International Plant & Animal Genome XXIV Conference

Applied innovation for the agriculture and food system

Ravi Ramadhar,
AgriGenomics Business Leader, Thermo Fisher Scientific

Novel workflow for SNP genotyping in sugar beet

Dr. Piergiorgio Stevanato, 
University of Padova, Italy

Sugar beet is one of the world’s most important crops, currently supplying around 20% of the sugar consumed worldwide. The development of varieties that require less technical inputs for cultivation is one of the main research goals in sugar beet. To achieve this, sugar beet breeding is focusing on genetic improvement programs assisted by SNP (single nucleotide polymorphisms) molecular markers. Several methods are available for SNP genotyping at University of Padova (Italy), with Applied Biosystems™ instruments that include Quantitative Real-Time PCR (qPCR), High Resolution Melting (HRM) and Digital PCR (dPCR) technologies. qPCR with TaqMan™ probes specific for each SNP allele is commonly used for SNP genotyping. The QuantStudio™ 12K Flex system coupled with TaqMan OpenArray™ technology constitutes a platform endowed with all the key elements required for high-throughput SNP genotyping, thus allowing rapid genotyping of large numbers of SNPs in many individuals in a relatively short time. HRM is generally used to identify SNP genotypes with unlabeled probes. QuantStudio 12K is the only real-time quantitative PCR system that combines 384-well plate compatibility with HRM analysis. An advantage of HRM over qPCR is that non-targeted mutations can also be revealed by HRM in the amplicon. Digital PCR is an emerging qPCR approach for detection of SNP polymorphisms in a diluted target sample. It is based on the partitioning of a standard PCR reaction mix in aliquots, thus allowing thousands of parallel PCR reactions. QuantStudio 3D Digital PCR System is able to identify SNP variants at about 1% allele frequency. dPCR is used for the SNP genotyping of DNA bulks, where plant DNA is pooled by group and genotyping is performed on the bulk rather than on individual plants. This option reduces the cost of SNP screening for removal of off-type plants. SNP genotyping approaches developed at University of Padova are making selection procedures in sugar beet more rapid, accurate and less expensive with relevant impact on breeding program decisions.

Parentage-based tagging for Pacific salmon

Terry Beacham, 
Molecular Genetics Lab, Pacific Biological Station

We have assembled a panel of primers for coho salmon that produces 360 amplicons containing approximately 440 single nucleotide polymorphisms (SNPs), with all sequences amplified via a single polymerase chain reaction. Application of the panel to coho salmon from hatchery populations in southern British Columbia (BC) has indicated substantial differentiation among populations, as well as differentiation among brood years and individuals within hatcheries. We have sampled all hatchery broodstock used in major hatcheries operating in southern BC, thereby achieving 100% ‘genetic tagging’ of hatchery production. Adipose-fin-clipping of juveniles upon hatchery release provides a visual marker to identify hatchery-produced coho salmon. Individual coho salmon (clipped and unclipped) that fail to assign to sampled parents will be identified to hatcheries and wild populations of origin through genetic stock identification using the SNP loci. Preliminary analysis has indicated very high resolution in stock composition estimates among populations. Assignment of individual coho salmon to parents using the SNP panel in a multi-generation tracked population has provided accurate assignments, thus confirming the correct assignment to both hatchery and brood year (and therefore providing age of the individual). Amplicon sequencing will be the routine method of choice for coho salmon stock identification in BC in the near future, as high-resolution estimates of stock composition, age, and family structure will be available. We have also assembled an amplicon panel for chinook salmon that amplifies 390 amplicons containing approximately 530 SNPs and have genotyped all hatchery broodstock in British Columbia hatcheries where coded wire tags are applied to some juveniles upon release. High resolution in identification of individuals to hatchery of origin has been achieved.

Genotypying by sequencing through Ion AmpliSeq technology: a tool for genetic trait selection

Alex Chubick, 
GeneSeek

Ion AmpliSeq™ technology is a next-generation sequencing protocol that is capable of processing low concentrations of DNA (≥10 ng) to yield results that have a high coverage uniformity, reproducibility, and specificity. Through the use of Ion AmpliSeq custom panels, GeneSeek—a Neogen Corporation subsidiary—has designed a primer pool that is able to target ~4000 SNP loci which allows certain traits such as tenderness and parentage to be identified in cattle. Within this process, GeneSeek has modified the Ion AmpliSeq protocol in order to sequence at least 192 samples in one run. In order to process such a high volume of samples, GeneSeek has begun to utilize the Tecan Fluent™ in conjunction with the Ion Chef™ and Ion Proton™. In the initial data calculated by the Ion Torrent™ browser, GeneSeek observed that on average there were 80 million reads being generated per run, with each sample generating 400,000 reads. The Ion Torrent browser also provided the mean depth and on-target percentage value for each sample in a run; and GeneSeek calculated that on average, these values are equal to 80% and 94%, respectively. The preliminary data that has been collected by GeneSeek suggests that Ion AmpliSeq technology can be utilized as a vital tool for genetic trait selection in cattle.

Targeted resequencing in maize using Ion AmpliSeq technology

Tiffany Jamann,
Assistant Professor, University of Illinois at Urbana-Champaign

Cost remains a prohibitory factor in obtaining whole genome sequencing across a large number of samples. Genotyping-by-sequencing is a cost-effective alternative for obtaining a large number of markers across a large sample size, but markers are randomly distributed throughout the genome. Alternately, enrichment coupled with next-generation sequencing is a cost-effective strategy for obtaining sequence information for loci of interest across a large sample. We will present our results using Ion AmpliSeq, a system for amplicon-based enrichment coupled with semi-conductor sequencing, on a validation set consisting of maize inbred lines, hybrids and landrace accessions. We report the use of a multiplexed panel of 319 PCR assays that target 20 candidate regions for photoperiod sensitivity and require less than 25 ng of starting DNA. Enriched regions had an average sequencing depth of 105X coverage with 98.67% of bases mapping to the B73 reference genome. We were successfully able to call heterozygotes in the hybrid materials and identified novel variation in the landrace accessions. In summary, we have validated Ion AmpliSeq for genotyping diverse maize germplasm.