The target fragment contained the DNA-(apurinic or apyrimidinic site) lyase (Apn2) gene approximately 800 bp including an intron region of 70–100 bp. The forward primer
(apn2fw2: GCMATGTTYGAMATYCTGGAG) and the reverse primer (apn2rw2: CTT GGTCTCCCAGCAGGTGAAC) were designed based on the proximal end of first exon and the distal end of the second exon region relatively conserved across the alignment. The selected primers were then evaluated for thermal properties, GC content, hairpin formation and self-complementarities using the online platforms of OligoCalc (http://www.basic.northwestern.edu/biotools/oligocalc.html) and the Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/pcr_primer_stats.html). Gradient PCR and reagent optimisations were used to develop the standard protocols for amplification. BI 2536 manufacturer click here Twelve reactions across an annealing temperature gradient
of 65–50 °C for each of the test isolates were performed in three replicates. The optimal annealing temperature was determined by the intensity of the amplicons visualised by agarose gel electrophoresis. Primers were initially tested against a panel of 20 species selected from a broad range of Diaporthe species and including the representative isolates of Ophiodiaporthe cyatheae (AR5192) and Mazzantia galii (AR4658). PCR products were purified and sequenced using the protocols detailed above. Sequence alignment and phylogenetic analysis Raw sequences were assembled with Sequencher 4.9 for Windows (Gene Codes Corp., Ann Arbor, Michigan). The consensus sequences were then initially aligned using MAFFTv.7 (Katoh and Standley 2013) (http://mafft.cbrc.jp/alignment/server/) and optimised in the SATEv.2.2.7 (Simultaneous Alignment and Tree Estimation) high throughput alignment platform (http://phylo.bio.ku.edu/software/sate/sate.html) (Liu et al. 2012). Newly generated ITS and EF1- α sequences were analysed with all available type-derived
sequences listed in Udayanga et al. (2011, 2012a) and Gomes et al. (2013) to determine initial identities of the isolates. ML gene-trees were estimated using the this website software RAxML 7.4.2 Black Box (Stamatakis 2006; CYTH4 Stamatakis et al. 2008) in the CIPRES Science Gateway platform (Miller et al. 2010). For the concatenated dataset all free modal parameters estimated by RAxML with ML estimate of 25 per site rate categories. The RAxML software accommodated the GTR model of nucleotide substitution with the additional options of modeling rate heterogeneity (Γ) and proportion invariable sites (I). These analyses utilised the rapid bootstrapping algorithm in RAxML. All isolates were subjected to a multi-gene analysis of seven genes including Apn2, EF1-α, CAL, HIS, FG1093, ACT and TUB regions, excluding the ITS region from the combined analysis.