Genetic and epigenetic alteration among three homoeologous genes of a class E MADS box gene in hexaploid wheat

Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.     

Cloning and characterization of wheat PDI (protein disulfide isomerase) homoeologous genes and promoter sequences

The genomic and cDNA sequences of three PDI homoeologous genes located on chromosomes 4A, 4B and 4D of bread wheat and their promoters were cloned and sequenced. The three sequences showed a very high conservation of the coding region and of the exon/intron structure, which consisted of ten exons. The comparison of wheat sequences with those of rice and Arabidopsis showed a significant conservation of the exon/intron structure across the three species. The expression of each gene was analysed by RT-PCR in different plant tissues (roots, coleoptiles, spikelets, leaves and developing caryopses). All the genes showed a higher expression in developing caryopses than in other analysed tissues, wherein some differences were detected. The promoter sequences of the three genes possessed some regulatory motifs typical of endosperm specific expression.     

Identification and chromosomal location of four subfamilies of the rubisco small subunit genes in common wheat

Summary Three different 3 noncoding sequences of wheat rubisco small subunit (SSU) genes (RbcS) were used as probes to identify the gene members of different RbcS subfamilies in the common wheat cultivar Chinese Spring (CS). All genes of the wheat RbcS multigene family were previously assigned to the long arm of homoeologous group 5 and to the short arm of homoeologous group 2 chromosomes of cv CS. Extracted DNA from various aneuploids of these homoeologous groups was digested with four restriction enzymes and hybridized with three different 3 noncoding sequences of wheat SSU clones. All RbcS genes located on the long arm of homoeologous group 5 chromosomes were found to comprise a single subfamily, while those located on the short arm of group 2 comprised three subfamilies. Each of the ancestral diploid genomes A, B, and D has at least one representative gene in each subfamily, suggesting that the divergence into subfamilies preceded the differentiation into species. This divergence of the RbcS genes, which is presumably accompanied by a similar divergence in the 5 region, may lead to differential expression of various subfamilies in different tissues and in different developmental stages, in response to different environmental conditions. Moreover, members of one subfamily that belong to different genomes may have diverged also in the coding sequence and, consequently, code for distinguishable SSU. It is assumed that such utilization of the RbcS multigene family increases the adaptability and phenotypic plasticity of common wheat over its diploid progenitors.     

Comparative Sequence Analysis of the <Emphasis Type="Italic">Phytochrome C</Emphasis> Gene and its Upstream Region in Allohexaploid Wheat Reveals New Data on the Evolution of its Three Constituent Genomes

Bread wheat is an allohexaploid with genome composition AABBDD. Phytochrome C is a gene involved in photomorphogenesis that has been used extensively for phylogenetic analyses. In wheat, the PhyC genes are single copy in each of the three homoeologous genomes and map to orthologous positions on the long arms of the group 5 chromosomes. Comparative sequence analysis of the three homoeologous copies of the wheat PhyC gene and of some 5 kb of upstream region has demonstrated a high level of conservation of PhyC, but frequent interruption of the upstream regions by the insertion of retroelements and other repeats. One of the repeats in the region under investigation appeared to have inserted before the divergence of the diploid wheat genomes, but was degraded to the extent that similarity between the A and D copies could only be observed at the amino acid level. Evidence was found for the differential presence of a foldback element and a miniature inverted-repeat transposable element (MITE) 5′ to PhyC in different wheat cultivars. The latter may represent the first example of an active MITE family in the wheat genome. Several conserved non-coding sequences were also identified that may represent functional regulatory elements. The level of sequence divergence (K_s) between the three wheat PhyC homoeologs suggests that the divergence of the diploid wheat ancestors occurred some 6.9 Mya, which is considerably earlier than the previously estimated 2.5–4.5 Mya. K_a/K_s ratios were <0.15 indicating that all three homoeologs are under purifying selection and presumably represent functional PhyC genes. RT-PCR confirmed expression of the A, B and D copies. The discrepancy in evolutionary age of the wheat genomes estimated using sequences from different parts of the genome may reflect a mosaic origin of some of the Triticeae genomes.     

