ovoshchiОвощи РоссииVegetable crops of Russia2072-91462618-7132Федеральный научный центр овощеводства10.18619/2072-9146-2026-3-57-61ovoshchi-2998Research ArticleСЕЛЕКЦИЯ, СЕМЕНОВОДСТВО И БИОТЕХНОЛОГИЯ РАСТЕНИЙBREEDING, SEED PRODUCTION AND PLANT BIOTECHNOLOGYИспользование маркеров M281 и М671 для выявления мужской фертильности чеснока Allium sativum L.Use of M281 and M671 markers for detection of male fertility in garlic Allium sativum L.https://orcid.org/0000-0003-1784-3429МартыновВ. В.MartynovV. V.

Виктор Викторович Мартынов – старший научный сотрудник, заведующий лабораторией ДНК маркеров растений 

Viktor V. Martynov – Senior Researcher, Head of the Laboratory of Plant DNA Markers

https://orcid.org/0000-0002-0951-0942ПоляковА. В.PolyakovA. V.

Алексей Васильевич Поляков – профессор, главный научный сотрудник сектора агробиотехнологий лаборатории репродуктивной биотехнологии предбридингового центра

Aleksey V. Polyakov – Professor, Chief Researcher of the Agrobiotechnology Sector of the Reproductive Biotechnology Laboratory of the Pre-breeding Center

https://orcid.org/0009-0009-7987-6286СтружкинЕ. И.StruzhkinE. I.

Евгений Игоревич Стружкин – младший научный сотрудник сектора агробиотехнологий лаборатории репродуктивной биотехнологии предбридингового центра, старший лаборант кафедры общей биологии и биоэкологии факультета естественных наук

Evgeny I. Struzhkin – Junior Researcher of the Agrobiotechnology Sector of the Laboratory of Reproductive Biotechnology of the Pre-breeding Center, Senior Laboratory Assistant at the Department of General Biology and Bioecology of the Faculty of Natural Sciences

ФГБНУ Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии; Всероссийский научно-исследовательский институт овощеводства – филиал ФГБНУ «Федеральный научный центр овощеводства»РоссияAll-Russia Research Institute of Agricultural Biotechnology; All-Russian Research Institute of Vegetable – Branch of the Federal State Budgetary Scientific Institution "Federal Scientific Vegetable Center"Russian FederationВсероссийский научно-исследовательский институт овощеводства – филиал ФГБНУ «Федеральный научный центр овощеводства»РоссияAll-Russian Research Institute of Vegetable – Branch of the Federal State Budgetary Scientific Institution "Federal Scientific Vegetable Center"Russian FederationВсероссийский научно-исследовательский институт овощеводства – филиал ФГБНУ «Федеральный научный центр овощеводства»; Государственный университет просвещенияРоссияAll-Russian Research Institute of Vegetable – Branch of the Federal State Budgetary Scientific Institution "Federal Scientific Vegetable Center"; State University of EducationRussian Federation202611082026035761Copyright © Мартынов В.В., Поляков А.В., Стружкин Е.И., 20262026Мартынов В.В., Поляков А.В., Стружкин Е.И.Martynov V.V., Polyakov A.V., Struzhkin E.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.vegetables.su/jour/article/view/2998

