Rapid genome evolution revealed by comparative sequence analysis of orthologous regions from four triticeae genomes

Bread wheat (Triticum aestivum) is an allohexaploid species, consisting of three subgenomes (A, B, and D). To study the molecular evolution of these closely related genomes, we compared the sequence of a 307-kb physical contig covering the high molecular weight (HMW)-glutenin locus from the A genome of durum wheat (Triticum turgidum, AABB) with the orthologous regions from the B genome of the same wheat and the D genome of the diploid wheat Aegilops tauschii (Anderson et al., 2003; Kong et al., 2004). Although gene colinearity appears to be retained, four out of six genes including the two paralogous HMW-glutenin genes are disrupted in the orthologous region of the A genome. Mechanisms involved in gene disruption in the A genome include retroelement insertions, sequence deletions, and mutations causing in-frame stop codons in the coding sequences. Comparative sequence analysis also revealed that sequences in the colinear intergenic regions of these different genomes were generally not conserved. The rapid genome evolution in these regions is attributable mainly to the large number of retrotransposon insertions that occurred after the divergence of the three wheat genomes. Our comparative studies indicate that the B genome diverged prior to the separation of the A and D genomes. Furthermore, sequence comparison of two distinct types of allelic variations at the HMW-glutenin loci in the A genomes of different hexaploid wheat cultivars with the A genome locus of durum wheat indicates that hexaploid wheat may have more than one tetraploid ancestor.     

Types and rates of sequence evolution at the high-molecular-weight glutenin locus in hexaploid wheat and its ancestral genomes

The Glu-1 locus, encoding the high-molecular-weight glutenin protein subunits, controls bread-making quality in hexaploid wheat (Triticum aestivum) and represents a recently evolved region unique to Triticeae genomes. To understand the molecular evolution of this locus region, three orthologous Glu-1 regions from the three subgenomes of a single hexaploid wheat species were sequenced, totaling 729 kb of sequence. Comparing each Glu-1 region with its corresponding homologous region from the D genome of diploid wheat, Aegilops tauschii, and the A and B genomes of tetraploid wheat, Triticum turgidum, revealed that, in addition to the conservation of microsynteny in the genic regions, sequences in the intergenic regions, composed of blocks of nested retroelements, are also generally conserved, although a few nonshared retroelements that differentiate the homologous Glu-1 regions were detected in each pair of the A and D genomes. Analysis of the indel frequency and the rate of nucleotide substitution, which represent the most frequent types of sequence changes in the Glu-1 regions, demonstrated that the two A genomes are significantly more divergent than the two B genomes, further supporting the hypothesis that hexaploid wheat may have more than one tetraploid ancestor.     

带芒草属低分子量谷蛋白基因的克隆及序列分析

在普通小麦中获得了大量的低分子量谷蛋白基因序列, 而在小麦近缘属物种中获得的同源基因则比较少, 导致对麦类低分子量谷蛋白基因家族成员间的关系还不清楚。因此, 进行近缘属物种低分子量谷蛋白基因的研究是非常必要的。此研究通过特殊设计的1对引物, 以小麦近缘属带芒草物种的基因组DNA为模板, 经过PCR和克隆, 从中得到了一条核苷酸序列长度为1 035 bp, 推测的氨基酸序列为343个氨基酸残基的低分子量谷蛋白基因, 该基因序列具有小麦低分子量谷蛋白基因的典型特征, 包括21个氨基酸残基的信号肽、13个氨基酸的N-端和由可重复的短肽单元组成的重复区以及1个C末端。序列比对结果揭示了来自带芒草的低分子量谷蛋白基因与小麦同源基因的差异及相互关系。此研究结果对从带芒草属以及其他小麦近缘属物种中分离未知低分子量谷蛋白基因有参考价值和借鉴意义。     

Cloning and Expression of Low Molecular Weight Glutenin Genes from the Chinese Elite Wheat Cultivar ＂Xiaoyan 54＂

