Genotyping of lines from the VIR sunflower genetic collection using allele-specific markers of the Rf1 locus

Background. The CMS-Rf genetic system based on the PET1-type cytoplasmic male sterility (CMS) is commonly used to create commercial sunflower (Helianthus annuus) hybrids. The Rf1 gene, of key importance for hybrid breeding, is necessary for restoring pollen fertility in F₁ plants. The molecular genetic markers tested on various genetic materials are an effective tool for identifying parental line genotypes at the Rf1 locus, controlling homogeneity, and determining the genetic purity of hybrid seed lots. In the present study, the allele-specific markers of the Rf1 candidate genes available from literature were used to genotype lines from the VIR sunflower genetic collection and F₂ hybrids. Material and methods. The study concentrated on two sample sets of genotypes, one of which contained 46 lines from the VIR sunflower genetic collection, previously characterized in field experiments for the pollen restoration ability, and the other 80 plants from segregating F₂ populations from crosses of the CMS VIR 116A line with fertility restorers VIR 740 and RIL 130, phenotyped for fertility/sterility. The lines differed in respect of the cytoplasm type and the presence of the SCAR marker HRG02 closely linked to the Rf1 locus. The lines have been genotyped using markers specific for the dominant (PPR621.5R, SRF833, 67N04_P_170) and recessive (PPR621.5M, 67N04_P_155) alleles of the Rf1 candidate genes. The PPR621.5M and PPR621.5 R, marker fragments amplified in six genotypes, have been isolated and sequenced. Results. The nucleotide sequences of PPR621.5M and PPR621.5R turned out to be different in four SNPs and completely identical to those presented in the published literature. The PPR621.5M and 67N04_P_155 markers specific for the rf1 allele were identified in CMS lines and the majority of sterility maintainers. Nineteen out of 21 lines characterized by sterile cytoplasm and the presence of the HRG02 marker had three markers specific for the dominant allele; two lines had two allele-specific markers. Four out of seven fertility restorers (sterile cytoplasm, without the HRG02 marker) were found to contain two or three markers specific for the dominant allele, while three lines had only markers for the recessive allele. The F₂ genotypes resulting from recombination between the SCAR marker HRG02 and allele specific markers were detected. Conclusion. The study confirmed efficiency of allele-specific markers of the Rf1 locus candidate genes for genotyping sunflower lines, as well as their diagnostic value for selecting target genotypes from segregating hybrid populations.

1. Abratti G., Bazzalo M.E., Leon A. Mapping a novel fertility restoration gene in sunflower. In: Proceedings of the 17th International Sunflower Conference; 2008 June 8-12; Cordoba, Spain. Consejería de Agricultura y Pesca; 2008. Vol. 2. P. 617-621. Available from https://www.isasunflower.org/fileadmin/documents/aaProceedings/17thISC_CordobaVol2/617sonia.pdf [accessed Nov. 15, 2024]

2. Anashchenko A.V., Duka M.V. Study of the genetic system of CMS-Rf in sunflower (Helianthus annuus L.). Communication II. Restoration of male fertility in hybrids based on CMS. Genetika. 1985;21(12):1999-2004. [in Russian]

3. Anisimova I.N., Karabitsina Yu.I., Alpatieva N.V., Kuznecova E.B., Titov N.V., Lyutko A.Yu., Gavrilova V.A. Diagnostic value of Rf1 gene molecular markers in sunflower. Biotechnology and Plant Breeding. 2021;4(2):28-37. [in Russian]. DOI: 10.30901/2658-6266-2021-2-o3

4. Badouin H., Gouzy J., Grassa C.J., Murat F., Staton S.E., Cottret L., Lelandais-Brière C., Owens G.L., Carrère S., Mayjonade B., Legrand L., Gill N., Kane N.C., Bowers J.E., Hubner S., Bellec A., Bérard A., Bergès H., Blanchet N., Boniface M-C., Brunel D., Catrice O., Chaidir N., Claudel C., Donnadieu C., Faraut T., Fievet G., Helmstetter N., King M., Knapp S.J., Lai Z., Le Paslier M-C., Lippi Y., Lorenzon L., Mandel J.R., Marage G., Marchand G., Marquand E., Bret-Mestries E., Morien E., Nambeesan S., Nguyen T., Pegot-Espagnet P., Pouilly N., Raftis F., Sallet E., Schiex T., Thomas J., Vandecasteele C., Varès D., Vear F., Vautrin S., Crespi M., Mangin B., Burke J.M., Salse J., Muños S., Vincourt P., Loren H. Rieseberg L.H., Langlade N.B. The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution. Nature. 2017;546:148-152. DOI: 10.1038/nature22380

