| | Introduction | Results and discussion | References
Introduction
Septoria speckled leaf blotch (SSLB) caused by the pathogen Septoria passerinii is a common and important leaf disease in barley in the Upper Midwest and adjacent Canadian provinces. The disease was severe in the 1950s in the north-central region of the United States and Prairie Provinces of Canada (Buchannon 1961; Green and Dickson 1957), with yield losses of 23-38% reported in Canada (Green and Bendelow, 1961). Recent yield losses due to SSLB reported by Toubia-Rahme and Steffenson (1999) are similar to those previously reported in Canada. The importance of SSLB is increased further due to its effects on grain quality such as reduced kernel size and malting quality (Green and Bendelow, 1961). All of the major malting and feed barley cultivars in the Upper Midwest region are susceptible to this pathogen. Development of resistant cultivars is the preferred method to prevent SSLB epidemics. Thus, it is necessary to map the genes controlling the resistance and develop molecular markers for use in screening breeding lines.
To date, three SSLB resistance loci designated Rsp1, Rsp2, and Rsp3 have been identified in CIho14300, CIho4780, and CIho10644 respectively (Rasmusson and Rogers, 1963). The genes, Rsp2 and Rsp3, are closely linked with about 3.8% recombination (Rasmusson and Rogers, 1963). The molecular mapping work for resistance genes has been published on Rsp2 and Rsp3 (Zhong et al. 2002). Two AFLP markers linked to Rsp2 were developed and mapped on the short arm of chromosome 1H(5). Information on map location and molecular markers for Rsp1 is still lacking. In addition, the AFLP technique has limited use in marker assisted selection (MAS) because it is laborious, time consuming, expensive, and technically difficult (Neil et al., 1997). Thus, the aim of this research was to identify randomly amplified polymorphic DNA (RAPD) markers linked to Rsp genes and convert them into sequence tagged site (STS) markers so that marker assisted selection (MAS) can be used to develop SSLB resistant cultivars in barley.
Results and Discussion
We developed six mapping populations by crossing the susceptible cultivars, Robust and Foster, with the resistant lines, CIho14300 (Rsp1), CIho4780 (Rsp2), and CIho10644 (Rsp3). Robust comes from the Minnesota barley breeding program and its pedigree is Morex x Manker and Foster comes from the North Dakota breeding program and its pedigree is Robust/6/Glenn/4/ Nordic//Dickson/Trophy/3/Azure/5/Glenn/Karl. F1 plants were selfed to obtain between 100 and 120 F2 plants. SSLB phenotypes were evaluated in F2 plants and F2.3 families at the seedling and adult stage in the greenhouse and in the field. Segregation analysis for Rsp1, Rsp2, and Rsp3 genes in two F2 populations of seedlings in the greenhouse showed an approximate segregation ratio of 3 resistant:1 susceptible in both genetic backgrounds (Table 1). This was confirmed in analysis of the F2:3 families from Robust x CIho 14300, Robust x CIho 4780, and Robust x CIho 10644 grown in the greenhouse and field at Langdon and Osnabrock, ND (Table 2).
Table 1. Segregation analysis for Septoria Speckled Leaf Blotch resistance genes Rsp1, Rsp2, and Rsp3 in F2 populations derived from two susceptible cultivars (Robust and Foster) and three resistant lines (CIho14300, CIho4780, CIho10644).
