Obtaining and evaluating breeding material for the creation of F1 hybrids of Chinese cabbage (B. rapa ssp. pekinensis) with resistance to stress factors

Relevance and purpose of the study. Chinese cabbage is a vegetable crop with a high content of vitamins and low calorie content, valued for its ability to receive several crops per year and high productivity. Recently, there has been a reduction in the supply of the vegetable to Russia. There are less than 70 varieties and hybrids of the crop in the State Register of Selection Achievements, many of them are susceptible to the most important diseases. It is necessary to meet consumer demand with new commercial hybrids with resistance to stressors. The aim of the study was to evaluate and obtain genotypes with a set of valuable economic traits, including resistance to clubroot, powdery mildew, and early bolting.
Materials and methods. The Chinese cabbage lines of various degrees of inbred and DH, as well as hybrid combinations from crossing these lines, were used as plant material. The lines were obtained in 2019-2020, the breeding material for them was selected on the ground infected with clubroot and with tolerance to tip burn of heads. To achieve the goal, the following methods were applied: plants cultivation; DNA isolation (CTAB method); PCR analysis; electrophoresis and visualization of results; productivity assessment and dispersion analysis; obtaining doubled haploids in culture of isolated microspores in vitro, assessment of resistance/susceptibility to powdery mildew.
Results. 2 genotypes with high productivity and alignment in turnover summer-autumn 2022 and a genotype exceeding 3 standards in head weight in turnover spring-summer 2022 were recommended; as a result of molecular genotyping, 41 lines were differentiated according to the clubroot resistance gene CRb; DH plants for 4 genotypes with resistance to early bolting were obtained, their responsiveness to embryogenesis was studied; 18 lines were evaluated for resistance to powdery mildew, 7 genotypes with resistance to the disease were identified.

1. Артемьева А.М., Соловьева А.Е. Генетическое разнообразие и биохимическая ценность капустных овощных растений рода Brassica L. Вестник НГАУ. Биология. 2018;4(49):50-61. DOI:10.31677/2072-6724-2018-49-4-50-61 [Artem'eva A.M., Soloveva A.E. Genetic diversity and biochemical value of cabbage vegetable plants of the genus Brassica L. Vestnik NGAU. Biologiya. 2018;4(49):50-61. DOI:10.31677/2072-6724-2018-49-4-50-61 (In Russ.)]

2. Yuan J., Shen C., Yuan R. et al. Identification of genes related to tipburn resistance in Chinese cabbage and preliminary exploration of its molecular mechanism. BMC Plant Biol. 2021;567(21):1-12. DOI:10.1186/s12870-021-03303-z

3. Kamiński P., Podwyszyńska, M., Starzycki M. et al. Interspecific hybridisation of cytoplasmic male-sterile rapeseed with Ogura cytoplasm and Brassica rapa var. pekinensis as a method to obtain male-sterile Chinese cabbage inbred lines. Euphytica. 2016;(208):519–534. DOI:10.1007/s10681-015-1595-9

4. Заставнюк А.Д., Монахос Г.Ф., Вишнякова А.В., Миронов А.А., Монахос С.Г. Генотипирование устойчивости к киле и оценка комбинационной способности капусты пекинской. Известия Тимирязевской сельскохозяйственной академии. 2022;(5):77- 91. DOI:10.26897/0021-342X-2022-5-77-91. EDN WDBKXQ. [Zastavnyuk A.D., Monakhos G.F., Vishnyakova A.V., Mironov A.A., Monakhos S.G. Chinese cabbage clubroot resistance genotyping and evaluation of combining ability. Izvestiya of Timiryazev Agricultural Academy. 2022;(5):77-91. (In Russ.)] EDN WDBKXQ.

5. Беренсен Ф.А., Антонова О.Ю., Артемьева А.М. Достижения и перспективы молекулярно-генетического маркирования устойчивости к некоторым патогенам у видов рода Brassica L. Вавиловский журнал генетики и селекции. 2019;23(6):656-666. DOI:10.18699/VJ19.538. [Berensen F.A., Antonova O.Yu., Artemeva A.M. Achievements and prospects of molecular genetic marking of resistance to certain pathogens in species of the genus Brassica L. Vavilov Journal of Genetics and Breeding. 2019;23(6):656-666. DOI:10.18699/VJ19.538 (In Russ.)]

