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

In this study, we report a simple procedure for developing and using new types of polymerase chain reaction (PCR) primers, named ‘‘high-frequency oligonucleotides–targeting active genes’’ (HFO-TAG). The HFOTAG primers were constructed by first using a ‘‘practical extraction and report language’’ script to identify oligonucleotides (8, 9, and 10 bases) that exist in high frequency in 4700 expressed sequence tag (EST)-unigenes of watermelon (Citrullus lanatus) fruit. This computer-based screening yielded 3162 oligonucleotides that exist 32 to 335 times in the 4700 EST-unigenes. Of these, 192 HFO-TAG primers (found 51 to 269 times in the 4700 EST-unigenes) were used to amplify genomic DNA of four closely related watermelon cultivars (Allsweet, Crimson Sweet, Charleston Gray, and Dixielee). The average number of DNA fragments produced by a single HFO-TAG primer among these four watermelon cultivars was considerably higher (an average of 5.74 bands per primer) than the number of fragments produced by intersimple sequence repeat (ISSR) or randomly amplified polymorphic DNA (RAPD) primers (an average of 2.32 or 4.15 bands per primer, respectively). The HFO-TAG primers produced a higher number of polymorphic fragments (an average of 1.77 polymorphic fragments per primer) compared with the ISSR and RAPD primers (an average of 0.89 and 0.47 polymorphic fragments per primer, respectively). Amplification of genomic DNA from 12 watermelon cultivars and two U.S. Plant Introductions with the HFO-TAG primers produced a significantly higher number of fragments than RAPD primers. Also, in PCR experiments examining the ability of primers to amplify fragments from a watermelon cDNA library, the HFO-TAG primers produced considerably more fragments (an average of 6.44 fragments per primer) compared with ISSR and RAPD primers (an average of 3.59 and 2.49 fragments per primer, respectively). These results indicate that the HFO-TAG primers should be more effective than ISSR or RAPD primers in targeting active gene loci. The extensive EST database available for a large number of plant and animal species should be a useful source for developing HFO-TAG primers that can be used in genetic mapping and phylogenic studies of important crop plants and animal species. Although watermelon has a wide range of fruit phenotypes (including fruit size and shape, skin color and rind thickness, flesh color and texture, sugar content, and seed number, size, color, and shape), a narrow genetic base exists among American heirloom cultivars. The use of randomly amplified polymorphic DNA (RAPD) primers produced a low number of polymorphic markers among watermelon heirloom cultivars (Levi et al., 2001). In a recent study, 1309 RAPD primers were screened to identify polymorphic markers between two watermelon breeding lines whose progeny segregate for race 1 fusarium wilt resistance (caused by Fusarium oxysporum f. sp. niveum). Of these 1309 primers, only 75 polymorphic markers ( 1 polymorphism per 17 primers) were identified between these two breeding lines of watermelon (Harris et al., 2008 and K. Harris and A. Levi, unpublished data). The phenotypic diversity among watermelon cultivars could be the result of point mutations in genes controlling fruit quality (Levi et al., 2001, 2006b, 2009), which may not be readily detected by random primers. In fact, DNA polymorphism among watermelon cultivars could be detected with expressed sequence tag (EST)-polymerase chain reaction Received for publication 4 Nov. 2009. Accepted for publication 23 Apr. 2010. The use of trade names in this publication does not imply endorsement by the USDA of the products named or criticism of similar ones not mentioned. We gratefully acknowledge the technical assistance of Laura Pence and Ellis Caniglia. Corresponding author. E-mail: [email protected]. J. AMER. SOC. HORT. SCI. 135(4):369–378. 