Keywords: African melon, Cucurbits, molecular markers, SSR, rate of allogamy

Transcription

1 Relationship between genetic diversity and reproduction strategy in a sexually-propagated crop in a traditional farming system, Citrullus lanatus var. citroides 1 L.-A. Minsart and P. Bertin Université catholique de Louvain (UCL), Unité d écophysiologie et d amélioration végétale ECAV, Croix du Sud 2 bte 11, B-1348 Louvain-la-Neuve, Belgium* Corresponding author laure_anne.minsart@yahoo.fr Keywords: African melon, Cucurbits, molecular markers, SSR, rate of allogamy Abstract Citrullus lanatus var. citroides (African melon) is a crop cultivated in sub- Saharan Africa for its dried seeds reported to be rich in nutrients. In this project the relationship between its mode of reproduction and its genetic diversity will be established. This relationship is particularly interesting as C. lanatus var. citroides has been evolving in a traditional farming system, under a low-anthropic selection. On the one hand, the accurate reproduction strategies of this species will be determined by studying the floral morphology, occurrence of autoincompatibility, rate of allogamy. On the other hand, the genetic structure inside different accessions, and the associated population genetic flow will be established. This genetic structure of the species, as well as the rate of allogamy, will be analyzed with the use of microsatellite (SSR) markers. The relevant microsatellite markers have been set up for the watermelon. The resolution of these SSR markers has been performed for the C. lanatus var. citroides, with optimal annealing temperature varying from 49 to 56 C. Seventeen markers that proved to amplify their respective loci have been selected. The associated polymorphism is currently being studied. INTRODUCTION Citrullus lanatus var. citroides (African melon) is prized in sub-saharan Africa for its seeds reported to be rich in nutrients ~ 60 % lipids and ~ 30 % proteins (Loukou 2007). This secondary crop constitutes a valuable income for a part of the population: the average price for one kg is 2.3 euros seven times higher than one kg of coffee beans (Zoro Bi et al. 2003). The plants are classified into two cultigroups according to the morphology of the seeds and the fruits in Ivory Coast. The first morphotype (wléwlé) is characterized by glossy seeds with a tapered proximal extremity. It includes three cultivars based on the size of the seeds. In the second morphotype (bebu), the seeds have a flat ovoid shape with rugged and thick ends (Djè Y, pers. commun.). African melon is generally considered as allogamous, monoecious, entomophilous and protandrous (Gusmini 2003). The occurrence of autoincompatibility has not been demonstrated. It is fair to say that the reproductive mechanisms of C. lanatus var. citroides are far from being completely understood. 1 Cucurbitaceae 2008, Proceedings of the IX th EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May th,

