Progress 06/01/07 to 05/31/12
Outputs
OUTPUTS: Activities in the 5th year (including 2 no-cost extensions to the original 3-years): This year has been devoted to continued analysis of earlier results, to publication, and to further experiments. Because of falling sequencing costs during the last two years, current experiments have been directed towards a genome sequencing project for R. pomonella. After an initial 2 lanes of short-read genomic DNA Illumina sequencing were performed on Rhagoletis pomonella, attention was shifted to the close species R. zephyria, which has far less genetic variation, and can thus be assembled more easily. Two lanes of short-read Illumina sequencing, and a 1.5 kb run, were carried out on R. zephyria, producing assemblies of up to 2.1 mb. Currently 5 and 10 kb libraries are being made at the Keck Center at the University of Illinois to allow a complete genome of R. zephyria to be assembled. Once this is complete, R. pomonella will be assembled against R. zephyria. Summary of the entire grant: The initial goal was to begin genomics analysis in the apple maggot (Rhagoletis pomonella), a major apple pest, by determining the DNA sequence of genes controlling attraction to host odors and life cycle timing via a large-scale expressed sequence tag (EST) study with 454 pyrosequencing technology. But because of new sequencing technologies and thus continually falling sequencing costs, a complete genome sequence was initiated, and as well experiments on pinpointing the specific genes involved in life history timing were carried out. In the initial, published 454 EST experiment, a total of three 454 runs at the Keck Center at the University of Illinois at Urbana-Champaign were carried out, with the first two producing a total number 24,073 contigs (continuous DNA sequences assembled from smaller sections that represent a wide variety of genes from the genome of R. pomonella), and the third run, using the new 500 base pair 454 reagents, producing around twice that number. Analysis of the genome data is still in progress, with olfactory receptors being the initial focus. The genome will be completely assembled by 2013. Dissemination: During the course of the award, co-PI Berlocher spoke about the research at the USDA CRI annual researchers meeting, as well as locally. Co-PI Feder gave numerous talks on Rhagoletis evolution and genomics over the course of the grant period. Co-PI Robertson also gave many talks at international conferences on insect genomics. Many of the sequences produced by the grant have already been made available to the research community and public at large via the GenBank Short Read Archive; files containing our raw sequences and quality scores are available for BLAST search at GenBank SRX001885, SRX001531, SRX001121, SRX001530, and SRX001529. The genomic DNA sequences are presently on a server purchased with the grant, and will be release to the public when all assembly is complete. PARTICIPANTS: Professor Stewart H. Berlocher has been the lead PI on the research. Together with co-PIs Robertson and Feder he has planned and executed the research, has analyzed many of the results, and has done writing for the results. Over the course of the grant he has received a summer salary for one or one half person month for most summers. Dr. Dietmar Schwarz was the postdoctoral fellow on the grant this first year, and did much of the research, analysis, and writing on the first paper. Dr. Schwarz left the University of Illinois at Urbana-Champaign at the end of August 2008 for an Assistant Professor position at Western Washington University. Professors H.M. Robertson (University of Illinois) and J.L. Feder (University of Notre Dame) are co-PIs and have been involved in all stages of the research. Neither received any salary from the grant. Kim Walden, technician in Robertson's lab, has received three person months of salary to do carry out DNA preparations and genome assembly work on the grant. Several collaborators and contacts have been instrumental in the research. Associate Professor M. Hudson and post-doc K. Varala of the University of Illinois aided in performing the analysis for Schwarz et al. 2009, on which they were also coauthors. Assistant Professor D. Hahn and his post-doc Dr. G. Ragland of the University of Florida performed some analysis on the 2009 paper, and were also deeply involved in the planning, execution, and writing of the large-scale study of gene expression