Molecular phylogeny of RPB2 gene reveals multiple origin, geographic differentiation of H genome, and the relationship of the Y genome to other genomes in Elymus species

It has been hypothesized from isozymic and cytological studies of Elymus species that the Old and New World taxa may be of separate origin of the H genome in the StH genome species. To test this hypothesis, and estimate the phylogenetic relationships of polyploid Elymus species within the Triticeae, the second largest subunit of RNA polymerase II (RPB2) sequence of 36 Elymus accessions containing StH or StY genomes was analyzed with those of Pseudoroegneria (St), Hordeum (H), Agropyron (P), Australopyrum (W), Lophopyrum(Ee), Thinopyrum(Eb) and Dasypyrum (V). Our data indicated that the H genome in Elymus species is differentiated in accordance with geographical origin, and that the Eurasian and American StH genome species have independent alloploid origins with different H-genome donors. Phylogenetic analysis of Y genome sequences with other genome donors (St, H, P, W) of Elymus revealed that W and P genomes are sister to Y genome with a 87% bootstrap support, and that StY and StH species group might have acquired their RPB2 St sequences from distinct Pseudoroegneria gene pools. Our data did not support the suggestion that the St and Y genomes have the same origin as put forward in a previous study using ITS data. Our result provides some insight on the origin of Y genome and its relationship to other genomes in Elymus.     

Phylogenetic relationships in Elymus (Poaceae: Triticeae) based on the nuclear ribosomal internal transcribed spacer and chloroplast trnL-F sequences

To estimate the phylogenetic relationship of polyploid Elymus in Triticeae, nuclear ribosomal internal transcribed spacer (ITS) and chloroplast trnL-F sequences of 45 Elymus accessions containing various genomes were analysed with those of five Pseudoroegneria (St), two Hordeum (H), three Agropyron (P) and two Australopyrum (W) accessions. The ITS sequences revealed a close phylogenetic relationship between the polyploid Elymus and species from the other genera. The ITS and trnL-F trees indicated considerable differentiation of the StY genome species. The trnL-F sequences revealed an especially close relationship of Pseudoroegneria to all Elymus species included. Both the ITS and trnL-F trees suggested multiple origins and recurrent hybridization of Elymus species. The results suggested that: the St, H, P, and W genomes in polyploid Elymus were donated by Pseudoroegneria, Hordeum, Agropyron and Australopyrum, respectively, and the St and Y genomes may have originated from the same ancestor; Pseudoroegneria was the maternal donor of the polyploid Elymus; and some Elymus species showed multiple origin and experienced recurrent hybridization.     

Molecular phylogeny revealed complex evolutionary process in Elymus species

Recent molecular phylogenetic studies on Elymus have added to our understanding of the origination of Elymus species. However, evolutionary dynamics and speciation of most species in Elymus are unclear. Molecular phylogeny has demonstrated that reticulate evolution has occurred extensively in the genus, as an example, the largest subunit of RNA polymerase II (rpb2) and phosphoenolpyruvate carboxylase (pepC) data revealed two versions of the St genome, St1 and St2contributing to speciation of E. caninus. Phylogenetic analyses of E. pendulinus uncovered additional genome-level complexity. Our data indicated that both chloroplast and nuclear gene introgression have occurred in the evolutionary process of E. pendulinus. Non-donor species genomes have been detected in severalElymus species, such as in allohexaploid E. repens (StStStStHH), a Taeniatherum-like (Ta genome in Triticeae) GBSSI sequence, Bromus- (Bromeae) and Panicum-like (Paniceae) ITS sequences have been detected. The chloroplast DNA data indicated that Pseudoroegneria is the maternal genome donor to Elymus species, but whether different Elymus species originated from different St donors remains an open question. The origin of the Y genome in Elymus is puzzling. It is clear that the Ygenome is distinct from the St genome, but unclear on the relationships of Y to other genomes in Triticeae. Introgressive hybridization may be an important factor complicating the evolutionary history of the species in Elymus. The extent of introgression and its role in creating diversity in Elymus species should be the objective of further investigations.     

