A linkage map of meadow fescue (<Emphasis Type="Italic">Festuca pratensis</Emphasis> Huds.) and comparative mapping with other Poaceae species

A genetic linkage map has been constructed for meadow fescue (Festuca pratensis Huds.) (2n=2x=14) using a full-sib family of a cross between a genotype from a Norwegian population (HF2) and a genotype from a Yugoslavian cultivar (B14). The two-way pseudo-testcross procedure has been used to develop separate maps for each parent, as well as a combined map. A total number of 550 loci have been mapped using homologous and heterologous RFLPs, AFLPs, isozymes and SSRs. The combined map consists of 466 markers, has a total length of 658.8 cM with an average marker density of 1.4 cM/marker. A high degree of orthology and colinearity was observed between meadow fescue and the Triticeae genome(s) for all linkage groups, and the individual linkage groups were designated 1F–7F in accordance with the orthologous Triticeae chromosomes. As expected, the meadow fescue linkage groups were highly orthologous and co-linear with Lolium, and with oat, maize and sorghum, generally in the same manner as the Triticeae chromosomes. It was shown that the evolutionary 4AL/5AL translocation, which characterises some of the Triticeae species, is not present in the meadow fescue genome. A putative insertion of a segment orthologous to Triticeae 2 at the top of 6F, similar to the rearrangement found in the wheat B and the rye R genome, was also observed. In addition, chromosome 4F is completely orthologous to rice chromosome 3 in contrast to the Triticeae where this rice chromosome is distributed over homoeologous group 4 and 5 chromosomes. The meadow fescue genome thus has a more ancestral configuration than any of the Triticeae genomes. The extended meadow fescue map reported here provides the opportunity for beneficial cross-species transfer of genetic knowledge, particularly from the complete genome sequence of rice.Communicated by P. Langridge     

Cbf14 copy number variation in the A, B, and D genomes of diploid and polyploid wheat

Freezing tolerance and winter hardiness are complex traits. In the Triticeae, two loci on the group 5 chromosome homoeologs are repeatedly identified as having major effects on these traits. Recently, we found that segments of the genomic region at one of these loci, Frost resistance-2 (Fr-2) is copy number variable in barley. Freezing-tolerant winter-hardy genotypes have greater tandem copy numbers of the genomic region encompassing the C-repeat binding factor genes Cbf2A and Cbf4B at Fr-H2 than the less freezing-tolerant nonwinter-hardy genotypes. Here we report that in wheat the Cbf14 gene at Fr-2 is copy number variable. Using DNA blot hybridizations, we estimated copy numbers of Cbf14 across the different genomes of diploid and polyploid wheat. Copy numbers of Cbf14 are lower in the B genome than in the A and D genomes across all ploidy levels. Among hexaploid red wheats, winter genotypes harbor greater Cbf14 copy numbers than spring genotypes. Cbf14 copy numbers also vary across the red winter wheats such that hard wheats harbor greater copy numbers than soft wheats. Analysis of hexaploid wheat chromosome 5 substitution lines indicates that Cbf14 copy numbers in the introgressions are stable in the different backgrounds. Taken together our data suggest that higher copy number states existed in the diploid wild ancestors prior to the polyploidization events and that the loss of Cbf14 copies occurred in the cultivated germplasm.     

Spatial and temporal expression of the two sucrose synthase genes in maize: immunohistological evidence

Summary The DNAs of two diploid species of Gossypium, G. herbaceum var. africanum (A₁ genome) and G. raimondii (D₅ genome), and the allotetraploid species, G. hirsutum (A_h and D_h genomes), were characterized by kinetic analyses of single copy and repetitive sequences. Estimated haploid genome sizes of A₁ and D₅ were 1.04 pg and 0.68 pg, respectively, in approximate agreement with cytological observations that A genome chromosomes are about twice the size of D genome chromosomes. This differences in genome size was accounted for entirely by differences in the major repetitive fraction (0.56 pg versus 0.20 pg), as single copy fractions of the two genomes were essentially identical (0.41 pg for A₁ and 0.43 pg for D₅). Kinetic analyses and thermal denaturation measurements of single copy duplexes from reciprocal intergenomic hybridizations showed considerable sequence similarity between A₁ and D₅ genomes (77% duplex formation with an average thermal depression of 6 °C). Moreover, little sequence divergence was detectable between diploid single copy sequences and their corresponding genomes in the allotetraploid, consistent with previous chromosome pairing observations in interspecific F₁ hybrids.Journal paper No. 4461 of the Arizona Agricultural Experiment Station     

