Abstract: 【Objective】The objective of this study is to determine the optimal DNA extraction methods for yak rumen bacterial genome and preliminarily identify the bacterial community of yak rumen.【Method】Physical method (bead-beating, freeze-thaw), chemical method(CTAB, SDS) and enzymatic method (lysozyme, protease K) were combined with the characteristics of rumen bacteria, 9 different methods (method 1, CTAB-SDS-Lysozyme; method 2, CTAB-SDS-Lysozyme-freeze-thaw; method 3, CTAB-SDS-Lysozyme-bead-beating; method 4, CTAB-Lysozyme; method 5, CTAB-Lysozyme-freeze-thaw; method 6, CTAB- Lysozyme-bead-beating; method 7, SDS-lysozyme; method 8, SDS-lysozyme- freeze-thaw; method 9, SDS-lysozyme-bead-beating) were generated to extract the rumen bacterial DNA, besides, method 10, QIA amp DNA Stool Mini Kit, was also used as reference. The above 9 methods were compared and analyzed based on DNA concentration, purity, DNA electrophoretogram and 16S rRNA high throughput sequencing results, and the bacterial community of yak rumen was preliminarily identified based on the sequencing results.【Result】The comparison results of different DNA extraction methods efficiency showed that the combined beat-beating and freeze-thaw could significantly increase cell lysis efficiency and DNA yield under the same chemical and enzymatic conditions. Methods 3 and 6 obtained high DNA concentration and purity, methods 7, 8, 9, and 10 had lower volume of DNA than other methods because of the lack of CTAB. In addition to methods 2 and 8 as their PCR products were too weak or not detected, the PCR products of rest methods all had suitable objective strap size and concentration which met the requirements of 16S rRNA high throughput sequencing. After high throughput sequencing, a total of 191 349 raw reads were obtained, and 171 231 clean reads were obtained after quality control. Rare faction curves analysis showed that the amount of reads reached the saturation level, which could completely reflect the bacterial community species of the samples. OTU-based taxology and the diversity index analysis showed that methods 6 and 10 contained more abundant bacteria. The comparison of the effect of different extraction methods on Gram-positive bacteria showed that method 6 presented a comparatively higher capability in lysing cell wall of Gram-positive bacteria. Above all, the DNA yield, OTUs number, bacteria diversity, and excellent cell-breaking capability of method 6 (CTAB-Lysozyme- bead-beating) was better than that with other methods. The bacterial community structure analysis showed that the number of taxa in yak rumen contained approximately 21 phyla, 35 classes, 75 families, 112 genera. The bacteria community of higher abundance included Bacteroidtes (64%), Firmicutes (20%), Spirochaetae (2.3%), Proteobacteri (1.8%) and Fibrobacter (1.7%). Comparative analysis showed that there are some variation in the bacterial community between yaks and cattle, which may be attributed to different diets and habitats.【Conclusion】This study screened out an optimal DNA extraction method (method 6,CTAB-Lysozyme-Bead-beating) for yak rumen bacterial genome using 16S rRNA high-throughput sequencing, and the bacterial community structure of yak rumen was preliminarily evaluated. Results of the study provide a foundation for identifying the specificity of rumen microbial community and discovering distinct gene resource in yak.

Key words: yak, rumen microbe, DNA , extraction methods, bacteria community structure