Syntenic Analysis of Cucurbit Cultivar Complex Using Microsatellite Markers

The Cucurbitaceae family is a monophyletic clade because of its morphological and biochemical distinctness and represents some of economically important species, particularly those with edible and medicinal fruits (Whitaker and Bemis, 1976; Batas and Robinson, 1995). Cucurbitaceae is known to have 90 genera and 700 species, of which there are several domesticated species. Cucurbit species include a variety of high value crops that play important role both in local diets and as export crops in the US. The cultivated watermelon belongs to Citrullus lanatus var. lanatus, which is considered as a descendent of the species C. colocynthis (Navot and Zamir, 1987; Levi et al. 2000).  Melons (Cucumis melo L.) are a diverse group. Summer squash is Cucurbita pepo, but winter squash may be C. pepo (e.g. 'Acorn’) or C. moschata (‘Butternut’). Microsatellites or Single Sequence Repeats (SSRs) are short repetitive nucleotide motifs found throughout the genome of all eukaryotes. Microsatellite markers are Co-dominant, multi-allelic and high polymorphic. Current research is focused to identify heterologous fruit related microsatellites that can be used to identify common fruit traits at molecular level across the Cucurbitaceae family.  DNA was extracted from leaf tissues using the method described in the QIAGEN DNeasy Plant Mini Kit. Fruit specific microsatellite sequences were obtained from the web site of International Cucurbit Genomics Initiative (http://www.icugi.org). Primers were designed using the software PRIMER 3.0 (http://frodo.wi.mit.edu). PCR conditions for SSRs were used as per Reddy et al., (2001) and gel electrophoresis was carried out using SFR high-resolution agarose (www.amresco-inc.com). Genetic similarities based on Jaccard’s coefficients (Jaccard 1908) were calculated using the SIMQUAL program of the Numerical Taxonomy Multivariate Analysis System (NTSYS-pc) Version 2.0 software package (Rohlf 1997). The resulting genetic diversity indices were used to generate a tree using the Neighbor Joining method (Saitou and Nei 1987). Principal Component Analysis (PCA) based on the genetic similarity matrices were performed using DCENTER and EIGEN algorithms of the NTSYS-pc software package. We identified 176 fruit related  EST specific SSRs developed from melon to amplify 750 different alleles across the 12 taxa that belong to  the sub-family Cucurbitae (Squash and Pumpkin)  and the subfamily Benincaseae (Watermelon, melon, bottler gourd and bitter gourd). A phylogenetic tree and PCA clearly resolved various Taxa according to their classical taxonomic relationshipsThe microsatellite markers that are characterized as heterologous can be of immense use for cucurbit breeding community for use in marker assisted selections and the other genetic/syntenic studies.