Validation of markers for resistance to Pyrenophora teres f. teres loci on barley chromosomes 3H, 4H, and 6H in the polygenic inheritance of the trait

The causal agent of net blotch Pyrenophora teres Drechs. f. teres (Ptt) is a dangerous pathogen of barley. The development of genetic protection against this disease is a necessary link in resource-saving and environmentally friendly barley cultivation technologies. Effective QTL markers controlling both qualitative and quantitative resistance are required for breeding for resistance to Ptt. As a result of GWAS, we identified barley accessions of different origins, the SNP haplotypes of which were associated with resistance loci simultaneously on different barley chromosomes (VIR catalogue numbers: k-5900, k-8829, k-8877, k-14936, k-30341 and k-18552). The aim of the study was to validate SNP markers (MM) of Ptt resistance loci on chromosomes 3H, 4H and 6H in F₂ from crossing six resistant accessions with the susceptible variety Tatum. The observed segregation for resistance in all crossing combinations confirmed the presence of several genetic determinants of resistance in the studied accessions. To study the polymorphism of the parents from the crosses and the correspondence between the phenotypes to the presence/absence of the markers in the segregating populations, primers with a specific 3’-end, CAPS markers, and KASP markers were developed. A significant association (p < 0.05) between the presence of the CAPS marker JHI-Hv50k-2016-391380 HindIII on chromosome 6H and the phenotype of resistance to Ptt in F₂ plants was revealed in crosses between the susceptible cultivar Tatum and accessions k-5900, k-8829, k-8877 and k-18552. On chromosome 4H, a significant association with the resistance phenotype in the F₂ population from the cross with accession k-8877 was revealed for marker JHI-Hv50k-2016-237924, and in that from the cross with accession k-5900, for marker SCRI_RS_181886. The presence of QTL on chromosome 6H, which controls qualitative resistance in four barley accessions, masks the expression of other genes, which explains the discrepancy between the resistance phenotype and the presence of molecular markers in the segregating populations. Resistance donors and molecular markers with proven efficacy can be used in marker-assisted selection (MAS) to develop barley cultivars resistant to net blotch.

1. Afanasenko О., Jalli M., Pinnschmidt H., Filatova O., Platz G. Deve lopment of an international standard set of barley differential geno types for Pyrenophora teres f. teres. Plant Pathol. 2009;58(4): 665-676. doi 10.1111/j.1365-3059.2009.02062.x

2. Afanasenko O.S., Koziakov A.V., Hedlay P.E., Lashina N.M., Ani simova A.V., Manninen O., Jalli M., Potokina E.K. Mapping of the loci controlling the resistance to Pyrenophora teres f. teres and Cochliobolus sativus in two double haploid barley popula tions. Russ J Genet Appl Res. 2015;5(3):242-253. doi 10.1134/S2079059715030028

3. Afanasenko O., Rozanova I., Gofman A., Lashina N., Novakazi F., Mironenko N., Baranova O., Zubkovich A. Validation of molecular markers of barley net blotch resistance loci on chromosome 3H for marker-assisted selection. Agriculture. 2022;12(4):439. doi 10.3390/agriculture12040439

4. Amezrou R., Verma R.P.S., Chao S., Brueggeman R.S., Belgadi L., Ar baoui M., Rehman S., Gyawali S. Genome-wide association studies of net form of net blotch resistance at seedling and adult plant stages in spring barley collection. Mol Breed. 2018;38:58. doi 10.1007/s11032-018-0813-2

5. Cakir M., Gupta S., Li C., Hayden M., Mather D.E., Ablett G.A., Platz G.J., Broughton S., Chalmers K.J., Loughman R., Jones M.G.K., Lance R.C.M. Genetic mapping and QTL analysis of disease resis tance traits in the barley population Baudin × AC Metcalfe. Crop Pasture Sci. 2011;62(2):152-161. doi 10.1071/cp10154

6. Clare S.J., Çelik Oğuz A., Effertz K., Sharma Poudel R., See D., Karakaya A., Brueggeman R.S. Genome-wide association map ping of Pyrenophora teres f. maculata and Pyrenophora teres f. teres resistance loci utilizing natural Turkish wild and landrace barley populations. G3. 2021;11(11):jkab269. doi 10.1093/g3journal/jkab269

