Summary auto-generated
This study investigated intron polymorphism in the small subunit (SSU) rDNA of Nectria galligena, a fungal pathogen causing European canker of apple. PCR amplification of SSU rDNA from 40 isolates revealed four length polymorphisms. Sequence analysis identified three distinct introns (363 bp, 1185 bp, and 1423 bp) inserted at the NS7 priming site, while a fourth category lacked an intron entirely. These introns do not exhibit characteristic features of typical group I introns, lacking conserved core elements and standard secondary structures. Nucleotide sequence homology suggests the three introns share a common lineage, likely originating through sequential deletions from an ancestral intron. PCR-RFLP analysis using four restriction enzymes grouped isolates into four categories similar to EcoRI RFLP patterns. The researchers developed N. galligena-specific primers (NS7 UP and ChIntrev) that amplify a product encompassing the intron region, enabling simultaneous confirmation of pathogen identity and 18S category determination in a single reaction. This molecular marker system provides a non-isotopic approach for rapidly detecting variation in N. galligena and potentially tracing infection sources in apple orchards.
Key findings
- Four distinct SSU rDNA length polymorphisms identified in N. galligena isolates, caused by introns of 363 bp, 1185 bp, 1423 bp, or absence of intron at the NS7 priming site
- The three introns lack characteristic group I intron features but show high sequence homology suggesting evolution through deletions from a common ancestral intron
- PCR-RFLP analysis divided isolates into four categories correlating with intron types, with geographic differences observed between English and Northern Irish isolates
- Specific PCR primers (NS7 UP and ChIntrev) amplify N. galligena-specific products that reveal both pathogen identity and 18S category in a single reaction
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Abstract
PCR amplification of the small subunit (SSU) rDNA gene of 40 isolates of Nectria galligena revealed four length polymorphisms. PCR-RFLP analysis of the SSU rDNA gene divided the isolates into four categories similar, but not identical, to categories identified by Southern-RFLP analysis. Nucleotide sequence analysis revealed that isolates in three of the four SSU rDNA (18S) categories possess an intron of 363 bp, 1185 bp or 1423 bp at the NS 7 priming site. Isolates in the fourth category do not possess an intron. The nucleotide sequences of these introns did not contain the core elements characteristic of typical group I introns, nor did they exhibit a group I intron secondary structure. Homology between the introns indicates a common lineage, all three possibly having come from a larger intron and having been formed by subsequent deletions. PCR primers upstream of the SSU rDNA intron region and from within the internal transcribed spacer 1 region amplify a product specific to N. galligena, which will confirm the identity of the pathogen and reveal its 18S category in a single reaction.