A pipeline for high throughput detection and mapping of SNPs from EST databases

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic variation that can be used as molecular markers. The SNPs that are hidden in sequence databases can be unlocked using bioinformatic tools. For efficient application of these SNPs, the sequence set should be error-free as much as possible, targeting single loci and suitable for the SNP scoring platform of choice. We have developed a pipeline to effectively mine SNPs from public EST databases with or without quality information using QualitySNP software, select reliable SNP and prepare the loci for analysis on the Illumina GoldenGate genotyping platform. The applicability of the pipeline was demonstrated using publicly available potato EST data, genotyping individuals from two diploid mapping populations and subsequently mapping the SNP markers (putative genes) in both populations. Over 7000 reliable SNPs were identified that met the criteria for genotyping on the GoldenGate platform. Of the 384 SNPs on the SNP array approximately 12% dropped out. For the two potato mapping populations 165 and 185 SNPs segregating SNP loci could be mapped on the respective genetic maps, illustrating the effectiveness of our pipeline for SNP selection and validation

SNP in starch biosynthesis genes associated with nutritional and functional properties of rice

Starch is a major component of human diets. The relative contribution of variation in the genes of starch biosynthesis to the nutritional and functional properties of the rice was evaluated in a rice breeding population. Sequencing 18 genes involved in starch synthesis in a population of 233 rice breeding lines discovered 66 functional SNPs in exonic regions. Five genes, AGPS2b, Isoamylase1, SPHOL, SSIIb and SSIVb showed no polymorphism. Association analysis found 31 of the SNP were associated with differences in pasting and cooking quality properties of the rice lines. Two genes appear to be the major loci controlling traits under human selection in rice, GBSSI (waxy gene) and SSIIa. GBSSI influenced amylose content and retrogradation. Other genes contributing to retrogradation were GPT1, SSI, BEI and SSIIIa. SSIIa explained much of the variation in cooking characteristics. Other genes had relatively small effects

Capture of assay template by multiplex PCR of long amplicons for genotyping SNPs and InDels with MALDI-TOF mass spectrometry

Mis-priming associated with uncharacterised single nucleotide polymorphisms (SNPs) may lead to failure of PCR for genotyping. This is particularly troublesome in high-throughput SNP genotyping applications relying on multiplex PCR (2-40-plex) generating many short amplicons (80-120 bp) of similar size, an approach best suited for whole genome scans. However, if the target SNPs are clustered within a few target genes one option to ameliorate this is to increase the amplicon length, effectively reducing the potential for primer/template interactions and mis-priming. We tested this approach in a diverse population of 372 Eucalyptus pilularis individuals (pi = 8.11 x 10(-3), H (e) = 0.75) using a modified Sequenom iPLEX gold assay. Four candidate genes (MYB1, MYB2, CAD and CCR) were amplified in a single long range multiplex capture PCR generating 6 long amplicons ranging in size from 907 to 2,225 bp. This contrasts with the standard approach which would have required the amplification of 98 short amplicons in 4 multiplex reactions. These 6 long amplicons provided the assay template for 98 assays (87 SNP and 11 InDel) within the 4 candidate genes. Reaction results indicated that longer amplicons could provide a suitable template for genotyping assays, with 90.8% of assays functional and 84.3% of assays suitable for downstream analysis. Additional advantages of this approach were the capacity for troubleshooting using gel electrophoresis and savings of 94% in capture primer synthesis costs. This approach will have the greatest relevance for candidate gene approaches for association testing in uncharacterised populations of organisms with high sequence diversity