16s-rRNA of guppy
Microbial community genomic DNA was extracted from guppy gut samples using the E.Z.N.A. soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturers instructions. The DNA extract was tested on 1% agarose gel, and DNA concentration and purity were determined with NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F (5'- ACTCCTACGGGAGGCAGCAG-3') and 806R(5'-GGACTACHVGGGTWTCTAAT-3') by an ABI GeneAmp 9700 PCR thermocycler (ABI, CA, USA). The PCR amplification of 16S rRNA gene was performed as follows: initial denaturation at 95  for 3 min, followed by 27 cycles of denaturing at 95  for 30 s, annealing at 55  for 30 s and extension at 72  for 45 s, and single extension at 72  for 10 min, and end at 4 . The PCR mixtures contain 5  TransStart FastPfu buffer 4 L, 2.5 mM dNTPs 2 L , forward primer (5 M) 0.8 L, reverse primer (5 M) 0.8 L, TransStart FastPfu DNA Polymerase 0.4 L, template DNA 10 ng, and finally ddH2O up to 20 L. PCR reactions were performed in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to manufacturers instructions and quantified using Quantus Fluorometer (Promega, USA).