New advances in DNA marker/QTL (gene) discovery and marker detection technology, through marker-assisted selection, have had an immense impact on crop improvement. To find and deploy the DNA markers in plant breeding programs, setting up inexpensive, large scale, high throughput and dependable and flexible marker genotyping platform is one of the main considerations.
Putting into place high-throughput workflows for SNP genotyping platforms such as KASP and TaqMan assays necessitates precise dispensing of assay master mixes and reagents. Speed and precision are crucial factors to achieve desirable assay performance while using a workflow that limits reagent utilization to enable studies on a large-scale.
This article illustrates the use of the MANTIS® instrument within the marker/QTL discovery and molecular breeding processes in the soybean program.
Materials
The MANTIS is a non-contact reagent dispenser, which is easy to program, low volume and low dead-volume. It is possible to configure the MANTIS with up to 6 different reagents, it has a dispense range of 100 nL - 2 mL, can dispense any quantity into any well, and is can work with any plate density below 1536.
Figure 1. MANTIS can be configured with up to (6) chip changer. Image Credit: FORMULATRIX
To limit dead volume, reagents can plug directly into microfluidic chips. By using pipette tips as reagent reservoirs, dead volumes can be reduced further to 6 μL. This feature is perfect for dispensing expensive PCR reagents.
Figure 2. Dead volumes can be further reduced to 6 μL by using pipette tips as reagent reservoirs. Image Credit: FORMULATRIX
At the center of the MANTIS® is a patented microfluidic valve cluster that measures and dispenses discrete amounts of liquid. Pressure and vacuum open and close each valve on the silicon valve cluster. Each chip has two micro – diaphragms (100 nL / 500 nL or 1 μL / 5 μL) that can fill and dispense as quickly as ten times per second.
Figure 3. MANTIS Chip with our patented Microdiaphragm Pump Technology. Image Credit: FORMULATRIX
The whole fluid path of a Mantis can be discarded to eradicate the risk of cross-contamination. The MANTIS® software offers a user friendly, easy to use, method of designing and running your assay. Add reagents to the dispense list, outline the amounts, and point and click on the virtual plate to define where you want your reagents to be dispensed.
The MANTIS software can design backfills and gradients, as well as importing .csv and .txt dispense lists.
Figure 4. User friendly MANTIS software allows point and click design. Image Credit: FORMULATRIX
Methods
Two BC1F2 populations that are segregating for FAD3A and FAD3C genes from the CX151244 source. Leaf samples were gathered from field-grown plants or greenhouse-grown seedlings and arranged into 96-well plates. For 24 hrs, the leaf samples were dried at 550 C and were then ground using GenoGrinder with a single BB (Daisy, Rogers AK) in the tube at 1600 rpm for two minutes. Then, DNA was extracted from leaf powders.
TaqMan assay primers and probes were developed utilizing Primer Express 3.0.1 software to find mutant SNPs in FAD3A and FAD3C (CX1512-44). All assays comprise of one primer pair to amplify amplicons range from 141-167 bp and two SNP-containing probes labeled with FAM (mutant) and VIC (wild type) dyes, respectively.
PCR reactions were performed in a 4.0 μL reaction system with 2 μL sample DNA, 2 μL combination of 2x TaqMan Universal master mix (Life Technologies, Carlsbad CA) and 0.2 μL of 5x assay mix (final concentration of 0.255 μM for each primer and 0.05 μM for each probe). Master and assay mixes were dispensed into 384 well plates which contain samples utilizing the Mantis Regent Dispenser.
After PCR, the plates were read with Tecan M1000 Pro Infinite Reader (Tecan Group Ltd, Morrisville NC) and the output files were imported into Kluster Caller software for SNP calling and visualization of clusters.
Results
Figure 5. SNP graphs of TaqMan assay. Image Credit: FORMULATRIX
Figure 6. Uniform dispensing of mastermix across the wells provided clear and easy SNP allele cells. Image Credit: FORMULATRIX
Conclusions
Soybean seed oil composition, particularly oleic, palmitic and linolenic acids, is of enormous interest to consumers due to its nutritional significance and industrial applications. Breeding low linolenic high oleic soybean cultivars becomes one of the most important breeding aims.
The SNP TaqMan marker assays that were designed to be able to select favorable oil composition from particular sources were strong in separating mutants, wild types and heterozygotes. With the help of the Mantis Reagent Dispenser during the genotyping process, it was possible to establish this genotyping workflow which is precise, cost effective and efficient. Therefore, the assays have been extensively applied in soybean breeding programs to enable the marker-assisted selection of fatty acid traits with enhanced efficiency and accuracy.
References
- http://formulatrix.com/mantis
Acknowledgments
The authors would like to thank the collaborators and others at The University of Georgia and Formulatrix for their contributions to this article.
About FORMULATRIX
FORMULATRIX® was established in 2002 to provide protein crystallization automation solutions. Since then, we've started developing other laboratory automation solutions including the next generation of liquid handlers using microfluidic technology.
Headquartered in Bedford, Massachusetts, we supply software and robotic automation solutions to leading pharmaceutical companies and academic research institutions around the world. Our team works tirelessly to provide the best products in the industry with support that is second to none.
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