Evaluation of Established Methods for DNA Extraction and Primer Pairs Targeting 16S rRNA Gene for Bacterial Microbiota Profiling of Olive Xylem Sap

Next-generation sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number factors can affect accuracy microbial community assessment, such as DNA extraction method, hypervariable region 16S rRNA gene targeted, or PCR primers used for amplification. The aim this study was assess influence commercially available kits different primer pairs provide non-biased vision bacterial communities present in olive xylem sap. For that purpose, branches from “Picual” “Arbequina” cultivars were sap using Scholander chamber device. protocol significantly affected assessment. That resulted significant differences alpha (Richness) beta diversity (UniFrac distances) metrics among protocols, with 12 evaluated being clustered four groups behaving differently. Although core taxa detected by all included phyla, seven classes, orders, 16 21 families, 14 genera when Greengenes Silva database taxonomic assignment, respectively, some taxa, particularly those identified at low frequency, only. most accurate depiction mock artificially inoculated on samples generated PowerPlant kit, combination 799F/1193R amplifying V5–V7 region, 132 assignment. DESeq2 analysis displayed abundance between tested. Thus, Enterobacter , Granulicatella Prevotella Brevibacterium presented higher protocols compared pair 799F/1193R, while opposite true Pseudomonas Pectobacterium . methodological approach followed be useful optimize plant-associated microbiome analysis, especially exploring new plant niches. Some selected will contribute better characterize inhabiting olives other woody crop species.