A 2600-locus chromosome bin map of wheat homoeologous group 2 reveals interstitial gene-rich islands and colinearity with rice

The complex hexaploid wheat genome offers many challenges for genomics research. Expressed sequence tags facilitate the analysis of gene-coding regions and provide a rich source of molecular markers for mapping and comparison with model organisms. The objectives of this study were to construct a high-density EST chromosome bin map of wheat homoeologous group 2 chromosomes to determine the distribution of ESTs, construct a consensus map of group 2 ESTs, investigate synteny, examine patterns of duplication, and assess the colinearity with rice of ESTs assigned to the group 2 consensus bin map. A total of 2600 loci generated from 1110 ESTs were mapped to group 2 chromosomes by Southern hybridization onto wheat aneuploid chromosome and deletion stocks. A consensus map was constructed of 552 ESTs mapping to more than one group 2 chromosome. Regions of high gene density in distal bins and low gene density in proximal bins were found. Two interstitial gene-rich islands flanked by relatively gene-poor regions on both the short and long arms and having good synteny with rice were discovered. The map locations of two ESTs indicated the possible presence of a small pericentric inversion on chromosome 2B. Wheat chromosome group 2 was shown to share syntenous blocks with rice chromosomes 4 and 7.     

Group 3 chromosome bin maps of wheat and their relationship to rice chromosome 1

The focus of this study was to analyze the content, distribution, and comparative genome relationships of 996 chromosome bin-mapped expressed sequence tags (ESTs) accounting for 2266 restriction fragments (loci) on the homoeologous group 3 chromosomes of hexaploid wheat (Triticum aestivum L.). Of these loci, 634, 884, and 748 were mapped on chromosomes 3A, 3B, and 3D, respectively. The individual chromosome bin maps revealed bins with a high density of mapped ESTs in the distal region and bins of low density in the proximal region of the chromosome arms, with the exception of 3DS and 3DL. These distributions were more localized on the higher-resolution group 3 consensus map with intermediate regions of high-mapped-EST density on both chromosome arms. Gene ontology (GO) classification of mapped ESTs was not significantly different for homoeologous group 3 chromosomes compared to the other groups. A combined analysis of the individual bin maps using 537 of the mapped ESTs revealed rearrangements between the group 3 chromosomes. Approximately 232 (44%) of the consensus mapped ESTs matched sequences on rice chromosome 1 and revealed large- and small-scale differences in gene order. Of the group 3 mapped EST unigenes approximately 21 and 32% matched the Arabidopsis coding regions and proteins, respectively, but no chromosome-level gene order conservation was detected.     

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.     

A chromosome bin map of 2148 expressed sequence tag loci of wheat homoeologous group 7

The objectives of this study were to develop a high-density chromosome bin map of homoeologous group 7 in hexaploid wheat (Triticum aestivum L.), to identify gene distribution in these chromosomes, and to perform comparative studies of wheat with rice and barley. We mapped 2148 loci from 919 EST clones onto group 7 chromosomes of wheat. In the majority of cases the numbers of loci were significantly lower in the centromeric regions and tended to increase in the distal regions. The level of duplicated loci in this group was 24% with most of these loci being localized toward the distal regions. One hundred nineteen EST probes that hybridized to three fragments and mapped to the three group 7 chromosomes were designated landmark probes and were used to construct a consensus homoeologous group 7 map. An additional 49 probes that mapped to 7AS, 7DS, and the ancestral translocated segment involving 7BS also were designated landmarks. Landmark probe orders and comparative maps of wheat, rice, and barley were produced on the basis of corresponding rice BAC/PAC and genetic markers that mapped on chromosomes 6 and 8 of rice. Identification of landmark ESTs and development of consensus maps may provide a framework of conserved coding regions predating the evolution of wheat genomes.     

