Preliminary Results for Use Ssr Markers in Bitter Vetch “Vicia Ervilia (L.) Willd”

Nov. 2013. Vol. 1, No.1 ISSN 2311-2476 International Journal of Research In Agriculture and Food Sciences © 2013 IJRAFS & K.A.J. All rights reserved http://www.ijsk.org/ijrafs.html PRELIMINARY RESULTS FOR USE SSR MARKERS IN BITTER VETCH “VICIA ERVILIA (L.) WILLD” Salama El Fatehi1,2, Gilles Béna2,4, Laïla Sbabou2,3, Abdelkarim Filali-Maltouf2,3 and Mohammed Ater1,2 1, Laboratoire de Diversité et Conservation des Systèmes Biologiques (LDICOSYB), Université Abdelmalek Essaâdi, P.B. 2121, Tétouan, Maroc. [email protected]. 2, Laboratoire Mixte International (LMI), Université Mohamed V-Agdal. Avenue Ibn Batouta BP 1014, Rabat, Maroc. 3, Laboratoire de Microbiologie et Biologie Moléculaire, Université Mohamed V-Agdal, Avenue Ibn Batouta BP 1014, Rabat, Maroc. 4, Laboratoire des Symbioses Tropicales et Méditerranéennes, Institut de Recherche pour le Développement (IRD), Campus International de Baillarguet, 34398 Montpellier Cedex 5, France. ABSTRACT A development of genomic DNA extraction protocol from the leaves of Vicia ervilia was conducted. The extraction from fresh material gives better outcomes than extraction from dried leaves. The DNA extracts obtained have significantly higher quality (ratio A260/280 is 1.99 and A260/230 is 1.92). The yields quantity of extracted DNA is also superior (1030 ng/µl). Using enriched genomic bank, sequences containing microsatellite were isolated. Twenty-four pairs of primers allowing the amplification products that are easy to read have been obtained, nine of which have revealed the polymorphism. The average value of the index PIC (Polymorphism Information Content) is 0.65 and the average number of alleles per locus is 7.6. These values prove to be more interesting compared to the available data in leguminous plants. Keywords : Vicia ervilia, DNA extraction, SSR, polymorphism resulted in ecotypes adapted to the local agro- 1. INTRODUCTION climatic conditions) whose genetic resources assessment has nearly never been done (but see Vicia ervilia (L.)Willd. is an ancient cultivated Francis et al. (1994); Van de Wouw et al. (2001) leguminous. Currently, it is a minor crop distributed for limited Vicia studies). Although it is a marginal in Southern of Europe, West and Central Asia and and minor culture, it represents significant genetic North Africa (GRIN, 2008). World production is resources which require efforts for conservation. estimated around 800,000 tons per year with an average yield of 1600Kg/ha (FAO, 2013). The On the other hand; the Mediterranean climate primary use of the bitter vetch seeds is animal regions have experienced sensitive rise in feeding (Enneking, 1995; Francis et al., 1999). The temperatures and periods of severe drought under use in human food is rare due to its toxicity the effect of climate change during the last decades (Sadeghi et al., 2009) and it is only mentioned (Bindi & Olesen, 2011; Supit et al., 2010). during starvation periods (Enneking et al., 1995). Thereby, rainfed agriculture is now facing high risks and climate uncertainties (Trnka et al., 2011). In Morocco, it is a minor crop in the northern Among the cultivated Vicia, V. ervilia is a very part of the country (Foury, 1954). Indeed, the interesting species with adaptive capacities, cultivated areas were already stagnated in the 80s especially tolerance to aridity and can be cultivated and 90s around 20,000 ha (Bounejmate, 1997) with poor rainfall (Foury, 1954 ; Maxted, 1995 ; reaching nowadays 10,000 ha (FAO, 2013). The Abd El-Moneim & Saxena, 1997). varieties used are populations locally maintained by traditional farming practices within traditional agro- Microsatellites (SSR) markers (Chambers & ecosystems of the Rif Mountain (Hmimsa & Ater, MacAvoy, 2000) have been shown to be excellent 2008; Ater & Hmimsa, 2008). This local selection molecular markers for the study of genetic diversity 40 Nov. 2013. Vol. 1, No.1 ISSN 2311-2476 International Journal of Research In Agriculture and Food Sciences © 2013 IJRAFS & K.A.J. All rights reserved http://www.ijsk.org/ijrafs.html (Karp et al. 1997; Luikart et al., 2003) and we will adapt DNA extraction protocol to our commonly used in studies of genetic diversity and material and then search for interpretable powders genotypic characterization. Indeed, they are for SSR markers. This is a relevant contribution to codominant and reveal high levels of polymorphism the assessment of genetic diversity of this species, because they are multi-allelic (Jarne & Lagoda, with a view to its valorization. 