Application of a new PCR primer for terminal restriction fragment length polymorphism analysis of the bacterial communities in plant roots.
Contamination with plastid small subunit (SSU) rDNA is a major drawback when analyzing the bacterial communities of plant roots using culture-independent methods. In this study, a polymerase chain reaction (PCR) primer, 783r, was designed and tested to specifically amplify the SSU rDNA of various bacterial species without amplifying the SSU rDNA of plant plastids. To confirm how useful the community analysis of rhizobacteria is using 783r, the terminal restriction fragment length polymorphism (T-RFLP) method was performed with wheat (Triticum aestivum) and spinach (Spinacea oleracea) root samples. Using the standard T-RFLP method, a large T-RF peak of plant plastid SSU rDNA interfered with the bacterial community analysis. In contrast, the T-RFLP method using the 783r primer was able to detect the bacterial DNA while directly eliminating the influence of the plant-derived DNA extracted from the plant roots. Primer 783r might, therefore, be a useful PCR primer for the culture-independent analysis of bacterial communities in plant roots using SSU rDNA.
Sakai
Masao
M
Department of Plant Resources, Faculty of Agriculture, Kyushu University, 6-10-1-Hakozaki, Higashi, Fukuoka 812-8581, Japan. msakai@agr.kyushu-u.ac.jp
Matsuka
Akira
A
Komura
Taichi
T
Kanazawa
Shinjiro
S
D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D018517:Plant Roots / Q000382:microbiology*; D018087:Plastids / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012231:Rhizobium / Q000235:genetics*; D018724:Spinacia oleracea; D014908:Triticum / Q000382:microbiology
D017931:DNA Primers; D004269:DNA, Bacterial; D004272:DNA, Mitochondrial