An integrated, systems-wide approach is needed for public-private partnerships to drive genetic innovation in crops.

Public-private partnerships are key to successfully translate knowledge to products, but current frameworks do not foster the systems-wide approach required to improve crops to meet the agricultural production challenges of the 21st century.

Fine mapping of a large-effect QTL conferring Fusarium crown rot resistance on the long arm of chromosome 3B in hexaploid wheat.

BACKGROUND: Fusarium crown rot (FCR) is a major cereal disease in semi-arid areas worldwide. Of the various QTL reported, the one on chromosome arm 3BL (Qcrs.cpi-3B) has the largest effect that can be consistently detected in different genetic backgrounds. Nine sets of near isogenic lines (NILs) for this locus were made available in a previous study. To identify markers that could be reliably used in tagging the Qcrs.cpi-3B locus, a NIL-derived population consisting of 774 F10 lines were generated and exploited to assess markers selected from the existing linkage map and generated from sequences of the 3B pseudomolecule. RESULTS: This is the first report on fine mapping a QTL conferring FCR resistance in wheat. By three rounds of linkage mapping using the NILs and the NIL-derived population, the Qcrs.cpi-3B locus was mapped to an interval of 0.7 cM covering a physical distance of about 1.5 Mb. Seven markers co-segregating with the locus were developed. This interval contains a total of 63 gene-coding sequences based on the 3B pseudomolecule, and six of them were known to encode disease resistance proteins. Several of the genes in this interval were among those responsive to FCR infection detected in an earlier study. CONCLUSIONS: The accurate localization of the Qcrs.cpi-3B locus and the development of the markers co-segregating with it should facilitate the incorporation of this large-effect QTL conferring FCR resistance into breeding programs as well as the cloning of the gene(s) underlying the QTL.

Meiotic gene evolution: can you teach a new dog new tricks?

Meiosis, the basis of sex, evolved through iterative gene duplications. To understand whether subsequent duplications have further enriched the core meiotic "tool-kit," we investigated the fate of meiotic gene duplicates following whole genome duplication (WGD), a common occurrence in eukaryotes. We show that meiotic genes return to a single copy more rapidly than genome-wide average in angiosperms, one of the lineages in which WGD is most vividly exemplified. The rate at which duplicates are lost decreases through time, a tendency that is also observed genome-wide and may thus prove to be a general trend post-WGD. The sharpest decline is observed for the subset of genes mediating meiotic recombination; however, we found no evidence that the presence of these duplicates is counterselected in two recent polyploid crops selected for fertility. We therefore propose that their loss is passive, highlighting how quickly WGDs are resolved in the absence of selective duplicate retention.

Wheat centromeric retrotransposons: the new ones take a major role in centromeric structure.

The physical map of the hexaploid wheat chromosome 3B was screened using centromeric DNA probes. A 1.1-Mb region showing the highest number of positive bacterial artificial chromosome (BAC) clones was fully sequenced and annotated, revealing that 96% of the DNA consisted of transposable elements, mainly long terminal repeat (LTR) retrotransposons (88%). Estimation of the insertion time of the transposable elements revealed that CRW (also called Cereba) and Quinta are the youngest elements at the centromeres of common wheat (Triticum spp.) and its diploid ancestors, with Quinta being younger than CRW in both diploid and hexaploid wheats. Chromatin immunoprecipitation experiments revealed that both CRW and Quinta families are targeted by the centromere-specific histone H3 variant CENH3. Immuno colocalization of retroelements and CENH3 antibody indicated that a higher proportion of Quinta than CRWs was associated with CENH3, although CRWs were more abundant. Long arrays of satellite repeats were also identified in the wheat centromere regions, but they lost the ability to bind with CENH3. In addition to transposons, two functional genes and one pseudogene were identified. The gene density in the centromere appeared to be between three and four times lower than the average gene density of chromosome 3B. Comparisons with related grasses also indicated a loss of microcollinearity in this region. Finally, comparison of centromeric sequences of Aegilops tauschii (DD), Triticum boeoticum (AA) and hexaploid wheat revealed that the centromeres in both the polyploids and diploids are still undergoing dynamic changes, and that the new CRWs and Quintas may have undertaken the core role in kinetochore formation.

Wheat syntenome unveils new evidences of contrasted evolutionary plasticity between paleo- and neoduplicated subgenomes.

