Evaluating the physical capture method of terminal restriction fragment length polymorphism for comparison of soil microbial communities

被引:18
作者
Blackwood, Christopher B. [1 ]
Buyer, Jeffrey S. [1 ]
机构
[1] USDA ARS, Beltsville Agr Res Ctr, Sustainable Agr Syst Lab, Beltsville, MD 20705 USA
关键词
terminal restriction fragment length polymorphism (T-RFLP); soil microorganisms; molecular community analysis; soil bacteria; soil fungi;
D O I
10.1016/j.soilbio.2006.09.010
中图分类号
S15 [土壤学];
学科分类号
0903 ; 090301 ;
摘要
Terminal restriction fragment length polymorphism (T-RFLP) is a popular method of comparative microbial community. analysis which is normally accomplished by tagging terminal restriction fragments (T-RFs) with a fluorescent primer. Here, we evaluate an alternative method of T-RFLP where T-RFs are physically captured using a biotinylated primer and streptavidin-coated beads. This eliminates one of the primary criticisms of T-RFLP, namely that T-RFs cannot be identified by sequence analysis, and also represents an alternative method for collecting T-RFLP profiles. Microbial communities from forest, agricultural, and turf soils were investigated using several sets of primers specific for different microbial groups. The physical capture method of T-RFLP resulted in similar profiles to those generated by fluorescent T-RFLP. The relationships among ecosystem types captured by both methods and revealed by ordination were virtually identical. The total variance in the profiles that was attributed to ecosystem type was approximately equal, or greater, when generated by the physical capture method, depending on the primers used. However, physical capture T-RFLP resolved fewer T-RFs than fluorescent T-RFLP, and this may reduce the sensitivity to changes in non-dominant populations within the community. Direct cloning and sequencing of physical capture T-RFs revealed that most bands were not comprised of sequences related to those in the database that would generate T-RFs of similar size. T-RFs should therefore be identified by sequencing, rather than by comparing the sizes of T-RFs to computer digests of database sequences. Physical capture T-RFLP should be a useful tool to identify T-RFs by sequencing, and for laboratories without economical access to equipment required to perform fluorescent T-RFLP. (c) 2006 Elsevier Ltd. All rights reserved.
引用
收藏
页码:590 / 599
页数:10
相关论文
共 29 条
[1]  
[Anonymous], MOL MICROBIAL ECOLOG
[2]   Minimal changes in rhizobacterial population structure following root colonization by wild type and transgenic biocontrol strains [J].
Bankhead, SB ;
Landa, BB ;
Lutton, E ;
Weller, DM ;
Gardener, BBM .
FEMS MICROBIOLOGY ECOLOGY, 2004, 49 (02) :307-318
[3]   Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities [J].
Blackwood, CB ;
Marsh, T ;
Kim, SH ;
Paul, EA .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2003, 69 (02) :926-932
[4]   Eubacterial community structure and population size within the soil light fraction, rhizosphere, and heavy fraction of several agricultural systems [J].
Blackwood, CB ;
Paul, EA .
SOIL BIOLOGY & BIOCHEMISTRY, 2003, 35 (09) :1245-1255
[5]   Phylum- and class-specific PCR primers for general microbial community analysis [J].
Blackwood, CB ;
Oaks, A ;
Buyers, JS .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2005, 71 (10) :6193-6198
[6]  
BLACKWOOD CB, 2006, IN PRESS MOL APPROAC
[7]   Reciprocal transfer effects on denitrifying community composition and activity at forest and meadow sites in the Cascade Mountains of Oregon [J].
Boyle, Stephanie A. ;
Rich, Jeremy J. ;
Bottomley, Peter J. ;
Cromack, Kermit, Jr. ;
Myrold, David D. .
SOIL BIOLOGY & BIOCHEMISTRY, 2006, 38 (05) :870-878
[8]   The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis [J].
Cole, JR ;
Chai, B ;
Farris, RJ ;
Wang, Q ;
Kulam, SA ;
McGarrell, DM ;
Garrity, GM ;
Tiedje, JM .
NUCLEIC ACIDS RESEARCH, 2005, 33 :D294-D296
[9]   Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities [J].
Dunbar, J ;
Ticknor, LO ;
Kuske, CR .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2001, 67 (01) :190-197
[10]   Fidelity of select restriction endonucleases in determining microbial diversity by terminal-restriction fragment length polymorphism [J].
Engebretson, JJ ;
Moyer, CL .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2003, 69 (08) :4823-4829