INTRODUCTION

Leaf scald, caused by the fungal pathogen Rhynchosporium secalis, is an important disease of barley in many regions of the world. Thirteen major resistance genes to R. secalis have been identified in barley (Jørgensen 1993; Abbott et al. 1992), but only five of them have been mapped to a specific chromosome. Dyck and Schaller (1961) identified five dominant genes (Rh2, Rh3, Rh4, Rh4², and Rh5) conferring resistance to the leaf scald pathogen and showed that Rh3 and Rh4 were closely linked to each other on the short arm of chromosome 3. The Rh (Rrs1) gene also was mapped to chromosome 3 and is regarded as part of a complex of closely linked resistance genes (Rrs1-Rrs3-Rrs4) (Habgood and Hayes 1971). Recently, RFLP markers were used to map Rh2 (Rrs2) to chromosome 1 (Schweizer et al. 1995) and Rrs13 to chromosome 6 (Abbott et al. 1995).

The Q21861/SM89010 doubled haploid population is polymorphic in response to a number of important barley pathogens including Puccinia graminis f. sp. tritici (wheat stem rust pathogen), P. g. f. sp. secalis (rye stem rust pathogen), P. hordei (leaf rust pathogen), and Blumeria graminisf. sp. tritici (powdery mildew pathogen) (Steffenson et al. 1995). It is also polymorphic in response to the leaf scald pathogen with parent SM89010 contributing the resistance. Genes for rust resistance have already been mapped in this population (Borovkova et al. 1995; unpublished). In this study, we used bulked segregant analysis to identify RAPD markers linked to the scald resistance gene.

MATERIALS AND METHODS

Doubled haploid (DH) progeny (126 total) from the Q218961/SM89010 population (Steffenson et al. 1995) were grown in a greenhouse (18±3°C) and then inoculated with isolate ND94L of R. secalis at the two leaf stage. This isolate exhibits low virulence on genotypes Atlas, Atlas 46, Brier, CI 2376, CI 5831, Kitchin, La Mesita, Modoc, Osiris, Trebi, Turk, and WWxGlabron and high virulence on CI 2266 and Wong. Inoculum (concentration of 4.5x10⁴ conidia/ml) was applied with an atomizer at a rate of 0.10 ml per plant. After inoculation, plants were placed in chambers without light at 15°C for 48 hours. The leaves of plants were kept saturated during this period by intermittent mistings from ultrasonic humidifiers. After the infection period, plants were incubated in a growth chamber at 15-18°C with a 13 hour photoperiod. Infection

responses were assessed on the second leaves of plants 14-16 days after inoculation using the rating scale of Jackson and Webster (1976).

DNA preparation procedures and PCR conditions were as described previously (Borovkova et al. 1995). Decamer primers used in the study were synthesized by Operon Technologies Inc., (Alameda, CA) and UBC (University of British Columbia). Bulked segregant analysis (Michelmore et al. 1991) was used to identify RAPD markers linked to the scald resistance gene. Polymorphic markers were analyzed on the entire population and linkage analysis was performed with MAPMAKER (Lander et al. 1987). Sequence Tagged Sites (STS) primers were kindly provided by Dr. Thomas Blake at Montana State University. Sequences of these primers are listed in the Graingenes database. Amplification protocols for these primers were the same as those described by Tragoonrung et al. (1992), except for BCD828 where the annealing temperature was increased to 61°C and number of cycles to 40. Restriction with HaeIII, HhaI, HinfI (Promega, Madison, WI) was performed according to the manufacturer's instructions.

RESULTS AND DISCUSSION

Two distinct reaction classes were observed in the DH population in response to isolate ND94L of R. secalis: one comprised of progeny exhibiting infection response 0 (occasionally 1) and the second of progeny exhibiting infection responses 3 or 4. The number of resistant (45) and susceptible progeny (81) observed did not fit either a one ((²=10.286; P=0.001) or two gene ((²=7.711; P=0.006) segregation ratio from the chi-square analysis. Segregation distortion in favor of Q21861 alleles has been previously reported for leaf rust resistance and the s locus (controlling rachilla hair length) in this population (Steffenson et al. 1995). The number of homozygous resistant (15), segregating (28), and homozygous susceptible (12) F_2:3 families from another Q21861/SM89010 population closely followed a 1:2:1 ratio ((²=0.345; P=0.841), indicating that resistance was conferred by a single gene. Skewed segregation has been reported for both RFLP and RAPD markers. In the case of RAPD markers, those displaying skewed segregation coincided with the skewed segregation of neighboring loci (Giese et al. 1994). The same was true for RAPD markers linked with the resistance locus in this study. This distorted segregation is not expected to affect the recombination values estimated between the loci (Kjær et al. 1995).

Since the exact source of the scald resistance gene and its possible chromosomal location were not known, RAPD analysis of bulks and parents was initiated with primers that were reported to be linked to the leaf scald resistance gene in the Blenheim/E224 population (Barua et al. 1993). We evaluated 8 of 11 primers (OPD-08, OPO-06, OPO-16, OPR-03, OPH-07, OPH-16, OPI-10, and OPJ-07) that were reported by Barua et al. (1993) to generate markers on chromosome 3. All of these primers either did not amplify fragments of the reported size in the Q21861/SM89010 population or produced markers that were not polymorphic. OPO-16 did, however, generate a 1.0 kb fragment (instead of a 0.9 kb fragment as reported for the Bleinheim/E224 cross), which was polymorphic in the Q21861/SM89010 population. This marker appeared to be linked to the resistance gene at a distance of 11.4 cM, which is closer than the 16.6 cM distance reported for OPO-16(900) in the Blenheim/E224 population.

Further analysis of additional 10-mer primers revealed three other RAPD markers linked to the scald resistance gene. The closest one identified was OPK-01(750) with a linkage distance of 6.9 cM. This marker also was polymorphic in the Steptoe/Morex population. PCR reactions were performed with the OPK-01 primers on the entire Steptoe/Morex population, and from this analysis, a linkage (8.3 cM) was detected between OPK-01(750) and ABG462, an RFLP marker located on chromosome 3 (Kleinhofs et al. 1993). These data suggested a possible chromosome 3 location for the resistance gene in SM89010. No other RAPD markers analyzed were polymorphic in the Steptoe/Morex population; therefore, the orientation of the other markers on the chromosome could not be determined. To further corroborate the putative chromosome 3 location of resistance gene, we evaluated four STS primers (ABC156, ABG396, ABC471, and BCD828) designed from RFLP clones that generated markers near the centromere of chromosome 3 in the Steptoe/Morex population. The first three pairs of STS primers did not produce polymorphic fragments after amplification and restriction with HaeIII, HhaI or HinfI. However, the BCD828 primers generated a polymorphic fragment in both the parents and the bulks. This result also suggested a chromosome 3 location for the resistance locus, possibly near the centromeric region. The relation of the resistance gene in SM89010 to ones previously reported on chromosome 3 is not known. Additional genetic crosses among the sources of Rrsloci mapping to this chromosome should be made to resolve this question.

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