Determination of the S-RNase gene allelic composition in pear accessions of the collection maintained at the Maikop Experiment Station of VIR

Background: Pear (Pyrus L.) is one of the most important fruit crops, widespread in the world. While in Europe and historically associated countries (USA, Australia), cultivars of one domesticated species Pyrus communis L. are grown, in the Asian region (China, Korea, Japan, Far East), cultivars were created based on individual botanical species, primarily Pyrus pyrifolia (Burm.fil.) Nakai, P. bretschneideri Rehder., P. ussuriensis Maxim., and P. × sinkiangensis T.T. Yu. The Caucasus region is considered as one of the most important centers for the formation of European pear cultivars. The concentration of a large number of species with overlapping ranges in the Caucasus promoted intensive interspecific hybridization and the emergence of polyhybrid forms that could be used in folk breeding. Commercial pear cultivation faces a number of difficulties, not the least of which is the self-incompatibility of cultivars. Gametophytic self-incompatibility in pear is controlled by the S-locus, which includes the S-RNase gene and multiple SFBB genes. These genes are highly polymorphic, so the study of the S-locus applies not only to the selection of pollinators in commercial pear orchards, but also to molecular S-genotyping. The aim of this work was to characterize the pear collection maintained at the VIR Maikop Experiment Station, and primarily landraces of Caucasian and Crimean origin, using various systems of S-locus allele markers. Materials and methods: we studied a subset of 194 accessions, including 182 cultivars and four hybrid forms from the collection of the Maikop Experiment Station of VIR, as well as eight accessions collected as part of the VIR expedition to the North Caucasus in 2022. The main analyzed groups were cultivars bred in Europe (49), those created by breeding institutions in the Caucasus (63), local Caucasian cultivars (46) and a group of Crimean cultivars (13). In the work, we used consensus PycomC1/PycomC5 and allele-specific primers selected from the literature data. Results: using both molecular marker systems, we were able to identify 25 S-alleles in the subset. Seven alleles (S₁₀₁, S₁₀₂, S₁₀₃, S_104-1, S_104-2, S₁₀₈, and S₁₂₂) were present with a frequency of ≥10%, the differences between frequencies of some of them were statistically significant in groups of local Caucasian, European and Crimean cultivars. These groups also differed in the presence of rare and unique alleles and in the presence of a large number of triploid forms. Conclusion: molecular screening of a large subset of pear accessions has established the allelic diversity of the S-locus and the uniqueness of the folk cultivars from the Caucasus and Crimea. Local cultivars with the original S-allele profile can be valuable material for breeding.

1. Aguiar B., Vieira J., Cunha A.E., Fonseca N.A., Reboiro-Jato D., Reboiro-Jato M., Fdez-Riverola F., Raspé O., Vieira C.P. Patterns of evolution at the gametophytic self-incompatibility Sorbus aucuparia (Pyrinae) S pollen genes support the non-self recognition by multiple factors model. Journal of Experimental Botany. 2013;64(8):2423-2434. DOI: 10.1093/jxb/ert098

2. Antonova O.Yu., Klimenko N.S., Rybakov D.A., Fomina N.A., Zheltova V.V., Novikova L.Yu., Gavrilenko T.A. SSR analysis of modern Russian potato varieties using DNA samples of nomenclatural standards. Plant Biotechnology and Breeding. 2020;3(4):77-96. [in Russian]. DOI: 10.30901/2658-6266-2020-4-o2

3. Bell R.L. Pears (Pyrus). Acta Horticulturae. 1991;290:657-697. DOI: 10.17660/ActaHortic.1991.290.15

4. Bennici S., Di Guardo M., Distefano G., Las Casas G., Ferlito F., De Franceschi P., Dondini L., Gentile A., La Malfa S. Deciphering S-RNase Allele Patterns in Cultivated and Wild Accessions of Italian Pear Germplasm. Forests. 2020;11(11):1228. DOI: 10.3390/f11111228

5. Claessen H., Keulemans W., Van de Poel B., De Storme N. Finding a compatible partner: self-incompatibility in European pear (Pyrus communis); molecular control, genetic determination, and impact on fertilization and fruit set. Frontiers in Plant Science. 2019;10:407. DOI: 10.3389/fpls.2019.00407

6. De Franceschi P., Pierantoni L., Dondini L., Grandi M., Sanzol J., Sansavini S. Cloning and mapping multiple S-locus F-box genes in European pear (Pyrus communis L.). Tree Genetics and Genomes. 2011a;7(2):231-240. DOI: 10.1007/s11295-010-0327-5

7. De Franceschi P., Pierantoni L., Dondini L., Grandi M., Sansavini S., Sanzol J. Evaluation of candidate F-box genes for the pollen S of gametophytic self-incompatibility in the Pyrinae (Rosaceae) on the basis of their phylogenomic context. Tree Genetics and Genomes. 2011b;7(4):663-683. DOI: 10.1007/s11295-011-0365-7

8. FAOSTAT. The Food and Agriculture Organization (FAO) of the United Nations. FAOstatistics. Available from: https://www.fao.org/faostat/en/#compare; https://www.fao.org/faostat/ru/#compare [accessed June 10, 2025].

