Sample collection and molecular analysis

To determine if these individual patches were derived from one or several genets, sampling was carried out on a grid pattern on two large patches at Dowd Morass (45 m x 120m and 30m x 68m) containing what appeared to be, from the circular patterning within the patches, three genets and two genets (Figure 3.3). The grid was arranged to ensure that samples were taken in the centre and edges of the patches and on either side of what appeared to be joins between the putative genets (Figure 3.3 a-b). A grid pattern was chosen in preference to random sampling as a grid more clearly determined the distribution of individual genets within a patch and clearly elucidates whether the plant is of guerrilla or phalanx growth habit (Chen et al. 2002). An additional five individual ring-shaped patches of plants were sampled to determine if these were individual genets as there was anecdotal evidence of sexual reproduction (true seedling having alternate leaves, ramets opposite leaves) within the senescent interiors of these large patches of M.

ericifolia (Figure 3.3 c).

Approximately 200 g of actively growing stem tips were collected for each sample from individual aerial branches, half of which was placed in sealed plastic bags kept on ice and transported to an -800 C freezer. The other half was sealed in plastic containers and desiccated with silica gel, also kept on ice and transported to a 40C refrigerator until processed. Frozen material was collected and saved to ensure availability of material should the preferred desiccated material prove unsuitable.

Figure 3.3 Location of sampled patches of M. ericifolia at Dowd Morass, Sale, Victoria. A = patches sampled for determination of genetic diversity of patches and intermingling of genets (close-up Figures 3.3a and 3.3b). B = Doughnut shaped patches sampled to determine if regeneration within the patch represents vegetative or sexual regeneration (close-up Figures 3.3c).

A1

A2

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Figure 3.3c Doughnut-shaped Melaleuca ericifolia patches (B) showing sample points. Lighter coloured edges represent actively expanding ‘front’, darker centres represent senescent older stems and area of regenerating young growths.

DNA isolation

DNA was isolated from leaf material dried in silica gel using QIAGEN Dneasy plant DNA extraction minikits (QIAGEN Pty. Ltd. Doncaster, Victoria. Aust.). Samples (20 mg) of dry tissue were ground with a small amount of acid-washed sand and DNA isolated according to the manufacturer’s instructions.

Primer screening

Primers considered promising for Melaleuca were screened and those that gave clear bands that were reproducible and could be scored readily were used to amplify DNA from all samples. In all, five primers were used: DatA, 888, BDBLz, HB15, and 814.

DNA amplification

DNA was amplified in 20 μl reactions containing 10 μl QIAGEN HotStart Master Mix (8

μl H2O, 1 μl 10 μM primer, 1 μl DNA (20 mg). Polymerase chain reactions (PCR) were

performed in an Eppendorf MasterCycler® gradient thermal cycler using the following profile: 950C for 15 min (1 cycle); 940C for 45 s, 720C for 1 min (35 cycles); final extension step of 720C for 10 min with an indefinite soak at 40C.

PCR products were visualised on 2.0% agarose gels stained with ethidium bromide and photographed under UV light. Gels were photographed with a Kodak EDAS 290 digital camera and Kodak 1D software. Each sample was scored manually using digital images

58 dominant markers such as ISSRs, the term phenotype is generally used rather than genotype (the underlying genetic basis). This is because a plant that, for example, is homozygous for the dominant allele (AA) cannot be distinguished from a plant that is heterozygous (Aa) but a homozygote recessive is distinguishable (aa).

PCR products were scored as present (1) or absent (0) for each individual which was then assigned a multilocus phenotype based on the combined PCR product patterns of all ISSR primers. The probablility of identical patterns having arisen independently via sexual reproduction was then calculated following the method described by Parks and Werth (1993). Due to slight amplification problems (not all fluoresced evenly) all primers were run twice. Poorly amplified samples were excluded from the scoring.

Data analysis

In clonal species such as M. ericifolia, the analysis of genetic diversity data can be misleading due to the individual genet being sampled multiple times although each sample is treated as if it were an separate individual. Determining a statistical basis for distinguishing if individual samples are derived from asexual reproduction from a single zygote or if the same phenotype was produced independently via sexual reproduction is difficult, particularly if individuals of the same phenotype are clustered (Parks and Werth 1993; Widen et al. 1994).

To overcome this difficulty, the approach used was to calculate the probability, P, (Eq. 1), once a particular phenotype was found, of obtaining that same phenotype, assuming

sexual reproduction, in (n-1) subsequently sampled individuals (Parks and Werth 1993; Sydes and Peakall 1998).

P = (Pgen)n –1

Where n is the total number of individuals with the same multilocus phenotype and Pgen = probability of obtaining the observed multilocus phenotype via sexual reproduction. If P

is small, it can be concluded that the most likely explanation for the observed cluster of individuals of the same phenotype is common derivation through asexual reproduction.

For dominant markers such as ISSRs where only two phenotypes are possible (presence or absence of a particular fragment), Pgen, represented as Pdgen (Sydes and Peakall 1998), is calculated using Eq. (2).

Pdgen =IIxi

Where xi is the frequency of whichever phenotype (band presence or absence) was

observed at locus i in the individual being considered. This approach has been used by a number of authors to analyse data from suspected clonal species (Parks and werth 1993; Widen et al. 1994; Sydes and Peakall 1998).

As M. ericifolia has been recorded as having multiple ramets arising from large inter-

connected roots (Figure 3.1), analysis of ISSR data was carried out according to the above equation to give the probability of phenotypes having arisen independently more than once. A complete set of the data and calculations is provided in Appendix 1.

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