First survey of the wheat chromosome 5A composition through a next generation sequencing approach

Wheat is one of the world's most important crops and is characterized by a large polyploid genome. One way to reduce genome complexity is to isolate single chromosomes using flow cytometry. Low coverage DNA sequencing can provide a snapshot of individual chromosomes, allowing a fast characterization of their main features and comparison with other genomes. We used massively parallel 454 pyrosequencing to obtain a 2x coverage of wheat chromosome 5A. The resulting sequence assembly was used to identify TEs, genes and miRNAs, as well as to infer a virtual gene order based on the synteny with other grass genomes. Repetitive elements account for more than 75% of the genome. Gene content was estimated considering non-redundant reads showing at least one match to ESTs or proteins. The results indicate that the coding fraction represents 1.08% and 1.3% of the short and long arm respectively, projecting the number of genes of the whole chromosome to approximately 5,000. 195 candidate miRNA precursors belonging to 16 miRNA families were identified. The 5A genes were used to search for syntenic relationships between grass genomes. The short arm is closely related to Brachypodium chromosome 4, sorghum chromosome 8 and rice chromosome 12; the long arm to regions of Brachypodium chromosomes 4 and 1, sorghum chromosomes 1 and 2 and rice chromosomes 9 and 3. From these similarities it was possible to infer the virtual gene order of 392 (5AS) and 1,480 (5AL) genes of chromosome 5A, which was compared to, and found to be largely congruent with the available physical map of this chromosome.     

Analysis of expressed sequence tag loci on wheat chromosome group 4

A total of 1918 loci, detected by the hybridization of 938 expressed sequence tag unigenes (ESTs) from 26 Triticeae cDNA libraries, were mapped to wheat (Triticum aestivum L.) homoeologous group 4 chromosomes using a set of deletion, ditelosomic, and nulli-tetrasomic lines. The 1918 EST loci were not distributed uniformly among the three group 4 chromosomes; 41, 28, and 31% mapped to chromosomes 4A, 4B, and 4D, respectively. This pattern is in contrast to the cumulative results of EST mapping in all homoeologous groups, as reported elsewhere, that found the highest proportion of loci mapped to the B genome. Sixty-five percent of these 1918 loci mapped to the long arms of homoeologous group 4 chromosomes, while 35% mapped to the short arms. The distal regions of chromosome arms showed higher numbers of loci than the proximal regions, with the exception of 4DL. This study confirmed the complex structure of chromosome 4A that contains two reciprocal translocations and two inversions, previously identified. An additional inversion in the centromeric region of 4A was revealed. A consensus map for homoeologous group 4 was developed from 119 ESTs unique to group 4. Forty-nine percent of these ESTs were found to be homoeologous to sequences on rice chromosome 3, 12% had matches with sequences on other rice chromosomes, and 39% had no matches with rice sequences at all. Limited homology (only 26 of the 119 consensus ESTs) was found between wheat ESTs on homoeologous group 4 and the Arabidopsis genome. Forty-two percent of the homoeologous group 4 ESTs could be classified into functional categories on the basis of blastX searches against all protein databases.     

Chromosome bin map of expressed sequence tags in homoeologous group 1 of hexaploid wheat and homoeology with rice and Arabidopsis

A total of 944 expressed sequence tags (ESTs) generated 2212 EST loci mapped to homoeologous group 1 chromosomes in hexaploid wheat (Triticum aestivum L.). EST deletion maps and the consensus map of group 1 chromosomes were constructed to show EST distribution. EST loci were unevenly distributed among chromosomes 1A, 1B, and 1D with 660, 826, and 726, respectively. The number of EST loci was greater on the long arms than on the short arms for all three chromosomes. The distribution of ESTs along chromosome arms was nonrandom with EST clusters occurring in the distal regions of short arms and middle regions of long arms. Duplications of group 1 ESTs in other homoeologous groups occurred at a rate of 35.5%. Seventy-five percent of wheat chromosome 1 ESTs had significant matches with rice sequences (E < or = e(-10)), where large regions of conservation occurred between wheat consensus chromosome 1 and rice chromosome 5 and between the proximal portion of the long arm of wheat consensus chromosome 1 and rice chromosome 10. Only 9.5% of group 1 ESTs showed significant matches to Arabidopsis genome sequences. The results presented are useful for gene mapping and evolutionary and comparative genomics of grasses.     

