A high density physical map of chromosome 1BL supports evolutionary studies,map-based cloning and sequencing in wheat

Background

As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning.

Results

Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome.

Conclusions

Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing.     

Characterisation of polymorphic microsatellite markers from Aegilops tauschii and transferability to the D-genome of bread wheat

Microsatellites were isolated from a Aegilops tauschii (the D-genome donor of bread wheat) library enriched for various motifs. Primers generated from the flanking region of the microsatellites were used successfully to amplify the corresponding loci in the D genome of bread wheat. Additional amplification sometimes also occurred from the A and B genomes. The majority of the microsatellites contained (GA)(n) and (GT)(n) motifs. GA and GT repeats appeared to be both more abundant in this library and more polymorphic than other types of repeats. The allele number for both types of dinucleotide repeats fitted a Poisson distribution. Deviance analysis showed that GA and GT were more polymorphic than other motifs in bread wheat. Within each motif type (di-, tri- and tetra-nucleotide repeats), repeat number has no influence on polymorphism. The microsatellites were mapped using the Triticum aestivum Courtot x Chinese Spring mapping population. A total of 100 markers was developed on this intraspecific map, mainly on the D genome. For polyploid species, isolation of microsatellites from an ancestral diploid donor seems to be an efficient way of developing markers for the corresponding genome in the polyploid plant.     

Studies of the transferability of microsatellites derived from Triticum tauschii to hexaploid wheat and to diploid related species using amplification,hybridization and sequence comparisons

Hexaploid wheat (Triticum aestivum L em Thell) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes, respectively. We recently isolated microsatellites from a T. tauschii library enriched for various motifs and evaluated the transferability of these markers to several diploid species carrying the A, B or D genomes. All of the primer pairs amplifying more than one locus on bread wheat and half of those giving D-genome-specific loci gave an amplification product on A-and/or B-diploid species. All of the markers giving a single amplification product for T. tauschii and no amplification on the other diploid species were D-genome-specific at the hexaploid level. The non-specific microsatellite markers (which gave an amplification product on diploid species carrying the A, B or D genome) gave either a complex amplification pattern on bread wheat (with several bands) or generated a single band which mapped to the D genome. Southern blot hybridizations with probes corresponding to the microsatellite flanking regions gave a signal on all diploid and hexaploid species, whatever the specificity of the microsatellite. The patterns observed on bread wheat were generally in accordance with those observed for diploid species, with slight rearrangements. This suggests that the specificity of microsatellite markers is probably due to mutations in microsatellite flanking regions rather than sequence elimination during polyploidization events and that genome stringency is higher at the polyploid than at the diploid level.     

Location of genes involved in ear compactness in wheat (Triticum aestivum) by means of molecular markers

Quantitative trait loci (QTLs) for three traits related to ear morphology (spike length, number of spikelets, and compactness as the ratio between number of spikelets and spike length) in wheat (Triticum aestivum L.) were mapped in a doubled-haploid (DH) population derived from the cross between the cultivars Courtot and Chinese Spring. A molecular marker linkage map of this cross that had previously been constructed based on 187 DH lines and 380 markers was used for QTL mapping. The genome was well covered (85%) except chromosomes 1D and 4D and a set of anchor loci regularly spaced (one marker each 15.5 cM) were chosen for marker regression analysis. The presence of a QTL was declared at a significance threshold = 0.001. The population was grown in one location under field conditions during three years (1994, 1995 and 1998). For each trait, 4 to 6 QTLs were identified with individual effects ranging between 6.9% and 21.8% of total phenotypic variation. Several QTLs were detected that affected more than one trait. Of the QTLs 50% were detected in more than one year and two of them (number of spikelets on chromosome 2B, and compactness on chromosome 2D) emerged from the data from the three years. Only one QTL co-segregated with the gene Q known to be involved in ear morphology, namely the speltoid phenotype. However, this chromosome region explained only a minor part of the variation (7.5–11%). Other regions had a stronger effect, especially two previously unidentified regions located on chromosomes 1A and 2B. The region on the long arm of chromosome 1A was close to the locus XksuG34-1A and explained 12% of variation in spike length and 10% for compactness. On chromosome 2B, the QTL was detected for the three traits near the locus Xfbb121-2B. This QTL explained 9% to 22% of variation for the traits and was located in the same region as the gene involved in photoperiod response (Ppd2). Other regions were located at homoeologous positions on chromosomes 2A and 2D.     

