Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): The objective of this research is to perform genome-wide analysis of several members of the Oriental fruit fly species complex with focus on: � Identification of gene regions for use in species-level diagnosis based on these genomes � Identification of gene regions that demonstrate poor performance for diagnostics (and should not be included in screening studies) � Identification of population-level markers for pathway analysis and population structure studies (these markers should be useful across species boundaries and provide markers for various bactrocera and fruit fly species) � Comparative phylogenomics analysis of the included species to direct future studies and test species boundaries Approach (from AD-416): Comparative genomic analysis within the dorsalis complex will be performed by using pyrosequencing technology to generate several �shallow� (low coverage) genomes from Bactrocera species and analyze them using bioinformatic tools. Using five-ten fruit fly specimens representing Bactrocera species in the dorsalis complex our team will generate genomic databases for each specimen using a 454 pyrosequencer operated at the University of Hawaii. This genomic work will be designed and supervised by a PBARC research entomologist who is currently in charge of annotating the completed Bactrocera dorsalis genome. The species included for genomic analysis will be selected based on similarity to B. dorsalis, economic significance, and value to SIT programs. The data generated using 454 technology will be edited, annotated and analyzed by CPHST and ARS staff. Documents Reimbursable with USDA-APHIS-CPHST. Log 42092. Formerly 5320-22430-023-04R; 6/2011. The goal of this research is to perform genome-wide analysis of several members of the oriental fruit fly species complex which contributes to objective 5 of the in-house project, "Develop basic understanding of the oriental fruit fly genome, annotation of functional proteins that regulate development, metabolism, sensory reception and sex determination". From high throughput sequencing of approximately 48 populations of Bactrocera dorsalis performed in the previous year, including samples from throughout South East Asia, as well as collections from the four major Hawaiian Islands, single nucleotide polymorphism (SNP) markers that can be used to discriminate Hawaiian populations from flies of Southeast Asian orgin were found. From this collection of SNPs (105 high quality SNPs), a subset are being used to create a SNP based diagnostic tool for USDA Animal and Plant Health Inspection Service (APHIS) to use to determine source of flies. In addition, there are many more markers that appear to be informative to identify populations of B. dorsalis within Southeast Asia, but have not been utilized in a SNP assay.
Impacts
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): The objective of this research is to perform genome-wide analysis of several members of the Oriental fruit fly species complex with focus on: � Identification of gene regions for use in species-level diagnosis based on these genomes � Identification of gene regions that demonstrate poor performance for diagnostics (and should not be included in screening studies) � Identification of population-level markers for pathway analysis and population structure studies (these markers should be useful across species boundaries and provide markers for various bactrocera and fruit fly species) � Comparative phylogenomics analysis of the included species to direct future studies and test species boundaries Approach (from AD-416): Comparative genomic analysis within the dorsalis complex will be performed by using pyrosequencing technology to generate several �shallow� (low coverage) genomes from Bactrocera species and analyze them using bioinformatic tools. Using five-ten fruit fly specimens representing Bactrocera species in the dorsalis complex our team will generate genomic databases for each specimen using a 454 pyrosequencer operated at the University of Hawaii. This genomic work will be designed and supervised by a PBARC research entomologist who is currently in charge of annotating the completed Bactrocera dorsalis genome. The species included for genomic analysis will be selected based on similarity to B. dorsalis, economic significance, and value to SIT programs. The data generated using 454 technology will be edited, annotated and analyzed by CPHST and ARS staff. The goal of this research is to perform genome-wide analysis of several members of the Oriental fruit fly species complex which contributes to Objective 5 of the in-house project. High throughput sequencing of approximately 48 populations of Bactrocera dorsalis have been completed. This includes samples from throughout South East Asia, as well as collections from the four major Hawaiian Islands through additional collecting that was completed this fiscal year. DNA extractions, library preparation, barcoding and sequencing was completed. Initial analysis of the data reveals about 20,000 loci that appear to be informative to identify populations of B. dorsalis, but more detailed analysis still needs to be completed. All of the data is loaded on a mysql database to facilitate data sharing and interactions. Throughout the year, communication between USDA-ARS, USDA-APHIS, and collaborators providing samples for sequencing have been ongoing through email communication and teleconferencing to manage progress and funding availability. This includes the arrangement of samples being sent from IAEA to USDA-ARS through the organization of USDA-APHIS. In addition, this project was discussed in detail at a workshop held at USDA-APHIS- CPHST, Edinburg, Texas, in January 2012.
