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NEVIMOVÁ, H., BEDNÁŘ, J., VYHNÁNEK, T.: Polymorphism of microsatellite markers on chromosomes 3H and 7H in barley genotypes resistant and susceptible to Rhynchosporium secalis. Acta univ. agric. et silvic. Mendel. Brun., 2009, LVII, No. 2, pp. 69–78 The objective of the present study was to explore the polymorphism of microsatellite markers localised on chromosomes 3H and 7H in 15 genotypes of barley (Hordeum vulgare L.), spring form (2n = 2x = 14 chromosomes, genome HVHV) from the collection of genetic resources of the Agricultural Research Institute Kroměříž, Ltd. showing various degrees of susceptibility to Rhynchosporium secalis. The selection of SSR markers was based on hitherto achieved knowledge according to which the greatest amount of resistance genes against Rhynchosporium secalis is localised on chromosomes 3H and 7H of barley. We selected 33 SSR markers for the analyses; 17 were localised on chromosome 3H of barley and 16 on chromosome 7H. Out of the total 33 SSR markers, 32 were polymorphous and one marker (Bmac0282) was monomorphic. In total we detected 172 alleles ranging between 101 and 235 bp; the average number of alleles per locus was 5.21. In terms of the polymorphism of the SSR markers localised on chromosomes 3H and 7H the highest polymorphism (60%) was detected in the Bmag0006 and Bmag0021 SSR markers; the lowest in the Bmag0877 and EBmac0713 markers, i.e. 20% and 13.3%, respectively. The average polymorphism based on analyses of 17 SSR markers on chromosome 3H was 37.6% and of 16 SSR markers on chromosome 7H was 31.3%. We also calculated the statistical indicators of the variability rate characteristics of the individual microsatellite markers: diversity index (DI) which ranged between 0.000 and 0.907 (on average 0.704); polymorphous information content (PIC) ranging between 0.000 and 0.906 (on average 0.679); and probability identity (PI) ranging between 0.006 and 1.000 (on average 0.137). On the basis of constructed dendrograms for SSR markers of both chromosomes together it was possible to divide the analysed set into cluster I of genotypes resistant and cluster II of genotypes susceptible and moderately susceptible to Rhynchosporium secalis, and was not possible in dendrograms of individual chromosomes. polymorphism, microsatellite markers, resistance, Rhynchosporium secalis At present, various molecular techniques are used to study DNA polymorphism, e.g. AFLP, RAPD, RFLP, SSR etc. Microsatellite markers (SSR – Simple Sequence Repeats) belong to DNA markers which are o en used for genetic mapping and population studies thanks to their frequency, capacity to detect co-dominant alleles, the high level of polymorphism, broad dispersal in various genomes, simple execution of PCR reactions and easy repeatability among various laboratories (Liu et al., 1996). Last but not least they require a minimal amount of analysed DNA (Powell et al., 1996). Leaf blotch (scald) is a fungal disease caused by the facultative pathogen Rhynchosporium secalis (Oudem.) J. J. Davis. It was described for the fi rst time in the late 19th century (Häni et al., 1993) and is spread particularly in the northern parts of Central Europe. It invades a number of cereals, espe70 H. Nevimová, J. Bednář, T. Vyhnánek cially barley (spring and winter). In colder and more humid regions a commercially more important and regular incidence can be expected, although a more widespread occurrence was monitored also in higher altitudes inland. In the past decades it was reported that the disease appeared even in localities of lower altitude and drier climate (Minaříková and Míša, 1995). Infections usually appear at temperatures of 10–20 °C, at a low intensity of solar radiation and 95% air humidity (Beer, 1991). At present the importance of this disease is on the increase because the varieties of spring barley which are used particularly in the malt industry are more susceptible to leaf scald. Yield losses at a local level may reach 20% or even more. Patil et al. (2003) reported that the genes determining resistance to R. secalis are localised primarily on chromosome 3H (Rrs1) and chromosome 7H (Rrs2) of barley. On the basis of analyses of dihaploid lines and backcrossing of barley loci determining re sistan ce to R. secalis were detected on the long arm of chromosome 3H (Rrs1 syn. Rh), on the short arm of chromosome 7H (Rrs2) and chromosome 4H (Rrs3). Donors of other genes of resistance Rrs12, Rrs13 a Rrs14 which are localised on the short arms of chromosomes 7H, 6H and 1H were described in wild barley (Hordeum vulgare ssp. spontaneum) (Gronnerod et al., 2002; Jensen et al., 2002; Genger et al., 2003; Patil et al., 2003). Genger et al. (2003) quoted loci on chromosomes 3HL and 7HS as majority loci. The objective of the present study was to evalua te the variability of microsatellite markers lo ca lised on chromosomes 3H and 7H of barley in genetic resources which have a diff erent susceptibility to