Genetic analysis of durable resistance to yellow rust in bread wheat

Yellow rust, caused by Puccinia striiformis, is one of the most damaging diseases affecting bread wheat in temperate regions. Although resistance to yellow rust is frequently overcome by new virulent races, a durable form of resistance in the French bread wheat Camp Rémy (CR) has remained effective since its introduction in 1980. We used 217 F₇ recombinant inbred lines (RILs) derived from the cross between CR and the susceptible cultivar Récital to identify and map quantitative trait loci (QTLs) involved in durable yellow rust resistance. Six significant QTLs that were stable over a 4-year period were detected. Two QTLs, denoted QYr.inra-2DS and QYr.inra-5BL.2, were located on the short arm of chromosome 2D and the long arm of chromosome 5B, respectively. Each explained on average 25–35% of the observed phenotypic variation and were probably inherited from Cappelle Desprez, a parent of CR that confers durable adult plant resistance to yellow rust. QYr.inra-2DS probably corresponds to the Yr16 gene. The most consistent QTL, designated QYr.inra-2BL, was located on the centromeric region of chromosome 2B and explained 61% of the phenotypic variation in 2003. This QTL was responsible for seedling-stage resistance and may correspond to a cluster of genes, including Yr7. The remaining QTLs were mapped to the short arm of chromosome 2B (R²=22–70%) and to the long arm of chromosomes 2A (R²=0.20–0.40) and 5B (R²=0.18–0.26). This specific combination of seedling and adult plant resistance genes found in CR and CD may constitute the key to their durable resistance against yellow rust.     

Transferable bread wheat EST-SSRs can be useful for phylogenetic studies among the Triticeae species

The genetic similarity between 150 accessions, representing 14 diploidand polyploid species of the Triticeae tribe, was investigated following the UPGMA clustering method. Seventy-three common wheat EST-derived SSR markers (EST-SSRs) that were demonstrated to be transferable across several wheat-related species were used. When diploid species only are concerned, all the accessions bearing the same genome were clustered together without ambiguity while the separation between the different sub-species of tetraploid as well as hexaploid wheats was less clear. Dendrograms reconstructed based on data of 16 EST-SSRs mapped on the A genome confirmed that Triticum aestivum and Triticum durum had closer relationships with Triticum urartu than with Triticum monococcum and Triticum boeoticum, supporting the evidence that T. urartu is the A-genome ancestor of polyploid wheats. Similarly, another tree reconstructed based on data of ten EST-SSRs mapped on the B genome showed that Aegilops speltoides had the closest relationship with T. aestivum and T. durum, suggesting that it was the main contributor of the B genome of polyploid wheats. All these results were expected and demonstrate thus that EST-SSR markers are powerful enough for phylogenetic analysis among the Triticeae tribe.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Contrasted Microcolinearity and Gene Evolution Within a Homoeologous Region of Wheat and Barley Species

We study here the evolution of genes located in the same physical locus using the recently sequenced Ha locus in seven wheat genomes in diploid, tetraploid, and hexaploid species and compared them with barley and rice orthologous regions. We investigated both the conservation of microcolinearity and the molecular evolution of genes, including coding and noncoding sequences. Microcolinearity is restricted to two groups of genes (Unknown gene-2, VAMP, BGGP, Gsp-1, and Unknown gene-8 surrounded by several copies of ATPase), almost conserved in rice and barley, but in a different relative position. Highly conserved genes between wheat and rice run along with genes harboring different copy numbers and highly variable sequences between close wheat genomes. The coding sequence evolution appeared to be submitted to heterogeneous selective pressure and intronic sequences analysis revealed that the molecular clock hypothesis is violated in most cases.     

