Project Number: 6204-21000-009-03
Project Type: Specific Cooperative Agreement

Start Date: Jun 01, 2003
End Date: May 31, 2008

Objective:
The objective of this cooperative research project is to generate sequence coverage to assemble the genome of the important honey bee pathogen Paenibacillus larvae subsp. larvae. The gram-positive bacterium, Paenibacillus larvae subsp. larvae, causes the most devastating larval honey bee disease, American Foulbrood (AFB). The second objective of this research project is to generate sequence coverage to assemble the genome of the important honey bee fungal pathogen Ascosphaera apis. A. apis causes another devastating honey bee disease, Chalkbrood. Genetic sequences for P. l. larve and A. apis would provide the first opportunity for interactive whole genome analysis of the insect host and its natural bacterial and fungal pathogens. This amendment involves the sequencing of three plasmids from P. larvae subsp. larvae, which are estimated collectively to have a length of roughly 200 kb.

Approach:
The strategy developed at BCM-HGSC will aim for 10X coverage of the ~5 MB P.l.larvae genome. A short-insert plasmid library (1-3 Kb, randomly sheared) will be constructed from a virulent isolate of the bacterial pathogen collected from a single honey bee colony with severe AFB symptoms (isolate #230010, Berkeley, CA). Other libraries will be made to more precise specifications for the P. larvae genome project. A total of 120,000 sequence reads are predicted. In addition to the construction of the draft DNA sequence, initial genome annotation and publication of the P.l.larvae genome are expected. The same strategy will be implemented to generate 5.5X coverage of the ~(22-24) MB A. apis genome. Virulent isolates of the fungal pathogen were collected from a single honey bee larvae infected with the Chalkbrood. A short-insert plasmid library (1-3 kb, randomly sheared) will be constructed from a single mating type of the fungus, isolated from the original culture. The second plasmid library will be constructed to generate 2X coverage of the genome isolated from the second fungal mating type. An additional plasmid library would be constructed to generate 1X coverage of the P. larvae native plasmid DNA.