Molecular Markers For Phylogenetic Studies Research Articles

Identification of a unique conserved region from a kinetoplastid genome of Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2 in Thailand

Mitochondrial DNAs (mtDNAs) appear in almost all eukaryotic species and are useful molecular markers for phylogenetic studies and species identification. Kinetoplast DNAs (kDNAs) are structurally complex circular mtDNA networks in kinetoplastids, divided into maxicircles and minicircles. Despite several kDNAs of many Leishmania species being examined, the kDNAs of the new species, Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2, have not been explored. This study aimed to investigate the maxicircle and minicircle DNAs of L. orientalis strain PCM2 using hybrid genome sequencing technologies and bioinformatic analyses. The kDNA sequences were isolated and assembled using the SPAdes hybrid assembler from the Illumina short-read and PacBio long-read data. Circular contigs of the maxicircle and minicircle DNAs were reconstructed and confirmed by BLASTn and rKOMICs programs. The kDNA genome was annotated by BLASTn before the genome comparison and phylogenetic analysis by progressiveMauve, MAFFT, and MEGA programs. The maxicircle of L. orientalis strain PCM2 (18,215 bp) showed 99.92% similarity and gene arrangement to Leishmania enriettii strain LEM3045 maxicircle with variation in the 12s rRNA gene and divergent region. Phylogenetics of the whole sequence, coding regions, divergent regions, and 12s rRNA gene also confirmed this relationship and subgenera separation. The identified 105 classes of minicircles (402–1177 bp) were clustered monophyletically and related to the Leishmania donovani minicircles. The kinetoplast maxicircle and minicircle DNAs of L. orientalis strain PCM2 contained a unique conserved region potentially useful for specific diagnosis of L. orientalis and further exploration of this parasite population genetics in Thailand and related regions.

5S Ribosomal DNA of Genus Solanum: Molecular Organization, Evolution, and Taxonomy.

The Solanum genus, being one of the largest among high plants, is distributed worldwide and comprises about 1,200 species. The genus includes numerous agronomically important species such as Solanum tuberosum (potato), Solanum lycopersicum (tomato), and Solanum melongena (eggplant) as well as medical and ornamental plants. The huge Solanum genus is a convenient model for research in the field of molecular evolution and structural and functional genomics. Clear knowledge of evolutionary relationships in the Solanum genus is required to increase the effectiveness of breeding programs, but the phylogeny of the genus is still not fully understood. The rapidly evolving intergenic spacer region (IGS) of 5S rDNA has been successfully used for inferring interspecific relationships in several groups of angiosperms. Here, combining cloning and sequencing with bioinformatic analysis of genomic data available in the SRA database, we evaluate the molecular organization and diversity of IGS for 184 accessions, representing 137 species of the Solanum genus. It was found that the main mechanisms of IGS molecular evolution was step-wise accumulation of single base substitution or short indels, and that long indels and multiple base substitutions, which arose repeatedly during evolution, were mostly not conserved and eliminated. The reason for this negative selection seems to be association between indels/multiple base substitutions and pseudogenization of 5S rDNA. Comparison of IGS sequences allowed us to reconstruct the phylogeny of the Solanum genus. The obtained dendrograms are mainly congruent with published data: same major and minor clades were found. However, relationships between these clades and position of some species (S. cochoae, S. clivorum, S. macrocarpon, and S. spirale) were different from those of previous results and require further clarification. Our results show that 5S IGS represents a convenient molecular marker for phylogenetic studies on the Solanum genus. In particular, the simultaneous presence of several structural variants of rDNA in the genome enables the detection of reticular evolution, especially in the largest and economically most important sect. Petota. The origin of several polyploid species should be reconsidered.