Актуальность. Мужская стерильность чеснока (Allium sativum L.) ограничивает семенное размножение культуры и существенно замедляет селекционный процесс. Одним из предполагаемых молекулярных механизмов стерильности является нарушение деградации каллозной оболочки микроспор, связанное со снижением активности гена AsaNRF1. Цель исследования — изучить полиморфизм промоторной области AsaNRF1 у сортов и селекционных образцов чеснока и оценить применимость маркеров M281 и M671 для предварительного отбора генотипов с различным репродуктивным потенциалом.Материал и методика. Исследована коллекция Allium sativum L., включавшая 3 сорта и 57 сортообразцов. Тотальную ДНК выделяли из молодых листьев двухнедельных растений набором «Сорб-ГМО-Б». Наличие вставок длиной 281 и 671 п.н. определяли методом ПЦР с использованием маркера M281 и разработанной авторами пары праймеров M671. Амплификацию проводили в течение 35 циклов при температуре отжига 58 °C; продукты разделяли электрофорезом в 1%ном агарозном геле. Каждый образец анализировали в двух аналитических повторностях.Результаты. Маркер M281 выявлен у 26 образцов (43,3%), M671 – у 23 (38,3%).Обе вставки детектированы у 17 генотипов (28,3%); только M281 – у 9 (15,0%), только M671 – у 6 (10,0%). У 28 образцов (46,7%) исследованные вставки не обнаружены. Сорт Император имел генотип M281-/M671+, что подтверждает неполное перекрытие диагностических групп.Совместное применение M281 и M671 повышает информативность предварительной молекулярной оценки, однако для надежного выявления фертильных форм результаты необходимо сопоставлять с морфологическими и цитологическими показателями развития репродуктивных органов. 

Relevance. Male sterility in garlic (Allium sativum L.) restricts sexual reproduction and slows breeding.One proposed molecular mechanism is impaired degradation of the callose wall surrounding microspores, associated with reduced activity of the AsaNRF1 gene. The study aimed to characterize polymorphism in the AsaNRF1 promoter region in garlic varieties and breeding accessions and to evaluate M281 and M671 markers for preliminary selection of genotypes with different reproductive potential.Material and methods. A collection of Allium sativum L. comprising 3 varieties and 57 breeding accessions was examined. Total DNA was isolated from young leaves of two-week-old plants using the Sorb-GMO-B kit. Insertions of 281 and 671 bp were detected by PCR with the published M281 marker and an M671 primer pair designed by the authors. Amplification included 35 cycles with annealing at 58 °C; products were separated in a 1% agarose gel. Each accession was analyzed in two analytical replicates.Results. M281 was detected in 26 accessions (43.3%), whereas M671 was detected in 23 (38.3%). Both insertions occurred in 17 genotypes (28.3%); M281 alone in 9 (15.0%), and M671 alone in 6 (10.0%). Neither insertion was detected in 28 accessions (46.7%). The variety Imperator had the M281-/M671+ genotype, confirming incomplete overlap between marker-defined groups.Combined use of M281 and M671 improves preliminary molecular screening; however, reliable identification of fertile garlic forms requires integration of marker data with morphological and cytological assessment of reproductive organs.

