TECHNICAL ADVANCE: Indel arrays: an affordable alternative for genotyping

Abstract

SummaryNatural variation and induced mutations are important resources for gene discovery and the elucidation of genetic circuits. Mapping such polymorphisms requires rapid and cost-efficient methods for genome-wide genotyping. Here we report the development of a microarray-based method that assesses 240 unique markers in a single hybridization experiment at a cost of less than US$50 in materials per line. Our genotyping array is built with 70-mer oligonucleotide elements representing insertion/deletion (indel) polymorphisms between the Arabidopsis thaliana accessions Columbia-0 (Col) and Landsberg erecta (Ler). These indel polymorphisms are recognized with great precision by comparative genomic hybridization, eliminating the need for array replicates and complex statistical analysis. Markers are present genome-wide, with an average spacing of approximately 500 kb. PCR primer information is provided for all array indels, allowing rapid single-locus inquiries. Multi-well chips allow groups of 16 lines to be genotyped in a single experiment. We demonstrate the utility of the array for accurately mapping recessive mutations, RIL populations and mixed genetic backgrounds from accessions other than Col and Ler. Given the ease of use of shotgun sequencing to generate partial genomic sequences of unsequenced species, this approach is readily transferable to non-model organisms.

Natural genetic variation caused by small insertions and deletions in the human genome

“…In further support of this conclusion, exome re-sequencing projects and the 1000 Genomes Project also have identified coding INDELs (Ng et al 2009(Ng et al , 2010; The 1000 Genomes Project Consortium 2010). Salathia et al (2007) previously described an approach for genotyping INDELs in the size range of 25 bp to 7260 bp in Arabidopsis thaliana. These INDELs were genotyped using 70-mer oligonucleotide probes and Comparative Genome Hybridization (CGH).…”

Array-based high-throughput DNA markers for crop improvement

“…The ML approach was used earlier in Arabidopsis, where in one study 20 diverse strains of this weed were genotyped for more than a million non-redundant SNPs for analyzing the patterns of DNA polymorphism , and in another study, a genotyping array based on a subset of 250 000 'tag' SNPs was used to study genomewide pattern of LD ). An 'InDel array' representing 240 unique InDel markers was also recently developed and used in Arabidopsis to genotype InDels of one or more nucleotides in an ultra-high-throughput manner (Salathia et al, 2007).…”

Isolation and characterization of a novel ammonium overly sensitive mutant, amos2, in Arabidopsis thaliana

“…To confirm the phenotypes of F 2 individuals, the F 3 progenies were further tested for both reduced lateral root phenotype and sensitivity to (NH 4 ) 2 SO 4 . For the initial mapping analysis, primers for InDel (insertion/deletion) markers were used (Salathia et al 2007). PCR was performed in a 20-ll volume containing 10-100 ng/ll genomic DNA (1 ll), 10 lM of each primer (1 ll), 2.5 mM dNTPs (1.6 ll), 25 mM Mg 2?…”