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

Koboldt DC, Staisch J, Thillainathan B, Haines K, Baird SE, Chamberlin HM, Haag ES, Miller RD, Gupta BP - BMC Genomics (2010)

Bottom Line: The utility of polymorphisms in genetic studies was demonstrated by successful mapping of 12 mutations, including 5 that were localized to sub-chromosomal regions.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada.

ABSTRACT

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.

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Validation of polymorphism assays in C. briggsae. (A) RFLP assays for snip-SNPs in three parental strains, namely AF16 (A), HK104 (H), and VT847 (V) by HindIII restriction digestion. (B) Medium indels in three parental strains showing a 100 bp deletion on fpc4171, a 145 bp insertion on fpc4140, and a 536 bp deletion on fpc0011. (C) Six additional medium indels (one for each chromosome) in AF16 and HK104 genotypes. The indels are (Chr. 1-5 and X, from left to right): cb-m142 (250 bp), cb-m21 (200 bp), cb-m205 (300 bp), cb-m172 (250 bp), cb-m103 (200 bp), and cb-m204 (250 bp). (D) Validation of small indels in AF16, HK104 and F1 heterozygotes (AH). Chr 1: bhP19 (32 bp), Chr 2: bhP21 (20 bp), Chr 3: bhP12 (39 bp), Chr 4: bhP9 (16 bp), Chr 5: bhP48 (22 bp) and Chr X: bhP24 (16 bp).
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Figure 2: Validation of polymorphism assays in C. briggsae. (A) RFLP assays for snip-SNPs in three parental strains, namely AF16 (A), HK104 (H), and VT847 (V) by HindIII restriction digestion. (B) Medium indels in three parental strains showing a 100 bp deletion on fpc4171, a 145 bp insertion on fpc4140, and a 536 bp deletion on fpc0011. (C) Six additional medium indels (one for each chromosome) in AF16 and HK104 genotypes. The indels are (Chr. 1-5 and X, from left to right): cb-m142 (250 bp), cb-m21 (200 bp), cb-m205 (300 bp), cb-m172 (250 bp), cb-m103 (200 bp), and cb-m204 (250 bp). (D) Validation of small indels in AF16, HK104 and F1 heterozygotes (AH). Chr 1: bhP19 (32 bp), Chr 2: bhP21 (20 bp), Chr 3: bhP12 (39 bp), Chr 4: bhP9 (16 bp), Chr 5: bhP48 (22 bp) and Chr X: bhP24 (16 bp).

Mentions: We selected a total of 20 RFLP assays (between 3 and 4 for each chromosome) based on HindIII, DraI and SalI snip-SNPs for validation in AF16 and HK104 parental DNA (Table 2). Roughly a third of the assays (6) failed PCR in one or both strains in repeated attempts. Although we did not investigate the issue of PCR failure, it is possible that redesigning primers (by moving them out or in) and testing different PCR conditions may produce desired products in some cases. All of the 14 assays successfully gave rise to strain-specific RFLP banding patterns, validating the predicted snip-SNP (Table 2, Figure 2A for two examples). Interestingly, two of these assays (cb55670 and cb20723) exhibited HK104 fragments that varied from in silico predictions, another possible consequence of unknown variants in this highly divergent strain. Consistent with C. briggsae clade structure [8], VT847 was not polymorphic (from AF16) for the snip-SNPs we examined.


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

Koboldt DC, Staisch J, Thillainathan B, Haines K, Baird SE, Chamberlin HM, Haag ES, Miller RD, Gupta BP - BMC Genomics (2010)

Validation of polymorphism assays in C. briggsae. (A) RFLP assays for snip-SNPs in three parental strains, namely AF16 (A), HK104 (H), and VT847 (V) by HindIII restriction digestion. (B) Medium indels in three parental strains showing a 100 bp deletion on fpc4171, a 145 bp insertion on fpc4140, and a 536 bp deletion on fpc0011. (C) Six additional medium indels (one for each chromosome) in AF16 and HK104 genotypes. The indels are (Chr. 1-5 and X, from left to right): cb-m142 (250 bp), cb-m21 (200 bp), cb-m205 (300 bp), cb-m172 (250 bp), cb-m103 (200 bp), and cb-m204 (250 bp). (D) Validation of small indels in AF16, HK104 and F1 heterozygotes (AH). Chr 1: bhP19 (32 bp), Chr 2: bhP21 (20 bp), Chr 3: bhP12 (39 bp), Chr 4: bhP9 (16 bp), Chr 5: bhP48 (22 bp) and Chr X: bhP24 (16 bp).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864247&req=5

Figure 2: Validation of polymorphism assays in C. briggsae. (A) RFLP assays for snip-SNPs in three parental strains, namely AF16 (A), HK104 (H), and VT847 (V) by HindIII restriction digestion. (B) Medium indels in three parental strains showing a 100 bp deletion on fpc4171, a 145 bp insertion on fpc4140, and a 536 bp deletion on fpc0011. (C) Six additional medium indels (one for each chromosome) in AF16 and HK104 genotypes. The indels are (Chr. 1-5 and X, from left to right): cb-m142 (250 bp), cb-m21 (200 bp), cb-m205 (300 bp), cb-m172 (250 bp), cb-m103 (200 bp), and cb-m204 (250 bp). (D) Validation of small indels in AF16, HK104 and F1 heterozygotes (AH). Chr 1: bhP19 (32 bp), Chr 2: bhP21 (20 bp), Chr 3: bhP12 (39 bp), Chr 4: bhP9 (16 bp), Chr 5: bhP48 (22 bp) and Chr X: bhP24 (16 bp).
Mentions: We selected a total of 20 RFLP assays (between 3 and 4 for each chromosome) based on HindIII, DraI and SalI snip-SNPs for validation in AF16 and HK104 parental DNA (Table 2). Roughly a third of the assays (6) failed PCR in one or both strains in repeated attempts. Although we did not investigate the issue of PCR failure, it is possible that redesigning primers (by moving them out or in) and testing different PCR conditions may produce desired products in some cases. All of the 14 assays successfully gave rise to strain-specific RFLP banding patterns, validating the predicted snip-SNP (Table 2, Figure 2A for two examples). Interestingly, two of these assays (cb55670 and cb20723) exhibited HK104 fragments that varied from in silico predictions, another possible consequence of unknown variants in this highly divergent strain. Consistent with C. briggsae clade structure [8], VT847 was not polymorphic (from AF16) for the snip-SNPs we examined.

Bottom Line: The utility of polymorphisms in genetic studies was demonstrated by successful mapping of 12 mutations, including 5 that were localized to sub-chromosomal regions.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada.

ABSTRACT

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.

Show MeSH