Allium sativum L.мужская фертильностьген AsaNRF1ДНК маркерыAllium sativum L.male fertilityAsaNRF1 geneDNA markersReferencesR. Garlic: botany and horticulture. In: Janick J., editor. Horticultural Reviews. Vol. 33. New York: John Wiley & Sons; 2007. p. 123-172.R. Garlic: botany and horticulture. In: Janick J., editor. Horticultural Reviews. Vol. 33. New York: John Wiley & Sons; 2007. p. 123-172.Rivlin R.S. Historical perspective on the use of garlic. The Journal of Nutrition. 2001;131(3):951S-954S. https://doi.org/10.1093/jn/131.3.951SRivlin R.S. Historical perspective on the use of garlic. The Journal of Nutrition. 2001;131(3):951S-954S. https://doi.org/10.1093/jn/131.3.951SShemesh-Mayer E., Kamenetsky-Goldstein R. Traditional and novel approaches in garlic (Allium sativum L.) breeding. In: Al-Khayri J.M., Jain S.M., Johnson D.V., editors. Advances in Plant Breeding Strategies: Vegetable Crops. Cham: Springer; 2021. p. 3-49. https://doi.org/10.1007/978-3-030-66965-2_1Shemesh-Mayer E., Kamenetsky-Goldstein R. Traditional and novel approaches in garlic (Allium sativum L.) breeding. In: Al-Khayri J.M., Jain S.M., Johnson D.V., editors. Advances in Plant Breeding Strategies: Vegetable Crops. Cham: Springer; 2021. p. 3-49. https://doi.org/10.1007/978-3-030-66965-2_1Kuznetsov A.V. Cultivated garlic. Moscow: Selkhozgiz; 1954. 119 p. (In Russ.)Kuznetsov A.V. Cultivated garlic. Moscow: Selkhozgiz; 1954. 119 p. (In Russ.)Kononkov P.F. Methods of obtaining garlic seeds. Sad i ogorod. 1953;(8):38-40. (In Russ.)Kononkov P.F. Methods of obtaining garlic seeds. Sad i ogorod. 1953;(8):38-40. (In Russ.)Katargin M.S., Katargina I.M. Generative reproduction of garlic. Trudy po prikladnoi botanike, genetike i selektsii. 1982;72(3):135-136. (In Russ.)Katargin M.S., Katargina I.M. Generative reproduction of garlic. Trudy po prikladnoi botanike, genetike i selektsii. 1982;72(3):135-136. (In Russ.)Hong C.-J., Etoh T. Fertile clones of garlic (Allium sativum L.) abundant around the Tien Shan Mountains. Breeding Science. 1996;46(4):349-353.Hong C.-J., Etoh T. Fertile clones of garlic (Allium sativum L.) abundant around the Tien Shan Mountains. Breeding Science. 1996;46(4):349-353.Etoh T. True seeds in garlic. Acta Horticulturae. 1997;433:247-255. https://doi.org/10.17660/ActaHortic.1997.433.24Etoh T. True seeds in garlic. Acta Horticulturae. 1997;433:247-255. https://doi.org/10.17660/ActaHortic.1997.433.24Etoh T., Simon P.W. Diversity, fertility and seed production of garlic. In: Rabinowitch H.D., Currah L., editors. Advances in Allium Science. Wallingford: CABI Publishing; 2002. p. 101-118.Etoh T., Simon P.W. Diversity, fertility and seed production of garlic. In: Rabinowitch H.D., Currah L., editors. Advances in Allium Science. Wallingford: CABI Publishing; 2002. p. 101-118.Winiarczyk K., Jaroszuk-Ściseł J., Kupisz K. Characterization of callase (β-1,3-D-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum. Sexual Plant Reproduction. 2012;25:123-131. https://doi.org/10.1007/s00497-012-0184-5Winiarczyk K., Jaroszuk-Ściseł J., Kupisz K. Characterization of callase (β-1,3-D-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum. Sexual Plant Reproduction. 2012;25:123-131. https://doi.org/10.1007/s00497-012-0184-5Shemesh-Mayer E., Winiarczyk K., Błaszczyk L., Kosmala A., Rabinowitch H.D., Kamenetsky R. Male gametogenesis and sterility in garlic (Allium sativum L.): barriers on the way to fertilization and seed production. Planta. 2013;237:103-120. https://doi.org/10.1007/s00425-012-1748-1Shemesh-Mayer E., Winiarczyk K., Błaszczyk L., Kosmala A., Rabinowitch H.D., Kamenetsky R. Male gametogenesis and sterility in garlic (Allium sativum L.): barriers on the way to fertilization and seed production. Planta. 