Gel consistency (GC) is a standard assay used in rice improvement programmes to determine whether rice cultivars/breeding lines of high amylose content are soft or firm textured when cooked. In this study, we show that sequence variation in exon 10 of the Waxy (Wx) gene associates with GC using RILs derived from parents with high amylose content that differ in GC. The association was validated using a diverse set of traditional varieties, selected on the basis of amylose content, from the generation challenge programme. Structural investigations to explain how the mutation leads to differences in GC showed a strong association between GC and the proportion of amylose that leaches. It was shown that cooked rices of hard GC do not change in hardness over 24 h, whereas rices of soft GC retrograde significantly over 24 h. This leads to the conclusion that the mutation on exon 10 of the Wx gene affects the proportion of amylose bound to amylopectin and the proportion able to leach, and these structural differences alter the composition of the gel, which affects the amount of time the gel takes to reach a final hardness. The SNP described here completes the set of markers required to genotype for the current traits of cooking quality, but selecting the allele for soft texture has the negative result of also selecting for retrogradation potential.

The feasibility of large-scale genome-wide association studies of complex human disorders depends on the availability of accurate and efficient genotyping methods for single nucleotide polymorphisms (SNPs). We describe a new platform of the invader assay, a biplex assay, where both alleles are interrogated in a single reaction tube. The assay was evaluated on over 50 different SNPs, with over 20 SNPs genotyped in study cohorts of over 1500 individuals. We assessed the usefulness of the new platform in high-throughput genotyping and compared its accuracy to genotyping results obtained by the traditional monoplex invader assay, TaqMan genotyping and sequencing data. We present representative data for two SNPs in different genes (CD36 and protein tyrosine phosphatase 1beta) from a study cohort comprising over 1500 individuals with high or low-normal blood pressure. In this high-throughput application, the biplex invader assay is very accurate, with an error rate of <0.3% and a failure rate of 1.64%. The set-up of the assay is highly automated, facilitating the processing of large numbers of samples simultaneously. We present new analysis tools for the assignment of genotypes that further improve genotyping success. The biplex invader assay with its automated set-up and analysis offers a new efficient high-throughput genotyping platform that is suitable for association studies in large study cohorts.

Short tandem repeats (STR) are common polymorphisms in the genome. The length of STR may influence gene transcription, exhibiting diverse phenotypes. Two STRs, one trinucleotide repeats in the androgen receptor (AR) gene and one dinucleotide repeats in the insulin-like growth factor-I (IGF-I) gene, have been studied for their role in cancer, and the results are conflicting. Although there are many reasons for inconsistent findings, laboratory issues are often overlooked. DNA sizing analysis is regularly used to determine the length of STR, but its analytic validity has not been evaluated in epidemiologic studies. To examine if sizing analysis can reliably determine dinucleotide STR, we compared the method with direct DNA sequencing in analyzing CA repeats in the IGF-I gene in a small case-control study. The study enrolled 75 breast cancer cases and 75 age- and race-matched controls. DNA was extracted from buffy coats and was analyzed for CA repeats by both DNA sizing and direct sequencing. Our comparison indicated that these methods detected the same number of repeats in the short allele but not in the long allele. There was a substantial discrepancy between the methods in determining homozygous alleles. Although the two methods showed &lt;10% of samples having an exact match on the number of repeats in both alleles, both techniques were able to detect a genotype-phenotype correlation and a racial disparity in the genotype. An association between breast cancer risk and IGF-I genotype was found in sequencing analysis but not in sizing analysis. Overall, the comparison suggests that laboratory analysis of dinucleotide STR may not be as reliable as originally thought. This unreliability in STR analysis may result in inconsistent study findings.