Computational protocol: Local origin of two vegetative compatibility groups of Fusarium oxysporum f. sp. vasinfectum in Australia

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Protocol publication

[…] AFLP fingerprints were generated using the protocol described by . DNA (250 ng) was co-digested with MseI and EcoRI at 37°C for 2 h and oligomer adapters ligated to DNA fragments at 37°C for 3 h in 40 μL of digestion-ligation buffer. Preselective amplification was performed with 5 μL of digestion-ligation reaction in 50 μL of polymerase chain reaction (PCR) buffer containing nonselective primers MseI+0 and EcoRI+0 (20 cycles of 30 s at 94°C, 60 s at 56°C, and 60 s at 72°C). Selective amplification was performed with 5 μL of 1:30 diluted preselective amplification reaction in 20 μL of PCR buffer containing primers MseI + A and 33P-labelled EcoRI + AGG (one cycle of 30 s at 94°C, 30 s at 65°C, and 60 s at 72°C; 12 cycles of 65°C with annealing temperature lowered by 0.7°C during each cycle; and 23 cycles of 30 s at 94°C, 30 s at 56°C, and 60 s at 72°C).Amplified DNA fragments were separated on a 6% polyacrylamide gel electrophoresed at 50 W for 2.5 h on an AFLexpress automatic sequencer (Amersham Pharmacia Biotech, Roosendaal, the Netherlands) flanked by a 30–330 plus 1668 bp AFLP DNA ladder. Autoradiographs were obtained by exposing Kodak BioMax MR film (Eastman Kodak Co., Rochester, NY, USA) to dried gels. All AFLP bands of medium to dark intensity were scored manually from the autoradiographs. Fragment sizes were inferred using Gene Profiler Eval. 4.03 (Scanalytics, Rockville, MD, USA). A common set of four reference strains were included on each gel to maintain consistency of scoring across gels. Identical profiles were obtained from different DNA preparations of the same isolates, confirming the reproducibility of the AFLP fingerprints.AFLP bands were scored as dominant markers (present/absent). The binary data matrix was analyzed using NTSYSpc 2.11X (Exeter Software, Setauket, NY, USA). Haplotypes were determined by calculating the Dice coefficient of genetic similarity in the SIMQUAL module and constructing an unweighted pair-group with arithmetic averages (UPGMA) dendrogram in the SAHN module. Bootstrap values (10 000 replicates) for each branch (%) of the dendrogram were calculated using Winboot (International Rice Research Institute, Manila, Philippines). [...] Amplification and sequencing primers are listed in . Portions of the translation elongation factor-1α (EF-1α) gene, the mitochondrial small subunit (mtSSU) rDNA, the nitrate reductase (NIR) gene, and the phosphate permease (PP) gene were amplified and sequenced from representative isolates (). The genes were amplified in 50 μL reaction mixtures containing 100 ng template DNA, 1.5 mm MgCl, 2 mm dNTPs, 10 pm primer, and 2 U Amplitaq DNA Polymerase (Applied Biosystems, Foster City, CA, USA) in 1×GeneAmp buffer (Applied Biosystems). PCR amplifications were performed in a Hybaid Express cycler (Thermo, San Diego, CA, USA) with the following program: initial denaturing (2 min at 95°C), 35 cycles of denaturing (30 s at 94°C), primer annealing, primer extension (45 s at 72°C), and final extension (5 min at 72°C). PCR products were purified using Amicon Montage PCR clean-up columns (Millipore, Bedford, MA, USA) and re-suspended in 100 μL of 10 mm TRIS.Sequencing reactions were conducted on the purified PCR products with 3.2 pm of the forward or reverse primer using the fluorescent-labeled BigDye kits v3.1 (Perkin-Elmer, Boston, MA, USA) in a Hybaid Express cycler (Thermo) with the program recommended by the manufacturer. Products were cleaned up by isopropanol precipitation and run on an ABI PRISM Genetic Analyzer capillary sequencer (Applied Biosystems).Forward and reverse sequences were assembled, edited using Sequencher 4.2 (Gene Codes, Ann Arbor, MI, USA), and deposited in GenBank (). Alignments were conducted using ClustalW as implemented in BioEdit (). In addition to the sequences generated in this study, representatives of F. oxysporum f. sp. vasinfectum race 1–8 and representative taxa from the order Hypocreales were downloaded from GenBank to augment the alignments (). Three sequence alignments were constructed: (i) concatenated EF-1α and mtSSU sequences from representative strains of Fov from Boggabilla and native F. oxysporum from soils associated with wild Gossypium populations to explore the genetic relationships between pathogenic and nonpathogenic (against G. hirsutum cotton) Australian strains (deposited in TreeBASE under the accession numbers SN2747-10816); (ii) concatenated EF-1α, mtSSU, NIR, and PP sequences from pathogenic and nonpathogenic Australian isolates and representatives of Fov races 1–8 to determine the genetic relationships between Australian F. oxysporum strains and Fov occurring elsewhere in the world (deposited in TreeBASE under the accession numbers SN3665-16634); and (iii) EF-1α sequences from all lineages of Australian F. oxysporum identified by the AFLP analyses and representatives of other key Fusarium lineages to assess the phylogenetic relationships of the Australian F. oxysporum to other Fusarium species and taxa (deposited in TreeBASE under the accession numbers SN3665-16635).Parsimony optimized topologies, partition homogeneity estimates, and bootstrap values were generated using PAUP 4.0 beta 10 (Sinauer Associates, Sunderland, MA, USA). Unweighted maximum parsimony was conducted using the heuristic search option and 100 random addition sequences with the tree-bisection-reconnection branch swapping and the MULTREES option on. Bayesian inference was used to estimate posterior probabilities for consensus nodes using MRBAYES 3.1 () and the most appropriate models of sequence evolution for the Bayesian analysis were identified using Modeltest 3.7 (). Trees were visualized using TreeView 1.6.6 (). […]

Pipeline specifications

Software tools NTSYSpc, Sequencher, Clustal W, BioEdit, PAUP*, MrBayes, ModelTest-NG, TreeViewX
Databases TreeBASE
Applications Phylogenetics, Population genetic analysis
Organisms Fusarium oxysporum, Gossypium hirsutum