PCR 101

Background for PCR:

DNA is the recipe cells use to create and sustain life. A location on a strand of DNA is called the gene locus and the different variations of the gene are called alleles. So a designated parking spot would be similar to the gene locus and the car parked there would be akin to an allele. If scientists want to study a particular locus in a set of samples, polymerase chain reaction (PCR) can be used to produce multiple copies of a specific region.

The basic steps to PCR:

First, the sample's DNA must be mixed with a combination of primers, a buffer, and a special enzyme named Taq that is heat resistant. Then the mix is heated and cooled repetitively to allow the primers and enzyme to create new copies of the DNA in a cycle. When finished, the DNA is multiplied/amplified and can be used for forensics, the detection of viruses, genotyping(observing which samples have which alleles), etc.

How PCR will help my project:

I learned PCR this summer because we have used it for genotyping our plants in progeny testing. We use a PCR technique called KASP PCR which amplifies the DNA as intended but also uses special primers that can be detected by a machine. So when the PCR is finished, a machine can use the primers to help us genotype (give us the allelic combination at certain locations) our plants. In our case, we are looking at genetic differences between our samples at a certain region to help narrow where we believe the chlorophyll locus is.

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Florida and DNA extraction

This week, my lab took a trip to Florida to harvest some tomatoes we had in Floridian fields. I learned some tomato terms like "breaker" (a tomato in between green/red) and "inflorescence" (a flowering group) while getting my field experience. The project works with the University of Florida which was kind enough to let us use some lab space while we were there. We chopped up tomatoes to observe their volatiles in GCMS for a Ph.D. student in our lab. I enjoyed the experience because it gave me another opportunity to explore a lab space dedicated to plant science and I was able to see interesting Floridian wildlife. We arrived at the fields in the early morning; these are the fields we harvested. Above is a bug that was blended into a plant in Florida that we saw on our trip. After returning to Georgia, we set to work to extract DNA from the F6 generation from the greenhouse in addition to the parental lines. By extracting one cotyledon from each plant, we had enoug

Selection of Plants for Transplanting

At this point, we have just confirmed our plant selection using marker-assisted KASP PCR genotyping. Using the DNA extracted earlier, we ran a KASP PCR. The then amplified DNA is read by the Teacan machine which works by reading the specific fluorescent dye in a FAM marker which will bind to the reference allele (determined by the Sol Genomics Network available for free on their website) while the HEX marker will bind to the alternative/SNP allele. The program, Klustercaller, is then able to interpret the Teacan reads which allows us to genotype the plants as either X:X(reference homozygous/FAM dye), X:Y(heterozygous/FAM & HEX), or Y:Y(alternative homozygous/HEX dye). Whether the BGV007900 or BGV007936 parental line has the reference or alternative allele depends on the location. For example, at locus "1", BGV007900 may have the same genotype as the reference genome, then at that location, X:X would correlate to having the BGV007900 allele. However, maybe at locus "2

Explaining the Project

For the general public: Leaf Color Genetics in Tomato Blossom-end rot (BER) is a physiological disorder in tomatoes that reduces the bottom end of the fruit to brown mush. My mentor studies BER, but the research involved takes many months. So to compensate for a lack of time, I am working with a simplified project involving leaf color/chlorophyll content. This project should mimic the process of BER trait testing on a smaller scale. This summer, I will aid in breeding projects aimed to confirm the location of the gene that controls leaf color/chlorophyll content. To do this, we will observe the genetic and expressed (color, incidence, etc.) traits of progeny that resulted from crosses between dark-leafed and light-leafed plants. By comparing the genetic and expressed traits, we should elucidate the gene's location. A picture of the greenhouse with my mentor's plants. The bottom left seed tray has my F2 generation. A picture of the greenhouse with my mentor's plants. The

HORT 484 Blog: UGA Crop Genetics and Genomics REEU

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