ACentre for Plant Biodiversity Research, CSIRO Plant Industry, GPO Box 1600, Canberra, ACT, 2601, Australia
BCSIRO Division of Marine Research, GPO Box 1538, Hobart TAS 7001, Australia
Scald is a foliar disease of barley that occurs throughout southern Australia, causing annual yield losses of 10-15%. Individual losses can be as high as 45% (Brown, 1985). Scald also reduces grain malting quality due to reduced size and plumpness (Khan and Crosbie, 1988). Rhynchosporium secalis, the fungal pathogen that causes scald, is highly variable, rapidly overcoming single resistance genes. Only a small number of disease resistance genes are available, and historically the durability of resistance to scald is low.
The wild progenitor of barley, Hordeum spontaneum, is a rich source of genes for scald resistance. Deployment of these genes into commercial cultivars will be accelerated by the use of linked molecular markers, which will be required to recognise the presence of multiple resistance genes if the genes are pyramided. We have previously used Hordeum spontaneum as the non-recurrent parent to generate scald resistant third backcross (BC3) lines in the susceptible barley cultivar Clipper. Here we report progress in developing markers for resistance genes in BC3 lines 240 and 245, using the AFLP technique.
Third backcross lines were generated from crosses between a range of H. spontaneum accessions and the scald susceptible cultivar Clipper as previously described (Brown et al., 1988; Garvin et al., 1997). Selected BC3 individuals were selfed to produce a segregating BC3F2 population. For both BC3 lines 240 and 245, single leaves were collected from each of 60 BC3F2 plants and DNA was extracted.
Seedling disease testing
Sixty BC3F2 plants each for lines 240 and 245 were tested for their reaction to scald isolate 109.1.1 using the seedling assay described in Abbott et al. (1991). BC3F3 seed was collected from these plants. The scald reaction of these BC3F3 plants was tested using the same seedling assay. To distinguish homozygous and heterozygous scald resistant BC3F2 individuals, twelve progeny were tested. Four progeny were tested for each scald susceptible BC3F2 individual, to confirm susceptibility.
Bulked segregant analysis
Two pools of DNA were created for each line, one from ten plants homozygous for resistance to scald, and one from ten plants homozygous for susceptibility to scald. These two pools, along with DNA of the recurrent parent Clipper, were screened using a set of 64 AFLP markers (Life Technologies AFLP Analysis System I) to identify bands polymorphic between the resistant and susceptible bulked DNA. The restriction enzymes used in preparation of the AFLP templates were EcoRI and MseI.
Polymorphisms identified between resistant and susceptible bulks were scored on the ten resistant and ten susceptible plants used to make the bulks, giving a preliminary assessment of linkage. Potentially linked polymorphisms were then scored on the remaining BC3F2 individuals, totalling 60 BC3F2 individuals for line 240 and 57 BC3F2 individuals for line 245. The Mapmaker program (version 3.0) was used to assess the strength of linkage between the resistance loci and the polymorphic bands.
Cloning and characterization
Polymorphic bands were excised, eluted and reamplified using the same AFLP primer combination. Fragments were cloned using a TA cloning kit (Promega), and at least 10 separate clones were sequenced (Big Dye, Perkin Elmer) on an ABI Prism Model 377 DNA sequencer. Several different clones were obtained for each AFLP band. Primers immediately internal to the AFLP primers were designed for cloned DNA for each band, and used for PCR on resistant and susceptible individuals.
A large number of polymorphisms (more than 100 for each line) was observed between the resistant and susceptible bulks for both lines 240 and 245 using a set of 64 AFLP primers. A subset of these polymorphisms was scored on BC3F2 individuals.
Twenty-six polymorphisms resulting from 8 AFLP primer combinations were scored on a set of 16 BC3F2 individuals, and mapped relative to the resistance gene in line. Six of the polymorphisms mapped with the resistance gene. Further linkage analysis was performed for 7 of the 26 polymorphisms. A total of 60 BC3F2 individuals were scored for these 7 polymorphisms. A cluster of five polymorphisms mapped close to the line 240 resistance gene (Figure 1). Of these 5 linked polymorphic bands, 2 segregated with resistance and 3 with susceptibility (Figure 1).