7. Dinglasan E., Hickey L., Ziems L., Fowler R., Anisimova A., Ba ranova O., Lashina N., Afanasenko O. Genetic characterization of resistance to Pyrenophora teres f. teres in the International barley differential Canadian lake Shore. Front Plant Sci. 2019;10:326. doi 10.3389/fpls.2019.00326

8. Friesen T.L., Faris J.D., Lai Z., Steffenson B.J. Identification and chro mosomal location of major genes for resistance to Pyrenophora teres in a doubled-haploid barley population. Genome. 2006;49(7):855 859. doi 10.1139/g06-024

9. Grewal T.S., Rossnagel B.G., Pozniak C.J., Scoles G.J. Mapping quantitative trait loci associated with barley net blotch resistance. Theor Appl Genet. 2008;116(4):529-539. doi 10.1007/s00122-007- 0688-9

10. Grewal T.S., Rossnagel B.G., Scoles G.J. Mapping quantitative trait loci associated with spot blotch and net blotch resistance in a doubled-haploid barley population. Mol Breed. 2012;30(1):267-279. doi 10.1007/s11032-011-9616-4

11. Gupta S., Li C.D., Loughman R., Cakir M., Platz G., Westcott S., Brad ley J., Broughton S., Lance R. Quantitative trait loci and epistatic interactions in barley conferring resistance to net type net blotch (Pyrenophora teres f. teres) isolates. Plant Breed. 2010;129(4):362 368. doi 10.1111/j.1439-0523.2009.01716.x

12. Jatayev S., Kurishbayev A., Zotova L., Khasanova G., Serikbay D., Zhu batkanov A., Botayeva M., Zhumalin A., Turbekova A., Soole K., Langridge P., Shavrukov Yu. Advantages of Amplifluor-like SNP markers over KASP in plant genotyping. BMC Plant Biol. 2017; 17(2):254. doi 10.1186/s12870-017-1197-x

13. Koladia V.M., Faris J.D., Richards J.K., Brueggeman R.S., Chao S., Friesen T.L. Genetic analysis of net-form net blotch resistance in barley lines CIho 5791 and Tifang against a global collection of P. teres f. teres isolates. Theor Appl Genet. 2017;130(1):163-173. doi 10.1007/s00122-016-2801-4

14. König J., Perovic D., Kopahnke D., Ordon F. Development of an ef f icient method for assessing resistance to the net type of net blotch (Pyrenophora teres f. teres) in winter barley and mapping of quan titative trait loci for resistance. Mol Breed. 2013;32:641-650. doi 10.1007/s11032-013-9897-x

15. König J., Perovic D., Kopahnke D., Ordon F. Mapping seedling resist ance to net form of net blotch (Pyrenophora teres f. teres) in barley using detached leaf assay. Plant Breed. 2014;133(3):356-365. doi 10.1111/pbr.12147

16. Lashina N.M., Mironenko N.V., Zubkovich A.A., Afanasenko O.S. Juvenile resistance of barley varieties and samples to net-, spot- and hybrid (net × spot) forms of Pyrenophora teres. Mycol Phyto pathol. 2023;57(1):48-59. doi 10.31857/S0026364823010099 (in Russian)

17. Liu Z., Ellwood S.R., Oliver R.P., Friesen T.L. Pyrenophora teres: pro f ile of an increasingly damaging barley pathogen. Mol Plant Pathol. 2011;12(1):1-19. doi 10.1111/j.1364-3703.2010.00649.x

18. Manninen O.M., Jalli M., Kalendar R., Schulman A., Afanasenko O., Robinson J. Mapping of major spot-type and net-type net-blotch resistance genes in the Ethiopian barley line CI 9819. Genome. 2006;49(12):1564-1571. doi 10.1139/g06-119

19. Martin A., Platz G.J., de Klerk D., Fowler R.A., Smit F., Potgieter F.G., Prins R. Identification and mapping of net form of net blotch resistance in South African barley. Mol Breed. 2018;38(5):53. doi 10.1007/s11032-018-0814-1

