S. Gupta

Mapping and validation of the genes for resistance to Pyrenophora teres f. teres in barley (Hordeum vulgare L.)

Identification and deployment of disease resistance genes are key objectives of Australian barley breeding programs. Two doubled haploid (DH) populations derived from Tallon × Kaputar (TK) and VB9524 × ND11231 (VN) crosses were used to identify markers for net type net blotch (NTNB) (Pyrenophora teres f. teres). The maps included 263 and 250 markers for TK and VN populations, respectively. The TK population was screened with 5 pathotypes and the VN population with 1 pathotype of NTNB as seedlings in the glasshouse. In addition, the TK population was subjected to natural infection in the field at Hermitage Research Station, Qld. Analyses of the markers were performed using the software packages MapManager and Qgene. One region on chromosome 6H was strongly associated with resistance to NTNB in both populations (R2 = 83% for TK and 66% for VN). In the TK population, 2 more quantitative trait loci (QTLs) were identified on chromosomes 2H and 3H, with R2 values of 30% and 31%, respectively. These associations were consistent over all pathotypes studied during the seedling stage. The same QTL on chromosome 6H was also found to be highly significantly associated (R2 = 65%) with the adult plant (field) response in the TK population. There are several very closely linked markers showing strong associations in these regions. Association of the 4 markers on chromosome 6H QTL with resistance to the NTNB has been validated in 2 other DH populations derived from barley crosses Pompadour × Stirling and WPG8412 × Stirling. These markers present an opportunity for marker assisted selection of lines resistant to NTNB in barley breeding programs.

Current virulence of Pyrenophora teres on barley in Western Australia

Studies on variation, occurrence, and distribution of virulence in Pyrenophora teres are helpful to identify effective sources of resistance that can be used for barley breeding in Western Australia. Seventy-nine isolates of Pyrenophora teres were collected from different barley fields of Western Australia in 1995-96. Seventy-four induced net type symptoms (P. teres f. teres) and five induced spot type symptoms (P. teres f. maculata). Net type isolate responses on 47 barley lines were similar to the range of responses induced by nine historical isolates collected in the region between 1975 and 1985. These net type isolates were classified into two distinct groups based on virulence to the cultivar Beecher. Isolates were further classified into eight groups based on minor pathogenic variation among the population. The virulence phenotype present in an eastern Australian isolate was not observed in any isolates collected from Western Australia. An analysis of variance on a subset of 12 net type isolates indicated a significant line × isolate interaction (P < 0.001), with the interaction term variance component four times larger than the error variance. Based on these studies, the virulence among net type isolates has remained stable in Western Australia for the last 19 years. Spot type isolates were collected from a wider geographic area than previously reported and varied in virulence based on response to barley line Herta. Variation in spot-type isolates is reported for the first time from the region. The results from this study are being used in the development of resistant varieties.

Validation of a new spot form of net blotch differential set and evidence for hybridisation between the spot and net forms of net blotch in Australia

A recently developed spot form of blotch differential set of 16 barley lines was tested for reaction response to 60 Pyrenophora teres f. maculata isolates from geographically disperse barley crops of Australia. Twelve barley lines (Arimont, Barque, Chebec, CI5286, CI5791, CI9214, CII6150, Dairokkaku, Esperance Orge 289, Galleon, Keel, Skiff, Torrens and TR250) provided differential response between the isolates. The susceptible controls Gairdner and Kombar provided indication of isolate virulence or avirulence. Abundant pathogenic diversity was revealed with 33 designated pathotypes, some of which related to geographic region. AFLP analysis also revealed abundant diversity with each of the isolates representing a unique genotype and one isolate that contained both AFLP bands unique to P. teres f. maculata and P. teres f. teres, the cause of spot form and net form of net blotch respectively, suggesting that sexual recombination between the net form and spot form isolates may have occurred naturally in the field.

Resistance in cultivated barleys to Pyrenophora teres f. teres and prospects of its utilisation in marker identification and breeding

Net type net blotch (NTNB) is a prevalent disease in Australia, causing significant losses in barley yield and quality. Its impact can be reduced with the identification and utilisation of effective sources of resistance. Sixty-nine cultivated barley lines were screened as seedlings against 9 isolates of Pyrenophora teres f. teres from Australia, and in the field in Western Australia. Resistance expressed in seedlings was frequently expressed in adult plants in the field, indicating that these sources are potentially useful for resistance breeding. Of these lines, 24 with the best overall resistance were identified, which could be used against virulence diversity present in P. teres f. teres in Australia. As a prelude to the evaluation of established mapping populations in the Australian Barley Molecular Marker Program, 42 parental lines were screened against a range of Australian isolates of P. teres f. teres. Variation in net blotch responses was observed among parents of the mapping populations. Ten principal mapping populations appear to provide opportunities to map resistances and identify molecular markers linked to NTNB resistance genes effective against Australian pathotypes.

