CEL I Nuclease Digestion for SNP Discovery and Marker Development in Common Bean (Phaseolus vulgaris L.)

Abstract

Single nucleotide polymorphisms (SNPs) are the most common sequence difference found in plant genomes, yet they have not been widely exploited for producing molecular markers in common bean (Phaseolus vulgaris L.). The objective of this study was to develop a SNP assay based on a type of heteroduplex mismatch cleavage called EcoTILLING for molecular marker development in this important legume, and apply the assay (i) to the conversion of a sequence‐characterized amplified region (SCAR) marker useful for selecting virus resistance (SR2) and (ii) to the screening of SNP polymorphisms in newly developed expressed sequence tag (EST)–based markers. The SNP assay involved heteroduplex mismatch cleavage by a single‐strand specific nuclease ‘CEL I’ which was used to uncover two SNPs in the SR2 fragment and 22 SNPs in 37 candidate ESTs, some of which were used in segregation analysis. While developing the SNP techniques we tested several platforms, including LI‐COR, nondenaturing polyacrylamide, and agarose gel detection. The agarose gel system was used for SNP genetic mapping in two common bean mapping populations, showing that heteroduplex cleavage is a useful technique for increasing molecular marker number for the crop. Examples are given of mapped SNP markers for the phytic acid pathway gene for myo‐inositol‐1‐phosphate synthase and a drought tolerance–related gene, S‐adenosylmethionine decarboxylase.

Diversity and genetic analysis through DArTseq in common bean(Phaseolus vulgaris L.) germplasm from Turkey

“…In common bean, SNP detection can be validated by several methods (Galeano et al, 2009;Hyten et al, 2010;Zhao et al, 2010) that each has advantages and disadvantages (Cortes et al, 2011). Recently, a new method was introduced based on a hybridization approach known as Diversity Arrays Technology (DArTseq) (Mace et al, 2008) using a method of genotyping by sequencing (GBS) that combines DArT complexity reduction methods with next-generation sequencing platforms (Phuong Phung et al, 2014).…”

Targeted mutation breeding of horticultural plants

“…For clarity in this review, TILLING will refer to detection through mismatchspecific nuclease cleavage, which was used for all TILLING screens shown in Table 1. EcoTILLING is a technically similar approach that has been used to identify natural polymorphisms in target genes of melon, bean, sunflower, and peppers (Nieto et al, 2007;Galeano et al, 2009;Fusari et al, 2010;Ibiza et al, 2010).…”

“…The new eIF4E alleles can be used as a genetic resource for potyvirus resistance in breeding programs. Natural SNPs in other virus resistance loci were identified in lettuce and common bean for application in marker-assisted selection (Simko et al, 2009;Galeano et al, 2009 (Cadle-Davidson, 2010). In addition to eIF4E, Mlo, and Prm6, there are other susceptibility gene candidates that may provide resistance when their pathogen-required function is disrupted (Pavan et al, 2010).…”

Nuevas herramientas en la lucha contra las virosis del pimiento

“…El EcoTILLING también se ha desarrollado en el cacahuete silvestre Arachis duranensis K. & G., para buscar nuevos alelos de un gen relacionado con reacciones alérgicas (Ramos et al, 2009). Adicionalmente, esta metodología puede utilizarse para la detección de SNPs (Single Nucleotide Polymorphisms) en poblaciones naturales (Gilchrist et al, 2006;Barkley et al, 2008;Elias et al, 2009;Galeano et al, 2009;Till et al, 2010). En melón, el EcoTILLING se ha utilizado con éxito en la detección de variantes alélicas del factor de iniciación a la traducción eIF4E, implicado en la resistencia a virus de distintas familias que ocasionan enfermedades de importancia económica en diferentes cultivos (Nieto et al, 2007).…”

“…Sólo se identificaron seis sitios polimórficos en 113 entradas de C. melo y una de Cucumis africanus L. A pesar de que nuestro ensayo se realizó únicamente a partir de la CDS (coding sequence) de los dos genes, nuestra plataforma de ADNc mostró un elevado nivel de polimorfismo en comparación con el melón, y fue similar al de otros trabajos que utilizaron otros genes y el ADN genómico como material de partida Mejlhede et al, 2006;Gilchrist et al, 2006;Barkley et al, 2008;Galeano et al, 2009;Wang et al, 2007). Por otra parte, a partir de los marcadores moleculares utilizados, AFLPs y SSRs, se identificó en las especies domesticadas del género Capsicum un nivel de polimorfismo elevado y sin diferencias significativas entre ellas.…”