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Mapping genes for resistiance to net form of net blotch and stripe rust in barley (HordeumvulgareL.)

Mehmet Cakir1, Nick Galwey1, David Poulsen2, Greg Platz2, Paul Johnston2, Garry Ablett3, Colin Wellings4 and Hugo Vivar5

1Plant Sciences, University of Western Australia, Nedlands, WA 6907
2
Queensland Department of Primary Industries, Hermitage Research Station, Warwick, QLD 4370
3
Center for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480
4
Plant Breeding Institute Cobbitty, University of Sydney, Camden, NSW 2570
5
ICARDA/CIMMYT, Apdo 370, Mexico 6, D.F. Mexico

Abstract

A dihaploid mapping population comprising 65 lines was developed between barley parent varieties Tallon and Kaputar and used to construct a genetic linkage map. This map, comprising 195 amplified fragment length polymorphism and 38 simple sequence repeat markers, was used to identify markers linked to the net form of net blotch (Pyrenophora teres f.sp. teres) and to stripe rust (Puccinia striiformis f.sp. hordei) in barley. The population was screened with five pathotypes of net blotch at the seedling stage in the glasshouse and subjected to a natural inoculation in Hermitage, Queensland. Stripe rust screening was conducted at the adult plant stage in Toluca, Mexico. Analyses of the markers were performed using Mapmanager and Qgene software. One region on chromosome 6H was highly significantly associated with resistance to the net blotch (R2 = 79%). This association was consistent for all pathotypes studied. One region on chromosome 5H was found to be highly significantly associated with resistance to stripe rust (R2= 65%). There are a number of very closely linked markers showing strong associations in these regions, and these markers present an opportunity for marker assisted selection of these traits in barley breeding programs.

Introduction

The net form of net blotch on barley, caused by Pyrenophora teres f.sp. teres, is a major disease in most barley growing areas around the world. Numerous studies have been conducted to explore the inheritance of this disease. In particular two dominant resistance genes have been identified in Australia (Khan and Boyd, 1969), two complementary genes in Sweden (Jonsson et al., 1999), and one dominant gene in Canada and Egypt (Buchannon and McDonald, 1965). Inheritance studies suggested that P. teres is a highly heterogenous fungus (Afanasenko et al., 2000). Reduced tillage farming systems have been reported to cause a great increase in the incidence of net blotch in Australia, with yield losses over 30% (Platz et. al., 2001). Therefore one of the major objectives of Australian barley breeding programs is to increase the resistance to this disease in locally-adapted barley varieties.

Several strains of stripe rust fungus (P. striiformis) have been detected over the last 20 years. Race 24, used in this study, belongs to the type specialised as a pathogen of barley (P. striiformis f.sp. hordei), which has not been detected in Australia. However, this pathogen caused significant damage in South America when first introduced there from Europe in 1975 (Dubin and Stubbs, 1986) and subsequently spread to North America in 1991 (Marshall and Sutton, 1995). Australian barleys tested at CIMMYT, Mexico, have proved to be generally susceptible to barley stripe rust. A research program funded by GRDC has recently been expanded to allow testing of breeding populations in an attempt to select for resistance in advance of the anticipated arrival of the barley form of stripe rust in Australia.

Molecular markers have been used as an aid during the selection of superior lines in crop breeding programmes. The use of markers is cost effective for the selection of traits that are difficult, time consuming and expensive to select for during tradational plant breeding schemes. Several studies have been conducted to identify markers linked to resistance to the net form of net blotch and resistance to stripe rust (Spaner et al., 1998; Richter et al., 1998; Chen et al., 1994; Toojinda et al., 2000) . The objective of this study is to identify markers closely liked to resistance to these diseases and make them available to Australian barley breeding programs.

Materials and Methods

Plant material

The barley parent varieties Tallon and Kaputar were used to construct a DH population using the anther culture technique. The population comprises 65 lines. Tallon is a malting barley, bred in and adapted to the Northern Region of Australia. Kaputar is a feed barley, bred by CIMMYT and released for the Northern Region.

Phenotyping

Net-type of Net Blotch. The DH lines and the parents were grown in replicated trials in pots in a glasshouse. Each pot contained 10 plants. The plants were inoculated with four strains prevalent in Queensland and one strain prevalent in South Australia.

