Evaluation of Septoria Nodorum Blotch (SNB) Resistance in Glumes of Wheat (Triticum aestivum L.) and the Genetic Relationship With Foliar Disease Response.

Septoria nodorum blotch (SNB) is a necrotrophic disease of wheat prominent in some parts of the world, including Western Australia (WA) causing significant losses in grain yield. The genetic mechanisms for resistance are complex involving multiple quantitative trait loci. In order to decipher comparable or independent regulation, this study identified the genetic control for glume compared to foliar resistance across four environments in WA against 37 different isolates. High proportion of the phenotypic variation across environments was contributed by genotype (84.0% for glume response and 82.7% for foliar response) with genotype-by-environment interactions accounting for a proportion of the variation for both glume and foliar response (14.7 and 16.2%, respectively). Despite high phenotypic correlation across environments, most of the eight and 14 QTL detected for glume and foliar resistance using genome wide association analysis (GWAS), respectively, were identified as environment-specific. QTL for glume and foliar resistance neither co-located nor were in LD in any particular environment indicating autonomous genetic mechanisms control SNB response in adult plants, regulated by independent biological mechanisms and influenced by significant genotype-by- environment interactions. Known Snn and Tsn loci and QTL were compared with 22 environment-specific QTL. None of the eight QTL for glume or the 14 for foliar response were co-located or in linkage disequilibrium with Snn and only one foliar QTL was in LD with Tsn loci on the physical map. Therefore, glume and foliar response to SNB in wheat is regulated by multiple environment-specific loci which function independently, with limited influence of known NE-Snn interactions for disease progression in Western Australian environments. Breeding for stable resistance would consequently rely on recurrent phenotypic selection to capture and retain favorable alleles for both glume and foliar resistance relevant to a particular environment.

Phenotypic Evaluation and Genetic Analysis of Seedling Emergence in a Global Collection of Wheat Genotypes (Triticum aestivum L.) Under Limited Water Availability.

The challenge in establishing an early-sown wheat crop in southern Australia is the need for consistently high seedling emergence when sowing deep in subsoil moisture (>10 cm) or into dry top-soil (4 cm). However, the latter is strongly reliant on a minimum soil water availability to ensure successful seedling emergence. This study aimed to: (1) evaluate 233 Australian and selected international wheat genotypes for consistently high seedling emergence under limited soil water availability when sown in 4 cm of top-soil in field and glasshouse (GH) studies; (2) ascertain genetic loci associated with phenotypic variation using a genome-wide association study (GWAS); and (3) compare across loci for traits controlling coleoptile characteristics, germination, dormancy, and pre-harvest sprouting. Despite significant (P < 0.001) environment and genotype-by-environment interactions within and between field and GH experiments, eight genotypes that included five cultivars, two landraces, and one inbred line had consistently high seedling emergence (mean value > 85%) across nine environments. Moreover, 21 environment-specific quantitative trait loci (QTL) were detected in GWAS analysis on chromosomes 1B, 1D, 2B, 3A, 3B, 4A, 4B, 5B, 5D, and 7D, indicating complex genetic inheritance controlling seedling emergence. We aligned QTL for known traits and individual genes onto the reference genome of wheat and identified 16 QTL for seedling emergence in linkage disequilibrium with coleoptile length, width, and cross-sectional area, pre-harvest sprouting and dormancy, germination, seed longevity, and anthocyanin development. Therefore, it appears that seedling emergence is controlled by multifaceted networks of interrelated genes and traits regulated by different environmental cues.

Multi-Location Evaluation of Global Wheat Lines Reveal Multiple QTL for Adult Plant Resistance to Septoria Nodorum Blotch (SNB) Detected in Specific Environments and in Response to Different Isolates.