2010. 369 (PCR) and sequence-related amplified polymorphism (SRAP) markers when analyzed using dye-based capillary electrophoresis technologies that have the capability to detect single nucleotide differences (Levi et al., 2006b, 2009). The polymorphism revealed by capillary electrophoresis is often the result of a difference in a single or a few base pairs that cannot be detected by standard agarose gel electrophoresis (Levi et al., 2006b). Also, our experiments with amplified fragment length polymorphism (AFLP) markers analyzed by capillary electrophoresis produced higher polymorphism among watermelon cultivars than RAPD primers (Levi et al., 2004). However, mapping experiments indicated that most of the AFLP markers were clustered on several linkage groups and did not cover all regions of the watermelon genome (Levi et al., 2006b). The new technologies developed in recent years for DNA sequencing have produced an extensive sequence database and have enabled the physical assembly of numerous plant and animal genomes (Wicker et al., 2009). Sequence data have been used for the development of simple sequence repeat (SSR) markers used in gene tagging (Gong et al., 2009; Katzir et al., 1996), as well as the development of single nucleotide polymorphism (SNP) markers (Ganal et al., 2009). Sequences with wide genomic distribution, like SSR motifs, have been useful for the development of genetic markers in a large number of plant genomes (Holland et al., 2001; Jarret et al., 1997; Katzir et al., 1996). Oligonucleotides that exist in high frequency in active gene sequences should be valuable anchors for producing polymorphic DNA markers and for genetic mapping and the identification of important gene loci in a large number of crop plants. We hypothesized that a bioinformatics approach could be used in identifying oligonucleotides that exist in high frequency in expressed genes. These oligonucleotides could then be used to design random yet species-targeted primers for the identification of polymorphic markers between closely related genotypes. In a recent study, we constructed a cDNA library representing early, maturing, and ripe stages of the watermelon fruit. The fruit cDNA library was normalized and subtracted with cDNAs from young watermelon leaf (Levi et al., 2006a; Wechter et al., 2008). Over 8800 cDNA clones were sequenced and assembled into 4700 EST-unigenes. These EST-unigene sequences are available on the International Cucurbit Genomics Initiative website (2009). In this study, we searched for oligonucleotides (8, 9, and 10 bp) that exist in high frequency in the 4700 watermelon EST-unigenes. We tested the potential of these oligonucleotides as PCR primers to produce polymorphic markers from genomic DNA of closely related heirloom watermelon cultivars (C. lanatus var. lanatus) and U.S. Plant Introductions (PIs) of C. lanatus var. lanatus and C. lanatus var. citroides. To substantiate the possibility that the high-frequency oligonucleotides–targeting