Comparative analysis of camelid mitochondrial genomes

Camelus dromedarius has played a pivotal role in both culture and way of life in the Arabian peninsula, particularly in arid regions where other domestic animals cannot be easily domesticated. Although, the mitochondrial genomes have recently been sequenced for several camelid species, wider phylogenetic studies are yet to be performed. The features of conserved gene elements, rapid evolutionary rate, and rare recombination make the mitochondrial genome a useful molecular marker for phylogenetic studies of closely related species. Here we carried out a comparative analysis of previously sequenced mitochondrial genomes of camelids with an emphasis on C. dromedarius, revealing a number of noticeable findings. First, the arrangement of mitochondrial genes in C. dromedarius is similar to those of the other camelids. Second, multiple sequence alignment of intergenic regions shows up to 90% similarity across different kinds of camels, with dromedary camels to reach 99%. Third, we successfully identified the three domains (termination-associated sequence, conserved domain and conserved sequence block) of the control region structure. The phylogenetic tree analysis showed that C. dromedarius mitogenomes were significantly clustered in the same clade with Lama pacos mitogenome. These findings will enhance our understanding of the nucleotide composition and molecular evolution of the mitogenomes of the genus Camelus, and provide more data for comparative mitogenomics in the family Camelidae.

Characterization of the complete mitochondrial genome of Uvitellina sp., representative of the family Cyclocoelidae and phylogenetic implications

Mitochondrial (mt) DNA has been useful in revealing the phylogenetic relationship of eukaryotic organisms including flatworms. Therefore, the use of mitogenomic data for the comparative and phylogenetic purposes is needed for those families of digenetic trematodes for which the mitogenomic data are still missing. Molecular data with sufficiently rich informative characters that can better resolve species identification, discrimination, and membership in different genera is also required for members of some morphologically difficult families of trematodes bearing few autapomorphic characters among its members. Here, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) and the complete mt genome of the trematode Uvitellina sp. (Cyclocoelidae: Haematotrephinae) was determined and annotated. The mt genome of this avian trematode is 14,217bp in length, containing 36 genes plus a single non-coding region. The ITS rDNA sequences were used for the pairwise sequence comparison of Uvitellina sp. with European cyclocoelid species, and the mitochondrial 12 protein-coding genes (PCGs) and two ribosomal RNA genes were used to evaluate the position of the family within selected trematodes. The ITS rDNA analysis of Uvitellina sp. showed less nucleotide differences with Hyptiasmus oculeus (16.77%) than with other European cyclocoelids (18.63-23.58%). The Bayesian inference (BI) analysis using the 12 mt PCGs and two rRNA genes supported the placement of the family Cyclocoelidae within the superfamily Echinostomatoidea (Plagiorchiida: Echinostmata). The availability of the mt genome sequences of Uvitellina sp. provides a novel resource of molecular markers for phylogenetic studies of Cyclocoelidae and other trematodes.

Phylogeny and genetic variation in the genus Eranthis using nrITS and cpIS single nucleotide polymorphisms

The relationships among species in the genus Eranthis Salisb. were investigated using single nucleotide polymorphisms (SNPs) of the nuclear DNA internal transcribed spacer 1, 2 region (nrITS) and the chloroplast trnL-trnF interspacer region (cpIS). Phylogenetic relationships based on the nrITS and cpIS were inferred with posterior probabilities with STRUCTURE analysis and the neighbor-joining method. Two major clades from nrITS and cpIS were consistent with species with yellow sepals in E. hyemalis, E. cilicica, E. longistipitata and the hybrid E. ×tubergenii; and white sepals in E. sibirica, E. longituba, E. albiflora, E. stellata, E. pungdoensis, E. byunsanensis, and E. pinnatifida. The phylogenetic tree of nrITS formed more subclades than the tree of cpIS, which suggested that nrITS SNPs are useful molecular markers for phylogenetic studies in the genus Eranthis. Only the SNPs of cpIS in E. pungdoensis accessions had a deletion at positions 259–420, and the posterior probability values (PPVs) assigned E. pungdoensis to population 4, which suggested that E. pungdoensis is different from E. byunsanensis. Therefore, E. byunsanensis and E. pungdoensis are considered to be true-to-type based on q-values because the PPVs were greater than 0.9 in both species based on the STRUCTURE analysis of nrITS SNPs. Significant genetic variation in E. stellata collected in Goesan-kun and Mt. Mugap, Korea indicated a potential gene flow among closely related E. byunsanensis, E. pinnatifida, E. sibirica, and E. stellata that could be due to geographic proximity in their distributions. E. stellata from Mt. Mugap showed mixed PPVs for E. stellata and E. byunsanensis, therefore, E. byunsanensis might be a possible hybrid origin for E. stellata collected from Goesan-kun and Mt. Mugap.