2 The genetic structure of C. lanatus has been investigated for several years (Navot and Zamir 1987; Guerra-Sanz 2002; Joobeur et al. 2006). In particular, Jarret et al. (1997) used SSR markers and found a higher genetic diversity among accessions of C. lanatus var. citroides as compared with accessions of C. lanatus var. lanatus (watermelon). This conclusion was also reached in another work based on RAPD markers (Levi et al. 2001). However, whether the distinction between bebu and wléwlé morphotypes corresponds to different genetic groups of the species is not known. More recently, an analysis relying on the use of ISSR markers (Djè et al. 2006) has shown the existence of distinct genetic groups inside a unique accession (Tahi 2006). Although this result has to be confirmed, it suggests that a review of our knowledge of the reproduction mechanisms of C. lanatus var. citroides is needed. In particular, a possible occurrence of geitonopollinisation is not excluded, as the male and female flowering overlap (private communication). This also shows that studies on diversity and reproduction mechanisms cannot be separated for the purpose of a global understanding of C. lanatus var. citroides. Thus the general objective of the project is to determine the relationship between the mode of reproduction and the genetic diversity. In the case of C. lanatus var. citroides, this relationship is particularly interesting as the plant has been evolving in a traditional farming system, under a low-anthropic selection. The accurate determination of the reproduction strategies of the species will be achieved through a study of the floral morphology, the possible occurrence of autoincompatibility and of the rate of allogamy. This rate, as well as the diversity structure, will be analyzed by the use of microsatellite markers. MATERIAL AND METHODS Plant material Ninety-six accessions were obtained from the collection of the University of Abobo-Adjamé (Ivory Coast). Two plants per morphotype were used for the set up of SSR markers. The seeds were germinated in germination chambers under controlled conditions (20-25 C; dark). DNA extraction A CTAB extraction procedure modified from Murray and Thompson (1980) was used (Pissard et al. 2006). DNA was isolated from the tissue of fresh young leaves. Small pieces (70-80 mg) were ground with liquid nitrogen. The resulting powder was immediately transferred to a microfuge tube with 600 µl extraction buffer [100 mm Tris HCl (ph 8.0), 2 M NaCl, 2 % cetyltrimethylammonium bromide (CTAB), 2 % polyvinyl pyrollidone (PVP), 20 mm EDTA (ph 8.2), 1 % β- mercaptoethanol] and mixed thoroughly. After 40 min incubation at 65 C, 600 µl of chlorophorm isoamyl alcohol (24:1) was added. After mixing, it was centrifuged at r/min for 10 min at room temperature. The aqueous phase was recovered in a new Eppendorf tube. The extraction step with chlorophorm-isoamyl alcohol and the centrifugation were repeated. After the addition of 350 µl isopropanol at -4 C, the DNA was left to precipitate for 15 min at -20 C, centrifuged at r/min for 10 min and then washed twice with 500 µl ethanol (70 %). Finally, the DNA was dissolved in 50 µl TE [10 mmol/l Tris-HCl, 1 mmol/l EDTA (ph 8.0)]. The DNA 342

3 concentration was estimated with a spectrophotometer. The DNA was stored at -20 C until use. PCR conditions The relevant microsatellite markers have been set up for watermelon (Guerra- Sanz 2002). The resolution of these 19 SSR markers has been performed for C. lanatus var. citroides, with optimal annealing temperature varying from 49 to 56 C. The best conditions were retained according to the intensity and sharpness of the bands. Amplification reactions were performed in a 15 µl volume containing ng genomic DNA, 100 µmol.l -1 of each dntp (Amersham Bioscience Corp., Piscataway, N.J.), 0.5 µmol.l -1 primer (Eurogentec SA, Seraing, Belgium), 0.6 U Taq polymerase (Amersham Bioscience Corp.), and 1 Taq DNA Poly buffer (Amersham Bioscience Corp.). The PCR amplifications were performed using a PTC 100 Thermal Cycler (MJ Research, Waltham, Mass.) programmed as: 4 min at 94 C; 40 cycles of 30 s at 94 C, 30 s at annealing temperature (varying from 49 to 56 C), and 30 s at 72 C; and a final elongation step of 5 min at 72 C. Electrophoresis and data analysis PCR products (7.5 µl) mixed with bromophenol blue markers (1 µl for each well) were analyzed by electrophoresis using 1.5 % w/v agarose gel (100 ml) with 1.5 % of ethidium bromide in 1x TBE Buffer (10 x TBE: 108 g.l -1 Trisbase, 55 g.l -1 Boric acid, 8.3 g.l -1 EDTA ph 8) carried out at a constant voltage of 90 V for 140 min. A DNA ladder (Smartladder, Eurogentec SA) was used in each electrophoresis gel as a molecular mass marker. Gels stained with ethidium bromide were visualized under UV light and recorded with a video image analyzer (Biocapt, Vilbert Lourmat, Marne-la-Vallée, France). The patterns were analyzed using the Gene Profiler software (Scanalytics, Inc.) and only clearly scorable and reproducible bands on multiple independent runs were considered. The intensity of the bands was not taken into account for the general scoring. RESULTS AND DISCUSSION Among the 19 markers, 17 have lead to one clear band on agarose gel and have therefore been selected. The appropriate annealing temperature was 53 C for all markers. As agarose gels were used for this development phase, preliminary results did not allow determining the polymorphism and the exact size of the SSR. This further step will be achieved in a second phase, through automatic sequencing. For this step, the PCR protocol will need to be optimized (e.g. MgCl 2 and primer concentration). CONCLUSION The first step of this project, i.e. the selection of SSR markers for C. lanatus var. citroides, has been presented in this paper. In the next step towards the determination of the genetic structure, the polymorphism of SSR of two specimens for each morphotype will be determined by using automatic sequencing. Then the genetic structure of 20 plants from 4 accessions characterized by different morphotype will be analyzed. 343

