Sequence Based Genotyping: from marker discovery to targeted detection
Michiel van Eijk, PhD - Keygene, USA
Next Generation Sequencing has revolutionized the life sciences including crop improvement. Over the past decade KeyGene has leveraged the power of NGS in its Accelerated Molecular Breeding platform to develop a portfolio of proprietary Sequence Based Genotyping approaches suiting the needs of today's molecular plant breeders in terms of quality, cost, flexibility and turn-around times. Sequence-Based Genotyping (SBG) technology enables simultaneous discovery and genotyping of novel polymorphisms in any germplasm using restriction enzyme-mediated complexity reduction approaches; at present many forms of SBG including GBS, RAD-Seq and related methods are widely used for crop genotyping. KeyGene® SNPSelect is complementary to SBG as it provides a fully customizable, highly flexible NGS-based targeted genotyping solution for preselected polymorphisms at unparalleled scale and competitive cost per data-point. Our whole genome re-sequencing approaches in combination with fast variant detection enable high resolution analysis of germplasm including detection of rare variants. Based on these methods, KeyGene offers tailor-made solutions to all plant breeder's genotyping needs. The SBG and KeyGene® SNPSelect technologies are protected by patents and patent applications owned by KeyGene N.V..
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“Genomic DNA extraction - 1 million cattle” Application of the oKtopure™ robot and sbeadex™ chemistry for high through-put DNA extraction
Romy Morrin, PhD - Weatherbys Ireland DNA Laboratory, Ireland
Weatherbys DNA Laboratory is currently the service provider to the largest global livestock genomics genotyping project for 1 million Irish beef cattle. Fundamental to the success of providing this service is the application of a high through-put DNA extraction procedure. Genotyping large numbers of samples over a short time frame requires a DNA extraction process to efficiently support the genotyping pipeline. The oktopure ™ automated DNA extraction robot in conjunction with sbeadex ™ chemistry provides DNA extraction to process over 3000 samples a day for downstream analysis on an Illumina® Infinium platform.
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Optimized marker sets for array-based plant genotyping – a continuous improvement process
Martin Ganal, PhD - TraitGenetics, Germany
Using genotyping arrays, large sets of varieties and SNP markers can be analyzed in many crop plants. Based on such data, it is now possible to generate marker databases that include mapping data, allele frequency data and haplotype information for many thousands of markers. Such information is crucial for the development of optimized array-based marker sets for plant breeding and genome research that provide a low amount of ascertainment bias and can be analyzed in a cost-effective way. For wheat as an example, we show that through a continuous analysis of new data generated from different genotyping arrays, it was possible to incrementally generate more comprehensive genotyping arrays which deliver a maximum of information. These optimized marker sets are ideal for marker-assisted selection and backcrossing, variety identification, the analysis genes associated with specific traits and genomic selection. With such arrays, TraitGenetics analyzes now many ten-thousands of samples each year for breeding companies and academic researchers.
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Rapid assessment of novel alleles and QTL through background genome selection: from sequence variation to functional characterization in tomato
David Francis, PhD - Horticulture and Crop Sciences, The Ohio State University, USA
The selection of novel alleles with superior phenotypes based on sequence comparison remains problematic without more complete information about the structural or regulatory consequences of variation. With over 400 sequenced genomes available for tomato (Solanum lycopersicum, L.) and several wild relatives, identification of new alleles for structural genes or regulatory regions is straight-forward. Assessing the function of this variation remains problematic. Classical breeding approaches augmented by marker-assisted selection strategies offer a means to rapidly assess the function of new alleles in uniform genetic backgrounds. We use the competitive allele specific primer extension (KASP™ ) genotyping technology to implement a marker-assisted (MAS) backcross breeding scheme leveraging genome-wide SNPs to rapidly develop genetic resources containing different QTL for disease resistance or alleles of the high β-carotene (B) promoter in a uniform genetic background. This approach is facilitated by high density SNP data for over 1,000 tomato accessions, permitting market-class and pedigree based strategies to optimize polymorphic marker sets. The flexibility of the KASP™ platform then allows for marker sets to be modified from one generation to the next in order to optimize informative data points. In our experience, marker-assisted backcrossing leds to the selection of plants that share 93-98% of the recurrent parent in only two generations, permitting rapid phenotypic evaluation. Using this approach we have identified functional allelic variation for β-carotene (pro-vitamin A) content and identified superior QTL for bacterial spot resistance.
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Flexible, low density SNP information in animal breeding
Kellie Watson, PhD - Roslin Institute, United Kingdom
Genomic selection has had a major impact on animal breeding programs, providing more accurate estimates for breeding value earlier in the life of breeding animals, giving more selection accuracy and allowing lower generation intervals in many species. Despite the rapid reduction in genotyping costs the economic investment required to implement genomic selection can still be considerable. For some species such as dairy cattle, the costs of genotyping can be easily offset against the increased selection response and improvements in the breeding programme design. However for agricultural species such as avian’s, the investment can considerable and strategies to reduce genotyping costs can be explored. Sharing experience and research outcomes, during the presentation the use of low density (LD) SNP panels in genomic selection and aligned projects will be covered. The use of LD-SNP panels for value adding to experimental populations will be outlined.
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