9. Fisher R.A. On the interpretation of χ2 from contingency tables, and the calculation of P. Journal of the Royal Statistical Society. 1922;85(1):87-94. Available from: http://hdl.handle.net/2440/15173 [accessed June 10, 2025].

10. Gharehaghaji A.N., Arzani K., Abdollahi H., Shojaeiyan A., Dondini L., De Franceschi P. Genomic characterization of self-incompatibility ribonucleases in the Central Asian pear germplasm and introgression of new alleles from other species of the genus Pyrus. Tree Genetics and Genomes. 2014;10:411-428. DOI: 10.1007/s11295-013-0696-7

11. Goldway M., Takasaki-Yasuda T., Sanzol J., Mota M., Zisovich A., Stern R.A., Sansavini S. Renumbering the S-RNase alleles of European pears (Pyrus communis L.) and cloning the S109 RNase allele. Scientia Horticulturae. 2009;119(4):417-422. DOI: 10.1016/j.scienta.2008.08.027

12. Ikeda K., Watari A., Ushijima K., Yamane H., Hauck N.R., Lezzoni A.F., Tao R. Molecular markers for the self-compatible S4’-haplotype, a pollen-part mutant in sweet cherry (Prunus avium L.). Journal of the American Society for Horticultural Science. 2004;129(5):724-728. DOI: 10.21273/JASHS.129.5.724

13. Inglis P.W., Pappas M.D.C.R., Resende L.V., Grattapaglia D. Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications. PLoS One. 2018;13(10):e0206085. DOI: 10.1371/journal.pone.0206085

14. Jaumien F. The causes of poor bearing of pear trees of the variety ‘Doyenne du Comice’. Acta Agrobotanica. 1968;21:75-106. DOI: 10.5586/aa.1968.003

15. Kubo K., Entani T., Takara A., Wang N., Fields A.M., Hua Z., Toyoda M., Kawashima S., Ando T., Isogai A., Kao T.H., Takayama S. Collaborative non-self recognition system in S-RNase-based self-incompatibility. Science. 2010;330:796-799. DOI: 10.1126/science.1195243

16. Lewis D., Modlibowska I. Genetical studies in pears. Journal of Genetics. 1942;43:211-222. DOI: 10.1007/BF02982754

17. Marchese A., Tobutt K.R., Raimondo A., Motisi A., Bošković R.I., Clarke J., Caruso T. Morphological characteristics, microsatellite fingerprinting and determination of incompatibility genotypes of Sicilian sweet cherry cultivars. Journal of Horticultural Science and Biotechnology. 2007;82:41-48. DOI: 10.1080/14620316.2007.11512197

18. Minamikawa M., Kakui H., Wang S., Kotoda N., Kikuchi S., Koba T., Sassa H. Apple S locus region represents a large cluster of related, polymorphic and pollen-specific F-box genes. Plant Molecular Biology. 2010;74:143-154. DOI: 10.1007/s11103-010-9662-z

19. Moriya Y., Yamamoto K., Okada K., Iwanami H., Bessho H., Nakanishi T., Takasaki T. Development of a CAPS marker system for genotyping European pear cultivars harboring 17 S alleles. Plant Cell Reports. 2007;26:345-354. DOI: 10.1007/s00299-006-0254-y

20. Nei M. Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences. 1973;70(12):3321-3323. DOI: 10.1073/pnas.70.12.3321

21. Okada K., Tonaka N., Taguchi T., Ichikawa T., Sawamura Y., Nakanishi T., Takasaki-Yasuda T. Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear. Journal of Experimental Botanics. 2011;62(6):1887-1902. DOI: 10.1093/jxb/erq381

22. Rubtsov G.A. Origin and evolution of cultivated pear (Proiskhozhdeniye i evolyutsiya kul'turnoy grushi). Proceedings of the USSR Academy of Sciences. 1940;28(4):351-354. [in Russian]

23. Sanzol J. Genomic characterization of self-incompatibility ribonucleases (S-RNases) in European pear cultivars and development of PCR detection for 20 alleles. Tree Genetics and Genomes. 2009a;5:393405. DOI: 10.1007/s11295-008-0194-5