Cross
Female (S) x Male (R) | Genes | Resistant plants | Susceptible plants | Expected ratio | X2 | Pa |
| Foster x CIho 14300 | Rsp1 | 93 | 25 | 3 : 1 | 0.92 | 0.34 |
| Robust x CIho 14300 | Rsp1 | 74 | 29 | 3 : 1 | 0.55 | 0.46 |
| Foster x CIho 4780 | Rsp2 | 93 | 27 | 3 : 1 | 0.40 | 0.53 |
| Robust x CIho 4780 | Rsp2 | 85 | 23 | 3 : 1 | 0.79 | 0.37 |
| Foster x CIho 10644 | Rsp3 | 82 | 32 | 3 : 1 | 0.57 | 0.45 |
| Robust x CIho 10644 | Rsp3 | 83 | 32 | 3 : 1 | 0.49 | 0.48 |
a P < 0.05 = significant deviation from the expected segregation ratio, P > 0.05 = fit to the expected segregation ratio of the F2 population
Table 2. Segregation analysis for Rsp1, 2, and 3 genes in F2:3 families derived from Robust × CIho14300, Robust × CIho4780, and Robust × CIho 10644.
| Cross | Location | Plant stages | RRb | Rr | rr | Expected ratio | X2 | Pc |
| Robust x CIho14300 | Langdona | Adult | 18 | 44 | 28 | 1:2:1 | 2.27 | 0.32 |
| Osnabrocka | Adult | 19 | 46 | 25 | 1:2:1 | 0.84 | 0.66 |
| Greenhouse | Seedling | 18 | 50 | 21 | 1:2:1 | 1.56 | 0.46 |
| Robust x CIho4780 | Langdona | Adult | 25 | 49 | 26 | 1:2:1 | 0.06 | 0.97 |
| Osnabrocka | Adult | 31 | 51 | 18 | 1:2:1 | 3.42 | 0.18 |
| Greenhouse | Seedling | 28 | 57 | 27 | 1:2:1 | 0.05 | 0.97 |
| Robust x CIho10644 | Langdona | Adult | 25 | 53 | 27 | 1:2:1 | 0.09 | 0.96 |
| Osnabrocka | Adult | 21 | 51 | 33 | 1:2:1 | 2.83 | 0.24 |
| Greenhouse | Seedling | 21 | 52 | 27 | 1:2:1 | 0.88 | 0.64 |
a Name of field location in ND, USA
b RR: Homozygous resistant, Rr: Heterozygous resistant, rr: Homozygous susceptible.
c P < 0.05 = significant deviation from the expected segregation ratio, P > 0.05 = fit to the expected segregation ratio of the F2:3 population
To develop molecular markers linked to Rsp genes, 480 10-mer RAPD primers (200 from University of British Columbia, and 280 from Operon Technologies Inc.) were used to screen polymorphisms between the two different DNA bulks from resistant and susceptible F2 plants, and between two susceptible parents and three resistant parents. The RAPD primers that showed a specific polymorphism between a resistant and susceptible bulk were selected to determine the genetic linkage between SSLB resistance genes and markers. Three RAPD markers, UBC285158R, OPAH5545C, and OPBA12314C, associated with Rsp genes, were identified using bulked segregant analysis in populations of 100-120 F2 individuals. Linkage analysis revealed RAPD markers UBC285158 (3.8±1.1cM) in repulsion linked to Rsp1, RAPD marker OPAH5545C (0.9±1.3) in coupling linked to Rsp2, and RAPD marker OPBA12314 (2.4cM) in coupling linked to Rsp3. A repulsion phase of dominant marker, UBC285158R, for Rsp1 and two coupling phase of dominant markers, OPAH5545C for Rsp2 and OPBA12314C for Rsp3, showed the expected segregation ratio 1 resistant:3 susceptible and 3 resistant:1 susceptible in F2 plants (Table 3).
For high reproducibility and ease to use, RAPD markers associated with Rsp genes were converted into sequence-tagged site (STS) markers, Rsp1158R, Rsp2545C, and Rsp3314C, (Figure 1).
Table 3. Chi-square test of segregation ratios with RAPD markers linked to Rsp genes in F2 populations derived from two susceptible cultivars (Robust and Foster) and three resistant lines (CIho14300, CIho4780, CIho10644).
