Computational protocol: Strong Genetic Differentiation of Submerged Plant Populations across Mountain Ranges: Evidence from Potamogeton pectinatus in Iran

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[…] Genomic DNA was extracted from leaf tissue using CTAB by Gawel and Jarret [] with some modifications. All individuals were genotyped at nine nuclear microsatellite loci:Potpect24, Potpect26, Potpect28, Potpect32, Potpect34, Potpect37, Potpect39, Potpect40, and Potpect42 []. Amplification of these nine loci was not performed in three multiplex PCR as described in Nies & Reusch [], but into a unique multiplex PCR. This single amplification was made possible by the use of the QIAGEN Multiplex PCR Kit (QIAGEN) in a final volume of 10.5 μL, as follow: 25 ng of DNA template, 5 μL 2× QIAGEN Multiplex PCR Master Mix [QIAGEN Multiplex PCR Buffer, pH 8.7, containing dNTPs, QIAGEN HotStar Taq DNA Polymerase, and 6 mM MgCl2 (for a final concentration of 3 mM)], 1 μL Q-Solution (59 concentrated proprietary QIAGEN PCR additive), 1 μL of a primer mix with 2 μM of each primer (for a 0.2 μM final concentration of each primer) and 1 μL of highly pure water obtained from a Milli-Q Synthesis A10 (Millipore, Molsheim, France). PCR were carried out in 96-well plates on a MyCycler TM thermal cycler (BIO-RAD) under the following conditions: 15 min denaturing at 95'C, [3000 denaturing at 94'C, 1.5 min annealing at 57'C and 1 min extension at 72'C] × 30 cycles and a final extension step at 72'C for 10 min. PCR were carried out in 96-well plates on a MyCycler TM thermal cycler (BIO-RAD) under the following conditions: 4 min denaturing at 94'C, [3000 denaturing at 94'C, 1 min annealing at 57'C and 1 min extension at 72'C] × 30 cycles and a final extension step at 72'C for 30 min. PCR products were run on ABI3730XL sequencer (Macrogen, Seoul, Korea) and fragments were scored with GeneMarker V2.20 (SoftGenetics LLC, State College, USA). Although P. pectinatus is hexaploid, the identified primer pairs amplify microsatellite loci that are ‘‘diploids”, confirming the observations of Nies & Reusch [, ]. To explain this phenomenon, these authors support the idea that the time that has elapsed after the polyploidisation event was sufficient to cause genetic divergence of the microsatellites on the different sets of homologous chromosomes []. After excluding forty eight repeated multilocus genotypes (MLGs) from the data set, we calculated genetic diversity measures on 133 individuals using FSTAT 2.9.3 [] and GenAlex 6.5 []. At locus level, we estimated the number of alleles (A), effective number of alleles (Ae), observed heterozygosity (Ho), expected and unbiased expected heterozygosity (He and uHe), Nei’s heterozygosity at subpopulation level (Hs) and total (Ht), Wright’s F statistics [] with inbreeding coefficient (FIS), total inbreeding (FIT), subpopulation differentiation (FST) and gene flow (Nm) using GenAlex. Weir & Cockerham [] estimation of FIT (CapF), FIS (small f) and FST (Theta) following Rousset [] were calculated with FSTAT. Inbreeding values for the total populations are given by small f and jackknifed over loci (FSTAT).We tested for recent bottlenecks in each population under the two-phase model (TPM) with 95% single-step mutations and 5% multiple-step mutations (Wilcoxon’s test 1-tailed) using bottleneck 1.2.02 []. Genetic structure was assessed with a three-level analysis of molecular variance (AMOVA) [] using hierarchical and standardized fixation indices for 5 regions, 10 populations and 68 individuals (GenAlex). Pairwise FST-values were calculated between all pairs of populations and tested for significant differentiation using 999 permutations. Isolation-by-distance between pairs of populations and their geographical distances was tested with ϴ/1- ϴ considering straight flight distances, log transformed [] between populations in a Mantel test using 1000 randomizations []. An estimator of actual differentiation Dest [] was calculated between all pairs of populations using SMOGD [] and used in a Mantel test with log transformed straight flight distances. To infer the population structure on basis of assignment of individual genotypes into groups, a Bayesian clustering method [] was carried out using STRUCTURE version 2.3. We tested for the number of clusters (K) in ten independent runs from 1 to 14 (10,000 burn-in, 10,000 Markov chain Monte Carlo replicates in each run), without using sampling site as a prior to assess convergence of the estimated ln probability of data, ln (PD). Runs were carried out assuming admixture and an independent model of allele frequencies. The number of clusters was determined from the K with the highest posterior probability and using the second-order rate of change of the likelihood function ΔK, as suggested by Evanno et al. []. For testing the evidence of scoring error, Evidence of large allele dropout and Evidence of null allele we used Micro-checker 2.2 software. Also we used POPTREE2 [] software for cluster analysis. Isolation by distance and RST was tested by calculating multilocus estimates of kinship coefficient (Fij) between all pairs of individuals implemented in the software SPAGeDi v1.4 []. […]

Pipeline specifications

Software tools GeneMarker, GenAlEx, POPTREE2, SPAGeDi
Applications Phylogenetics, Population genetic analysis
Organisms cellular organisms, Pristis pectinata