SUMMARY. The Crucifers family (Brassicaceae) includes a large number of economically important crops, particularly Brassica rapa, which is a widely-used model plant for molecular genetic studies of oilseeds. B. rapa is a highly polymorphic species that includes a large number of genetically distinct subspecies. Considering this fact, intraspecific hybridization of B. rapa subspecies is considered a promising breeding approach aimed on in-creasing genetic diversity of the crop. Previously, we have shown that one of such hybrids, oil tyfon (B. rapa subsp. oleifera f. biennis × (subsp. rapifera × pekinensis)), could be a valuable oil feedstock due to its increased productivity. However, obtaining hybrids and their subsequent breeding would require the involvement of diverse molecular marker systems. So far, the method of estimating the length polymorphism of the first (TBP) and second (cTBP) introns of β-tubulin has demonstrated its high accuracy and reliability in the identification (DNA-barcoding) of flowering plants taxonomic units at different levels. In the present study, we evaluated the productivity of such hybrid oilseed crop as tyfon, as well as carried out DNA-barcoding of various hybrid lines of tyfon and its parental B. rapa subspecies using on β-tubulin intron length polymorphism assessment approach. Based on the data of the molecular genetic analysis, which included the assessment of length polymorphism of 1st and 2nd introns of β-tubulin genes, we were able to confirm the origin of oil tyfon hybrid from Dutch leaf tyfon (B. rapa subsp. rapifera × pekinensis) and winter turnip rape (B. rapa subsp. oleifera) with a high confidence. Along with that, it was possible to differentiate var. glabra and var. laxa accession of napa cabbage (B. rapa subsp. pekinensis) for the first time using the combined TBP and cTBP analyses. A variation in the number of amplified regions of β-tubulin introns was noted in different genotypes, however these differences did not appear to be a specific feature a particular subspecies/hybrid. This suggests that B. rapa hybrids most likely do not differ in ploidy, compared to their parental genotypes. In addition, it was shown that the mentioned oil tyfon hybrid lines of Ukrainian breeding show a significant level of morphological variation, despite their common breeding pedigree.
Keywords: Brassicaceae, Brassica rapa, ILP, genotyping, DNA-barcoding, hybrid oilseed crops
Full text and supplemented materials
References
Allainguillaume, J., Alexander, M., Bullock, J.M., et al., Fitness of hybrids between rapeseed (Brassica napus) and wild Brassica rapa in natural habitats, Mol. Ecol., 2006, vol. 15, no. 4, pp. 1175–1184. https://doi.org/10.1111/j.1365-294X.2006.02856.x
Bardini, M., Lee, D., Donini, P., et al., Tubulin-based polymorphism (TBP): a new tool, based on functionally relevant sequences, to assess genetic diversity in plant species, Genome, 2004, vol. 47, pp. 281–291. https://doi.org/10.1139/g03-132
Benbouza, H., Jean-Marie, J., and Jean-Pierre, B., Optimization of a reliable, fast, cheap and sensitive silver staining method to detect SSR markers in polyacrylamide gels, Biotechnol., Agron., Soc. Environ., 2006, vol. 10, no. 2, pp. 77–81.
Blume, R.Ya., Boychuk, Yu.M., Yemets, A.I., et al., Comparative analysis of fatty acid composition for oils from seeds of tyfon, oil radish and camelina breeding forms and varieties as perspective source for biodiesel production, Factors Exp. Evol. Org., 2016, vol. 18, pp. 61–66.
Blume R.Ya., Lantukh G.V., Yemets A., et al., Comparative analysis of productive potential and fatty acid composition of oil from seeds of spring and winter turnip rape as perspective source for production of diesel biofuel compounds, Factors., 2017, vol. 21, pp. 96–101.
