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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", BGV007936 has the same genotype as the reference genome and BGV007900 has a single nucleotide polymorphism (SNP), or an alternation in the genetic code, then X:X would correlate to having the BGV007936 allele. We will use this information to find and select 6 heterozygous and 9 homozygous for each parental trait.  We will later phenotype them to compare the genotypes and phenotypes.




The image above shows the fixed (green/red) and non-fixed (white) areas of the four families we are using. If the box is non-fixed/heterozygous, then the progeny have a chance of taking after either parent or being heterozygous themselves (think of crossing AaxAx to have AA:Aa:Aa:aa in the next generation). The top row indicates markers used and their position, it is in order, so it is essentially a linear map. This information was known when the families were chosen, but it will help us understand which areas our gene of interest could be in. For example, if we have 9 progeny of 21SS237 that take after the BGV7900 parent(it would have the green/BGV7900 allele across the region pictured above) and 9 progeny 21SS237 that take after the BGV7936 parent(it would have red/BGV7936 along the top region until it hits the 20EP130 marker where it would enter the green fixed region), we can compare their phenotypes when they get older. If they have the same phenotype, then the gene controlling chlorophyll would not be in that area. However, if the genotypes present different chlorophyll-related phenotypes, then the gene of interest must be in that area!


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