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'.

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.

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.