Abstract:

Objective: This study analyzed the homology of 27 strains of lactic acid bacterial genotype from Tibetan yak
cheese by randomly amplified polymorphic DNA (RAPD) method. Methods: To establish a reliable RAPD protocol with
5 primers, the optimal primers and PCR conditions for the quality and reproducibility of RAPD profiles were investigated,
including Mg2+ concentration, dNTP amount, annealing temperature, and DNA template/primer ratio. The optimal PCR
procedure and reaction reagent concentration were employed for RAPD amplification of 27 isolates from yak milk cheeses
and 4 standard strains of lactic acid bacteria. Electrophoretic profiles were analyzed by NTsys 2.10e software, genetic
similarity coefficients were calculated and a cluster dendrogram was plotted. Results: Genetic similarity coefficients of 31
strains ranged from 0.72 to 1.00. The cluster dendrogram showed that 31 isolates were classified into 8 groups using the
similarity coefficient of 0.82 as a cut-off point. The group discrimination corresponded well with the species assignment by
16S rRNA sequence analysis, and Lactobacillus casei and Lactobacillus paracasei were separated successfully. Conclusion:
The RAPD technique was useful for relationship analysis of lactic acid bacteria isolated from Tibetan yak milk cheeses.

Key words: Tibet, yak milk cheese, lactic acid bacteria, randomly amplified polymorphic DNA (RAPD), cluster analysis