51. Development of O. minuta specific clones using representational difference analysis (RDA)

1) Department of Ecology, Systematics and Evolution, University of Paris, France
2) International Rice Research Institute, Los Banos, Philippines

Representational difference analysis (RDA) is a subtractive cloning method that allows us to clone DNAs that are different between two complex genomes, thus isolating clones that are specific to one species and absent from the second one. This technique is based on a PCRbased representation step that reduces the complexity of the samples and facilitates the subtractive step based on genomic hybridization. It is a powerful technique to isolate genome specific markers. Such markers would be important to detect alien introgression into the rice genome. This technique has been used to analyze genomic differentiation between human and great apes (Toder et al. 1998) and in plants to determine phylogenetic relationships within Quercus genus (Zoldos et al. 2001) and to study the genomic differences between rice and foxtail millet (Panaud et al. 2002). No report is available on the application of RDA to isolate genome specific clones and detect alien introgression in rice.

We report on the preliminary results on the development of O. minuta specific clones using RDA. We followed the RDA protocol as described by Panaud et al (2002). The RDA library was constructed using wild species O. minuta as tester and rice O. sativa, cv. new plant type (NPT) as driver in order to clone sequences that are specific to O. minuta. Three cycles of hybridization/amplification were performed and carefully checked for increasing the amount of positive clones after each cycles. Southern blot experiments on hybridization/amplification products show that, as the hybridization signal obtained with NPT labeled representation remains strong after each cycle, the corresponding signal obtained with O. minuta labeled representation decreases. This can be interpreted as an enrichment of O. minuta specific clones following the cycles, showing that the subtractive step is efficient.

The PCR product from the third cycle was cloned and 1500 clones were selected for further analysis. Among them, 600 were taken randomly and checked for specificity through southern blotting. NPT rice and O. minuta representative were labeled using the non-radioactive procedure described by Panaud et al. (1993). Stringency conditions of the final wash prior to band detection were 0.1 x SSC at 65C. Only clones showing a strong hybridization signal with O. minuta probe and none with NPT probe were considered as specific to O. minuta. The result of a Southern hybridization experiment is presented in Figure 1. Out of the 600 clones analyzed, we found 458 clones that gave a signal with O. minuta labeled probe and none with

NPT labeled probe. These clones were considered as positive, leading to an estimation of around 84% positive clones specific to O. minuta genome.

For validation and large scale analysis of the clones, we attempted to use microarray. The PCR products of 1500 clones were precipitated and dissolved in 5 micro l of water. When ready to array, 5 micro l of 100% dimethyl solfoxide (DMSO) were added to each sample and mixed. The mix was then transferred to a 384-well plate. The library generated was printed onto a Corning CM-GAPS II amino-silane coated glass slide using the GeneTACTMG3 Workstation connected to a Microarrayer application program called High Density Arrayer (HDA). Each 11 x 11 patch contains 2 replicates of each clone and one spot is allocated for the controls NPT and O. minuta amplicons, which alternate in each patch. The arrayed DNA was fixed onto the slide through UV cross-linkage using the UVTEC CL-508 set to 0.120 Joules/cm2 at a wavelength of 312 nm.

NPT and O. minuta representation were labeled with Cyanine 5 (Cy5) and Cyanine 3 (Cy3) respectively through PCR amplification: 1 micro l of amplicon DNA was amplified using the R-Csp 24 primer in a total reaction volume of 100 micro l containing 1 micro l of Cy3 or Cy5. Both PCR products were mixed, precipitated and dissolved in 130 ml 1X GeneTac HybBuffer. Final mix was kept in the dark until it was used for hybridization. Efficiency of the labeling was checked by loading 2 micro l of each of the labeled product before and after the precipitation on a Mupid gel and scanned using Cy3 and Cy5 respective wavelengths. The probes were denatured and loaded along with the slides using the GeneTACTM HybStation set to 65C for 16 hours hybridization. After the completion of hybridization and washes, the slides were spindried in a 50 ml Corning tube at 3000 g for 2 min. The final microarray slides were imaged for the Cy3 and Cy5 fluorescence using GeneTAC LS IV scanner. The preliminary analyses were done using GT software and a more in-depth analysis was performed using Analyzer software.

We used microarray to monitor positive clones among the 1500 clones generated. O. minuta and NPT representative were labeled with green fluorescence (Cy3) and red fluorescence (Cy5). Oryza minuta specific clones were expected to appear green on the hybridized slide (Fig. 2) whereas non-specific clones were expected to appear yellow, in case the sequence is equally represented in the two genomes or red if the sequence is more represented in NPT genome than in O. minuta. NPT positive control showed a red signal whereas O. minuta showed a yellow signal (Fig. 2).

These genome-specific clones are a valuable genetic resource for future research on characterization of introgression from O. minuta into the rice genome and in identification of monosomic alien addition lines (MAALs) and detection of translocated segments. They can be used as probes for fluorescent in situ hybridization (FISH) analysis in order to monitor fragments introgressed from O. minuta into the NPT genetic background and as probes to monitor the presence of an O. minuta fragment through Southern blot analysis. Designing of primers specific to those clones would also make possible a high-throughput analysis using PCR amplification. It thus offers new potential to screen large numbers of putative specific clones. One application is to use as probe DNA from derived lines corresponding to progenies of any initial O. sativa x O. minuta cross: one can print a slide containing our RDA library and test the presence of the clones in breeding lines using its DNA as probe.


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