The slow rate of genetic gain for improving resistance to Septoria nodorum blotch (SNB) is due to the inherent complex interactions between host, isolates, and environments. Breeding for improved SNB resistance requires evaluation and selection of wheat genotypes consistently expressing low SNB response in different target production environments. The study focused on evaluating 232 genotypes from global origins for resistance to SNB in the flag leaf expressed in different Western Australian environments. The aim was to identify resistant donor germplasm against historical and contemporary pathogen isolates and enhance our knowledge of the genetic basis of genotype-by-environment interactions for SNB response. Australian wheat varieties, inbred lines from Centro Internacional de Mejoramiento de Maiz y Trigo (CIMMYT), and International Center for Agricultural Research in the Dry Areas (ICARDA), and landraces from discrete regions of the world showed low to moderate phenotypic correlation for disease response amongst genotypes when evaluated with historical and contemporary isolates at two locations across 3 years in Western Australia (WA). Significant (P < 0.001) genotype-by-environment interactions were detected regardless of same or different isolates used as an inoculum source. Joint regression analysis identified 19 genotypes that consistently expressed low disease severity under infection with different isolates in multi-locations. The CIMMYT inbred lines, 30ZJN09 and ZJN12 Qno25, were particularly pertinent as they had low SNB response and highest trait stability at two locations across 3 years. Genome wide association studies detected 20 QTL associated with SNB resistance on chromosomes 1A, 1B, 4B, 5A, 5B, 6A, 7A, 7B, and 7D. QTL on chromosomes 1B and 5B were previously reported in similar genomic regions. Multiple QTL were identified on 1B, 5B, 6A, and 5A and detected in response to SNB infection against different isolates and specific environments. Known SnTox-Snn interactions were either not evident or variable across WA environments and SNB response may involve other multiple complex biological mechanisms.

High-density SNP mapping reveals closely linked QTL for resistance to Stagonospora nodorum blotch (SNB) in flag leaf and glume of hexaploid wheat.

The genetic control of adult plant resistance to Stagonospora nodorum blotch (SNB) is complex, consisting of genes with minor effects interacting in an additive manner. Earlier studies detected quantitative trait loci (QTL) for flag leaf resistance in successive years on chromosomes 1B, 2A, 2D, and 5B using SSR- and DArT-based genetic maps of progeny from the crosses EGA Blanco/Millewa, 6HRWSN125/WAWHT2074, and P92201D5/P91193D1. Similarly, QTL for glume resistance detected in successive years and multiple environments were identified on chromosomes 2D and 4B from genetic maps of P92201D5/P91193D1 and 6HRWSN125/WAWHT2074, respectively. The SSR- and DArT-based genetic maps had an average distance of 6.5, 7.8, and 9.7 cM between marker loci for populations EGA/Millewa, P92201D5/P91193D1, and 6HRWSN125/WAWHT2074, respectively. This study used single nucleotide polymorphism (SNP) markers from the iSelect Infinium 90K genotyping array to fine-map genomic regions harbouring QTL for flag leaf and glume SNB resistance, reducing the average distance between markers to 2.9, 3.3, and 3.4 cM for populations P92201D5/P91193D1, EGA/Millewa, and 6HRWSN125/WAWHT2074, respectively. Increasing the marker density of the genetic maps with SNPs did not identify any new QTL for SNB resistance but discriminated previously identified co-located QTL into separate but closely linked QTL.

Phylogenetic relationships and protein modelling revealed two distinct subfamilies of group II HKT genes between crop and model grasses.

Molecular evolution of large protein families in closely related species can provide useful insights on structural functional relationships. Phylogenetic analysis of the grass-specific group II HKT genes identified two distinct subfamilies, I and II. Subfamily II was represented in all species, whereas subfamily I was identified only in the small grain cereals and possibly originated from an ancestral gene duplication post divergence from the coarse grain cereal lineage. The core protein structures were highly analogous despite there being no more than 58% amino acid identity between members of the two subfamilies. Distinctly variable regions in known functional domains, however, indicated functional divergence of the two subfamilies. The subsets of codons residing external to known functional domains predicted signatures of positive Darwinian selection potentially identifying new domains of functional divergence and providing new insights on the structural function and relationships between protein members of the two subfamilies.

A comparative gene analysis with rice identified orthologous group II HKT genes and their association with Na(+) concentration in bread wheat.

