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Abstract
Hypericum perforatum, H. umbellatum, H. maculatum, and H. hircinum accessions originating from botanical gardens across Europe were examined by flow cytometry and molecular markers. 2C DNA content of 17 Hypericum perforatum accessions (Hp) and the H. perforatum cultivar Topaz amounted between 1.56 pg and 1.62 pg. In four Hp accessions few individual plants were found with a DNA content corresponding to 6Cx (2.34 - 2.39 pg). All plants of accession Hp8 showed a DNA content of 6Cx (2.41 pg). In root tips of Hp plants with an average DNA amount of 1.58 pg, 32 chromosomes were detected corresponding to 2n = 4x. H. umbellatum and H. maculatum, each contained 0.76 pg DNA and 16 chromosomes were counted. The 2C DNA content of H. hircinum is described for the first time and is 1.00 pg. Additionally, a combined marker analysis, based on inter-simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP), was conducted to gain a better understanding of diversity especially within the accessions of H. perforatum. A total of 27 (11 ISSR and 16 SRAP) markers were screened, showing 699 bands of which 661 were polymorphic. UPGMA clustering revealed that accessions from the same geographic area tended to be more closely related, while H. maculatum was grouped separately from all H. perforatum accessions. Both methods have shown similar sensitivities in detecting the genetic diversity of the analyzed genotypes. Our results may be useful for Hypericum breeding programs and the development of effective conservation strategies.
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Copyright (c) 2022 Anca Butiuc Keul, Ana Coste, Holger Budahn, Frank Dunemann, Anca Farkas, Dragos Postolache, Evelyn Klocke
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References
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- Brutovská, R., Cellárová, E., Schubert, I., 2000: Cytogenetic characterization of three Hypericum species by in situ hybridization. Theoretical and Applied Genetics 101, 46–50.
- Castro, M., Rosselló, J.A., 2006: New chromosome numbers for plant taxa endemic to the Balearic Islands. Folia Geobotanica 41, 433–451.
- Chen, S.Y., Dai, T.X., Chang, Y.T., Wang, S.S., Ou, S.L., Chuang, W.L., Cheng, C.Y., Lin, Y.H., Lin, L.Y., Ku, H.M., 2013: Genetic diversity among Ocimum species based on ISSR, RAPD and SRAP markers. Australian Journal of Crop Science 7, 1463–1471.
- Corral, J.M., Molins, M.P., Aliyu, O.M., Sharbel, T.F., 2011: Isolation and characterization of microsatellite bands from apomictic Hypericum perforatum (Hypericaceae). American Journal of Botany 98, e167–169.
- Coste, A., Vlase, L., Halmagyi, A., Deliu, C., Coldea, G., 2011: Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and Hypericum maculatum. Plant Cell, Tissue and Organ Culture 106(2), 279–288.
- Doležel, J., Bartoš, J., 2005: Plant DNA flow cytometry and estimation of nuclear genome size. Annals of Botany 95, 99–110.
- Doležel, J., Sgorbati, S., Lucretti, S., 1992: Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum 85, 625–631.
- Doyle, J., Doyle, J.L., 1987: A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19, 11–15.
- Franklin, G., Beerhues, L., Čellárová, E., 2016: Molecular and biotechnological advancements in Hypericum species. Frontiers in Plant Sciences 7(1687) doi: 10.3389/fpls.2016.01687.
- Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P., Firoozabady, E., 1983: Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051.
- Galla, G., Barcaccia, G., Schallau, A., Puente Molins, M., Bäumlein, H., Sharbel, T.F., 2011: The cytohistological basis of apospory in Hypericum perforatum L. Sexual Plant Reproduction 24, 47–61.
- He, M., Wang, Z., 2013: Genetic diversity of Hypericum perforatum collected from the Qinling Mountains of China. Biochemical Systematics and Ecology 50, 232–239.
- Jaccard, P., 1908: Nouvelles recherches sur la distribution florale, Bulletin Societé Vaudoise des Sciences Naturelles 44, 223–270.
- Karioti, A., Bilia, A.R., 2010: Hypericins as potential leads for new therapeutics. International Journal of Molecular Sciences 11, 562–594.