6. Монахос С.Г., Воронина А.В., Байдина А.В., Зубко О.Н. Селекция растений на устойчивость–основа защиты от болезней в органическом земледелии. Картофель и овощи. 2019;(6):38-40. DOI: 10.25630/PAV.2019.92.83.009 [Monahos S.G., Voronina A.V., Bajdina A.V., Zubko O.N. Plant breeding for sustainability is the basis of protection against diseases in organic farming. Potato and vegetables. 2019;6:38-40 (In Russ.). DOI: 10.25630/PAV.2019.92.83.009 (In Russ.)]

7. Монахос Г.Ф., Монахос С.Г. Капуста пекинская. Биологические особенности, генетика, селекция и семеноводство. Монография. Москва: Издательство РГАУ-МСХА имени КА Тимирязева; 2009. [Monakhos G.F., Monakhos S.G. Chinese cabbage Brassica rapa L.Em. Metzg. ssp. pekinensis (Lour.) Hanelt. Biological characteristics, genetics, breeding and seed production. Monograph]. M.: Izd-vo RGAU-MSKhA imeni K.A. Timiryazeva. 2009. 182 р. (In Russ.)]

8. Malinowski R., Truman W., Blicharz S. Genius architect or clever thief—How Plasmodiophora brassicae reprograms host development to establish a pathogen oriented physiological sink. Molecular Plant-Microbe Interactions. 2019;32(10):1259–1266. DOI: 10.1094/MPMI-03-19-0069-CR

9. Wenjing R., Zhiyuan L., Fengqing H. et al. Utilization of Ogura CMS germplasm with the clubroot resistance Y. gene by fertility restoration and cytoplasm replacement in Brassica oleracea L. Horticulture research. 2020;(7):61. DOI:10.1038/s41438-020-0282-8

10. Yoshikawa H. Studies on breeding of clubroot resistance in cole [Cruciferae] crops. Bulletin of the National Research Institute of Vegetables, Ornamental Plants and Tea. Series A.(Japan). 1993;(7):1–165. ISSN: 0916-684X.

11. Matsumoto E., Yasui С., Ohi M., Tsukada M. Linkage analysis of RFLP markers for clubroot resistance and pigmentation in Chinese cabbage (Brassica rapa ssp. pekinensis. Euphytica. 1998;104:79:86. DOI: 10.1023/A:1018370418201

12. Kuginuki Y., Yoshikawa H., Hirai M. Variation in virulence of Plasmodiophora brassicae in Japan tested with clubroot-resistant cultivars of Chinese cabbage (Brassica rapa L. ssp. pekinensis). European Journal of Plant Pathology. 1999;(105):327-332. DOI: 10.1023/A:1008705413127

13. Hirai M. Genetic analysis of clubroot resistance in Brassica rapa. Breeding science. 2006;(56):223–229. DOI: 10.1270/jsbbs.56.223

14. Monakhos S.G., Li N.M. Breeding value of clubroot resistance genes of Brassica rapa L. lines and effectiveness of molecular markers of mapped loci. Izvestiya of Timiryazev Agricultural Academy (TAA). 2013;(6):68-81. (In Russ.)]

15. Ueno H.. et al. Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa. Plant molecular biology. 2012;80:621–629. DOI: 10.1007/s11103–012–9971–5.