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     

Complex genome rearrangements reveal evolutionary dynamics of pericentromeric regions in the Triticeae.

Most pericentromeric regions of eukaryotic chromosomes are heterochromatic and are the most rapidly evolving regions of complex genomes. The closely related genomes within hexaploid wheat (Triticum aestivum L., 2n=6x=42, AABBDD), as well as in the related Triticeae taxa, share large conserved chromosome segments and provide a good model for the study of the evolution of pericentromeric regions. Here we report on the comparative analysis of pericentric inversions in the Triticeae, including Triticum aestivum, Aegilops speltoides, Ae. longissima, Ae. searsii, Hordeum vulgare, Secale cereale, and Agropyron elongatum. Previously, 4 pericentric inversions were identified in the hexaploid wheat cultivar 'Chinese Spring' ('CS') involving chromosomes 2B, 4A, 4B, and 5A. In the present study, 2 additional pericentric inversions were detected in chromosomes 3B and 6B of 'CS' wheat. Only the 3B inversion pre-existed in chromosome 3S, 3Sl, and 3Ss of Aegilops species of the Sitopsis section, the remaining inversions occurring after wheat polyploidization. The translocation T2BS/6BS previously reported in 'CS' was detected in the hexaploid variety 'Wichita' but not in other species of the Triticeae. It appears that the B genome is more prone to genome rearrangements than are the A and D genomes. Five different pericentric inversions were detected in rye chromosomes 3R and 4R, 4Sl of Ae. longissima, 4H of barley, and 6E of Ag. elongatum. This indicates that pericentric regions in the Triticeae, especially those of group 4 chromosomes, are undergoing rapid and recurrent rearrangements.     

A New Class of Wheat Gliadin Genes and Proteins

The utility of mining DNA sequence data to understand the structure and expression of cereal prolamin genes is demonstrated by the identification of a new class of wheat prolamins. This previously unrecognized wheat prolamin class, given the name δ-gliadins, is the most direct ortholog of barley γ3-hordeins. Phylogenetic analysis shows that the orthologous δ-gliadins and γ3-hordeins form a distinct prolamin branch that existed separate from the γ-gliadins and γ-hordeins in an ancestral Triticeae prior to the branching of wheat and barley. The expressed δ-gliadins are encoded by a single gene in each of the hexaploid wheat genomes. This single δ-gliadin/γ3-hordein ortholog may be a general feature of the Triticeae tribe since examination of ESTs from three barley cultivars also confirms a single γ3-hordein gene. Analysis of ESTs and cDNAs shows that the genes are expressed in at least five hexaploid wheat cultivars in addition to diploids Triticum monococcum and Aegilops tauschii. The latter two sequences also allow assignment of the δ-gliadin genes to the A and D genomes, respectively, with the third sequence type assumed to be from the B genome. Two wheat cultivars for which there are sufficient ESTs show different patterns of expression, i.e., with cv Chinese Spring expressing the genes from the A and B genomes, while cv Recital has ESTs from the A and D genomes. Genomic sequences of Chinese Spring show that the D genome gene is inactivated by tandem premature stop codons. A fourth δ-gliadin sequence occurs in the D genome of both Chinese Spring and Ae. tauschii, but no ESTs match this sequence and limited genomic sequences indicates a pseudogene containing frame shifts and premature stop codons. Sequencing of BACs covering a 3 Mb region from Ae. tauschii locates the δ-gliadin gene to the complex Gli-1 plus Glu-3 region on chromosome 1.     