Genome discrimination by in situ hybridization in Icelandic species of Elymus and Elytrigia (Poaceae: Triticeae).

The genome constitution of Icelandic Elymus caninus, E. alaskanus, and Elytrigia repens was examined by fluorescence in situ hybridization using genomic DNA and selected cloned sequences as probes. Genomic in situ hybridization (GISH) of Hordeum brachyantherum ssp. californicum (diploid, H genome) probe confirmed the presence of an H genome in the two tetraploid Elymus species and identified its presence in the hexaploid Elytrigia repens. The H chromosomes were painted uniformly except for some chromosomes of Elytrigia repens which showed extended unlabelled pericentromeric and subterminal regions. A mixture of genomic DNA from H. marinum ssp. marinum (diploid, Xa genome) and H. murinum ssp. leporinum (tetraploid, Xu genome) did not hybridize to chromosomes of the Elymus species or Elytrigia repens, confirming that these genomes were different from the H genome. The St genomic probe from Pseudoroegneria spicata (diploid) did not discriminate between the genomes of the Elymus species, whereas it produced dispersed and spotty hybridization signals most likely on the two St genomes of Elytrigia repens. Chromosomes of the two genera Elymus and Elytrigia showed different patterns of hybridization with clones pTa71 and pAes41, while clones pTa1 and pSc119.2 hybridized only to Elytrigia chromosomes. Based on FISH with these genomic and cloned probes, the two Elymus species are genomically similar, but they are evidently different from Elytrigia repens. Therefore the genomes of Icelandic Elymus caninus and E. alaskanus remain as StH, whereas the genomes of Elytrigia repens are proposed as XXH.     

Distinct origin of the Y and St genome in Elymus species: evidence from the analysis of a large sample of St genome species using two nuclear genes

Background

Previous cytological and single copy nuclear genes data suggested the St and Y genome in the StY-genomic Elymus species originated from different donors: the St from a diploid species in Pseudoroegneria and the Y from an unknown diploid species, which are now extinct or undiscovered. However, ITS data suggested that the Y and St genome shared the same progenitor although rather few St genome species were studied. In a recent analysis of many samples of St genome species Pseudoroegneria spicata (Pursh) À. Löve suggested that one accession of P. spicata species was the most likely donor of the Y genome. The present study tested whether intraspecific variation during sampling could affect the outcome of analyses to determining the origin of Y genome in allotetraploid StY species. We also explored the evolutionary dynamics of these species.

Methodology/Principal Findings

Two single copy nuclear genes, the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor G (EF-G) sequences from 58 accessions of Pseudoroegneria and Elymus species, together with those from Hordeum (H), Agropyron (P), Australopyrum (W), Lophopyrum (E^e), Thinopyrum (E^a), Thinopyrum (E^b), and Dasypyrum (V) were analyzed using maximum parsimony, maximum likelihood and Bayesian methods. Sequence comparisons among all these genomes revealed that the St and Y genomes are relatively dissimilar. Extensive sequence variations have been detected not only between the sequences from St and Y genome, but also among the sequences from diploid St genome species. Phylogenetic analyses separated the Y sequences from the St sequences.

Conclusions/Significance

Our results confirmed that St and Y genome in Elymus species have originated from different donors, and demonstrated that intraspecific variation does not affect the identification of genome origin in polyploids. Moreover, sequence data showed evidence to support the suggestion of the genome convergent evolution in allopolyploid StY genome species.     