Bread wheat derives from a grass ancestor structured in seven protochromosomes followed by a paleotetraploidization to reach a 12 chromosomes intermediate and a neohexaploidization (involving subgenomes A, B and D) event that finally shaped the 21 modern chromosomes. Insights into wheat syntenome in sequencing conserved orthologous set (COS) genes unravelled differences in genomic structure (such as gene conservation and diversity) and genetical landscape (such as recombination pattern) between ancestral as well as recent duplicated blocks. Contrasted evolutionary plasticity is observed where the B subgenome appears more sensitive (i.e. plastic) in contrast to A as dominant (i.e. stable) in response to the neotetraploidization and D subgenome as supra-dominant (i.e. pivotal) in response to the neohexaploidization event. Finally, the wheat syntenome, delivered through a public web interface PlantSyntenyViewer at http://urgi.versailles.inra.fr/synteny-wheat, can be considered as a guide for accelerated dissection of major agronomical traits in wheat.

Characterization of polyploid wheat genomic diversity using a high-density 90,000 single nucleotide polymorphism array.

High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker-trait associations in mapping experiments. We developed a genotyping array including about 90,000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence-absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.

Frequent gene movement and pseudogene evolution is common to the large and complex genomes of wheat, barley, and their relatives.

All six arms of the group 1 chromosomes of hexaploid wheat (Triticum aestivum) were sequenced with Roche/454 to 1.3- to 2.2-fold coverage and compared with similar data sets from the homoeologous chromosome 1H of barley (Hordeum vulgare). Six to ten thousand gene sequences were sampled per chromosome. These were classified into genes that have their closest homologs in the Triticeae group 1 syntenic region in Brachypodium, rice (Oryza sativa), and/or sorghum (Sorghum bicolor) and genes that have their homologs elsewhere in these model grass genomes. Although the number of syntenic genes was similar between the homologous groups, the amount of nonsyntenic genes was found to be extremely diverse between wheat and barley and even between wheat subgenomes. Besides a small core group of genes that are nonsyntenic in other grasses but conserved among Triticeae, we found thousands of genic sequences that are specific to chromosomes of one single species or subgenome. By examining in detail 50 genes from chromosome 1H for which BAC sequences were available, we found that many represent pseudogenes that resulted from transposable element activity and double-strand break repair. Thus, Triticeae seem to accumulate nonsyntenic genes frequently. Since many of them are likely to be pseudogenes, total gene numbers in Triticeae are prone to pronounced overestimates.

High-resolution analysis of a QTL for resistance to Stagonospora nodorum glume blotch in wheat reveals presence of two distinct resistance loci in the target interval.

Stagonospora nodorum glume blotch (SNG), caused by the necrotrophic fungus Stagonospora nodorum, is one of the economically important diseases of bread wheat (Triticum aestivum L.). Resistance to SNG is known to be quantitative and previous studies of a recombinant inbred line (RIL) population identified a major quantitative trait locus (QTL) for resistance to SNG on the short arm of chromosome 3B. To localize this QTL (QSng.sfr-3BS) with high resolution, we constructed a genetic map for the QTL target region using information from sequenced flow-sorted chromosomes 3B of the two parental cultivars 'Arina' and 'Forno', the physical map of chromosome 3B of cultivar 'Chinese Spring' and BAC-clone sequences. The mapping population of near-isogenic lines (NIL) was evaluated for SNG resistance in field infection tests. NILs segregated for disease resistance as well as for plant height; additionally, we observed a high environmental influence on the trait. Our analysis detected a strong negative correlation of SNG resistance and plant height. Further analysis of the target region identified two linked loci associated with SNG resistance. One of them was also associated with plant height, revealing an effect of QSng.sfr-3BS on plant height that was hidden in the RIL population. This result demonstrates an unexpectedly high genetic complexity of resistance controlled by QSng.sfr-3BS and shows the importance of the study of QTL in mendelized form in NILs.

Megabase level sequencing reveals contrasted organization and evolution patterns of the wheat gene and transposable element spaces.

To improve our understanding of the organization and evolution of the wheat (Triticum aestivum) genome, we sequenced and annotated 13-Mb contigs (18.2 Mb) originating from different regions of its largest chromosome, 3B (1 Gb), and produced a 2x chromosome survey by shotgun Illumina/Solexa sequencing. All regions carried genes irrespective of their chromosomal location. However, gene distribution was not random, with 75% of them clustered into small islands containing three genes on average. A twofold increase of gene density was observed toward the telomeres likely due to high tandem and interchromosomal duplication events. A total of 3222 transposable elements were identified, including 800 new families. Most of them are complete but showed a highly nested structure spread over distances as large as 200 kb. A succession of amplification waves involving different transposable element families led to contrasted sequence compositions between the proximal and distal regions. Finally, with an estimate of 50,000 genes per diploid genome, our data suggest that wheat may have a higher gene number than other cereals. Indeed, comparisons with rice (Oryza sativa) and Brachypodium revealed that a high number of additional noncollinear genes are interspersed within a highly conserved ancestral grass gene backbone, supporting the idea of an accelerated evolution in the Triticeae lineages.

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.

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.

Whole Genome Profiling provides a robust framework for physical mapping and sequencing in the highly complex and repetitive wheat genome.