The low molecular weight （LMW） glutenln subunlts account for 40% of wheat gluten protein content by mass and these proteins are considered to significantly affect dough quality characteristics. Five new full-length LMW glutenln genes （designated LMW-5, LMW-7, LMW-42, LMW-58, and LMW-34） were isolated from the Chinese elite wheat cultivar ＂Xlaoyan 54＂ by PCR amplification of genomlc DNA using a pair of degenerate primers designed from the conserved sequences of the N- and C-terminal regions of published LMW glutenln genes. Deduced amino acid sequence analysis showed that LMW-5 belongs to the LMW-i type genes and that the other four belong to LMW-m type genes. Sequence comparisons revealed that point mutations occasionally occurred in signal peptide and N-terminus domains and often existed in domain III and domain V. Small insertions and deletions are represented in the repetitive domain. There is a stop codon after amino acid position 110 In the repetitive domain of LMW.34, indicating that It is a pseudogene. The other four genes have complete open reading frames and the putative mature regions of these genes were subcloned Into pET-30a expression vector and successfully expressed in Escherlchla coll. Protein sodium dodecyl sulfate-polyacrylamlde gel electro- phoresls analysis showed that all proteins expressed in E. coil by the four genes could be related to B-group LMW glutenln subunits of wheat.     

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.     

Dynamics of the evolution of orthologous and paralogous portions of a complex locus region in two genomes of allopolyploid wheat

Two overlapping bacterial artificial chromosome (BAC) clones from the B genome of the tetraploid wheat Triticum turgidum were identified, each of which contains one of the two high-molecular-weight (HMW) glutenin genes, comprising the complex Glu-B1 locus. The complete sequence (285 506 bp of DNA) of this chromosomal region was determined. The two paralogous x-type ( Glu-1-1 ) and y-type ( Glu-1-2 ) HMW-glutenin genes of the complex Glu-B1 locus were found to be separated by ca. 168 000 bp instead of the 51 000 bp separation previously reported for the orthologous Glu-D1 locus of Aegilops tauschii, the D-genome donor of hexaploid wheat. This difference in intergene spacing is due almost entirely to be the insertion of clusters of nested retrotransposons. Otherwise, the orientation and order of the HMW glutenins and adjacent genes were identical in the two genomes. A comparison of these orthologous regions indicates modes and patterns of sequence divergence, with implications for the overall Triticeae genome structure and evolution. A duplicate globulin gene, found 5' of each HMW-glutenin gene, assists to tentatively define the original duplication event leading to the paralogous x- and y-type HMW-glutenin genes. The intergenic regions of the two loci are composed of different patterns and classes of retrotransposons, indicating that insertion times of these retroelements were after the divergence of the two wheat genomes. In addition, a putative receptor kinase gene near the y-type HMW-glutenin gene at the Glu-B1 locus is likely active as it matches recently reported ESTs from germinating barley endosperm. The presence of four genes represented only in the Triticeae endosperm ESTs suggests an endosperm-specific chromosome domain.     

Characterization of SBEIIa homoeologous genes in bread wheat

To elucidate some of the molecular mechanisms involved in genome differentiation and evolution of cultivated wheats, we compared orthologous genes encoding starch branching enzyme IIa (SBEIIa). Bread wheat is an allohexaploid species comprising the three genomes A, B and D, each of which contributes a copy of the SBEIIa gene, involved in starch biosynthesis and known to control important quality traits related to technological and nutritional value of wheat-based food products. Alignment of the nucleotide sequences of these three genes revealed variation, both at the level of single nucleotides and indels. Multiple transposon elements were identified in the intragenic regions, some of which appear to have inserted before the divergence of the wheat diploid genomes. The B genome homoeologue was the most divergent of the three genes. Two MITE transposon insertions were detected within the intronic sequence of SBEIIa-B and two other transposons within SBEIIa-D. The presence/absence of these transposons in a panel of diploid and polyploid Triticum and Aegilops species provided some insights into the phylogeny of wheat.     

Genetic polymorphism in low-molecular-weight glutenin genes from Triticum aestivum, variety Chinese Spring

Low-molecular-weight (LMW) glutenin subunits consist mainly of two domains, one at the N- terminus which contains repeats of short amino-acid motifs, and a non-repetitive one rich in cysteine, at the C- terminal region. In previous reports, polyacrylamide-gel electrophoresis has been used to show that large size variation exists among LMW and HMW glutenin subunits, and it has been suggested that deletions and insertions within the repetitive region are responsible for these variations in length. In this study, PCR-amplification of genomic DNA (Triticum aestivum variety Chinese Spring) was used to isolate three full-length LMW glutenin genes: LMWG-MB1, LMWG-MB2 and LMWG-MB3. The deduced amino-acid sequences show a high similarity between these ORFs, and with those of other LMW glutenin genes. Comparisons indicate that LMWG-MB1 has probably lost a 12-bp fragment through deletion and that LMWG-MB1 and LMWG-MB2 have an insertion of 81 bp within the repetitive domain. The current study has shown direct evidence that insertions and/or deletions provide a mechanistic explanation for the allelic variation, and the resultant evolution, of prolamin genes. Single-base substitutions at identical sites generate stop codons in both LMWG-MB2 and LMWG-MB3 indicating that these clones are pseudogenes. Received: 7 May 1999 / Accepted: 17 June 1999     