4 For the investigation of the mode of reproduction, accessions will be cultivated both in greenhouses and fields. The different modes of controlled fecundation will be carried out during two generations: - Bagging of female flowers in order to check the potential occurrence of apomixis, - In case of presence of bisexual flowers, bagging of these flowers and histological study to discriminate between self-fertilization and apomixis, - Fertilization between flowers of both sexes carried by the same plant (geitonopollinisation), - Cross-fertilization between flowers of both sexes carried by different plants. The rate of allogamy will be established in situ (Côte d Ivoire) through a study of allelic composition on 25 mothers and 10 descendants for each of them (Ritland 2002), by the use of the above described microsatellite markers. The results of this project could help to establish adequate genetic improvement and conservation strategies of African melon. ACKNOWLEDEMENTS This research is supported by the FSR (Fonds Spéciaux de Recherche, UCL) and by the FRIA (Fonds pour la formation à la Recherche dans l Industrie et dans l Agriculture, Belgium). This project is done in collaboration with J.-P. Baudoin (Faculté Universitaire des Sciences Agronomiques de Gembloux, Belgium), Y. Djè (Université d'abobo-adjamé, Côte d'ivoire), I. Zoro Bi (Université d'abobo-adjamé, Côte d'ivoire), P. Baret, X. Draye, and A.-L. Jacquemart (UCL). Literature cited Djè Y, Tahi G, Zoro Bi A, Malice M, Baudoin J-P, Bertin P (2006) Optimization of ISSR marker for African edible-seeded Cucurbitaceae species genetic diversity analysis. Afr J Biotechnol 5: Guerra-Sanz J (2002) Citrullus simple sequence repeats markers from sequence databases. Molecular Ecology Notes 2: Gusmini G (2003) Watermelon (Citrullus lanatus) breeding handbook. Raleigh, NC State University (NC, USA) 90 pp Jarret R, Memck L, Holms T, Evans T, Aradhya M (1997) Simple sequence repeats in watermelon (Citrullus lanatus (Thumb.) Matsum. & Nakai). Genome 40: Joobeur T, Zhang X, Xu Y, Wehner T, Gusmini G, Levi A, Oliver M, Dean R (2006) Construction of a watermelon BAC library and identification of SSRs anchored to melon or Arabidopsis genomes. Theor Appl Genet 112: Levi A, Thomas C E, Wehner T C, Zhang X (2001) Low genetic diversity indicates the need to broaden the genetic base of cultivated watermelon. HortScience 36: Loukou AL, Gnakri D, Djè Y, Kippré AV, Malice M, Baudoin J-P, Zoro Bi I (2007) Macronutrient composition of three cucurbit species cultivated for seed consumption in Côte d Ivoire. Afr J Biotechnol 6: Murray M, Thompson W (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research 8: Navot N, Zamir D (1987) Isozyme and seed protein phylogeny of the genus Citrullus (Cucurbitaceae). Plant Syst Evol 156: Pissard A, Ghislain M, Bertin P (2006) Genetic diversity of the Andean tuber bearing species, oca (Oxalis tuberosa Mol.), investigated by inter-simple sequence repeats. Genome 49:

5 Ritland K (2002) Extensions of models for the estimation of mating systems using independent loci. Heredity 88: Tahi G (2006) Genetic structure of African edible seeds Citrullus lanatus (Thunberg) Matsumara & Nakai var. citroides using ISSR molecular markers.université catholique de Louvain, Faculté d'ingénierie biologique, agronomique et environnementale. Master report, 45 pp Zoro Bi I, Koffi K, Djè Y (2003) Caractérisation botanique et agronomique de trois espèces de cucurbites consommées en sauce en Afrique de l'ouest: Citrullus sp., Cucumeropsis mannii Naudin et Lagenaria siceraria (Molina) Standl. Biotech Agron Soc Environ 7:

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