20. Mascher M., Wicker T., Jenkins J., Plott C., Lux T., Koh C.S., Ens J., … Šimková H., Moscou M.J., Grimwood J., Schmutz J., Stein N. Long read sequence assembly: A technical evaluation in barley. Plant Cell. 2021;33(6):1888-1906. doi 10.1093/plcell/koab077

21. Novakazi F., Afanasenko O., Anisimova A., Platz G.J., Snowdon R., Kovaleva O., Zubkovich A., Ordon F. Genetic analysis of a world wide barley collection for resistance to net form of net blotch disease (Pyrenophora teres f. teres). Theor Appl Genet. 2019;132(9):2633 2650. doi 10.1007/s00122-019-03378-1

22. O’Boyle P.D., Brooks W.S., Barnett M.D., Berger G.L., Steffenson B.J., Stromberg E.L., Maroof M.A.S., Liu S.Y., Griffey C.A. Mapping net blotch resistance in ‘Nomini’ and CIho 2291 barley. Crop Sci. 2014;54(6):2596-2602. doi 10.2135/cropsci2013.08.0514

23. Pandis N. The chi-square test. Am J Orthodontics Dentofa cial Orthopaedics. 2016;150(5):898-899. doi 10.1016/j.ajodo.2016.08.009

24. Potokina E.K., Hedley P., Afanasenko O.S., Lashina N.M., Anisimo va A.V., Kozyakov A.V., Yalli M., Manninen O. Mapping of QTL (Quantitative Trait Loci) determining resistance to net blotch of bar ley. In: Technologies for creating and using varieties and hybrids with group and complex resistance to pests in plant protection. St. Petersburg, 2010;229-236 (in Russian)

25. Rehman S., Al-Jaboobi M., Verma R.P.S., Sanchez-Garcia M., Vi sioni A. Genome-wide association mapping of net form net blotch resistance in barley at seedling and adult plant stages. Front Agron. 2025;7:1525588. doi 10.3389/fagro.2025.1525588

26. Richards J.K., Friesen T.L., Brueggeman R.S. Association mapping utilizing diverse barley lines reveals net form net blotch seedling resistance/susceptibility loci. Theor Appl Genet. 2017;130(5):915 927. doi 10.1007/s00122-017-2860-1

27. Richter K., Schondelmaier J., Jung C. Mapping of quantitative trait loci affecting Drechslera teres resistance in barley with molecular markers. Theor Appl Genet. 1998;97:1225-34. doi 10.1007/s001220051014

28. Rozanova I.V., Lashina N.M., Mustafin Z.S., Gorobets S.A., Efi mov V.M., Afanasenko O.S., Khlestkina E.K. SNPs associated with barley resistance to isolates of Pyrenophora teres f. teres. BMC Genomics. 2019;20:292. doi 10.1186/s12864-019-5623-3

29. Steffenson B.J., Hayes P.M., Kleinhofs A. Genetics of seedling and adult plant resistance to net blotch (Pyrenophora teres f. teres) and spot blotch (Cochliobolus sativus) in barley. Theor Appl Genet. 1996;92:552-558. doi 10.1007/bf00224557

30. Tekauz A. A numerical scale to classify reactions of barley to Pyre nophora teres. Can J Plant Pathol. 1985;7(2):181-183. doi 10.1080/07060668509501499

31. Wonneberger R., Ficke A., Lillemo M. Identification of quantitative trait loci associated with resistance to net form net blotch in a col lection of Nordic barley germplasm. Theor Appl Genet. 2017;130: 2025-2043. doi 10.1007/s00122-017-2940-2

32. Yun S.J., Gyenis L., Bossolini E., Hayes P.M., Matus I., Smith K.P., Steffenson B.J., Tuberosa R., Muehlbauer G.J. Validation of quan titative trait loci for multiple disease resistance in barley using advanced backcross lines developed with a wild barley. Crop Sci. 2006;46(3):1179-1186. doi 10.2135/cropsci2005.08-0293