Molecular and FISH analyses of a 53-kbp intact DNA fragment inserted by biolistics in wheat (Triticum aestivum L.) genome.

KEY MESSAGE: A large, 53-kbp, intact DNA fragment was inserted into the wheat ( Triticum aestivum L.) genome. FISH analyses of individual transgenic events revealed multiple insertions of intact fragments. Transferring large intact DNA fragments containing clusters of resistance genes or complete metabolic pathways into the wheat genome remains a challenge. In a previous work, we showed that the use of dephosphorylated cassettes for wheat transformation enabled the production of simple integration patterns. Here, we used the same technology to produce a cassette containing a 44-kb Arabidopsis thaliana BAC, flanked by one selection gene and one reporter gene. This 53-kb linear cassette was integrated in the bread wheat (Triticum aestivum L.) genome by biolistic transformation. Our results showed that transgenic plants harboring the entire cassette were generated. The inheritability of the cassette was demonstrated in the T1 and T2 generation. Surprisingly, FISH analysis performed on T1 progeny of independent events identified double genomic insertions of intact fragments in non-homoeologous positions. Inheritability of these double insertions was demonstrated by FISH analysis of the T1 generation. Relative conclusions that can be drawn from molecular or FISH analysis are discussed along with future prospects of the engineering of large fragments for wheat transformation or genome editing.

Fine mapping of LrSV2, a race-specific adult plant leaf rust resistance gene on wheat chromosome 3BS.

KEY MESSAGE: Fine mapping permits the precise positioning of genes within chromosomes, prerequisite for positional cloning that will allow its rational use and the study of the underlying molecular action mechanism. Three leaf rust resistance genes were identified in the durable leaf rust resistant Argentinean wheat variety Sinvalocho MA: the seedling resistance gene Lr3 on distal 6BL and two adult plant resistance genes, LrSV1 and LrSV2, on chromosomes 2DS and 3BS, respectively. To develop a high-resolution genetic map for LrSV2, 10 markers were genotyped on 343 F2 individuals from a cross between Sinvalocho MA and Gama6. The closest co-dominant markers on both sides of the gene (3 microsatellites and 2 STMs) were analyzed on 965 additional F2s from the same cross. Microsatellite marker cfb5010 cosegregated with LrSV2 whereas flanking markers were found at 1 cM distal and at 0.3 cM proximal to the gene. SSR markers designed from the sequences of cv Chinese Spring BAC clones spanning the LrSV2 genetic interval were tested on the recombinants, allowing the identification of microsatellite swm13 at 0.15 cM distal to LrSV2. This delimited an interval of 0.45 cM around the gene flanked by the SSR markers swm13 and gwm533 at the subtelomeric end of chromosome 3BS.

Radiation hybrid QTL mapping of Tdes2 involved in the first meiotic division of wheat.

Since the dawn of wheat cytogenetics, chromosome 3B has been known to harbor a gene(s) that, when removed, causes chromosome desynapsis and gametic sterility. The lack of natural genetic diversity for this gene(s) has prevented any attempt to fine map and further characterize it. Here, gamma radiation treatment was used to create artificial diversity for this locus. A total of 696 radiation hybrid lines were genotyped with a custom mini array of 140 DArT markers, selected to evenly span the whole 3B chromosome. The resulting map spanned 2,852 centi Ray with a calculated resolution of 0.384 Mb. Phenotyping for the occurrence of meiotic desynapsis was conducted by measuring the level of gametic sterility as seeds produced per spikelet and pollen viability at booting. Composite interval mapping revealed a single QTL with LOD of 16.2 and r (2) of 25.6 % between markers wmc326 and wPt-8983 on the long arm of chromosome 3B. By independent analysis, the location of the QTL was confirmed to be within the deletion bin 3BL7-0.63-1.00 and to correspond to a single gene located ~1.4 Mb away from wPt-8983. The meiotic behavior of lines lacking this gene was characterized cytogenetically to reveal striking similarities with mutants for the dy locus, located on the syntenic chromosome 3 of maize. This represents the first example to date of employing radiation hybrids for QTL analysis. The success achieved by this approach provides an ideal starting point for the final cloning of this interesting gene involved in meiosis of cereals.

[A comparative genetic and cytogenetic mapping of wheat chromosome 5B using introgression lines].