- Koch, M.A., Scheriau, C., Betzin, A., Hohmann, N., Sharbel, T.F., 2013: Evolution of cryptic gene pools in Hypericum perforatum: the influence of reproductive system and gene flow. Annals of Botany 111, 1083–1094.
- Kovach, W.L., 2007: MVSP - A Multivariate Statistical Packages for Windows ver 3.21. Pentraeth, Wales, UK, Kovach Computing Services.
- Li, G., Quiros, C.F., 2001: Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: Its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics 103, 455–461.
- Loon, J.C., Jong, H., 1978: IOPB chromosome number reports LIX. Taxon 27, 57–60.
- Mártonfi, P., Mártonfiová, L., 2011: Reproduction mode in Hypericum and its consequences. Medicinal and Aromatic Plant Science and Biotechnology 5, 48–52.
- Matzk, F., Hammer, K., Schubert, I., 2003: Coevolution of apomixis and genome size within the genus Hypericum. Sexual Plant Reproduction 16, 51–58.
- Matzk, F., Meister, A., Brutovská, R., Schubert, I., 2001: Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis. Plant Journal 26, 275–282.
- Mayo, G.M., Langridge, P., 2003: Modes of reproduction in Australian populations of Hypericum perforatum L. (St. John's wort) revealed by DNA fingerprinting and cytological methods. Genome 46, 573-579.
- Morshedloo, M.R., Moghadam, M.R.F., Ebadi, A., Yazdani, D., 2014: Genetic relationships of Iranian Hypericum perforatum L. wild populations as evaluated by ISSR markers. Plant Systematics and Evolution 301, 657–665.
- Nürk, N.M., 2011: Phylogenetic analyses in St. John’s wort (Hypericum). Inferring character evolution and historical biogeography. PhD thesis. Free University, Berlin, Germany.
- Nürk, N.M., Blattner, F.R., 2010: Cladistic analysis of morphological characters in Hypericum (Hypericaceae). Taxon 59, 1495–1507.
- Peakall, R., Smouse, P.E., 2012: GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research - An update. Bioinformatics 28, 2537–2539.
- Pilepić, K.H., Balić, M., Blažina, N., 2011: Estimation of phylogenetic relationships among some Hypericum (Hypericaceae) species using internal transcribed spacer sequences. Plant Biosystems 145(1), 81–87.
- Prevost, A., Wilkinson, M.J., 1999: A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theoretical and Applied Genetics 98, 107–112.
- Qu, L., Widrlechner, M.P., Rigby, S.M., 2010: Analysis of breeding systems, ploidy, and the role of hexaploids in three Hypericum perforatum L. populations. Industrial Crops and Products 32, 1–6.
- Robson, N.K.B., 1981: Studies in the genus Hypericum L. (Guttiferae). 2. Characters of the genus. Bulletin of the British Museum (Natural History) Botany 8, 55–226.
- Robson, N.K.B., 2003: Hypericum botany. In: Ernst, E. (ed.), Hypericum. The Genus Hypericum, 1–22. Taylor and Francis, London.
- Rostami-Ahmadvandi, H., Cheghamirza, K., Kahrizi, D., Bahraminejad, S., 2013: Comparison of morpho-agronomic traits versus RAPD and ISSR markers in order to evaluate genetic diversity among Cuminum cyminum L. accessions. Australian Journal of Crop Science 7, 361–367.
- Savaş Tuna, G., Duyu, G., Uzun, K., Yücel, G., Tuna, M., 2017: Determination of nuclear DNA content and ploidy of Hypericum perforatum L. accessions collected from Western Turkey. The Journal of Agricultural Science 23, 395–403.
- Scheriau, C.L., Nuerk, N.M., Sharbel, T.F., Koch, M.A., 2017: Cryptic gene pools in the Hypericum perforatum-H. maculatum complex: diploid persistence versus trapped polyploid melting. Annals of Botany 120, 955–966.
- Sehgal, D., Rajpal, V.R., Raina, S.N., Sasanuma, T., Sasakuma, T., 2009: Assaying polymorphism at DNA level for genetic diversity diagnostics of the safflower (Carthamus tinctorius L.) world germplasm resources. Genetica 135, 457–470.
