浙江农业学报 ›› 2022, Vol. 34 ›› Issue (7): 1377-1385.DOI: 10.3969/j.issn.1004-1524.2022.07.04
吕倩1,2(), 骆巧1, 罗雪1, 陈久兵1, 马莉1, 罗正中2, 姚学萍1, 余树民1, 沈留红1, 曹随忠1,*()
收稿日期:
2020-08-26
出版日期:
2022-07-25
发布日期:
2022-07-26
通讯作者:
曹随忠
作者简介:
* 曹随忠,E-mail: suizhongcao@sicau.edu.cn基金资助:
LYU Qian1,2(), LUO Qiao1, LUO Xue1, CHEN Jiubing1, MA Li1, LUO Zhengzhong2, YAO Xueping1, YU Shumin1, SHEN Liuhong1, CAO Suizhong1,*()
Received:
2020-08-26
Online:
2022-07-25
Published:
2022-07-26
Contact:
CAO Suizhong
摘要:
为探究奶牛场沙土垫层和橡胶垫层菌群特征及差异,分别采集四川省两个规模化奶牛场的沙土垫层样本和橡胶垫层样本各6份,利用高通量测序技术对16S rRNA基因的V4区进行测序和生物信息学分析。结果显示,两个奶牛场的卧床垫层12个样本共获得1 443 818条优化序列(clean reads),将相似度为97%的有效序列进行操作分类单元(operational taxonomic units,OTUs)聚类,共得到2 311个OTUs。在属分类水平上,沙土垫层优势菌属为不动杆菌属(Acinetobacter)、海杆菌属(Marinobacter)、里氏杆菌属(Riemerella)、棒状杆菌属(Corynebacterium)、节杆菌属(Arthrobacter);橡胶垫层优势菌属为棒状杆菌属(Corynebacterium)、藤黄单胞菌属(Luteimonas)、梭杆菌属(Fusobacterium)、盐单胞菌属(Halomonas)、弧菌属(Vibrio)。沙土垫层样本中不动杆菌属(Acinetobacter)、里氏杆菌属(Riemerella)的相对丰度显著高于橡胶垫层样本(P<0.05),橡胶垫层样本中梭杆菌属(Fusobacterium)的相对丰度显著高于沙土垫层样本(P<0.05)。Chao指数、Ace指数、Shannon指数、Simpson指数在两组之间差异均不显著(P>0.05)。结果表明,沙土样本间的菌群差异大于橡胶垫层样本,无论是沙土垫层还是橡胶垫层,均存在环境条件致病菌。
中图分类号:
吕倩, 骆巧, 罗雪, 陈久兵, 马莉, 罗正中, 姚学萍, 余树民, 沈留红, 曹随忠. 基于高通量测序技术分析奶牛场垫沙和橡胶垫卧床中的菌群差异[J]. 浙江农业学报, 2022, 34(7): 1377-1385.
LYU Qian, LUO Qiao, LUO Xue, CHEN Jiubing, MA Li, LUO Zhengzhong, YAO Xueping, YU Shumin, SHEN Liuhong, CAO Suizhong. Analysis of microbial community difference between sand and rubber bedding in dairy farm by high throughput sequencing technology[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1377-1385.
垫料选择标准Bedding selection standard | 风干河沙Sand bedding | 橡胶垫Rubber bedding |
---|---|---|
水分Moisture | ≤5% | — |
含土率Soil content | ≤5% | — |
杂质Impurities | ≤5% | — |
厚度Thickness | ≥15 cm | 上层为1 cm橡胶,下层为3~4 cm海绵The upper layer is 1 cm rubber and the lower layer is 3-4 cm sponge |
清理频次Cleaning frequency | 每天2次Twice per day | 每天2次Twice per day |
表1 卧床垫层选择标准及清洁标准
Table 1 Bedding selection standard and cleaning standard
垫料选择标准Bedding selection standard | 风干河沙Sand bedding | 橡胶垫Rubber bedding |
---|---|---|
水分Moisture | ≤5% | — |
含土率Soil content | ≤5% | — |
杂质Impurities | ≤5% | — |
厚度Thickness | ≥15 cm | 上层为1 cm橡胶,下层为3~4 cm海绵The upper layer is 1 cm rubber and the lower layer is 3-4 cm sponge |
清理频次Cleaning frequency | 每天2次Twice per day | 每天2次Twice per day |
月份 Month | 平均温度Average temperature/ ℃ | 平均湿度Average humidity/% | ||
---|---|---|---|---|
F1 | F2 | F1 | F2 | |
7月July | 25.2±1.8 | 26.0±1.7 | 80.8±3.1 | 80.1±2.1 |
8月August | 26.2±2.1 | 27.4±2.1 | 78.9±3.4 | 81.0±1.6 |
9月September | 21.3±1.5 | 21.7±1.9 | 76.0±2.9 | 81.5±2.4 |
表2 两奶牛场7—9月平均温度和湿度
Table 2 Average temperature and humidity from July to September in two dairy farms
月份 Month | 平均温度Average temperature/ ℃ | 平均湿度Average humidity/% | ||
---|---|---|---|---|
F1 | F2 | F1 | F2 | |
7月July | 25.2±1.8 | 26.0±1.7 | 80.8±3.1 | 80.1±2.1 |
8月August | 26.2±2.1 | 27.4±2.1 | 78.9±3.4 | 81.0±1.6 |
9月September | 21.3±1.5 | 21.7±1.9 | 76.0±2.9 | 81.5±2.4 |
图3 属水平菌群组成柱状图 横坐标是样本名称(F1、F2代表场1和场2),纵坐标是注释到的物种相对丰度。该分类水平未注释到的合并为Unclassified,丰度在所有样本均低于0.5%的物种全部合并成其他。
Fig.3 Composition of microbial community at genus level The abscissa is the sample name (F1 and F2 represent field 1 and field 2), and the ordinate is the relative abundance of the annotated species. The classification level is that the annotations are merged into Unclassified, and the species whose abundance is less than 0.5% in all samples merged into Others.
图4 关键物种差异比较柱状图 横坐标是丰度为前10的物种,纵坐标是其相对丰度,*代表差异检验的显著性(如P<0.001,则用“***”标记;如0.001≤P<0.01,则用“**”标记;如0.01≤P≤0.05,则用“*”标记;如果P>0.05,则不标记)。
Fig.4 Histogram of key species differences The abscissa is the species of the top 10 abundances, the ordinate is the relative abundance, and * represents the significance of the difference test (if P<0.001, marked by “***; for example, 0.001≤P<0.01, marked by “**”; 0.01≤P≤0.05, marked by “*”;P>0.05, no mark.
图5 Alpha多样性盒型图 由下到上的五条线分别为:最小值,第一个四分位数,中位数,第三个四分位数和最大值。异常值以“。”表示。
Fig.5 Alpha diversity estimates of the bacterial communities The five lines from bottom to top are: minimum, first quartile, median, third quartile and maximum. Outliers are represented by “。”.
图6 Alpha多样性稀释曲线图 横坐标代表测序数据量,纵坐标代表Chao指数对应的Alpha多样性指标。
Fig.6 Alpha diversity dilution curve The abscissa represents the amount of sequencing data, and the ordinate represents the Alpha diversity index corresponding to the Chao index.
图7 β多样性热图 指数越大,样本间差异越大。进化树距离越近,样本相似性越大。
Fig.7 Heatmap of β diversity The larger the index, the greater the difference between samples. The closer the evolutionary tree, the greater the similarity of the samples.
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