DNA sequence analyses of the ribosomal spacer regions in theTriticeae

Two regions of the ribosomal DNA (rDNA) were sequenced from a range of species from the tribeTriticeae. One region, the central spacer, was found to be more divergent in sequence than the other, the 18 S-spacer junction. Both regions contained sequences 20–30 bp long which were more highly conserved than the remainder of the region and their possible significance in rDNA expression is discussed. Phenetic relationships based on the sequence data were generally consistent with the relationships based on other criteria. Species possessing the S, E, J₁J₂, D, and B genomes clustered together, with the H genome species being the most distinct of those examined. The R, P, and V genome species occupy an intermediate position in the overall pattern of relationships. Some relationships differed in detail from those established by other parameters, for example the position of the N genome species, and explanations for discrepancies of this type are discussed.     

Comparison of Acetyl-CoA carboxylase 1 (Acc-1) gene diversity among different Triticeae genomes

It has widely been documented that life form and mating system have significant influences on genetic diversity. In the tribe Triticeae, several genera contain both annual and perennial species, whereas other genera comprise strictly annual or perennial species. It was suggested that Triticeae annuals have originated from Triticeae perennials. The present study aims to analyze nucleotide diversity of Acc-1 gene among different Triticeae genomes, and attempts to link effects of life history (annuals and perennials) and mating systems. The nucleotide diversity of 364 Acc-1 sequences in Triticeae species was characterized. The highest estimates of nucleotide diversity values (π = 0.01919, θ = 0.03515) were found for the Ns genome among the genomes analyzed. Nucleotide diversities in the D genome and Ns genome of polyploids are higher than those in respective genomes of diploids, while in the St genome of polyploids, it is lower than that in the St genome of diploids. The averaged π value (0.013705) in the genomes of perennials is more than twice of the value (0.00508) in the genomes of annuals. The averaged π value (0.01323) in the genomes of outcrossing species is two-fold of the value (0.005664) in the genomes of selfer. Our results suggested that the evolutionary history and mating system may play an important role in determining nucleotide diversity of Acc-1 gene in each genome.     

Intron loss in the chalcone-flavanone isomerase gene of rye

The Chi gene encodes the flavonoid synthesis enzyme chalcone-flavanone isomerase. The complete coding sequence of the Chi gene was isolated by PCR from four cultivars of cereal rye (Secale cereale L.). Unlike most monocot and dicot plant species, S. cereale has one, rather than three introns in the Chi gene. Screening of a panel of 63 Triticeae accessions, representing 31 species, showed two intron loss events in the Triticeae tribe. One intron loss occurred early in the evolution of the Triticeae tribe, while another intron loss was only detected in S. cereale Chi. A new rye-specific PCR marker was developed based on Chi intron loss polymorphism and was shown to be effective for analysis of a wide range of intergenera Triticeae hybrids for the presence of rye genome. In addition, precise genetic mapping of the rye Chi gene was carried out based on insertion/deletion polymorphism between parents of a rye mapping population. The Chi gene was mapped on the long arm of chromosome 5R 9.3 cM distal to the restriction fragment length polymorphism marker Xscb35 and 4.4 cM proximal to the locus 3Rt encoding another flavonoid synthesis enzyme, anthocyanidin-3-glucoside rhamnosyltransferase.     

Phylogenetic analysis of the dimeric alpha-amylase inhibitor sequences from an orthologous region in 21 different genomes of the tribe Triticeae (Poaceae)

Six hundred and thirty gene sequences from 21 different genomes in Triticeae tribe were obtained and subjected to phylogenetic analysis. The sequences showed high homology in both nucleotide sequences and length variation, and had a common conserved cysteine skeleton C–Xn–C–Xn–C–Xn–CC–Xn–C–X–C–Xn–C–Xn–C–Xn–C. The sequences from common wheat formed three clusters; two were close to Aegilops tauschii and Aegilops speltoides sequences, respectively, and the third cluster was complex with sequences from Ae. speltoides, Aegilops searsii, and Aegilops bicornis. Different S genome(s) of Aegilops contributed α-amylase inhibitor loci to polyploid wheat by gene introgression in interspecific hybridizations. No sequence from common wheat was similar to that from einkorn wheat. We conclude that the occurrence of multiple chromosomal translocations or inversions in the different genomes of Triticeae had not dramatically affected the primary structure of dimeric α-amylase inhibitors. The results revealed important information on genome shaping events and processes occurring at the dimeric α-amylase inhibitor genes loci and their bearing on the phylogenetic relationships in the tribe Triticeae (Poaceae).     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.     

