Identification of molecular markers linked to the Agropyron elongatum-derived leaf rust resistance gene Lr24 in wheat

The objective of this study was to identify molecular markers linked to the wheat leaf rust resistance gene Lr24 derived from Agropyron elongatum (3DL/3Ag translocation). Two near isogenic lines (NILs), ‘Arina’ and Lr24/7 ^* “Arina”, were screened for polymorphism at the DNA level with 115 RFLP probes. Twenty-one of these probes map to the homoeologous group 3. In addition, 360 RAPD primers were tested on the NILs. Six RFLP probes showed polymorphism between the NILs, and 11 RAPD primers detected one additional band in the resistant NIL. The genetic linkage of the polymorphic markers with Lr24 was tested on a segregating F₂ population (150 plants) derived from a cross between the leaf rust resistant Lr24/7 ^* “Arina” and the susceptible spelt (Triticum spelta) variety ‘Oberkulmer’. All 6 RFLP markers were completely linked to Lr24: one was inherited as a codominant marker (PSR1205), one was in coupling phase (PSR1203) and 4 were in repulsion phase (PSR388, PSR904, PSR931, PSR1067) with Lr24. The localization of these probes on chromosome 3D was confirmed by nulli-tetrasomic analysis. Distorted genotypic segregation was found for the Codominant RFLP marker PSR1205. This distortion can be explained by the occurrence of hemizygous plants. One of the 11 RAPD markers (OPJ-09) also showed complete linkage to theLr24 resistance gene. The polymorphic RAPD fragment was cloned and sequenced. Specific primers were synthesized, and they produced an amplification product only in the resistant plants. This specific marker allows a reliable and rapid screening of a large number of genotypes in practical breeding. Analysis of 6 additional lines containing Lr24 revealed that 3 lines have a smaller chromosomal segment of A. elongatum than lines derived from ‘Agent’, a commonly used gene donor for the Lr24 resistance gene.     

Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat

More than 100 resistance genes against wheat rust pathogens have been described in wheat and its relatives. Although many of them have been extensively used in wheat resistance breeding, none of these resistance loci has yet been analyzed at the molecular level. By screening a set of near-isogenic lines carrying different leaf rust resistance genes with a wheat probe encoding a serine/threonine protein kinase, we detected a polymorphic DNA fragment in the line with the Lr10 resistance gene. This fragment mapped to the Lr10 disease resistance locus and encodes a receptor-like protein kinase which we called LRK10. LRK10 contains a new type of extracellular domain not found in known plant or animal receptor kinases. Several conserved amino acids in S-domain glycoproteins and receptor-like kinases were also found in LRK10, suggesting that LRK10 and S-domain proteins belong to the same superfamily of specific recognition molecules in plants. Lrk10 was expressed at low levels in young seedlings and belongs to a gene family. Analysis of wheat lines with and without the Lr10 gene demonstrated that Lrk10 and Lr10 belong to the same genetic locus. We conclude that gene isolation based on protein kinase homology can identify new receptor domains and provide candidates for disease resistance genes in the complex wheat genome.     

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.     

Multi-environment analysis and improved mapping of a yield-related QTL on chromosome 3B of wheat

Improved mapping, multi-environment quantitative trait loci (QTL) analysis and dissection of allelic effects were used to define a QTL associated with grain yield, thousand grain weight and early vigour on chromosome 3BL of bread wheat (Triticum aestivum L.) under abiotic stresses. The QTL had pleiotropic effects and showed QTL x environment interactions across 21 diverse environments in Australia and Mexico. The occurrence and the severity of water deficit combined with high temperatures during the growing season affected the responsiveness of this QTL, resulting in a reversal in the direction of allelic effects. The influence of this QTL can be substantial, with the allele from one parent (RAC875) increasing grain yield by up to 12.5 % (particularly in environments where both heat and drought stress occurred) and the allele from the other parent (Kukri) increasing grain yield by up to 9 % in favourable environments. With the application of additional markers and the genotyping of additional recombinant inbred lines, the genetic map in the QTL region was refined to provide a basis for future positional cloning.     

