基于16S rDNA高通量测序技术分析湖北黑头羊瘤胃细菌的多样性

Analysis of rumen bacterial diversity of Hubei black-head goat based on 16S rDNA MiSeq sequencing technology

为了解湖北黑头羊瘤胃细菌多样性及群落结构,选择体重(35±2)kg、安装有永久性瘤胃瘘管的12月龄湖北黑头羊羯羊15只,正常饲喂20 d后采集瘤胃液样品,提取样品总DNA后对细菌16S rDNA序列的V4区进行PCR扩增,利用Illumina MiSeq PE300平台对扩增子进行高通量测序,并采用QIIME等软件对测序序列进行生物信息学分析。结果表明:15个样本共获得高质量有效序列51 762条,基于97%相似性共归为851个操作分类单位(OTU);α-多样性指数表明Chao1、Ace指数分别为509.39,502.40,Shannon、Simpson指数分别为6.23,0.958 2;经过物种注释和统计,15个门、24个纲、24个目、34个科以及87个属被鉴定;拟杆菌门(Bacteroidetes,66.94%)、厚壁菌门(Firmicutes,23.41%)、柔膜菌门(Tenericutes,3.19%)为优势门,拟杆菌纲(Bacteroidia,66.09%)、梭菌纲(Clostridia,20.74%)为优势纲,拟杆菌目(Bacteroidales,66.09%)、梭菌目(Clostridiales,20.74%)、厌氧原体目(Anaeroplasmatales,2.75%)为优势目,普雷沃氏菌科(Prevotellaceac,43.88%)、理研菌科(Rikenellaceac,12.33%)、毛螺旋菌科(Lachonospiraceae,10.47%)为优势科,普雷沃代菌属1(Prevotella1,35.37%)、普雷沃氏菌科未分类属(unclassified,17.73%)、理菌属RC9(RikenellaceaeRC9gutgroup,12.00%)为优势菌属。说明湖北黑头羊瘤胃细菌多样性较低,最优势菌门为拟杆菌门,厚壁菌门次之;普雷沃氏菌属1是湖北黑头羊瘤胃中最优势细菌属。

In order to understand the rumen bacterial diversity and community structure of Hubei black-head goat,fifteen black-head sheep(one-year-old)with a weight of(35±2)kg and a permanent rumen fistula were selected.After normal feeding of 20 d,the rumen fluid was collected and the total DNA was extracted for PCR amplification of the bacterial 16 S rDNA gene,and then high-throughput sequencing was performed using the Illumina MiSeq PE300 platform and QIIME software was used for bioinformatics analysis of sequences.The results showed that the valid sequence from 15 samples was classified into 851 operational taxa unit(OTU)according to 97%similarity.Theα-diversity index showed that the Chao1 and Ace indices were 509.39 and 502.40,respectively,and the Shannon and Simpson indices were 6.23 and 0.958 2,respectively.After species annotation and statistics,15 phyla,24 classes,24 orders,34 families,and 87 genera were identified.Bacteroidetes(66.94%),Firmicutes(23.41%)and Tenericutes(3.19%)were superior phyla.Bacteroidia(66.09%)and Clostridia(20.74%)were superior classes.Bacteroidales(66.09%),Clostridiales(20.74%)and Anaeroplasmatales(2.75%)were superior orders.Prevotellaceae(43.88%),Rikenellaceae(12.33%)and Lachnospiraceae(10.47%)were superior families.Preootella1(35.37%),unclassified(17.73%),RikenellaceaeRC9gutgroup(12.00%)were superior genera.These results indicated that the rumen bacterial diversity of Hubei blackhead goat was low.Bacteroidetes was the most superior phylum,followed by Firmicutes.Prevolla was the most dominant bacterial genus in the rumen of Hubei black-head goat.