Quantitative evolution of transposable and satellite DNA sequences in Picea species

Clones containing tandemly arranged repeats belonging to two distinct sequence families, (i) PAG004P22F (2F) and PAG004E03C (3C) or (ii) Ty3/gypsy- (8R; PAG004B08R) and Ty1/copia-like sequences (9R; PAG007F19R), were selected from a randomly sheared total genomic DNA library of Picea abies . The inserts were used as probes in dot-blot hybridizations to genomic DNA of P. abies, Picea orientalis , Picea pungens , and Picea pungens var. glauca. All these entities are diploid and share the same chromosome number (2n = 24), but the genome sizes differ largely. The redundancy (copy number per 1C DNA) of sequences related to each probe varied greatly between the genomes. No significant correlation was found between the genome size and the copy number of sequences in any family. The quantitative ratios varied greatly (in each genome) between the two families of satellite DNA, between the sequences that represented copia or gypsy retrotransposons, and between tandemly arranged sequences and retroelements as a whole, suggesting that there is no common factor that controls the quantitative evolution of repeats belonging to different sequence families during speciation in Picea.     

Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum

Background

Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis–associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome.

Results

Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45–767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5′ and 3′ ends of chromosome 6 and the gp60 region, largely the result of genetic recombination.

Conclusions

The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1517-1) contains supplementary material, which is available to authorized users.     

Distribution of Plasmids in Distinct Leptospira Pathogenic Species

Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups—pathogens, non-pathogens, and intermediates—based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological significance might contribute to our understanding of biology and infectivity of pathogenic spirochetes.     

Towards a whole‐genome sequence for rye (Secale cereale L.)

We report on a whole‐genome draft sequence of rye (Secale cereale L.). Rye is a diploid Triticeae species closely related to wheat and barley, and an important crop for food and feed in Central and Eastern Europe. Through whole‐genome shotgun sequencing of the 7.9‐Gbp genome of the winter rye inbred line Lo7 we obtained a de novo assembly represented by 1.29 million scaffolds covering a total length of 2.8 Gbp. Our reference sequence represents nearly the entire low‐copy portion of the rye genome. This genome assembly was used to predict 27 784 rye gene models based on homology to sequenced grass genomes. Through resequencing of 10 rye inbred lines and one accession of the wild relative S. vavilovii, we discovered more than 90 million single nucleotide variants and short insertions/deletions in the rye genome. From these variants, we developed the high‐density Rye600k genotyping array with 600 843 markers, which enabled anchoring the sequence contigs along a high‐density genetic map and establishing a synteny‐based virtual gene order. Genotyping data were used to characterize the diversity of rye breeding pools and genetic resources, and to obtain a genome‐wide map of selection signals differentiating the divergent gene pools. This rye whole‐genome sequence closes a gap in Triticeae genome research, and will be highly valuable for comparative genomics, functional studies and genome‐based breeding in rye.     

Conserved globulin gene across eight grass genomes identify fundamental units of the loci encoding seed storage proteins