Figure 1: Linkage map of 7 AFLP polymorphisms scored on 60 line 240 BC3F2 individuals
No. of fragments cloned
Fragment sizes (bp)
249; 250; 251; 251; 253
163; 163; 150*
244; 245; 248
Table 1: Cloned fragments identified for 4 polymorphic bands linked to the line 240 resistance gene; * probable contaminant; ** one MseI-MseI fragment; discarded
Polymorphic AFLP bands of approximately 150 bp and above were targeted for analysis. Of the cluster of 5 polymorphic bands linked to the resistance gene, the AFLP band for aaccaa4 was 105 bp long, and analysis of this band was discontinued. The remaining four polymorphic bands were cloned, and at least 10 separate clones for each band were sequenced. For each band, more than one fragment was obtained. Fragments differed in sequence and in size (Table One).
To identify the correct DNA fragment from the several cloned for each band, primers were designed to the ends of several of the sequenced fragments for each band. PCR on DNA from resistant and susceptible individuals showed no polymorphisms for the fragments analysed. This suggested that the polymorphisms between resistant and susceptible individuals may occur in the selective nucleotides at the extreme ends of the fragments. To test this, PCR was performed using one primer of each pair, with the reciprocal AFLP primer, using preamplified resistant and susceptible bulked DNA as a template. For one primer set each for bands aaccaa3, aaccac5 and aaccac8, polymorphisms were seen between resistant and susceptible bulks (Figure Two), suggesting that the correct fragment has been cloned for these bands and that the polymorphism occurs in the extreme ends of the cloned fragments.
Figure 2: Polymorphisms between resistant and susceptible preamplified bulks detected by PCR using one primer internal to the AFLP band and the reciprocal AFLP primer (R – line 240 resistant bulk; S – line 240 susceptible bulk; C – Clipper)
Polymorphisms from 22 AFLP primer combinations were scored on a set of 20 BC3F2 individuals. Eight polymorphisms from 5 primer combinations were mapped relative to the line 245 resistance gene, providing preliminary linkage estimates using Mapmaker version 3.0. Polymorphisms from the other 17 primer combinations proved to be unlinked to the resistance gene.
Two AFLP primer combinations were scored on a further 37 BC3F2 individuals. Two polymorphic bands from the first primer combination were loosely linked to the line 245 resistance gene (data not shown), but the polymorphic bands from the second primer combination were unlinked.
Bulk segregant analysis using the AFLP technique identified large numbers of polymorphisms between resistant and susceptible bulks for both lines 240 and 245. Although many of these polymorphisms proved not to be linked to resistance, five polymorphisms closely linked to the line 240 resistance gene, and two polymorphisms loosely linked to the line 245 resistance gene, have been identified.
Four markers are being developed for line 245. Two of these are linked to the resistance phenotype, and two to the susceptible phenotype. Thus, even if the polymorphisms cannot be individually developed as codominant markers, use of paired resistance-linked and susceptible-linked markers may provide a codominant system. Three polymorphisms map close together on one side of the resistance locus, while the other flanks the resistance locus. Flanking markers will provide a more accurate system for marking the resistance gene than a single marker.
Preliminary evidence indicates that the polymorphisms between resistant and susceptible individuals occur in the 3 selective nucleotides at the extreme end of the fragment. If this proves to be the case, it will be necessary to isolate larger fragments of DNA containing the polymorphic sequences, in order to design primers which will distinguish them. As no closely linked polymorphisms have yet been identified for line 245, analysis of polymorphisms identified in bulk segregant analysis will continue for this line.
Judy Cassells provided excellent technical assistance. This research was supported by the GRDC, Australian Maltsters and Brewers, and CSIRO, through the Malting Barley Improvement Program.
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