Characterisation and diversity of Pyrenophora teres f. maculata isolates in Western Australia

Spot type net blotch caused by Pyrenophora teres f. maculata (Ptm) has become a prominent disease in Western Australia, as has also occurred elsewhere. The disease has a negative impact on both grain yield and quality resulting from reduced grain size. Lack of resistance and stubble retention are the likely factors in the increased severity of the disease in barley growing areas of Western Australia. Because of the increasing importance of spot type net blotch and the need to improve barley resistance, understanding pathogen virulence is a high priority as this has direct impact on the identification and utilization of resistance genes in breeding programs. Ninety nine isolates of Ptm were collected from geographically dispersed barley fields of Western Australia during 2001 and 2002. Forty nine sporulating isolates of Ptm were classified into seven isolate groups (IGs) on the basis of their infection responses on 26 differential barley lines. The 26 lines were likewise classified into four line groups (LGs) based on their distinguishing response to the spot type net blotch isolates. The varied infection responses among the differential barley lines demonstrated a wide geographic dispersal of IGs as well as previously undetected virulence in Ptm in Western Australia. The commercially grown barley cultivars Baudin and Gairdner are regarded as susceptible to spot type net blotch, but showed a range of reactions to the various Ptm isolates as seedling plants. The variability in the pathogen and the resistance identified in some genotypes used in this study are being investigated further to develop superior, adapted germplasm for use in barley breeding programs in Australia.

Large Population with Low Marker Density Verse Small Population with High Marker Density for QTL Mapping: A Case Study for Mapping QTL Controlling Barley Net Blotch Resistance

The development of array-based high-throughput genotyping methods created significant opportunities to increase the number of genetic populations for linkage analysis. In the present study, a strategy was proposed for mapping QTLs (quantitative trait loci) based on DArT (diversity arrays technology) genotyping system. A consensus linkage map was constructed with both DArT and SSR markers by utilizing a subgroup DH population, and a second linkage map was constructed with SSR markers alone and a more extensive full DH population. Resistance to barley net-type net blotch disease was analyzed using the subpopulation data with the high-density consensus linkage map and the full-population data with the low-density SSR linkage map, respectively. Two interactive QTLs were detected either by the sub- or full population. Simulation studies were conducted to validate the strategy presented in this chapter. In addition, a computer program written in C++ is freely available on the web to deal with the data files. Based on both real data analysis and simulation studies, we concluded that high-density molecular markers, small population size, and precise phenotyping can improve the precision of mapping major-effect QTL and the efficiency of conducting QTL mapping experiment.

A major QTL controlling adult plant resistance for barley leaf rust

Race-specific resistance genes (Rph) for leaf rust (Puccinia hordei) are often overcome by new pathotypes with matching virulence. Adult plant resistance (APR) is considered potentially more durable for controlling barley leaf rust. Previous studies established that the cultivar Pompadour carried APR to leaf rust. A doubled haploid population (DH) of 200 lines developed from a cross Pompadour/Stirling, and the parents were phenotyped for leaf rust resistance at five field experimental sites in three agricultural zones in Australia. Using a linkage map of SSR and DArT molecular markers, a major QTL associated with the leaf rust resistance was identified on the short arm of chromosome 5H. This QTL explained between 31% and 86% of the phenotypic variation for the APR at different sites. A PCR-based molecular marker was developed and mapped at 1.6 cM to the APR gene. The present study provides new genetic material and a molecular tool for breeding new varieties with adult plant leaf rust resistance using marker-assisted selection.

A locus on barley chromosome 5H affects adult plant resistance to powdery mildew

Adult plant resistance against plant pathogens is of interest as a means to achieve durable resistance. Prior to this research, the barley lines CLE210 (from Uruguay) and Denar (from the Czech Republic) had been reported to exhibit adult-plant resistance against powdery mildew. Here, populations of doubled haploid lines from crosses of these lines with the susceptible cultivar Baudin were evaluated for powdery mildew resistance in field experiments. Using linkage maps constructed from genotyping-by-sequencing (GBS) data, it was determined that differences in resistance were largely attributable to a region on the long arm of chromosome 5H (5HL). Therefore, KASP™ assays were developed based on GBS tag sequences mapped on that chromosome, providing more reliable genetic maps. In each population, a large-effect QTL was mapped on 5HL. As no sequence variation was detected between CLE210 and Denar in this region of 5HL, the two sources of resistance may be identical by descent in the QTL region and carry the same resistance gene. Marker assays from the QTL region were evaluated on a panel of barley lines, providing information that breeders could use to select assays for use in marker-assisted selection.

Intravenous glucose utilization in marasmus

Glucose utilization coefficient was determined in 50 marasmic patients and 25 age matched healthy controls after administering glucose intravenously in three dosage schedules. The coefficient was directly related to the amount of glucose administered and the values were comparable in the marasmic and control subjects in their respectve schedules indicating normal glucose utilization in marasmus. Fasting blood glucose levels were significantly low in the patients compared to the controls. However, in only two patients fasting blood glucose value fell in the hypoglycemic range (less than 50 mg/dl).