RAPD-ANALYSIS IN THE SYSTEM OF COMPLEX ECOLOGICAL AND ANALYTICAL MONITORING OF RED CLOVER POPULATIONS OF MEADOW GRASS PHYTOCOENOSIS OF THE SUMY REGION
Keywords:
Trifolium pratense L., RAPD analysis, natural herbaceous phytocoenoses, populations, genetic diversity, primer
Abstract
The article is devoted to the study of populations of red clover (Trifolium pratense L.) in natural herbaceous phytocoenoses in Sumy city and Sumy region (Nature Reserve Mykhailivska Tsilyna) using molecular biological methods, in particular PCR diagnostics with the participation of oligonucleotide RAPD primers, in order to establish the genetic diversity of the species at the population level. The study of the genetic structure of species populations is a component of a comprehensive population analysis, which, among other things, includes the study of age and ontogenetic, sexual, size and vitality structures, reproduction and growth processes, which allows us to reveal the mechanisms of species resistance in certain growth conditions, assess the prospects for its existence and draw conclusions about the genetic diversity of its populations. From an agronomic point of view, red clover is an extremely valuable field crop, as it is used as fodder for livestock, and its ability to fix atmospheric nitrogen in the soil provides companion grasses with this nutrient. For this reason, meadow clover is a good precursor for cereals. Within natural phytocoenoses, the share of meadow clover can be up to 20% of the grass stand, which, together with other grass species, forms highly productive meadows. The study of natural communities of red clover at the molecular (genomic) level allows us to assess the degree of biodiversity of the species, the breeding potential of varieties as a source of target genes for selection in particular and genetic plasma in general. Molecular analysis will also make it possible to estimate the loss of genetic resources of the red clover as a result of aggressive military operations. The object of the study was red clover samples collected in different locations of herbaceous phytocoenoses in Sumy and the Nature Reserve Mykhailivska Tsilyna. Genetic material (DNA) was extracted using salt-enzyme extraction, followed by purification and nucleic acid precipitation. DNA amplification was performed using RAPD primers Ver_1 AATCGGGCTG and Ver_2 GTTGCGATCC followed by visualization of reaction products in an agarose gel in the presence of ethidium bromide. The nature of the amplicons indicates the satisfactory quality of the nucleic acid preparations. Amplification of fragments revealed a high degree of polymorphism at both markers (17 polymorphic loci out of 18), and the study of the affinity of red clover samples using cluster analysis established a close relationship between samples growing within the city of Sumy in adjacent locations, as well as the Nature Reserve Mykhailivska Tsilyna.
References
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3. Bondarieva, L.M., Kyrylchuk, K.S., Skliar, V.H., Tikhonova, O.M., Zhatova, H.O. & Bashtovyi, M.G. (2019). Population dynamics of the typical meadow species in the conditions of pasture digression in flooded meadows. Ukrainian Journal of Ecology, 9 (1), 204–211.
4. Bondarieva, L. M., & Kyrylchuk, K. S. (2023). Struktura populiatsii luchnykh roslyn na zaplavnykh lukakh lisostepovoi zony za umov vypasannia ta sinokosinnia [Structure of meadow plant populations in flood meadows of the forest-step zone under grazing and mowing conditions]. Bulletin of Sumy National Agrarian University. The Series: Agronomy and Biology, 51(1), 3–13 (in Ukrainian). doi: 10.32782/agrobio.2023.1.1
5. Collins, R. P., Helgadóttir, Á., Frankow-Lindberg, B. E., Skøt, L., Jones, C. & Skøt, K. P. (2012). Temporal changes in population genetic diversity and structure in red and white clover grown in three contrasting environments in northern Europe. Annals of Botany, 110, 1341–1350. doi: 10.1093/aob/mcs058
6. De Vega, J. J., Sarah Ayling, Matthew Hegarty, Dave Kudrna, Jose L. Goicoechea, Åshild Ergon, Odd A. Rognli, Charlotte Jones, Martin Swain, Rene Geurts, Chunting Lang, Klaus F. X. Mayer, Stephan Rössner, Steven Yates, Kathleen J. Webb, Iain S. Donnison, Giles E. D. Oldroyd, Rod A. Wing, Mario Caccamo, Wayne Powell, Michael T. Abberton & Leif Skøt. (2015). Red clover (Trifolium pratense L.) draft genome provides a platform for trait improvement. Scientific Reports, 5(17394), 1–2. doi: 10.1038/srep17394
7. Dluhošová, J., Ištvánek, J., Nedĕlník, J., & Řepková, J. (2017). Red Clover (Trifolium pratense) and Zigzag Clover (T. medium) – A Picture of Genomic Similarities and Differences. Frontiers in Plant Science, 9(724), 1–14. doi: 10.3389/fpls.2018.00724
8. Drebot, O. V., Kudryk, A. P., Pitsil, A. O. & Lukianenko, O. P. (2018). Pidvyshchennia bioriznomanittia roslynnykh formatsii pid chas zemleustroiu ahrolandshaftu [Increasing the biodiversity of plant formations during land management of the agrolandscape]. Naukovyi visnyk NLTU Ukrainy, 28(3), 18–21, (in Ukrainian).