5. Baute G.J., Kane N.C., Grassa C.J., Lai Z., Rieseberg L.H. Genome scans reveal candidate domestication and improvement genes in cultivated sunflower, as well as post-domestication introgression with wild relatives. New Phytologist. 2015;206(2):830-838. DOI: 10.1111/nph.13255

6. Choudhari A.K., Bagade A.B. Diverse cytosteriles in sunflower: a review. International Journal of Current Microbiology and Applied Sciences. 2019;8(11):1641-1644. DOI: 10.20546/ijcmas.2019.811.189

7. Feng J., Jan C.C. Introgression and molecular tagging of Rf4, a new male fertility restoration gene from wild sunflower Helianthus maximiliani L. Theoretical and Applied Genetics. 2008;117(2):241. DOI: 10.1007/s00122-008-0769-4

8. Gavrilova V.A., Anisimova I.N., Alpatyeva N.V., Rozhkova V.T, Stupnikova T.G., Karabitsina Yu.I., Kuznetsova E.B. Catalogue of the VIR global collection. Iss. 853. Genetic collection of sunflower (Geneticheskaya kollektsiya podsolnechnika). St. Petersburg: VIR; 2017. [in Russian]

9. Goryunov D.V., Anisimova I.N., Gavrilova V.A., Chernova A.I., Sotnikova E.A., Martynova E.U., Boldyrev S.V., Ayupova A.F., Gubaev R.F., Mazin P.V., Gurchenko E.A., Shumskiy A.A., Petrova D.A., Garkusha S.V., Mukhina Z.M., Benko N.I., Demurin Y.N., Khaitovich P.E., Goryunova S.V. Association mapping of fertility restorer gene for CMS PET1 in sunflower. Agronomy. 2019;9(2):49. DOI: 10.3390/agronomy9020049

10. Heiser C.B. Hybridization in the annual sunflowers: Helianthus annuus × H. debilis var. cucumerifolius. Evolution. 1951;5(1):42-51. DOI: 10.2307/2405429

11. Horn R., Köhler R.H., Zetsche K. A mitochondrial 16 kDa protein is associated with cytoplasmic male sterility in sunflower. Plant Molecular Biology. 1991;17(1):29-36. DOI: 10.1007/BF00036803

12. Horn R., Kusterer B., Lazarescu E., Prüfe M., Friedt W. Molecular mapping of the Rf1 gene restoring fertility in PET1-based F1 hybrids in sunflower (Helianthus annuus L.). Theoretical and Applied Genetics. 2003;106:599-606. DOI: 10.1007/s00122-002-1078-y

13. Horn R., Radanovic A., Fuhrmann L., Sprycha Y., Hamrit S., Jockovic M., Miladinovic D., Jansen C. Development and validation of markers for the fertility restorer gene Rf1 in sunflower. International Journal of Molecular Sciences. 2019:20(6):1260. DOI: 10.3390/ijms20061260

14. Jan C.C., Vick B.A., Miller J.F., Kahler A.L., Butler E.T. Construction of an RFLP linkage map for cultivated sunflower. Theoretical and Applied Genetics. 1998;96(1):15-22. DOI: 10.1007/s001220050703

15. Jan C.C., Vick B.A. Inheritance and allelic relationships of fertility restoration genes for seven new sources of male-sterile cytoplasm in sunflower. Plant Breeding. 2007;126(2):213-217. DOI: 10.1111/j.1439-0523.2007.01350.x

16. Karabitsina Y.I., Gavrilova V.A., Alpatieva N.V., Kuznetsova E.B., Anisimova I.N. Peculiarities of inheritance of pollen fertility restoration trait in sunflower with cytoplasmic male sterility. Russian Journal of Genetics. 2019;55:1375-1382. DOI: 10.1134/S1022795419110073

17. Kinman M.L. New developments in the USDA and state experiment station sunflower breeding programs. In: Proceedings of the 4th International Sunflower Conference; 1970 June 23-25; Memphis, Tennessee, USA. 1970. P. 181-183. Available at: https://www.isasunflower.org/ fileadmin/documents/Proceedings/4thISC1970/T1970BRE08.pdf [accessed Nov. 15, 2024].

18. Leclerq P. Une sterilite cytoplasmique chez le tournesol. Annales de l'Amélioration des Plantes. 1969;19(2):99-106. [In French]

19. Li J.T., Yang J., Chen D.C., Zhang X.I., Tang Z.S. An optimized mini-preparation method to obtain high-quality genomic DNA from mature leaves of sunflower. Genetics and Molecular Research. 2007;6(4):1064-1071.