BACKGROUND: Although the HKT transporter genes ascertain some of the key determinants of crop salt tolerance mechanisms, the diversity and functional role of group II HKT genes are not clearly understood in bread wheat. The advanced knowledge on rice HKT and whole genome sequence was, therefore, used in comparative gene analysis to identify orthologous wheat group II HKT genes and their role in trait variation under different saline environments. RESULTS: The four group II HKTs in rice identified two orthologous gene families from bread wheat, including the known TaHKT2;1 gene family and a new distinctly different gene family designated as TaHKT2;2. A single copy of TaHKT2;2 was found on each homeologous chromosome arm 7AL, 7BL and 7DL and each gene was expressed in leaf blade, sheath and root tissues under non-stressed and at 200 mM salt stressed conditions. The proteins encoded by genes of the TaHKT2;2 family revealed more than 93% amino acid sequence identity but ≤52% amino acid identity compared to the proteins encoded by TaHKT2;1 family. Specifically, variations in known critical domains predicted functional differences between the two protein families. Similar to orthologous rice genes on chromosome 6L, TaHKT2;1 and TaHKT2;2 genes were located approximately 3 kb apart on wheat chromosomes 7AL, 7BL and 7DL, forming a static syntenic block in the two species. The chromosomal region on 7AL containing TaHKT2;1 7AL-1 co-located with QTL for shoot Na(+) concentration and yield in some saline environments. CONCLUSION: The differences in copy number, genes sequences and encoded proteins between TaHKT2;2 homeologous genes and other group II HKT gene families within and across species likely reflect functional diversity for ion selectivity and transport in plants. Evidence indicated that neither TaHKT2;2 nor TaHKT2;1 were associated with primary root Na(+) uptake but TaHKT2;1 may be associated with trait variation for Na(+) exclusion and yield in some but not all saline environments.

Metabolomic profiling and genomic analysis of wheat aneuploid lines to identify genes controlling biochemical pathways in mature grain.

Metabolomics is becoming an increasingly important tool in plant genomics to decipher the function of genes controlling biochemical pathways responsible for trait variation. Although theoretical models can integrate genes and metabolites for trait variation, biological networks require validation using appropriate experimental genetic systems. In this study, we applied an untargeted metabolite analysis to mature grain of wheat homoeologous group 3 ditelosomic lines, selected compounds that showed significant variation between wheat lines Chinese Spring and at least one ditelosomic line, tracked the genes encoding enzymes of their biochemical pathway using the wheat genome survey sequence and determined the genetic components underlying metabolite variation. A total of 412 analytes were resolved in the wheat grain metabolome, and principal component analysis indicated significant differences in metabolite profiles between Chinese Spring and each ditelosomic lines. The grain metabolome identified 55 compounds positively matched against a mass spectral library where the majority showed significant differences between Chinese Spring and at least one ditelosomic line. Trehalose and branched-chain amino acids were selected for detailed investigation, and it was expected that if genes encoding enzymes directly related to their biochemical pathways were located on homoeologous group 3 chromosomes, then corresponding ditelosomic lines would have a significant reduction in metabolites compared with Chinese Spring. Although a proportion showed a reduction, some lines showed significant increases in metabolites, indicating that genes directly and indirectly involved in biosynthetic pathways likely regulate the metabolome. Therefore, this study demonstrated that wheat aneuploid lines are suitable experimental genetic system to validate metabolomics-genomics networks.

Identification of a member of the catalase multigene family on wheat chromosome 7A associated with flour b* colour and biological significance of allelic variation.

Carotenoids (especially lutein) are known to be the pigment source for flour b* colour in bread wheat. Flour b* colour variation is controlled by a quantitative trait locus (QTL) on wheat chromosome 7AL and one gene from the carotenoid pathway, phytoene synthase, was functionally associated with the QTL on 7AL in some, but not all, wheat genotypes. A SNP marker within a sequence similar to catalase (Cat3-A1snp) derived from full-length (FL) cDNA (AK332460), however, was consistently associated with the QTL on 7AL and implicated in regulating hydrogen peroxide (H2O2) to control carotenoid accumulation affecting flour b* colour. The number of catalase genes on chromosome 7AL was investigated in this study to identify which gene may be implicated in flour b* variation and two were identified through interrogation of the draft wheat genome survey sequence consisting of five exons and a further two members having eight exons identified through comparative analysis with the single catalase gene on rice chromosome 6, PCR amplification and sequencing. It was evident that the catalase genes on chromosome 7A had duplicated and diverged during evolution relative to its counterpart on rice chromosome 6. The detection of transcripts in seeds, the co-location with Cat3-A1snp marker and maximised alignment of FL-cDNA (AK332460) with cognate genomic sequence indicated that TaCat3-A1 was the member of the catalase gene family associated with flour b* colour variation. Re-sequencing identified three alleles from three wheat varieties, TaCat3-A1a, TaCat3-A1b and TaCat3-A1c, and their predicted protein identified differences in peroxisomal targeting signal tri-peptide domain in the carboxyl terminal end providing new insights into their potential role in regulating cellular H2O2 that contribute to flour b* colour variation.