9. Duhar, Iu. M. (2013). Henetychni vzaiemovidnosyny ukrainskykh sortiv koniushyny luchnoi za DNK-markeramy [Genetic relationships of Ukrainian meadow clover varieties according to DNA markers]. Biolohichni systemy, 5(4), 479–483 (in Ukrainian).
10. Duhar, Iu. M. & Popov, V. N. (2013). Genetic structure and diversity of Ukrainian red clover cultivars revealed by microsatellite markers. Open Journal of Genetics, 3, 235–242. doi: 10.4236/ojgen.2013.34026
11. Ištvánek, J., Dluhošová, J., Dluhoš, P., Pátková, L., Nedĕlník, J., & Řepková, J. (2017). Gene Classification and Mining of Molecular Markers Useful in Red Clover (Trifolium pratense) Breeding. Frontiers in Plant Science, 8, 1–16. doi: 10.3389/fpls.2017.00367
12. Jones, C., De Vega, J., Lloyd, D., Hegarty, M., Ayling, S., Powell, W. & Skøt, L. (2020). Population structure and genetic diversity in red clover (Trifolium pratense L.) germplasm. Scientific Reports, 10 (8364), 1 – 12. doi: 10.1038/s41598-020-64989-z
13. Kim C.S., Lee C.H., Shin J.S., Chung Y.S., Hyung N.I. (1997). A simple and rapid method for isolation of high-quality genomic DNA from fruit trees and conifers using PVP. Nucleic Acids Research. 25 (5), 1085–1086.
14. Kovalenko, I., Skliar, Iu., Klymenko, H. & Kovalenko, N. (2019). Vitality Structure of the Populations of Vegetative Motile Plants of Forest Ecosystems of the North-East of Ukraine. The Open Agriculture Journal, 13, 125–132. doi: 10.2174/1874331501913010125.
15. Kyrylchuk, K., Skliar, V., Tykhonova, O. & Kobzhev, O. (2021). Vitality dynamics of populations of some legume species in floodplain meadows of the Psel river basin under grazing and haymaking (Ukraine). Scientific Papers. Series B, Horticulture, LXV(1), 406–414.
16. Kyrylchuk, K.S. & Bashtovyi, M.H. (2018). Kompleksnyi analiz populiatsii Trifolium pratense L. na zaplavnykh lukakh lisostepovoi zony Ukrainy [Comprehensive analysis of Trifolium pratense L. populations in floodplain meadows of the forest-steppe zone of Ukraine]. Naukovyi visnyk Skhidnoievropeiskoho natsionalnoho universytetu imeni Lesi Ukrainky, 4 (377), 5–15. doi: 10.29038/2617-4723-2018-377-5-15
17. Kyrylchuk K.S. (2017). Vitalitetna struktura populiatsii Trifolium pratense L. ta Trifolium repens L. na zaplavnykh lukakh v umovakh hospodarskoho korystuvannia [Vitality structure of Trifolium pratense L. and Trifolium repens L. populations on floodplain meadows under conditions of economic use]. Visnyk SNAU: Seriia «Ahronomiia i biolohiia», 2 (33), 12–16 (in Ukrainian).
18. Kyrylchuk K.S. (2007). Vikova ta vitalitetna struktury populiatsii bobovykh na zaplavnykh lukakh r. Psel (Lisostepova zona) v umovakh hospodarskoho korystuvannia [Age and vitality structures of legume populations on the floodplain meadows of the Psel River (Forest-Steppe zone) under conditions of economic use]. Ukrainskyi botanichnyi zhurnal, 64(3), 418–425 (in Ukrainian).
19. Naydenova, G. & Vasileva, V. (2019). Comparative evaluation of diploid and tetraploid red clover genotypes in a flat area of Northern Bulgaria. Journal of Central European Agriculture, 20 (3), 919 – 927. DOI: /10.5513/JCEA01/20.3.2231
20. Osterman, J., Hammenhag, C., Ortiz, R. & Geleta, M. (2021). Insights Into the Genetic Diversity of Nordic Red Clover (Trifolium pratense) Revealed by SeqSNP-Based Genic Markers. Frontiers in Plant Science, 12, 1 – 18. doi: 10.3389/fpls.2021.748750
21. Osterman, J., Hammenhag, C., Ortiz, R. & Geleta, M. (2022). Discovering candidate SNPs for resilience breeding of red clover. Frontiers in Plant Science, 13, 1 – 17. doi: 10.3389/fpls.2022.997860
22. Petkovic, B., Przulj, N., Radic, V. & Mirosavljevic, M. (2017). Comparative study of seed yield and seed quality of advanced lines and commercial varieties of red clover (Trifolium pratense L.). Legume Research, 40(6), 1066–1071. doi: 10.18805/LR-360
23. Petrauskas, G., Norkevičienė, E. & Baistruk-Hlodan, L. (2023). Genetic Differentiation of Red Clover (Trifolium pratense L.) Cultivars and Their Wild Relatives. Agriculture, 13 (1008), 1 – 13. doi: 10.3390/agriculture13051008
24. Petrauskas, G., Stukonis, V. & Norkevičienė, E. (2020). Defining a Phenotypic Variability and Productivity in Wild Type Red Clover Germplasm. Journal of Agricultural Science, 12 (9), 52–61. doi: 10.5539/jas.v12n9p52
25. Polevoy, A. M., Bozhko, L. E. & Barsukova, E. A. (2021). The influence of weather conditions on the formation of meadow clover productivity on the right bank of the Forest-Steppe of Ukraine. Bulletin of Poltava State Agrarian Academy, 2, 38–45. doi: 10.31210/visnyk2021.02.04
26. Řepková, J. & Nedělník, J. (2014). Modern Methods for Genetic Improvement of Trifolium pratense. Czech Journal of Genetics and Plant Breeding, 50 (2), 92–99.