differences between the apple and hawthorn host races of the apple maggot fly (Ragland et al. 2011). Training and development opportunities were provided for the post-doc, D. Schwarz, and also for masters student Lynn Fenema and PhD student Fred Larabee, who while supported on a different grant, was involved in conversations with grant participants. TARGET AUDIENCES: The research was essentially entirely basic research, and the primary target audience was and is other basic and applied entomological researchers, in particular those working in insect olfaction and insect-plant interactions. However, as the remaining analysis is completed and more information on the genomics of olfaction and diapause control in Rhagoletis is summarized, this information will become disseminated to additional, more applied researchers, such as plant breeders who wish to develop apples that are unattractive to the apple maggot fly. However, the research is also broadly known in biology because Rhagoletis pomonella provides an excellent example of evolution in historical time, and thus is of interest to high school and undergraduate instructors. Throughout the award period co-PI Berlocher co-taught two courses at the University of Illinois, including Rhagoletis in both courses, and participated in the Integrative Biology Honors program. Co-PI Feder is currently director of GLOBES (Global Linkages of Biology, the Environment and Society), an interdisciplinary graduate training program launched in 2005 with funding from an IGERT grant award from the National Science Foundation, and also uses Rhagoletis in his teaching. PROJECT MODIFICATIONS: As described in previous annual reports, one important modification in approach was the use of 454 pyrosequencing instead of the conventional cloning and Sanger sequencing using ABI machines described in the original report. We estimate that by using the new technology we obtained 100 times the total DNA sequence as we would have using the old technology, for the same amount of money. Another important modification was expanding the scope of the grant (with permission from the director of NRI) to include obtaining a complete genome of Rhagoletis pomonella, using Illumina technology. The ability to determine a complete genome for Rhagoletis pomonella from the funding of the original grant is an amazing demonstration of the power of new sequencing technologies, and is clearly a desirable modification of the original grant. Finally, the rapid rate of acquisition of sequences allowed an expression study of genes involved in diapause to be started and partially completed during the grant period.
Impacts
The primary outcome of this research is a great increase in knowledge of the genetics of this key apple pest, knowledge that can potentially be exploited in the future to find better ways to manage this pest. Much of this knowledge has to do with olfactory receptors, which are key elements in how insects find both their natural food plants as well as crops. When our analysis is complete, our research can be generalized to the genetics of phytophagous insects in general. We have thus far found hundreds of genes that have the potential to affect how, why, and when the apple maggot chooses to lay eggs into apples. A number of specific findings have emerged from the research. Perhaps most significantly, a set of genes that precisely determine the onset of development after diapause, a key trait in allowing R. pomonella to emerge earlier in the year and thus infest apple, have been identified (see Ragland et al. 2011). Another significant finding is that introns are much longer in R. pomonella than in Drosophila; the first intron in odorant receptor gene Or40a is approximately 2,000 base pairs, versus 200 in Drosophila. Yet another significant finding is that many of the odorant receptors in R. pomonella can be successfully homologized with those in Drosophila, which will facilitate understanding of the ligands of the Rhagoletis receptors. When the genome is complete and available to the all researchers, it will be a valuable resource in understanding how phytophagous insects adapt to their host plants.
Publications
- Ragland, G.J., Egan, S.P., Feder, J.L., Berlocher, S.H. and Hahn, D.A. 2011. Developmental trajectories of gene expression reveal candidates for diapause termination: A key life-history transition in the apple maggot fly Rhagoletis pomonella. The Journal of Experimental Biology 214: 3948-3959.
