A. Karakousis

Cloning and characterisation of a family of disease resistance gene analogs from wheat and barley

Abstract The most common class of plant disease resistance (R) genes cloned so far belong to the NBS-LRR group which contain nucleotide-binding sites (NBS) and a leucine-rich repeat (LRR). Specific primer sequences derived from a previously isolated NBS-LRR sequence at the Cre3 locus, which confers resistance to cereal cyst nematode (CCN) in wheat (Triticum aestivum L.) were used in isolating a family of resistance gene analogs (RGA) through a polymerase chain reaction (PCR) cloning approach. The cloning, analysis and genetic mapping of a family of RGAs from wheat (cv ‘Chinese Spring’) and barley (Hordeum vulgare L. cvs ‘Chebec’ and ‘Harrington’) are presented. The wheat and barley RGAs contain other conserved motifs present in known R genes from other plants and share between 55–99% amino acid sequence identity to the NBS-LRR sequence at the Cre3 locus. Phylogenetic analysis of the RGAs with other cloned R genes and RGAs from various plant species indicate that they belong to a superfamily of NBS-containing genes. Two of the barley derived RGAs were mapped onto loci on chromosomes 2H (2), 5H (7) and 7H (1) using barley doubled haploid (DH) mapping populations. Some of these loci identified are associated with regions carrying resistance to CCN and corn leaf aphid.

Construction of three linkage maps in bread wheat, Triticum aestivum

Genetic maps were compiled from the analysis of 160-180 doubled haploid lines derived from 3 crosses: Cranbrook × Halberd, CD87 × Katepwa, and Sunco × Tasman. The parental wheat lines covered a wide range of the germplasm used in Australian wheat breeding. The linkage maps were constructed with RFLP, AFLP, microsatellite markers, known genes, and proteins. The numbers of markers placed on each map were 902 for Cranbrook × Halberd, 505 for CD87 × Katepwa, and 355 for Sunco × Tasman. Most of the expected linkage groups could be determined, but 10-20% of markers could not be assigned to a specific linkage group. Homologous chromosomes could be aligned between the populations described here and linkage groups reported in the literature, based around the RFLP, protein, and microsatellite markers. For most chromosomes, colinearity of markers was found for the maps reported here and those recorded on published physical maps of wheat. AFLP markers proved to be effective in filling gaps in the maps. In addition, it was found that many AFLP markers defined specific genetic loci in wheat across all 3 populations. The quality of the maps and the density of markers differs for each population. Some chromosomes, particularly D genome chromosomes, are poorly covered. There was also evidence of segregation distortion in some regions, and the distribution of recombination events was uneven, with substantial numbers of doubled haploid lines in each population displaying one or more parental chromosomes. These features will affect the reliability of the maps in localising loci controlling some traits, particularly complex quantitative traits and traits of low heritability. The parents used to develop the mapping populations were selected based on their quality characteristics and the maps provide a basis for the analysis of the genetic control of components of processing quality. However, the parents also differ in resistance to several important diseases, in a range of physiological traits, and in tolerance to some abiotic stresses.

A consensus linkage map of barley

A consensus linkage map of the barley genome was constructed. The map is based on six doubled haploid and one F2 population. The mapping data for three of the doubled haploid populations was obtained via the GrainGenes database. To allow merger of the maps, only RFLP markers that produce a single scorable band were included. Although this reduced the available markers by about half, the resultant map contains a total of 587 markers including 87 of known function. As expected, gene order was highly conserved between maps and all but two discrepancies were found in closely linked markers and are likely to result from the small population sizes used for some maps. The consensus map allows the rapid localisation of markers between published maps and should facilitate the selection of markers for high-density mapping in defined regions.

