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Divergence in cis-regulatory sequences surrounding the opsin gene arrays of African cichlid fishes.

O'Quin KE, Smith D, Naseer Z, Schulte J, Engel SD, Loh YH, Streelman JT, Boore JL, Carleton KL - BMC Evol. Biol. (2011)

Bottom Line: We also found several microRNA target sites within the 3'-UTR of each opsin, including two 3'-UTRs that differ significantly between O. niloticus and M. zebra.We found that all regions were highly conserved with some evidence of CRX transcription factor binding site turnover.We also found three SNPs within two opsin promoters and one non-coding element that had weak association with cichlid opsin expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Maryland, College Park, MD 20742, USA.

ABSTRACT

Background: Divergence within cis-regulatory sequences may contribute to the adaptive evolution of gene expression, but functional alleles in these regions are difficult to identify without abundant genomic resources. Among African cichlid fishes, the differential expression of seven opsin genes has produced adaptive differences in visual sensitivity. Quantitative genetic analysis suggests that cis-regulatory alleles near the SWS2-LWS opsins may contribute to this variation. Here, we sequence BACs containing the opsin genes of two cichlids, Oreochromis niloticus and Metriaclima zebra. We use phylogenetic footprinting and shadowing to examine divergence in conserved non-coding elements, promoter sequences, and 3'-UTRs surrounding each opsin in search of candidate cis-regulatory sequences that influence cichlid opsin expression.

Results: We identified 20 conserved non-coding elements surrounding the opsins of cichlids and other teleosts, including one known enhancer and a retinal microRNA. Most conserved elements contained computationally-predicted binding sites that correspond to transcription factors that function in vertebrate opsin expression; O. niloticus and M. zebra were significantly divergent in two of these. Similarly, we found a large number of relevant transcription factor binding sites within each opsin's proximal promoter, and identified five opsins that were considerably divergent in both expression and the number of transcription factor binding sites shared between O. niloticus and M. zebra. We also found several microRNA target sites within the 3'-UTR of each opsin, including two 3'-UTRs that differ significantly between O. niloticus and M. zebra. Finally, we examined interspecific divergence among 18 phenotypically diverse cichlids from Lake Malawi for one conserved non-coding element, two 3'-UTRs, and five opsin proximal promoters. We found that all regions were highly conserved with some evidence of CRX transcription factor binding site turnover. We also found three SNPs within two opsin promoters and one non-coding element that had weak association with cichlid opsin expression.

Conclusions: This study is the first to systematically search the opsins of cichlids for putative cis-regulatory sequences. Although many putative regulatory regions are highly conserved across a large number of phenotypically diverse cichlids, we found at least nine divergent sequences that could contribute to opsin expression differences in cis and stand out as candidates for future functional analyses.

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Interspecific polymorphism in eight putative cis-regulatory regions from 18 Lake Malawi cichlid species. A - H) Minor allele frequency (MAF; in red) and nucleotide diversity (π; in black) calculated in a sliding window across the proximal promoter regions of five opsins (A - E), CNE 7 (LWS-LCR) (F), and two opsin 3'-UTRs (G - H) using 18 Lake Malawi cichlid species. Numbers above peaks of MAF and π denote the position of SNPs analyzed for allelic-association with opsin expression (see Table 6); asterisks (*) denote polymorphisms that interrupt CRX binding sites.
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Figure 4: Interspecific polymorphism in eight putative cis-regulatory regions from 18 Lake Malawi cichlid species. A - H) Minor allele frequency (MAF; in red) and nucleotide diversity (π; in black) calculated in a sliding window across the proximal promoter regions of five opsins (A - E), CNE 7 (LWS-LCR) (F), and two opsin 3'-UTRs (G - H) using 18 Lake Malawi cichlid species. Numbers above peaks of MAF and π denote the position of SNPs analyzed for allelic-association with opsin expression (see Table 6); asterisks (*) denote polymorphisms that interrupt CRX binding sites.

Mentions: Although the 18 Lake Malawi cichlid species we use have been previously characterized with regard to opsin gene expression, we confirmed these gene expression results by measuring the expression of each opsin in all species via RT-qPCR (see Additional File 8 for opsin expression results). These expression results were highly concordant with previous measurements [26]. Following qPCR, we re-sequenced the entire 1 kb region upstream of both the SWS1 and SWS2A opsins, 956 bp upstream of the LWS opsin, 951 bp upstream of the RH2B opsin, and 694 bp upstream of the SWS2B opsin. We also re-sequenced 900 bp surrounding the LWS-LCR (CNE 7) and 450 bp downstream of the SWS2B and LWS opsins. As expected given the young age of Lake Malawi cichlids, we found that all regions were highly conserved among the species sampled. Overall, we identified fewer than 15 single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) per region examined (Table 5). In each case, most SNPs were found in only one individual. Other diversity statistics--including the total number of segregating sites (S), total number of singletons (s), number of haplotypes (H), nucleotide diversity (π), sequence conservation (C), and Tajima's D (TD)--also indicate low levels of polymorphism, despite our use of alternate species and genera as sampling units (see Additional file 8 for a list of all polymorphisms found among the 18 species sampled). Nevertheless, following a sliding window analysis of nucleotide diversity (π) and minor allele frequency (MAF), we were able to identify several peaks of relatively highπ and MAF within each region (Figure 4). These peaks correspond to SNPs and indels segregating at high frequency within the species and genera sampled, and therefore represent potential cis-regulatory alleles.