active genes (HFO-TAG) primers are better suited than ISSR or RAPD primers to target active gene sequences, we also examined their ability to amplify fragments from a cDNA library representing watermelon fruit. Materials and Methods PLANT MATERIAL AND ISOLATION OF DNA. Twelve heirloom cultivars that were previously shown to have a low rate of crosscultivar polymorphism when analyzed using standard RAPD primers (Levi et al., 2001) were selected for analysis. These cultivars are Allsweet, Crimson Sweet, Charleston Gray, Dixielee, Congo, Mickylee, Minilee, Sugar Baby, Jubilee, Black Diamond, Calhoun Gray, and New Hampshire Midget. In addition, U.S. PI 203551 (C. lanatus var. lanatus) and PI 296341 (C. lanatus var. citroides) also were used for analysis. Seedlings of the watermelon cultivars and PIs were grown in the greenhouse at 26/20 C (day/night temperatures). Young leaves were collected from 2to 3-week-old plants and were stored at –80 C for later DNA isolation. The DNA was isolated from the frozen leaves using the method described by Levi and Thomas (1999). BIOINFORMATIC ANALYSIS FOR IDENTIFICATION OF PREVALENT SHORT SEQUENCES. An in-house computer script was written in practical extraction and report language (Perl) and was used to query for the most frequent 8to10-bp oligonucleotides (GC content >75%) in the 4700 watermelon EST-unigenes published on the International Cucurbit Genomics Initiative (ICuGI) website (International Cucurbit Genomics Initiative, 2009). One-hundred ninety-two oligonucleotides with melting temperatures of 34 to 42 C were selected for constructing the HFO-TAG primers that were tested in this study (Table 1). RAPD AND ISSR PRIMERS. We used RAPD and ISSR primers as a comparative reference to assess the effectiveness of the HFO-TAG primers in amplifying watermelon genomic DNA. Forty-nine RAPD primers (with a GC content of 60%– 90%) were from the AK and AL RAPD primer sets (Operon Technologies, Alameda, CA), and the UBC 500 and 700 primer sets (University of British Columbia, Vancouver, BC, Canada). The ISSR primers (98 primers) were from the UBC 800 primer set. DNA AMPLIFICATION CONDITIONS AND GEL ELECTROPHORESIS. The PCR reaction and thermal cycling conditions for HFOTAG primers selected in this study (Table 1) were as described for RAPD primers by Levi et al. (1993, 2001): The PCR reaction cocktail (25 mL) contained 20 mM NaCl, 50 mM TrisHCl, pH 9, 1% Triton X-100, 0.01% gelatin, 1.6 mM MgCl2, 200 mM each of dNTPs (Sigma-Aldrich, St. Louis), 100 mM primer, 5 units of GoTaq DNA polymerase (Promega, Madison, WI), and 7 ng of template DNA. Amplifications were carried out for 40 cycles in a PTC-200 thermocycler (MJ Research, Watertown, MA) for 60 s to denature the DNA at 92 C, 70 s for primer annealing at 35, 40, 45, 48, 50, 55, or 60 C [as determined for each primer in Table 1, based on primer melting temperature (Tm)], and 120 s for primer extension at 72 C. Amplification products were separated by electrophoresis in 1.4% agarose gel in 0.5· Tris borate buffer (Sambrook et al., 1989). Before loading on agarose gels, the DNA samples were stained with the EZ-Vision -Three dye (Amresco, Solon, OH). DNA fragments were visualized under ultraviolet light and were photographed using a still video system (Gel Doc 2000; Bio-Rad, Hercules, CA). The size of the amplification products was determined by comparison with a 1-kb DNA ladder (Gibco-BRL, Gaithersburg, MD). COMPARISON OF AMPLICONS GENERATED