Molecular evolution and genome divergence at <Emphasis Type="Italic">RPB2</Emphasis> gene of the St and H genome in <Emphasis Type="Italic">Elymus</Emphasis> species

Molecular evolution of the second largest subunit of low copy nuclear RNA polymerase II (RPB2) in allotetrploid StH genomic species of Elymus is characterized here. Our study first reported a 39-bp MITE stowaway element insertion in the genic region of RPB2 gene for all tetraploid Elymus St genome and diploid Pseudoroegneria spicata and P. stipifolia St genome. The sequences on 3′-end are highly conserved, with AGTA in all sequences but H10339 (E. fibrosis), in which the AGTA was replaced with AGAA. All 12 Stowaway-containing sequences encompassed a 9 bp conserved TIRs (GAGGGAGTA). Interestingly, the 5′-end sequence of GGTA which was changed to AGTA or deleted resulted in Stowaway excision in the H genome of Elymus sepcies, in which Stowaway excision did not leave footprint. Another two large insertions in all St genome sequences are also transposable-like elements detected in the genic region of RPB2 gene. Our results indicated that these three transposable element indels have occurred prior to polyploidization, and shaped the homoeologous RPB2 loci in St and H genome of Eymus species. Nucleotide diversity analysis suggested that the RPB2 sequence may evolve faster in the polyploid species than in the diploids. Higher level of polymorphism and genome-specific amplicons generated by this gene indicated that RPB2 is an excellent tool for investigating the phylogeny and evolutionary dynamics of speciation, and the mode of polyploidy formation in Elymus species.     

Molecular evolution and phylogeny of the RPB2 gene in the genus Hordeum

Background and Aims

It is known that the miniature inverted-repeat terminal element (MITE) preferentially inserts into low-copy-number sequences or genic regions. Characterization of the second largest subunit of low-copy nuclear RNA polymerase II (RPB2) has indicated that MITE and indels have shaped the homoeologous RPB2 loci in the St and H genome of Eymus species in Triticeae. The aims of this study was to determine if there is MITE in the RPB2 gene in Hordeum genomes, and to compare the gene evolution of RPB2 with other diploid Triticeae species. The sequences were used to reconstruct the phylogeny of the genus Hordeum.

Methods

RPB2 regions from all diploid species of Hordeum, one tetraploid species (H. brevisubulatum) and ten accessions of diploid Triticeae species were amplified and sequenced. Parsimony analysis of the DNA dataset was performed in order to reveal the phylogeny of Hordeum species.

Key Results

MITE was detected in the Xu genome. A 27–36 bp indel sequence was found in the I and Xu genome, but deleted in the Xa and some H genome species. Interestingly, the indel length in H genomes corresponds well to their geographical distribution. Phylogenetic analysis of the RPB2 sequences positioned the H and Xa genome in one monophyletic group. The I and Xu genomes are distinctly separated from the H and Xa ones. The RPB2 data also separated all New World H genome species except H. patagonicum ssp. patagonicum from the Old World H genome species.

Conclusions

MITE and large indels have shaped the RPB2 loci between the Xu and H, I and Xa genomes. The phylogenetic analysis of the RPB2 sequences confirmed the monophyly of Hordeum. The maximum-parsimony analysis demonstrated the four genomes to be subdivided into two groups.Key words: Molecular evolution, RPB2, Hordeum, transposable element, phylogeny     

Genetic variability and genomic divergence of Elymus repens and related species

Summary In order to study the genetic variation and phylogenetic relationship in Elymus repens, amplified fragment length polymorphism (AFLP) were used, together with sequence data for the nuclear gene phytochrome B, phyB, and the chloroplast ribosomal protein encoding gene rps4. A total of 83 collected E. repens samples, 3 E. repens reference samples and 18 related species accessions were analysed and compared with 13 GenBank sequences. AMOVA analysis revealed a moderate genetic differentiation between the populations of E. repens in the three Swedish provinces investigated, while no differentiation was observed due to landscape type. A moderate genetic differentiation was also found when samples from different fields in one province were compared to samples from a selected field. A common female origin was found in E. repens and seven other Elymus species, Pseudoroegneria spicata, Thinopyrum intermedium, T. junceum, Hordeum bogdanii and H. stenostachys. The latter two both harbour the H genome. Taken together, the data suggest that the Swedish E. repens population is slightly heterogeneous and comprises multiple origins of genome donors; a nuclear genome with contributions from Pseudoroegneria (St), Hordeum (H), Thinopyrum (E) and Y with an unknown donor together with a maternal genome donated from Pseudoroegneria.     

