2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Confirm and demonstrate the utility of Sequencing Individuals in Reduced Representation Libraries (SIRRL) as a tool for linkage mapping and population studies of the European corn borer, Ostrinia nubilalis, a major lepidopteran pest of agriculture. Objective 2: Verify that SIRRL can be used to generate markers for non-model lepidopteran species, which are increasing in pest status (western bean cutworm, Striacosta albicosta) or may become a significant new pest (prairie cordgrass caterpillar, Tortricidae).
1b.Approach (from AD-416):
Up to 50 barcode-tagged "A" adaptors bearing HindIII overhangs will be designed for Illumina sequencing chemistry. Up to 50 European corn borer (ECB) siblings will be used to prepare a SIRRL sample, each bearing an adaptor with a different barcode. Individual DNA samples will be pooled prior to size selection. The SIRRL sample will then be prepared according to the optimized conditions and sequenced using one of the two regions of a PicoTiter plate. Software and published algorithms will be used to determine the reads that derive from each individual, based on the barcodes. Barcode sequences will be masked prior to using the newbler software to group reads into genomic regions. The association between grouped reads and individual insects will be used to determine individual genotypes. To verify repeatability of the SIRRL method, the experiment will be repeated in its entirety using the same set of DNA samples and the results compared.
The suitability of SIRRL markers for the construction of linkage maps will be demonstrated using two F1 families of ECB. A sample of 50 individuals per family will be labelled with 50 barcoded SIRRL adaptors and each sequenced on one region of a PicoTiter plate (i.e., one full instrument run per family). The genotypes of each individual will be determined using the methods developed in the experiments described above, and linkage relationships between polymorphisms established.
The SIRRL method also will be applied to samples of approximately 200 ECB collected from field populations in IA and PA. Batches of up to 50 individuals will be prepared, each labeled with a different barcoded adaptor. Each batch will be sequenced using one region of a PicoTiter plate (i.e., a total of two instrument runs). The genotypes of each individual will be determined at each polymorphic site identified. The genetic variation revealed by SIRRL can be analyzed like that from any other co-dominant genetic marker. Basic population genetic analyses, such as tests for departures from Hardy-Weinberg genotypic proportions and allele frequency differences between locations will be conducted to evaluate the utility of SIRRL in population studies.
We will test the ease with which SIRRL can be applied to species with little or no prior genetic characterization. We will apply SIRRL to samples of up to 100 individuals of two such species, the western bean cutworm and the prairie cordgrass caterpillar. Initially, 50 individuals from each species will be labeled with barcoded adaptors and the sample from each will be sequenced on one region of a PicoTiter plate (i.e., one full instrument run for both species). The data from this first run may indicate that modifications to the sample preparation are needed (e.g., a narrower or broader size selection). If so, these adjustments will be made and the experiment repeated using the same samples. If the results of the first experiment indicate that no modifications are needed, an additional 50 individuals from different populations from each species will be examined, and the degree of genetic divergence between populations will be examined.
Protocols were successfully optimized to prepare DNA samples to generate genetic markers using a new method we developed that is called Sequencing Individuals in Reduced Representation Libraries (SIRRL). The European corn borer was used as the test species. The new method includes a DNA fragment size selection step and a restriction/ligation reaction step where genomic DNA is completely digested. DNA from an individual corn borer was digested and sequenced to verify the distribution of distinct clusters of single copy, moderately repetitive and highly repetitive DNA. The results indicated that over 1000 markers were consistent with single-copy loci, a prerequisite for useful markers. Two families of 50 each European corn borer siblings were produced and used to prepare a SIRRL marker sample with each bar-coded for sequencing. This test run was successful, demonstrating that the method for library preparation and paired-end sequencing in fact work as well as conceived. Data quality was excellent, as was the quantity of paired reads: 89 million from a single lane. Given this success, barcoded adapters for a full run on a wild population sample of 200 European corn borers were purchased, and full sequencing was completed. This will allow testing of SIRRL markers for use in population genetics, including a genome scan to find one or more markers linked to crop rotation resistance. Three libraries from western bean cutworm, prepared using different restriction enzymes, have been sequenced and analysis of the resulting data are underway. Collections of a little known pest of biofuel grasses, the prairie cordgrass caterpillar, were made by collaborators in Illinois, and transferred to University of Nebraska. Genomic DNA has been extracted from these samples. Sequencing libraries are being made and will soon be sent for sequencing.