27. Skliar, V., Kovalenko, I., Skliar, Iu. & Sherstiuk, M. (2019). Vitality structure and its dynamics in the process of natural reforestation of Quercus robur L. AgroLife Journal, 8(1), 233–241. Access mode: https://agrolifejournal.usamv.ro/index.php/agrolife/article/view/441
28. Tucak, M., Čupić, T., Popović, S., Stjepanović, M., Gantner, R. & Meglič, V. (2009). Agronomic Evaluation and Utilization of Red Clover (Trifolium pratense L.) Germplasm. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 37 (2), 206–210.
29. Trněný, O., Vlk, D., Macková, E., Matoušková, M., Řepková, J., Nedělník, J., Hofbauer, J., Vejražka, K., Jakešová, H., Jansa, J., Piálek, L. & Knotová, D. (2019). Allelic Variants for Candidate Nitrogen Fixation Genes Revealed by Sequencing in Red Clover (Trifolium pratense L.). International Journal of Molecular Science, 20 (5470), 1 – 26. doi: 10.3390/ijms20215470
30. Tykhonova O., Skliar V., Sherstiuk M., Kyrylchuk K., Butenko A., Bashtovyi M. (2021). Analysis of Setaria glauca (L.) P. Beauv. Population’s Vital Parametres in grain Agrophytocenoses. Environmental Research, Engineering and Management, 2021, 77(1), 33‒46. doi: 10.5755/j01.erem.77.1.25489
31. Zanotto, S. (2022). A study of freezing tolerance in red clover (Trifolium pratense L.). Norwegian University of Life Sciences, Faculty of Biosciences, Department of Plant Science, 160.
32. Zanotto, S., Amdahl, H. & Ergon, Å. (). Red clover adaptation to a Nordic climate. Vleugels Exploiting genetic diversity of forages to fulfil their economic and environmental roles. Proceedings of the 34th Meeting of the EUCARPIA Fodder Crops and Amenity Grasses Sectionin cooperation with the EUCARPIA Festulolium Working GroupFreising 6–8 September, 2021, 33 – 36. doi: 10.5507/vup.21.24459677.08
33. Zanotto, S., Palmé, A., Helgadóttir, Á., Daugstad, K., Isolahti, M., Öhlund, L., Marum, P., Ahlin, M., Merja, M., Odd, V., Rognli, A. & Ergon, Å. (2021). Trait characterization of genetic resources reveals useful variation for the improvement of cultivated Nordic red clover. Journal of Agronomy and Crop Science, 3, 492–503. doi: 10.1111/jac.12487
34. Verwimp, C., Ruttink, T., Muylle, H., Glabeke, S. V., Cnops, G., Quataert, P., Olivier, H. & Roldán-Ruiz, I. (2018). Temporal changes in genetic diversity and forage yield of perennial ryegrass in monoculture and in combination with red clover in swards. Plos One, 8, 1–25. doi: 10.1371/journal.pone.0206571
35. Yan, Z., Sang, L., Ma, Y., He, Y., Sun, J., Ma, L., Li, S., Miao, F., Zhang, Z., Huang, J., Wang, Z. & Yang, G. (2022). A de novo assembled high‑quality chromosome‑scale Trifolium pratense genome and fine‑scale phylogenetic analysis. BMC Plant Biology, 22(332), 1 – 12. doi: 10.1186/s12870-022-03707-5
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Published
2024-10-23
How to Cite
Kyrylchuk, K., Bakumenko, O., & Vereshchahin, I. V. (2024). RAPD-ANALYSIS IN THE SYSTEM OF COMPLEX ECOLOGICAL AND ANALYTICAL MONITORING OF RED CLOVER POPULATIONS OF MEADOW GRASS PHYTOCOENOSIS OF THE SUMY REGION. Bulletin of Sumy National Agrarian University. The Series: Agronomy and Biology, 56(2), 34-41. https://doi.org/10.32782/agrobio.2024.2.5
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