Impacts
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) The objective of this research is to perform genome-wide analysis of several members of the Oriental fruit fly species complex with focus on: � Identification of gene regions for use in species-level diagnosis based on these genomes � Identification of gene regions that demonstrate poor performance for diagnostics (and should not be included in screening studies) � Identification of population-level markers for pathway analysis and population structure studies (these markers should be useful across species boundaries and provide markers for various bactrocera and fruit fly species) � Comparative phylogenomics analysis of the included species to direct future studies and test species boundaries Approach (from AD-416) Comparative genomic analysis within the dorsalis complex will be performed by using pyrosequencing technology to generate several �shallow� (low coverage) genomes from Bactrocera species and analyze them using bioinformatic tools. Using five-ten fruit fly specimens representing Bactrocera species in the dorsalis complex our team will generate genomic databases for each specimen using a 454 pyrosequencer operated at the University of Hawaii. This genomic work will be designed and supervised by a PBARC research entomologist who is currently in charge of annotating the completed Bactrocera dorsalis genome. The species included for genomic analysis will be selected based on similarity to B. dorsalis, economic significance, and value to SIT programs. The data generated using 454 technology will be edited, annotated and analyzed by CPHST and ARS staff. The methodology for preparing high throughput DNA sequencing libraries for RAD-Tag (restriction site associated DNA tag) sequencing was developed at USDA-ARS-PBARC. This included development of methodologies for extracting high quality DNA from preserved specimens, developing methodologies for library sheering and size selection and developing barcoding methods to allow multiplexing of multiple samples of a single sequencing run. Initial sequencing libraries were prepared from 6 IAEA laboratory strains and 2 USDA-ARS laboratory strains as a proof of concept demonstration of the methodology with 4 samples barcoded on each of 2 sequencing runs. These samples are currently being sequenced on an Illumnia GAIIx high throughput sequencer and results are pending. Additionally, over 30 distinct populations of B. dorsalis are being made available to this project for RAD-Tag sequencing from a range of geographic locations in Southeast Asia and the Hawaiian Islands. Once confirmation of the proof of concept data is verified, these libraries will be sequenced. To facilitate the analysis of the large amount of data that will be produced in this project, a database has been initiated to house genomic information and can be accessed in house at USDA-ARS-PBARC as well as can be made available to collaborators at www.bactrobase.org. Throughout the year, communication between USDA-ARS, USDA-APHIS, and collaborators providing samples for sequencing have been ongoing through email communication and teleconferencing. This includes the arrangement of samples being sent from IAEA to USDA-ARS through the organization of USDA-APHIS.
Impacts
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The objective of this research is to perform genome-wide analysis of several members of the Oriental fruit fly species complex with focus on: � Identification of gene regions for use in species-level diagnosis based on these genomes � Identification of gene regions that demonstrate poor performance for diagnostics (and should not be included in screening studies) � Identification of population-level markers for pathway analysis and population structure studies (these markers should be useful across species boundaries and provide markers for various bactrocera and fruit fly species) � Comparative phylogenomics analysis of the included species to direct future studies and test species boundaries Approach (from AD-416) Comparative genomic analysis within the dorsalis complex will be performed by using pyrosequencing technology to generate several �shallow� (low coverage) genomes from Bactrocera species and analyze them using bioinformatic tools. Using five-ten fruit fly specimens representing Bactrocera species in the dorsalis complex our team will generate genomic databases for each specimen using a 454 pyrosequencer operated at the University of Hawaii. This genomic work will be designed and supervised by Dr. Scott Geib who is currently in charge of annotating the completed Bactrocera dorsalis genome. The species included for genomic analysis will be selected based on similarity to B. dorsalis, economic significance, and value to SIT programs. The data generated using 454 technology will be edited, annotated and analyzed by CPHST and ARS staff. Documents Reimbursable with USDA-APHIS-CPHST. Log 42092 Initial plans were discussed between APHIS-CPHST and PBARC on the specific approaches to use for genome wide sequencing of members of the Bactrocera dorsalis complex. A reduced representation restriction site associated DNA marker sequencing (RAD sequencing) approach is being taken and methods for sample prep are currently being developed at PBARC. Samples from multiple locations in East Asia were selected for analysis (B. dorsalis, B. papayae, B. phillipinensis, and B. carambolae) and are planned to be sent to PBARC for analysis. Access of up to 264 fly samples within this complex is being made available to the project by Karen Armstrong at Lincoln University, Canterbury, New Zealand. Funds were transferred to University of Hawaii Manoa Sequencing Facility to provide sequencing services for this project on a 454 pyrosequencer and Illumnia GAIIx over the next year.
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