Progress 10/01/12 to 09/30/15
Outputs
Target Audience:The ultimate target audience includes scientists interested in European Corn Borer and farmers who grow crops for which this insect is a pest. Changes/Problems:We have not made as much progress as we hoped, in part because of problems obtaining new samples (see above), but also because of the rapid changes in appropriate methods for DNA sequencing and genotyping. The Harrison lab has been adopting new methods that will ultimately be more efficient and effective for data collection, but this has slowed us down a bit. What opportunities for training and professional development has the project provided?Henry Kunerth, now a second year graduate student, is working on the project and has learned sequencing and genotyping approaches and methods for analyzing DNA sequence data. We also trained an undergraduate (Jeeyun Lee) in a variety of techniques used for DNA sequence analysis of insect populations. Jeeyun learned to extract and PCR amplify DNA; she prepared a library for Illumina sequencing and analyzed the data thus obtained. Jeeyun graduated from Cornell and is now in graduate school in evolutionary biology. How have the results been disseminated to communities of interest?The thesis produced by Ben Hamilton has not yet been published, delayed by the fact that Ben moved from Ithaca. I am currently working with Ben to publish his work, which will be submitted within the next 6 months. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We worked with the New York State Sweet Corn Trapping Network to gain access to male ECB that were trapped during the summers of 2014 and 2015. However, because of the widespread planting of Bt corn, ECB numbers at most trap sites have been low.Because of small sample sizes from most sites, we have used moths collected in previous years and have developed a multiplex PCR assay, through which we can assay 25-35 gene loci and obtain sequences across all loci for 96 individuals on the Illumina Mi-Seq (2 x 300 bp - paired-end sequencing). Because this approach is far more efficient than the approach we initially proposed, we have relied on high-throughput sequencing rather than SNP genotyping to obtain the data on genotypes for E and Z ECB in populations where the two pheromone strains occur together. Developing the new approach has slowed data collection, and we have initially focused on Z-linked loci (that is, markers on the sex chromosome). We have analyzed sympatric populations of E and Z borers from four localities: New York, Delaware, North Carolina, and Italy. The initial panel of ECB used for sequencing consisted of 384 moths. For each sample, two multiplexed PCR runs were carried out, utilizing genomic DNA primers for a total of 46 target loci (all Z-linked). Targets consisted of fragments ranging from 350-500 base pairs. Reactions were carried out using reagents from a Qiagen multiplex PCR kit and generic DNA primers. We filtered out individual samples with substantial missing data, and the final dataset consisted of 350 moths with low levels (< 10%) of missing data. We then estimated pairwise FST for each population and pheromone strain group comparison. FST measures differentiation between populations, with higher values indicating that the populations are genetically distinct. Initial results showed a clear pattern of elevated FST between sympatric pheromone strains within one region of the Z chromosome. This pattern held for two of the four population comparisons (New York and North Carolina); in the other two populations (Italy and Delaware) FST was not elevated, consistent with evidence that the two strains hybridize at these sites. Our data clearly show that at least for a major region on the Z chromosome, the two pheromone strains of ECB are exchanging genes (hybridizing) only at a very low rate. We now must expand our analyses to look at autosomal loci. We have now developed primers that target 50 kb genomic intervals across the divergent region of the Z chromosome and across an autosomal region that includes the pgFAR locus (this has been shown to be the gene locus that determines whether female moths produce the E or Z blend). This ongoing project should help to define the boundaries of the FST peak and the extent to which the pgFAR locus has an influence on the surrounding autosomal region.
Publications
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2015
Citation:
Hamilton, Benjamin 2015. COORDINATION AND DISRUPTION OF TRAITS CONTRIBUTINGTO REPRODUCTIVE ISOLATION IN OSTRINIA NUBILALIS, THE EUROPEAN CORN BORER. M.S. Thesis, Cornell University.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems: The two problems we face are the low number of ECB in pheromone traps throughout much of NY state and the degradation of DNA in moth samples when pheromone traps are only checked once each week. In 2015, we intend to identify a few sites, and monitor traps on a more regular basis. What opportunities for training and professional development has the project provided? A new graduate student in the Harrison lab (Henry Kunerth) is working to analyze the initial data set. This is an excellent opportunity for training in genomics and bioinformatics, and Henry has already learned a lot about how to process and analyze large DNA sequence data sets. Training in these areas is essential for students who want to use molecular population genetic or molecular phylogenetic approaches to understand the genetics of natural populations. How have the results been disseminated to communities of interest? Data are still preliminary. These preliminary results have been presented in seminars to colleagues, but are not ready for publication. What do you plan to do during the next reporting period to accomplish the goals? We will expand the scope of our sequencing project - to include both more gene loci and more individuals. We will participate in the pheromone trapping network during the summer of 2015, with modified protocols that hopefully will give us better quality DNA samples when extractions are done from pheromone trapped males.