Genetic characterization and mapping of the Rht-1 homoeologs and flanking sequences in wheat

The introgression of Reduced height (Rht)-B1b and Rht-D1b into bread wheat (Triticum aestivum) varieties beginning in the 1960s led to improved lodging resistance and yield, providing a major contribution to the ‘green revolution’. Although wheat Rht-1 and surrounding sequence is available, the genetic composition of this region has not been examined in a homoeologous series. To determine this, three Rht-1-containing bacterial artificial chromosome (BAC) sequences derived from the A, B, and D genomes of the bread wheat variety Chinese Spring (CS) were fully assembled and analyzed. This revealed that Rht-1 and two upstream genes were highly conserved among the homoeologs. In contrast, transposable elements (TEs) were not conserved among homoeologs with the exception of intronic miniature inverted-repeat TEs (MITEs). In relation to the Triticum urartu ancestral line, CS-A genic sequences were highly conserved and several colinear TEs were present. Comparative analysis of the CS wheat BAC sequences with assembled Poaceae genomes showed gene synteny and amino acid sequences were well preserved. Further 5′ and 3′ of the wheat BAC sequences, a high degree of gene colinearity is present among the assembled Poaceae genomes. In the 20 kb of sequence flanking Rht-1, five conserved non-coding sequences (CNSs) were present among the CS wheat homoeologs and among all the Poaceae members examined. Rht-A1 was mapped to the long arm of chromosome 4 and three closely flanking genetic markers were identified. The tools developed herein will enable detailed studies of Rht-1 and linked genes that affect abiotic and biotic stress response in wheat.     

Identification and characterization of high temperature stress responsive genes in bread wheat (Triticum aestivum L.) and their regulation at various stages of development

To elucidate the effect of high temperature, wheat plants (Triticum aestivum cv. CPAN 1676) were given heat shock at 37 and 42°C for 2 h, and responsive genes were identified through PCR-Select Subtraction technology. Four subtractive cDNA libraries, including three forward and one reverse subtraction, were constructed from three different developmental stages. A total of 5,500 ESTs were generated and 3,516 high quality ESTs submitted to Genbank. More than one-third of the ESTs generated fall in unknown/no hit category upon homology search through BLAST analysis. Differential expression was confirmed by cDNA macroarray and by northern/RT-PCR analysis. Expression analysis of wheat plants subjected to high temperature stress, after 1 and 4 days of recovery, showed fast recovery in seedling tissue. However, even after 4 days, recovery was negligible in the developing seed tissue after 2 h of heat stress. Ten selected genes were analyzed in further detail including one unknown protein and a new heat shock factor, by quantitative real-time PCR in an array of 35 different wheat tissues representing major developmental stages as well as different abiotic stresses. Tissue specificity was examined along with cross talk with other abiotic stresses and putative signalling molecules.     

Isolation and comparative expression analysis of six MBD genes in wheat

The 5-methylcytosines (m5C) play critical roles in epigenetic control, often being recognized by proteins containing an MBD. In this study, we isolated six wheat cDNAs with open reading frame encoding putative methyl-binding domain proteins, which were designated as TaMBD1-TaMBD6, respectively. BLASTX searches and phylogenetic analysis suggested that the six TaMBD genes belonged to four (I, II, III and VIII) of the eight subclasses of MBD family. Genomic analysis showed that a 1386 bp intron was included in TaMBD1 and a 12-bp intron was found in TaMBD4. The expression profiles of the six TaMBDs were studied via Q-RT-PCR and the results indicated that the TaMBDs were differentially expressed in detected wheat tissues. It was interesting to note that 3 TaMBDs were highly expressed in dry seeds and endosperms. Moreover, the differential expression patterns of TaMBDs were observed in leaves and roots under water-stress. We concluded that multiple wheat MBD genes were present and they might play important roles in wheat growth and development, as well as in the water-stress response.     

