Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination.

The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.

A toolkit for rapid gene mapping in the nematode Caenorhabditis briggsae.

BACKGROUND: The nematode C. briggsae serves as a useful model organism for comparative analysis of developmental and behavioral processes. The amenability of C. briggsae to genetic manipulations and the availability of its genome sequence have prompted researchers to study evolutionary changes in gene function and signaling pathways. These studies rely on the availability of forward genetic tools such as mutants and mapping markers. RESULTS: We have computationally identified more than 30,000 polymorphisms (SNPs and indels) in C. briggsae strains AF16 and HK104. These include 1,363 SNPs that change restriction enzyme recognition sites (snip-SNPs) and 638 indels that range between 7 bp and 2 kb. We established bulk segregant and single animal-based PCR assay conditions and used these to test 107 polymorphisms. A total of 75 polymorphisms, consisting of 14 snip-SNPs and 61 indels, were experimentally confirmed with an overall success rate of 83%. The utility of polymorphisms in genetic studies was demonstrated by successful mapping of 12 mutations, including 5 that were localized to sub-chromosomal regions. Our mapping experiments have also revealed one case of a misassembled contig on chromosome 3. CONCLUSIONS: We report a comprehensive set of polymorphisms in C. briggsae wild-type strains and demonstrate their use in mapping mutations. We also show that molecular markers can be useful tools to improve the C. briggsae genome sequence assembly. Our polymorphism resource promises to accelerate genetic and functional studies of C. briggsae genes.

Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny.

To determine whether the distinctive features of Caenorhabditis elegans chromosomal organization are shared with the C. briggsae genome, we constructed a single nucleotide polymorphism-based genetic map to order and orient the whole genome shotgun assembly along the six C. briggsae chromosomes. Although these species are of the same genus, their most recent common ancestor existed 80-110 million years ago, and thus they are more evolutionarily distant than, for example, human and mouse. We found that, like C. elegans chromosomes, C. briggsae chromosomes exhibit high levels of recombination on the arms along with higher repeat density, a higher fraction of intronic sequence, and a lower fraction of exonic sequence compared with chromosome centers. Despite extensive intrachromosomal rearrangements, 1:1 orthologs tend to remain in the same region of the chromosome, and colinear blocks of orthologs tend to be longer in chromosome centers compared with arms. More strikingly, the two species show an almost complete conservation of synteny, with 1:1 orthologs present on a single chromosome in one species also found on a single chromosome in the other. The conservation of both chromosomal organization and synteny between these two distantly related species suggests roles for chromosome organization in the fitness of an organism that are only poorly understood presently.

Distribution of human SNPs and its effect on high-throughput genotyping.

Utilizing the results of extensive single nucleotide polymorphism (SNP) studies in humans, stimulated by the International HapMap Project, we present evidence that SNPs are not randomly spaced across the genome, but are somewhat clustered. This observation has important consequences for assay design, since hidden variants in primer sites can affect the accuracy of data. Indeed, using data from the calibration exercises of the HapMap Project, we found instances in which primer site mutations caused allele dropout and other genotyping failures. Given the dynamic nature of SNP discovery, it was inevitable that SNPs would be identified in the primer sites of many assays used for HapMap genotyping. We found that assays with such primer site mutations were correlated with elevated rates of genotype failure and allele dropout. This suggests that taking nearby SNPs into account is important for optimal genotyping assay design.

Primer design for PCR and sequencing in high-throughput analysis of SNPs.

To achieve high-throughput analysis of allele frequencies in human SNPs, we have developed automated methodsfor designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP, SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated. some assays filed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23,000 assays, including 96% of those recently analyzed, have been experimentally successfuL Similar design methods could be usedfor PCR assays in any organism with substantial available sequence.

The immunogenetics of smallpox vaccination.

We hypothesized that individuals who develop fever after smallpox vaccination have genetically determined differences in their immune responses to vaccinia virus. We looked for an association between the development of fever and single-nucleotide polymorphisms (SNPs) in 19 candidate genes in 346 individuals previously assessed for clinical responses to smallpox vaccination. Fever after smallpox vaccination is associated with specific haplotypes in the interleukin (IL)-1 gene complex and in the IL18 gene. A haplotype in the IL4 gene was highly significant for reduced susceptibility to the development of fever after vaccination among vaccinia-naive individuals. Our results indicate that certain haplotypes in the IL-1 gene complex and in IL18 and IL4 predict an altered likelihood of the development of fever after smallpox vaccination. Our findings also raise the possibility that these same haplotypes may identify individuals at risk for the development of fever after receipt of other live virus vaccines, providing information that could be useful in anticipating and preventing more-serious adverse events.

High-density single-nucleotide polymorphism maps of the human genome.