USING HIGHLY SIMILAR HFO-TAG PRIMERS. Eight 8-mer primers differing at a single nucleotide at the 3# or 5# end, and a 9-mer primer differing at the last two nucleotides of the 3# end (Table 2) were tested with DNA from a single watermelon cultivar (Charleston Gray). Amplification conditions and post-amplification DNA visualization were performed as described above. PCR AMPLIFICATION CONDITIONS FOR HFO-TAG, ISSR, AND RAPD PRIMERS USING CDNA AS A TEMPLATE. The cDNA library was constructed previously using mRNA representing watermelon fruit of the heirloom cultivar Illiniwake Red (as described by Levi et al., 2006a). PCR amplification conditions of 370 J. AMER. SOC. HORT. SCI. 135(4):369–378. 2010. Table 1. The 192 high-frequency oligonucleotides–targeting active genes (HFO-TAG) primers that were tested in PCR experiments with watermelon DNA. Oligonucleotide frequency among the 4700 watermelon EST-unigenes (FRQ), number of nucleotide bases for each primer (B), GC content (1 = 100%), melting temperature (Tm), and annealing temperature (Ta) used in PCR reactions with genomic or cDNA templates. In addition, the total number of fragments (TF) and the number of polymorphic fragments (PF) produced by each of the HFO-TAG primers among the closely related watermelon cultivars Allsweet, Crimson Sweet, Charleston Gray, and Dixielee. Primer Oligos FRQ (no.) B (no.) GC Tm ( C) Ta ( C) TF (no.) PF (no.) HFO-1 CGGCGGCG 269 8 1 43 48 9 2 HFO-2 CGCCGCCG 269 8 1 43 48 8 2 HFO-3 CCGCCGCC 240 8 1 41.9 48 9 1 HFO-4 GGCGGCGG 240 8 1 41.9 48 7 2 HFO-5 CCACCGCC 172 8 0.875 34 40 15 1 HFO-6 GGCGGTGG 172 8 0.875 34 40 12 1 HFO-7 GCCGCCGC 170 8 1 43.5 50 9 2 HFO-8 GCGGCGGC 170 8 1 43.5 50 10 0 HFO-9 CGGCGGAG 159 8 0.875 34 40 10 3 HFO-10 CTCCGCCG 159 8 0.875 34 40 8 2 HFO-11 GGCGGAGG 144 8 0.875 32.6 40 9 3 HFO-12 CCTCCGCC 144 8 0.875 32.6 40 8 2 HFO-13 TCCGCCGC 140 8 0.875 38.4 45 3 1 HFO-14 GCGGCGGA 140 8 0.875 38.4 45 6 1 HFO-15 CACCGCCG 136 8 0.875 35.4 40 3 1 HFO-16 CGGCGGTG 136 8 0.875 35.4 40 9 1 HFO-17 CGACGGCG 132 8 0.875 35.9 40 8 1 HFO-18 CGCCGTCG 132 8 0.875 35.9 40 7 2 HFO-19 TCGCCGCC 127 8 0.875 38.4 45 2 1 HFO-20 GGCGGCGA 127 8 0.875 38.4 45 7 1 HFO-21 CCGCCGCCG 120 9 1 49.1 55 9 6 HFO-22 CGGCGGCGG 120 9 1 49.1 55 4 1 HFO-23 ACGGCGGC 119 8 0.875 39.1 45 9 2 HFO-24 CCGCCACC 119 8 0.875 34 40 9 0 HFO-25 GGTGGCGG 119 8 0.875 34 40 7 0 HFO-26 GCCGCCGT 119 8 0.875 39.1 45 7 0 HFO-27 CCGCCGTC 118 8 0.875 34.7 40 6 1 HFO-28 GACGGCGG 118 8 0.875 34.7 40 9 2 HFO-29 CGCCGCCGC 114 9 1 50.4 55 0 0 HFO-30 GCGGCGGCG 114 9 1 50.4 55 6 3 HFO-31 CGCCGCCA 105 8 0.875 39.1 45 5 0 HFO-32 TGGCGGCG 105 8 0.875 39.1 45 1 1 HFO-33 CGCCGGAG 104 8 0.875 34 40 5 2 HFO-34 CTCCGGCG 104 8 0.875 34 40 5 2 HFO-35 GCCGCCGCC 96 9 1 49.7 55 6 4 HFO-36 GGCGGCGGC 96 9 1 49.7 55 8 6 HFO-37 CGGCGCCG 92 8 1 40.9 45 4 3 HFO-38 GCCGCCGG 85 8 1 41.9 48 11 3 HFO-39 CCGGCGGC 85 8 1 41.9 48 9 1 HFO-40 CCGCCTCC 85 8 0.875 32.6 40 10 1 HFO-41 GCGGTGGC 85 8 0.875 35.8 40 6 1 HFO-42 GCCACCGC 85 8 0.875 35.8 40 6 1 HFO-43 GGAGGCGG 85 8 0.875 32.6 40 12 2 HFO-44 CGCCGGCG 84 8 1 40.9 45 1 1 HFO-45 CGGCGACG 84 8 0.875 35.9 40 7 1 HFO-46 CGTCGCCG 84 8 0.875 35.9 40 5 1 HFO-47 GCCGCCAC 83 8 0.875 35.8 40 14 2 HFO-48 GTGGCGGC 83 8 0.875 35.8 40 10 2 HFO-49 GCGGCGGT 82 8 0.875 39.1 45 5 1 HFO-50 ACCGCCGC 82 8 0.875 39.1 45 7 2 HFO-51 TCGCCGCCG 81 9 0.889 46.1 50 8 0 HFO-52 CGGCGGCGA 81 9 0.889 46.1 50 9 1 HFO-53 GGTGGGGG 81 8 0.875 30.6 35 7 0 HFO-54 CCCCCACC 81 8 0.875 30.6 35 8 2