Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

doi:10.1016/S2095-3119(13)60529-8

Journal of Integrative Agriculture ›› 2014, Vol. 13 ›› Issue (6): 1340-1348.DOI: 10.1016/S2095-3119(13)60529-8

Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

SU Yong, Hauke Smidt , ZHU Wei-Yun

1、Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095,
P.R.China
2、Laboratory of Microbiology, Agrotechnology and Food Sciences Group, Wageningen University, Wageningen 6703 HB, The Netherlands

收稿日期:2013-01-17 出版日期:2014-06-01 发布日期:2014-06-06
通讯作者: ZHU Wei-yun, Tel: +86-25-84395523, Fax: +86-25-84395314, E-mail: zhuweiyun@njau.edu.cn
基金资助:
Basic Research Program of China (2012CB124705, 2013CB127603), the European Community’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement n° 227549, the Royal Dutch Academy of Sciences (KNAW) through Project 09CDP006, and the Fundamental Research Funds for the Central Universities, China (KYZ201153).

Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

SU Yong, Hauke Smidt , ZHU Wei-Yun

1、Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095,
P.R.China
2、Laboratory of Microbiology, Agrotechnology and Food Sciences Group, Wageningen University, Wageningen 6703 HB, The Netherlands

Received:2013-01-17 Online:2014-06-01 Published:2014-06-06
Contact: ZHU Wei-yun, Tel: +86-25-84395523, Fax: +86-25-84395314, E-mail: zhuweiyun@njau.edu.cn
Supported by:
Basic Research Program of China (2012CB124705, 2013CB127603), the European Community’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement n° 227549, the Royal Dutch Academy of Sciences (KNAW) through Project 09CDP006, and the Fundamental Research Funds for the Central Universities, China (KYZ201153).

摘要/Abstract

摘要： Erhualian and Landrace breeds are typical genetically obese and lean pigs, respectively. To compare the fecal methanogenic Archaeal community between these two pig breeds, fecal samples from different growth phase pigs were collected and used for PCR-denaturing gradient gel electrophoresis (DGGE) with two primer pairs (344fGC/519r and 519f/915rGC) and real-time PCR analysis. Results showed that a better separation and higher quality of bands pattern were obtained in DGGE profiles using primers 344fGC/519r as compared with primers 519f/915rGC. Sequencing of DGGE bands showed that the predominant methanogens in the feces of Erhualian and Landrace pigs belonged to Methanobrevibacter spp. and Methanosphaera spp. Real-time PCR analysis revealed that there was no significant difference in the numbers of fecal total methanogens between Erhualian and Landrace pigs; however, pig growth phase affected the numbers of 16S rRNA genes of total methanogens and Methanobrevibacter smithii. Dissociation curves of methyl coenzyme-M reductase subunit A (mcrA) gene fragments amplified with real-time PCR showed all samples possessed a single peak at 82°C, which might be associated with M. smithii. Samples from the same growth phase of each breed showed good replicative dissociation curves. The results suggest that the growth phase (including diet factor) other than genotype of pig may affect the fecal methanogenic Archaeal community of pigs.

关键词: methanogenic Archaea , Erhualian pig , Landrace pig , Methanobrevibacter smithii

Abstract: Erhualian and Landrace breeds are typical genetically obese and lean pigs, respectively. To compare the fecal methanogenic Archaeal community between these two pig breeds, fecal samples from different growth phase pigs were collected and used for PCR-denaturing gradient gel electrophoresis (DGGE) with two primer pairs (344fGC/519r and 519f/915rGC) and real-time PCR analysis. Results showed that a better separation and higher quality of bands pattern were obtained in DGGE profiles using primers 344fGC/519r as compared with primers 519f/915rGC. Sequencing of DGGE bands showed that the predominant methanogens in the feces of Erhualian and Landrace pigs belonged to Methanobrevibacter spp. and Methanosphaera spp. Real-time PCR analysis revealed that there was no significant difference in the numbers of fecal total methanogens between Erhualian and Landrace pigs; however, pig growth phase affected the numbers of 16S rRNA genes of total methanogens and Methanobrevibacter smithii. Dissociation curves of methyl coenzyme-M reductase subunit A (mcrA) gene fragments amplified with real-time PCR showed all samples possessed a single peak at 82°C, which might be associated with M. smithii. Samples from the same growth phase of each breed showed good replicative dissociation curves. The results suggest that the growth phase (including diet factor) other than genotype of pig may affect the fecal methanogenic Archaeal community of pigs.

Key words: methanogenic Archaea , Erhualian pig , Landrace pig , Methanobrevibacter smithii

SU Yong, Hauke Smidt , ZHU Wei-Yun. Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis[J]. Journal of Integrative Agriculture, 2014, 13(6): 1340-1348.

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Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

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