Genetic Diversity and Molecular Phylogeny of Iranian Goats Based on Cytochrome Oxidase I (COXI) Gene Sequences (KERAGAMAN GENETIK DAN FILOGENI MOLEKULER KAMBING-KAMBING IRAN BERDASARKAN SEKUENS GEN CYTOCHROME OXIDASE I (COXI))

Mitochondrial DNA has been one of the most widely used molecular markers for phylogenetic studies in animals because of its simple genomic structure. This study examines the genetic characteristic of domestic goat using sequence analysis of mitochondrial DNA Cytochrome oxidase subunit I (COXI) to identify and differentiate among three common breeds (Adani, Najdi and Markhoz) of Iran. The genomic DNA was isolated by salting out method and amplified cytochrome oxidase I gene using Polymerase Chain Reaction (PCR) method with a pair of primer. Phylogenetic trees and pairwise calculations were obtained by using Mega 6 software. A partial sequence of cytochrome oxidase I gene of Iranian goats is 1286 bp and contained four variable sites and three haplotypes. Phylogenetic analysis of haplotype in the combination with the goat from GenBank showed that Iranian goatclustered in a separate lineage. This study was found informative for establishing relationships between breeds from different parts of the world. This study may facilitate the future researchers and breeders for better understanding the genetic interactions and breed differentiation for devising future breeding and conservation strategies to preserve the rich animal genetic reservoir of the country.

Sequencing and analysis of the complete mitochondrial genome in Anopheles sinensis (Diptera: Culicidae)

BackgroundAnopheles sinensis (Diptera: Culicidae) is a primary vector of Plasmodium vivax and Brugia malayi in most regions of China. In addition, its phylogenetic relationship with the cryptic species of the Hyrcanus Group is complex and remains unresolved. Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes, of which mitochondrial genome data of An. sinensis is not available.MethodsAn. sinensis samples was collected from Shandong, China, and identified by molecular marker. Genomic DNA was extracted, followed by the Illumina sequencing. Two complete mitochondrial genomes were assembled and annotated using the mitochondrial genome of An. gambiae as reference. The mitochondrial genomes sequences of the 28 known Anopheles species were aligned and reconstructed phylogenetic tree by Maximum Likelihood (ML) method.FindingsThe length of complete mitochondrial genomes of An. sinensis was 15,076 bp and 15,138 bp, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and an AT-rich control region. As in other insects, most mitochondrial genes are encoded on the J strand, except for ND5, ND4, ND4L, ND1, two rRNA and eight tRNA genes, which are encoded on the N strand. The bootstrap value was set as 1000 in ML analyses. The topologies restored phylogenetic affinity within subfamily Anophelinae. The ML tree showed four major clades, corresponding to the subgenera Cellia, Anopheles, Nyssorhynchus and Kerteszia of the genus Anopheles.ConclusionsThe complete mitochondrial genomes of An. sinensis were obtained. The number, order and transcription direction of An. sinensis mitochondrial genes were the same as in other species of family Culicidae.

The complete chloroplast genome sequences of three Adenophora species and comparative analysis with Campanuloid species (Campanulaceae)

We report the complete chloroplast genomes of three Adenophora species, and analyzed these compared them to five published Campanuloid plastomes. The total genome length of Adenophora divaricata, Adenophora erecta, and Adenophora stricta ranged from 159,759 to 176,331 bp. Among the eight Campanuloid species, many inversions were found to be only in the LSC region. IR contraction was also identified in the plastid genome of Adenophora stricta. Phylogenetic analyses based on 76 protein coding genes showed that Campanuloids are monophyletic, and are composed of two major groups: Campanula s. str. and Rapunculus. When we compared each homologous locus among the four Adenophora species, ten regions showed high nucleotide divergence value (>0.03). Among these, nine loci, excepting ycf3-rpoB, are considered to be useful molecular markers for phylogenetic studies and will be helpful to resolve phylogenetic relationships of Adenophora.

Venom Gland Transcriptomic and Proteomic Analyses of the Enigmatic Scorpion Superstitionia donensis (Scorpiones: Superstitioniidae), with Insights on the Evolution of Its Venom Components.

