To date, there are only a few sequenced-tagged site (STS) markers associated with genes controlling fruit quality in watermelon. A normalized cDNA library for watermelon fruit (Citrullus lanatus var. lanatus) was constructed. Sequence analysis of the cDNA clones resulted in the development of 4700 non-redundant ESTs (EST unigenes) expressed in watermelon fruit (http://www.ncbi.nlm.nih.gov, http://www.icugi.org). One hundred of these EST unigenes [including 40 EST unigenes that contain simple sequence repeat (SSR) motives (EST-SSRs) and 60 customary EST unigenes (not containing SSR motives)] were used for designing primer pairs for PCR experiments. The EST primer pairs were tested in PCR experiments with genomic DNA of 25 watermelon heirloom cultivars and 13 United States Plant Introductions (US PIs) of Citrullus sp., including four C. lanatus var. lanatus, five C. lanatus var. citroides PIs and four Citrullus colocynthis PIs. The 40 EST-SSR and 60 EST primer pairs produced 108 and 142 EST-PCR markers, respectively, among the Citrullus PIs and watermelon cultivars. A large number of the EST-PCR markers were polymorphic between the Citrullus PIs and the watermelon cultivars, but significantly less polymorphic among the cultivars. Of the 108 EST-PCR markers associated with EST-SSRs, 103 exist in the Citrullus PIs and 64 in the cultivars. Of these 64 markers, 45 (70.3%) were polymorphic among the cultivars. Of the 142 EST-PCR markers associated with customary ESTs, 134 exist in the Citrullus PIs and 108 in the cultivars. Of these 108 markers, 86 (79.6%) were polymorphic among cultivars. The results in this study indicate that polymorphism exists in coding regions of genes expressed in fruits of watermelon cultivars. In total, 131 polymorphic EST-PCR markers (45 associated with EST-SSRs and 86 associated with customary ESTs) related to watermelon fruit genes were generated using the above data. These markers should be useful for DNA fingerprinting of cultivars and breeding lines, for assessing genetic relationships and for genetic mapping of watermelon.
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