Computational protocol: The Use of DNA Barcoding on Recently Diverged Species in the Genus Gentiana (Gentianaceae) in China

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Protocol publication

[…] The original trace files were checked and verified by searches with NCBI’s web-based BLASTn. Sequences were assembled and inspected with Sequencher 4.1 [], aligned with the MUSCLE aligner implemented in Mega 5.0 [], and modified manually using Se-al version 2.0a11 []. Due to the presence of indels, trnH-psbA was aligned by section, and intraspecific inversions were found in this region. To reduce costs, we retrieved ITS2 from ITS data and re-amplified failed ITS samples.Genetic divergence was calculated for the five markers according to the Kimura 2-Parameter (K2P) model using MEGA 5.0. Six distance parameters were estimated, including three inter-specific distance parameters (average inter-specific distance, average theta prime and smallest inter-specific distance) and three intra-specific parameters (average intra-specific distance, theta and largest intra-specific distance) []. We calculated the mean K2P distances for each of the six sections and explored the difference in evolutionary divergence among sections in Gentiana.Three methods, namely a genetic distance-based method, the analysis of Best Close Match and a tree-based method, were employed to evaluate the five single markers and their combinations. The first two methods were conducted using the R package SPIDER []. For the tree-based method, two phylogenetic trees were inferred to calculate the rate of monophyletic clusters. Neighbor-Joining (NJ) trees were built using the software PAUP* version 4b10 with the K2P model. Node supports were assessed by 1000 bootstrap replicates. A Bayesian inference (BI) analysis was implemented using MrBayes on XSEDE (v3.2.6) [], and the optimal models for each marker were determined under the Akaike Information Criterion (AIC) using jModelTest2 on XSEDE (v2.1.6) []. Both were conducted on the CIPRES supercomputer cluster [] with parameter sets according to Yan et al. []. Species were considered successfully identified if the monophyletic cluster of sequences representing a species was grouped with a bootstrap value above 70% or a posterior probability above 0.95. Singleton species (species with one specimen) were included and considered as the source of resolution failure. […]

Pipeline specifications

Software tools BLASTN, Sequencher, MEGA, Se-Al, MrBayes, jModelTest
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Gentiana crassicaulis, Gentiana straminea