Systematic Studies on the Family Cucurbitaceae of Eastern Bihar - Download as DOC by XvkK39d


									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

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 (, 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:                                      (
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

Cucurbit Genetics Cooperative Report 28-29: 19-21 (2005-2006)                                                                                              21

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