Origin of the H genome in StH-genomic Elymus species based on the single-copy nuclear gene DMC1

Previous studies have suggested that the H haplome in Elymus could originate from different diploid Hordeum species, however, which diploid species best represent the parental species remains unanswered. The focus of this study seeks to pinpoint the origin of the H genome in Elymus. Allopolyploid Elymus species that contain the StH genome were analyzed together with diploid Hordeum species and a broad sample of diploid genera in the tribe Triticeae using DMC1 sequences. Both parsimony and maximum likelihood analyses well separated the American Hordeum species, except Hordeum brachyantherum subsp. californicum, from the H genome of polyploid Elymus species. The Elymus H-genomic sequences were formed into different groups. Our data suggested that the American Horedeum species, except H. brachyantherum subsp. californicum, are not the H-genomic donor to the Elymus species. Hordeum brevisubulatum subsp. violaceum was the progenitor species to Elymus virescens, Elymus confusus, Elymus lanceolatus, Elymus wawawaiensis, and Elymus caninus. Furthermore, North American H. brachyantherum subsp. californicum was a progenitor of the H genome to Elymus hystrix and Elymus cordilleranus. The H genomes in Elymus canadensis, Elymus sibiricus, and Elymus multisetus were highly differentiated from the H genome in Hordeum and other Elymus species. The H genome in both North American and Eurasian Elymus species was contributed by different Hordeum species.     

Origin and Reticulate Evolutionary Process of Wheatgrass Elymus trachycaulus (Triticeae: Poaceae)

To study origin and evolutionary dynamics of tetraploid Elymus trachycaulus that has been cytologically defined as containing StH genomes, thirteen accessions of E. trachycaulus were analyzed using two low-copy nuclear gene Pepc (phosphoenolpyruvate carboxylase) and Rpb2 (the second largest subunit of RNA polymerase II), and one chloroplast region trnL–trnF (spacer between the tRNA Leu (UAA) gene and the tRNA-Phe (GAA) gene). Our chloroplast data indicated that Pseudoroegneria (St genome) was the maternal donor of E. trachycaulus. Rpb2 data indicated that the St genome in E. trachycaulus was originated from either P. strigosa, P. stipifolia, P. spicata or P. geniculate. The Hordeum (H genome)-like sequences of E. trachycaulus are polyphyletic in the Pepc tree, suggesting that the H genome in E. trachycaulus was contributed by multiple sources, whether due to multiple origins or introgression resulting from subsequent hybridization. Failure to recovering St copy of Pepc sequence in most accessions of E. trachycaulus might be caused by genome convergent evolution in allopolyploids. Multiple copies of H-like Pepc sequence from each accession with relative large deletions and insertions might be caused by either instability of Pepc sequence in H- genome or incomplete concerted evolution. Our results highlighted complex evolutionary history of E. trachycaulus.     

Karyotypes of Elymus scabrifolius (Poaceae: Triticeae) from South America studied by banding techniques and in situ hybridization