Blume, R.Ya., Lantukh, G.V., Levchuk, I.V., et al., Evaluation of perspectivity of use of a new hybrid oil culture of Tyfon in comparison with its parental species as raw material for biodiesel production, Faktory Eksp. Evol. Org., 2019, vol. 24, pp. 33–39. https://doi.org/10.7124/FEEO.v24.1074
Blume, R.Ya., Rabokon, A.N., and Pirko, Ya.V., β-tubulin intron length polymorphism among forms var. glabra and var. laxa of napa cabbage, Faktory Eksp. Evol. Org., 2020a, vol. 26, pp. 87–92. https://doi.org/10.7124/FEEO.v26.1247
Blume, R.Y., Lantukh, G.V., Levchuk, I.V., et al., Evaluation of potential biodiesel feedstocks: camelina, turnip rape, oil radish and tyfon, Open Agric. J., 2020b, vol. 14, pp. 299–320. https://doi.org/10.2174/1874331502014010299
Blume, R.Y., Rabokon, A.N., Postovitova, A.S., et al., Evaluating diversity and breeding perspectives of Ukrainian spring camelina genotypes, Cytol. Genet., 2020c, vol. 54, no. 5, pp. 420–436. https://doi.org/10.3103/S0095452720050084
Braglia, L.B., Manca, A.M., Mastromauro, F.M., and Breviario, D., cTBP: A successful intron length polymorphism (ILP)–based genotyping method targeted to well defined experimental needs, Diversity, 2010, vol. 2, pp. 572–585. https://doi.org/10.3390/d2040572
Braglia, L., Gavazzi, F., Morello, L., et al., On the applicability of the Tubulin-Based Polymorphism (TBP) genotyping method: a comprehensive guide illustrated through the application on different genetic resources in the legume family, Plant Methods, 2020, vol. 16, p. 86. https://doi.org/10.1186/s13007-020-00627-z
Braglia, L., Lauria, M., Appenroth, K.J., et al., Duckweed species genotyping and interspecific hybrid discovery by tubulin-based polymorphism fingerprinting, Front. Plant Sci., 2021, vol. 12, p. 625670. https://doi.org/10.3389/fpls.2021.625670
Braglia, L., Gavazzi, F., Gianì, S., et al., Tubulin-based polymorphism (TBP) in plant genotyping, in Methods in Molecular Biology, vol. 2638: Plant genotyping, Shavrukov, Y., Ed., New York: Humana, 2023, pp. 387–401. https://doi.org/10.1007/978-1-0716-3024-2_28
Breviario, D., Baird, W.V., Sangoi, S., et al., High polymorphism and resolution in targeted finger-printing with combined β-tubulin introns, Mol. Breed., 2007, vol. 20, pp. 249–259. https://doi.org/10.1007/s11032-007-9087-9
Breviario, D., Giani, S., and Morello, L., Multiple tubulins: evolutionary aspects and biological implications, Plant J., 2013, vol. 75, pp. 202–218. https://doi.org/10.1111/tpj.12243
Cassida, K.A., Barton, B.A., Hough, R.L., et al., Feed intake and apparent digestibility of hay-supplemented brassica diets for lambs, J. Anim. Sci., 1994, vol. 72, no. 6, pp. 1623–1629. https://doi.org/10.2527/1994.7261623x
Cheng, F., Wu, J., Cai, C., et al., Genome resequencing and comparative variome analysis in a Brassica rapa and Brassica oleracea collection, Sci. Data, 2016, vol. 3, p. 160119. https://doi.org/10.1038/sdata.2016.119
Chu, P.L., Vanderghem, C., MacLean, H.L., and Saville, B.A., Financial analysis and risk assessment of hydroprocessed renewable jet fuel production from camelina, carinata and used cooking oil, Appl. Energy, 2017, vol. 198, pp. 401–409. https://doi.org/10.1016/j.apenergy.2016.12.001
Crowell, H., Gamble, A.V., Feng, Y., et al., Impacts of winter grazing on soil health in southeastern cropping systems, Agrosyst. Geosci. Environ., 2022, vol. 5, p. e20240. https://doi.org/10.1002/agg2.20240
Dalkiewicz-Baranowska, H. and Wilczyńska, M., Morphology and anatomy of vegetative Perko organs with reference to their fodder value, Acta Agrobot., 1981, vol. 34, no. 1, pp. 69–88. https://doi.org/10.5586/aa.1981.005
Downey, R.K., The origin and description of the Brassica oilseed crops, in High and Low Erucic Acid Rapeseed Oils Production, Usage, Chemistry, and Toxicological Evaluation, Kramer, J.K.G., Sauer, F.D., Pigden, W.J., Eds., Toronto: Academic, 1983, pp. 1–20.