Mapping genes affecting flowering time and frost resistance on chromosome 5B of wheat

Two populations of single chromosome recombinant lines were used to map genes controlling flowering time on chromosome 5B of wheat, and one of the populations was also used to map a new frost resistance gene. Genetic maps were developed, mainly using microsatellite markers, and QTL analysis was applied to phenotypic data on the performance of each population collected from growth-room tests of flowering time and frost tolerance. Using a recombinant substitution-line mapping population derived from a cross between the substitution-line 'Chinese Spring' ('Cheyenne' 5B) and 'Chinese Spring' (CS), the gene Vrn-B1, affecting vernalization response, an earliness per se locus, Eps-5BL1, and a gene, Fr-B1, affecting frost resistance, were mapped. Using a 'Hobbit Sib' ('Chinese Spring' 5BL) x 'Hobbit Sib' recombinant substitution line mapping population, an earliness per se locus, Eps-5BL2 was mapped. The Vrn-B1 locus was mapped on the distal portion of the long arm of chromosome 5B, to a region syntenous with the segments of chromosomes 5A and 5D containing Vrn-A1 and Vrn-D1 loci, respectively. The two Eps-5BL loci were mapped close to the centromere with a 16-cM distance from each other, one in agreement with the position of a homoeologous locus previously mapped on chromosome 5H of barley, and suggested by the response of 'Chinese Spring' deletion lines. The Fr-B1 gene was mapped on the long arm of chromosome 5B, 40 cM from the centromeric marker. Previous comparative mapping data with rice chromosome 9 would suggest that this gene could be orthologous to the other Fr genes mapped previously by us on chromosomes 5A or 5D of wheat, although in a more proximal position. This study completes the mapping of these homoeoallelic series of vernalization requirement genes and frost resistance genes on the chromosomes of the homoeologous group 5 in wheat.     

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.     

Targeted mapping of ESTs linked to the adult plant resistance gene Lr46 in wheat using synteny with rice

The gene Lr46 has provided slow-rusting resistance to leaf rust caused by Puccinia triticina in wheat (Triticum aestivum), which has remained durable for almost 30 years. Using linked markers and wheat deletion stocks, we located Lr46 in the deletion bin 1BL (0.84–0.89) comprising 5% of the 1BL arm. The distal part of chromosome 1BL of wheat is syntenic to chromosome 5L of rice. Wheat expressed sequence tags (ESTs) mapping in the terminal 15% of chromosome 1BL with significant homology to sequences from the terminal region of chromosome 5L of rice were chosen for sequence-tagged site (STS) primer design and were mapped physically and genetically. In addition, sequences from two rice bacterial artificial chromosome clones covering the targeted syntenic region were used to identify additional linked wheat ESTs. Fourteen new markers potentially linked to Lr46 were developed; eight were mapped in a segregating population. Markers flanking (2.2 cM proximal and 2.2 cM distal) and cosegregating with Lr46 were identified. The physical location of Lr46 was narrowed to a submicroscopic region between the breakpoints of deletion lines 1BL-13 [fraction length (FL)=0.89–1] and 1BL-10 (FL=0.89–3). We are now developing a high-resolution mapping population for the positional cloning of Lr46.     

