Exploring genotyping technologies for low to medium-throughput single nucleotide polymorphism detection based on candidate gene approach in common sunflower (H. annuus)

FUSARI CM; NISHINAKAMASU V; PUEBLA AF; MALIGNE A; HOPP E; HEINZ RA; LIA VV; PANIEGO NB

Rosario

Simposio; VII Simposio Nacional de Biotecnología REDBIO Argentina. II Congreso Internacional-REDBIO-Argentina; 2009

REBIO

The analysis of DNA sequence variation is of major importance in genetic studies. To achieve this challenge, different types of molecular markers have been employed in genetic analysis of crop plants. In sunflower, analyses of genetic diversity were based on traditional techniques such as allozymes, AFLPs, SSRs and recently on single nucleotide polymorphism and short insertion/deletion (SNPs and InDels). The abundance, ubiquity and interspersed nature of SNPs together with the potential of automatic high-throughput analysis make them ideal candidates as molecular markers for construction of high density genetic maps, QTL fine mapping, marker assisted plant breeding and genetic association studies. In addition, SNPs located in known genes provide a fast alternative to analyse the fate of agronomically important alleles in breeding populations, thus providing functional markers. Recent advances in DNA sequence analysis and the establishment of high-throughput assays have provided the framework for large-scale discovery and analysis of SNPs. Despite the many methodologies available for SNP genotyping, most of these methods are expensive and often beyond the budget of the low to medium throughput academic laboratory. Many laboratories are pursuing candidate gene approaches rather than a genome scan, in order to limit the extent of markers that need to be typed. Those projects demand simple, accurate and cost-effective technology for SNPs typed both on hundreds of individuals or on pooled samples for a candidate gene region. In this study, we tested 10 out of 28 candidate gene characterized previously in our lab with two genotyping methods, denaturing high-performance liquid chromatography (dHPLC) and CEL1 endonuclease cleavage of missmatch heteroduplex. In order to set up a systematic detection platform for medium/high-throughput SNP genotyping we redesigned primers to amplify 10 regions in a 200-500 bp fragment range. Amplifications were performed using DNA from a set of sunflower inbred lines of known and unknown genotypes. Since inbred lines are homozygous for any particular allele, it is necessary to create heterozygous samples to discriminate between homoduplex and heteroduplex molecules. Hence, PCR products from known genotypes were added in equimolar concentration to all the known genotypes to form the heteroduplex. Samples were either analysed by dHPLC or incubated with CEL1 endonuclease followed by capillary electrophoresis to detect the corresponding fragments. For dHPLC: melting temperature, gradient conditions, flow rate and quantity of heteroduplex were optimized. For the CEL1 cleavage: purification of the enzyme, quantity of enzyme and quantity of heteroduplex were also tested to reach the best detection. Results from these experiments indicate that both dHPLC and CEL1 cleavage can be efficiently employed in analysing SNPs on a medium/high-throughput scale for genotyping in sunflower.