In order to prepare the inoculum, single conidial isolates of each strain were increased on Peanut Oatmeal agar (Speakman and Pommer 1986) at 19C. After nine days in culture conidia were washed from the agar surface, filtered through a 330m strainer and made up into an aqueous suspension containing 12,500 conidia mL-1. Approximately 1.125mL of this suspension was applied per pot with a Krebs airless paint sprayer (Oldfields Pty. Limited) when plants were at an average growth stage of 13.5 (Zadoks et al 1974). Inoculated plants were immediately placed in a fogging chamber and maintained at 100% relative humidity for 24 hours (14D; 10L) at 19C, then returned to the glasshouse for disease development. Notes on infection type (IT) were taken 9 days after inoculation using the scale of Tekauz (1985). Reaction type was scored using a 0-9 scale (0=resistant, 9 susceptible).

Stripe Rust. The DH lines and parents were assessed for adult plant resistance in Toluca, Mexico, 2600 meters above sea level, in 1999 and 2000. They were planted in unreplicated double rows 1 m in length. A field epidemic was initiated by inoculating spreader rows (formed from a mixture of 15 extremely susceptible genotypes) with a stripe rust isolate whose virulence pattern corresponds to the race 24 Varunda-Mazurka type described by Dubin and Stubbs (1986). Severity of stripe rust was rated at DGS59 (Feekes stage 10.5) as a percentage on a plot basis. Reaction type was scored using a 0-9 scale (0=resistant, 9 susceptible). Mapping was performed based on both severity and infection type data, and also on a combined measure of severity and infection type. This was obtained by multiplying the severity value by a value related to the infection type, namely 0.2 (resistant), 0.4 (moderately resistant), 0.6 (moderately susceptible) or 1.0 (susceptible).

Marker analysis

The genetic map with 232 DNA markers was used to identify marker loci associated with these disease traits. QTL analyses were performed using Mapmanager (Manly, 2001) and Qgene (Nelson, 1997) software. A threshold LOD (logarithm of odds ratio) score of 3.0 was chosen for declaring the existence of a QTL. Wherever appropriate simple regression and interval mapping analysis were used to find the associations. Separate analysis for each strain or site and a joint analysis over all strains/sites were performed for each trait.

Results and Discussion

The parent lines differed widely in their resistance to strains NB54 and NB52B of net blotch and in their resistance to stripe rust. The DH population showed wide segregation for resistance to all strains of net blotch, and for resistance to stripe rust in both years. In the case of net blotch strains NB50, NB81 and NB97 there appeared to be transgressive segregation – that is, some DH lines lay substantially outside the range of the parents (Table 1).

Table 1. Summary statistics for resistance to stripe rust and the net form of net blotch in a doubled-haploid population of barley

Character

Parental Means

DH Line Means

 

Tallon

Kaputar

Min

Max

Mean

Net blotch strain

         

NB50

8.5

6.5

5.5

10.0

8.5

NB81

3.0

3.5

1.5

6.5

3.3

NB97

7.0

5.0

2.5

9.5

5.8

NB54

9.0

5.0

3.0

10.0

6.9

NB52B

8.0

4.0

2.5

10.0

6.1

Stripe rust

         

1999 season

10.0

80.0

1.0

100.0

45.7

2000 season

10.0

80.0

1.0

90.0

50.9

Markers were found to be linked to both disease resistance traits. In particular, one region on chromosome 6H was highly significantly associated with resistance to the net form of net blotch (R2 = 79%) (Fig 1). This association was consistent for the five net blotch strains, and offers a good opportunity to Australian plant breeders for the implementation of marker assisted selection for resistance to net blotch. One region on chromosome 5H was found to be highly significantly associated with resistance to stripe rust (R2= 65%). The close linkage of the markers in this region with resistance to disease was consistent in the two years (Fig 2).

Fig 1. Chromosomal locations of the major genes for resistance to the net form of net blotch. Each contour represents a strain of net blotch fungus.

Fig 2. Location of the major gene for resistance to stripe rust chromosome on 5H. Each contour represents data from an individual year (1999 or 2000).

Conclusions

DNA markers are being used as tools in marker assisted selection of barley in breeding programs throughout Australia. The current project has identified a number of markers that are associated with resistance to the net form of net blotch and to stripe rust. The regions of the chromosomes in which these markers are located will be focal points of further research for validation and implementation of the markers for routine selection in breeding programs. The markers located in these regions could also be used in pedigree-based association mapping studies using diverse barley genetic resources. This process will allow the identification of markers associated with these disease resistance traits in different genetic backgrounds.

Acknowledgements

Funding for this research is provided by GRDC through the National Barley Molecular Marker Programme. We are grateful to the Western Australian State Agricultural Biotechnology Center for providing laboratory facilities and the Scottish Crop Research Institute for proving the SSR markers.

References

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