BACKGROUND: Sequencing projects using a clone-by-clone approach require the availability of a robust physical map. The SNaPshot technology, based on pair-wise comparisons of restriction fragments sizes, has been used recently to build the first physical map of a wheat chromosome and to complete the maize physical map. However, restriction fragments sizes shared randomly between two non-overlapping BACs often lead to chimerical contigs and mis-assembled BACs in such large and repetitive genomes. Whole Genome Profiling (WGP™) was developed recently as a new sequence-based physical mapping technology and has the potential to limit this problem. RESULTS: A subset of the wheat 3B chromosome BAC library covering 230 Mb was used to establish a WGP physical map and to compare it to a map obtained with the SNaPshot technology. We first adapted the WGP-based assembly methodology to cope with the complexity of the wheat genome. Then, the results showed that the WGP map covers the same length than the SNaPshot map but with 30% less contigs and, more importantly with 3.5 times less mis-assembled BACs. Finally, we evaluated the benefit of integrating WGP tags in different sequence assemblies obtained after Roche/454 sequencing of BAC pools. We showed that while WGP tag integration improves assemblies performed with unpaired reads and with paired-end reads at low coverage, it does not significantly improve sequence assemblies performed at high coverage (25x) with paired-end reads. CONCLUSIONS: Our results demonstrate that, with a suitable assembly methodology, WGP builds more robust physical maps than the SNaPshot technology in wheat and that WGP can be adapted to any genome. Moreover, WGP tag integration in sequence assemblies improves low quality assembly. However, to achieve a high quality draft sequence assembly, a sequencing depth of 25x paired-end reads is required, at which point WGP tag integration does not provide additional scaffolding value. Finally, we suggest that WGP tags can support the efficient sequencing of BAC pools by enabling reliable assignment of sequence scaffolds to their BAC of origin, a feature that is of great interest when using BAC pooling strategies to reduce the cost of sequencing large genomes.

DNA repair and crossing over favor similar chromosome regions as discovered in radiation hybrid of Triticum.

BACKGROUND: The uneven distribution of recombination across the length of chromosomes results in inaccurate estimates of genetic to physical distances. In wheat (Triticum aestivum L.) chromosome 3B, it has been estimated that 90% of the cross over events occur in distal sub-telomeric regions representing 40% of the chromosome. Radiation hybrid (RH) mapping which does not rely on recombination is a strategy to map genomes and has been widely employed in animal species and more recently in some plants. RH maps have been proposed to provide i) higher and ii) more uniform resolution than genetic maps, and iii) to be independent of the distribution patterns observed for meiotic recombination. An in vivo RH panel was generated for mapping chromosome 3B of wheat in an attempt to provide a complete scaffold for this ~1 Gb segment of the genome and compare the resolution to previous genetic maps. RESULTS: A high density RH map with 541 marker loci anchored to chromosome 3B spanning a total distance of 1871.9 cR was generated. Detailed comparisons with a genetic map of similar quality confirmed that i) the overall resolution of the RH map was 10.5 fold higher and ii) six fold more uniform. A significant interaction (r = 0.879 at p = 0.01) was observed between the DNA repair mechanism and the distribution of crossing-over events. This observation could be explained by accepting the possibility that the DNA repair mechanism in somatic cells is affected by the chromatin state in a way similar to the effect that chromatin state has on recombination frequencies in gametic cells. CONCLUSIONS: The RH data presented here support for the first time in vivo the hypothesis of non-casual interaction between recombination hot-spots and DNA repair. Further, two major hypotheses are presented on how chromatin compactness could affect the DNA repair mechanism. Since the initial RH application 37 years ago, we were able to show for the first time that the iii) third hypothesis of RH mapping might not be entirely correct.

Variation in crossover rates across a 3-Mb contig of bread wheat (Triticum aestivum) reveals the presence of a meiotic recombination hotspot.

In bread wheat (Triticum aestivum L.), initial studies using deletion lines indicated that crossover (CO) events occur mainly in the telomeric regions of the chromosomes with a possible correlation with the presence of genes. However, little is known about the distribution of COs at the sequence level. To investigate this, we studied in detail the pattern of COs along a contig of 3.110 Mb using two F2 segregating populations (Chinese Spring × Renan (F2-CsRe) and Chinese Spring × Courtot (F2-CsCt)) each containing ~2,000 individuals. The availability of the sequence of the contig from Cs enabled the development of 318 markers among which 23 co-dominant polymorphic markers (11 SSRs and 12 SNPs) were selected for CO distribution analyses. The distribution of CO events was not homogeneous throughout the contig, ranging from 0.05 to 2.77 cM/Mb, but was conserved between the two populations despite very different contig recombination rate averages (0.82 cM/Mb in F2-CsRe vs 0.35 cM/Mb in F2-CsCt). The CO frequency was correlated with the percentage of coding sequence in Cs and with the polymorphism rate between Cs and Re or Ct in both populations, indicating an impact of these two factors on CO distribution. At a finer scale, COs were found in a region covering 2.38 kb, spanning a gene coding for a glycosyl transferase (Hga3), suggesting the presence of a CO hotspot. A non-crossover event covering at least 453 bp was also identified in the same interval. From these results, we can conclude that gene content could be one of the factors driving recombination in bread wheat.