The genetic map of chromosome 5B has been constructed by using microsatellite (SSR) analysis of 381 plants from the F2 population produced by cross of the Chinese Spring (CS) and Renan cultivars. Initially, 180 SSR markers for the common wheat 5B chromosome have been used for analysis of these cultivars. The 32 markers able to detect polymorphism between these cultivars have been located on the genetic map of chromosome 5B. Cytogenetic mapping has involved a set of CS 5B chromosome deletion lines. Totally, 51 SSR markers have been located in ten regions (deletion bins) of this chromosome by SSR analysis of these deletion lines. Five genes--TaCBFIIIc-B10, Vrn--B1, Chi--B1, Skr, and Ph1--have been integrated into the cytogenetic map of chromosome 5B using the markers either specific of or tightly linked to the genes in question. Com- parison of the genetic and cytogenetic maps suggests that recombination is suppressed in the pericentromeric region of chromosome 5B, especially in the short arm segment. The 18 markers localized to deletion bins 5BL16-0.79-1.00 and 5BL8-0.66-0.79 have been used to analyze common wheat introgression lines L842, L5366-180, L73/00i, and L21-4, carrying fragments of alien genomes in the terminal region of 5B long arm. L5366-180 and L842 lines carry a fragment of the Triticum timopheevii 5GL chromosome, while L73/00i ? L21-4 lines, a fragment of the Aegilopsspeltoides 5SL chromosome. As has been shown, the translocated fragments in these four lines are of different lengths, allowing bin 5BL18-0.66-0.79 to be divided into three shorter regions. The utility of wheat introgression lines carrying alien translocations for increasing the resolution of cytogenetic mapping is discussed.

Integrating cereal genomics to support innovation in the Triticeae.

The genomic resources of small grain cereals that include some of the most important crop species such as wheat, barley, and rye are attaining a level of completion that now is contributing to new structural and functional studies as well as refining molecular marker development and mapping strategies for increasing the efficiency of breeding processes. The integration of new efforts to obtain reference sequences in bread wheat and barley, in particular, is accelerating the acquisition and interpretation of genome-level analyses in both of these major crops.

Genetic diversity and linkage disequilibrium studies on a 3.1-Mb genomic region of chromosome 3B in European and Asian bread wheat (Triticum aestivum L.) populations.

Genetic diversity and linkage disequilibrium (LD) were investigated in 376 Asian and European accessions of bread wheat (Triticum aestivum L.). After a first and rapid screening about diversity and genetic structure at the whole genome scale using 70 simple sequence repeats (SSRs), we focused on a sequenced contig (ctg954) of 3.1 Mb located on the short arm of chromosome 3B of cv. Chinese Spring, using 32 SSRs and 10 single nucleotide polymorphisms. This contig is part of a multiple fungal resistance region. Mean polymorphism information content value on the 32 SSRs was slightly higher in the Asian genepool (0.396) than that for the European (0.329) pool. Compared with results at the whole genome scale, data from this 3.1-Mb region indicated similar trends in genetic diversity indices between both genepools. Population structure and molecular variance analyses demonstrated significant genetic differentiation and geographical subdivision in both groups of accessions. Concerning LD at the contig level, the European population had a significantly higher mean r(2) value (0.23) than the Asian population (0.18), indicating a stronger LD in the European material. With a mean of 1 marker every 74 kb, the resolution reached here allowed to perform a detailed comparative analysis of the LD and genetic diversity along the complete 3.1-Mb region in both genepools. A sliding-window approach revealed some interesting regions of the contig where LD is increasing when genetic diversity is decreasing. This study provides an in-depth understanding of molecular population genetics in European and Asian wheat gene pools, and prospects for association mapping of important sources of fungal disease resistance.

Evaluation of the genetic variability of homoeologous group 3 SSRs in bread wheat.

Thorough characterization of the genetic variability in bread wheat (Triticum aestivum L.) is important for a better improvement of this key crop and to increase cereal yield in the context of sustainable agriculture to face human needs in the next decades. To study the genetic variability of SSRs on wheat homoeologous group 3 chromosomes, we characterized 38 hexaploid and two tetraploid wheat lines using a set of 165 microsatellites that we cytogenetically assigned to the 17 deletion bins for chromosomes group 3. A comparative analysis of the genetic variability through the PIC value study, allele numbers and SSR lengths indicated that there were no statistically significant differences (p > 0.05) between the three chromosomes of this homoeologous group despite the fact that SSRs from chromosome 3B exhibited slightly more alleles per locus compared to chromosomes 3A and 3D as well as slightly higher PIC values compared to chromosome 3D. However, there was a stronger correlation between SSR length and allele number on the short arms compared to the long arms and the correlation increased from the centromeres toward the telomeres. We did not find statistically significant differences in allele numbers and PIC values for SSRs located in more distal bins on 3A and 3B chromosomes. On the contrary, for chromosome 3D, we observed significant differences (p < 0.05) between the PIC values determined for SSRs assigned to deletion bin 3DL3-0.81-1.00 bin that is located distal compare to the more proximal region (C-3DL3-0.81). These results suggest that recombination which is higher in the telomeric regions does not contribute to increase a lot the variability of the SSRs.

Biosafety Level 3 setup for multiphoton microscopy in vivo.

