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Genetic studies of tropical ornamental fish using RAPD-PCR technology
Author
Chin, Christopher Tet Lipp
Supervisor
Tan, Leo Wee Hin
Gan-Yap, Yik Yuen
Abstract
RAPD-PCR is a useful new molecular technique to characterize and study genomes. In this study, in order to ensure the repeatability and reproducibility of RAPD-PCR results within and between laboratories, various factors influencing the reproducibility of amplification were investigated systematically. Results indicate that template DNA and magnesium factors were important parameters which affected reproducibility. Variation in these factors could cause discrepancies in amplification of certain of certain RAPD bands. It was important to use relatively pure DNA samples for RAPD-PCR amplifications. Although a standard protocol for genomic DNA extraction was employed, purity of DNA extracts varied from sample to sample. Impure DNA samples had to be re-purified before they were used for amplifications.
Results indicated that higher template concentrations were favourable for stable amplification of higher molecular weight products. On the other hand, higher primer concentrations were favourable for stable amplification of lower molecular weight products. There were different optimal concentrations of magnesium in RAPD-PCR amplifications using long or short primers. The usage of high magnesium concentrations was necessary for longer primers.
By performing studies using mixture of templates, results suggests that competition for amplification by background or contaminating DNA could lead to discrepancy or error in scoring some bands. However, the results also implied that the effect of background or contaminating DNA was insignificant when the template of interest was present in a much higher proportion.
To test the potential use of RAPD-PCR in analysing genetic diversity in subpopulations, zebrafish families were used as artificial subdivided populations. Two sets of RAPD data were used. The first set was made up of 71 monomorphic and polymorphic bands which were detected by 4 primers. The second set comprised of 41 polymorphic bands detected by 12 primers. A similarity index was used to quantify genetic identity while a Fst index was used to measure the partitioning of genetic diversity in these "subpopulations". The Fst values calculated from both sets of data were comparably similar. Results indicated that about 46.5% of the genetic diversity was attributable to the restricted breeding groups or "subdivided populations". In comparing to a group of random fish, the between-groups diversity dropped from 37.3% to 6.9% when the three groups of families were taken as one group (no longer subdivided). The sharp drop indicated that RAPD-PCR assay was a sensitive method to detect partitioning of genetic diversity caused by population subdivision or inbreeding.
The RAPD technique was also applied in the identification of 3 species in the genus Danio. D. frankei was postulated to be not a true species by some fish biologists. Analysis of similarity index was performed for the banding profiles obtained from these 3 species with 7 primers. The genetic distance was then calculated. In this study, eight species-specific bands were also identified after screening with 32 primers. The RAPD results consistently implied that D. rerio and D. frankei were of the same species and D. albolineatus was a different species. This molecular identification was in agreement with the observation of extensive interbreeding between D. rerio and D. frankei. The origin of D. frankei is still unknown but nonetheless, D. frankei should now be considered as D. rerio.
Results indicated that higher template concentrations were favourable for stable amplification of higher molecular weight products. On the other hand, higher primer concentrations were favourable for stable amplification of lower molecular weight products. There were different optimal concentrations of magnesium in RAPD-PCR amplifications using long or short primers. The usage of high magnesium concentrations was necessary for longer primers.
By performing studies using mixture of templates, results suggests that competition for amplification by background or contaminating DNA could lead to discrepancy or error in scoring some bands. However, the results also implied that the effect of background or contaminating DNA was insignificant when the template of interest was present in a much higher proportion.
To test the potential use of RAPD-PCR in analysing genetic diversity in subpopulations, zebrafish families were used as artificial subdivided populations. Two sets of RAPD data were used. The first set was made up of 71 monomorphic and polymorphic bands which were detected by 4 primers. The second set comprised of 41 polymorphic bands detected by 12 primers. A similarity index was used to quantify genetic identity while a Fst index was used to measure the partitioning of genetic diversity in these "subpopulations". The Fst values calculated from both sets of data were comparably similar. Results indicated that about 46.5% of the genetic diversity was attributable to the restricted breeding groups or "subdivided populations". In comparing to a group of random fish, the between-groups diversity dropped from 37.3% to 6.9% when the three groups of families were taken as one group (no longer subdivided). The sharp drop indicated that RAPD-PCR assay was a sensitive method to detect partitioning of genetic diversity caused by population subdivision or inbreeding.
The RAPD technique was also applied in the identification of 3 species in the genus Danio. D. frankei was postulated to be not a true species by some fish biologists. Analysis of similarity index was performed for the banding profiles obtained from these 3 species with 7 primers. The genetic distance was then calculated. In this study, eight species-specific bands were also identified after screening with 32 primers. The RAPD results consistently implied that D. rerio and D. frankei were of the same species and D. albolineatus was a different species. This molecular identification was in agreement with the observation of extensive interbreeding between D. rerio and D. frankei. The origin of D. frankei is still unknown but nonetheless, D. frankei should now be considered as D. rerio.
Date Issued
1995
Call Number
SH155.5 Chi
Date Submitted
1995