A haplotype map of allohexaploid wheat reveals distinct patterns of selection on homoeologous genomes

BackgroundBread wheat is an allopolyploid species with a large, highly repetitive genome. To investigate the impact of selection on variants distributed among homoeologous wheat genomes and to build a foundation for understanding genotype-phenotype relationships, we performed population-scale re-sequencing of a diverse panel of wheat lines.ResultsA sample of 62 diverse lines was re-sequenced using the whole exome capture and genotyping-by-sequencing approaches. We describe the allele frequency, functional significance, and chromosomal distribution of 1.57 million single nucleotide polymorphisms and 161,719 small indels. Our results suggest that duplicated homoeologous genes are under purifying selection. We find contrasting patterns of variation and inter-variant associations among wheat genomes; this, in addition to demographic factors, could be explained by differences in the effect of directional selection on duplicated homoeologs. Only a small fraction of the homoeologous regions harboring selected variants overlapped among the wheat genomes in any given wheat line. These selected regions are enriched for loci associated with agronomic traits detected in genome-wide association studies.ConclusionsEvidence suggests that directional selection in allopolyploids rarely acted on multiple parallel advantageous mutations across homoeologous regions, likely indicating that a fitness benefit could be obtained by a mutation at any one of the homoeologs. Additional advantageous variants in other homoelogs probably either contributed little benefit, or were unavailable in populations subjected to directional selection. We hypothesize that allopolyploidy may have increased the likelihood of beneficial allele recovery by broadening the set of possible selection targets.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0606-4) contains supplementary material, which is available to authorized users.     

Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae

Key message

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.

Abstract

To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.     

Deletion mapping of homoeologous group 6-specific wheat expressed sequence tags

To localize wheat (Triticum aestivum L.) ESTs on chromosomes, 882 homoeologous group 6-specific ESTs were identified by physically mapping 7965 singletons from 37 cDNA libraries on 146 chromosome, arm, and sub-arm aneuploid and deletion stocks. The 882 ESTs were physically mapped to 25 regions (bins) flanked by 23 deletion breakpoints. Of the 5154 restriction fragments detected by 882 ESTs, 2043 (loci) were localized to group 6 chromosomes and 806 were mapped on other chromosome groups. The number of loci mapped was greatest on chromosome 6B and least on 6D. The 264 ESTs that detected orthologous loci on all three homoeologs using one restriction enzyme were used to construct a consensus physical map. The physical distribution of ESTs was uneven on chromosomes with a tendency toward higher densities in the distal halves of chromosome arms. About 43% of the wheat group 6 ESTs identified rice homologs upon comparisons of genome sequences. Fifty-eight percent of these ESTs were present on rice chromosome 2 and the remaining were on other rice chromosomes. Even within the group 6 bins, rice chromosomal blocks identified by 1-6 wheat ESTs were homologous to up to 11 rice chromosomes. These rice-block contigs were used to resolve the order of wheat ESTs within each bin.     

Comparison of the genetic organization of the early salt-stress-response gene system in salt-tolerant Lophopyrum elongatum and salt-sensitive wheat

Lophopyrum elongatum is a facultative halophyte related to wheat. Eleven unique clones corresponding to genes showing enhanced mRNA accumulation in the early stages of salt stress were previously isolated from a L. elongatum salt-stressed-root cDNA library. The chromosomal distribution of genes complementary to these clones in several genomes of the tribe Triticeae and their copy number in the L. elongatum and wheat genomes are reported. Genes complementary to clones pESI4, pESI14, pESI15, pESI28, and pESI32 were found in homoeologous group 5, those complementary to pESI18 and pESI35 in homoeologous group 6, and those complementary to pESI47, pESI48, pESI3, and pESI2 in homoeologous groups 1, 3, 4, and 7, respectively. The genes are present in a single copy per genome in L. elongatum with the exception of those complementary to pESI2 and pESI18 which are present in at least two and five copies, respectively. Since similar copy numbers per genome were found in wheat (except for pESI2), the ability of L. elongatum to accumulate higher mRNA levels than wheat in response to salt shock apears to have evolved by changes in the regulation of these genes.     