Cross
Female(S) x Male(R) | Genes |  | RRa | Rr | rr | Expected segregation | X2 | Pb | Geneticc distance (cM) |
| Robust x CIho14300 | Rsp1 | UBC285158R | 22 | 81 | | 25.75:77.25 | 0.55 | 0.46 | 3 |
| Foster x CIho14300 | Rsp1 | UBC285158R | 25 | 91 | | 29:87 | 0.74 | 0.39 | 4.5 |
| Robust x CIho4780 | Rsp2 | OPAH5545C | 75 | | 25 | 75:25 | 0 | 1 | 0 |
| Foster x CIho4780 | Rsp2 | OPAH5545C | 93 | | 27 | 90:30 | 0.28 | 0.6 | 1.8 |
| Robust x CIho10644 | Rsp3 | OPBA12314C | 84 | | 29 | 84.75:28.25 | 0.03 | 0.87 | 2.4 |
a RR: Homozygous resistant, Rr: Heterozygous resistant, rr: Homozygous susceptible.
b P < 0.05 = significant deviation from the expected segregation ratio, P > 0.05 = fit to the expected segregation ratio of the F2 population
c Genetic distances (cM) were analysed by MAPMAKER, LOD>3.0.
Figure 1. Identification of polymorphisms associated with SSLB resistance genes, Rsp2 and Rsp3, using RAPD markers (A) OPBA12314C and (C) UBC285158R. The RAPD markers were converted into sequence tagged site (STS) markers, (B) Rsp3314C and (C) Rsp1158R. Lanes are as follows: (A), (B), and (D) = M, DNA size markers in bp; 1, resistant parent CIho10644 (A and D) and CIho14300 (D); 2, susceptible parent Robust; 3, susceptible parent Foster; 4, F1 plant of Robust×CIho10644 (A and B) and Robust×CIho14300 (C); 5, F1 plant of Foster×CIho10644 (A and B) and Foster×CIho14300 (C); 6 through 10, F2 resistant plants; 11 through 15, F2 susceptible plants. (C) = M, DNA size markers in bp; 1, susceptible parent Robust; 2, susceptible parent Foster; 3, resistant parent CIho14300; 4, F1 plant of Robust×CIho14300; 5, F1 plant of Foster×CIho14300; 6 through 15, F2 susceptible plants; 16 through 24, F2 resistant plants.
To determine the existence of STS markers linked to Rsp genes, 21 resistant and 16 susceptible barley lines identified as resistant or susceptible by Toubia-Rahme and Steffenson (2004) were evaluated (Table 4). It is not known which resistance genes are present in these lines. Two STS markers, Rsp1158R in repulsion and Rsp2545C in coupling showed the expected presence or absence of bands in resistant lines. However, unexpected results were obtained in 7/18 (Rsp1158R) and 9/18 (Rsp2545C) susceptible lines. This may be due to our previous finding that the markers are not within, but separated from the genes of interest. The STS marker Rsp3314C linked to Rsp3, amplified a band only in the four resistant lines, Bolron, Feebar, Flynn1, and Vaughn. The pedigree of CIho10644 containing Rsp3 is Feebar/Kindred. The pedigree of Feebar is Peatland/Vaughn, and the pedigree of Vaughn is Mariout/Leiorrhynchium or Club Mariout/Lion. Flynn also comes from a cross between Club Mariout/Lion and Flynn1 is a selection from Flynn. Thus, the lines CIho10644, Feebar, Vaughn, and Flynn1 which gave a band with the STS marker Rsp3314C contain a genetic background with Rsp3 in one of the parents. This result gives hope that with further testing the STS marker Rsp3314C can be effectively used in MAS to identify lines containing Rsp3. The other line testing positive to Rsp3314C, Bolron, is from a cross Bolivia/Chevron, and has a different genetic background. Bolivia and Chevron need to be tested with the STS marker Rsp3314C and allelism tests done to determine if they contain Rsp3.
Table 4. Validation of sequence-tagged site (STS) markers and reaction to SSLB for 24 resistant and 18 susceptible barley lines.