Divergence in cis-regulatory sequences surrounding the opsin gene arrays of African cichlid fishes.

O'Quin KE, Smith D, Naseer Z, Schulte J, Engel SD, Loh YH, Streelman JT, Boore JL, Carleton KL - BMC Evol. Biol. (2011)

Interspecific polymorphism in eight putative cis-regulatory regions from 18 Lake Malawi cichlid species. A - H) Minor allele frequency (MAF; in red) and nucleotide diversity (π; in black) calculated in a sliding window across the proximal promoter regions of five opsins (A - E), CNE 7 (LWS-LCR) (F), and two opsin 3'-UTRs (G - H) using 18 Lake Malawi cichlid species. Numbers above peaks of MAF and π denote the position of SNPs analyzed for allelic-association with opsin expression (see Table 6); asterisks (*) denote polymorphisms that interrupt CRX binding sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116502&req=5

Figure 4: Interspecific polymorphism in eight putative cis-regulatory regions from 18 Lake Malawi cichlid species. A - H) Minor allele frequency (MAF; in red) and nucleotide diversity (π; in black) calculated in a sliding window across the proximal promoter regions of five opsins (A - E), CNE 7 (LWS-LCR) (F), and two opsin 3'-UTRs (G - H) using 18 Lake Malawi cichlid species. Numbers above peaks of MAF and π denote the position of SNPs analyzed for allelic-association with opsin expression (see Table 6); asterisks (*) denote polymorphisms that interrupt CRX binding sites.
Mentions: Although the 18 Lake Malawi cichlid species we use have been previously characterized with regard to opsin gene expression, we confirmed these gene expression results by measuring the expression of each opsin in all species via RT-qPCR (see Additional File 8 for opsin expression results). These expression results were highly concordant with previous measurements [26]. Following qPCR, we re-sequenced the entire 1 kb region upstream of both the SWS1 and SWS2A opsins, 956 bp upstream of the LWS opsin, 951 bp upstream of the RH2B opsin, and 694 bp upstream of the SWS2B opsin. We also re-sequenced 900 bp surrounding the LWS-LCR (CNE 7) and 450 bp downstream of the SWS2B and LWS opsins. As expected given the young age of Lake Malawi cichlids, we found that all regions were highly conserved among the species sampled. Overall, we identified fewer than 15 single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) per region examined (Table 5). In each case, most SNPs were found in only one individual. Other diversity statistics--including the total number of segregating sites (S), total number of singletons (s), number of haplotypes (H), nucleotide diversity (π), sequence conservation (C), and Tajima's D (TD)--also indicate low levels of polymorphism, despite our use of alternate species and genera as sampling units (see Additional file 8 for a list of all polymorphisms found among the 18 species sampled). Nevertheless, following a sliding window analysis of nucleotide diversity (π) and minor allele frequency (MAF), we were able to identify several peaks of relatively highπ and MAF within each region (Figure 4). These peaks correspond to SNPs and indels segregating at high frequency within the species and genera sampled, and therefore represent potential cis-regulatory alleles.

Bottom Line: We also found several microRNA target sites within the 3'-UTR of each opsin, including two 3'-UTRs that differ significantly between O. niloticus and M. zebra.We found that all regions were highly conserved with some evidence of CRX transcription factor binding site turnover.We also found three SNPs within two opsin promoters and one non-coding element that had weak association with cichlid opsin expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Maryland, College Park, MD 20742, USA.

ABSTRACT

Background: Divergence within cis-regulatory sequences may contribute to the adaptive evolution of gene expression, but functional alleles in these regions are difficult to identify without abundant genomic resources. Among African cichlid fishes, the differential expression of seven opsin genes has produced adaptive differences in visual sensitivity. Quantitative genetic analysis suggests that cis-regulatory alleles near the SWS2-LWS opsins may contribute to this variation. Here, we sequence BACs containing the opsin genes of two cichlids, Oreochromis niloticus and Metriaclima zebra. We use phylogenetic footprinting and shadowing to examine divergence in conserved non-coding elements, promoter sequences, and 3'-UTRs surrounding each opsin in search of candidate cis-regulatory sequences that influence cichlid opsin expression.

Results: We identified 20 conserved non-coding elements surrounding the opsins of cichlids and other teleosts, including one known enhancer and a retinal microRNA. Most conserved elements contained computationally-predicted binding sites that correspond to transcription factors that function in vertebrate opsin expression; O. niloticus and M. zebra were significantly divergent in two of these. Similarly, we found a large number of relevant transcription factor binding sites within each opsin's proximal promoter, and identified five opsins that were considerably divergent in both expression and the number of transcription factor binding sites shared between O. niloticus and M. zebra. We also found several microRNA target sites within the 3'-UTR of each opsin, including two 3'-UTRs that differ significantly between O. niloticus and M. zebra. Finally, we examined interspecific divergence among 18 phenotypically diverse cichlids from Lake Malawi for one conserved non-coding element, two 3'-UTRs, and five opsin proximal promoters. We found that all regions were highly conserved with some evidence of CRX transcription factor binding site turnover. We also found three SNPs within two opsin promoters and one non-coding element that had weak association with cichlid opsin expression.

Conclusions: This study is the first to systematically search the opsins of cichlids for putative cis-regulatory sequences. Although many putative regulatory regions are highly conserved across a large number of phenotypically diverse cichlids, we found at least nine divergent sequences that could contribute to opsin expression differences in cis and stand out as candidates for future functional analyses.

Show MeSH
Related in: MedlinePlus