Genetic diversity in Australian wheat varieties and breeding material based on RFLP data

Restriction fragment length polymorphisms (RFLPs) have been used to characterise the genetic diversity of wheat (Triticum aestivum) germplasm. One hundred and twenty-four accessions comprising all major Australian wheat varieties and lines important for breeding purposes were assayed for RFLPs with clones of known genetic location and selected to give uniform genome coverage. The objectives of this study were to determine RFLP-based genetic similarity between accessions and to derive associations between agronomically significant traits and RFLP phenotypes. Ninety-eight probes screened against genomic DNA digested with five restriction endonucleases detected a total of 1968 polymorphic fragments. Genetic similarity (GS) calculated from the RFLP data ranged from 0.004 to 0.409 between accessions, with a mean of 0.18. Cluster analysis based on GS estimates produced four groupings that were generally consistent with available pedigree information. Comparisons of the RFLP phenotypes of accessions containing disease resistance genes present on introgressed alien segments enabled the identification of specific alleles characteristic of these regions. Associations were derived for a range of stem-rust, leaf-rust and yellow-rust resistance genes. These results suggest that RFLP analysis can be used for the characterisation and grouping of elite breeding material of wheat and RFLP profiling can identify chromosome segments associated with agronomic traits.

A consensus map of barley integrating SSR, RFLP, and AFLP markers

A consensus map of barley combining simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) markers has been developed by combining 5 Australian barley linkage maps, Galleon × Haruna Nijo, Chebec × Harrington, Clipper × Sahara, Alexis × Sloop, and Amaji Nijo × WI2585, using the software package JOINMAP 2.0. The new consensus map consists of 700 markers, with 136 being SSRs, and has a total genetic distance of 933 cM. The consensus map order appears to be in good agreement with the Australian barley linkage maps, with the exception of a small inversion located close to the centromere of chromosome 5H. Similarly, the SSR map orders are in good agreement with SSR markers integrated into the doubled haploid linkage map of Lina × Hordeum spontaneum, Canada Park. The new consensus map provides a framework to cross examine and align partial and complete barley linkage maps using markers common to many barley maps. This map will allow researchers to rapidly and accurately select SSR markers for chromosome regions of interest for barley genetic and plant breeding studies.

Mapping of chromosome regions conferring boron toxicity tolerance in barley (Hordeum vulgare L.)

Abstract Boron toxicity has been recognised as an important problem limiting production in the low-rainfall regions of southern Australia, West Asia and North Africa. Genetic variation for boron toxicity tolerance in barley has been characterised but the mode of inheritance and the location of genes controlling tolerance were not previously known. A population of 150 doubled-haploid lines from a cross between a boron toxicity tolerant Algerian landrace, Sahara 3771, and the intolerant Australian cultivar Clipper was screened in four tolerance assays. An RFLP linkage map of the Clipper×Sahara population was used to identify chromosomal regions associated with boron tolerance in barley. Interval regression-mapping allowed the detection of four chromosomal regions involved in the boron tolerance traits measured. A region on chromosome 2H was associated with leaf-symptom expression, a region on chromosome 3H was associated with a reduction of the affect of boron toxicity on root growth suppression, a region on chromosome 6H was associated with reduced boron uptake, and a region on chromosome 4H was also associated with the control of boron uptake as well as being associated with root-length response, dry matter production and symptom expression. The benefits and potential of marker-assisted selection for boron toxicity tolerance are discussed.

Mapping and validation of chromosome regions conferring boron toxicity tolerance in wheat (Triticum aestivum)

Abstract Boron is an essential plant micro-nutrient which can be phytotoxic to plants if present in soils in high concentration. Boron toxicity has been recognised as an important problem limiting production in the low rainfall areas of southern Australia, West Asia and North Africa. Genetic variation for boron toxicity tolerance in wheat has been well-characterised. The efficiency of breeding for boron toxicity tolerance could be greatly enhanced by the development of molecular markers associated with QTLs for tolerance in wheat. A population of 161 doubled haploids from a cross between the tolerant cultivar Halberd and the moderately sensitive cultivar Cranbrook was used to identify chromosomal regions involved in boron tolerance. A combined RFLP and AFLP linkage map of the Cranbrook x Halberd population was used to identify chromosomal regions involved in the boron tolerance traits measured. Regions on chromosome 7B and 7D were associated with leaf symptom expression. The region on chromosome 7B was also associated with the control of boron uptake and with a reduction in the effect of boron toxicity on root-growth suppression. RFLP markers at the chromosome 7B and 7D loci were shown to be effective in selecting for improved boron tolerance in an alternative genetic background. Halberd alleles at the chromosome 7B locus were associated with the concentration of boron in whole shoots and grain. The concentration of boron in whole shoots and in grain were both related to grain yield in a field trial conducted on soil containing toxic levels of boron. Implications relating to marker-assisted selection for boron toxicity tolerance in wheat are discussed.