16. Kato T., Hatakeyama K., Fukino N. et al. Fine mapping of the clubroot resistance gene CRb and development of a useful selectable marker in Brassica rapa. Breeding science. 2013;63(1):116- 124. DOI: 10.1270/jsbbs.73-1cover

17. Suwabe K., Tsukazaki H., Iketani H., Hatakeyama K., Fujimura M., Nunome T., Fukuoka H., Matsumoto S., Hirai M. Identification of two loci for resistance to clubroot (Plasmodiophora brassicae Woronin) in Brassica rapa L. Theoretical Application Genetic. 2003.107:997-1002. DOI: 10.1007/s00122-003-1309-x

18. Nguen M.L., Monakhos G.F., Komahin R.A., Monakhos S.G. The new clubroot resistance locus is located on chromosome A05 in Chinese Cabbage (Brassica rapa L.). Genetika. 2018;54:296–304 (In Russ.) DOI: 10.7868/S0016675818030037.

19. Monakhos S.G., Li N.M. Breeding of Chinese cabbage using biotechnological methods. Potato and vegetables. 2014;9:34-35. (In Russ.).

20. Dong Y.Q. Influencing factors and physiochemical changes of embryogenesis through in vitro isolated microspore culture in Brassica species. Biologia. 2021;(76):2629-2654. DOI:10.1007/s11756-021-00721-0

21. Шмыкова Н.А., Шумилина Д.В., Супрунова Т.П. Получение удвоенных гаплоидов у видов рода Brassica L. Вавиловский журнал генетики и селекции. 2015;19(1):111-120. DOI:10.18699/VJ15.014. [Shmykova N. A., Shumilina D.V., Suprunova T.P. Obtaining doubled haploids in species of the genus Brassica L. Vavilov Journal of Genetics and Breeding. 2015;19(1):111-120. DOI:10.18699/VJ15.014 (In Russ.)]

22. Monakhos S., Uwiragiye A., Zhao J., Zhang N., Bonnema G. Generation of doubled haploids through microspore culture from vegetable and oilseed Brassica rapa crops. Izvestiya of Timiryazev Agricultural Academy (TAA). 2010;7:128-135 (In Russ.).

23. Krishnia S.K., Saharan G.S.; Singh D. Genetics of Alternaria blight resistance in inter and intraspecific crosses of Brassica juncea and Brassica carinata. Annals of Biology. 2000;16(2):211- 216. ISSN: 0970-0153.

24. Conn K.L., Tewari J.P., Awasthi R.P. A disease assessment key for Alternaria blackspot in rapeseed and mustard. Disease des plantes Survey'au Canada. 1990;70(1):19–22. ISSN: 0008-476X

25. Monakhos S.G. Creation of pure lines-doubled cabbage haploids in the culture of isolated microspores and selection of F1 hybrids based on modern biotechnology methods: methodological recommendations]. M.. 2014. 44 р. (In Russ.)

26. Custers J.B.M. Microspore culture in rapeseed (Brassica napus L.). Doubledhaploid production in crop plants. Academic Publisher. 2003. Р.185-194. DOI: 10.1007/978-94-017-1293-4_29

27. Murray M.G. and Thompson W.F. Rapid isolation of high molecular weight plant DNA. Nucleic acids research. 1980;8(19):4321- 4326. DOI: 10.1093/nar/8.19.4321

28. Kato T., Hatakeyama K., Fukino N. & Matsumoto S. Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.). Breeding science. 2012;62(3):282-287. DOI:10.1270/jsbbs.62.282

29. Griffing B.А. Concept of general and specific combining ability in relation to diallel crossing systems. Australian journal of biological sciences. 1956;9(4):463-493. DOI: 10.1071/BI9560463

30. Pechan P.M., Keller W.A. Identification of potentially embryogenic microspores in Brassica napus. Physiologia Plantarum. 1988;74(2):377-384. DOI: 10.1111/j.1399-3054.1988.tb00646.x

31. Monakhos S.G. Integration of modern biotechnological and classical methods in vegetable crop breeding. Moscow, Russian State Agrarian University – Moscow Timiryazev Agricultural Academy. 2016. 335 р. (In Russ.).

32. Buczacki S., Toxopeus H., Mattusch P., Johnston T., Dixon G. & Hobolth L. Study of physiologic specialization in Plasmodiophora brassicae: proposals for attempted rationalization through an international approach. Transactions of the British Mycological Society. 1975;65(2):295-303. DOI:10.1016/S0007-1536(75)80013-1