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

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.

A 3,000-loci transcription map of chromosome 3B unravels the structural and functional features of gene islands in hexaploid wheat.

To improve our understanding of the organization and regulation of the wheat (Triticum aestivum) gene space, we established a transcription map of a wheat chromosome (3B) by hybridizing a newly developed wheat expression microarray with bacterial artificial chromosome pools from a new version of the 3B physical map as well as with cDNA probes derived from 15 RNA samples. Mapping data for almost 3,000 genes showed that the gene space spans the whole chromosome 3B with a 2-fold increase of gene density toward the telomeres due to an increase in the number of genes in islands. Comparative analyses with rice (Oryza sativa) and Brachypodium distachyon revealed that these gene islands are composed mainly of genes likely originating from interchromosomal gene duplications. Gene Ontology and expression profile analyses for the 3,000 genes located along the chromosome revealed that the gene islands are enriched significantly in genes sharing the same function or expression profile, thereby suggesting that genes in islands acquired shared regulation during evolution. Only a small fraction of these clusters of cofunctional and coexpressed genes was conserved with rice and B. distachyon, indicating a recent origin. Finally, genes with the same expression profiles in remote islands (coregulation islands) were identified suggesting long-distance regulation of gene expression along the chromosomes in wheat.

Reconstruction of monocotelydoneous proto-chromosomes reveals faster evolution in plants than in animals.

Paleogenomics seeks to reconstruct ancestral genomes from the genes of today's species. The characterization of paleo-duplications represented by 11,737 orthologs and 4,382 paralogs identified in five species belonging to three of the agronomically most important subfamilies of grasses, that is, Ehrhartoideae (rice) Panicoideae (sorghum, maize), and Pooideae (wheat, barley), permitted us to propose a model for an ancestral genome with a minimal size of 33.6 Mb structured in five proto-chromosomes containing at least 9,138 predicted proto-genes. It appears that only four major evolutionary shuffling events (alpha, beta, gamma, and delta) explain the divergence of these five cereal genomes during their evolution from a common paleo-ancestor. Comparative analysis of ancestral gene function with rice as a reference indicated that five categories of genes were preferentially modified during evolution. Furthermore, alignments between the five grass proto-chromosomes and the recently identified seven eudicot proto-chromosomes indicated that additional very active episodes of genome rearrangements and gene mobility occurred during angiosperm evolution. If one compares the pace of primate evolution of 90 million years (233 species) to 60 million years of the Poaceae (10,000 species), change in chromosome structure through speciation has accelerated significantly in plants.

Cereal breeding takes a walk on the wild side.

Elite cultivated crop gene pools of the Triticeae tribe (wheat, barley and rye) exhibit limited genetic diversity, raising concerns about our ability to increase or simply sustain crop yield and quality in the face of dynamic environmental and biotic threats. Although exploiting their wild relatives as a source of novel alleles is challenging, it has provided notable successes in cereal improvement for >100 years. Increasingly facile gene discovery, improved enabling technologies for genetics and breeding and a better understanding of the factors limiting practical exploitation of exotic germplasm promise to transform existing, and accelerate the development of new, strategies for efficient and directed germplasm utilization.

Combined meta-genomics analyses unravel candidate genes for the grain dietary fiber content in bread wheat (Triticum aestivum L.).

Grain dietary fiber content in wheat not only affects its end use and technological properties including milling, baking and animal feed but is also of great importance for health benefits. In this study, integration of association genetics (seven detected loci on chromosomes 1B, 3A, 3D, 5B, 6B, 7A, 7B) and meta-QTL (three consensus QTL on chromosomes 1B, 3D and 6B) analyses allowed the identification of seven chromosomal regions underlying grain dietary fiber content in bread wheat. Based either on a diversity panel or on bi-parental populations, we clearly demonstrate that this trait is mainly driven by a major locus located on chromosome 1B associated with a log of p value >13 and a LOD score >8, respectively. In parallel, we identified 73 genes differentially expressed during the grain development and between genotypes with contrasting grain fiber contents. Integration of quantitative genetics and transcriptomic data allowed us to propose a short list of candidate genes that are conserved in the rice, sorghum and Brachypodium chromosome regions orthologous to the seven wheat grain fiber content QTL and that can be considered as major candidate genes for future improvement of the grain dietary fiber content in bread wheat breeding programs.