Haplotyping was performed using the microsatellites D7S1824, D7S1826 up-stream of KCNH2 and D7S636, D7S3070, D7S483 and D7S1803 downstream of KCNH2 (Figure 1 ). CentiMorgan distances were obtained from the Map-O-Mat database for microsatellites (http://compgen.rutgers.edu/Mapomat/ ). PCR amplicons were generated using fluorescently end-labelled primers (available at NCBI UniSTS) at 0.4 μM per primer, per reaction. A loading mix of 0.5 μl amplicon, 9 μl HiDi formamide (Applied Biosystems, Foster City, CA, USA) and 0.5 μl 600LIZ size standard (Applied Biosystems) was prepared, and DNA products were electrophoresed on an ABI PRISM® 3100 Genetic Analyser. Data were analysed using ABI GeneMapper software v4.0 (Applied Biosystems).
600 liz size standard
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 600 LIZ size standard is a molecular biology tool used for sizing DNA fragments. It provides a set of DNA fragments of known sizes that can be used as a reference to determine the size of unknown DNA samples during electrophoretic separation.
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Lab products found in correlation
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (3 mentions)
Hi di formamide, by Thermo Fisher Scientific (2 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (2 mentions)
Abi prism 3100 genetic analyser, by Thermo Fisher Scientific (1 mentions)
Genemapper ver 4, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Peak scanner software v1, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Peak scanner software version 1, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Genemarker version 1, by SoftGenetics (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Genemapper v5, by Thermo Fisher Scientific (1 mentions)
Genemapper, by Thermo Fisher Scientific (1 mentions)
8 protocols using 600 liz size standard
1
Microsatellite Haplotyping of KCNH2 Gene
2
Genetic Characterization of Alexandrium ostenfeldii
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Fifty exponentially growing clones were randomly selected from each sampling date, 0.5–10 ml of each culture was harvested and centrifuged at 2,000 g for 10 min. The obtained cell pellet was stored at −20°C until further processing and DNA was extracted in 5% Chelex solution as described by Nagai et al. (2012 (link)). Primer pairs and the PCR (polymerase chain reaction) conditions were described by Nagai et al. (2014 (link)) and nine primer pairs (excluding primers for locus Aosten144) were used in this study. PCR products were electrophoresed on an ABI 3730xl DNA Analyzer (Applied Biosystems) and allele sizes were determined using a 600 LIZ size standard (Applied Biosystems) and genemapper version 4.0 (ABI). Allele numbers at the 10 loci ranged from two to 12 (average of 5.3), and estimates of gene diversity (Nei, 1978 (link)) from 0.10 to 0.92, suggesting that the microsatellites are suitable to characterize the genetic structure of A. ostenfeldii at the population level (Sildever et al., 2019 (link)).
3
Microsatellite Genotyping of Alexandrium ostenfeldii
4
Quantitative Analysis of ygaV Expression in E. coli
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The E. coli ygaV mutant was grown in LB medium until the mid-log phase (OD600 = 0.3) was harvested, and total RNA was extracted using the RNeasy Mini kit (Qiagen). Reverse transcription was then conducted using QuantiTect Reverse Transcription Kit (QIAGEN) with 7 mg purified RNA and 100 µM of the 6-FAM–labeled primer (5′-GTTCGGCACTGGCCTGTAATTGCGCGAG-3′). The mixture was subsequently incubated at 42 °C for 15 min, then incubated again at 95 °C for 3 min to stop the reaction. Afterward, synthesized cDNA was purified by ethanol precipitation, and resuspended in 12 µL Hi-Di formamide (Thermo Fisher Scientific). Then, 0.5 µL of 600 LIZ Size Standard (Applied biosystem) was added to the purified sample (~15 µL), which was analyzed using a 3730xl DNA analyzer (Applied biosystems). Finally, fragment analysis was conducted with Peak scanner software v.1.0 (Thermo Fisher Scientific).