Cultivars/Lines | Reactiona to SSLB | STS marker |
| | Rsp1158R | Rsp2545C | Rsp3314C |
| Atlas54 | R | +b | - | - |
| Atlas | R | + | - | - |
| Bolron | R | - | - | + |
| CIho4428 | R | - | - | - |
| CIho4439 | R | + | + | - |
| CIho6398 | R | - | - | - |
| CIho9831 | R | - | + | - |
| Custer | R | + | - | - |
| Feebar | R | + | - | + |
| Flynn1 | R | + | - | + |
| Glacier | R | - | - | - |
| Hor2683-84 | R | + | + | - |
| Hor 9471-87 | R | - | + | - |
| Nomini | R | - | - | - |
| ND16092 | R | + | + | - |
| PC11 | R | - | + | - |
| PC84 | R | + | + | - |
| Sp.No:1 | R | - | - | - |
| Starling | R | - | + | - |
| Sussex | R | - | - | - |
| Vaughn | R | - | - | + |
| Bowman | S | + | + | - |
| Carlsberg | S | - | - | - |
| CIho13581 | S | - | + | - |
| CIho4753 | S | + | + | - |
| CIho592 | S | - | - | - |
| CIho0182 | S | - | + | - |
| CIho2947 | S | + | - | - |
| CIho8096 | S | - | + | - |
| Heimdal | S | - | + | - |
| Hiland | S | + | + | - |
| Kindred | S | + | - | - |
| Olli | S | - | - | - |
| Supi 1 | S | + | + | - |
| Trebi | S | + | + | - |
| Velvon | S | + | - | - |
| ZAU7 | S | + | - | - |
| Robust | S | + | - | - |
| Foster | S | + | - | - |
| CIho14300 (Rsp1) | R | - | - | - |
| CIho4780 (Rsp2) | R | + | + | - |
| CIho10644 (Rsp3) | R | + | - | + |
a SSLB inoculation and disease assessment was taken as described by Toubia-Rahme and Steffenson (2004).
R: resistant, S: susceptible.
b Existence of alleles (+) and absence of alleles (-).
In future work, other RAPD markers linked to Rsp genes will be converted into STS markers, with emphasis on Rsp1 and Rsp2. The evaluation of selection effectiveness in F3 populations and the allelism test for Rsp genes with STS markers will be undertaken. The screening of other resistant sources for resistance genes to SSLB will be performed and validated with the STS markers associated with Rsp genes.
References
Buchannon, K.W. 1961. Inheritance of reaction to Septoria passerinii Sacc., and Pyrenophora teres (Died.) Drechsl., and of row number, in barley. Ph.D. thesis, University of Saskatchewan, Saskatoon, Sask.
Green, G. J. and Bendelow, V. M. 1961. Effect of speckled leaf blotch, Septoria passerinii Sacc., on the yield and malting quality of barley. Can. J. Plant Sci. 41: 431-435.
Green, G.J., and Dickson, J.G. 1957. Pathological histology and varietal reactions in Septoria leaf blotch of barley. Phytopathology, 47: 73–79.
Neil, J., Helen, O. and Howard, T. 1997. Markers and Mapping: We are all geneticists now. New Phytologist 137(1):165-177.
Rasmusson, D. C. and Rogers, W. E. 1963. Inheritance of resistance to Septoria in barley. Crop Sci. 3: 161-162.
Toubia-Rahme, H., and Steffenson, B. J. 2004. Sources of resistance to septoria speckled leaf blotch caused by Septoria passerinii in barley. Can. J. Plant Pathol. 26: 358–364.
Zhong, S., Toubia-Rahme, H., Steffenson, B. J. and Waugh, R. 2002. Molecular mapping of Septoria speckled leaf blotch resistance in barley. Plant, Animal and Microbe Genomes. X. Poster. 22.
S.H. Lee and S.M. Neate
North Dakota State University, Dept. Plant Pathology, Fargo, ND 58105
Presented at the 18th North American Barley Researchers Workshop, July 17-20, 2005 |
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