Progress 05/01/04 to 04/30/09
Outputs
Progress Report Objectives (from AD-416) To establish a database of Boophilus microplus ESTs, and a gene index consisting of putatively identified ESTs and full-length sequenced cDNAs; and to obtain basic information on the structure of the B. microplus genome, through sequencing of genomic library and BAC library clones. Approach (from AD-416) Establish a collection of expressed sequence tags (ESTs) and a gene index of ESTs and sequenced cDNAs. A normalized B. microplus cDNA library is available from ARS and at least 40,000 clones will be sequenced using both forward and reverse sequencing primers. Obtain basic information on the structure of the B. microplus genome, establishing whether B. microplus genes are arranged using long or short interspersed patterns and obtaining indications of the general sizes of introns. A B. microplus BAC library is to be synthesized and probed by ARS, and selected BAC clones will be sequenced by TIGR. Additionally, small and medium insert sized B. microplus genomic libraries will be synthesized at TIGR using ARS-supplied genomic DNA and sequenced at TIGR by shotgun DNA sequencing methodology. Significant Activities that Support Special Target Populations The "high Cot" fraction of highly purified tick genomic DNA was used for 454 pyrosequencing. This fraction of genomic DNA contains the gene coding regions of the genome and is largely void of the highly repetitive genomic DNA regions which are very difficult to sequence and assemble. The 454 sequencing protocol returned 2.7 million sequencing reads (average read length of 237 bp) from the "high Cot" DNA and was analyzed by bioinformatic analysis to identify gene coding regions. A manuscript has been written and is undergoing peer review under ARS and JCVI procedures. The ADODR monitored progress of the project through emails and teleconferences with personnel of the JCVI.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) To establish a database of Boophilus microplus ESTs, and a gene index consisting of putatively identified ESTs and full-length sequenced cDNAs; and to obtain basic information on the structure of the B. microplus genome, through sequencing of genomic library and BAC library clones. Approach (from AD-416) Establish a collection of expressed sequence tags (ESTs) and a gene index of ESTs and sequenced cDNAs. A normalized B. microplus cDNA library is available from ARS and at least 40,000 clones will be sequenced using both forward and reverse sequencing primers. Obtain basic information on the structure of the B. microplus genome, establishing whether B. microplus genes are arranged using long or short interspersed patterns and obtaining indications of the general sizes of introns. A B. microplus BAC library is to be synthesized and probed by ARS, and selected BAC clones will be sequenced by TIGR. Additionally, small and medium insert sized B. microplus genomic libraries will be synthesized at TIGR using ARS-supplied genomic DNA and sequenced at TIGR by shotgun DNA sequencing methodology. Significant Activities that Support Special Target Populations This report documents research conducted under a grant agreement between ARS and J. Craig Venter Intitute. Additional details of research can be found in the report for the in-house associated project CRIS 6205-32000- 026-00D, Molecular Biology of Boophilus microplus. Extremely high quality genomic DNA has been prepared from R. microplus eggs and the "high Cot" fraction purified and prepared for Roche 454 sequence analysis. This fraction of genomic DNA contains the gene coding regions of the genome and is largely void of the highly repetitive genomic DNA regions that are very difficult to sequence and assemble. The 454 sequencing protocol is a cutting edge protocol that will return several million sequencing reads from the "high Cot" DNA and will greatly expand the available sequence from protein coding and gene promoter regions in the R. microplus genome. The technique of serial expression of gene expression (SAGE) was used to examine differential gene expression induced by infection with Babesia bovis, the causative agent of cattle fever. The identification of up- and down-regulated infection-associated genes should assist in identifying those that are critical in the infection and transmission process in the ovary. The ADODR monitors project activities by phone and conference calls, site visits, and meetings.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under an assistance- type cooperative agreement between ARS and The Institute for Genomic Research (TIGR). Additional details of research can be found in the report for the parent CRIS 6205-32000-026-00D, Molecular Biology of Boophilus microplus. Approximately 4000 expressed gene sequences have been obtained from a subtracted cDNA library synthesized from dissected guts and ovaries of B. microplus females feeding upon a calf infected with Babesia bovis, the causative agent of cattle fever. These sequences should assist in identifying genes that are critical in the infection process in the critical gut and ovary tissues. A BAC clone encoding CzEST9, a pyrethroid-degrading metabolic esterase, has been completed and a manuscript covering this research is currently in the ARS peer review process.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under an assistance- type cooperative agreement between ARS and The Institute for Genomic Research (TIGR). Additional details of research can be found in the report for the parent project, 6205-32000-026-00D, Molecular Biology of Boophilus microplus. Approximately 45,000 DNAs from expressed B. microplus genes have been sequenced and assembled into a Gene Index of approximately 13,000 coding regions covering an estimated 35-60% of the tick's genes. Additionally, approximately 1,000 expressed gene sequences have been obtained from a gene library synthesized from B. microplus larvae infected with Babesia bovis, the causative agent of cattle fever. These sequences should assist in identifying genes that are critical in the tick-pathogen interaction allowing disease transmission. Sequencing of genomic DNA clones has begun and is confirming that a high amount of repetitive DNA exists in the tick
genome. The work with the Gene Index has been documented in the first of several anticipated publications.
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? D. Progress Report This report serves to document research conducted under an assistance- type cooperative agreement between ARS and The Institute of Genomic Research (TIGR). Additional details of research can be found in the report for the parent CRIS 6205-32000-024-00D, Detection and Management of Pesticide Resistance in Populations of Boophilus microplus and the Horn Fly. The availability of a B. microplus genomic and expressed gene database would maximize the capability of modern molecular approaches to advance tick studies, both basic and applied. We propose to: 1) Establish a database of B. microplus expressed sequence tags (ESTs) and a gene index consisting of annotated cDNA sequences. 2) Obtain basic information on the structure of the B. microplus genome, through sequencing of genomic library and BAC library clones. This is a new project, and there is no activity to report as yet.
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