Comparative sequence analysis of wheat and barley powdery mildew fungi reveals gene colinearity, dates divergence and indicates host-pathogen co-evolution

The two fungal pathogens Blumeria graminis f. sp. tritici (B.g. tritici) and hordei (B.g. hordei) cause powdery mildew specifically in wheat or barley. They have the same life cycle, but their growth is restricted to the respective host. Here, we compared the sequences of two loci in both cereal mildews to determine their divergence time and their relationship with the evolution of their hosts. We sequenced a total of 273.3kb derived from B.g. tritici BAC sequences and compared them with the orthologous regions in the B.g. hordei genome. Protein-coding genes were colinear and well conserved. In contrast, the intergenic regions showed very low conservation mostly due to different integration patterns of transposable elements. To estimate the divergence time of B.g. tritici and B.g. hordei, we used conserved intergenic sequences including orthologous transposable elements. This revealed that B.g. tritici and B.g. hordei have diverged about 10 million years ago (MYA), two million years after wheat and barley (12 MYA). These data suggest that B.g. tritici and B.g. hordei have co-evolved with their hosts during most of their evolutionary history after host divergence, possibly after a short phase of host expansion when the same pathogen could still grow on the two diverged hosts.     

Molecular characterization of a diagnostic DNA marker for domesticated tetraploid wheat provides evidence for gene flow from wild tetraploid wheat to hexaploid wheat

All forms of domesticated tetraploid wheat (Triticum turgidum, genomes AABB) are nearly monomorphic for restriction fragment length polymorphism (RFLP) haplotype a at the Xpsr920 locus on chromosome 4A (Xpsr920-A1a), and wild tetraploid wheat is monomorphic for haplotype b. The Xpsr920-A1a/b dimorphism provides a molecular marker for domesticated and wild tetraploid wheat, respectively. Hexaploid wheat (Triticum aestivum, genomes AABBDD) is polymorphic for the 2 haplotypes. Bacterial artificial chromosome (BAC) clones hybridizing with PSR920 were isolated from Triticum urartu (genomes AA), Triticum monococcum (genomes AmAm), and T. turgidum ssp. durum (genomes AABB) and sequenced. PSR920 is a fragment of a putative ATP binding cassette (ABC) transporter gene (designated ABCT-1). The wheat ABCT-1 gene is more similar to the T. urartu gene than to the T. monococcum gene and diverged from the T. urartu gene about 0.7 MYA. The comparison of the sequence of the wheat A genome BAC clone with that of the T. urartu BAC clone provides the first insight into the microsynteny of the wheat A genome with that of T. urartu. Within 103 kb of orthologous intergenic space, 37 kb of new DNA has been inserted and 36 kb deleted leaving 49.7% of the region syntenic between the clones. The nucleotide substitution rate in the syntenic intergenic space has been 1.6 x 10(-8) nt(-1) year(-1), which is, respectively, 4 and 3 times as great as nucleotide substitution rates in the introns and the third codon positions of the juxtaposed gene. The RFLP is caused by a miniature inverted transposable element (MITE) insertion into intron 18 of the ABCT-A1 gene. Polymerase chain reaction primers were developed for the amplification of the MITE insertion site and its sequencing. The T. aestivum ABCT-A1a haplotype is identical to the haplotype of domesticated tetraploid wheat, and the ABCT-A1b haplotype is identical to that of wild tetraploid wheat. This finding shows for the first time that wild tetraploid wheat participated in the evolution of hexaploid wheat. A cline of the 2 haplotype frequencies exists across Euro-Asia in T. aestivum. It is suggested that T. aestivum in eastern Asia conserved the gene pool of the original T. aestivum more than wheat elsewhere.     

Structural organization of the barley D-hordein locus in comparison with its orthologous regions of wheat genomes.