Characterization of the multigene family TaHKT 2;1 in bread wheat and the role of gene members in plant Na(+) and K(+) status.

BACKGROUND: A member of the TaHKT2;1 multigene family was previously identified as a Na(+) transporter with a possible role in root Na(+) uptake. In the present study, the existing full-length cDNA of this member was used as a basis to query the International Wheat Genome Survey Sequence to identify all members of the TaHKT2;1 family. Individual TaHKT2;1 genes were subsequently studied for gene and predicted protein structures, promoter variability, tissue expression and their role in Na(+) and K(+) status of wheat. RESULTS: Six TaHKT2;1 genes were characterized which included four functional genes (TaHKT2;1 7AL-1, TaHKT2;1 7BL-1, TaHKT2;1 7BL-2 and TaHKT2;1 7DL-1) and two pseudogenes (TaHKT2;1 7AL-2 and TaHKT2;1 7AL-3), on chromosomes 7A, 7B and 7D of hexaploid wheat. Variability in protein domains for cation specificity and in cis-regulatory elements for salt response in gene promoters, were identified amongst the functional TaHKT2;1 members. The functional genes were expressed under low and high NaCl conditions in roots and leaf sheaths, but were down regulated in leaf blades. Alternative splicing events were evident in TaHKT2;1 7AL-1. Aneuploid lines null for each functional gene were grown in high NaCl nutrient solution culture to identify potential role of each TaHKT2;1 member. Aneuploid lines null for TaHKT2;1 7AL-1, TaHKT2;1 7BL-1 and TaHKT2;1 7BL-2 showed no difference in Na(+) concentration between Chinese Spring except for higher Na(+) in sheaths. The same aneuploid lines had lower K(+) in roots, sheath and youngest fully expanded leaf but only under high (200 mM) NaCl in the external solution. There was no difference in Na(+) or K(+) concentration for any treatment between aneuploid line null for the TaHKT2;1 7DL-1 gene and Chinese Spring. CONCLUSIONS: TaHKT2;1 is a complex family consisting of pseudogenes and functional members. TaHKT2;1 genes do not have an apparent role in controlling root Na(+) uptake in bread wheat seedlings under experimental conditions in this study, contrary to existing hypotheses. However, TaHKT2;1 genes or, indeed other genes in the same chromosome region on 7AL, are candidates that may control Na(+) transport from root to sheath and regulate K(+) levels in different plant tissues.

Chromosomal location of wheat genes of the carotenoid biosynthetic pathway and evidence for a catalase gene on chromosome 7A functionally associated with flour b* colour variation.

Knowledge of molecular and genetic mechanisms controlling wheat grain quality characteristics is significant for improving flour for end-product functionality. Flour b* colour is an important quality trait for breeding wheat varieties to produce grain for specific market requirements. The degree of flour yellowness is due to the accumulation of carotenoids in grain, particularly lutein. Flour b* is under polygenic control and quantitative trait loci (QTL) have frequently been reported on chromosome 7AL. Analysis of carotenoid genes showed that phytoene synthase (PSY) co-located to the QTL on 7AL but other genes at this locus are also thought to contribute flour b* colour variation. This study used the wheat genome survey sequence and identified the chromosomal location of all wheat carotenoid genes, but none other than PSY were located on 7AL and, therefore, other genes may control flour b* colour variation including oxidative genes that degrade carotenoids. An investigation of EST bin mapped to 7AL identified a gene encoding a catalase enzyme (Cat3-A1) that was phylogenetically related to other plant class III enzymes, co-located to the QTL for flour b* colour variation on 7AL in three mapping populations and expressed during seed development. Therefore, Cat3-A1 was functionally associated with flour b* colour variation. Catalase acts upon hydrogen peroxide as a substrate and it was postulated that Cat3-A1 alleles control varying degrees of bleaching action on lutein in developing wheat grain. Markers for Cat3-A1 developed in this study can be used in conjunction with other candidate gene markers including phytoene synthase and lycopene-ε-cylase to develop a molecular signature for selecting lines with specific flour b* colour values in wheat breeding.

Identification, characterization and interpretation of single-nucleotide sequence variation in allopolyploid crop species.

