My work here at Mahyco

Some quick updates:
We have nearly finished our initial project on Bacterial Leaf Blight in rice, and we are beginning the process once again with Sheath Blight. If you want to learn more about what I'm doing, just google search "molecular markers". Also, I sent my overview project to Ms. Fleming last week. For those of you reading this that don't specialize in molecular biology, here is that broad summary:

In the long run, we hope to identify those plants with multiple immunity QTLs so that the breeders can create a line of rice with lasting resistance to disease. It is much less likely that a pathogen can adapt to infect a plant with multiple resistance genes.

For my two month period in India, I am working in the Molecular Biology lab along side Sheetal Bhosle, my mentor and teacher. We are working to either validate previous studies on rice’s molecular markers, or to analyze genotypes based on pre-validated molecular markers. More specifically, we are focusing on analyzing Mahyco’s 34 rice genotypes for resistance genes to bacterial and sheath blight. Molecular analysis is necessary because to develop lasting resistance, a plant should have multiple different resistance genes. If a breeder were to attempt this the conventional way, there would be no way to differentiate between those plants with multiple resistance genes and those with only one.

A molecular marker is a piece of DNA sequence that is located near or on a gene that we wish to confirm in the genotype. For almost a century scientists have been tagging and arranging genes and gene-groups (known as Quantitative Trait Loci, or QTLs) that control favorable traits so that they can be identified on a molecular level, saving many generations of time for the breeder. The basic principle is this: the closer a marker is located to the gene you want within a chromosome, the higher the probability that one will not exist without the other. For example, a high-yielding plant can be tagged with two known genetic markers. In the next generation, if 1 in 100 plants have the first marker, but are no longer high yielding, while 10 of 100 plants contain the second marker but are not high yielding, we can conclude that the first marker is closer on the chromosome to the desired trait of high yield.

To begin our test across primers and genotypes, we first collected DNA samples from seeds. DNA extraction involves the crushing of the outer shell, disturbance of the cell wall, binding of the DNA to silica powder, then the use of water and ethanol to remove all polar and non polar contaminants.

Once the DNA is prepared, we can begin to test for the existence of our molecular markers that are linked with resistance to bacterial leaf or sheath blight. Each well in a 96-sample plate is given 5 micro liters of DNA. Then, a master mix including the forward and reverse primers, water a buffer, dNTP and Taq polymerase enzyme is added to each sample. The process that takes place in the PCR machine is thus: the samples are heated to denature the DNA. Next, the Taq enzyme replicated the process of DNA replication in our own cells as is facilitates the reforming of the sequence within our forward and reverse primers. In other words, as the system cools, the primers cut a segment of DNA which is then replicated. After 35 cycles, we should have 2 to the 35th copies of our desired sequence.

After the samples have completed their PCR amplification, we must test to ensure that the sequence was amplified before sending the samples to more precise equipment for analysis. To test for amplification, we place a row of randomly selected samples into an agarose gel. We place the gel in a buffer solution within a battery. DNA is negatively charged and will head one side as the cathode and anode create charged areas. Once completely diffused, the EtBr within the gel allows us to see the DNA under a black light. If it is clear that there are strands of more than 50 base pairs amplified, the PCR was successful.

For a more thorough analysis, the samples that are known to be amplified are placed in a MultiNA machine. This device works the same way as the agarose gel, but the computer can analyze the exact concentration of each specific base pair length within the sample. Though time consuming, we can use MultiNA results to confirm the existence of our desired marker and whether or not it was polymorphic.