Impacts
What was accomplished under these goals?
We have worked with the New York State Sweet Corn Trapping Network to gain access to male ECB that were trapped during the summer of 2014. Because of the widespread planting of Bt corn, ECB numbers at most trap sites were low throughout the summer. One site (Seneca Castle) is adjacent to a large organic grower and trap numbers here were substantial. We have obtained most of these moths (through our collaborators Abby Seaman and Marion Zuefle), although some remain frozen elsewhere. DNA extracted from these moths initially produced poor results, but subsequent cleanup of these preps has resulted in good template for PCR and sequencing. Because of small sample sizes from most sites, we have used moths collected in previous years and have developed a multiplex PCR assay, through which we can assay 25-35 gene loci and obtain sequences across all loci for 96 individuals on the Illumina Mi-Seq (2 x 300 bp - paired-end sequencing). Because this approach is far more efficient than the approach we initially proposed, we will now rely on high-throughput sequencing rather than SNP genotyping to obtain the data we need. In developing the new approach, we have initially focused on Z-linked loci (that is, markers on the sex chromosome), have obtained sequence data for sympatric (co-occurring) moths of the E and Z pheromone strains, and are currently analyzing these data. The data analyses are still in preliminary stage, but it is already clear that for many markers on the Z chromosome, the two pheromone strains remain distinct.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems: We were disappointed in the numbers of ECB collected over the past summer (although that is good news for farmers). We did obtain good population samples from 2-3 populations (had hoped for 5). This is not a major problem, and we have samples from previous years that we can use if numbers are again low. We will not use the Sequenom MassARRAY genotyping assay that we initially proposed, because this approach has essentially become "outmoded." We can obtain more data for less money using other techniques (using mutliplex PCR with barcoding to generate templates for sequencing on the Illumina platform). What opportunities for training and professional development has the project provided? We have trained an undergraduate (Jeeyun Lee) in a variety of techniques used for DNA sequence analysis of insect populations. Jeeyun extracted and amplified DNA, prepared a library for Illumina sequencing (this is the most efficient of the next generation sequencing techniques), and analyzed the data obtained from Illumina sequencing. Jeeyun has graduated from Cornell, is working in the Entomology Department at North Carolina State, and is preparing to go to graduate school. How have the results been disseminated to communities of interest? The results of our pheromone trap monitoring were included each week in the report of trap catches from many sites as part of the Sweet Corn Pheromone Trap Network. What do you plan to do during the next reporting period to accomplish the goals? We intend to again be involved in monitoring pheromone traps and collecting ECB rom natural populations. We will further develop our methods for identifying and characterizing SNPs and will develop a "new" multiplex PCR approach for obtaining sequence data from multiple individuals for a large number of SNPs. Because technology is changing rapidly, new and better techniques are appearing.
Impacts
What was accomplished under these goals?
We monitored pheromone traps at two sites (Baldwinsville, NY and Kirkville, NY) as part of the Sweet Corn Pheromone Trapping Network and collected/froze moths of both the E and Z strain from these sites during the entire season. Collaborators monitored other sites and collected moths (by pheromone strain) at those sites. Probably because of the widespread use of Bt corn, the numbers of ECB were low at most sites in NY this past season. DNA has been extracted from the moths that we collected from Baldwinsville and Kirkville. We have used two approaches to identify SNPs that differentiate the two pheromone strains of ECB. One involves comparison of transcriptome sequences (sequences of genes that are expressed in various life stages); the second is a relatively new technique, using next-generation sequencing,that allows sequencing of a defined subset of the genome and comparison of those sequences among individuals from populations. We have used data generated by the second method (called ddRAD) and identified hundreds of SNPs. Analysis with a widely used software package (STACKS) has proved somewhat unsatisfactory - and we are currently exploring other methods for SNP identification and characterization of allele frequencies.
Publications
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