An understanding of nature and extent of nucleotide sequence variation is required for programmes of discovery and characterization of single nucleotide polymorphisms (SNPs), which provide the most versatile class of molecular genetic marker. A majority of higher plant species are polyploids, and allopolyploidy, because of hybrid formation between closely related taxa, is very common. Mutational variation may arise both between allelic (homologous) sequences within individual subgenomes and between homoeologous sequences among subgenomes, in addition to paralogous variation between duplicated gene copies. Successful SNP validation in allopolyploids depends on differentiation of the sequence variation classes. A number of biological factors influence the feasibility of discrimination, including degree of gene family complexity, inbreeding or outbreeding reproductive habit, and the level of knowledge concerning progenitor diploid species. In addition, developments in high-throughput DNA sequencing and associated computational analysis provide general solutions for the genetic analysis of allopolyploids. These issues are explored in the context of experience from a range of allopolyploid species, representing grain (wheat and canola), forage (pasture legumes and grasses), and horticultural (strawberry) crop. Following SNP discovery, detection in routine genotyping applications also presents challenges for allopolyploids. Strategies based on either design of subgenome-specific SNP assays through homoeolocus-targeted polymerase chain reaction (PCR) amplification, or detection of incremental changes in nucleotide variant dosage, are described.

Development of wheat-Lophopyrum elongatum recombinant lines for enhanced sodium 'exclusion' during salinity stress.

Lophopyrum elongatum (tall wheatgrass), a wild relative of wheat, can be used as a source of novel genes for improving salt tolerance of bread wheat. Sodium 'exclusion' is a major physiological mechanism for salt tolerance in a wheat-tall wheatgrass amphiploid, and a large proportion ( approximately 50%) for reduced Na(+) accumulation in the Xag leaf, as compared to wheat, was earlier shown to be contributed by genetic effects from substitution of chromosome 3E from tall wheatgrass for wheat chromosomes 3A and 3D. Homoeologous recombination between 3E and wheat chromosomes 3A and 3D was induced using the ph1b mutant, and putative recombinants were identified as having SSR markers specific for tall wheatgrass loci. As many as 14 recombinants with smaller segments of tall wheatgrass chromatin were identified and low-resolution breakpoint analysis was achieved using wheat SSR loci. Seven recombinants were identified to have leaf Na+ concentrations similar to those in 3E(3A) or 3E(3D) substitution lines, when grown in 200 mM NaCl in nutrient solution. Phenotypic analysis identified recombinants with introgressions at the distal end on the long arm of homoeologous group 3 chromosomes being responsible for Na(+) 'exclusion'. A total of 55 wheat SSR markers mapped to the long arm of homoeologous group 3 markers by genetic and deletion bin mapping were used for high resolution of wheat-tall wheatgrass chromosomal breakpoints in selected recombinants. Molecular marker analysis and genomic in situ hybridisation confirmed the 524-568 recombinant line as containing the smallest introgression of tall wheatgrass chromatin on the distal end of the long arm of wheat chromosome 3A and identified this line as suitable for developing wheat germplasm with Na(+) 'exclusion'.

Comparison of genetic and cytogenetic maps of hexaploid wheat (Triticum aestivum L.) using SSR and DArT markers.

A number of technologies are available to increase the abundance of DNA markers and contribute to developing high resolution genetic maps suitable for genetic analysis. The aim of this study was to expand the number of Diversity Array Technology (DArT) markers on the wheat array that can be mapped in the wheat genome, and to determine their chromosomal location with respect to simple sequence repeat (SSR) markers and their position on the cytogenetic map. A total of 749 and 512 individual DArT and SSR markers, respectively, were identified on at least one of four genetic maps derived from recombinant inbred line (RIL) or doubled haploid (DH) populations. A number of clustered DArT markers were observed in each genetic map, in which 20-34% of markers were redundant. Segregation distortion of DArT and SSR markers was also observed in each mapping population. Only 14% of markers on the Version 2.0 wheat array were assigned to chromosomal bins by deletion mapping using aneuploid lines. In this regard, methylation effects need to be considered when applying DArT marker in genetic mapping. However, deletion mapping of DArT markers provides a reference to align genetic and cytogenetic maps and estimate the coverage of DNA markers across the wheat genome.

Isolation of wheat-rye 1RS recombinants that break the linkage between the stem rust resistance gene SrR and secalin.

