Shaver, Amanda, Hafeez, Zaki, Edison, Arthur, Anderson, Lindsay, Mohamed, Youssef, Spencer, Deontis, Shah, Man, Asif, Muhammad Zaka, Muzio, Cole
[
International Worm Meeting,
2021]
The Caenorhabditis elegans Natural Diversity Resource (CeNDR) maintains a library of strains of C. elegans collected from every continent except Antarctica (Caenorhabditis elegans Natural Diversity Resource [CeNDR], 2020). However, currently, there is only one C. elegans strain from the state of Georgia cataloged in CeNDR (CeNDR, 2020). In an attempt to expand upon the diversity of these collected strains and in an effort to study the distribution of C. elegans in Georgia, we set forth to collect a number of wild nematode isolates. Samples of rotting and decaying vegetation were collected from a variety of locations across Georgia. Data such as temperature, location, and sample type were recorded along with images of each collection site using the Nematode Field Sampling app within the data collection app Fulcrum. Nematodes were isolated from these samples and screened by visual inspection for morphological similarity to C. elegans. Potential C. elegans strains were then cultivated. To confirm that an isolate is indeed C. elegans, we will perform PCR and gel electrophoresis. For worms with an appropriately sized PCR band, we intend to submit samples of the PCR product for Sanger sequencing. We will then use NCBI BLAST to compare the sequencing results of the wild isolates with known species. Finally, we will submit frozen isolates of C. elegans to CeNDR for cataloging and whole-genome sequencing. Caenorhabditis elegans Natural Diversity Resource. (2020, August 30). Global Strain Map [Interactive Map]. Retrieved March 24, 2021 from https://www.elegansvariation.org/strain/global-strain-map
[
International C. elegans Meeting,
1999]
We would like to reconstruct the history of the C. elegans species at the genome level, therefore we sampled the genomes of four natural isolates (strain CB4857 isolated in Claremont, California, RC301 from Freiburg, Germany, TR403 from Madison, Wisconsin and AB1 from Adelaide, Australia) for single nucleotide polymorphisms (SNPs). Random genomic DNA fragments from the 4 strains were shotgun cloned and sequenced. There was no selection for transcribed or non-transcribed regions of the genome. In total we sequenced 1572 clones resulting in over 1 Mb of sequence information. The sequences are compared to the canonical Bristol N2 sequence to ask the question whether the clone maps to a unique sequence, and -if so- whether it contains polymorphisms. Once a SNP is identified we check other strains for the presence of the same polymorphism by PCR amplification and sequence analysis. In an initial experiment we found approximately one SNP per 3000 bp sequenced. The SNPs are randomly spread over the genome. Based on these observations we expect to find approximately 500 SNPs, one in every 200 kb. In the initial experiment we found, as expected, that several SNPs initially detected in one strain were also present in some but not all other strains. For example: a T in the Australian AB1, is a G at the same position in Bristol N2 in cosmid K10D2 at position 27946, and we found it to be like AB1 in the Californian CB4857 strain and the German RC301 strain, while the TR403 strain from Wisconsin resembles the Bristol N2 strain. Thus different patches of the genome have different ancestors. With our high density SNP map we will generate a genome map for each isolate which will show how each genome is patched together from a limited set of parental strains. The SNP's will be added to ACeDB, and can also be used as markers on the genetic map. They can be recognised by PCR followed by sequencing, but we also found that the SNPs we looked at could be visualised by SSCP analysis. We thank Jane Rogers and Amanda McMurray for their assistance in sequencing the clones.