The wheat high molecular weight (HMW) glutenins are important seed storage proteins that determine bread-making quality in hexaploid wheat (Triticum aestivum). In this study, detailed comparative sequence analyses of large orthologous HMW glutenin genomic regions from eight grass species, representing a wide evolutionary history of grass genomes, reveal a number of lineage-specific sequence changes. These lineage-specific changes, which resulted in duplications, insertions, and deletions of genes, are the major forces disrupting gene colinearity among grass genomes. Our results indicate that the presence of the HMW glutenin gene in Triticeae genomes was caused by lineage-specific duplication of a globulin gene. This tandem duplication event is shared by Brachypodium and Triticeae genomes, but is absent in rice, maize, and sorghum, suggesting the duplication occurred after Brachypodium and Triticeae genomes diverged from the other grasses ~35 Ma ago. Aside from their physical location in tandem, the sequence similarity, expression pattern, and conserved cis-acting elements responsible for endosperm-specific expression further support the paralogous relationship between the HMW glutenin and globulin genes. While the duplicated copy in Brachypodium has apparently become nonfunctional, the duplicated copy in wheat has evolved to become the HMW glutenin gene by gaining a central prolamin repetitive domain.     

Whole-genome synthesis and characterization of viable S13-like bacteriophages

Background

Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template.

Methodology/Principal Findings

The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl₂, the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition.

Conclusions/Significance

The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.     

Microsatellite mapping of <Emphasis Type="Italic">Ae. speltoides</Emphasis> and map-based comparative analysis of the S,G, and B genomes of Triticeae species

The first microsatellite linkage map of Ae. speltoides Tausch (2n = 2x = 14, SS), which is a wild species with a genome closely related to the B and G genomes of polyploid wheats, was developed based on two F₂ mapping populations using microsatellite (SSR) markers from Ae. speltoides, wheat genomic SSRs (g-SSRs) and EST-derived SSRs. A total of 144 different microsatellite loci were mapped in the Ae. speltoides genome. The transferability of the SSRs markers between the related S, B, and G genomes allowed possible integration of new markers into the T. timopheevii G genome chromosomal maps and map-based comparisons. Thirty-one new microsatellite loci assigned to the genetic framework of the T. timopheevii G genome maps were composed of wheat g-SSR (genomic SSR) markers. Most of the used Ae. speltoides SSRs were mapped onto chromosomes of the G genome supporting a close relationship between the G and S genomes. Comparative microsatellite mapping of the S, B, and G genomes demonstrated colinearity between the chromosomes within homoeologous groups, except for intergenomic T6A^tS.1G, T4AL.5AL.7BS translocations. A translocation between chromosomes 2 and 6 that is present in the T. aestivum B genome was found in neither Ae. speltoides nor in T. timopheevii. Although the marker order was generally conserved among the B, S, and G genomes, the total length of the Ae. speltoides chromosomal maps and the genetic distances between homoeologous loci located in the proximal regions of the S genome chromosomes were reduced compared with the B, and G genome chromosomes.     

Characterization of two novel γ-gliadin genes encoded by K genome of Crithopsis delileana and evolution analysis with those from Triticeae

By acid polyacrylamide gel electrophoresis (A-PAGE) analysis, it was indicated that the electrophoresis mobility of gliadins from Crithopsis delileana (Schult) Roshev (2n=2x=14, KK) had obvious difference with those from common wheat in α, γ and ω region. Using homologous primers, two γ-gliadin genes (gli-Kr1 and gli-Kr2) were isolated from C. delileana, which had been deposited in the GenBank under accession numbers EU283818 and EU283821, respectively. Two γ -gliadin genes of C. delileana had the similar primary structures to the corresponding gene sequences from other wheat related species. The differences were mainly resulted from substitutions, insertions and deletions involving single amino acid residues or motifs of γ-gliadins. The repetitive domains of gli-Kr1 and gli-Kr2 from C. delileana are shorter than most of other sequences. By the alignment of γ-gliadin genes from A, B, D, A^m, A^u, S, S^l, S^sh, S^s and S^b genomes of Triticum and Aegilops, R genome of Secale (γ-secalin), Ee genome of Lophopyrum and K genome of Crithopsis in Triticeae, phylogenetic analysis indicated that two γ-gliadin genes of C. delileana could be clustered together with a γ-gliadin genefrom Ssh genome of Aegilops by an interior paralleled branch. It was the first time that the γ-gliadin genes encoded by K genome of C. delileana were characterized. These could offer precious information for better understanding the qualities associated with gliadins, the response in coeliac disease and studying the evolutionary relationship of gliadins in Triticeae.