Computational protocol: Molecular characterization and the effect of salinity on cyanobacterial diversity in the rice fields of Eastern Uttar Pradesh, India

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Protocol publication

[…] A multiple alignment was produced using the CLUSTAL_X ver. 2 [] and manually corrected using JalView. Bands with identical mobility on DGGE gel were considered to have identical sequences. Sequence similarity between the 31 different partial 16S rRNA gene sequences resulting from DGGE analysis were deposited in GenBank and assessed by BLASTN [] homology searches using the nonredundant NCBI GenBank database. In addition to this, 43 16S rRNA gene sequences from GenBank, which showed the closest similarity with the different DGGE-PCR products, were also included in the multiple alignment. Pair-wise distance matrices were calculated using the Tajima-Nei method []. Character positions with gaps were deleted. The 16S rRNA gene sequences of cyanobacteria were classified into phylogenetic groups as proposed by Desikachary [] for the determination of genetic variability within and between the groups. Phylogenetic trees were constructed using the neighbor-joining algorithm [] provided in MEGA4 []. One thousand bootstrap replicates of the alignment data were also performed and the consensus tree was constructed. [...] Results of the soil analysis were statistically analyzed using one-way ANOVA followed by correlation coefficient (r) analysis using SPSS 10.0. Principal component analysis was performed using Statistica 8.0. For PCA analysis, the soil analysis data presented in Tables and were considered. The clustering in the PCA was performed as per Coeyne et al. [] using cluster analysis (similarity measure: Pearson or product-moment correlation coefficient; clustering method: UPGMA). Further, to correlate cyanobacterial abundance and the physicochemical properties of soil (EC and available nitrogen), each band on a DGGE gel was treated as an individual species. The total number of bands present in any individual lane was considered to be the cyanobacterial diversity in that soil sample. Further, for accurate estimation of diversity, microscopic observations were also compared with the molecular data. Three independent variables were used for each experiment. […]

Pipeline specifications

Software tools Clustal W, Jalview, BLASTN, MEGA, SPSS, Statistica
Applications Miscellaneous, Phylogenetics
Organisms Oryza sativa Indica Group, Oryza sativa
Chemicals Ammonia, Nitrogen