From 8 μl of pooled product,
2.5 μl was mixed with 0.25 μl of GeneScan-500 Liz molecular size standard (Applied Biosystems Cat #4322682A) and 7.25 μl of Hi-Di Formamide (Applied Biosystems Cat. #4311320). The mixture of products was then loaded onto a Genetic Analyzer (Applied Biosystems, Foster City, CA) equipped with the 36 cm 16-capillary array filled with POP-7 polymer (Applied Biosystems, Foster City, CA). Data acquisition and fragment mTOR inhibitor size determinations were carried out by GeneMapper v4.0 software (Applied Biosystems, Foster City, CA). Genotypes and genetic diversity analysis Genotypes were identified based on combination of allelic data from multiloucs microsatellite loci. A clone-corrected (removing repeated genotypes within a population) data set was built and used for the analysis of genetic diversity, linkage disequilibrium and genetic structure. GenAlEx Version 6.3 [37] was used to calculate the average number of alleles (Na) and haploid genetic diversity (H) at each locus as well as across all loci for each of the populations. Linkage disequilibrium analysis A global test (Fisher’s method) implemented in GENEPOP web version 4.0.10 [38] was used to test for the genotyping linkage disequilibrium (LD) between all pair learn more of loci across all
samples in this study. Genetic structure analysis To determine the genetic relationships of ‘Ca. L. asiaticus’isolates, a UPGMA dendrogram was constructed based on Nei’s genetic distance [22]. The trees were calculated using POPULATION software package Tyrosine-protein kinase BLK Version 1.2.31 (Olivier Langella, CNRS UPR9034, France
found at web: http://bioinformatics.org/~tryphon/populations/) and graphically displayed with MEGA4 software [39]. Confidence in specific clusters of the resulting topology was estimated by bootstrap analysis with 1,000 replicates. The program STRUCTURE 2.3.1 [40] was also used for a this website clustering algorithm based on a Bayesian model to assign individual isolate of ‘Ca. L. asiaticus’ to a specified number of clusters. This algorithm assumes a model in which there are K clusters (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. No linkage disequilibrium was detected between all pairs of loci across all samples with the clonal corrected data set. Therefore, the program STRUCTURE 2.3.1 [40] was rationally used to estimate the number of clusters (K) within ‘Ca. L. asiaticus’ where 10 independent runs of K = 1-10 were performed without any prior information as to the origin (location) of individual samples. For each run, a burn-in period of 25,000 iterations was used followed by a run length of 50,000 Markov chain Monte Carlo iterations, and a model with correlated allele frequencies and admixture among populations. The model was run with 10 independent simulations for each K.