Varietal classification of rice (Oryza sativa L.) by oligomer hybridization to genomic DNA arrays

The feasibility of using the microarray platform, with the novel adaptation of arraying sheared genomic DNA instead of genes or cDNA as targets and using indel-based oligomers as problems in classifying major types of indica and japonica rice is reported. Seven indica (93-11)-specific and 4 japonica (cv. Nipponbare)-specific oligonucleotide probes were designed based on indels identified by genomic sequence alignments between japonica rice cv. Nipponbare and indica rice cv. 93-11. Additionally, 2 oligonucleotides that are complementary to sequences that are present in multiple copies within the Nipponbare and 93-11 genome were designed. Alongside this, genomic DNA arrays composed of 384 accessions in 5 replicates was developed. Shearing of the genomic DNAs improved the spread and binding of the samples to the glass slides. Hybridization of the control probes to the genomic arrays gave a uniform fluorescent signal across the array, providing an experimental validation of the specificity of the probes performed in silico. On the other hand, the variety-specific probes gave different patterns of hybridization at variable temperature. In all hybridization experiments, the high-copy control probes consistently gave higher signal intensity values compared to the single-copy variety-specific probes. Classification of 384 rice accessions into variety groups by oligomer hybridization resulted in the clear separation of the samples into indica or japonica. The classification obtained gave 38-39% concordance with the reported classification of the same samples using SSRs. Intrinsic variation within cultivars belonging to the same variety group as well as non-specific cross-hybridization between the probes and non-targets account for the low concordance obtained. To enhance the efficiency of the technique for genotyping, targeting longer indels using multiple short probes is suggested to ensure hybridization events between the probes and the complementary sequence in the indel of interest. Alternatively, targeting multiple indel events across the genome to enhance the uniqueness of the signature of each accession is suggested. Selecting probes with high discriminating power will allow the genotyping of a large set of accessions using only a small set of probes. The results demonstrate the potential of the current method in classifying rice cultivars into variety groups.