Venom gland transcriptomic and proteomic analyses have improved our knowledge on the diversity of the heterogeneous components present in scorpion venoms. However, most of these studies have focused on species from the family Buthidae. To gain insights into the molecular diversity of the venom components of scorpions belonging to the family Superstitioniidae, one of the neglected scorpion families, we performed a transcriptomic and proteomic analyses for the species Superstitionia donensis. The total mRNA extracted from the venom glands of two specimens was subjected to massive sequencing by the Illumina protocol, and a total of 219,073 transcripts were generated. We annotated 135 transcripts putatively coding for peptides with identity to known venom components available from different protein databases. Fresh venom collected by electrostimulation was analyzed by LC-MS/MS allowing the identification of 26 distinct components with sequences matching counterparts from the transcriptomic analysis. In addition, the phylogenetic affinities of the found putative calcins, scorpines, La1-like peptides and potassium channel κ toxins were analyzed. The first three components are often reported as ubiquitous in the venom of different families of scorpions. Our results suggest that, at least calcins and scorpines, could be used as molecular markers in phylogenetic studies of scorpion venoms.

Mitochondrial Genomes ofAnopheles(Kerteszia) (Diptera: Culicidae) From the Atlantic Forest, Brazil

Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes, such as the Anopheles albitarsis complex. Except for a few studies that employed a limited number of nuclear or mitochondrial loci to address the genetic structure and species status of Anopheles cruzii, Anopheles bellator, and Anopheles homunculus, little is known about genetic markers that can be employed in studies focusing on Kerteszia species. The complete mitochondrial genomes of seven specimens of An. bellator, An. cruzii, An. homunculus, and Anopheles laneanus were sequenced using long-range polymerase chain reaction and Illumina sequencing. The mitochondrial genomes varied from 15,446 to 15,738 bp in length and contained 37 genes (13 protein-encoding genes, 2 rRNA genes [12S rRNA and 16S rRNA] and 22 tRNA genes), and the AT-rich control region, as all do other Anopheles mitochondrial genomes sequenced to date. Specimens from four populations of An. cruzii showed differences in codon composition.

Mitochondrial markers for identification and phylogenetic studies in insects – A Review

AbstractSimilar morphology and high genetic diversity poses problems in phylogenetic studies of insects. To solve these problems, mitochondrial based markers have been adopted and are increasingly used as molecular markers for phylogenetic studies. Varied markers have been used for different species of insects, viz., markers for 16S r RNA, 12S r RNA, ND (1-6 genes), ATPase and control regions. Among which protein coding gene, CO-1 is found to be best because of its advantage over others whereas, AT rich region of mitochondrial DNA is the least used marker. A recent advanced technology in phylogenetic analysis; namely mitogenomics have greatly improved this research field. This short review attempted to summarize recent studies on the application of various mitochondrial molecular markers for phylogenetic study of insects.

Genetic Diversity of Bile Salt Hydrolases Among Human Intestinal Bifidobacteria

This study analyzes the application of degenerative primers for the screening of bile salt hydrolase-encoding genes (bsh) in various intestinal bifidobacteria. In the first stage, the design and evaluation of the universal PCR primers for amplifying the partial coding sequence of bile salt hydrolase in bifidobacteria were performed. The amplified bsh gene fragments were sequenced and the obtained sequences were compared to the bsh genes present in GenBank. The determined results showed the utility of the designed PCR primers for the amplification of partial gene encoding bile salt hydrolase in different intestinal bifidobacteria. Moreover, sequence analysis revealed that bile salt hydrolase-encoding genes may be used as valuable molecular markers for phylogenetic studies and identification of even closely related members of the genus Bifidobacterium.

Application of EST-SSRs to examine genetic diversity in eggplant and its close relatives

Within the genus Solanum, the term 'eggplant' encompasses several cultivated species that are used for food and, to a lesser extent, for medicine. The use of one common name to describe more than one species and the existence of many related wild species have led to taxonomic confusion which, in turn, have complicated analyses of evolutionary relationships and genetic diversity within this groups of species. A further challenge for eggplant research is that, despite the fact that the use of molecular markers for phylogenetic studies is well-established, very few studies have described the development of new markers for eggplant. In our work, genic microsatellite (SSR) markers were identified from an expressed sequence tag library of S. melongena and used for analysis of 47 accessions of eggplant and closely related species. The markers had very good polymorphism in the 18 species tested including 8 S. melongena accessions. Moreover, genetic analysis performed with these markers showed concordance with previous research and knowledge of eggplant domestication. These markers are expected to be a valuable resource for studies of genetic relationships, fingerprinting, and gene mapping in eggplant.

