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SNP Discovery and Mapping in Melon (Cucumis melo L.) Maya Lotan-Pompan, Galil Tzuri, Vitaly Portnoy, Rotem Beja and Nurit Katzir Agricultural Research Organization, Department of Genetics and Vegetable Crops, Newe Ya’ar Research Center, P. O. Box 1021, Ramat Yishay 30-095, Israel email@example.com Amir Sherman, Shahar Cohen, Nir Dai and Arthur A. Schaffer Agricultural Research Organization, Dept. of Genetics and Vegetable Crops, P. O. Box 6, Bet Dagan, 50-250, Israel Jim Giovannoni Boyce Thompson Institute for Plant Research, Tower Road, Cornell University, Ithaca, NY 14853, U.S.A. Introduction: Several studies have applied SNP discovery. From the melon EST various marker types (including RFLP, database (http://melon.bti.cornell.edu), three RAPD, AFLP and SSR) for mapping and different methods were used for SNP assessing genetic variation within C. melo detection: (i) comparative analysis of (1, 3, 6, 7). A partial linkage map of melon sequences of different genotypes, (ii) from a cross between PI 414723 (C. melo primers designed for the end sequences of Group Acidulus) and ‘Dulce’ (C. melo interesting ESTs were used to amplify the Group Reticulatus) was previously described genomic DNA of both parental lines by us (2). Single nucleotide polymorphisms (‘Dulce’ and PI 414723); this was followed (SNPs) are an abundant form of genetic by screening of the amplicons using either variation in the genome of various species DHPLC (Denaturing High Performance that are extremely useful for gene mapping Liquid Chromatography) or SSCP (Single and phylogenetic studies. In recent years, Strand Conformational Polymorphisms), in SNPs have become important as genomic order to find polymorphism between the markers with numerous technical methods parental amplicons. The polymorphic developed for their detection (4). In this amplicons were re-sequenced in order to study, SNP markers for genes belonging to validate the SNPs, (iii) direct sequencing of major fruit metabolic pathways were parental amplicons. developed. In order to link between these markers and fruit quality traits, these SNPs Genotyping. SNP genotyping was are currently being located on the PI 414723 conducted using the Sequenom ‘Dulce’ map. MassARRAY® platform, by the high- throughput genotyping assays: hME and Materials and Methods: Plant material. iPLEX. These assays allowed multiple PCR The F2 mapping population included 112 reactions in a single well (5). JoinMap 3.0 individuals derived from a cross between (Kyazma, Holland) was used for linkage ‘Dulce’ and PI 414723 (2). The ‘Dulce’ fruit analysis and map calculations. is aromatic, sweet, of high pH, with orange- colored flesh and a netted rind. The PI Results: SNP Markers for ca. 100 genes 414723 fruit is non-aromatic, non-sweet, belonging to major fruit metabolic pathways and acidic, with salmon-colored flesh and no were developed (Table 1). These include net. genes encoding for key enzymatic and transport steps in carbohydrate, acid, volatile and carotenoid metabolism in melon and Cucurbit Genetics Cooperative Report 28-29: 19-21 (2005-2006) 19 other cucurbits. SNP frequency was found to 4. Gut, I. G. 2001. Automation in vary between one in 5000 bp to ten in 300 genotyping of single nucleotide bp. A search for linkages between SNPs and polymorphisms. Hum. Mutat. 17: 475- traits is underway using a RIL population 492. derived from the F2 population. 5. Oeth, P., M. Beaulieu, C. Park, D. Kosman, G. del Mistro, D. van den Acknowledgements: Boom, and C. Jurinke. 2005. iPLEXTM Assay: Increased plexing efficiency and Contribution no. 108/2007 of the Institute of flexibility for MassARRAY® System Plant Sciences, Agricultural Research through single base primer extension Organization, Israel. with mass-modified terminators. Sequenom application note Literature Cited: (http://www.sequenom.com/Seq_genoty ping.html). 