Transferability of wheat microsatellites to diploid Triticeae species carrying the A, B and D genomes

Hexaploid wheat (Triticum aestivum L em Thell) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. In this study, we evaluated the ability of microsatellite sequences from T. aestivum to be revealed on different ancestral diploid species more or less closely related, i.e. to test for their transferability. Fifty five primer pairs, evenly distributed all over the genome, were investigated. Forty three of them mapped to single loci on the hexaploid wheat genetic map although only 20 (46%) gave single PCR products; the 23 others (54%) gave more than one band with either only one being polymorphic, the others remaining monomorphic, or with several co-segregating polymorphic bands. The other 12 detected two (9) or three (3) different loci. From the 20 primer pairs which gave one amplification pro- duct on hexaploid wheat, nine (45%) also amplified products on only one of the diploid species, and seven (35%) on more than one. Four microsatellites (20%) which mapped to chromosomes from the B genome of wheat, did not give any amplification signal on any of the diploid species. This suggests that some regions of the B genome have evolved more rapidly compared to the A or D genomes since the emergence of polyploidy, or else that the donor(s) of this B genome has(have) not yet been identified. Our results confirm that Triticum monococcum ssp. urartu and Triticum tauschii were the main donors of the A and D genomes respectively, and that Aegilops speltoides is related to the ancestor(s) of the wheat polyploid B genome. Received: 21 June 2000 / Accepted: 15 November 2000     

Chromosome synteny of the a genome of two evolutionary wheat lines

In order to estimate synteny between A^t and A polyploid wheat genomes belonging to different evolutionary lines (Timopheevi and Emmer), saturation of chromosome maps of Triticum timopheevii A^t genome by molecular markers has been conducted. Totally, 179 EST-SSR and 48 genomic SSR-markers have been used with the following integration of 13 and 7 markers correspondingly into chromosome maps of A^t genome. ESTSSR showed higher transferability and lower polymorphism than genomic SSR markers. The chromosome maps designed were compared to maps of homoeologous chromosome group of the T. aestivum A genome. No disturbances of colinearity, i.e., of the order of markers within the chromosome segments on which they had been previously mapped, were observed. According to the quantity assessment of markers amplifying in homoeologous chromosomes, the maximum divergence was detected in two groups (4A^t/4A and 3A^t/3A) among the seven chromosomes examined in the A ^t and A genomes. Comparison of molecular genetic mapping results with the published results of studying meiosis of F₁ hybrids and the frequency of chromosomes substitution in introgressive T. aestivum × T. timopheevii lines suggest that individual chromosomes of the A^t and A genomes evolve differently. Translocations were shown to introduce the major impact on the divergence of 4A^t/4A and 6A^t/6A chromosomes, while mutations of the primary DNA structure, on the divergence of homoeologous group 3 chromosomes. The level of reorganization of other chromosomes during the evolution in the A^t and A genomes was significantly lower.     

Detailed Recombination Studies Along Chromosome 3B Provide New Insights on Crossover Distribution in Wheat (Triticum aestivum L.)

In wheat (Triticum aestivum L.), the crossover (CO) frequency increases gradually from the centromeres to the telomeres. However, little is known about the factors affecting both the distribution and the intensity of recombination along this gradient. To investigate this, we studied in detail the pattern of CO along chromosome 3B of bread wheat. A dense reference genetic map comprising 102 markers homogeneously distributed along the chromosome was compared to a physical deletion map. Most of the COs (90%) occurred in the distal subtelomeric regions that represent 40% of the chromosome. About 27% of the proximal regions surrounding the centromere showed a very weak CO frequency with only three COs found in the 752 gametes studied. Moreover, we observed a clear decrease of CO frequency on the distal region of the short arm. Finally, the intensity of interference was assessed for the first time in wheat using a Gamma model. The results showed m values of 1.2 for male recombination and 3.5 for female recombination, suggesting positive interference along wheat chromosome 3B.