High-resolution genotyping via whole genome hybridizations to microarrays containing long oligonucleotide probes

Abstract

To date, microarray-based genotyping of large, complex plant genomes has been complicated by the need to perform genome complexity reduction to obtain sufficiently strong hybridization signals. Genome complexity reduction techniques are, however, tedious and can introduce unwanted variables into genotyping assays. Here, we report a microarray-based genotyping technology for complex genomes (such as the 2.3 GB maize genome) that does not require genome complexity reduction prior to hybridization. Approximately 200,000 long oligonucleotide probes were identified as being polymorphic between the inbred parents of a mapping population and used to genotype two recombinant inbred lines. While multiple hybridization replicates provided ∼97% accuracy, even a single replicate provided ∼95% accuracy. Genotyping accuracy was further increased to >99% by utilizing information from adjacent probes. This microarray-based method provides a simple, high-density genotyping approach for large, complex genomes.

Figure 1. Comparisons of analytical approaches for CGH-based mapping.

Data for B>M probes located on chromosome 1 that exhibit at least a 2-fold change (n = 26,953) were plotted following the use of different data analysis approaches. The upper set of plots display data for the RIL M0022 while the lower panels show data for the RIL M0023. The plots on the left and right display probes from the entire chromosome 1 and a close-up view of a 20 Mb region of chromosome 1 (positions 200 Mb–220 MB), respectively. For each set of plots the first panel provides visualization genotyping calls and log2 (RIL/B73) ratios following normalization and analysis using a linear model of multiple replications (Method I). The second and third panels show the genotyping calls and log₂(RIL/B73) ratios based on the analysis of a single replicate of data that was normalized using standard NimbleScan approaches (Method II). The final plot shows the genotyping calls for each BAC (n = 1,369) using data from replicate 1 (Method III).

Figure 2. Visualization of whole genome genotypes of two RILs.

The genotype of each BAC with at least 5 filtered B>M probes (n = 7,978 that were called in both lines) was determined and color coded (B73 – blue; Mo17 – red). BACs were then plotted according to their physical positions along a chromosome (x-axis) and by chromosome (y-axis). This visualization was created using a single replicate of data.

Figure 3. High-resolution of recombination break-points.

Several plots show detailed views of the CGH mapping data near recombination events in the RIL M0022. The log2(M0022/B73) value is plotted along the y-axis for each of the B>M probes (q<0.0001 and fold-change>2) in five genomic regions. The arrowheads indicate the position of the recombination event and the label indicates the chromosome and the base pair resolution of the recombination event.