Isolation of mutations with dumpy-like phenotypes and of collagen genes in the nematode Pristionchus pacificus.

The nematode Pristionchus pacificus was developed as a satellite system in evolutionary developmental biology and forward and reverse genetic approaches allow a detailed comparison of various developmental processes between P. pacificus and Caenorhabditis elegans. To facilitate map-based cloning in P. pacificus, a genome map was generated including a genetic linkage map of approximately 300 molecular markers and a physical map of 10,000 BAC clones. Here, we describe the isolation and characterization of more than 40 morphological mutations that can be used as genetic markers. These mutations fall into 12 Dumpy genes and one Roller gene that represent morphological markers for all six P. pacificus chromosomes. Using an in silico approach, we identified approximately 150 hits of P. pacificus collagen genes in the available EST, BAC-end, and fosmid-end sequences. However, 1:1 orthologs could only be identified for fewer than 20 collagen genes.

A bacterial artificial chromosome-based genetic linkage map of the nematode Pristionchus pacificus.

To understand the evolution of developmental processes, nonmodel organisms in the nematodes, insects, and vertebrates are compared with established model systems. Often, these comparisons suffer from the inability to apply sophisticated technologies to these nonmodel species. In the nematode Pristionchus pacificus, cellular and genetic analyses are used to compare vulva development to that of Caenorhabditis elegans. However, substantial changes in gene function between P. pacificus and C. elegans limit the use of candidate gene approaches in studying P. pacificus mutations. To facilitate map-based cloning of mutations in P. pacificus, we constructed a BAC-based genetic linkage map. A BAC library of 13,440 clones was generated and completely end sequenced. By comparing BAC end and EST sequences between the "wild-type" strain P. pacificus var. California and the polymorphic strain P. pacificus var. Washington, 133 single-stranded conformational polymorphisms were identified. These markers were tested on a meiotic mapping panel of 46 randomly picked F(2) animals after a cross of the two strains, providing the first genetic linkage map of P. pacificus. A mapping strategy using two selected markers per chromosome was devised and the efficiency of this approach was illustrated by the mapping of the Ppa-unc-1/Twitchin gene.