- Shannon, C.E., Weaver, W., 1949: The mathematical theory of communication. University of Illinois Press, Urbana.
- Temsch, E.M., Temsch, W., Ehrendorfer-Schratt, L., Greilhuber, J., 2010: Heavy metal pollution, selection, and genome size: The species of the Žerjav study revisited with flow cytometry. Journal of Botany, ID 596542.
References
Alan, A.R., Murch, S.J., Saxena, P.K., 2015: Evaluation of ploidy variations in Hypericum perforatum L. (St. John’s wort) germplasm from seeds, in vitro germplasm collection, and regenerants from floral cultures. In Vitro Cellular & Developmental Biology – Plant 51, 452–462.
Barcaccia, G., Bäumlein, H., Sharbel, T.F., 2007: Apomixis in St. John’s wort (Hypericum perforatum L.): An overview and glimpse towards the future. In: Hörandl, E., Grossniklaus, U., Sharbel, T., van Dijk, P. (eds.), Apomixis: Evolution, mechanism and perspectives, 259–280. Ganter Verlag, Regnum Vegetabile, Ruggell.
Brutovská, R., Cellárová, E., Schubert, I., 2000: Cytogenetic characterization of three Hypericum species by in situ hybridization. Theoretical and Applied Genetics 101, 46–50.
Castro, M., Rosselló, J.A., 2006: New chromosome numbers for plant taxa endemic to the Balearic Islands. Folia Geobotanica 41, 433–451.
Chen, S.Y., Dai, T.X., Chang, Y.T., Wang, S.S., Ou, S.L., Chuang, W.L., Cheng, C.Y., Lin, Y.H., Lin, L.Y., Ku, H.M., 2013: Genetic diversity among Ocimum species based on ISSR, RAPD and SRAP markers. Australian Journal of Crop Science 7, 1463–1471.
Corral, J.M., Molins, M.P., Aliyu, O.M., Sharbel, T.F., 2011: Isolation and characterization of microsatellite bands from apomictic Hypericum perforatum (Hypericaceae). American Journal of Botany 98, e167–169.
Coste, A., Vlase, L., Halmagyi, A., Deliu, C., Coldea, G., 2011: Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and Hypericum maculatum. Plant Cell, Tissue and Organ Culture 106(2), 279–288.
Doležel, J., Bartoš, J., 2005: Plant DNA flow cytometry and estimation of nuclear genome size. Annals of Botany 95, 99–110.
Doležel, J., Sgorbati, S., Lucretti, S., 1992: Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum 85, 625–631.
Doyle, J., Doyle, J.L., 1987: A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19, 11–15.
Franklin, G., Beerhues, L., Čellárová, E., 2016: Molecular and biotechnological advancements in Hypericum species. Frontiers in Plant Sciences 7(1687) doi: 10.3389/fpls.2016.01687.
Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P., Firoozabady, E., 1983: Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051.
Galla, G., Barcaccia, G., Schallau, A., Puente Molins, M., Bäumlein, H., Sharbel, T.F., 2011: The cytohistological basis of apospory in Hypericum perforatum L. Sexual Plant Reproduction 24, 47–61.
He, M., Wang, Z., 2013: Genetic diversity of Hypericum perforatum collected from the Qinling Mountains of China. Biochemical Systematics and Ecology 50, 232–239.
Jaccard, P., 1908: Nouvelles recherches sur la distribution florale, Bulletin Societé Vaudoise des Sciences Naturelles 44, 223–270.
Karioti, A., Bilia, A.R., 2010: Hypericins as potential leads for new therapeutics. International Journal of Molecular Sciences 11, 562–594.
Koch, M.A., Scheriau, C., Betzin, A., Hohmann, N., Sharbel, T.F., 2013: Evolution of cryptic gene pools in Hypericum perforatum: the influence of reproductive system and gene flow. Annals of Botany 111, 1083–1094.
Kovach, W.L., 2007: MVSP - A Multivariate Statistical Packages for Windows ver 3.21. Pentraeth, Wales, UK, Kovach Computing Services.