1996). Upgrading to SSR markers points conducted in several species in the legumes (Rong et al., 2012; 2. MATERIAL AND METHODS Sun et al., 2012; Ohtsuki et al., 2011) and genus Vicia (Chung et al., 2013); especially in Vicia faba 2.1. Vegetal Material (Gong et al., 2010; Gong et al., 2011; Yang et al., 2012) species of great interest for Mediterranean A collection made up of 19 accessions collected agriculture. Vicia ervilia use of SSR markers was in the North-West of Morocco was carried (Tab. 1). not yet performed. Each accession is composed of 10 individuals sampled directly into the fields; thus, the collection Hence the goals of this study were to provide a has a total of 190 individuals. first assessment to characterizing the genetic diversity with SSR markers among local populations of Vicia ervilia in Morocco. To do this, Table 1: Localization of the sampled populations Locality name Longitude East Latitude North Altitude (m) Kramat 05’34,780 35’40,638 301 Dharlahdida 05’34,307 35’39,893 373 Rouman 05’38,098 35’32,080 175 Ahrit 05’33,511 35’38,990 224 Adrou 05’33,236 35’25,308 566 Achakrad 05’29,985 35’23,729 468 Tafifoute 05’22,355 35’23,507 647 Bouatou 05’25,355 35’221,507 713 Jnanate 05’23,301 35’17,876 720 Iabasan 05’25,504 35’16,551 819 Talamboute 05’18,888 35’18,262 504 Pont Talamboute 05’20,992 35’18,979 419 Khizana 05’14,750 35’03,583 825 Bab el hourrate 05’04,150 34’59,348 492 Asserdoune 05’03,178 34’59,797 644 Ain Beida 05’24,647 35’01,393 185 Tiamma 05’32,011 34’55,654 136 Douaher 05’33,317 34’52,120 228 El Jabriyine 05’32,703 34’52,914 170 2.2. DNA extraction i. According to Doyle & Doyle (1987): Preheat 4 ml of CTAB isolation buffer Extraction of genomic DNA of Vicia ervilia is (2% hexadecyltrimethyl ammonium made from dried leaves harvested in the field. bromide [CTAB: Sigma H-5882], 1.4 M Alone, the last pairs of leaves are collected and then NaCl, 20 mM EDTA, 100 mM Tris-HCl, dried by Silica gel. A second extract made from the pH 8.0). Grind 35 mg dry leaf tissue with fresh plant material. The seeds are germinated in 300µl CTAB isolation buffer. Incubate the Petri dishes and the seedlings are used for the sample at 60°C for 60min. with optional extraction of DNA. occasional gentle swirling. Extract once with chloroform-isoamyl alcohol Two protocols have been tested: Doyle & Doyle (400µl), mixing gently but thoroughly, (1987) and Khanuja et al. (1999). this produces two phases, an upper 41 Nov. 2013. Vol. 1, No.1 ISSN 2311-2476 International Journal of Research In Agriculture and Food Sciences © 2013 IJRAFS & K.A.J. All rights reserved http://www.ijsk.org/ijrafs.html aqueous phase which contains the DNA, 2.4. Development of nuclear microsatellite and a lower chloroform phase that primers and amplification contains some degraded proteins, lipids, and many secondary compounds. The The genomic bank was constructed from the interface between these two phases DNA of 7 individuals belonging to the collection, contains most of the "junk" cell debris, according to the pyrosequencing method many degraded proteins, etc. Spin in (GENOSCREEN, Lille, France). 24 primer pairs clinical centrifuge at room temperature at were tested (Tabl. 2). 6000 x g for 10min.. Remove aqueous PCR amplification was performed with 20 ng of phase with wide pipette, transfer to clean total DNA and in a final volume of 10μl containing glass centrifuge tube, add 2/3 (200µl) 37.5 pmol of MgCl2, 6 pmol of dNTPs, 1 pmol of volumes cold isopropanol, and mix each primer and one unit of Taq DNA polymerase. gently to precipitate nucleic acids. The DNA amplification program adopted is as Incubate sample at 4°C for 60min.. After follows: 95°C for 10 min., followed by 40 cycles of spin in centrifuge at 6000 x g for 10 min, 30s at 95°C, 30s at 55°C and 1min. at 72°C, the supernatant removed and the pellet followed by a final extension for 10min. at 72°C. was washed with ethanol 80% and dried. Resuspend nucleic acid pellet in 70µl TE The lengths of the PCR products are revealed by (10 mM Tris-HCl, 1 mM EDTA, pH capillary electrophoresis at sequencer ABI 3730, by 7.4). Add 5 µl RNAase and incubate using the size marker Liz500 (Applied Biosystems). 30min. at 37°C. The reading of lengths of the fragments is performed using the GeneMapperR software ii. According to Khanuja et al. (1999): The (Applied Biosystems). extraction buffer is composed of 2,5% CTAB, 1.5 M NaCl, 25 mM EDTA, 100 The PIC value (polymorphism information mM Tris-HCl, pH 8.0 et 0,2% β- content) was calculated according to the formula mercaptophénol. After grinding with the (Anderson et al., 1993): PIC= 1 – ∑ , where buffer solution. Samples were incubated Pij is the frequency of the jth allele at the ith locus. at 60°C for 90 min. Proteins were removed by the chloroform-isoamyl 3. RESULTS AND DISCUSSIONS alcohol, and then by centrifugation at 8000g for 10 min, DNA is located in the 3.1.

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