PCR-based Single-strand Conformation Polymorphism (SSCP) Analysis to Clone Nine Aquaporin Genes in Cucumber

Combining the use of PCR and single-strand conformation polymorphisms (SSCP), nine sequences from the cucumber genome were successfully identified and cloned that encoded two well-conserved asparagine-proline-alanine (NPA) domain homologues to aquaporin genes. The sensitivity and detection efficiency of SSCP and restriction enzyme analysis for detecting DNA sequence variation were evaluated using similar-sized DNA fragments. The SSCP analysis was more sensitive and efficient for discriminating different clones than restriction enzyme analysis, although some sequence variation inside similar-sized DNA fragments could be identified by restriction analysis. Consideration of the results of SSCP analysis with DNA sequence information indicated that one or two base pair changes in the amplified regions could be detected. Moreover, the SSCP analysis results of genomic DNA PCR products that were amplified by degenerate primers can provide rough information about the number of member genes. If the SSCP bands of a cloned fragment (such as CRB7) did not have the corresponding bands from genomic DNA PCR products, that fragment might be a misamplified product. The PCR-based SSCP method with degenerate oligonucleotide primers should facilitate the cloning of member genes. for rapid analysis of mutations (Orita et al., 1989). Single-strand conformation polymorphisms has been used extensively in human genetics to detect mutations within genes, and was able to detect single-point mutations (Hayashi, 1992). Single-strand conformation polymorphisms analysis was considered as a sensitive, inexpensive, and rapid method for detecting sequence variation (Sekiya, 1993; Slabaugh et al., 1997). This technique was also employed to characterize expressed products between two highly related member genes (Hedley et al., 1994), analyze the difference among members of a gene family (Hagiwara et al., 1998; Slabaugh et al., 1997), and produce SSCP markers for gene mapping (Slabaugh et al., 1997). Recently, the usefulness of this approach has been extended through the use of end-labeled primers and PCR amplification, with a small amount of genomic DNA or RNA being analyzed (Peterson et al., 1995; Slabaugh et al., 1997). Aquaporins (AQPs) are a family of intrinsic membrane proteins with many homologous genes that function as water-selective channels in the plasma membranes of many water-transporting tissues (Agre et al., 1993). We needed to identify a rootspecific aquaporin gene in cucumber for our root-knot nematode resistance work. Using PCR-based SSCP analysis, nine sequences have been successfully cloned from the cucumber genome that encoded two well-conserved NPA domains homologous to the aquaporin gene. Materials and Methods DNA PREPARATION. Seedlings of Cucumis sativus L. ‘Sumter’, a monoecious inbred pickling cucumber, were grown in the greenhouse. Leaves of four-week-old seedlings were harvested for DNA isolation. Total DNA was prepared by grinding frozen leaf tissue and suspending the powder in CTAB buffer containing 50 mM EDTA, 10 mM Tris-HCl (pH 8.0), 1% CTAB, 0.7 M NaCl, and 0.2% β-mercaptoethanol. DNA was precipitated by ice-cold 100% iso-propanol, and then washed with 76% ethanol with 0.2 M NaOAc and 76% ethanol with 10 mM NH4OAc, respectively. SYNTHESIS OF OLIGONUCLEOTIDE PRIMERS. Multiple alignments Received for publication 1 May 2002. Accepted for publication 2 Sept. 2002. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service (NCARS), Raleigh, NC 27695-7643 of the products named, or criticism of similar ones not mentioned. This work was supported by a grant from Pickle Packers International. The authors gratefully acknowledge the technical assistance of J.P. Fellers, Y. Li, G.N. Acedo, and W.L. Liu. Postdoctoral research associate. Currently researcher, Vector Tobacco Ltd., 700 Main Street, Durham, NC 27701. Professor and corresponding author; email [email protected]. Professor. Currently vice president, Vector Tobacco Ltd., 700 Main Street, Durham, NC 27701. The polymerase chain reaction (PCR) using degenerate oligonucleotide primers has made it possible to clone genes more quickly and easily (Takumi and Lodish, 1994; You and Scholl, 1998). With the accumulation of gene sequences, it becomes easy to design the degenerate primers for cloning new members of a gene family from related species. However, for members of a gene family amplified using degenerate primers, the PCR products for these genes can be similar in length. Thus, the different genes will be hidden when they are run on an agarose gel. Direct DNA sequencing would be the best method for distinguishing different DNA fragments, and sequencing as many clones as possible may permit the identification of some or all members of a gene family, but it is time-consuming and expensive. After cloning, different restriction enzymes that cut DNA infrequently are used to identify the new homologue, especially if using enzymes with recognition sites of 6 to 8 bp (Preston, 1993). The problem is that restriction analysis for small (300 to 500 bp) DNA bands requires that the bands have a recognition site close to the middle. DNA bands without enzyme recognition sites, or with two outside recognition sites may not appear in the analysis. Therefore, a more sensitive method is needed for detection of DNA fragments of similar length to identify the member genes during cloning. Single-strand conformation polymorphisms (SSCP) are based on electrophoretic detection of conformational changes in singlestranded DNA molecules resulting from point mutations or other forms of small nucleotide changes. It was originally developed