Here we report a large, extensively characterized set of single-nucleotide polymorphisms (SNPs) covering the human genome. We determined the allele frequencies of 55,018 SNPs in African Americans, Asians (Japanese-Chinese), and European Americans as part of The SNP Consortium's Allele Frequency Project. A subset of 8333 SNPs was also characterized in Koreans. Because these SNPs were ascertained in the same way, the data set is particularly useful for modeling. Our results document that much genetic variation is shared among populations. For autosomes, some 44% of these SNPs have a minor allele frequency > or =10% in each population, and the average allele frequency differences between populations with different continental origins are less than 19%. However, the several percentage point allele frequency differences among the closely related Korean, Japanese, and Chinese populations suggest caution in using mixtures of well-established populations for case-control genetic studies of complex traits. We estimate that approximately 7% of these SNPs are private SNPs with minor allele frequencies <1%. A useful set of characterized SNPs with large allele frequency differences between populations (>60%) can be used for admixture studies. High-density maps of high-quality, characterized SNPs produced by this project are freely available.

BIOTYPING CONFIRMS A NEARLY CLONAL POPULATION STRUCTURE IN ESCHERICHIA COLI.

A. reference collection of 72 natural isolates of Escherichia coli (the ECOR collection) has been examined with respect to eight metabolic capabilities (biotype characters) plus motility and resistance or sensitivity to five common antibiotics. Data from biotype characters were analyzed by means of unweighted pair-group cluster analysis, and the genetic variation among the strains defines three major clusters of strains with substantial variation within each cluster but greater genetic similarity of strains within a cluster than between clusters. These clusters define an infraspecific population structure in E. coli, which reflects the predominantly clonal mode of reproduction in this organism. The clusters identified by the biotype characters are in good agreement with those resulting from an analysis of 11 enzyme polymorphisms (allozymes) among the strains, and these are in good agreement with the infraspecific structure detected by factor analysis of allozyme data. The clusters of strains also differ in several genetic characteristics that are independent of those used in making the classification.

Efficient high-throughput resequencing of genomic DNA.

Targeted resequencing of genomic DNA from organisms such as humans is an important tool enabling experimental access to variation within the species and between similar species. Taking full advantage of the reference genome sequences in designing robust, specific PCR assays and using stringent conditions, resequencing can be done efficiently without purification of the PCR product. By using a 10-fold greater amount of one primer when setting up the PCR initially in a new version of asymmetric PCR, one simply adds the rest of the sequencing reagents at the end of PCR and allows the sequencing reaction to proceed, with the excess PCR primer serving as the sequencing primer. We demonstrated that this streamlined protocol can be used with PCR products up to 1300 bp and had up to a 97% success rate in high-throughput analysis of allele frequencies for >30,000 single-nucleotide polymorphisms (SNPs). SNP primers and characterization results are provided at http://snp.wustl.edu.

Regulator of sex-limitation (Rsl) encodes a pair of KRAB zinc-finger genes that control sexually dimorphic liver gene expression.

Sexually dimorphic expression of a broad array of liver proteins involved in reproduction and xenobiotic metabolism is induced at puberty by sex-specific growth hormone patterns. An additional control of sex-dependent gene expression is conferred by Regulator of sex-limitation (Rsl) alleles. In variant rsl mice, females inappropriately express the male Sex-limited protein, Slp. We recently showed that a panel of male-specific liver genes is repressed by Rsl, accentuating sex differences in a hormone-independent manner. Here we map rsl to a region on Chromosome 13 comprised exclusively of KRAB (Kruppel-associated box) zinc-finger protein (ZFP) genes. Among eight Rsl candidate (Rslcan) genes within the critical genetic interval, the recent duplicates Rslcan-4 and Rslcan-9 both harbor mutations in rsl mice (partial deletion and splice-site inactivation, respectively). Transgenesis with bacterial artificial chromosome (BAC) clones encompassing Rslcan-4 restores male-specific MUP (major urinary protein) expression to rsl mice, whereas a BAC containing Rslcan-9 rescues sex-specific expression of Slp and cytochrome P450 Cyp2d9. Thus, the Rslcan-4 and Rslcan-9 paralogs partitioned regulation of their target genes during evolution. This demonstrates the first biological role for a set of KRAB zinc-finger repressor proteins and reveals the molecular basis of a gene-silencing pathway critical for sexual dimorphism.

Analysis of candidate genes for prostate cancer.

Considerable evidence demonstrates that genetic factors are important in the development and aggressiveness of prostate cancer. To identify genetic variants that predispose to prostate cancer we tested candidate SNPs from genomic regions that show linkage to prostate cancer susceptibility and/or aggressiveness, as well as genes that show a significant difference in mRNA expression level between tumor and normal tissue. Cases had histologically verified prostate cancer. Controls were at least 65 years old, never registered a PSA above 2.5 ng/ml, always had digital rectal examinations that were not suspicious for cancer, and have no known family history of prostate cancer. Thirty-nine coding SNPs and nine non-coding SNPs were tested in up to 590 cases and 556 controls resulting in over 40,000 SNP genotypes. Significant differences in allele frequencies between cases and controls were observed for ID3 (inhibitor of DNA binding), p = 0.05, HPN (hepsin), p = 0.009, BCAS1 (breast carcinoma amplified sequence 1), p = 0.007, CAV2 (caveolin 2), p = 0.007, EMP3 (epithelial membrane protein 3), p < 0.0001, and MLH1 (mutL homolog 1), p < 0.0001. SNPs in three of these genes (BCAS1, EMP3 and MLH1) remained significant in an age-matched subsample.