Rhynchosporium secalis. MATERIAL AND METHODS Detection of polymorphism of microsatellite markers was conducted in 15 genotypes of barley (Hordeum vulgare L.), spring form (2n = 2x = 14 chromosomes, genome HVHV) from the collection of genetic resources of the Agricultural Research Institute Kroměříž, Ltd. of diff erent susceptibility to Rhynchosporium secalis (Tab. I). A total of 33 SSR markers obtained from the GrainGenes database (Ramsay et al., 2000) and from literature (Liu et al., 1996; Li et al., 2003; Varshney et al., 2007) were selected for the analyses. The total genomic DNA was isolated by DNeasy Plant Mini Kit (Qiagen, GE) from the fi rst leaf (6–7 days old). The isolated DNA was quantifi ed spectrophotometrically by measuring the absorbance of DNA at 260 nm. The total volume of the PCR reaction mixture was 25 μl and contained 18.4 μl deionised H2O; 2.5 μl reaction buff er for polymerase Green GoTaq (Promega, USA); 1.0 μl of 100 mM dNTPs mixture (Promega, USA); 1.0 μl forward primer; 1.0 μl reverse primer; 0.1 μl of thermo-stable Taq DNA po ly mera se – 5 U (Promega, USA); and 1.0 μl template DNA. The PCR was conducted in the T3 Thermocycler combi (Biometra, GE) where the PCR program consisted of initial denaturation for 2 min at 93 °C, followed by 30 cycles of 1 min at 93 °C, 2 min at 54 °C, 2 min at 72 °C. The resulting products of the PCR reaction were separated electrophoretically on hori zon tal electrophoresis at 70 V on 1% aga ro se gels stained with ethidium bromide. Positive samples were separated on vertical electrophoresis at 300 V on 10% non-denaturation polyacrylamide gels in a TBE buff er stained with silver nitrate (0.2% AgNO3) were selected. Polymorphic DNA products were scored as presence (1) or absence (0) of an allele and used to construct a binary matrix. The genetic si mila ri ty matrix of all genotypes was analysed using unweighted pair group method with arithmetic average (UPGMA) algorithm performed using FreeTree version 9.1 (Jaccard similarity coeffi cient); by means of TreeView version 1.6 dendrograms were drawn illustrating the genetic similarity of the tested genotypes. Further three statistical characteristics were calculated – the diversity index (DI), the pro ba bi li ty identity (PI) and the polymorphous information content (PIC) according to Russell et al. (1997). The polymorphism value (%) was calculated as the ratio of alleles detected for a microsatellite marker and the number of analysed genotypes. RESULTS AND DISCUSSION Out of the 33 SSR markers 32 were polymorphous and one was monomorphic (Bmac0282 – 7H). 1–9 alleles per locus were detected. Altogether 172 alleles, i.e. on average 5.21 alleles per 1 locus were detected. The size of the amplifi ed products (alleles) ranged between 101 bp and 235 bp. Hamza et al. (2004) used 17 microsatellite markers in 26 barley varieties and they discovered that the average number of alleles per locus was 3.6; a value lower than the results of the present study. To the contrary Struss and Plieske (1998) detected 130 alleles in 163 barley genotypes using 15 SSR markers; they found 5 to 14 alleles per locus, averaging 8.60 alleles per locus. Leišová et al. (2007) studied the genetic variability in 176 barley genotypes using 26 SSR markers and they detected as many as 328 alleles averaging 12.6 alleles per locus. It is apparent that the total number of alleles and the average number per locus is dependent on how extensive is the collection of genotypes to be analysed, including the diversity of their origin, and also on the number of the SSR markers. On chromosome 3H the polymorphism of 17 microsatellite markers and on chromosome 7H of 16 SSR markers were explored. Graph 1 shows the polymorphism detected in SSR markers lo ca lised on chromosome 3H. The SSR marker Bmag0006 showed the highest degree of polymorphism (60%) and marker Bmag0877 the lowest (20%). The average degree of polymorphism determined on the basis of analyses of 17 SSR markers on chromosome 3H was 37.6%. Polymorphism of microsatellite markers on chromosomes 3h and 7h in barley genotypes 71 I: Characteristics of the barley genotypes Genotype taxon country of origin * Pedigree resistance genes susceptibility to Rhynchosporium secalis Abyssinian Hordeum vulgare L. subsp. distichon (L.) KOERN. var. nutans SCHUEBL ETH SCI 668 Rh susceptible Atlas Hordeum vulgare L. subsp. vulgare var. hybernum VIL USA SCoast Rh2 resistant Atlas 46 Hordeum vulgare L. subsp. vulgare var. hybernum VIL. USA Hanna/Atlas Rh, Rh2, Rh3 resistant Atlas 57 Hordeum vulgare L. subsp. vulgare var. hybernum VIL. USA Lion/Atlas Rh2 resistant Bay Hordeum vulgare L. subsp. vulgare var. hybernum VIL. USA Minnesota 450/Spartan Rh3 resistant Cambrinus Hordeum vulgare L. subsp. distichon (L.) KOERN. var. nutans SCHUEBL. NED Balder/Streg Franken III not described susceptible Clipper Hordeum vulgare L. subsp. distichon (L.) KOERN. var. nutans SCHUEBL. AUS Prior/Proctor not described moderately susceptible Jet Hordeum vulgare L. subsp