecular evidence for the synonymy of two species of Apatemon Szidat, 1928, A. gracilis (Rudolphi, 1819) and A. annuligerum (von Nordmann, 1832) (Digenea: Strigeidae) parasitic as metacercariae in British fishes

The current study examined rDNA (internal transcribed spacer regions, ITS1 and ITS2) and cytochrome c oxidase subunit 1 (CO1) sequence data of Apatemon annuligerum (originating from two geographical locations) and A. gracilis metacercariae (originating from four natural piscine hosts) to determine the systematic status of these two strigeid digeneans. With the exception of short repeat motifs, the ITS1 regions sequenced demonstrated no intra- or inter-specific sequence variation. ITS2 sequences were 292 bp and CO1 sequences 366 bp in length and identical for both nominal Apatemon species. These sequence data provide strong evidence that the two species are con-specific and that A. annuligerum should be regarded as a junior synonym of A. gracilis.     

黑龙江省大豆菌核病菌核盘菌遗传多样性分析

了解黑龙江省不同地区侵染大豆核盘菌菌株分离物间的主要特性差异,利用PDA培养基对核盘菌进行分离和纯化,同时利用RAPD和rDNA-ITS标记方法对核盘菌进行遗传多样性分析,获得了50株纯化的核盘菌,用RAPD标记确定的遗传相似系数范围为0.54-0.98,平均相似系数为0.76,说明供试的核盘菌菌株的基因型具有一定的差异。对50个测定序列有差异的32个核盘菌ITS和5.8S rDNA片段的多序列对位分析,在ITS1区域的1-40bp种间变化较大,主要以碱基颠换和转换为进化形式。ITS2区域非常保守没有变异位点。黑龙江省核盘菌菌株在DNA水平上和ITS间隔区上具有较显著的遗传变异,显示出丰富的遗传多样性。     

苦瓜种质遗传多样性的RAPD和ISSR分析

采用RAPD和ISSR分子标记技术对38份苦瓜种质进行遗传多样性分析。结果表明:10个RAPD和10个ISSR引物分别扩增出93条和81条带,多态性比率分别为50.54%和61.29%;RAPD和ISSR标记检测供试材料的遗传相似性系数(GS)范围,分别为0.287~1和0.221~1,ISSR(平均GS值0.672)检测多态性效果高于RAPD(平均GS值0.694)。RAPD标记聚类分析将供试种质分为3个类群6组,分类结果与苦瓜瓜瘤的表型分类比较相似;ISSR标记聚类分析将供试种质分为3个类群7组,ISSR标记划分类群与形态上以颜色分类比较接近。RAPD和ISSR标记的遗传相似性系数呈显著相关(r=0.550)。两个标记整合后聚类分析可检测到更大的遗传变异,结果与苦瓜的农艺性状分类和地理分布有一定的相关性。     

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。     

RAPD分析与ITS序列分析在拟茎点霉分类鉴定上的应用

采用随机扩增多态性DNA(RAPD)技术和核糖体RNA基因(rDNA)转录间隔区(ITS)序列分析对22种拟茎点霉共34个菌株进行了系统发育研究。RAPD分析构建的UPGMA聚类图所反映的种间、种内关系与形态学分类结果基本一致,可以清楚地将分自7科寄主植物上的不同的种分别区分开来,但分自同科或同属寄主植物上的不同的种并不具有相近的亲缘关系。ITS序列分析结果不支持将Phomopsis mangiferae Ahmad和P. cytosporella Penz. et Sacc. 合并为同一个种的观点,同时还显示出P. mangiferae与P. psidii de Camara的亲缘关系非常近, 可能是异名同物;而为害木棉叶的拟茎点霉与杨梅枝枯病菌P. myricae Y.J.Huang et P.K.Chi之间的碱基差异亦属于种下不同菌株间的正常差别范围,很可能就是同一个种。对相同的供试菌株两技术所反映的亲缘关系趋势相同,表明两技术用于拟茎点霉的亲缘关系分析和种类鉴定均是可行的。     