Comparative mapping of wheat chromosome 1AS which contains the tiller inhibition gene (<Emphasis Type="Italic">tin</Emphasis>) with rice chromosome 5S

The capacity to tiller is a key factor that determines plant architecture. Using molecular markers, a single major gene reducing tiller number, formally named the tiller inhibition gene (tin), was mapped to the short arm of chromosome 1A in wheat. We identified a tightly linked microsatellite marker (Xgwm136) that may be useful in future marker-assisted selection. The tin gene was mapped to the distal deletion bin of chromosome 1AS (FLM value 0.86) and wheat ESTs which were previously mapped to the same deletion bin were used to identify 18 closely related sequences in the syntenic region of rice chromosome 5. For a subset of wheat ESTs that detected flanking markers for tin, we identified closely related sequences within the most distal 300 kb of rice chromosome 5S. The synteny between the distal chromosome ends of wheat 1AS and rice 5S appeared to be disrupted at the hairy glume locus and seed storage protein loci. We compared map position of tin with other reduced tillering mutants characterised in other cereals to identify possible orthologous genes.     

A 2500-locus bin map of wheat homoeologous group 5 provides insights on gene distribution and colinearity with rice

We constructed high-density deletion bin maps of wheat chromosomes 5A, 5B, and 5D, including 2338 loci mapped with 1052 EST probes and 217 previously mapped loci (total 2555 loci). This information was combined to construct a consensus chromosome bin map of group 5 including 24 bins. A relatively higher number of loci were mapped on chromosome 5B (38%) compared to 5A (34%) and 5D (28%). Differences in the levels of polymorphism among the three chromosomes were partially responsible for these differences. A higher number of duplicated loci was found on chromosome 5B (42%). Three times more loci were mapped on the long arms than on the short arms, and a significantly higher number of probes, loci, and duplicated loci were mapped on the distal halves than on the proximal halves of the chromosome arms. Good overall colinearity was observed among the three homoeologous group 5 chromosomes, except for the previously known 5AL/4AL translocation and a putative small pericentric inversion in chromosome 5A. Statistically significant colinearity was observed between low-copy-number ESTs from wheat homoeologous group 5 and rice chromosomes 12 (88 ESTs), 9 (72 ESTs), and 3 (84 ESTs).     

Molecular characterization of a set of wheat deletion stocks for use in chromosome bin mapping of ESTs

The objective of this study was molecular characterization of a set of deletion stocks and other aneuploids for use in chromosome bin mapping of ESTs in wheat. Wheat aneuploid stocks including 21 nullisomic-tetrasomic (NT), 24 ditelosomic (Dt), and 101 deletion (del) lines were screened with 526 EST clones. A total of 1,951 loci were detected by 493 informative EST clones and tagged 150 of the 159 deletion intervals or chromosome bins. Previously described deletion lines del1AS-4, del6AL-2, del6BS-6, and del7DS-6 were found to have normal chromosome constitution. The short arm deletion in del3AS-3 may be translocated from an unknown chromosome as this stock is nullisomic for the 3AS arm. Thirty-five new deletions were detected in 26 lines. Most of the new deletions occurred in terminal regions of chromosomes and probably resulted from the loss of very small terminal fragments that were difficult to detect cytologically. Eleven chromosome aberrations were also detected in two NT and five Dt lines. Overall, the chromosome bin map provides a resolution of around 28 Mb for an anchor map of a basic set of seven chromosomes of the Triticeae. Any target gene can be allocated to a specific 28-Mb bin and associated ESTs, anchored to the other Triticeae/grass maps including rice and, therefore, amenable to molecular cloning by comparative and wheat-based positional cloning methods. Electronic Publication     

The Ph2 pairing homoeologous locus of wheat (Triticum aestivum): identification of candidate meiotic genes using a comparative genetics approach