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Progress 06/01/10 to 05/31/11
Outputs
OUTPUTS: Activities: The fourth year of the grant has been devoted to continued analysis of the results from the first three years of the grant, to publication, and to further experiments. The initial goal was to begin genomics analysis in the apple maggot (Rhagoletis pomonella), a major apple pest, by determining the DNA sequence of genes controlling attraction to host odors and genes controlling coordination of fly life cycle with apple fruiting time. In the first year of the research, we conducted a large-scale Expressed Sequence Tag (EST) study using 454 pyrosequencing technology. A total of three 454 runs at the Keck Center at the University of Illinois at Urbana-Champaign were eventually carried out, with the first two producing a total number 24,073 contigs (continuous DNA sequences assembled from smaller sections that represent a wide variety of genes from the genome of R. pomonella), and the third run, using the new 500 base pair 454 reagents, producing around twice that number. The results greatly exceeded the goals of the original grant proposal: to date 9 complete or essentially complete and 13 partial olfactory and gustatory receptor genes were recovered, as well as approximately 40 odorant binding protein genes. Many genes involved in diapause were cataloged. The diapause genes we discovered are being analyzed in collaborative work with Dr. Daniel Hahn of the University of Florida. Microarrays are being used to measure expression differences between the apple and hawthorn host races relating to when they emerge from the ground. Finally, because of falling sequencing costs during the last year, we have initiated a genome sequencing project for R. pomonella, which is now well under way. Two lanes of genomic DNA sequencing were performed using Illumina technology, which yielded a total of some 48 x 10^9 base pairs. Assembly of these data is now underway. Initial assembly focused on genes that had been earlier sequenced by Dr. J.L. Feder of Notre Dame University (a co-PI) to calibrate assembly parameters. Current work is focusing on olfactory receptors, with the analog of Or40a of Drosophila melanogaster being the initial focus. One of the findings thus far is that introns are much longer in R. pomonella than in Drosophila; the first intron in the gene is approximately 2,000 base pairs, versus 200 in Drosophila. Greater intron length will complicate the search for genes that control host selection in Rhagoletis. During the 2010-2011 award year I co-taught two courses at the University of Illinois, including Integrative Biology Honors Ecology and Evolution. Products: The primary product of the grant is a very large number of gene sequences. Many of the sequences have been made available to the research community and public at large via the GenBank Short Read Archive; files containing our raw sequences and quality scores are available for BLAST search at GenBank SRX001885 and SRX001531 (heads), SRX001121 (larvae), SRX001530 (bodies minus heads), and SRX001529 (pupae). Dissemination: Results of the last year of the research will be presented at the National Entomology meetings in Reno in November 2011. PARTICIPANTS: Professor Stewart H. Berlocher has been the lead PI on the research. Together with co-PIs Robertson and Feder he has planned and executed the research, has analyzed many of the results, and has done writing for the results. He has received one person month of salary in the 2009-2010 award year. Dr. Dietmar Schwarz was the postdoctoral fellow on the grant this first year, and did much of the research, analysis, and writing on the first paper. Dr. Schwarz left the University of Illinois at Urbana-Champaign at the end of August 2008 for an Assistant Professor position at Western Washington University. Professors H.M. Robertson (University of Illinois) and J.L. Feder (University of Notre Dame) are co-PIs and have been involved in all stages of the research. Neither received any salary from the grant. Several collaborators and contacts have been instrumental in the research. Assistant Professor D. Hahn and his post-doc Dr. G. Ragland of the University of Florida performed some analysis on the BMC Genomics paper, and are also involved in the planning and execution of a large-scale study of gene expression differences between the apple and hawthorn host races of the apple maggot fly. Assistant Professor M. Hudson and post-doc K. Varala of the University of Illinois aided in performing the analysis for the above paper. Training and development opportunities were provided for the post-doc, D. Schwarz, and also for masters student Lynn Fenema and PhD student Fred Larabee, who while supported on a different grant, were involved in conversations with grant participants. TARGET AUDIENCES: The research is 100% basic research, and the direct target audience is other basic researchers, in particular those working in insect olfaction and insect-plant interactions. However, as the work proceeds and basic information on olfaction and diapause control in Rhagoletis is obtained, this information will become disseminated to more basic researchers such as plant breeders who wish to develop apples that are unattractive to the apple maggot fly. PROJECT MODIFICATIONS: As described in the 2007, 2008, and 2009 reports, the main modification in approach has been the use of 454 pyrosequencing instead of the conventional cloning and Sanger sequencing using ABI machines described in the original report. We estimate that by using the new technology we obtained 100 times the total DNA sequence as we would have using the old technology for the same amount of money. The other modification is that we are currently exploiting Illumina technology to allow a complete genome of Rhagoletis pomonella to be sequenced. The ability to determine a complete genome for Rhagoletis pomonella from the funding of the original grant is an amazing demonstration of the power of new sequencing technologies, and is clearly a desirable modification of the original grant.
Impacts
The primary outcome of this research remains a change in knowledge. But it is a profound change in knowledge. This research has produced a huge increase in information of the genetics of the apple maggot, and by extension, about the genetics of insects in general. We have thus far found hundreds of genes that have the potential to affect how the apple maggot chooses to lay eggs into apples. Over the next year, as researchers on plant pests and plant breeders become aware of this work, we expect this research to have significant impacts on both basic and applied research on plant pests. This knowledge can be exploited in the future to provide new and better ways to control this and related pests.
