David M. Francis, The Ohio State University
Hi, my name is Caleb Orchard; I'm an undergraduate student working with the Solanaceae Co-ordinated Agricultural Project. This video gives an overview of how DNA sequence variation can be used to complement plant breeding by helping us identify plants with improved characteristics using a process called marker-assisted selection. This video uses tomato plants as an example.
We start by collecting leaf tissue from young plants. DNA extraction from tissue can be performed at different scales. The process is most efficient when performed in batches of 96. Metal balls are added to tubes to help break up the plant material and an extraction buffer containing detergent is added. Racks with 96 samples are agitated to grind the tissue. The racks are then spun in a centrifuge to reduce foam. Samples are then heated for 10 to 15 minutes at 68°C. This step helps the detergent break up the membranes and release the DNA. An organic solvent, chloroform, is added to denature proteins and help remove the lipids. The tubes are then mixed well and then centrifuged to separate the organic and aqueous phases. When the phases are separated, the top phase will contain DNA and salts and water. This aqueous phase is then removed to a new plate. Samples are then mixed with alcohol to help precipitate the DNA. Centrifuging the samples forces the DNA to the bottom of the tube or plate. The alcohol is poured off, leaving DNA and residual alcohol, which is evaporated by letting the sample dry in air for a brief period. The DNA is then resuspended in a water-based solution containing buffer, salts, and chemicals to protect the DNA.
This DNA serves as a template for the polymerase chain reaction, or PCR. PCR is a technique that allows us to amplify specific fragments of DNA. These DNA fragments are referred to as markers when they distinguish plant varieties and breeding parents. Following PCR, DNA samples are mixed with a dye and loaded onto a gel. The gel shown here is made of polyacrylamide, which allows us to separate DNA fragments that differ in size by as few as two nucleotides. The gel provides a matrix to sieve the DNA fragments. An electrical field is applied and the negatively charged DNA migrates towards the positive pole. Small fragments move faster in the matrix. Each lane on the gel contains PCR amplification products from a single plant. We can estimate the size of the fragments relative to a standard, shown here in blue. When we have determined a specific fragment size that is associated with a desirable trait, such as a plant's ability to resist a disease, we can select on the basis of the marker. This association is determined by genetic studies that demonstrate linkage between sequence variation giving rise to fragment size differences and the desirable trait.
Selecting on the basis of fragment size allows us to identify resistant individuals at the seedling stage. In this panel resistant individuals contain the larger fragment. We want to keep plants three and four, but not one and two. In this way our breeding populations can be biased toward plants with desirable characteristics. Plants with the susceptible marker are eliminated and only the best individuals are transplanted to the field, where they are further evaluated on the basis of adaptation to growing environment, performance, and quality.
Modern plant breeding benefits from emerging sequence data by expanding the number of markers available as tools for selection.
Development of this page was supported in part by the National Institute of Food and Agriculture (NIFA) Solanaceae Coordinated Agricultural Project, agreement 2009-85606-05673, administered by Michigan State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the United States Department of Agriculture.