20. Liu Z., Mulpuri S., Feng J., Vick B.A., Jan C.-C. Molecular mapping of the Rf3 fertility restoration gene to facilitate its utilization in breeding confection sunflower. Molecular Breeding. 2012;29:275-284. DOI: 10.1007/s11032-011-9563-0

21. Liu Z., Wang D., Feng J., Seiler G.J., Cai X., Jan C.-C. Diversifying sunflower germplasm by integration and mapping of a novel male fertility restoration gene. Genetics. 2013;193(3):727-37. DOI: 10.1534/genetics.112.146092

22. Liu Z., Zhang L., Seiler G.J., Jan C.-C. Molecular mapping of theRf9 gene from RCMG 1 for CMS ANN3 derived from wild sunflower (Helianthus annuus L.). Euphytica. 2023;219:46. DOI: 10.1007/s10681-023-03176-3

23. NCBI. National Center for Biotechnology Information. Available from: https://www.ncbi.nlm.nih.gov [accessed Nov. 15, 2024].

24. Oil seed sunflower description of released restorer line germplasm. Available from: https://www.ag.ndsu.edu/fss North Dakota Foundation Seedstocks [accessed Nov. 15, 2024].

25. Polivanova O.B., Sivolapova A.B., Goryunov D.V., Fedorova A.V., Sotnikova E.A., Chebanova Y.V., Karabitsina Y.U.; Benko N.I., Demurin Y.N., Goryunova S.V. Structural diversity of sunflower (Helianthus annuus L.) candidate Rf1 loci based on gene-specific PCR. Research on Crops. 2021;22(1):40-46. DOI: 10.31830/2348-7542.2021.034

26. Qi L.L., Seiler G.J., Hulke B.S., Vick B.A., Gulya T.J. Genetics and mapping of the R11 gene conferring resistance to recently emerged rust races, tightly linked to male fertility restoration, in sunflower (Helianthus annuus L.). Theoretical and Applied Genetics. 2012;125:921-932. DOI: 10.1534/genetics.112.146092

27. Radanovi´c A.; Sprycha Y.; Jockovi´c M.; Sundt M.; Miladinovi´c D.; Jansen C.; Horn R. KASP markers specific for the fertility restorer locus Rf1 and application for genetic purity testing in sunflowers (Helianthus annuus L.). Genes. 2022;13:465. DOI: 10.3390/genes13030465

28. Sajer O., Schirmak U., Hamrit S., Horn R. Mapping of the new fertility restorer gene Rf-PET2 close to Rf1 on linkage group 13 in sunflower (Helianthus annuus L.). Genes. 2020;11:269. DOI: 10.3390/genes11030269

29. Schnabel U., Engelmann U., Horn R. Development of markers for the use of the PEF1 cytoplasm in sunflower hybrid breeding. Plant Breeding. 2008;127(6):587-591. DOI: 10.1111/j.1439-0523.2008.01516.x

30. Serieys H. Identification, study, utilization in breeding programs of new CMS sources. In: Proceedings of the Sunflower Subnetwork Progress Report; 2005 July 17-20; Novi Sad, Serbia and Montenegro. Rome, Italy: FAO. 2005. P. 47-53.

31. Sivolapova A.B., Polivanova O.B., Goryunov D.V., Chebanova Y.V., Fedоrova A.V., Sotnikova E.A., Karabitsina Y.I., Benko N.I., Mukhina Z.M., Anisimova I.N., Demurin Y.N., Goryunova S.V. Refinement of Rf1-gene localization and development of the new molecular markers for fertility restoration in sunflower. Molecular Biology Reports. 2023;50(9):7919-7926. DOI: 10.1007/s11033-023-08646-4

32. Talukder Z., Ma G., Hulke B., Jan C.-C., Qi L. Linkage mapping and genome-wide association studies of the Rf gene cluster in sunflower (Helianthus annuus L.) and their distribution in world sunflower collections. Frontiers in Genetics. 2019;10:216. DOI: 10.3389/fgene.2019.00216

33. Trubacheeva N.V., Salina E.A., Shumny V.K., The use of CMS/Rf system for sunflower hybrid breeding. Pisma v Vavilovskii Zhurnal Genetiki i Selektsii = Letters to Vavilov Journal of Genetics and Breeding. 2024;10(2):119-131. [in Russian]. DOI: 10.18699/letvjgb-2024-10-14

34. Yue B., Vick B.A., Cai X., Hu J. Genetic mapping for the Rf1 (fertility restoration) gene in sunflower (Helianthus annuus L.) by SSR and TRAP markers. Plant Breeding. 2010;129:24-28. DOI: 10.1111/j.14390523.2009.01661.x