Publications
- No publications reported this period
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Progress 06/01/09 to 05/31/10
Outputs
OUTPUTS: Activities: The third year of the grant has been devoted to continued analysis of the results from the first two years of the grant, to publication, and to further experiments. The initial goal was to begin genomics analysis in the apple maggot (Rhagoletis pomonella), a major apple pest, by determining the DNA sequence of genes controlling attraction to host odors and genes controlling coordination of fly life cycle with apple fruiting time. In the first year of the research, we conducted a large-scale Expressed Sequence Tag (EST) study using 454 pyrosequencing technology. Two 454 runs at the Keck Center at the University of Illinois at Urbana-Champaign produced a total number 24,073 contigs (continuous DNA sequences assembled from smaller sections that represent a wide variety of genes from the genome of R. pomonella). The results greatly exceeded our original goals; 13 olfactory and gustatory receptor genes (including the newly discovered ionotropic receptors) were recovered, as well as 25 odorant binding protein genes. Many genes involved in diapause were cataloged. The diapause genes we discovered are being analyzed in collaborative work with Dr. Daniel Hahn of the University of Florida. Microarrays are being used to measure expression differences between the apple and hawthorn host races relating to when they emerge from the ground. In an additional EST experiment carried out during the 2009-2010 award year, heads of flies of the apple and hawthorn host races collected from two sites in Urbana, IL were analyzed using the new 500 base pair 454 sequencing technology. This experiment produced a very significant increase in amount of information on olfactory genes in R. pomonella over the first, published 454 run, with 9 complete odorant receptor coding sequences recovered, as well as a large number of partial sequences that are still being analyzed. Finally, because of falling sequencing costs during the last year, we have initiated a genome sequencing project for R. pomonella, which is now in progress. This enormous increase in genetic information is a bonus produced by improved technology at greatly reduced cost. During the 2009-2010 award year I co-taught two courses at the University of Illinois, including Integrative Biology Honors Ecology and Evolution. During the last year I employed a graduate student, Fred Larabee, who has been working on another grant, but who has benefited from the Rhagoletis genomics in this grant. Products: The primary product of the grant is a very large number of gene sequences that represent the beginnings of the genomics of the apple maggot. The sequences have been made available to the research community and public at large via the GenBank Short Read Archive; files containing our raw sequences and quality scores are available for BLAST search at GenBank SRX001885 and SRX001531 (heads), SRX001121 (larvae), SRX001530 (bodies minus heads), and SRX001529 (pupae). Dissemination: Results of the last year of the research were presented by me at the NRI Awardee workshop March 23 in Washington D.C., and will be presented at the National Entomology meetings in San Diego in November 2010. PARTICIPANTS: Professor Stewart H. Berlocher has been the lead PI on the research. Together with co-PIs Robertson and Feder he has planned and executed the research, has analyzed many of the results, and has done writing for the results. He has received one person month of salary in the 2009-2010 award year. Dr. Dietmar Schwarz was the postdoctoral fellow on the grant this first year, and did much of the research, analysis, and writing on the first paper. Dr. Schwarz left the University of Illinois at Urbana-Champaign at the end of August 2008 for an Assistant Professor position at Western Washington University. Professors H.M. Robertson (University of Illinois) and J.L. Feder (University of Notre Dame) are co-PIs and have been involved in all stages of the research. Neither received any salary from the grant. Several collaborators and contacts have been instrumental in the research. Assistant Professor D. Hahn and his post-doc Dr. G. Ragland of the University of Florida performed some analysis on the BMC Genomics paper, and are also involved in the planning and execution of a large-scale study of gene expression differences between the apple and hawthorn host races of the apple maggot fly. Assistant Professor M. Hudson and post-doc K. Varala of the University of Illinois aided in performing the analysis for the above paper. Training and development opportunities were provided for the post-doc, D. Schwarz, and also for masters student Lynn Fenema and PhD student Fred Larabee, who while supported on a different grant, was involved in conversations with grant participants. TARGET AUDIENCES: The research is 100% basic research, and the direct target audience is other basic researchers, in particular those working in insect olfaction and insect-plant interactions. However, as the work proceeds and basic information on olfaction and diapause control in Rhagoletis is obtained, this information will become disseminated to more basic researchers such as plant breeders who wish to develop apples that are unattractive to the apple maggot fly. PROJECT MODIFICATIONS: As described in the 2007 and 2008 reports, the only modification in approach has been the use of 454 pyrosequencing instead of the conventional cloning and Sanger sequencing using ABI machines described in the original report. We estimate that by using the new technology we obtained 100 times the total DNA sequence as we would have using the old technology, for the same amount of money. We are now exploiting new technologies such as Solexa sequencing to allow a complete genome of Rhagoletis pomonella to be sequenced.
