Microsatellite DNA

Microsatellite DNA from Rufous Treecreeper (RTC) individuals were amplified using 8 locus specific primers, originally designed for the Brown Treecreeper, Climacteris picumnus. The microsatellites were detected using a DNA 3730 capillary sequencer and their fragment lengths calculated (scored) using GeneMapper® software by Applied Biosystems (ABI).

Primer Development

Microsatellite amplification experiments were conducted on the Rufous Treecreeper (RTC), Yellow-plumed Honeyeater and Western Yellow Robins, with microsatellite primers specifically designed for Australian passerines and primers that amplified

across-species (see Appendix 1). However, locus specific primers designed for the Brown Treecreeper by Doer, 2004 (unpublished), were used for the final amplification of microsatellite DNA in the RTC. DNA sequencing of these microsatellites (via a cloning procedure) was conducted to test if the same locus was being amplified in both species and to generate sequence information for designing new, more specific primers for the RTC. Amplification of PCR products using primers (with a universal

fluorescent tag, attached to an M13(-21) tail, as described by (Schuelke, 2000). This method proved to be unsuccessful, therefore, the BTC primers were synthesised with a standard labelled fluorescent tag. These were NED (black), PET (red), 6FAM (blue) and VIC (green).

PCR reagents and reaction conditions

A 100uL PCR (Polymerase Chain Reaction) mix was prepared with 50ng DNA, 5p mole of each primer, 10 mM (of each) dNTP, a X1 PCR buffer and 1U HotStarTaq® DNA polymerase. A gradient of magnesium chloride concentration (20-25mM) and annealing temperatures of 45◦C to 60◦C was used to optimise PCR reaction conditions.

Separation of products on an agarose gel

A 1% DNA agarose gel with 1% TAE buffer was prepared and 10uL of PCR product was loaded into each well to separate products. A 1000 bp DNA ladder with 100bp increments was used to scale the PCR products. The DNA agarose gel was run at 80V for 40 minutes (see Appendix 2 for photos).

PCR Clean-up

With the remaining 90uL PCR products, a sodium acetate method was used to remove excess reagents that may interfere with the subsequent cloning processes.

Cloning procedure

This procedure was undertaken using the Promega pGEM®-T Easy Vector Systems kit with JM109 competent cells. Ligation and Transformation of vector was carried out as to Manufacture’s protocol. Eight tubes of transformation vectors, (each containing a particular microsatellite DNA) were inoculated onto LB/ampicillin/IPTG/X-Gal plates, using 3 different dilutions of 1uL, 20uL and 100uL per plate. These plates were incubated over night at 37◦C and the next day transformant cells were selected by their white colour or antibiotic resistance. Cells that did not successfully ligate with Rufous Treecreeper DNA, were blue in colour.

Culture of clonal cells

A single white colony (transformant) that was isolated and regular in size and shape was selected, cultured in 5mL of LB broth and incubated at 37◦C overnight. This selection was repeated for 6 individual colonies, for each different microsatellite DNA. After overnight incubation, each broth was checked for turbidity (bacterial growth) and any broths that had little bacterial growth were further incubated for another 7 hours.

Extraction of plasmid DNA

As the Rufous Treecreeper microsatellite DNA is incorporated into the bacterial plasmid DNA by the previous ligation/transformation procedure, an extraction of plasmid DNA from the bacteria is necessary. This procedure was carried out using a

QIAGEN plasmid extraction kit (QIAprep® Spin Miniprep Kit) and following the recommended protocol.

Sequencing of plasmid DNA

The sequencing was carried out on an ABI 3730 48 capillary machine. The protocol is based on recommendations made from the Big Dye Terminator v3.1 Cycle Sequencing protocol from ABI. A PCR sequencing reaction was performed using 10ng DNA extracted from plasmid DNA kit, 2uL Dye terminator mix, 1.6 pmoles of reverse primer and made up to 5uL volume with de ionised water.

Sequence Comparisons of Microsatellite DNA

The Rufous Treecreeper sequences produced were aligned using CLUSTALW (V1.83). Sequences of the Brown Treecreeper were found in Genbank (Accession Numbers AY894981-AY894988).

Modification of New Primers

Modified primers were then designed using the RTC cloned sequences and the Brown Treecreeper microsatellite sequences found in GENBANK (NCBI). Flanking the repeat motifs, sequence information was screened for improvement of the G:C ratios

(sequences of 45-50% GC content in 18-22bp primer). Only some of original the primer sets were modified in order to improve primer binding to template DNA (see Appendix 3).

Final PCR Reaction

The modified microsatellite primers were optimised using QIAGEN HotStarTaq®_{. A} 50uL PCR reaction was prepared according to instructions. Loci 3, 4, 6 and 8 were amplified using 1.5mM MgCl2 and a 57ºC annealing temperature. Locus 7 was

amplified with 2.5mM MgCl2 and a 45ºC annealing temperature, locus 1 with 2.0mM

MgCl2 at 58ºC and Loci 5 and 2 primers were amplified using 2.0 mM MgCl2 and a

59ºC annealing temperature.

Separation of microsatellites on an agarose gel

A 1% DNA agarose gel with 1% TAE buffer was prepared and 10uL of PCR product was loaded into each well with a 1000 bp DNA ladder. The agarose gel was run at 90V for 30 minutes. This step is used as a final check for microsatellite products, before continuing with the fragment length analysis.

Fragment Length Analysis

The PCR product from each individual was prepared into a 96 well plate and sealed with a septum. Each well contains 2uL PCR product, 15uL Formamide and 0.1uL LIZ size standard dye. Fragment Analysis of each microsatellite allele, for each individual was conducted on a 3730 Capillary DNA Sequencer and Genemapper® software was used to call allele sizes.