Multiphoton microscopy has revealed important insights into cellular behavior in vivo. However, its application in infectious settings often encounters technical, safety and regulatory limitations that prevent its wider use with highly virulent human pathogens. Herein, we present a method that renders multiphoton microscopy in vivo compatible with biosafety level 3 regulations and present an example of its application and potential to visualize a Mycobacterium tuberculosis infection of the mouse lung.

Colinearity and gene density in grass genomes.

Grasses are the single most important plant family in agriculture. In the past years, comparative genetic mapping has revealed conserved gene order (colinearity) among many grass species. Recently, the first studies at gene level have demonstrated that microcolinearity of genes is less conserved: small scale rearrangements and deletions complicate the microcolinearity between closely related species, such as sorghum and maize, but also between rice and other crop plants. In spite of these problems, rice remains the model plant for grasses as there is limited useful colinearity between Arabidopsis and grasses. However, studies in rice have to be complemented by more intensive genetic work on grass species with large genomes (maize, Triticeae). Gene-rich chromosomal regions in species with large genomes, such as wheat, have a high gene density and are ideal targets for partial genome sequencing.

A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification.

The plant aromatic alcohol dehydrogenase, cinnamyl alcohol dehydrogenase (CAD2 from Eucalyptus) was found by sequence analysis of its cloned gene to be homologous to a range of dehydrogenases including alcohol dehydrogenases, L-threonine-3-dehydrogenase, D-xylose reductase and sorbitol dehydrogenase. A homology model of CAD2 was built using the X-ray crystallographic coordinates of horse-liver alcohol dehydrogenase to provide the template, with additional modelling input from other analogous regions of structure from similar enzymes where necessary. The structural model thus produced rationalised the Zn-binding properties of CAD2, indicated the possession of a Rossmann fold (GXGXXG motif), and explained the class A stereospecificity (pro-R hydrogen removal from substrate alcohol) and aromatic substrate specificity of the enzyme. A range of potential ligands was designed based on the homology model and tested as inhibitors of CAD2 and horse liver alcohol dehydrogenase.

A large rearrangement involving genes and low-copy DNA interrupts the microcollinearity between rice and barley at the Rph7 locus.

Grass genomes differ greatly in chromosome number, ploidy level, and size. Despite these differences, very good conservation of the marker order (collinearity) was found at the genetic map level between the different grass genomes. Collinearity is particularly good between rice chromosome 1 and the group 3 chromosomes in the Triticeae. We have used this collinearity to saturate the leaf rust resistance locus Rph7 on chromosome 3HS in barley with ESTs originating from rice chromosome 1S. Chromosome walking allowed the establishment of a contig of 212 kb spanning the Rph7 resistance gene. Sequencing of the contig showed an average gene density of one gene/20 kb with islands of higher density. Comparison with the orthologous rice sequence revealed the complete conservation of five members of the HGA gene family whereas intergenic regions differ greatly in size and composition. In rice, the five genes are closely associated whereas in barley intergenic regions are >38-fold larger. The size difference is due mainly to the presence of six additional genes as well as noncoding low-copy sequences. Our data suggest that a major rearrangement occurred in this region since the Triticeae and rice lineage diverged.

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.

High transferability of bread wheat EST-derived SSRs to other cereals.

The increasing availability of expressed sequence tags (ESTs) in wheat (Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. In this study, 300 primer pairs were designed from 265 wheat ESTs that contain microsatellites in order to develop new markers for wheat. Their level of transferability in eight related species [Triticum durum, T. monococcum, Aegilops speltoides, Ae. tauschii, rye (Secale cereale), barley (Hordeum vulgare), Agropyron elongatum and rice (Oryza sativa)] was assessed. In total, 240 primer pairs (80%) gave an amplification product on wheat, and 177 were assigned to wheat chromosomes using aneuploid lines. Transferability to closely related Triticeae species ranged from 76.7% for Ae. tauschii to 90.4% for T. durum and was lower for more distant relatives such as barley (50.4%) or rice (28.3%). No clear putative function could be assigned to the genes from which the simple sequence repeats (SSRs) were developed, even though most of them were located inside ORFs. BLAST: analysis of the EST sequences against the 12 rice pseudo-molecules showed that the EST-SSRs are mainly located in the telomeric regions and that the wheat ESTs have the highest similarity to genes on rice chromosomes 2, 3 and 5. Interestingly, most of the SSRs giving an amplification product on barley or rice had a repeated motif similar to the one found in wheat, suggesting a common ancestral origin. Our results indicate that wheat EST-SSRs show a high level of transferability across distantly related species, thereby providing additional markers for comparative mapping and for following gene introgressions from wild species and carrying out evolutionary studies.

High gene density is conserved at syntenic loci of small and large grass genomes.