Karyotypes of 4 accessions of Elymus scabrifolius (2n = 4x = 28) were investigated by Giemsa C- and N-banding, GAA-banding (one accession), AgNO3-staining and in situ hybridization with the rDNA probe pTa71. Two additional accessions were studied in less detail. The chromosomes were large (9-14 microns). The complements included 11 pairs of metacentrics, one with conspicuous satellites on the short arms, and 3 pairs of submetacentrics. Two of 4 accessions from Eastern Argentina and Uruguay had minute or small satellites on a submetacentric pair. No such satellites were observed in the other two accessions. In two accessions from the Cordoba province, a non-homologous submetacentric pair had very long satellites. AgNO3-staining established the presence of 4 nucleoli, two larger and two small ones, in 5 accessions. The C-banding patterns comprised from one to 12 conspicuous bands per chromosome at no preferential positions. The amount of constitutive heterochromatin (19-21%) was the highest hitherto established in the Triticeae. Similarities in banding patterns and chromosome morphology identified homologous and discriminated between non-homologous chromosomes within and, except for two chromosomes, between plants. Heteromorphic chromosome pairs were identified in satellite-carrying chromosomes only. N-banding produced conspicuous bands overall at the same positions as C-banding. GAA-banding patterns were similar to N-banding patterns. The rDNA probe hybridized to chromosome segments at nucleolar constrictions only. The production of C- and N-banding patterns in both genomes of E. scabrifolius suggests the presence of two H genomes and the absence of the pivotal St genome of Elymus. On account of the uncertain identity of one genome, and the overall similar gross morphology of E. scabrifolius and other tetraploid South American species referred to Elymus, E. scabrifolius is retained in Elymus.     

Phylogeny of Hystrix and related genera (Poaceae: Triticeae) based on nuclear rDNA ITS sequences

The taxonomic status of Hystrix and phylogenetic relationships among Hystrix and its related genera of Pseudoroegneria (St), Hordeum (H), Psathyrostachys (Ns), Elymus (StH), Leymus (NsXm), Thinopyrum bessarabicum (E(b)) and Lophopyrum elongatum (E(e)) were estimated from sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The type species of Hystrix, H. patula, clustered with species of Pseudoroegneria, Hordeum, Elymus, Th. bessarabicum and Lo. elongatum, while H. duthiei ssp. duthiei, H. duthiei ssp. longearistata, H. coreana and H. komarovii were grouped with Psathyrostachys and Leymus species. The results indicate that: (i) H. patula is distantly related to other species of Hystrix, but is closely related to Elymus species; (ii) H. duthiei ssp. duthiei, H. duthiei ssp. longearistata, H. coreana and H. komarovii have a close affinity with Psathyrostachys and Leymus species, and H. komarovii might contain the NsXm genome of Leymus; and (iii) the St, H and Ns genomes in Hystrix originate from Pseudoroegneria, Hordeum and Psathyrostachys, respectively, while the Xm in Hystrix and Leymus has a complex relationship with the E or St genomes. According to the genomic system of classification in Tiritceae, it is reasonable to treat Hystrix patula as Elymus hystrix L, and the other species of Hystrix as species of a section of Leymus, Leymus Sect. Hystrix.     

Discovery of the genus Pseudoroegneria (Triticeae,Poaceae) in the Western Mediterranean on exploring the generic boundaries of Elymus

In this study, we review the classification of two species, Elymus hispanicus and E. marginatus, which are restricted to highly valuable and sensitive Mediterranean ecosystems. The genomic composition of the two species is analysed by in situ hybridization. In addition, lodicule morphology and foliar anatomy of both species are compared with those of E. caninus, E. repens, E. sibiricus (i.e., the type species of Elymus s.s.) and Pseudoroegneria strigosa (i.e., the type species of Pseudoroegneria). The genomic formula 2n = 8x = 56; HStStSt is proposed for E. hispanicus and 2n = 4x = 28; StSt for E. marginatus. In this latter species, the absence of the ribosomal genes in one of the two St genomes suggests that diploidization may have occurred during the evolution the species. Regarding foliar anatomy, E. hispanicus, E. caninus, E. repens, and E. sibiricus shared several characteristics, but the leaf blades of E. marginatus proved anatomically more similar to those of Ps. strigosa. The data compiled support the contention that: (1) E. hispanicus belongs to Elymus s.s.; (2) E. marginatus should be transferred to Pseudoroegneria; and (3) the morphology of the lodicules should be carefully reconsidered for appropriately describing the boundaries between Elymus s.s. and Pseudoroegneria. The new combination Ps. marginata is proposed and a detailed iconography of the plant is provided.     

Analysis of tetraploid Elymus species using wheat microsatellite markers and RAPD markers.

