Taxon-specific oligonucleotide primers for detection of Glomus etunicatum

详细信息    查看全文

• 作者：P. D. Millner ; W. W. Mulbry and S. L. Reynolds
• 关键词：Keywords  ; Arbuscular mycorrhizal fungi ; Internal transcribed spacer ; Ribosomal DNA ; Polymerase chain reaction primers ; Phylogenetic tree
• 刊名：Mycorrhiza
• 出版年：2001
• 出版时间：April 2001
• 年：2001
• 卷：10
• 期：6
• 页码：259-265
• 全文大小：301 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Microbiology
  Plant Sciences
  Ecology
  Agriculture
  Forestry
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-1890

文摘

The 5.8S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus etunicatum MD107, MD127, TN101, and FL329 were amplified by polymerase chain reaction (PCR) using ITS1Kpn and ITS4Pst as primers. The amplification products (597, 599, 598, and 613 bp, respectively) were cloned and sequenced. The similarity among ITS region sequences from MD107, MD127, and TN101 was 99%, whereas the sequence similarity between the ITS regions of these three DNAs and that from FL329 was 91%. The 5.8S rDNA sequences of all four G. etunicatum isolates were identical. In contrast, major dissimilarities in the corresponding rDNA sequence regions of other glomalean taxa were observed. Oligonucleotide sequences unique to G. etunicatum were tested for their specificity in PCR amplification of genomic DNA from spores of 55 isolates comprising 29 glomalean fungi: 18 isolates of G. etunicatum, five G. intraradices, three G. claroideum, 16 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The G. etunicatum isolates were from a broad range of geographic regions and soils. The oligonucleotide pair GETU1:GETU2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all G. etunicatum. This primer pair did not prime PCR when template consisted of DNA from any of the other glomalean fungi or any of the non-mycorrhizal controls, including roots of corn (Zea mays). In addition, the pair successfully detected G. etunicatum in nested PCR using a primary PCR product amplified from highly diluted extracts of colonized corn roots using modified ITS1:ITS4 primers. In the phylogenetic analysis of Glomus 5.8S and ITS2 rDNA region sequences, which included 500 bootstrap data sets, confidence in the G. etunicatum branch was very strong (90%) and clearly independent of G. claroideum and G. intraradices, to which it is very closely related.