Sequence variation of the internal transcribed spacer (ITS) region of ribosomal DNA in Cerastoderma species (Bivalvia: Cardiidae)

The internal transcribed spacer (ITS) region of ribosomal DNA (ITS1, 5.8S rRNA gene and ITS2) constitutes one of the most widely applied molecular markers in phylogenetic studies and species differentiation. Here, the ITS region of the cockles Cerastoderma edule from NW Spain and C. glaucum from the Mediterranean and Baltic Seas was characterized and its variation examined. The length of the ITS was 762–783 bp (ITS1, 226–251 bp; 5.8S rRNA gene, 158–161 bp; ITS2, 305–325 bp) and the GC content ranged between 57% and 60% (ITS1, 52–62%; 5.8S rRNA gene, 56–57%; ITS2, 61–63%). All individuals showed variation among ITS repeats, mostly in the spacers, with nucleotide diversity values of 0.0005–0.07. Among the C. glaucum sequences, two types of ITS (ITSI and ITSII) were distinguished, based on the percentage of differences observed in the spacers, sequence of the 5.8S gene, GC content of the ITS1 and clustering in phylogenetic trees. A PCR assay using specific primers for each ITS type demonstrated that both types coexist in all C. glaucum individuals. While type I seems to contain the bona fide 5.8S rRNA gene, type II could contain a pseudogene. Cerastoderma edule sequences grouped separately on phylogenetic trees, as expected for a differentiated species, but those of C. glaucum from the Mediterranean and Baltic Seas were intermixed in the two clades defining the ITS types, which supports the absence of systematic division previously inferred from other molecular markers.

Duplications and Expression of DIVARICATA-Like Genes in Dipsacales

The genetics underlying flower symmetry shifts between radial and bilateral symmetry has been intensively studied in the model Antirrhinum majus. Understanding the conservation or diversification of this genetic pathway in other plants is of special interest in understanding angiosperm evolution and ecology. Evidence from Antirrhinum indicates that TCP and MYB transcription factors, especially CYCLOIDEA (CYC), DICHOTOMA (DICH), DIVARICATA (DIV), and RADIALIS (RAD) play a role in specifying dorsal identity (CYC, DICH, and RAD) and ventral identity (DIV) in the corolla and androecium of monosymmetric (bilateral) flowers. Previous data indicate that the ECE clade of TCP genes (including CYC and DICH) underwent two duplication events around the diversification of the core eudicots. In this study, we examined the duplication events within Dipsacales, which contains both radially and bilaterally symmetrical flowered species. Additionally, we report here the phylogenetic relationships of the DIV-like genes across core eudicots. Like TCP genes, we found three core eudicot clades of DIV-like genes, with duplications occurring around the diversification of the core eudicots, which we name DIV1, DIV2, and DIV3. The Antirrhinum genes, DIVARICATA and its sister DVL1, fall into the DIV1 clade. We also found additional duplications within these clades in Dipsacales. Specifically, the Caprifoliaceae (bilaterally symmetrical clade) duplicated independently in each of the three core eudicot DIV clades. Using reverse transcription polymerase chain reaction (rtPCR), we showed that most of these copies are expressed across floral tissues in the Dipsacales species Heptacodium miconioides. One copy, DipsDIV1A (orthologous to DIV and DVL1), was expressed in a dorsal-ventral pattern. DipsDIV1A was expressed only in petal tissue, in both dorsal and ventral regions but was lacking from lateral petals. We argue that this suggests that DipsDIV1A may be expressed in a similar pattern to DIV in Antirrhinum, suggesting a broad conservation of this pathway. Finally, DIV contains a large intron near the beginning of the second MYB domain, which shows promise as a highly variable molecular marker for phylogenetic studies.