Colinearity in gene content and order between rice and closely related grass species has emerged as a powerful tool for gene identification. Using a comparative genetics approach, we have identified the rice genomic region syntenous to the region deleted in the wheat chromosome pairing mutant ph2a, with a view to identifying genes at the Ph2 locus that control meiotic processes. Utilising markers known to reside within the region deleted in ph2a, and data from wheat, barley and rice genetic maps, markers delimiting the region deleted on wheat chromosome 3DS in the ph2a mutant were used to locate the syntenous region on the short arm of rice chromosome 1. A contig of rice genomic sequence was identified from publicly available sequence information and used in blast searches to identify wheat expressed sequence tags (ESTs) exhibiting significant similarity. Southern analysis using a subset of identified wheat ESTs confirmed a syntenous relationship between the rice and wheat genomic regions and defined precisely the extent of the deleted segment in the ph2a mutant. A 6.58-Mb rice contig generated from 60 overlapping rice chromosome 1 P1 artificial chromosome (PAC) clones spanning the syntenous rice region has enabled identification of 218 wheat ESTs putatively located in the region deleted in ph2a. What seems to be a terminal deletion on chromosome 3DS is estimated to be 80 Mb in length. Putative candidate genes that may contribute to the altered meiotic phenotype of ph2a are discussed.     

Structural and functional analyses of the wheat genomes based on expressed sequence tags (ESTs) related to abiotic stresses.

To gain insights into the structure and function of the wheat (Triticum aestivum L.) genomes, we identified 278 ESTs related to abiotic stress (cold, heat, drought, salinity, and aluminum) from 7671 ESTs previously mapped to wheat chromosomes. Of the 278 abiotic stress related ESTs, 259 (811 loci) were assigned to chromosome deletion bins and analyzed for their distribution pattern among the 7 homoeologous chromosome groups. Distribution of abiotic stress related EST loci were not uniform throughout the different regions of the chromosomes of the 3 wheat genomes. Both the short and long arms of group 4 chromosomes showed a higher number of loci in their distal regions compared with proximal regions. Of the 811 loci, the number of mapped loci on the A, B, and D genomes were 258, 281, and 272, respectively. The highest number of abiotic stress related loci were found in homoeologous chromosome group 2 (142 loci) and the lowest number were found in group 6 (94 loci). When considering the genome-specific ESTs, the B genome showed the highest number of unique ESTs (7 loci), while none were found in the D genome. Similarly, considering homoeologous group-specific ESTs, group 2 showed the highest number with 16 unique ESTs (58 loci), followed by group 4 with 9 unique ESTs (33 loci). Many of the classified proteins fell into the biological process categories associated with metabolism, cell growth, and cell maintenance. Most of the mapped ESTs fell into the category of enzyme activity (28%), followed by binding activity (27%). Enzymes related to abiotic stress such as beta-galactosidase, peroxidase, glutathione reductase, and trehalose-6-phosphate synthase were identified. The comparison of stress-responsive ESTs with genomic sequences of rice (Oryza sativa L.) chromosomes revealed the complexities of colinearity. This bin map provides insight into the structural and functional details of wheat genomic regions in relation to abiotic stress.     

Detailed comparison between the wheat chromosome group 7 short arms and the rice chromosome arms 6S and 8L with special reference to genes involved in starch biosynthesis

Rice bacterial artificial chromosome (BAC) clones have been identified that contain sequences orthologous to each EST localized to wheat chromosome 7AS deletion stocks by Southern blot hybridization. This information has been used to relate the DNA sequence included in each wheat deletion stock to a complement of rice BACs. A virtual contig was used that covered 90 cM (21 Mb) of DNA sequence (with a gap for the 6S/8L junction). Comparison of the positions of orthologous genes on the rice virtual contig and on wheat chromosome 7AS showed that there was an unexpectedly low level of synteny (31.4%) and a high level of chromosome rearrangements (68.6%). The non-syntenous loci were of two classes: wheat and rice genes found at different locations in the genome (32.6%), and ESTs in wheat not present in rice (36.0%). Four starch synthetic genes, GBSSI, SSI, SSIIa and DBEI, were located at similar positions on wheat chromosome 7AS and the virtual rice contig covering wheat chromosome 7AS. A preliminary comparison between the short arms of chromosome 7A and 7D in wheat showed that both chromosomes had a similar level of sequence synteny with rice. Therefore, there appears to be considerable variation in gene order between wheat chromosome 7S and rice chromosome 6S and 8L.