Genetic marker resources for application in Cyclopia species

Niemandt, Marione (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Cyclopia species are endemic to the Fynbos Biome of South Africa and have been utilised for many years as a health drink known as honeybush tea. Despite the commercial importance of Cyclopia, no molecular resources are available to characterise this genus. The polyploid nature furthermore limits the use of molecular markers as some species exhibit up to 14 sets of chromosomes (Cyclopia intermedia and Cyclopia meyeriana: 2n = 14x = 126). This study optimised a DNA extraction protocol for various Cyclopia species in order to obtain high quality DNA as the first crucial step during molecular genetic studies. The use of young, fresh leaves as starting material for DNA extraction presents a challenge when sampling from distant locations; therefore, a CTAB/NaCl buffer was optimised to preserve the leaves for up to two weeks prior to DNA extraction under laboratory conditions. Microsatellite markers were developed in the commercially important C. subternata and transferred to six other Cyclopia species with a success rate of 81-88%. The Agricultural Research Council (ARC) maintain a field gene bank for several of the Cyclopia species and a set of six DNA fingerprinting markers were developed to characterise the accessions. This will facilitate the correct management of the gene bank, such as keeping track of clones for seed orchards or commercial release and the identification of duplicates in the gene bank. The genetic diversity of C. subternata wild populations was investigated and compared to the accessions to ensure that the gene bank accurately reflects the natural diversity. As such, the C. subternata accessions were representative of the wild samples, excluding the genetically distinct Haarlem population. The genetic resource tools developed in this study can be applied to detect the extent of cross-contamination of cultivated material to wild populations of Cyclopia as well as the characterisation of wild populations of all known species that could be included in the field gene bank. Further conservation strategies include the monitoring of wild-harvesting as well as the in situ conservation of genetically distinct populations.

AFRIKAANS OPSOMMING: Cyclopia spesies is endemies aan die Fynbos Bioom van Suid-Afrika en word al vir baie jare as ‘n gesondheidsdrankie, naamlik heuningbos tee, gebruik. Ten spyte van die kommersiële belangrikheid van Cyclopia, is daar geen molekulêre hulpbronne beskikbaar om die genus te karakteriseer nie. Die poliploïdie vlak van sommige spesies, wat tot en met 14 stelle chromosome kan insluit (Cyclopia intermedia en Cyclopia meyeriana: 2n = 14x = 126) plaas verdere beperkings op die gebruik van molekulêre merkers. Hierdie studie het ‘n DNA ekstraksie protokol vir verskeie Cyclopia spesies geoptimiseer wat reeds tydens die eerste stap van molekulêre studies ‘n hoë kwaliteit DNA verseker. Die gebruik van jong, vars blare vir DNA ekstraksies is nie prakties wanneer monsterneming in afgeleë areas geskied nie, daarom is ‘n CTAB/NaCl buffer metode geoptimiseer om die blare tot enmet twee weke te preserveer voordat DNA ekstraksie in ‘n laboratorium omgewing geskied. Mikrosatellietmerkers is vir die kommersieel belangrike spesie, C. subternata, ontwikkel en met ‘n sukseskoers van 81-88% na ses ander Cyclopia spesies oorgedra. Die Landbou Navorsingsraad (LNR) besit ʼn veld genebank vir verskeie van die heuningbosspesies en ‘n stel van ses DNA vingerafdrukmerkers is ontwikkel om die plantaanwinste te karakteriseer. Dit sal die korrekte bestuur van die genebank verseker soos om tred te hou met klone vir saadboorde en kommersiële vrystelling asook identifisering van duplikate. Die genetiese diversiteit van wilde populasies van C. subternata is ondersoek en vergelyk met die verboude varieteite in die genebank om te verseker dat genoeg diversiteit vasgevang en behoue bly. Dit is gevind dat die C. subternata plantaanwinste verteenwoordigend is van die wilde populasies se genetiese diversiteit, behalwe vir die Haarlem populasie wat geneties van die ander twee populasies verskil. Die molekulêre hulpbronne wat tydens die studie ontwikkel is, kan gebruik word om genetiese kontaminasie tussen verboude plante en wilde populasies waar te neem, asook om wilde populasies van al die verskillende spesies te karakteriseer om later by die genebank in te sluit. Verdere bewaringstrategieë sluit in die kontrolering van veldoes, sowel as die in situ bewaring van unieke genetiese populasies in hulle natuurlike habitat.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100166
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