Phylogenetic comparison of local length plasticity of the small subunit of nuclear rDNAs among all Hexapoda orders and the impact of hyper-length-variation on alignment

The SSU nrDNA (18S), is one of the most frequently sequenced molecular markers in phylogenetic studies. However, the length-hyper-variation at multiple positions of this gene can affect the accuracy of alignment greatly and this length variation makes alignment across arthropod orders a serious problem. The analyses of Hexapoda phylogeny is such a case. A more clear recognition of the distribution of the length-variable-regions is needed. In this study, the secondary structure of some length-variable-regions in the SSU nrRNA of Arthropoda was adjusted by the principle of co-variation. It is found that the extent of plasticity of some length-variable-region can extraordinarily be higher than 600 bases in hexapods. And the numbers of hyper length-variable-regions are largest in Strepsiptera and Sternorrhyncha (Hemiptera). Our study shows that some length-variable-regions can serve as synapomorphies for some groups. The phylogenetic comparison also suggested that the expansion of a lateral bulge could be the origin of a helix.

Intron and Polypeptide Evolution of Conserved NPA to NPA Motif Regions in Plant Aquaporins

Aquaporin proteins are part of an ancient family that functions as water transporting facilitators in all organisms. Phylogenetic and physiological analyses have revealed that plant aquaporins consist of two groups: the plasma membrane intrinsic proteins (PIPs) and the tonoplast intrinsic proteins (TIPs). Using the conserved asparagineproline-alanine (NPA) to NPA motif regions, we studied the evolution of 35 plant aquaporins that included nine of our newly cloned cucumber aquaporins and 26 from the GenBank database. Results indicated that NPA repeated regions were effective for phylogenetically characterizing the plant aquaporin family, and to accurately localize the introns. Phylogenetic analysis showed that 35 plant aquaporins fell into two distinct groups (except for the Arabidopsis gene AtMip)—PIPs and TIPs. The nine cucumber aquaporins belong to the PIP group that were localized further into two different sub-groups. The intron analysis showed that introns of plant aquaporins mainly consist of two types. Eighteen PIPs shared identical intron positions localized in connecting loop C between amino acids 95 and 96. Nine TIPs shared the other identical intron positions localized in connecting loop D between amino acids 44 and 45. Cucumber aquaporins CRB9 and CRB10 (with no intron in the repeated NPA regions) may be the result of intron loss events, while intronless rice (Orzya sativa) Os-TIP1 and Os-TIP2 may have resulted from other intron loss events. PIP11 and Os-PIP do not have the same amino acid number as major PIP members, but combined phylogenetic analysis results along with intron positions and phases showed that they belong to the PIP group. The phylogenetic tree and intron position information suggest that AtMip was mis-annotated as a member of aquaporin, and is a homologue of the glycerol facilitator-like protein. Introns share identical positions and phases within the PIP group (except PIP13) or the TIP group, but differ between the plasma and the tonoplast membrane aquaporins matching the phylogenetic analysis results. Intron positions of the repeated NPA regions of plant aquaporins that have stable inheritance can act as molecular markers for phylogenetic studies.

Interspecific polymorphism at non-coding regions of chloroplast, mitochondrial DNA and rRNA IGS region in Elymus species.

Several published universal primers for amplification of non-coding regions of chloroplast, mitochondrial and ribosomal (rRNA) IGS region were tested whether they can amplify respective regions in Elymus species. PCR-RFLP analysis of the chloroplast, mitochondral DNA, and rRNA IGS region of the genus Elymus was used to determine if the method could be employed to detect inter-specific variation in this genus. Published universal primers for amplification of trnK [tRNA-Lys (UUU) exon 1]-trnK [tRNA-Lys (UUU) exon2], and mitochondrial nad1 exon B-nadl exon C intron successfully amplified the respective regions in Elymus species. However, the primers for amplification of chloroplast trnD-trnT intron and rRNA IGS failed to amplify the respective region in Elymus species. New primer pairs were designed and successfully amplified the cpDNA trnD-trnT intron and rRNA IGS region in Elymus species. The amplification products were digested with seven restriction enzymes. The results showed that the investigated regions of chloroplast and mitochondrial genomes are variable in most of the tested taxa and contain multiple variable regions. These regions should serve as useful molecular markers in phylogenetic studies of closely related species, at least at the interspecific level in Elymus. It is likely that further studies, including larger sample sizes, more regions of these genomes and/or more powerful methods for the detection of cpDNA and mt DNA variation will reveal additional variation for this genus. Highly inter- and intra-specific polymorphisms for rRNA IGS region were detected, suggesting the IGS will be a useful molecular marker for population studies of Elymus species.