24. Sanzol J. Pistil-function breakdown in a new S-allele of European pear, S21 confers self-compatibility. Plant Cell Reports. 2009b;28(3):457-467. DOI: 10.1007/s00299-008-0645-3

25. Sanzol J. Two neutral variants segregating at the gametophytic self‐incompatibility locus of European pear (Pyrus communis L.) (Rosaceae, Pyrinae). Plant Biology. 2010.;12(5):800-805. DOI: 10.1111/j.1438-8677.2009.00277.x

26. Sanzol J., Herrero M.B. Identification of self-incompatibility alleles in pear cultivars (Pyrus communis L.). Euphytica. 2002;128:325-331. DOI: 10.1023/A:1021213905461

27. Sanzol J., Robbins T.P. Combined analysis of S-Alleles in European pear by pollinations and PCR-based S-genotyping; correlation between S-phenotypes and S-RNase genotypes. Journal of American Society for Horticultural Science. 2008;133(2):213-224. DOI: 10.21273/JASHS.133.2.213

28. Sanzol J., Sutherland B.G., Robbins T.P. Identification and characterization of genomic DNA sequences of the S‐ribonuclease gene associated with self‐incompatibility alleles S1 to S5 in European pear. Plant Breeding. 2006;125(5):513-518. DOI: 10.1111/j.1439-0523.2006.01269.x

29. Sassa H., Kakui H., Miyamoto M., Suzuki Y., Hanada T., Ushijima K., Kusaba M., Hirano H., Koba T. S Locus F-Box Brothers: multiple and pollen-specific F-Box genes with S haplotype-specific polymorphisms in apple and Japanese pear. Genetics. 2007;175:1869-1881. DOI: 10.1534/genetics.106.068858

30. Suprun I.I., Tokmakov S.V., Makarkina M.V. Analysis of allelic polymorphism of self-incompatibility in some Russian varieties of pears (Pyrus communis L.) using consensus and S5, S8, allele-specific DNA markers. Polythematic Online Electronic Scientific Journal of Kuban State Agrarian University. 2014;103(09):607-618. [in Russian]

31. Takasaki T., Moriya Y., Okada K., Yamamoto K., Iwanami H., Bessho H., Nakanishi T. cDNA cloning of nine S alleles and establishment of a PCR-RFLP system for genotyping European pear cultivars. Theoretical and Applied Genetics. 2006;112:1543-1552. DOI: 10.1007/s00122-006-0257-7

32. Tuz A.S. Pyrus L. – Pear (Pyrus L. – Grusha). In: Likhonos F.D., Tuz A.S., Lobachev A.J. The USSR flora of cultivated plants. Vol. 14. Pome fruits (Kulturnaya flora SSSR. T. 14. Semechkovyie). Moscow; 1983. p.126-225. [in Russian]

33. Vavilov N.I. Wild progenitors of the fruit trees of Turkestan and the Caucasus and the problem of the origin of fruit trees. Proceedings on Applied Botany, Genetics and Breeding. 1931;26(3):85-107. [in Russian]

34. Wu J., Li M., Li T. Genetic features of the spontaneous self-compatible mutant, ‘Jin Zhui’ (Pyrus bretschneideri Rehd.). PloS One. 2013;8(10):e76509. DOI: 10.1371/journal.pone.0076509

35. Wu J., Wang Y., Xu J., Korban S.S., Fei Z., Tao S., Ming R., Tai S., Khan A.M., Chao Gu C., Yin H., Zheng D., Qi K., Li Y., Wang R., Deng C.H., Kumar S., Chagné D., Li X., Wu J., Huang X., Zhang H., Xie Z., Li X., Zhang M., Li Y., Yue Z., Fang X., Li J., Li L., Jin C., Qin M., Zhang J., Wu X., Ke Y., Wang J., Yang H., Zhang S. Diversification and independent domestication of Asian and European pears. Genome Biology. 2018;19:1-16. DOI: 10.1186/s13059-018-1452-y

36. Zisovich A.H., Stern R., Sapir G., Shafir S., Goldway M. The RHV region of S-RNase in the European pear (Pyrus communis) is not required for the determination of specific pollen rejection. Sexual Plant Reproduction. 2004;17:151-156. DOI: 10.1007/s00497-004-0225-9

37. Zuccherelli S., Tassinari P., Broothaerts W., Tartarini S., Dondini L., Sansavini S. S-Allele characterization in self-incompatible pear (Pyrus communis L.). Sexual Plant Reproduction. 2002;15:153-158. DOI: 10.1007/s00497-002-0145-5