2013;237:103-120. https://doi.org/10.1007/s00425-012-1748-1Ben Michael T.E., Rozenblat L., Faigenboim A., Shemesh-Mayer E., Forer I., Peters R., Klein J.D., Rabinowitch H.D., Kamenetsky-Goldstein R. From embryo to adult: low temperatures affect phase transitions of Allium sativum L. from germination to flowering. Agronomy. 2020;10(11):1651. https://doi.org/10.3390/agronomy10111651.Ben Michael T.E., Rozenblat L., Faigenboim A., Shemesh-Mayer E., Forer I., Peters R., Klein J.D., Rabinowitch H.D., Kamenetsky-Goldstein R. From embryo to adult: low temperatures affect phase transitions of Allium sativum L. from germination to flowering. Agronomy. 2020;10(11):1651. https://doi.org/10.3390/agronomy10111651.Piazena H. The effect of altitude upon the solar UV-B and UVA irradiance in the tropical Chilean Andes. Solar Energy. 1996;57(2):133-140. https://doi.org/10.1016/S0038-092X(96)00049-7Piazena H. The effect of altitude upon the solar UV-B and UVA irradiance in the tropical Chilean Andes. Solar Energy. 1996;57(2):133-140. https://doi.org/10.1016/S0038-092X(96)00049-7Llorens L., Badenes-Pérez F.R., Julkunen-Tiitto R., Zidorn C., Fereres A., Jansen M.A.K. The role of UV-B radiation in plant sexual reproduction. Perspectives in Plant Ecology, Evolution and Systematics. 2015;17(3). https://doi.org/10.1016/j.ppees.2015.03.001Llorens L., Badenes-Pérez F.R., Julkunen-Tiitto R., Zidorn C., Fereres A., Jansen M.A.K. The role of UV-B radiation in plant sexual reproduction. Perspectives in Plant Ecology, Evolution and Systematics. 2015;17(3). https://doi.org/10.1016/j.ppees.2015.03.001Feng H., An L., Tan L., Hou Z., Wang X. Effect of enhanced ultraviolet-B radiation on pollen germination and tube growth of 19 taxa in vitro. Environmental and Experimental Botany. 2000;43(1):45-53.Feng H., An L., Tan L., Hou Z., Wang X. Effect of enhanced ultraviolet-B radiation on pollen germination and tube growth of 19 taxa in vitro. Environmental and Experimental Botany. 2000;43(1):45-53.Shemesh-Mayer E., Ben-Michael T., Kimhi S., Forer I., Rabinowitch H.D., Kamenetsky R. Effects of different temperature regimes on flower development, microsporogenesis and fertility in bolting garlic (Allium sativum). Functional Plant Biology. 2015;42:514-526.Shemesh-Mayer E., Ben-Michael T., Kimhi S., Forer I., Rabinowitch H.D., Kamenetsky R. Effects of different temperature regimes on flower development, microsporogenesis and fertility in bolting garlic (Allium sativum). Functional Plant Biology. 2015;42:514-526.McCormick S. Control of male gametophyte development. The Plant Cell. 2004;16:S142-S153. https://doi.org/10.1105/tpc.016659McCormick S. Control of male gametophyte development. The Plant Cell. 2004;16:S142-S153. https://doi.org/10.1105/tpc.016659Mamun E.A., Cantrill L.C., Overall R.L., Sutton B.G. Cellular organisation in meiotic and early post-meiotic rice anthers. Cell Biology International. 2005;29:903-913. https://doi.org/10.1016/j.cellbi.2005.08.001Mamun E.A., Cantrill L.C., Overall R.L., Sutton B.G. Cellular organisation in meiotic and early post-meiotic rice anthers. Cell Biology International. 2005;29:903-913. https://doi.org/10.1016/j.cellbi.2005.08.001Wan L., Zha W., Cheng X., Liu C., Lv L., Liu C., et al. A rice β-1,3-glucanase gene Osg1 is required for callose degradation in pollen development. Planta. 2011;233:309-323. https://doi.org/10.1007/s00425-010-1301-zWan L., Zha W., Cheng X., Liu C., Lv L., Liu C., et al. A rice β-1,3-glucanase gene Osg1 is required for callose degradation in pollen development. Planta. 2011;233:309-323. https://doi.org/10.1007/s00425-010-1301-zNovák F.J. Tapetal development in the anthers of Allium sativum L. and Allium longicuspis Regel. Experientia. 1972;28(11):1380-1381. https://doi.org/10.1007/BF01965358Novák F.J. Tapetal development in the anthers of Allium sativum L. and Allium longicuspis Regel. Experientia. 