High-density mapping and comparative analysis of agronomically important traits on wheat chromosome 3A

Bread wheat chromosome 3A has been shown to contain genes/QTLs controlling grain yield and other agronomic traits. The objectives of this study were to generate high-density physical and genetic-linkage maps of wheat homoeologous group 3 chromosomes and reveal the physical locations of genes/QTLs controlling yield and its component traits, as well as agronomic traits, to obtain a precise estimate of recombination for the corresponding regions and to enrich the QTL-containing regions with markers. Physical mapping was accomplished by 179 DNA markers mostly representing expressed genes using 41 single-break deletion lines. Polymorphism survey of cultivars Cheyenne (CNN) and Wichita (WI), and a substitution line of CNN carrying chromosome 3A from WI [CNN(WI3A)], with 142 RFLP probes and 55 SSR markers revealed that the extent of polymorphism is different among various group 3 chromosomal regions as well as among the homoeologs. A genetic-linkage map for chromosome 3A was developed by mapping 17 QTLs for seven agronomic traits relative to 26 RFLP and 15 SSR chromosome 3A-specific markers on 95 single-chromosome recombinant inbred lines. Comparison of the physical maps with the 3A genetic-linkage map localized the QTLs to gene-containing regions and accounted for only about 36% of the chromosome. Two chromosomal regions containing 9 of the 17 QTLs encompassed less than 10% of chromosome 3A but accounted for almost all of the arm recombination. To identify rice chromosomal regions corresponding to the particular QTL-containing wheat regions, 650 physically mapped wheat group 3 sequences were compared with rice genomic sequences. At an E value of E < or = 10(-5), 82% of the wheat group 3 sequences identified rice homologs, of which 54% were on rice chromosome 1. The rice chromosome 1 region collinear with the two wheat regions that contained 9 QTLs was about 6.5 Mb.     

Intraspecific sequence comparisons reveal similar rates of non-collinear gene insertion in the B and D genomes of bread wheat

ABSTRACT: BACKGROUND: Polyploidization is considered one of the main mechanisms of plant genome evolution. The presence of multiple copies of the same gene reduces selection pressure and permits sub-functionalization and neo-functionalization leading to plant diversification, adaptation and speciation. In bread wheat, polyploidization and the prevalence of transposable elements resulted in massive gene duplication and movement. As a result, the number of genes which are non-collinear to genomes of related species seems markedly increased in wheat. RESULTS: We used new-generation sequencing (NGS) to generate sequence of a Mb-sized region from wheat chromosome arm 3DS. Sequence assembly of 24 BAC clones resulted in two scaffolds of 1,264,820 and 333,768 bases. The sequence was annotated and compared to the homoeologous region on wheat chromosome 3B and orthologous loci of Brachypodium distachyon and rice. Among 39 coding sequences in the 3DS scaffolds, 32 have a homoeolog on chromosome 3B. In contrast, only fifteen and fourteen orthologs were identified in the corresponding regions in rice and Brachypodium, respectively. Interestingly, five pseudogenes were identified among the non-collinear coding sequences at the 3B locus, while none was found at the 3DS locus. CONCLUSION: Direct comparison of two Mb-sized regions of the B and D genomes of bread wheat revealed similar rates of non-collinear gene insertion in both genomes with a majority of gene duplications occurring before their divergence. Relatively low proportion of pseudogenes was identified among non-collinear coding sequences. Our data suggest that the pseudogenes did not originate from insertion of non-functional copies, but were formed later during the evolution of hexaploid wheat. Some evidence was found for gene erosion along the B genome locus.