Galasso, I., Manca, A., Braglia, L., et al., Genomic fingerprinting of Camelina species using cTBP as molecular marker, Am. J. Plant Sci., 2015, vol. 6, pp. 1184–1200. https://doi.org/10.4236/ajps.2015.68122
Gavazzi, F., Pigna, G., Braglia, L., et al., Evolutionary characterization and transcript profiling of β-tubulin genes in flax (Linum usitatissimum L.) during plant development, BMC Plant Biol., 2017, vol. 17, p. 237. https://doi.org/10.1186/s12870-017-1186-0
Gotlin Čuljak, T., Pernar, R., Juran, I., et al., Impact of oilseed rape crop management systems on the spatial distribution of Brassicogethes aeneus (Fabricius 1775): Implications for integrated pest management, Crop Prot., 2016, vol. 89, pp. 129–138. https://doi.org/10.1016/j.cropro.2016.07.017
Gowers, S., Swedes and turnips, in Root and Tuber Crops. Handbook of Plant Breeding, Bradshaw, J., Ed., New York: Springer-Verlag, 2010, vol. 7, pp. 245–289. https://doi.org/10.1007/978-0-387-92765-7_8
Guadalupi, C., Braglia, L., Gavazzi, F., et al., A combinatorial Q-locus and tubulin-based polymorphism (TBP) approach helps in discriminating Triticum species, Genes, 2022, vol. 13, p. 633. https://doi.org/10.3390/genes13040633
Guillard, K. and Allinson, D.W., Yield and nutrient content of summer- and fall-grown forage Brassica crops, Can. J. Plant Sci., 1988, vol. 68, no. 3, pp. 721–731. https://doi.org/10.4141/cjps88-085
Hillis, D.M. and Bull, J.J., An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis, Syst. Biol., 1993, vol. 42, pp. 182–192. https://doi.org/10.1093/sysbio/42.2.182
Kaneko, Y. and Bang, S.W., Interspecific and intergeneric hybridization and chromosomal engineering of Brassicaceae crops, Breed. Sci., 2014, vol. 64, pp. 14–22. https://doi.org/10.1270/jsbbs.64.14
Mandáková, T. and Lysak, M.A., Chromosomal phylogeny and karyotype evolution in x=7 crucifer species (Brassicaceae), Plant Cell, 2008, vol. 20, no. 10, pp. 2559–2570. https://doi.org/10.1105%2Ftpc.108.062166
Mikić, A., Mihailović, V., Marjanović-Jeromela, A., and Terzić, S., Certain aspects of breeding forage Brassicas, in Quantitative Traits Breeding for Multifunctional Grasslands and Turf, Sokolović, D., Huyghe, C., and Radović, J., Eds., Dordrecht: Springer-Verlag, 2014, pp. 163–166. https://doi.org/10.1007/978-94-017-9044-4_24
Nei, M., Genetic distance between populations, Am Nat., 1972, vol. 106, pp. 283–292.
Nei, M. and Li, W.H., Mathematical model for studying genetic variation in terms of restriction endonucleases, Proc. Natl. Acad. Sci. U. S. A., 1979, vol. 76, pp. 5269–5273. https://doi.org/10.1073%2Fpnas.76.10.5269
Park, H.R., Kang, T., Yi, G., et al., Genome divergence in Brassica rapa subspecies revealed by whole genome analysis on a doubled-haploid line of turnip, Plant Biotechnol. Rep., 2019, vol. 13, pp. 677–687. https://doi.org/10.1007/s11816-019-00565-w
Pavlicek, A., Hrda, S., and Flegr, J., FreeTree – Freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD analysis of the genus Frenkelia, Folia Biol., 1999, vol. 45, pp. 97–99.
Rabokon, À.Ì., Intron length polymorphism of tubulin genes as an effective tool for genetic plant differentiation, Proc. Natl. Acad. Sci. Ukr., 2021, vol. 10, pp. 30–35. https://doi.org/10.15407/visn2021.10.030
Rabokon, A.N., Pirko, Ya.V., Demkovych, A.Ye., and Blume, Ya.B., Comparative analysis of the efficiency of intron-length polymorphism of β-tubulin genes and microsatellite loci for flax varieties genotyping, Cytol. Genet., 2018, vol. 52, no. 1, pp. 3–15. https://doi.org/10.3103/S0095452718010115
Rakhmetov, D.B. and Rakhmetova, S.Î., Summary of introduction and breeding of tyfon (Brassica rapa L. × B. campestris f. biennis DC.) in M.M. Griyshko National Botanical Garden of the NAS of Ukraine, Plant Introd., 2015, vol. 4, pp. 18–30.
