A novel method for screening species-specific gDNA probes for species identification

Abstract

We report a method called SSH array which combines the suppression subtraction hybridization (SSH) and DNA array techniques to find species-specific DNA probes from genomic DNA (gDNA) for species identification. The method first obtains the differential gDNA fragments between two species by SSH and then hybridizes the differential gDNA fragments with arrays made of multiple whole genomes from several species to screen the unique gDNA fragments for one species. The screened unique gDNA fragments can be used as species-specific probes to differentiate the species they represent from all other species. We used five species of the genus Dendrobrium, D.aurantiacum Kerr, D.officinale Kimura et Migo, D.nobile Lindl., D.chrysotoxum Lindl. and D.fimbriatum Hook., as experimental materials to study the feasibility of the method. The results showed that the method could efficiently obtain different species-specific probes for each of the five species.

Figure 1

Images and signal intensity plots (including t-tests) of hybridization of the species-specific probes to the gDNAs array of five species. Columns of spots on each array from left to right are D.aurantiacum Kerr, D.officinale Kimura et Migo, D.nobile Lindl., D.chrysotoxum Lindl. and D.fimbriatum Hook. D.A-SP1 (A) and D.A-SP2 (C) are D.aurantiacum Kerr-specific probes, D.O-SP1 (E) and D.O-SP2 (G) are D.officinale Kimura et Migo-specific probes, D.N-SP1 (I) and D.N-SP2 (K) are D.nobile Lindl.-specific probes, D.C-SP1 (M) and D.C-SP2 (O) are D.chrysotoxum Lindl.-specific probes and D.F-SP1 (Q) and D.F-SP2 (S) are D.fimbriatum Hook.-specific probes. (B), (D), (F), (H), (J), (L), (N), (P), (R) and (T) are signal intensities of (A), (C), (E), (G), (I), (K), (M), (O), (Q) and (S), respectively. All of the t-tests were significant (P < 0.01). A, O, N, C and F on the x-axis of the signal intensity plots represent D.aurantiacum Kerr, D.officinale Kimura et Migo, D.nobile Lindl., D.chrysotoxum Lindl. and D.fimbriatum Hook., respectively.

Figure 1

Images and signal intensity plots (including t-tests) of hybridization of the species-specific probes to the gDNAs array of five species. Columns of spots on each array from left to right are D.aurantiacum Kerr, D.officinale Kimura et Migo, D.nobile Lindl., D.chrysotoxum Lindl. and D.fimbriatum Hook. D.A-SP1 (A) and D.A-SP2 (C) are D.aurantiacum Kerr-specific probes, D.O-SP1 (E) and D.O-SP2 (G) are D.officinale Kimura et Migo-specific probes, D.N-SP1 (I) and D.N-SP2 (K) are D.nobile Lindl.-specific probes, D.C-SP1 (M) and D.C-SP2 (O) are D.chrysotoxum Lindl.-specific probes and D.F-SP1 (Q) and D.F-SP2 (S) are D.fimbriatum Hook.-specific probes. (B), (D), (F), (H), (J), (L), (N), (P), (R) and (T) are signal intensities of (A), (C), (E), (G), (I), (K), (M), (O), (Q) and (S), respectively. All of the t-tests were significant (P < 0.01). A, O, N, C and F on the x-axis of the signal intensity plots represent D.aurantiacum Kerr, D.officinale Kimura et Migo, D.nobile Lindl., D.chrysotoxum Lindl. and D.fimbriatum Hook., respectively.

Figure 2

Images and signal intensity plots (including t-tests) of hybridization of the species-specific probes to the unknown gDNAs arrays for species identification. (A) Results of probe D.O-SP1 hybridizing with the unknown gDNAs array, suggesting that the gDNAs comprising column 4 of the array should come from the species D.officinale Kimura et Migo. (C) Image of probe D.F-SP2 hybridizing to the unknown gDNAs array, suggesting that the gDNAs comprising column 1 of the array should be from species D.fimbriatum Hook. (B and D) Signal intensities of (A) and (C), respectively. The two t-tests were significant (P < 0.01).

Figure 3

Images of the species-specific probes hybridizing with the gDNAs array consisting of 72 Dendrobrium samples. The gDNAs of each sample were printed in double spots on the array. (A) Hybridization of probe D.O-SP2 to the array. All 21 samples of D.officinale Kimura et Migo were correctly detected. (B) Hybridization of probe D.C-SP2 to the array. Eleven samples of D.chrysotoxum Lindl. were definitely detected.