1. Danin-Poleg, Y., N. Reis, S. Baudracco- 6. Périn, C., L. Hagen, V. De Conto, N. Arnas, M. Pitrat, J.E. Staub, M. Oliver, Katzir, Y. Danin-Poleg, V. Portnoy, S. P. Arus, C.M. deVicente, and N. Katzir. Baudracco-Arnas, J. Chadoeuf, C. 2000. Simple Sequence Repeats in Dogimont, and M. Pitrat. 2002. A Cucumis mapping and map merging. reference map of Cucumis melo based on Genome 43: 963-974. two recombinant inbred line populations. 2. Danin-Poleg, Y., Y. Tadmor, G. Tzuri, Theor. Appl. Genet. 104: 1017-1034. N. Reis, J. Hirschberg, and N. Katzir. 7. Silberstein, L.,I. Kovalski, Y. Brotman, 2002. Construction of a genetic map of C. Perin, C. Dogimont, M. Pitrat, J. melon with molecular markers, Klingler, G. Thompson, V. Portnoy, N. horticultural traits and ZYMV Katzir, and R. Perl-Treves, 2003. resistance. Euphytica 125: 373-384. Linkage map of Cucumis melo including 3. Gonzalo, M. J., M. Oliver, J. Garcia- phenotypic traits and sequence- Mas, A. Monfort, R. Dolcet-Sanjuan , N. characterized genes. Genome 46: 761- Katzir, P. Arús, and A. J. Monforte. 773. 2005. Simple-sequence repeat markers used in merging linkage maps of melon (Cucumis melo L.). Theor. Appl. Genet. 110: 802-811. 20 Cucurbit Genetics Cooperative Report 28-29: 19-21 (2005-2006) Table 1. Genes associated with carbohydrate, carotenoid and organic acid metabolism. SNPs were developed for all the listed genes. Stars mark the genes located on the melon map. Carbohydrate genes Carotenoid genes Organic acid genes Alkaline alpha Hexose * Geranylgeranyl Phosphoenol pyruvate NAD malate * Vacuolar pump galactosidase I transporter 1 Pyrophosphate reductase carboxykinase dehydrogenase (mitochondria) V0-a subunit * Alkaline alpha * Hexose transporter Geranylgeranyl * Phosphoenol pyruvate NAD malate * Vacuolar pump V0-c' galactosidase 2 6 pyrophosphate synthase carboxylase (a) dehydrogenase (glyoxysome) subunit * Acid alpha * Acid * Phytoene * Phosphoenol pyruvate * NADP malic * Vacuolar pump galactosidase 1 invertase 1 synthase 1 carboxylase (b) enzyme (chloroplast) V0-c subunit * Putative galactose * Acid * Phytoene * Citrate synthase * NAD malate * Vacuolar pump V0-d kinase invertase 2 synthase 2 (glyoxysome) dehydrogenase (chloroplast) subunit Galactose-1-phosphate *Alkaline/ * Phytoene desaturase * Citrate synthase * Malate synthase * Vacuolar pump uridyltransferase neutral invertase 1 (mitochondria) (glyoxysome) V0-e subunit * UDP-glucose 4- *Alkaline/ * Zeta carotene * Aconitase * NADP Isocitrate lyase * Vacuolar pump V1-a epimerase 1 neutral invertase 2 desaturase subunit UDP-glucose 4- * Sucrose * Lycopene beta- * NAD Isocitrate ATP citrate lyase (b) * Vacuolar pump epimerase 2 synthase cyclase dehydrogenase (β) V1-b subunit UDP-glucose 4- Sucrose * Βeta-carotene * α keto glutarate NADP malic enzyme Vacuolar pump epimerase 3 synthase 1 hydroxilase dehydrogenase (E2) (cytosol) V1-C subunit UDP-glucose * Sucrose cleavage * Carotenoid isomerase * Succinyl CoA * NAD Isocitrate dehydrogenase * Vacuolar pump V1-d pyrophosphorylase protein-like synthetase (α) subunit *Phosphoglucomutase Hexokinase 2 * Succinyl CoA * Citrate transporter (mitochondria) * Vacuolar pump (cytosol) synthetase (β) V1-e subunit * Sucrose-phosphate Fructokinase 3 * Succinate *Citrate transporter * Vacuolar pump synthase 1 dehydrogenase (α) (glyoxysome) V1-f subunit Putative sucrose * Succinate Vacuolar pump transporter dehydrogenase (β) V1-G subunit Sugar transporter * Fumarase Vacuolar H[+]- superfamily translocating inorganic pyrophosphatase Cucurbit Genetics Cooperative Report 28-29: 19-21 (2005-2006) 21