Rao, S.C. and Horn, F.P., Planting season and harvest date effects on dry matter production and nutritional value of Brassica spp. in the Southern Great Plains, Agron. J., 1986, vol. 78, pp. 327–333. https://doi.org/10.2134/agronj1986.00021962007800020023x
Robinson, D., Non-traditional forages for grazing: turnips and other brassicas, Proceedings of the Heart of America Grazing Conference, 2006, pp. 62–64.
Ruiter, J., Wilson, D., Maley, S., et al., Management Practices for Forage Brassicas, Hamilton: Forage Brassica Development Group, 2009
Sakharova, V.G., Blume, R.Ya., Rabokon, A.N., et al., Efficiency of genetic diversity assessment of littlepod false flax (Camelina microcarpa Andrz. ex DC.) in Ukraine using SSR- and TBP-marker systems, Rep. Natl. Acad. Sci. Ukr., 2023, vol. 4, pp. 93–102. https://doi.org/10.15407/dopovidi2023.04.093
Sambrook, J. and David, W.R., Molecular Ñloning: A Laboratory Manual, Cold Spring Harbor: Cold Spring Harbor Lab., 2001, vol. 2.
Sohn, S.I., Oh, Y.J., Lee, K.R., et al., Characteristics analysis of F1 hybrids between genetically modified Brassica napus and B. rapa, PLoS One, 2016, vol. 11, no. 9, p. e0162103. https://doi.org/10.1371/journal.pone.0162103
Tamura, K., Stecher, G., and Kumar, S., MEGA11: Molecular evolutionary genetics analysis version 11, Mol. Biol. Evol., 2021, vol. 38, pp. 3022–3027. https://doi.org/10.1093/molbev/msab120
Tao, L., Milbrandt, A., Zhang, Y., and Wang, W.-C., Techno-economic and resource analysis of hydroprocessed renewable jet fuel, Biotechnol. Biofuels, 2017, vol. 10, p. 261. https://doi.org/10.1186/s13068-017-0945-3
Tsaruk, I.V. and Rakhmetov, D.B., Peculiarities of the seed productivity formation of Tyfon (Brassica campestris var. oleifera f. biennis D.C. × B. rapa L.) plants under the effect of cultivation technology, Adv. Agritechnol., 2022, vol. 10 no. 1, p. 265592. https://doi.org/10.47414/na.10.1.2022.265592
Villalobos, L.A. and Brummer, J.E., Forage Brassicas stockpiled for fall grazing: yield and nutritive value, Crop, Forage Turfgrass Manage., 2015, vol. 1, pp. 1–6. https://doi.org/10.2134/cftm2015.0165
Warwick, S.I., Brassicaceae in agriculture, in Genetics and Genomics of the Brassicaceae, vol. 9: Plant genetics and genomics: crops and models, Schmidt, R. and Bancroft, I., Eds., New York: Springer-Verlag, 2011, pp. 33–65. https://doi.org/10.1007/978-1-4419-7118-0_2
Warwick, S.I., Simard, M.J., Légère, A., et al., Hybridization between transgenic Brassica napus L. and its wild relatives: Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L., and Erucastrum gallicum (Willd.) O.E. Schulz, Theor. Appl. Genet., 2003, vol. 107, pp. 528–539. https://doi.org/10.1007/s00122-003-1278-0
Wiedenhoeft, M.H., Management and environment effects on dry matter yields of three Brassica species, Agron. J., 1993, vol. 85, pp. 549–553. https://doi.org/10.2134/agronj1993.00021962008500030006x
Zhang, Y.W., Jin, D., Xu, C., et al., Regulation of bolting and identification of the α-tubulin gene family in Brassica rapa L. ssp pekinensis, Genet. Mol. Res., 2016, vol. 15, no. 1, p. 15017507. https://doi.org/10.4238/gmr.15017507
Zhang, L., Cai, X., Wu, J., et al., Improved Brassica rapa reference genome by single-molecule sequencing and chromosome conformation capture technologies, Hortic. Res., 2018, vol. 5, p. 50. https://doi.org/10.1038/s41438-018-0071-9