Preliminary testing of SRAP primers in four Ranunculaceae species from Romania for conservation strategies Cristina Daniela Kelemen, PhD student Department of Horticulture, Faculty of Horticulture, University of Agricultural Sciences and Veterinary Medicine, Manastur Street 3-5, 400372, Cluj-Napoca, Romania; Biological diversity = variation within the living world. Benefits of biodiversity Biodiversity is essential Biodiversity provides : natural pest control Food maintenance of the fibers=clothes. various populations types of oil nutrient cycling conservation and . drugs and medicines purification of water formation of soil etc. Biodiversity depletion: • urbanization • expansion of agriculture • deforestation • exploitation of resources • climatic changes • pollution • invasive plants • diseases etc Biological diversity = variation within the living world. Diversity can occur at three levels Genetic diversity A better understanding of genetic diversity is essential for conservation purposes. Role of genetic diversity data: taxonomy selection what/ where to conserve germplasm conservation management Genetic diversity molecular Each marker has different markers: RAPD, AFLP, SRAP, ISSR or properties and will reflect different SSR. aspects of genetic diversity. RAPD -random amplified polymorphic DNA AFLP-amplified fragment length polymorphism SRAP-sequence-related amplified polymorphism ISSR-inter-simple sequence repeat SSR-simple sequence repeats The main aim of the study To assess the genetic diversity of 4 medicinal Ranunculaceae species using SRAP markers. Aconitum moldavicum Anemone transsilvanica Ranunculus carpaticus Ranunculus platanifolius • Preliminary testing of SRAP primers was necessary to be able to select the appropriate primer combinations on a large-scale use Preliminary for future analysis. SRAP testing • SRAP primers: • more suitable to reveal genetic diversity among related species (Budak et al., 2004, Ferriol et al. 2003) than other markers; Why SRAP • presented a higher reproducibility than RAPD and AFLP; primers? • simple and efficient marker system can be adapted for various purposes; • involves low costs. Ranunculaceae family is represented by ca 2,500 species in 56 genera distributed worldwide with a great genetic variability; in Romania are 23 genera and ca 110 species including rare and endemic species. A. transsilvanica Nigella sativa Local species have been used as: medicinal plants ornamentals locally consumed as food Little is known concerning their Clematis sp. Ranunculus ficaria genetic diversity and conservation. Plant Material Plants were collected in July and August 2016 from two locations (Mt. Stramba and Mt. Postavaru) from Romania; Voucher specimens have been stored in the Herbarium collection at USAMV of Cluj- Napoca, Romania; Plants were selected following the folk medicinal use. Table 1: (Ethno)botanical data on Ranunculaceae species tested Species name VSNb) Folk medicine use Aconitum moldavicum Hacq. CLA30049 Analgesic, antitussive Antibronchitis, anti-hepatitis, Anemone transsilvanica FUSS. CLA30047 antiinfectives, Ranunculus carpaticus Herbich. CLA30044 Analgesic, antirheumatic Ranunculus platanifolius L. CLA30040 Homeopathy DNA isolation CTAB* method as described by Lodhi et al. (1994) and improved by Pop et al. (2003). http://moleculargeneticsatuofr.blogspot.com/2005/01/ctab-cetyltrimethylammonium-bromide.htm Extraction buffer - 2% CTAB - 100 mM Tris Base The quality and quantity of DNA were estimated by - 50 mM EDTA - 1.4 M NaCl measuring its absorbance at 260 and 280 nm using a - 2% PVP NanoDrop1000 spectrophotometer. - 10 mM ascorbic acid - 5 mM DIECA CTAB* (cetyl-trimethylammonium bromide) SRAP reactions SRAP analysis was carried out according to Talebi et al. (2012) protocol. 64 primer combinations were employed (eight forward (Me1-8) and eight reverse (Em1-8) primers. 