Impacts
The primary outcome of this research remains a change in knowledge. But it is a profound change in knowledge. This research has produced a huge increase in information of the genetics of the apple maggot, and by extension, about the genetics of insects in general. We have thus far found hundreds of genes that have the potential to affect how the apple maggot chooses to lay eggs into apples. Over the next year, as researchers on plant pests and plant breeders become aware of this work, we expect this research to have signficant impacts on both basic and applied research on plant pests. This knowledge can be exploited in the future to provide new and better ways to control this and related pests.
Publications
- Ramsdell, K.M.M., Lyons-Sobaski, S.A., Robertson, H.M., Walden, K.K.O., Feder, J.L., Wanner, K. and Berlocher, S.H. 2009. Expressed sequence tags from cephalic chemosensory organs of the northern walnut husk fly, Rhagoletis suavis, including a putative canonical odorant receptor. Journal of Insect Science 2010. 10:51 (3 June 2010).
- Schwarz, D., Robertson, H.M., Feder J.L., Hudson, M.E., Ragland, G.J., Hahn, D.A. and Berlocher S.H. 2009. Sympatric ecological speciation meets pyrosequencing: Sampling the transcriptome of the apple maggot Rhagoletis pomonella. BMC Genomics 2009, 10:633 (27 December 2009).
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Progress 06/01/08 to 05/31/09
Outputs
OUTPUTS: Activities: The second year of the grant has been devoted to analysis of the results from the first year, preparing them for publication, and to initiating further experiments. The initial goal was to begin genomics analysis in the apple maggot (Rhagoletis pomonella), a major apple pest, by determining the DNA sequence of genes controlling attraction to host odors and genes controlling coordination of fly life cycle with apple fruiting time. In the first year of the research, we conducted a large-scale Expressed Sequence Tag (EST) study using 454 pyrosequencing technology. We sampled all life stages of R. pomonella except for eggs, and sampled both the apple and hawthorn host races. Two 454 runs at the Keck Center at the University of Illinois at Urbana-Champaign produced a total number 24,073 contigs (continuous DNA sequences assembled from smaller sections, that represent a wide variety of genes from the genome of R. pomonella). The results greatly exceeded our original goals; 13 olfactory and gustatory receptor genes (including the newly discovered ionotropic receptors) were recovered, as well as 25 odorant binding protein genes (many of which are represented by complete or nearly complete transcipts). Many genes involved in diapause were cataloged. The diapause genes we discovered are being analyzed in collaborative work with Dr. Daniel Hahn of the University of Florida. Microarrays are being used to measure expression differences between the apple and hawthorn host races relating to when they emerge from the ground. In a different experiment also begun during the 2008-2009 award year, heads of flies of the apple and hawthorn host races are being processed so that the new 500 base pair 454 sequencing technology can be carried out in fall 2009. This experiment will produce greatly increased amounts of information on genes involved in olfaction in R. pomonella. During the 2008-2009 award year I co-taught two courses at the University of Illinois, including IB Honors Ecology and Evolution. I also mentored a masters student, Lynn Fenema, who completed her masters in June 2008 on the period locus in R. pomonella. Products: The primary product of the grant is a large number of gene sequences that represent the beginnings of the genomics of the apple maggot. The sequences have been made available to the research community and public at large in two ways. First, the basic DNA sequences have been uploaded to the GenBank Short Read Archive. Files containing our raw sequences and quality scores are available for BLAST search at GenBank SRX001885 and SRX001531 (heads), SRX001121 (larvae), SRX001530 (bodies minus heads), and SRX001529 (pupae). The second way in which the sequences have been made available is at http://www.life.illinois.edu/berlocher/454_pyrosequencing_files/, in files "Rhagoletis_pom_contig_blast.xls", "Rhagoletis_pom_single_blast.xls", "Rhagoletis_pom_all_seq.txt", and "Rhagoletis_pom_contig_seq.txt". Dissemination: Results of the first year and a half of the research were presented by co-PI Jeffrey Feder at the USDA pre-meeting symposium at the 2008 meetings and at the 2008 National Entomology meeting in Reno, Nevada. PARTICIPANTS: Professor Stewart H. Berlocher has been the lead PI on the research. Together with co-PIs Robertson and Feder he has planned and executed the research, has analyzed many of the results, and has done writing for the results. He has recieved one person month of salary in the 2008-2009 award year. Dr. Dietmar Schwarz was the postdoctoral fellow on the grant this first year, and did much of the research, analysis, and writing on the first paper. Dr. Schwarz left the University of Illinois at Urbana-Champaign at the end of August 2008 for an Assistant Professor position at Western Washington University. In the 2008-2009 award year he recieved three person months of salary. Professors H.M. Robertson (University of Illinois) and J.L. Feder (University of Notre Dame) are co-PIs and have been involved in all stages of the research. Neither recieved any salary from the grant. Several collaborators and contacts have been instrumental in the research. Assistant Professor D. Hahn and his post-doc Dr. G. Ragland of the University of Florida performed some analysis on the BMC Genomics paper, and are also involved in the planning and execution of a large-scale study of gene expression differences between the apple and hawthorn host races of the apple maggot fly. Assistant Professor M. Hudson and post-doc K. Varala of the University of Illinois aided in performing the analysis for the above paper. Training and development opportunities were provided for the post-doc, D. Schwarz, and also for masters student Lynn Fenema, who while supported on a different grant, was involved in conversations with grant participants. TARGET AUDIENCES: The research is 100% basic research, and the direct target audience is other basic researchers, in particular those working in insect olfaction and insect-plant interactions. However, as the work proceeds and basic information on olfaction and diapause control in Rhagoletis is obtained, this information will become disseminated to more basic researchers such as plant breeders who wish to develop apples that are unattractive to the apple maggot fly. PROJECT MODIFICATIONS: As described in the 2007 report, the only modification in approach has been the use of 454 pyrosequencing instead of the conventional cloning and Sanger sequencing using ABI machines described in the original report. We estimate that by using the new technology we obtained 10 times the total DNA sequence as we would have using the old technology, for the same amount of money. We intend to continue to exploit new technologies such as Solexa sequencing to study gene expression differences in olfactory receptors.
Impacts
The primary outcome of this research remains a change in knowledge. But it is a profound change in knowledge. This research has produced a huge increase in information of the genetics of the apple maggot, and by extension, about the genetics of insects in general. We have thus far found hundreds of genes that have the potential to affect how the apple maggot chooses to lay eggs into apples. Over the next year, as researchers on plant pests and plant breeders become aware of this work, we expect this research to have signficant impacts on both basic and applied research on plant pests. This knowledge can be exploited in the future to provide new and better ways to control this and related pests.
Publications
- Ramsdell, K.M.M., Lyons-Sobaski, S.A., Robertson, H.M., Walden, K.K.O., Feder, J.L., Wanner, K. and Berlocher, S.H. 2009. Expressed sequence tags from cephalic chemosensory organs of the northern walnut husk fly, Rhagoletis suavis, including a putative canonical odorant receptor. Journal of Insect Science (In Press).
- Schwarz, D., Robertson, H.M., Feder J.L., Hudson, M.E., Ragland, G.J., Hahn, D.A. and Berlocher S.H. 2009. Sympatric ecological speciation meets pyrosequencing: Sampling the transcriptome of the apple maggot Rhagoletis pomonella. BMC Genomics (Pending).