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.     

Characterization of low-molecular-weight glutenin genes in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

This paper reports the characterization of the low-molecular-weight (LMW) glutenin gene family of Aegilops tauschii (syn. Triticum tauschii), the D-genome donor of hexaploid wheat. By analysis of bacterial artificial chromosome (BAC) clones positive for hybridization with an LMW glutenin probe, seven unique LMW glutenin genes were identified. These genes were sequenced, including their untranslated 3 and 5 flanking regions. The deduced amino acid sequences of the genes revealed four putative active genes and three pseudogenes. All these genes had a very high level of similarity to LMW glutenins characterized in hexaploid wheat. The predicted molecular weights of the mature proteins were between 32.2 kDa and 39.6 kDa, and the predicted isoelectric points of the proteins were between 7.53 and 8.06. All the deduced proteins were of the LMW-m type. The organization of the seven LMW glutenin genes appears to be interspersed over at least several hundred kilo base pairs, as indicated by the presence of only one gene or pseudogene per BAC clone. Southern blot analysis of genomic DNA of Ae. tauschii and the BAC clones containing the seven LMW glutenin genes indicated that the BAC clones contained all LMW glutenin-hybridizing bands present in the genome. Two-dimensional gel electrophoresis of an LMW glutenin extract from Ae. tauschii was conducted and showed the presence of at least 11 distinct proteins. Further analysis indicated that some of the observed proteins were modified gliadins. These results suggest that the actual number of typical LMW glutenins may in fact be much lower than previously thought, with a number of modified gliadins also being present in the polymeric fraction.     

Molecular characterization of a low-molecular-weight glutenin cDNA clone from Triticum durum

Summary A full-length, low-molecular-weight (LMW) glutenin cDNA clone, pTdUCD1, has been isolated from a Triticum durum cv Mexicali wheat cDNA library. The complete sequence was determined and compared to the LMW glutenin genes that have been isolated from hexaploid wheat, Triticum aestivum. This cDNA codes for a protein of 295 amino acids (33,414 daltons) including a 20-amino acid signal peptide as deduced from the DNA sequence. Northern analysis showed that this cDNA hybridizes to a family of related sequences ranging in length from 1,200 to 1,000 nucleotides. This gene is similar but not identical to previously published LMW glutenin gene sequences. The most striking characteristic of all cloned LMW glutenin genes is the conservation of eight cysteine residues, which could be involved in potential secondary or tertiary structure, disulfide bond interactions. This paper presents a structural map defining distinct regions of the LMW glutenin gene family.     

Comparative DNA sequence analysis of mapped wheat ESTs reveals the complexity of genome relationships between rice and wheat

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to related large-genome species has revolutionized molecular genetics and breeding strategies for improving those crops. Comparative sequence analysis methods can be used to cross-reference genes between species maps, enhance the resolution of comparative maps, study patterns of gene evolution, identify conserved regions of the genomes, and facilitate interspecies gene cloning. In this study, 5,780 Triticeae ESTs that have been physically mapped using wheat (Triticum aestivum L.) deletion lines and segregating populations were compared using NCBI BLASTN to the first draft of the public rice (Oryza sativa L.) genome sequence data from 3,280 ordered BAC/PAC clones. A rice genome view of the homoeologous wheat genome locations based on sequence analysis shows general similarity to the previously published comparative maps based on Southern analysis of RFLP. For most rice chromosomes there is a preponderance of wheat genes from one or two wheat chromosomes. The physical locations of non-conserved regions were not consistent across rice chromosomes. Some wheat ESTs with multiple wheat genome locations are associated with the non-conserved regions of similarity between rice and wheat. The inverse view, showing the relationship between the wheat deletion map and rice genomic sequence, revealed the breakdown of gene content and order at the resolution conferred by the physical chromosome deletions in the wheat genome. An average of 35% of the putative single copy genes that were mapped to the most conserved bins matched rice chromosomes other than the one that was most similar. This suggests that there has been an abundance of rearrangements, insertions, deletions, and duplications eroding the wheat-rice genome relationship that may complicate the use of rice as a model for cross-species transfer of information in non-conserved regions.     

PCR-based analysis of the intergenic spacers of the Nor loci on the A genomes of Triticum diploids and polyploids.