Chromosome 1R of rye is a useful source of genes for disease resistance and enhanced agronomic performance in wheat. One of the most prevalent genes transferred to wheat from rye is the stem rust resistance gene Sr31. The recent emergence and spread of a stem rust pathotype virulent to this gene has refocused efforts to find and utilize alternative sources of resistance. There has been considerable effort to transfer a stem rust resistance gene, SrR, from Imperial rye, believed to be allelic to Sr31, into commercial wheat cultivars. However, the simultaneous transfer of genes at the Sec-1 locus encoding secalin seed storage proteins and their association with quality defects preclude the deployment of SrR in some commercial wheat breeding programs. Previous attempts to induce homoeologous recombination between wheat and rye chromosomes to break the linkage between SrR and Sec-1 whilst retaining the tightly linked major loci for wheat seed storage proteins, Gli-D1 and Glu-D3, and recover good dough quality characteristics, have been unsuccessful. We produced novel tertiary wheat-rye recombinant lines carrying different lengths of rye chromosome arm 1RS by inducing homoeologous recombination between the wheat 1D chromosome and a previously described secondary wheat-rye recombinant, DRA-1. Tertiary recombinant T6-1 (SrR+ Sec-1-) carries the target gene for stem rust resistance from rye and retains Gli-D1 but lacks the secalin locus. The tertiary recombinant T49-7 (SrR- Sec-1+) contains the secalin locus but lacks the stem rust resistance gene. T6-1 is expected to contribute to wheat breeding programs in Australia, whereas T49-7 provides opportunities to investigate whether the presence of secalins is responsible for the previously documented dough quality defects.

Arabidopsis-rice-wheat gene orthologues for Na+ transport and transcript analysis in wheat-L. elongatum aneuploids under salt stress.

Lophopyrum elongatum is a wild relative of wheat that provides a source of novel genes for improvement of the salt tolerance of bread wheat. Improved Na(+) 'exclusion' is associated with salt tolerance in a wheat-L. elongatum amphiploid, in which a large proportion (ca. 50%) of the improved regulation of leaf Na(+) concentrations is controlled by chromosome 3E. In this study, genes that might control Na(+) accumulation, such as for transporters responsible for Na(+) entry (HKT1) and exit (SOS1) from cells, or compartmentalisation within vacuoles (NHX1, NHX5, AVP1, AVP2) in the model plant, Arabidopsis thaliana, were targeted for comparative analyses in wheat. Putative rice orthologues were identified and characterised as a means to bridge the large evolutionary distance between genomes from the model dicot and the more complex grass species. Wheat orthologues were identified through BLAST searching to identify either FL-cDNAs or ESTs and were subsequently used to design primers to amplify genomic DNA. The probable orthologous status of the wheat genes was confirmed through demonstration of similar intron-exon structure with their counterparts in Arabidopsis and rice. The majority of exons for Arabidopsis, rice and wheat orthologues of NHX1, NHX5 and SOS1 were conserved except for those at the amino and carboxy terminal ends. However, additional exons were identified in the predicted NHX1 and SOS1 genes of rice and wheat, as compared with Arabidopsis, indicating gene rearrangement events during evolution from a common ancestor. Nullisomic-tetrasomic, deletion and addition lines in wheat were used to assign gene sequences to chromosome regions in wheat and L. elongatum. Most sequences were assigned to homoeologous chromosomes, however, in some instances, such as for SOS1, genes were mapped to other unpredicted locations. Differential transcript abundance under salt stress indicated a complex pattern of expression for wheat orthologues that may regulate Na(+) accumulation in wheat lines containing chromosomes from L. elongatum. The identification of wheat orthologues to well characterized Arabidopsis genes, map locations and gene expression profiles increases our knowledge on the complex mechanisms regulating Na(+) transport in wheat and wheat-L. elongatum lines under salt stress.

Fructosyltransferase and invertase genes evolved by gene duplication and rearrangements: rice, perennial ryegrass, and wheat gene families.