35 Cycles http://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/pcr.html DNA electroforetic profile Amplified products were separated 1.6 % (w/v) agarose gels in 1.0 × TAE buffer at 115 V, for 2 hours stained with 1µg/mL EtBr for 20 to 30 minutes; The obtained images were processed with Total Lab 120 software Sequence-related amplified polymorphism (SRAP) profiles obtained from 8 primer combinations of 4 samples Results: Primer combination screening 27 primers pairs were selected as the most efficient combinations which generated a total number of 553 DNA fragments with an average number of 20.5. clarity Main selection criteria reproducibility rate of polymorphism The most representative Me1/Em1, SRAP profiles were obtained Me3/Em3, with the primer combinations Me7/Em4, Me7/Em5, A total number of 201 Me7/Em8, DNA fragments Me8/Em1 Me8/Em5 Results: Primer combination screening Electrophoretic profiles of four species (A. moldavicum, A. transsilvanica, R. carpaticus and R. platanifolius) obtained with 8 primer combinations. Results: Selected primer pairs for A. moldavicum A. moldavicum samples show good results with the primer combinations: Me7/Em8, Me8/Em1, Me8/Em5, Me7/Em4, Me6/Em6, Results: Selected primer pairs for A. transsilvanica For A. transsilvanica the most effective primers pairs were founded to be: Me3/Em5, Me6/Em5, Me7/Em8, Me8/Em5. Results: Selected primer pairs for R. carpaticus For R. carpaticus the following combinations were the most informative: Me3/Em3, Me4/Em1, Me7/Em5, Me8/Em1, Me8/Em5. Results: Selected primer pairs for R. platanifolius The most effective primer combinations for R. platanifolius were Me7/Em4, Me7/Em6, Me7/Em8, Me8/Em1, Me8/Em6. Similar studies Species The most representative References SRAP primer pairs Carthamus species Me4/Em1, Me5/Em2 (Mokhtari et al., 2013) Citrus species Me3/Em2, Me6/Em4 (Uzun et al., 2009) Vitis species Me1/Em7, Me3/Em2, Me5/Em6 (Guo et al., 2012) Stylosanthes species Me1/Em2, Me6/Em7, Me8/Em1 (Huang et al., 2017) Carthamus tinctorius Vitis vinifera Stylosanthes hamata SRAP markers were used for the first time to analyze genetic diversity of species belonging to the Ranunculaceae family. The screening of SRAP primer combinations is needed to ensure a high polymorphic content for further genetic analyses. These results provide: useful information of the most efficient SRAP marker combinations of the tested species the most representative primer combinations were Me1/Em1, Me3/Em3, Me7/Em4, Me7/Em5, Me7/Em8, Me8/Em1 and Me8/Em5; highly contribute to perform future genetic diversity analyses; in situ and ex-situ conservation perspective. REFERENCES 1. Budak H., Shearman R.C., Parmaksiz I., Gaussoin R.E., Riosdan T.P., Dweikat I., 2004. Molecular characterization of buffalograss germplasm using sequence-related amplified polymorphism markers. Theoretical and Applied Genetics, 108(2):328–334. 2. Guo D., Zhang J., Liu C., Zhang G., Li M., Zhang Q., 2012. Genetic variability and relationships between and within grape cultivated varieties and wild species based on SRAP markers. Tree Genetics and Genomes, 8(4):789–800. 3. Heywood H., Brummitt R. K., Culham A., Seberg O.V., 2007. Flowering Plant Families of the World. Edited by R.V.H. Heywood. Firefly Bo. Ontario. 4. Huang C., Liu G., Bai C., 2017. Polymorphism analysis in identification of genetic variation and relationships among Stylosanthes species. 3 Biotech Journal, 7(1):1–10. 5. 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(2–3):455–461. 6. Lodhi M.A., Ye G.N., Weeden N.F., Reisch B.I., 1994. A simple and efficient method for DNA extraction from grapevine cultivars Vitis species. Plant Molecular Biology Reporter, 12(1):6-13. 7. Mokhtari N., Rahimmalek M., Talebi M., Khorrami M., 2013. Assessment of genetic diversity among and within Carthamus species using sequence-related amplified polymorphism (SRAP) markers. Plant Systematics and Evolution, 299(7):1285–1294. 8. Pop R., Ardelean M., Pamfil D., Gaboreanu I.M., 2003. The efficiency of different DNA isolation and purification in ten cultivars of Vitis vinifera. Bulletin USAMV Biotechnology, 59:259-261. 9. Prati D., Peintinger M., Fischer M., 2016. genetic composition, genetic diversity and small-scale environmental variation matter for the experimental reintroduction of a rare plant. Journal of Plant Ecology, 9(6):805–13. 10. Talebi, M., Kazemi, M., & Sayed-Tabatabaei, B. E. 2012. Molecular diversity and phylogenetic relationships of Pistacia vera, Pistacia atlantica subsp. mutica and Pistacia khinjuk using SRAP markers. Biochemical Systematics and Ecology, 44, 179–185. 11. Tămaș M., 2005. Botanică Farmaceutică: Sistematica-Cormobionta, 3th edn. Editura Medicală Universitară Publishing House, Cluj Napoca, 40-44. 12. Uzun A., Yesiloglu T., Aka-Kacar Y., Tuzcu O., Gulsen O. 2009. Genetic diversity and relationships within Citrus and related genera based on sequence related amplified polymorphism markers (SRAPs). Scientia Horticulturae, 121(3):306–312. .