5
Electrophoretic Mobility Shift Assay for Promoter Analysis
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PCR provided Cy5.5-labeled DNA fragments of the ygaV and katG operators/promoters using the labeled primer pair as described above (electrophoretic mobility shift assay). For the analysis of these operators/promoters, each labeled DNA fragment (100 nM) was mixed with purified apo-YgaV at various concentrations in a 22 μL buffer, containing 12.5 mM HEPES/NaOH (pH 8.0), 5 mM K-acetate, 2.5 mM Mg-acetate, 1 mM CaCl2, 12.5 µg mL−1 bovine serum albumin, 1 mM DTT, and 0.3 mg mL−1 heparin. Next, the mixtures were incubated at room temperature (~22 °C) for 30 min, after which 3 μL of 80 times diluted DNase I (TaKaRa) was added to the reaction mixtures and further incubated for 15 min. The reaction was then stopped by adding 25 µL of 0.5 M EDTA (pH 8.0). DNA fragments obtained were purified using a MinElute PCR purification kit (QIAGEN). Afterward, 0.5 µL of 600 LIZ Size Standard (Applied biosystem) was added to each purified sample (~15 µL), followed by an analysis of samples with a 3730xl DNA analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Finally, fragment analysis was conducted using a Peak scanner software version 1.0 (Thermo Fisher Scientific).
6
Microsatellite Genotyping Protocol
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We amplified microsatellites in sets of 10 μl multiplex reactions using a Qiagen multiplex PCR kit and 50–100 ng of DNA. We used negative controls in each PCR and ran a subset of the samples as repeats to verify the genotyping calls (i.e., positive controls). We performed genotyping runs on an ABI 3100 S automated sequencer (Applied Biosystems) at the San Diego State University Microbiology Core Facility using a LIZ600 size standard (Applied Biosystems) and scored alleles using GeneMarker version 1.85 (Softgenetics LLC). We obtained a total of 261 alleles with 0.39% missing data.
7
Microsatellite Analysis for Clonal Diversity
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Eleven microsatellite markers (Barnes et al., 2008) were used to determine the multilocus haplotype of each isolate. Multiplex PCR of the markers (Doth_DS1, Doth_DS2, Doth_E, Doth_F, Doth_G, Doth_I, Doth_J, Doth_K, Doth_L, Doth_M, Doth_O) and fragment analysis were conducted as described by Mullett et al. (2015) . The two panels of PCR products were analysed using an Applied Biosystems 3130XL genetic analyser along with a LIZ 600 size standard (Applied Biosystems), and alleles scored using GENEMAPPER v5.0 (Applied Biosystems, Carlsbad, USA).
Gene diversity was plotted against the number of loci using MULTILOCUS 1.3b (Agapow and Burt, 2001) in order to assess whether scoring more loci would enable greater discrimination of gene diversity. Individuals with identical multilocus haplotypes (MLHs, i.e.
alleles identical at all 11 loci) were considered clones. Two data sets were created: one containing all individuals (non-clone-corrected data set), the second containing only one individual of each multilocus haplotype per population (clone-corrected data set).
Gene diversity was plotted against the number of loci using MULTILOCUS 1.3b (Agapow and Burt, 2001) in order to assess whether scoring more loci would enable greater discrimination of gene diversity. Individuals with identical multilocus haplotypes (MLHs, i.e.
alleles identical at all 11 loci) were considered clones. Two data sets were created: one containing all individuals (non-clone-corrected data set), the second containing only one individual of each multilocus haplotype per population (clone-corrected data set).
8
Genetic Diversity Analysis via AFLP Markers
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Data regarding selectively amplified DNA fragments were separated by capillary electrophoresis using an ABI3730xl DNA Analyzer (Applied Biosystems). The polymorphic AFLP loci were analyzed using GeneMapper, version 4.0, with the LIZ 600 size standard (Applied Biosystems). Polymorphic AFLP fragments were scored as present (1) or absent (0) based on the AFLP pattern amplified by each primer combination. The ability of the most informative primer pairs to differentiate among strains was assessed by calculating the percentage of polymorphic bands and the resolving power (Rp) (Prevost and Wilkinson, 1998) . Rp has been described to correlate strongly with the ability to distinguish between species according to the following formula (Gilbert et al., 1999; Chennaoui-Kourda et al., 2012) :
where Ib = 1 -(2 x |0.5 -p|) and p is the proportion of species containing the 1 band. POPGENE 1.3.1 was used to calculate the genetic diversity. Genetic distances were calculated using the formula of Nei and Li (1979) . The obtained matrix was then used for cluster analysis assessed by unweighted pair group mean of arithmetic method analysis (UPGMA) implemented in the NTSYS-pc software (version 2.1) (Rohlf, 2000) .
where Ib = 1 -(2 x |0.5 -p|) and p is the proportion of species containing the 1 band. POPGENE 1.3.1 was used to calculate the genetic diversity. Genetic distances were calculated using the formula of Nei and Li (1979) . The obtained matrix was then used for cluster analysis assessed by unweighted pair group mean of arithmetic method analysis (UPGMA) implemented in the NTSYS-pc software (version 2.1) (Rohlf, 2000) .
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