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Single nucleotide polymorphism genotyping by heteroduplex analysis in sunflower (Helianthus annuus L.)

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Abstract

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 the budget 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 in several 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 differences in 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 bank or 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 medium-scale laboratories to scale up the number of both SNPs and individuals to be included in genetic studies and targeted germplasm diversity characterization (EcoTILLING).

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Acknowledgments

This research was supported by ANPCyT/FONCYT, PID 2007 00073, INTA-PRR AEBIO 245001 and 245005, INTA-PE AEBIO 24554711 and 241351. The authors thank the Germplasm Bank from Estación Experimental Agropecuaria INTA Manfredi for kindly providing sunflower inbred lines seeds. C.M.F. is a Ph.D. student supported by a fellowship from Instituto Nacional de Tecnología Agropecuaria (INTA). Drs. V.V.L., R.A.H. and N.B.P. are career members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Dr. H.E.H. is a career member of the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC) and Professor at the Facultad de Ciencias Exactas y Naturales, University of Buenos Aires (UBA).

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  1. Instituto Nacional de Tecnología Agropecuaria (INTA), Instituto de Biotecnología (CNIA), CC 25, Hurlingham, Buenos Aires, 1686, Argentina

    Corina M. Fusari, Verónica V. Lia, Verónica Nishinakamasu, Jeremías E. Zubrzycki, Andrea F. Puebla, Alberto E. Maligne, H. Esteban Hopp, Ruth A. Heinz & Norma B. Paniego

  2. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, Ciudad Universitaria, 1428, Argentina

    Verónica V. Lia, H. Esteban Hopp & Ruth A. Heinz

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  1. Corina M. Fusari
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  2. Verónica V. Lia
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  3. Verónica Nishinakamasu
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Correspondence to Norma B. Paniego.

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Fusari, C.M., Lia, V.V., Nishinakamasu, V. et al. Single nucleotide polymorphism genotyping by heteroduplex analysis in sunflower (Helianthus annuus L.). Mol Breeding 28, 73–89 (2011). https://doi.org/10.1007/s11032-010-9462-9

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