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Progress 06/01/07 to 05/31/08
Outputs
OUTPUTS: Activities: The goal of the proposal was to begin genomics analysis in the apple maggot (Rhagoletis pomonella), a major apple pest, by identifying and determining the DNA sequence of genes controlling attraction to host odors and genes controlling coordination of fly life cycle with apple fruiting time. To do this we conducted a large-scale Expressed Sequence Tag (EST) study. We sampled life stages of R. pomonella ranging from larvae to adults, from either apple or hawthorn, and from two geographic sites, Illinois and Michigan. The adult sample was subdivided into heads and bodies. RNA was extracted from samples so that there were four samples representing larvae, pupae, adult bodies, and adult heads. mRna was extracted from the total RNA, cDNA was prepared, and then the cDNA was sent to the Keck Center at the University of Illinois at Urbana-Champaign for two separate cycles of 454 pyrosequencing. On each run the plate was divided into four parts for the four samples. The combined analysis of both runs produced a total number 24,073 contigs (continuous DNA sequences assembled from smaller sections). When aligning all contigs against each other with BLASTN we obtained 526 contigs (2.2% of all contigs) that produced alignments with 100% identity to another contig. The median length of contigs produced by our study is 240bp. The range of contig length stretches from ca. 100 bp to a maximum of 2823 bp. Contig lengths between 100 and 300 bp are most frequent, but contigs > 300 bp still constitute 32% of all contigs. The results have greatly exceeded our original goals of obtaining genes involved in olfaction and diapause. At least 13 olfactory and gustatory receptor genes have been recovered, as well as ~25 odorant binding protein genes. Genes involved in diapause are still being cataloged, but it is clear that may genes that are upregulated in diapause, such as heat shock proteins, were recovered in our study. Over 9,000 different genes have been identified. Events: Results of the first six months research were presented by the postdoctoral fellow on the grant, Dietmar Schwarz, at the USDA pre-meeting symposium at the 2007 meetings. The major results described above for the first full year of the grant will be presented at the 2008 National Entomology at Reno, Nevada. Services: At this early stage in the grant, the primary service has been to tutor the postdoctoral fellow, Dietmar Schwarz, and my graduate student, Lynn Fennima, who is doing behavioral and population genetics research on the apple maggot while supported on my National Science Foundation grant. Products: The primary product of the grant is fundamental research on the genomics of the apple maggot. A database of the 9,000+ genes is being prepared and will be made public when the project is nearer completion. Dissemination: As noted in Events, results of the first six months research were presented at the USDA pre-meeting symposium at the 2007 National Entomology Society meetings, and results of the first year will be presented at the 2008 Entomology meetings. As noted in Products, the database of our results will be available on the internet in the next year. PARTICIPANTS: The postdoctoral fellow on the grant this year has been Dr. Dietmar Schwarz, who has been very productive and has contributed greatly to the success of the project. Dr. Schwarz will be leaving the University of Illinois at Urbana-Champaign at the end of August 2008 for an assistant professor position at Western Washington University. A search for a replacement is underway at present. TARGET AUDIENCES: The research is 100% basic research, and the direct target audience is other basic researchers, in particular those working in insect olfaction and insect-plant interactions. However, as the work proceeds and basic information on olfaction and diapause control in Rhagoletis is obtained, this information will become disseminated to more basic researchers such as plant breeders who wish to develop apples that are unattractive to the apple maggot fly. PROJECT MODIFICATIONS: The primary change in approach has been the use of 454 pyrosequencing instead of conventional cloning and Sanger sequencing using ABI machines. We estimate that by using the new technology we obtained 10 times the total DNA sequence as we would have using the old technology, for the same amount of money. We intend to continue to exploit new technologies such as Solexa sequencing to study gene expression differences in olfactory receptors.
Impacts
The primary outcome of the research at this early stage is a change in knowledge. As a result of the research, a huge increase in knowledge of the genomics of the apple maggot has been achieved. The 9,000+ genes that we now have partially sequenced include a very large number of genes that affect the ability of the flies to find and lay eggs into apples. This knowledge can be exploited in the future to provide new and better ways to control this and related pests. A side outcome of this research is the realization that new sequencing technologies such as 454 pyrosequencing will completely change the way that the genomics of insects is viewed.
Publications
- Ramsdell, K., Lyons-Sobaski, S.A., Walden, K., Robertson, H.R., Wanner, K. and Berlocher, S.H. 2008. Results of an EST study of Rhagoletis suavis (Diptera: Tephritidae) antennal genes. Journal of Insect Science (Pending).
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