Statistics
As the sequenced library size was variable among samples, ASV count was
normalised by the Trimmed Mean of M values method in the edgeR package
in R (Robinson et al., 2010). Bray-Curtis distance was calculated
between samples to indicate the differentiation of microbiome
composition. Based on Bray-Curtis distance, non-metric multidimensional
scaling (NMDS) plot was generated to visualize the microbial community
from the Vegan Package (Oksanen et al., 2007). Permutational
multivariate analyses of variance (PERMANOVA) with maximum 999
permutations (adonis function from Vegan package) was conducted
to test the effects of soil type, chickpea genotype, rhizocompartment,
root segment, and their interactions on microbiome composition, and root
segment and rhizocompartment were nested in soil and genotype. Shannon
index of samples was estimated using iNEXT in R package (Hsieh et al.,
2016). PERMANOVA and unconstrained principal coordinate analyses (PCoA)
using Bray-Curtis distance were also conducted to analyse the
composition of root exudate compounds. The Statistical Analysis of
Metagenomic Profiles (STAMP) package was used to determine statistical
differences of taxonomic groups between experimental treatments with
Benjamini-Hochberg FDR method to correct for the P value (Parks et al.,
2014). For the variables of Bray-Curtis distance, Shannon index, CSR,
bacterial number, and root exudates, General Linear Model was applied to
test the effects of experimental factors and interactions using Minitab
(Minitab Inc., State College, PA, USA). Normal distribution of each of
the variables was checked and confirmed. Tukey HSD post-hoc pairwise
comparison test was performed to compare the treatments.