1972;28(11):1380-1381. https://doi.org/10.1007/BF01965358Gonzalez L.G., Ford-Lloyd B.V. Facilitation of wide-crossing through embryo rescue and pollen storage in interspecific hybridization of cultivated Allium species. Plant Breeding. 1987;98:318-322. https://doi.org/10.1111/j.1439-0523.1987.tb01136.xGonzalez L.G., Ford-Lloyd B.V. Facilitation of wide-crossing through embryo rescue and pollen storage in interspecific hybridization of cultivated Allium species. Plant Breeding. 1987;98:318-322. https://doi.org/10.1111/j.1439-0523.1987.tb01136.xOhsumi C., Kojima A., Hinata K., Etoh T., Hayashi T. Interspecific hybrid between Allium cepa and Allium sativum. Theoretical and Applied Genetics. 1993;85:969-975. https://doi.org/10.1007/BF00215036.Ohsumi C., Kojima A., Hinata K., Etoh T., Hayashi T. Interspecific hybrid between Allium cepa and Allium sativum. Theoretical and Applied Genetics. 1993;85:969-975. https://doi.org/10.1007/BF00215036.Kamenetsky R., Rabinowitch H.D. Florigenesis. In: Rabinowitch H.D., Currah L., editors. Allium Crop Science: Recent Advances. Wallingford: CABI Publishing; 2002. p. 31-57.Kamenetsky R., Rabinowitch H.D. Florigenesis. In: Rabinowitch H.D., Currah L., editors. Allium Crop Science: Recent Advances. Wallingford: CABI Publishing; 2002. p. 31-57.Leubner-Metzger G., Meins F. Jr. Functions and regulation of plant β-1,3-glucanases (PR-2). In: Datta S.K., Muthukrishnan S., editors. Pathogenesis-Related Proteins in Plants. Boca Raton: CRC Press; 1999. p. 49-76.Leubner-Metzger G., Meins F. Jr. Functions and regulation of plant β-1,3-glucanases (PR-2). In: Datta S.K., Muthukrishnan S., editors. Pathogenesis-Related Proteins in Plants. Boca Raton: CRC Press; 1999. p. 49-76.Fei H., Sawhney V.K. MS32-regulated timing of callose degradation during microsporogenesis in Arabidopsis is associated with the accumulation of stacked rough ER in tapetal cells. Sexual Plant Reproduction. 1999;12:188-193.Fei H., Sawhney V.K. MS32-regulated timing of callose degradation during microsporogenesis in Arabidopsis is associated with the accumulation of stacked rough ER in tapetal cells. Sexual Plant Reproduction. 1999;12:188-193.Shi X., Han X., Lu T.-G. Callose synthesis during reproductive development in monocotyledonous and dicotyledonous plants. Plant Signaling & Behavior. 2016;11:e1062196. https://doi.org/10.1080/15592324.2015.1062196Shi X., Han X., Lu T.-G. Callose synthesis during reproductive development in monocotyledonous and dicotyledonous plants. Plant Signaling & Behavior. 2016;11:e1062196. https://doi.org/10.1080/15592324.2015.1062196Pu Y., Hou L., Guo Y., Ullah I., Yang Y., Yue Y. Genome-wide analysis of the callose enzyme families of fertile and sterile flower buds of the Chinese cabbage (Brassica rapa L. ssp. pekinensis). FEBS Open Bio. 2019;9:1432-1449. https://doi.org/10.1002/2211-5463.12685Pu Y., Hou L., Guo Y., Ullah I., Yang Y., Yue Y. Genome-wide analysis of the callose enzyme families of fertile and sterile flower buds of the Chinese cabbage (Brassica rapa L. ssp. pekinensis). FEBS Open Bio. 2019;9:1432-1449. https://doi.org/10.1002/2211-5463.12685Liu Z., Duan N., Yang Z., Yue L., Fei Z., Kong S. Identification of male fertility gene AsaNRF1 and molecular marker development in cultivated garlic (Allium sativum L.). Frontiers in Plant Science. 2024;15:1419260. https://doi.org/10.3389/fpls.2024.1419260Liu Z., Duan N., Yang Z., Yue L., Fei Z., Kong S. Identification of male fertility gene AsaNRF1 and molecular marker development in cultivated garlic (Allium sativum L.). Frontiers in Plant Science. 2024;15:1419260. https://doi.org/10.3389/fpls.2024.1419260

The authors declare that there are no conflicts of interest present.