In silico comparative analysis reveals a mosaic conservation of genes within a novel colinear region in wheat chromosome 1AS and rice chromosome 5S

Comparative RFLP mapping has revealed extensive conservation of marker order in different grass genomes. However, microcolinearity studies at the sequence level have shown rapid genome evolution and many exceptions to colinearity. Most of these studies have focused on a limited size of genomic fragment and the extent of microcolinearity over large distances or across entire genomes remains poorly characterized in grasses. Here, we have investigated the microcolinearity between the rice genome and a total of 1,500 kb from physical BAC contigs on wheat chromosome 1AS. Using ESTs mapped in wheat chromosome bins as an additional source of physical data, we have identified 27 conserved orthologous sequences between wheat chromosome 1AS and a region of 1,210 kb located on rice chromosome 5S. Our results extend the orthology described earlier between wheat chromosome group 1S and rice chromosome 5S. Microcolinearity was found to be frequently disrupted by rearrangements which must have occurred after the divergence of wheat and rice. At the Lr10 orthologous loci, microrearrangements were due to the insertion of mobile elements, but also originated from gene movement, amplification, deletion and inversion. These mechanisms of genome evolution are at the origin of the mosaic conservation observed between the orthologous regions. Finally, in silico mapping of wheat genes identified an intragenomic colinearity between fragments from rice chromosome 1L and 5S, suggesting an ancestral segmental duplication in rice.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

A highly conserved gene island of three genes on chromosome 3B of hexaploid wheat: diverse gene function and genomic structure maintained in a tightly linked block

Background  

The complexity of the wheat genome has resulted from waves of retrotransposable element insertions. Gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat have resulted in disruption of colinearity at a micro (sub-megabase) level among the cereals. In view of genomic changes that are possible within a given time span, conservation of genes between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. The ctg1034 contig completed in this paper was initially studied because it was assigned to the Sr2 resistance locus region, but detailed mapping studies subsequently assigned it to the long arm of 3B and revealed its unusual features.     

Cross-genome map based dissection of a nitrogen use efficiency ortho-metaQTL in bread wheat unravels concerted cereal genome evolution

Monitoring nitrogen use efficiency (NUE) in plants is becoming essential to maintain yield while reducing fertilizer usage. Optimized NUE application in major crops is essential for long-term sustainability of agriculture production. Here, we report the precise identification of 11 major chromosomal regions controlling NUE in wheat that co-localise with key developmental genes such as Ppd (photoperiod sensitivity), Vrn (vernalization requirement), Rht (reduced height) and can be considered as robust markers from a molecular breeding perspective. Physical mapping, sequencing, annotation and candidate gene validation of an NUE metaQTL on wheat chromosome 3B allowed us to propose that a glutamate synthase (GoGAT) gene that is conserved structurally and functionally at orthologous positions in rice, sorghum and maize genomes may contribute to NUE in wheat and other cereals. We propose an evolutionary model for the NUE locus in cereals from a common ancestral region, involving species specific shuffling events such as gene deletion, inversion, transposition and the invasion of repetitive elements.     

Crop genome sequencing: lessons and rationales

2010 marks the 10th anniversary of the completion of the first plant genome sequence (Arabidopsis thaliana). Triggered by advancements in sequencing technologies, many crop genome sequences have been produced, with eight published since 2008. To date, however, only the rice (Oryza sativa) genome sequence has been finished to a quality level similar to that of the Arabidopsis sequence. This trend to produce draft genomes could affect the ability of researchers to address biological questions of speciation and recent evolution or to link sequence variation accurately to phenotypes. Here, we review the current crop genome sequencing activities, discuss how variability in sequence quality impacts utility for different studies and provide a perspective for a paradigm shift in selecting crops for sequencing in the future.     

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.Edwige Isidore and Beatrice Scherrer contributed equally to the work.