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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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Conservation between O. niloticus opsin-containing BAC regions and four fish genomes. A) SWS1 opsin-containing region. B) SWS2-LWS opsin-containing region. C) RH2 opsin-containing region. Top line represents O. niloticus BAC sequence. Conserved non-coding elements (CNEs) are numbered and highlighted in red; repetitive sequences are highlighted in green; promoter sequences later examined for interspecific polymorphism are highlighted in blue.
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Figure 1: Conservation between O. niloticus opsin-containing BAC regions and four fish genomes. A) SWS1 opsin-containing region. B) SWS2-LWS opsin-containing region. C) RH2 opsin-containing region. Top line represents O. niloticus BAC sequence. Conserved non-coding elements (CNEs) are numbered and highlighted in red; repetitive sequences are highlighted in green; promoter sequences later examined for interspecific polymorphism are highlighted in blue.

Mentions: The goal of this study is to identify candidate cis-regulatory sequences that control opsin gene expression in African cichlid fishes. Opsins are a group of G protein-coupled receptors that confer sensitivity to light and mediate color vision [20]. African cichlids comprise a diverse clade of freshwater, teleost fish found throughout the lakes and rivers of Africa, including the three African Great Lakes, Lakes Tanganyika, Malawi, and Victoria [21,22]. Cichlids from Lakes Tanganyika and Malawi exhibit dramatic variation in their sensitivity to colored light [23-25]. Species from these lakes exhibit retinal sensitivities that are maximally sensitive to short, middle, or long-wavelength spectra; in some cases, closely related species can differ in their maximal retinal sensitivity by over 100 nm [25-27]. This striking variation makes the cichlid visual system one of the most diverse vertebrate visual systems so far identified. Most variation in cichlid color sensitivity is due to changes in the regulation of their cone opsin genes [26,27]. Cichlids have seven cone opsin genes used for color vision; these opsins are SWS1 (ultraviolet-sensitive), SWS2B (violet-sensitive), SWS2A (blue-sensitive), RH2B (blue-green-sensitive), RH2A and RH2A (green-sensitive), and LWS (red-sensitive) [28]. Additionally, these opsins are located in three regions of the cichlid genome: SWS1 is found on cichlid linkage group (LG) 17; RH2B, RH2A and RH2A are found together in a tandem array on LG 5; and SWS2A, SWS2B, and LWS form a second tandem array on LG 5 (Lee et al. 2005) (Figure 1). Among different cichlid species, these opsins are alternatively co-expressed in three predominant groups, or palettes, to produce the three common visual pigment sets: SWS1-RH2B-RH2A (short wavelength-sensitive), SWS2B-RH2B-RH2A (middle wavelength-sensitive), and SWS2A-RH2A-LWS (long wavelength-sensitive) [26]. Cichlids exhibit several correlations between the expression of their opsins and important ecological variables, including foraging preference and ambient light intensity [26,27]. These correlations suggest that opsin gene expression varies adaptively in cichlids, especially since some expression-ecology correlations have evolved independently among cichlids in different lakes [27]. A recent quantitative genetic analysis of opsin expression in two Lake Malawi cichlids found a quantitative trait locus (QTL) located near the opsin genes [29]. The proximity of this QTL to the opsins suggests that mutations within one or more cis-regulatory sequences may contribute to variation in cichlid opsin expression. But like many non-model systems, few genomic resources are currently available for cichlids, making it difficult to identify potential cis-regulatory alleles and test their association with opsin gene expression.


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)

Conservation between O. niloticus opsin-containing BAC regions and four fish genomes. A) SWS1 opsin-containing region. B) SWS2-LWS opsin-containing region. C) RH2 opsin-containing region. Top line represents O. niloticus BAC sequence. Conserved non-coding elements (CNEs) are numbered and highlighted in red; repetitive sequences are highlighted in green; promoter sequences later examined for interspecific polymorphism are highlighted in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Conservation between O. niloticus opsin-containing BAC regions and four fish genomes. A) SWS1 opsin-containing region. B) SWS2-LWS opsin-containing region. C) RH2 opsin-containing region. Top line represents O. niloticus BAC sequence. Conserved non-coding elements (CNEs) are numbered and highlighted in red; repetitive sequences are highlighted in green; promoter sequences later examined for interspecific polymorphism are highlighted in blue.
Mentions: The goal of this study is to identify candidate cis-regulatory sequences that control opsin gene expression in African cichlid fishes. Opsins are a group of G protein-coupled receptors that confer sensitivity to light and mediate color vision [20]. African cichlids comprise a diverse clade of freshwater, teleost fish found throughout the lakes and rivers of Africa, including the three African Great Lakes, Lakes Tanganyika, Malawi, and Victoria [21,22]. Cichlids from Lakes Tanganyika and Malawi exhibit dramatic variation in their sensitivity to colored light [23-25]. Species from these lakes exhibit retinal sensitivities that are maximally sensitive to short, middle, or long-wavelength spectra; in some cases, closely related species can differ in their maximal retinal sensitivity by over 100 nm [25-27]. This striking variation makes the cichlid visual system one of the most diverse vertebrate visual systems so far identified. Most variation in cichlid color sensitivity is due to changes in the regulation of their cone opsin genes [26,27]. Cichlids have seven cone opsin genes used for color vision; these opsins are SWS1 (ultraviolet-sensitive), SWS2B (violet-sensitive), SWS2A (blue-sensitive), RH2B (blue-green-sensitive), RH2A and RH2A (green-sensitive), and LWS (red-sensitive) [28]. Additionally, these opsins are located in three regions of the cichlid genome: SWS1 is found on cichlid linkage group (LG) 17; RH2B, RH2A and RH2A are found together in a tandem array on LG 5; and SWS2A, SWS2B, and LWS form a second tandem array on LG 5 (Lee et al. 2005) (Figure 1). Among different cichlid species, these opsins are alternatively co-expressed in three predominant groups, or palettes, to produce the three common visual pigment sets: SWS1-RH2B-RH2A (short wavelength-sensitive), SWS2B-RH2B-RH2A (middle wavelength-sensitive), and SWS2A-RH2A-LWS (long wavelength-sensitive) [26]. Cichlids exhibit several correlations between the expression of their opsins and important ecological variables, including foraging preference and ambient light intensity [26,27]. These correlations suggest that opsin gene expression varies adaptively in cichlids, especially since some expression-ecology correlations have evolved independently among cichlids in different lakes [27]. A recent quantitative genetic analysis of opsin expression in two Lake Malawi cichlids found a quantitative trait locus (QTL) located near the opsin genes [29]. The proximity of this QTL to the opsins suggests that mutations within one or more cis-regulatory sequences may contribute to variation in cichlid opsin expression. But like many non-model systems, few genomic resources are currently available for cichlids, making it difficult to identify potential cis-regulatory alleles and test their association with opsin gene expression.

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