EXPERIMENTAL HISTORY In the past I was involved with a project similar to this one. In that project, fish from three populations were collected and tissue samples taken. DNA was isolated using the GeneClean method. Representative fish from each population were then subjected to RAPD primers in the PCR process and a genetic tree was constructed from the data through computer analysis. Data from that experiment tell us that indeed there is enough genetic variance to indicate possible speciation between populations. As of now these three populations are classified as one species. Conclusions based on that experimental data show longear sunfish in the Gasconade river may possibly be sub-species megalotis. Fish from the Osage river may possibly to be nuchalis and fish from the White river may be a mixture of the two. This is where my project begins. In further testing of the speciation scenario previously stated, I have attempted to produce independent results based on experiments dealing with mitochondrial DNA instead of nuclear DNA. Mitochondrial DNA is less sensitive to evolutionary change but it still reveals relationships among populations. Enzymatic digestion of mitochondrial DNA will tell us similarities between populations and either strengthen or contradict the previous conclusion.
METHODS DNA templates had been collected in the field and isolated using the GeneClean“ method by Shawn Banks and Casey Dillman during a previous project. These isolations have been stored at -20*C. These isolations contain mitochondrial DNA which happen to be circular. Amplification of mitochondrial DNA requires a forward and reverse primer. These primers L(5’-AATAGTTTATCC,A/G,TTGGTCTTAGG-3’) and H(5’-GTTGAATGACAATGGTGGTTCTTC-3’), which the sequence was known and specially made, target a region of mitochondrial DNA known as ND5/6. By the PCR process, tens of thousands of a 2.4kb fragment of DNA result. These single pieces of DNA are then subjected to enzymatic digestions in which the fragments are cut into smaller pieces according to an enzyme’s specific binding sequence. In context to this project mitochondrial DNA of two fish from each of three populations were attempted to be amplified. 5ml of each PCR product was then added to .2ml of enzyme, 2ml of the respective 10X buffer, 12.8ml of dH2O, and placed in a 37*C water bath for three hours. Two samples from each population were digested by four enzymes; DraI, TaqaI, RsaI, and AvaI. After the restriction digests were complete, they were run on an agarose gel to reveal various bands.
DISCUSSION These longear sunfish of southern Missouri along with other regions of the country have caught alot of attention and gained alot of interest. Indeed the real question is whether these fish have enough genetic variability to deserve further speciation between populations in southern Missouri. Although the final decision for this particular region may have impacts on decisions made for other regions concerning further speciation. When analyzing the results from this project, I believe it is important to shed light on a previous study and conclusion surrounding a project similar to this. That conclusion states as follows; Gasconade river is indeed megalotis, Osage river is indeed nuchalis, and White river is rather megalotis. Despite of the small amount of data collected due to problems running PCR, the results from my project show no initial evidence to reject the hypothesis that there would be a difference to that of the previous conclusion. I was only able to get two fish from population A which is from the Gasconade river, two fish from population C which is from the Osage river, and zero fish from population B which is from the White river. For each one of these four fish I ran four different resriction digests. For each enzyme the banding pattern was the same for all four fish despite the fact that they were from two separate populations. This is evidence from an independent source that the Gasconade and Osage river populations may be the same which contradicts the RAPD data. This mitochondrial DNA data does not support the RAPD data. As for the White river population B, it should be expected that the mitochondrial DNA will be the same as A and C.
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