Lactobacillus Diversity Analysis via PCR-DGGE

PCR-DGGE analyses were performed to investigate lactobacilli populations; for each sampling location, 17 (out of 20) DNA extracted from individual guts were processed. The PCR and subsequent denaturing gradient gel electrophoresis (DGGE) analysis, using the Dcode Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA), were performed as described by Alberoni et al. (2018) (link). Denaturing gradient was established at 35–65%. Fingerprinting analyses were carried out using the Bionumerics v 7.1 (Applied Maths, St. Martens-Latem, Belgium) and the UPGMA algorithm based on the Pearson correlation coefficient with an optimization of 1% was applied. Microbial diversity was analyzed with the following parameters: Shannon–Wiener index (H), Simpson index (S), and band evenness (EH), calculated according to Hill et al. (2003) (link). Moreover, principal components analysis (PCA) was carried out by using Bionumerics. Relevant bands were excised from the gels and processed to achieve purified amplicons to be sequenced (Gaggìa et al., 2015 ). Sequencing was carried out by Eurofins Genomics (Ebersberg, Germany) and obtained sequences were assigned to bacterial species using megablast algorithm.²