RFLP mapping of the Ha 2 cereal cyst nematode resistance gene in barley

Abstract The cereal cyst nematode (CCN), Heterodera avenae Woll., is an economically damaging pest of barley in many of the world’s cereal-growing areas. The development of CCN-resistant cultivars may be accelerated through the use of molecular markers. A number of resistance genes against the pest are well known; one of them, the single dominant Ha 2 resistance gene, has been shown to be effective against the Australian pathotype and maps to chromosome 2 of barley. Segregation analysis identified two restriction fragment length polymorphism (RFLP) markers flanking the resistance gene in two doubled-haploid populations of barley. AWBMA 21 and MWG 694 mapped 4.1 and 6.1 cM respectively from the Ha 2 locus in the Chebec×Harrington cross and 4.0 and 9.2 cM respectively in the Clipper×Sahara cross. Analysis of a further seven sources of CCN resistance in the form of near-isogenic lines (NILs) indicates that all available sources of resistance to the Australian pathotype of CCN in barley represent the Ha 2 locus.

Identification and mapping of a gene conferring resistance to the spot form of net blotch (Pyrenophora teres f maculata) in barley

Abstract Spot form of net blotch (SFNB) (Pyrenophora teres f maculata) is an economically damaging foliar disease of barley in many of the world’s cereal growing areas. The development of SFNB-resistant cultivars may be accelerated through the use of molecular markers. A screen for SFNB resistance in 96 lines identified four new sources of resistance, including a feed variety, ‘Galleon’, for which a fully mapped doubled haploid population was available. Segregation data indicated SFNB resistance was conferred by a single gene in the ‘Galleon’בHaruna Nijo’ cross, positioned on the long arm of chromosome 7H. This gene is designated Rpt4 and is flanked by the RFLP loci Xpsr117(D) and Xcdo673 at distances of 6.9 cM and 25.9 cM, respectively. The marker Xpsr117(D) was validated using another population segregating for Rpt4, correctly predicting SFNB resistance with more than 90% accuracy.

Potential of SSR markers for plant breeding and variety identification in Australian barley germplasm

SSR markers closely linked to 18 loci that control 16 important barley traits were assessed for their applicability in Australian barley breeding programs. A panel of 40 genotypes routinely used by the South Australian Barley Improvement Program (SABIP) was used to examine the usefulness of these SSR markers for marker assisted selection (MAS). The success of monitoring a trait locus from donor to recipient lines ranged from 10 to 98%, depending on the marker. SSRs with a high polymorphic information content (PIC) value were found to be the most useful for application in MAS. The assessment also indicated that SSRs derived from genomic sequences were more successful for MAS than those designed from expressed sequence tags. A total of 130 SSR markers were screened among 2 panels of Australian barley genotypes to determine which markers would be the most useful for discriminating Australian germplasm. PIC values generated by this screening were also compared with those generated using a panel of European barley genotypes. Using ordinary correlations (parametric), rank correlations (non-parametric), and partial correlations (multi-variate), a strong association was found between the 2 Australian panels, but no or weak correlation was observed between the 2 Australian panels and the European dataset. It can therefore be concluded that PIC values generated by SSR markers screened with European genotypes cannot be used to predict the usefulness of an SSR marker for discriminating Australian genotypes. From PIC values generated in this study, 36 SSR markers have been selected for the discrimination of Australian genotypes. These markers all show high and/or consistent PIC values among Australian and European barley genotypes.