Single Nucleotide Polymorphism Genotyping by Heteroduplex Analysis in Sunflower (Helianthus annuus L.)

FUSARI C.; LIA V.V.; NISHINAKAMSU V.; ZUBRZYCKI J.; PUEBLA A.; MALIGNE A.; HOPP H.E.; HEINZ R.A.; PANIEGO N.

MOLECULAR BREEDING

SPRINGER

Lugar: Berlin; Año: 2011 vol. 28 p. 73 - 89

1380-3743

Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) are increasingly used for cultivar identification, construction of genetic maps, genetic diversity assessment, association mapping and marker-assisted breeding. Although there are several highly sensitive methods for the detection of polymorphisms, most of them are often beyond thebudget of medium-throughput academic laboratories or seed companies. Heteroduplex analysis by enzymatic cleavage (CEL1CH) or denaturing high-performance liquid chromatography (dHPLC) has been successfully used to examine genetic variation inseveral plant and animal species. In this work, we assess and compare the performance of both methods in sunflower by genotyping SNPs from a set of 24 selected polymorphic candidate genes. The CEL1CH method allowed us to accurately detect allele differencesin 10 out of 24 regions using an in-house prepared CEL1 enzyme (celery single strand endonuclease 1, Apium graveolens L.). Similarly, a total of 11 regions were successfully optimized for dHPLC analysis. As a scaling-up approach, both strategies were tested to genotype either 42 SNPs/indels in 22 sunflower accessions from the local germplasm bankor 33 SNPs/indels in 90 recombinant inbred lines (RILs) for genetic mapping purposes. Summarizing, a total of 601 genotypes were efficiently analyzed either with CEL1CH (110) or dHPCL (491). In conclusion, CEL1CH and dHPLC proved to be robust, complementary methods, allowing mediumscale laboratories to scale up the number of both SNPs and individuals to be included in genetic studies and targeted germplasm diversity  characterization (EcoTILLING).