High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping

Abstract

BackgroundCucurbita pepo is a member of the Cucurbitaceae family, the second- most important horticultural family in terms of economic importance after Solanaceae. The "summer squash" types, including Zucchini and Scallop, rank among the highest-valued vegetables worldwide. There are few genomic tools available for this species.The first Cucurbita transcriptome, along with a large collection of Single Nucleotide Polymorphisms (SNP), was recently generated using massive sequencing. A set of 384 SNP was selected to generate an Illumina GoldenGate assay in order to construct the first SNP-based genetic map of Cucurbita and map quantitative trait loci (QTL).ResultsWe herein present the construction of the first SNP-based genetic map of Cucurbita pepo using a population derived from the cross of two varieties with contrasting phenotypes, representing the main cultivar groups of the species' two subspecies: Zucchini (subsp. pepo) × Scallop (subsp. ovifera). The mapping population was genotyped with 384 SNP, a set of selected EST-SNP identified in silico after massive sequencing of the transcriptomes of both parents, using the Illumina GoldenGate platform. The global success rate of the assay was higher than 85%. In total, 304 SNP were mapped, along with 11 SSR from a previous map, giving a map density of 5.56 cM/marker. This map was used to infer syntenic relationships between C. pepo and cucumber and to successfully map QTL that control plant, flowering and fruit traits that are of benefit to squash breeding. The QTL effects were validated in backcross populations.ConclusionOur results show that massive sequencing in different genotypes is an excellent tool for SNP discovery, and that the Illumina GoldenGate platform can be successfully applied to constructing genetic maps and performing QTL analysis in Cucurbita. This is the first SNP-based genetic map in the Cucurbita genus and is an invaluable new tool for biological research, especially considering that most of these markers are located in the coding regions of genes involved in different physiological processes. The platform will also be useful for future mapping and diversity studies, and will be essential in order to accelerate the process of breeding new and better-adapted squash varieties.

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

“…An efficient transformation system for other Cucurbita species may be established by optimization of the factors described above. Genetic and genomic tools for Cucurbita species are advancing (Blanca et al 2011;Esteras et al 2012). We hope that this transformation study contributes to molecular breeding and basic knowledge.…”

Angolan vegetable crops have unique genotypes of potential value for future breeding programmes

“…5 DNA concentrations were measured with a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and adjusted to 50 ng/µl. Twenty-six microsatellite loci were selected from those previously identified in an in silico screen of an Expressed Sequence Tag (EST) collection of C.pepo.…”

“…First, the amplification of the whole set of SSRs was checked in a subset of 5 accessions belonging to various accessions of the main cultivated species (C.pepo, C.moschata and C.maxima), including the parentals of the C. pepo genetic map from which these SSRs were derived. 5 All the SSRs that amplified in the three species and which indicated variability were analysed in the full collection. Amplifications were carried out in 15 …”

Desarrollo y aplicación de herramientas genómicas para la mejora de especies cucurbitáceas por calidad y resistencia a enfermedades.

“…Los trabajos incluidos en la presente Tesis referentes a C. pepo incluyen, por una parte, la validación de parte de esta colección de EST-SSRs y su aplicación en un estudio de variabilidad en comparación con los SSRs genómicos previamente desarrollados por : "Genetic diversity of Spanish Cucurbita pepo landraces: an unexploited resource for summer squash breeding") y, por otra parte, la aplicación de 384 EST-SNPs para la construcción del primer mapa genético en el género basado en este tipo de marcadores (Esteras et al, 2012a: "High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping"). Los mapas previos estaban basados principalmente en marcadores dominantes y poco transferibles a otras poblaciones (Lee et al, 1995y Brown y Myers, 2002, basados en el cruce interespecífico C. pepo x C. moschata; Zraidi et al, 2007), o basados en cruces intraespecíficos e inter-subespecíficos dentro de C. pepo), y sólo el publicado por incluía gSSRs, algunos de los cuales se anclan al mapa presentado aquí (Esteras et al, 2012a). La aplicación de estos marcadores de alta calidad no sólo ha impulsado los estudios de diversidad en la especie, incluyendo tipos comerciales y tradicionales, sino que ha permitido la puesta a punto de técnicas de genotipado de alto rendimiento como la plataforma GoldenGate de Illumina y un primer análisis de QTLs (Esteras et al, 2012a).…”

“…Los mapas previos estaban basados principalmente en marcadores dominantes y poco transferibles a otras poblaciones (Lee et al, 1995y Brown y Myers, 2002, basados en el cruce interespecífico C. pepo x C. moschata; Zraidi et al, 2007), o basados en cruces intraespecíficos e inter-subespecíficos dentro de C. pepo), y sólo el publicado por incluía gSSRs, algunos de los cuales se anclan al mapa presentado aquí (Esteras et al, 2012a). La aplicación de estos marcadores de alta calidad no sólo ha impulsado los estudios de diversidad en la especie, incluyendo tipos comerciales y tradicionales, sino que ha permitido la puesta a punto de técnicas de genotipado de alto rendimiento como la plataforma GoldenGate de Illumina y un primer análisis de QTLs (Esteras et al, 2012a).…”