An analysis of Amplification fragment polymorphism of DNA from 27 accessions of 19 tetraploid Elymus species was carried out using 18 wheat microsatellite (WMS) primer pairs and 10 decamer primers. Ten WMS primer pairs produced multiple polymorphism on all accessions tested. Two independent phenograms, one based on WMS-PCR and one on RAPDs, separated the 19 tetraploid species into two main groups, viz., the SH genome species group and the SY genome species group. The results coincide with the genomic classification of these species and hence support previous studies showing that Elymus is not a monophyletic genus. The assays indicated that accessions within a species cluster together, which concurs with the morphological classification. Interspecific and intraspecific polymorphisms were detected by the WMS-PCR and RAPD analyses. Variation was observed among accessions of Elymus caninus. The WMS-PCR detected a much higher level of polymorphism than the RAPD analysis. WMSs seem to be more efficient markers than RAPD markers for studying the population diversity of Elymus species. The potential of cross-species amplification of microsatellite markers as an additional source for genetic analysis and applications in Elymus is discussed in the context of these results.     

Physical mapping of repetitive sequences and genome analysis in six Elymus species by in situ hybridization

Genome constitution and genetic relationships between six Elymus species were assessed by physical mapping of different repetitive sequences using a technique of sequential fluorescence in situ hybridization and genomic in situ hybridization.The six Elymus species are all naturally growing species in northwest China,namely,E.sibiricus,E.nutans,E.barystachyus,E.xiningensis,E.excelsus,and E.dahuricus.An StStHH genome constitution was revealed for E.sibiricus and StStHHYY for the remainder species.Each chromosome could be clearly characterized by physical mapping with 18S-26S rDNA,5S rDNA,Afa-family,and AAG repeats,and be allocated to a certain genome by genomic in situ hybridization.Two 5S rDNA sites,each in the H and St genomes,and three 18S-26S rDNA sites,two in the St genome and one in the Y genome,were uncovered in most of the species.The strong Afa-family hybridization signals discriminated the H genome from the St and Y genomes.The H and Y genome carried more AAG repeats than St.A common non-Robertsonian reciprocal translocation between the H and Y genomes was revealed in E.barystachyus,E.xiningensis,E.excelsus and E.dahuricus.Comparison of molecular karyotypes strongly suggests that they can be classified into three groups,namely,E.sibiricus,E.nutans,and others.     

Phylogenetic Analysis of Leymus (Poaceae: Triticeae) Inferred from Nuclear rDNA ITS Sequences

To investigate the phylogenetic relationships of polyploid Leymus (Poaceae: Triticeae), sequences of the nuclear rDNA internal transcribed spacer region (ITS) were analyzed for 34 Leymus accessions representing 25 species, together with three Psathyrostachys species (Ns genome), two Pseudoroegneria (St genome) species, Lophopyrum elongatum (E(e) genome), and Thinopyrum bessarabicum (E(b) genome). The phylogenetic analyses (maximum likelihood and Bayesian inference) supported two major clades, one including 21 Leymus species and three Psathyrostachys species, the other with nine Leymus species and four diploid species. The ITS RNA secondary structure of the Leymus species was compared with that of their putative diploid donor. It is suggested that (1) the species from the same areas or neighboring geographic regions are closely related to each other; (2) L. coreanus, L. duthiei, L. duthiei var. longearistatus, and L. komarovii are closely related to other Leymus species, and it is reasonable to transfer these species from the genus Hystrix to Leymus; (3) the ITS sequences of Leymus are evolutionarily distinct; (4) the different Leymus species and different distribution of a species derived their Ns genome from different Psathyrostachys species; and (5) there is a close relationship among Leymus, Pseudoroegneria, Lophopyrum, and Thinopyrum, but it is difficult to presume that the St, E(e), and E(b) genome may be the Xm genome donor of the Leymus species.     

