Assessment of genetic diversity and DNA fingerprinting of the Cape parrot (Poicephalus robustus) using randomly amplified polymorphic DNA (RAPD)
Blue, Gillian Margaret.
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The Cape parrot (Poicephalus robustus) is South Africa's only endemic parrot. It has become increasingly rare in recent years, with fewer than 500 birds left in the wild, and is now regarded as endangered. Possible factors contributing to this rapid decline in numbers include habitat loss, food shortage, disease and illegal trafficking and trading in the species. Habitat loss and food shortage have been brought about by the rapid destruction of the yellowwood trees in the afromontane forests in South Africa and have played a role in reducing the population numbers. The Psittacine beak and feather disease virus (PBFDV) has also contributed to the loss of some individuals, however it is the illegal trafficking of this rare and valuable species that has become of great concern. As the Cape parrot is becoming increasingly rare and therefore highly sought after, its commercial value has multiplied to the extent that illegal black market trapping is on the rise. The industry involved in breeding and conservation of endangered bird species, has a need for the proper establishment of studbooks, containing all available information on captive as well as tagged birds. Most of the information found in studbooks is based on morphological attributes of individual birds. Although this is useful, there is a need to add molecular information in order for complete identification of individuals, particularly in a species threatened by illegal trading and theft. A preliminary analysis of the amount of variation present in the population of interest is therefore required so that appropriate methods and techniques can be developed to identify individual birds. A RAPD analysis was conducted to assess the amount of variation in the Cape parrot and lay the foundations for the establishment of individual identification in the species. Blood samples from 30 parrots, consisting of both related and unrelated individuals, were obtained from three separate locations: Amazona in Assagay, Rehoboth Farm in Dargle, as well as from the Eastern Cape. 15 random primers were selected and used to conduct a randomly amplified polymorphic DNA (RAPD) analysis. RAPDs are extremely useful in situations where relatively inexpensive first approximations of the genetic variation are needed, such as in rare and endangered species. After successful optimisation of the technique in the species, the 15 primers were screened for all 30 individuals and the individual DNA fingerprints, analysed. Clear, distinctive and reliable DNA fingerprints were obtained for all individuals however, it was interesting to note despite the analysis of 85 loci using the 15 primers almost identical DNA fingerprints were produced between the individual birds. A population analysis into the amount of variation present between and within the three populations, as well as for the representative population as a whole, was conducted. Using various statistical programmes such as POPGENE and ARLEQUIN, heterozygosities, genetic distance measures, diversity indices, Wright's fixation index and AMOVAs were estimated. The amount of polymorphism detected in this investigation was 33 % and the heterozygosity, 0.37, which is a relatively high value for the uniformity displayed in the DNA profiles. The high GC content of the primers however, could be a possible explanation thereof. Relationship and kinship determination, sex determination as well as population assignment was possible despite not being able to identify each individual based on unique DNA fingerprints. The AMOVA analysis indicated significant variation on both the between (5.59 %) and within (94.41 %) levels of analysis. Little variation or differentiation was observed between the three subpopulations, which was confirmed with an FST value of 0.056. The variation experienced within each subpopulation was analysed using Shannon's index of phenotypic diversity. The Amazona population displayed the most variation with a value of 0.286 and the Rehoboth population, the least with 0.195. This was expected, with the individuals from the latter population comprising one extended family. Nei's measures of genetic identity revealed that the individuals from Amazona were more similar to the Eastern Cape population, which was again expected with regular exchanging of chicks between the two breeders. RAPD technology was successful in laying the foundations for individual identification in the Cape parrot. It was also successful in producing reproducible DNA fingerprints in the species that were able to determine relatedness to some extent, determine the sex of individuals and identify individuals from a particular subpopulation. Furthermore RAPD analysis gave a good indication of the variation found in the Cape parrot population, which is important for conservation purposes. In order to maximize conservation efforts and strategies in an endangered species, determining the level of genetic diversity and variation found in the remaining individuals of the population is of great importance. This information could provide powerful insight for conservation purposes and depending on the level of diversity detected, appropriate breeding programmes could be set up in order to increase the genetic variation and thereby reduce the chance of extinction of the species. The following important findings emerged from this investigation: • RAPD technology, once optimised for the species of interest, is successful in producing clear and reliable DNA fingerprints, provided the same protocol is followed carefully throughout the investigation. • An optimised protocol for fingerprinting the Cape parrot using RAPDs was established. • Possible sex identification, population assignment and a degree of kinship determination was determined using RAPDs. • Little variation was found within the representative Cape parrot population as a whole due to small population size and possible inbreeding. • As expected for an avian species, little genetic sub-division or differentiation was observed between the three populations analysed.