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Title: High-frequency Oligonucleotides in Watermelon Expressed Sequenced Tag-unigenes Are Useful in Producing Polymorphic Polymerase Chain Reaction Markers among Watermelon Genotypes

Author
item Levi, Amnon
item Wechter, William - Pat
item Harris-Shultz, Karen
item Davis, Angela
item FIE, ZHANGJUN - Boyce Thompson Institute

Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/26/2010
Publication Date: 7/1/2010
Citation: Levi, A., Wechter, W.P., Harris-Shultz, K.R., Davis, A.R., Fie, Z. 2010. High-frequency Oligonucleotides in Watermelon Expressed Sequenced Tag-unigenes Are Useful in Producing Polymorphic Polymerase Chain Reaction Markers among Watermelon Genotypes. Journal of the American Society for Horticultural Science. 135(4):369-378.

Interpretive Summary: Advanced DNA technologies are producing enormous amounts of information with respect to gene sequences of animals and plants. Still, it is difficult to employ the gene sequence information in studies aiming to find linkage between the gene sequences and the traits they control in crop plants. For example in watermelon, there is still a great need to genetically map and identify genes that confer disease or pest resistance and incorporate them into watermelon cultivars. In this study, scientists at the USDA, ARS, U.S. Vegetable Laboratory (Charleston, SC) collaborated with scientists at the Boyce Thompson Institute (Ithaca, NY) in developing a procedure to identify and map gene sequences on a genetic linkage map constructed for watermelon. The scientists used computational technology to identify 3,200 DNA sequences that are prevalent in genes of watermelon and named them “high frequency gene sequences (HFGS)”. These HFGS proved useful in the development of DNA markers used in genetic mapping of watermelon. The HFGS procedure developed in this study provides a new opportunity in genetic mapping, and should be useful for molecular biologists and plant breeders interested in identifying genes controlling valuable horticultural traits and in incorporating them into elite cultivars in variety of crop plants.

Technical Abstract: In this study, we report a simple procedure for developing gene targeted primers, named high-frequency gene sequence (HFGS) primers, based on oligonucleotides that exist in high frequency in watermelon expressed sequence tag (EST)-unigenes. These HFGS primers were constructed by first screening for 8, 9, and 10 base sequences that exist in high frequency in 4,700 watermelon fruit EST-unigenes [International Cucurbit Genomics Initiative (ICuGI); http://www.icugi.org]. The screening, using an in house Perl script, yielded 3,200 oligonucleotides that exist 32 to 335 times in the 4,700 EST-unigens. Then, one-hundred and ninety-two HFGS oligonucleotide (87.5-100% GC content) were used for primer construction. The 192 HFGS-primers were tested in PCR reactions with genomic DNA of the watermelon cultivars Allsweet, Crimson Sweet, Charleston Gray and Dixie Lee, known to have close genetic similarity, based on analysis with 85 RAPD primers. The average number of bands produced by the HFGS primers among these four watermelon cultivars was comparable (5.1 bands per primer) to the number of bands produced by RAPD primers (5.6 bands per primer). However, the HFGS primers produced a higher number of polymorphic bands (an average of 1.77 bands per primer) than RAPD primers (an average of 0.73 polymorphic bands per primer). Because they are derived from EST-unigenes, the HFGS primers may reveal new genomic loci and be more useful in gene tagging and genetic mapping than RAPD primers.