Bigdye terminator 3.1 kit

Growing mycelia of individual fungal strains were maintained in pure cultures for 7 days on PDA medium for genomic DNA extraction. Genomic DNA was extracted using a modified CTAB (hexadecyltrimethylammonium bromide) method, described earlier [27 ]. The DNA extracts were stored at – 20 °C until used. Species identification was done on the basis of the sequences of the Internal Transcribed Spacers of the ribosomal DNA region (ITS1-ITS2).
Polymerase chain reactions were performed as described previously by Kozłowska et al. [9 ], using DreamTaq Green DNA polymerase (Thermo Scientific, Espoo, Finland). The PCR amplification was done using ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) primers. Amplicons were electrophoresed in 1.5% agarose gel (Invitrogen) with GelGreen Nucleic Acid Stain (Biotium, Inc.).
For sequence analysis, PCR-amplified DNA fragments were purified as described previosly by Kozłowska et al. [9 ], DNA fragments were labelled using a forward primer and the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the producer’s recommendations, and precipitated with 96% ethanol. Sequence reading was performed using Applied Biosystems equipment. Sequences were analysed using the BLASTn algorithm.

A modified method using CTAB (hexadecyltrimethylammonium bromide) was applied for genomic DNA extraction, as described earlier^{58 (link)}. Species identification was performed on the basis of the sequence analysis of the Internal Transcribed Spacers of the ribosomal DNA region (ITS1-ITS2).
Polymerase chain reactions (PCRs) were performed as described earlier^{27 (link)} using DreamTaq Green DNA polymerase (Thermo Scientific, Espoo, Finland). For the PCR amplification specific primers were used: ITS4 – forward primer (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 – reverse primer (5′-GGAAGTAAAAGTCGTAACAAGG-3′)⁵⁹ . Amplicons were separated in 1.5% agarose gel (Invitrogen) with GelGreen Nucleic Acid Stain (Biotium, Inc.).
For sequence analysis, PCR-amplified DNA fragments were purified as described earlier^{60 (link)}. DNA fragments were labelled using a forward primer and the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the producer’s recommendations and precipitated with 96% ethanol. Sequence reading was performed using Applied Biosystems equipment. Sequences were analysed using the BLASTn algorithm against the GenBank database-deposited reference sequences.

For sex-determination of moose, five sex-specific SNPs are included in the SNP panel; three of these markers were developed de novo. DNA from six moose individuals were included in the development. Fragments of the Sry region were PCR amplified with the primers CerSRYf (5'-TGAACGAAGACGAAAGGTGGCTCT-3') and CerSRYr (5'-TACCCTATTGTGGCCCAGGTTTGT-3') following the method described in Lindsay and Belant [33 ]. The PCR products were Sanger sequenced using the BigDye Terminator 3.1 kit on a 3730 xl DNA analyzer (Applied Biosystems, Foster City, USA) at the department of Medical Biosciences, Umeå universitet, Sweden. The generated sequences were aligned and a consensus sequence was constructed with BioEdit 7.0.5 (Hall, T. Ibis Therapeutics, Carlsbad, USA) that was manually scanned for sex-specific SNPs. The other two SNP markers for sex-determination: Ce10ay and Ce12ay were previously developed by Nichols and Spong [34 ]. The sex-specific SNPs were designed to detect the presence of the Y-chromosome (thus failing to provide positive amplification results in females). The minimum threshold for acceptable sex-determination was a positive amplification at three out of five SNPs. Samples amplifying at fewer loci, but not zero, would have been considered ambiguous. The sex-specific SNPs were validated by genotyping of 59 individuals of known sex.

The amplification product of ORF2 was purified using reagents and protocols of the commercial Wizard SV Gel kit and PCR Cleaning System (Promega, USA). Sequencing reactions were performed using reagents and protocols of Big Dye Terminator 3.1 kit (Applied Biosystems, EUA). Phylogenetic analyses were conducted with Bayesian inference using Markov Chain Monte Carlo (MCMC) statistical framework implemented in the program BEAST v1.8.1 [30 (link)] under TRN+G nucleotide substitution model. A phylogenetic tree, based on the HEV ORF2 region (302bp), was constructed with sequences retrieved from GenBank, including prototype sequences from HEV genotypes 3 and 4.

The coding region of human AMPKγ1 (NM_002733), AMPKγ2 (NM_016203.3), and AMPKγ3 (NM_017431) was amplified from muscle RNA (Agilent) using a Superscript III 1 step RT PCR kit (Invitrogen). The resulting PCR products were either ligated into intermediate vector (pSC Agilent) or digested with the relevant restriction enzymes and ligated directly into the mammalian expression vector (pCMV5 acc. AF239249), which was modified to incorporate a NH₂-terminal FLAG tag. Sequences of all clones were verified in house at the Nestlé Institute of Health Sciences using the BigDye Terminator 3.1 kit and 3500XL Genetic analyzer (Applied Biosystems).

Genomic DNA was extracted using the 2 × CTAB protocol, modified from Doyle and Doyle [66 ]. Three DNA regions (nuclear PHYC gene, and the ribosomal external and internal transcribed spacers (ETS and ITS)) were selected based on their variability in previous Malpighiaceae studies [2 (link),5 (link),6 (link),8 (link),67 (link)]. Protocols to amplify and sequence ETS and ITS followed Almeida et al. [8 (link)]. For amplification, we used the TopTaq (Qiagen) mix following the manufacturer’s standard protocol, with the addition of betaine (1.0 M final concentration) and 2% DMSO for the ETS region. PCR products were purified using PEG (polyethylene glycol) 11% and sequenced directly with the same primers used for PCR amplification. Sequence electropherograms were produced on an automatic sequencer (ABI 3130XL genetic analyzer) using the Big Dye Terminator 3.1 kit (Applied Biosystems). Additional sequences for PHYC were retrieved from GenBank (Table 3). Newly generated sequences were edited using the Geneious software [68 (link)], and all datasets were aligned using Muscle [69 ], with subsequent adjustments in the preliminary matrices made by eye. The complete data matrices are available at TreeBase (accession number S21218). This study was authorized by the Genetic Heritage and Associated Traditional Knowledge Management National System of Brazil (SISGEN #A3B8F19).

We sampled individuals from 31 localities, including 20 locations of C. fernambucensis subsp. fernambucensis, seven of C. fernambucensis subsp. sericifer, and four from C. insularis, covering the entire documented distribution of our ingroup (Table 1; Figure 1). Total Genomic DNA was extracted from root tissues with DNeasy Plant Mini Kit (Qiagen). Exon 1 from nuclear phytochrome C (PHYC) gene and the plastid intergenic spacer trnS‐trnG were used as molecular markers. These segments were selected based on previous variability screening for Cereus (Romeiro‐Brito, Moraes, Taylor, Zappi, & Franco, 2016; Silva et al., 2016). Amplification reactions for trnS‐trnG and PHYC were performed following Bonatelli, Zappi, Taylor, and Moraes (2013) and Helsen, Browne, Anderson, Verdyck, and Dongen (2009), respectively. The direct sequencing was prepared using the Big Dye terminator 3.1 kit (Applied Biosystems) and conducted in Gene Amp PCR System 9700 (Applied Biosystems). The forward and reverse sequences were assembled in Chromas 1.5 software, and the alignments were performed in ClustalW (Thompson, Higgins, & Gibson, 1994). No heterozygous site was identified for PHYC by considering the absence of potential double peaks after inspection of the sequencing chromatograms.