We present DNA sequence data showing population variation in the intergenic spacer (IGS) regions of the ribosomal DNAs (rDNAs) on the A genomes of 27 diploid and polyploid wheats. PCRs (polymerase chain reactions) specific for the A(m) genome gave products with five populations of Triticum monococcum but did not give products with AABB or AABBDD wheats. PCRs specific to the A(u) genome of T. urartu gave products with all the AABB and AABBDD polyploids that were tested, but not with T. monococcum. AAGG tetraploids gave products only with the A(u)-specific primers, but the AAAAGG hexaploid T. zhukovskyi gave products with both the A(u) and A(m) primers. Phylogenetic analysis showed a substantial degree of IGS divergence for both the A(m) and A(u) genomes in diploids and polyploids compared with other genomes of Triticum and Aegilops. The rate of evolution of the IGS is much greater than previously reported for the internal transcribed region of the rDNAs but the view that the IGS only gives random noise is rejected, the IGS sequences presented here reflecting the general evolutionary trends affecting the wheat genome as a whole.     

The amino-acid sequence of a purothionin from Triticum monococcum, a diploid wheat

Purothionins were extracted and purified from the diploid wheat Triticum monococcum. Two proteins were obtained, one of which was present in only very small amounts. The major purothionin of T. monococcum was sequenced and it had an amino acid sequence identical with that of the beta-purothionin of Triticum aestivum (hexaploid bread wheat). It is known that T. monococcum contains the wheat A genome, so the structural gene coding for the beta-purothionin must comprise a part of the A genome. There have been no observable (as amino acid replacements) changes in the DNA comprising either the beta-purothionin gene of T. aestivum or the purothionin gene of T. monococcum, since T. monococcum (or its wild equivalent, Triticum boeoticum) hybridized with the diploid wheat B genome progenitor and started the evolution from diploid to allohexaploid wheat. All of the investigated characteristics of the purothionin-like protein isolated in small amounts suggested that it was essentially identical in amino acid sequence with the T. monococcum purothionin. It may be a dimerized form of beta-purothionin.     

Variation in the HMW and LMW glutenin subunits from Spanish accessions of emmer wheat (Triticum turgidum ssp. dicoccum Schrank)

Emmer wheat (Triticum turgidum ssp. dicoccum Schrank) is hulled wheat that survives in marginal areas of the Mediterranean Region. The HMW and LMW glutenin subunit composition of 97 accessions of emmer wheat from Spain have been analysed by SDS-PAGE. For the HMW glutenin subunits, four allelic variants were detected for the Glu-A1 locus; one of them has not been previously described. For the Glu-B1 locus, three of the nine alleles detected have not been found before. A high degree of variation was evident for the LMW glutenin subunits, and up to 23 different patterns were detected for the B-LMW glutenin subunits. Considering both types of proteins (HMW and LMW), 30 combinations were found between all the evaluated lines. This wide polymorphism can be used to transfer new quality genes to wheat, and to widen its genetic basis. Received: 13 June 2000 / Accepted: 3 July 2000     

Evolution of Conserved Non-Coding Sequences Within the Vertebrate Hox Clusters Through the Two-Round Whole Genome Duplications Revealed by Phylogenetic Footprinting Analysis

As a result of two-round whole genome duplications, four or more paralogous Hox clusters exist in vertebrate genomes. The paralogous genes in the Hox clusters show similar expression patterns, implying shared regulatory mechanisms for expression of these genes. Previous studies partly revealed the expression mechanisms of Hox genes. However, cis-regulatory elements that control these paralogous gene expression are still poorly understood. Toward solving this problem, the authors searched conserved non-coding sequences (CNSs), which are candidates of cis-regulatory elements. When comparing orthologous Hox clusters of 19 vertebrate species, 208 intergenic conserved regions were found. The authors then searched for CNSs that were conserved not only between orthologous clusters but also among the four paralogous Hox clusters. The authors found three regions that are conserved among all the four clusters and eight regions that are conserved between intergenic regions of two paralogous Hox clusters. In total, 28 CNSs were identified in the paralogous Hox clusters, and nine of them were newly found in this study. One of these novel regions bears a RARE motif. These CNSs are candidates for gene expression regulatory regions among paralogous Hox clusters. The authors also compared vertebrate CNSs with amphioxus CNSs within the Hox cluster, and found that two CNSs in the HoxA and HoxB clusters retain homology with amphioxus CNSs through the two-round whole genome duplications.