Comparative genomic analysis at the genetic-map level has shown extensive conservation of the gene order between the different grass genomes in many chromosomal regions. However, little is known about the gene organization in grass genomes at the microlevel. Comparison of gene-coding regions between maize, rice, and sorghum showed that the distance between the genes is correlated with the genome size. We have investigated the microcolinearity at Lrk gene loci in the genomes of four grass species: wheat, barley, maize, and rice. The Lrk genes, which encode receptor-like kinases, were found to be consistently associated with another type of receptor-like kinase (Tak) on chromosome groups 1 and 3 in Triticeae and on chromosomes homoeologous to Triticeae group 3 in the other grass genomes. On Triticeae chromosome group 1, Tak and Lrk together with genes putatively encoding NBS/LRR proteins form a cluster of genes possibly involved in signal transduction. Comparison of the gene composition at orthologous Lrk loci in wheat, barley, and rice revealed a maximal gene density of one gene per 4-5 kb, very similar to the gene density in Arabidopsis thaliana. We conclude that small and large grass genomes contain regions that are highly enriched in genes with very little or no repetitive DNA. The comparison of the gene organization suggested various genome rearrangements during the evolution of the different grass species.

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.

Two haplotypes of resistance gene analogs have been conserved during evolution at the leaf rust resistance locus Lr10in wild and cultivated wheat.

The isolation of genes of agronomic interest such as disease resistance genes is a central issue in wheat research. A good knowledge of the organization and evolution of the genome can greatly help in defining the best strategies for efficient gene isolation. So far, very few wheat disease resistance loci have been studied at the molecular level and little is known about their evolution during polyploidization and domestication. In this study, we have analyzed the haplotype structure at loci orthologous to the leaf rust resistance locus Lr10in hexaploid wheat which spans 350 kb in diploid wheat. Two haplotypes (H1, H2) were defined by the presence (H1) or the absence (H2) of two different resistance gene analogs ( rga1, rga2) at this locus on chromosome 1AS. Both haplotypes were found in a collection of 113 wild and cultivated diploid and polyploid wheat lines and they do not reflect phylogenetic relationships. This indicates an ancient origin for this disease resistance locus and the independent conservation of the two haplotypes throughout the evolution of the wheat genome. Finally, the coding regions of the H1 haplotype RGAs are extremely conserved in all the species. This suggests a selective pressure for maintaining the structural and functional configuration of this haplotype in wheat. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0051-9.

Differential characteristics of cell suspension cultures initiated from Eucalyptus gunnii clones differing by their frost tolerance.

Cell suspension cultures were initiated from two clones of Eucalyptus gunnii differing by their frost resistance.During cold treatments viability of the individual cell lines and of their protoplasts was correlated to the degree of frost resistance of the starting clones.Moreover, at moderate temperature (10°C) the growth rate was higher for the tolerant cells than for the sensitive ones.Free proline content was ten-fold higher in the resistant cell line than in the sensitive one whereas concentrations of other free amino-acids were equivalent.

Genetic and physical characterization of the LR1 leaf rust resistance locus in wheat (Triticum aestivum L.).

The objective of this study was to characterize the leaf rust resistance locus Lr1 in wheat. Restriction fragment length polymorphism (RFLP) analysis was performed on the resistant line Lr1/6* Thatcher and the susceptible varieties Thatcher and Frisal, as well as on the segregating F2 populations. Seventeen out of 37 RFLP probes mapping to group 5 chromosomes showed polymorphism between Lr1/6* Thatcher and Frisal, whereas 11 probes were polymorphic between the near-isogenic lines (NILs) Lr1/6* Thatcher and Thatcher. Three of these probes were linked to the resistance gene in the segregating F2 populations. One probe (pTAG621) showed very tight linkage to Lr1 and mapped to a single-copy region on chromosome 5D. The map location of pTAG621 at the end of the long arm of chromosome 5D was confirmed by the absence of the band in the nulli-tetrasomic line N5DT5B of Chinese Spring and a set of deletion lines of Chinese Spring lacking the distal part of 5DL. Twenty-seven breeding lines containing the Lr1 resistance gene in different genetic backgrounds showed the same band as Lr1/6* Thatcher when hybridized with pTAG621. The RFLP marker was converted to a sequence-tagged-site marker using polymerase chain reaction (PCR) amplification. Sequencing of the specific fragment amplified from both NILs revealed point mutations as well as small insertion/deletion events. These were used to design primers that allowed amplification of a specific product only from the resistant line Lr1/6* Thatcher. This STS, specific for the Lr1 resistance gene, will allow efficient selection for the disease resistance gene in wheat breeding programmes. In addition, the identification of a D-genome-specific probe tightly linked to Lr1 should ultimately provide the basis for positional cloning of the gene.