Li, G., Quiros, C.F., 2001: Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: Its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics 103, 455–461.
Loon, J.C., Jong, H., 1978: IOPB chromosome number reports LIX. Taxon 27, 57–60.
Mártonfi, P., Mártonfiová, L., 2011: Reproduction mode in Hypericum and its consequences. Medicinal and Aromatic Plant Science and Biotechnology 5, 48–52.
Matzk, F., Hammer, K., Schubert, I., 2003: Coevolution of apomixis and genome size within the genus Hypericum. Sexual Plant Reproduction 16, 51–58.
Matzk, F., Meister, A., Brutovská, R., Schubert, I., 2001: Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis. Plant Journal 26, 275–282.
Mayo, G.M., Langridge, P., 2003: Modes of reproduction in Australian populations of Hypericum perforatum L. (St. John's wort) revealed by DNA fingerprinting and cytological methods. Genome 46, 573-579.
Morshedloo, M.R., Moghadam, M.R.F., Ebadi, A., Yazdani, D., 2014: Genetic relationships of Iranian Hypericum perforatum L. wild populations as evaluated by ISSR markers. Plant Systematics and Evolution 301, 657–665.
Nürk, N.M., 2011: Phylogenetic analyses in St. John’s wort (Hypericum). Inferring character evolution and historical biogeography. PhD thesis. Free University, Berlin, Germany.
Nürk, N.M., Blattner, F.R., 2010: Cladistic analysis of morphological characters in Hypericum (Hypericaceae). Taxon 59, 1495–1507.
Peakall, R., Smouse, P.E., 2012: GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research - An update. Bioinformatics 28, 2537–2539.
Pilepić, K.H., Balić, M., Blažina, N., 2011: Estimation of phylogenetic relationships among some Hypericum (Hypericaceae) species using internal transcribed spacer sequences. Plant Biosystems 145(1), 81–87.
Prevost, A., Wilkinson, M.J., 1999: A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theoretical and Applied Genetics 98, 107–112.
Qu, L., Widrlechner, M.P., Rigby, S.M., 2010: Analysis of breeding systems, ploidy, and the role of hexaploids in three Hypericum perforatum L. populations. Industrial Crops and Products 32, 1–6.
Robson, N.K.B., 1981: Studies in the genus Hypericum L. (Guttiferae). 2. Characters of the genus. Bulletin of the British Museum (Natural History) Botany 8, 55–226.
Robson, N.K.B., 2003: Hypericum botany. In: Ernst, E. (ed.), Hypericum. The Genus Hypericum, 1–22. Taylor and Francis, London.
Rostami-Ahmadvandi, H., Cheghamirza, K., Kahrizi, D., Bahraminejad, S., 2013: Comparison of morpho-agronomic traits versus RAPD and ISSR markers in order to evaluate genetic diversity among Cuminum cyminum L. accessions. Australian Journal of Crop Science 7, 361–367.
Savaş Tuna, G., Duyu, G., Uzun, K., Yücel, G., Tuna, M., 2017: Determination of nuclear DNA content and ploidy of Hypericum perforatum L. accessions collected from Western Turkey. The Journal of Agricultural Science 23, 395–403.
Scheriau, C.L., Nuerk, N.M., Sharbel, T.F., Koch, M.A., 2017: Cryptic gene pools in the Hypericum perforatum-H. maculatum complex: diploid persistence versus trapped polyploid melting. Annals of Botany 120, 955–966.
Sehgal, D., Rajpal, V.R., Raina, S.N., Sasanuma, T., Sasakuma, T., 2009: Assaying polymorphism at DNA level for genetic diversity diagnostics of the safflower (Carthamus tinctorius L.) world germplasm resources. Genetica 135, 457–470.
Shannon, C.E., Weaver, W., 1949: The mathematical theory of communication. University of Illinois Press, Urbana.
Temsch, E.M., Temsch, W., Ehrendorfer-Schratt, L., Greilhuber, J., 2010: Heavy metal pollution, selection, and genome size: The species of the Žerjav study revisited with flow cytometry. Journal of Botany, ID 596542.