Programmable microfluidic genotyping of plant DNA samples for marker-assisted selection

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.

Facile and scalable tubing-free sample loading for droplet microfluidics

“…2 ). Similar to our previous devices 41 – 45 , this device adopts the two-layer architecture with the bottom valve layer (Fig. 2 , red) housing “push-up” microvalves 41 , 44 – 46 to regulate the fluid flow in the top fluidics layer (Fig.…”

Customizing droplet contents and dynamic ranges via integrated programmable picodroplet assembler

“…By employing a spatial (and hence, temporal) indexing scheme, we can easily distinguish signals from each picodroplet group without the need for complex optical indexing schemes. In this work, we demonstrate our platform’s capability by generating five distinct combinations of reagents using two inlets, but we note that our scalable device architecture has been previously used to assemble as many as 650 unique nanoplug combinations by taking advantage of 12 inlets within a single device 30,37–39 .…”

“…iPPA devices were fabricated with polydimethylsiloxane (PDMS) via multilayer soft lithography 30,31,37,39 . Our devices employ a “push-up” architecture, wherein the valve control layer is sandwiched between the fluidic layer and a glass slide.…”

Emerging Trends in Microfluidics Based Devices

“…The use of MFs chips for high throughput genotyping analysis of plants is a well‐known technique. [ 81,82 ] It has revolutionized the field of NA analysis by providing a large number of plant species genotype data in lesser time and cost‐effective manner, which has helped in designing genetically modified crops. In addition to genotyping MFs chips, the phenotypic analysis has recently shifted from traditional culturing of seeds in pots or petrie dishes or greenhouses to MFDs.…”