RAPD分析与ITS序列分析在拟茎点霉分类鉴定上的应用*

采用随机扩增多态性DNA(RAPD)技术和核糖体RNA基因(rDNA)转录间隔区(ITS)序列分析对22种拟茎点霉共34个菌株进行了系统发育研究。RAPD分析构建的UPGMA聚类图所反映的种间、种内关系与形态学分类结果基本一致,可以清楚地将分自7科寄主植物上的不同的种分别区分开来,但分自同科或同属寄主植物上的不同的种并不具有相近的亲缘关系。ITS序列分析结果不支持将Phomopsis mangiferae Ahmad和P. cytosporella Penz. et Sacc. 合并为同一个种的观点,同时还显示出P. mangiferae与P. psidii de Camara的亲缘关系非常近, 可能是异名同物;而为害木棉叶的拟茎点霉与杨梅枝枯病菌P. myricae Y.J.Huang et P.K.Chi之间的碱基差异亦属于种下不同菌株间的正常差别范围,很可能就是同一个种。对相同的供试菌株两技术所反映的亲缘关系趋势相同,表明两技术用于拟茎点霉的亲缘关系分析和种类鉴定均是可行的。     

Evolution and phylogenetic information content of the ribosomal DNA repeat unit in the Blattodea (Insecta)

The organization, structure, and nucleotide variability of the ribosomal repeat unit was compared among families, genera, and species of cockroaches (Insecta:Blattodea). Sequence comparisons and molecular phylogenetic analyses were used to describe rDNA repeat unit variation at differing taxonomic levels. A reverse similar 1200 bp fragment of the 28S rDNA sequence was assessed for its potential utility in reconstructing higher-level phylogenetic relationships in cockroaches. Parsimony and maximum likelihood analyses of these data strongly support the expected pattern of relationships among cockroach groups. The examined 5' end of the 28S rDNA is shown to be an informative marker for larger studies of cockroach phylogeny. Comparative analysis of the nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among closely related species of Blattella and Periplaneta reveals that ITS sequences can vary widely in primary sequence, length, and folding pattern. Secondary structure estimates for the ITS region of Blattella species indicate that variation in this spacer region can also influence the folding pattern of the 5.8S subunit. These results support the idea that ITS sequences play an important role in the stability and function of the rRNA cluster.     

Intraspecific Concerted Evolution of the rDNA ITS1 in Anopheles farauti Sensu Stricto (Diptera: Culicidae) Reveals Recent Patterns of Population Structure

We examined the intraindividual variation present in the first ribosomal internal transcribed spacer (ITS1) of Anopheles farauti to determine the level of divergence among populations for this important malarial vector. We isolated 187 clones from 70 individuals and found regional variation among four internal tandem repeats. The data were partitioned prior to analysis given the presence of a paralogous ITS2 sequence, called the 5'-subrepeat, inserted in the ITS1 of most clones. A high level of homogenization and population differentiation was observed for this repeat, which indicates a higher rate of turnover relative to the adjacent 'core' region. Bayesian analysis was performed using several substitutional models on both a combined and a partitioned data set. On the whole, the ITS1 phylogeny and geographic origin of the samples appear to be congruent. Some interesting exceptions indicate the spread of variant repeats between populations and the retention of ancestral polymorphism. Our data clearly demonstrate concerted evolution at the intraspecific level despite intraindividual variation and a complex internal repeat structure from a species that occupies a continuous coastal distribution. A high rate of genomic turnover in combination with a high level of sequence divergence appears to be a major factor leading to its concerted evolution within these populations.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

Random Amplified Polymorphic DNA and Polymerase Chain Reaction Markers for the Differentiation and Detection of Stenocarpella maydis in Maize Seeds

The genetic relationship of 34 isolates of Stenocarpella maydis from different geographic regions in South Africa was analysed by random amplified polymorphic DNA (RAPD) and ribosomal DNA markers. Two genetic groups were differentiated by using three RAPD primers and correlated to the cultural morphology of the isolates. Of all the isolates tested, 79.4% were clustered into RAPD group I (RG I), which did not sporulate when cultured on potato dextrose agar (PDA) at 25°C for 10 days. The rest of the isolates designated as RG II sporulated on PDA medium and showed a higher genetic variation. Ribosomal DNA (rDNA) was amplified using polymerase chain reaction (PCR) with the universal primers, internal transcribed spacer (ITS) 1 and ITS 4. Restriction digestion of PCR products displayed three types (RF A, RF B and RF C) of profiles. RF A was in accordance with RG I. RF B was consistent with RG II except for one isolate, U5. However, U5 displayed a unique profile and had no restriction sites for Hpa II and Hae III. The results indicate that two distinct genetic groups exist among S. maydis isolates from maize in S. Africa. The ITS1 and ITS2 regions of rDNA were sequenced and primers were designed. The designed primer pair P1/P2 permitted a sensitive and specific detection of S. maydis .     

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.