Abstract
As demands to maintain the global food production continue to mount, multinational seed companies are turning to new DNA marker technologies to accelerate the rate of plant breeding and crop improvement. The key to widespread adoption of molecular breeding is the availability of flexible and cost-effective tools that can perform combinatorial and high-throughput genotyping of single-nucleotide polymorphisms (SNPs) to guide the crop development process. Toward this end, we have developed a programmable, droplet-based microfluidic device for genotyping maize genomic DNA. A unique feature of the microfluidic platform is the nano sample processors (NSPs), which allow the device to sequentially load an unrestricted number of unique DNA samples using only two inlets, overcoming the current limitation to the number of sample inputs due to small device footprint. Direct and programmable droplet generation within the device allows each sample to be genotyped against a panel of markers on demand. Moreover, we have successfully implemented the Invader assay for SNP genotyping in flowing, 50-nL droplets, thus achieving significant reduction in consumption of reagents per reaction as compared with conventional genotyping platforms. As a demonstration, we performed 240 Invader reactions (testing 8 DNA samples against 10 SNP markers) and achieved greater than 93% accuracy in SNP calling of plant DNA samples in a single droplet-based experiment.
Highlights
Recent years have witnessed increasing prevalence of genomic selection technologies in the agricultural industry to accelerate plant breeding and crop improvement for maintaining stable global food supply in the face of growing human population, shrinking arable land, emerging new pests, and changing climate patterns[1,2]
The agriculture industry has been focusing on high-throughput detection of single-nucleotide polymorphism (SNP) markers[5,6] because of their abundance throughout the genome, stability over generations[7,8], and binary-like information that simplifies genotyping and data storage[4]
SNP genotyping microfluidic platform ð1Þ To achieve programmable, multiplexed SNP genotyping of a library of maize DNA samples against a panel of SNP markers, we a nano sample processors (NSPs) 1 – Sample 1 NSP 2 – Sample 2
Summary
Recent years have witnessed increasing prevalence of genomic selection technologies in the agricultural industry to accelerate plant breeding and crop improvement for maintaining stable global food supply in the face of growing human population, shrinking arable land, emerging new pests, and changing climate patterns[1,2]. Widespread adoption of genomic selection in plant-breeding hinges on rapid, flexible, high-throughput, and cost-effective genotyping technologies[4] To this end, the agriculture industry has been focusing on high-throughput detection of single-nucleotide polymorphism (SNP) markers[5,6] because of their abundance throughout the genome, stability over generations[7,8], and binary-like information that simplifies genotyping and data storage[4]. Among the genotyping systems that have been introduced to the market[12,13,14,15], Array TapeTM Technology (Douglas Scientific) is considered state-of-the-art and is used by large multinational seed companies[4] This technology processes spools of Array TapeTM for assay set-up, polymerase chain reaction (PCR), and fluorescent detection—genotyping up to 150 000 data points per day. There remains a strong interest in the agricultural industry to develop alternative genotyping technologies that are rapid, flexible, costeffective, high-throughput, and tenable for automation
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