BAC library construction, screening and clone sequencing of lake whitefish (Coregonus clupeaformis, Salmonidae) towards the elucidation of adaptive species divergence

Genomic DNA sequences and other genomic resources are essential towards the elucidation of the genomic bases of adaptive divergence and reproductive isolation. Here, we describe the construction, characterization and screening of a nonarrayed BAC library for lake whitefish (Coregonus clupeaformis). We then show how the combined use of BAC library screening and next-generation sequencing can lead to efficient full-length assembly of candidate genes. The lake whitefish BAC library consists of 181,050 clones derived from a single heterozygous fish. The mean insert size is 92 Kb, representing 5.2 haploid genome equivalents. Ten BAC clones were isolated following a quantitative real-time PCR screening approach that targeted five previously identified candidate genes. Sequencing of these clones on a 454 GS FLX system yielded 178,000 reads with a mean length of 358 bp, for a total of 63.8 Mb. De novo assembly and annotation then allowed retrieval of contigs corresponding to each candidate gene, which also contained up- and/or downstream noncoding sequences. These results suggest that the lake whitefish BAC library combined with next-generation sequencing technologies will be key resources to achieve a better understanding of both adaptive divergence and reproductive isolation in lake whitefish species pairs as well as salmonid evolution in general.     

Discovery of single-nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete Ophiognomonia clavigignenti-juglandacearum from 454 sequence data

The benefits from recent improvement in sequencing technologies, such as the Roche GS FLX (454) pyrosequencing, may be even more valuable in non-model organisms, such as many plant pathogenic fungi of economic importance. One application of this new sequencing technology is the rapid generation of genomic information to identify putative single-nucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. The focus of this research was to sequence, assemble, discover and validate SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. Genomic DNA from eight isolates of Ophiognomonia clavigignenti-juglandacearum was pooled in one region of a four-region sequencing run on a Roche 454 GS FLX. This yielded 71 million total bases comprising 217,000 reads, 80% of which collapsed into 16,125,754 bases in 30,339 contigs upon assembly. By aligning reads from multiple isolates, we detected 298 SNPs using Roche's GS Mapper. With no reference sequence available, however, it was difficult to distinguish true polymorphisms from sequencing error. Eagleview software was used to manually examine each contig that contained one or more putative SNPs, enabling us to discard all but 45 of the original 298 putative SNPs. Of those 45 SNPs, 13 were validated using standard Sanger sequencing. This research provides a valuable genetic resource for research into the genus Ophiognomonia, demonstrates a framework for the rapid and cost-effective discovery of SNP markers in non-model organisms and should prove especially useful in the case of asexual or clonal fungi with limited genetic variability.