The invertase enzyme family is responsible for carbohydrate metabolism in rice, perennial ryegrass, and wheat. Fructan molecules accumulate in cell vacuoles of perennial ryegrass and wheat and are associated with abiotic stress tolerance. High levels of amino acid similarity between the fructosyltransferases responsible for fructan accumulation indicates that they may have evolved from invertase-like ancestral genes. In this study, we have applied comparative genomics to determine the mechanisms that lead to the evolution of fructosytransferase and invertase genes in rice, perennial ryegrass, and wheat. Duplications and rearrangements have been inferred to generate variant forms of the rice invertases since divergence from a common grass progenitor. The occurrence of multiple copies of fructosyltransferase genes indicated that duplication events continued during evolution of the wheat and perennial ryegrass lineages. Further gene rearrangements were evident in perennial ryegrass genes, albeit at a reduced level compared with the rice invertases. Gene orthologs were largely static after duplication during evolution of the wheat lineage. This study details evolutionary events that contribute to fructosyltransferase and invertase gene variation in grasses.

A resistance-like gene identified by EST mapping and its association with a QTL controlling Fusarium head blight infection on wheat chromosome 3BS.

Fusarium head blight (FHB) is a major disease in the wheat growing regions of the world. A quantitative trait locus (QTL) on the short arm of chromosome 3B controls much of the variation for resistance. The cloning of candidate disease-resistance genes for FHB QTLs on chromosome 3B can provide further elucidation of the mechanisms that control resistance. However, rearrangements and divergence during plant genome evolution often hampers the identification of sequences with similarity to known disease-resistance genes. This study focuses on the use of wheat expressed sequence tags (ESTs) that map to the region on chromosome 3B containing the QTL for FHB resistance and low-stringency BLAST searching to identify sequences with similarity to known disease-resistance genes. One EST rich with leucine repeats and low similarity to a protein kinase domain of the barley Rpg1 gene was identified. Genetic mapping using a Ning894037 x Alondra recombinant inbred (RI) population showed that this EST mapped to the QTL on the short arm of chromosome 3B and may represent a portion of a newly diverged gene contributing to FHB resistance. The EST is a new marker suitable for marker-assisted selection and provides a starting point to begin map-based cloning for chromosome walking and investigate new diverged genes at this locus.

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.

Application of comparative genomics to narrow-leafed lupin (Lupinus angustifolius L.) using sequence information from soybean and Arabidopsis.

The completion of genome-sequencing initiatives for model plants and EST databases for major crop species provides a large resource for gaining fundamental knowledge of complex gene interactions and the functional significance of proteins. There are increasingly numerous opportunities to transfer this information to other plant species with uncharacterized genomes and make advances in genome analysis, gene expression, and predicted protein function. In this study, we have used DNA sequences from soybean and Arabidopsis to determine the feasibility of applying comparative genomics to narrow-leafed lupin. We have used transcribed sequences from soybean and showed that a high proportion cross hybridize to lupin DNA, identifying similar genes and providing landmarks for estimating the degree of chromosomal synteny between species. To further investigate comparative relationships in this study, a detailed analysis of three lupin genes and comparison of orthologs from soybean and Arabidopsis shows that, in some cases, gene structure and expression are highly conserved and their proteins may have similar function. In other cases, genes show variation in expression profiles indicating alternative functions across species. The advantages and limitation of using soybean and Arabidopsis sequences for comparative genomics in lupins are discussed.

Differential expression of a novel gene during seed triacylglycerol accumulation in lupin species ( Lupinus angustifolius L. and L. mutabilis L.).

Seed triacylglycerols (TAGs) are stored as energy reserves and extracted for various end-product uses. In lupins, seed oil content varies from 16% in Lupinus mutabilisto 8% in L. angustifolius. We have shown that TAGs rapidly accumulate during mid-stages of seed development in L. mutabilis compared to the lower seed oil species, L. angustifolius. In this study, we have targeted the key enzymes of the lipid biosynthetic pathway, acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DAGAT), to determine factors regulating TAG accumulation between two lupin species. A twofold increase in ACCase activity was observed in L. mutabilis relative to L. angustifolius and correlated with rapid TAG accumulation. No difference in DAGAT activity was detected. We have identified, cloned and partially characterised a novel gene differentially expressed during TAG accumulation between L. angustifolius and L. mutabilis. The gene has some identity to the glucose dehydrogenase family previously described in barley and bacteria and the significance of its expression levels during seed development in relation to TAG accumulation is discussed. DNA sequence analysis of the promoter in both L. angustifolius and L. mutabilis identified putative matrix attachment regions and recognition sequences for transcription binding sites similar to those found in the Adh1 gene from Arabidopsis. The identical promoter regions between species indicate that differential gene expression is controlled by alternative transcription factors, accessibility to binding sites or a combination of both.