Allohexaploidy, introgression, and the complex phylogenetic history of Elymus repens (Poaceae)

The phylogenetic position of hexaploid Elymus repens within the tribe Triticeae (Poaceae) was examined using cloned sequences from the low-copy nuclear genes encoding phosphoenolpyruvate carboxylase (pepC) and beta-amylase. A previous analysis of E. repens using data from the nuclear granule-bound starch synthase I (GBSSI) gene had yielded five phylogenetically distinct gene copies, two more than expected from hexaploidy alone. The three gene trees share three distinct E. repens clades, suggesting that E. repens contains three phylogenetically divergent genomes, contributed by Hordeum, Pseudoroegneria, and an unknown donor. The two additional GBSSI sequences, including one that was apparently derived from outside of the tribe, appear to reflect past introgression of GBSSI sequences into the E. repens genome. On all three trees, the Hordeum-like E. repens sequences are polyphyletic within Hordeum, and the trees are in conflict with regard to the placement of these sequences within Hordeum, highlighting multiple contributions from Hordeum to E. repens.     

Chromosomal Distribution of the 18S-5.8S-26S rDNA Loci and Heterogeneity of Nuclear ITS Regions in Thinopyrum intermedium （Poaceae： Triticeae）

Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal location of 18S-5.8S-26S rDNA loci in Thinopyrum intermedium (Host) Barkworth et Dewey (2n=6x=42). In all accessions and individuals studied, 3 or 4 pairs of major loci were detected. Subsequent genomic in situhybddization (GISH) analyses revealed that one pair was located on the ends of the short arms of one pair of homologous chromosomes of the St genome, while the other 2 or 3 pairs of major loci were located in the E genomes (including the E^o and E^b). It is suggested that 2 to 3 pairs of major loci were probably lost during the evolution of this hexaploid species. The variation in rDNA positions and copy numbers between the diploid donors and Th. interrnedium, as well as the diversity among the accessions of Th. intermedium confirmed that the rDNA gene family conveyed the characters of DNA mobile elements. The internal transcribed spacer (ITS) regions of the rDNA in Th. intermedium were also investigated. Sequence data of seven positive clones from one individual suggested high degree of individual heterogeneity exists among ITS repeats. Phylogenetic analyses showed that there were two distinct types of ITS sequences in Th. intermedium, one with homology to that of Pseudoroegneria species (St genome) and the other to that of the E genome diploid species. This showed that the ITS paralogues in Th. intermedium have not been uniformly homogenized by concerted evolution. The limitation of using the chromosomal location of rDNA loci for phylogenetic analysis is discussed.     

Sequence Variations and Haplotype Identification of Wheat Dimeric α-Amylase Inhibitor Genes in Einkorn Wheats

This study characterizes 80 dimeric alpha-amylase inhibitor genes from 68 accessions of the einkorn wheats Triticum urartu, T. boeoticum, and T. monococcum. The mature protein coding sequences of WDAI genes were analyzed. Nucleotide sequence variations in these regions resulted from base substitution and/or indel mutations. Most of the WDAI gene sequences from T. boeoticum and all sequences from T. monococcum had one nucleotide insertion in the coding region, such that these alpha-amylase inhibitor sequences could not encode the correct mature proteins. We identified 21 distinct haplotypes from the diploid wheat WDAI gene sequences. A main haplotype was found in 15 gene samples from the A(u) genome and 35 gene samples from the A(m) genome. The T. monococcum and T. boeoticum accessions shared the same main haplotype, with 25 samples from T. monococcum and 10 from T. boeoticum. The WDAI gene sequences from the A(u) and A(m) genomes could be obviously clustered into two clades, but the sequences from the A(m) genome of T. boeoticum and T. monococcum could not be clearly distinguished. The phylogenetic analysis revealed that the WDAI gene sequences from the A(m) genome had accumulated fewer variations and evolved at a slower rate than the sequences from the A(u) genome. Although some accessions from only one or two areas had unique mutations at the same position, the diversity of WDAI gene sequences in diploid wheat showed little relationship to the origin of the accessions.