Article, Resources

dbCNS: a new database for conserved noncoding sequences
Jun Inoue1,2 and Naruya Saitou1,3Population Genetics Laboratory, Department of Genomics and Evolutionary Biology
National Institute of Genetics, Mishima, Japan
2
Center for Earth Surface System Dynamics, Atmosphere and Ocean Research Institute,
University of Tokyo, Kashiwa, Japan
3
Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
1

Abstract
We developed dbCNS (http://yamasati.nig.ac.jp/dbcns), a new database for conserved
noncoding sequences (CNSs). CNSs exist in many eukaryotes and are assumed to be involvedin protein expression control. Version 1 of dbCNS, introduced here, includes a powerful andprecise CNS identification pipeline for multiple vertebrate genomes. Mutations in CNSs may
induce morphological changes, but also cause genetic diseases. For this reason, manyvertebrate CNSs have been identified, with special reference to primate genomes. We
integrated ~6.9 million CNSs from many vertebrate genomes into dbCNS, which allows users
to extract CNSs near genes of interest using keyword searches. In addition to CNSs, dbCNS
contains published genome sequences of 161 species. With purposeful taxonomic sampling ofgenomes, users can employ CNSs as queries to reconstruct CNS alignments and phylogenetic
trees, to evaluate CNS modifications, acquisitions, and losses, and to roughly identify species
with CNSs having accelerated substitution rates. dbCNS also produces links to dbSNP for
searching pathogenic SNPs in human CNSs. Thus, dbCNS connects morphological changes
with genetic diseases. A test analysis using 38 gnathostome genomes was accomplished within30s. dbCNS results can evaluate CNSs identified by other stand-alone programs using genomescale data.
Keywords: dbCNS, conserved noncoding sequences, vertebrates, single nucleotide
polymorphisms, cis-regulatory elements© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided theoriginal work is properly cited.

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Corresponding author: Naruya Saitou
Email: saitounr@nig.ac.jp
Phone/FAX: +81-55-981-6790/-6789
Postal address: 1111 Yata, Mishima 411-8540, Japan

Introduction
It has long been speculated that protein noncoding regions are involved in protein expression
control (King and Wilson 1975). Genomic sequence comparisons between humans and fugu
conservation among vertebrate genomes (Aparicio et al. 1995). Although conserved sequencesof noncoding regions are identified in the literature with different names, such as CNEs
(conserved noncoding elements: Woolfe et al. 2005) or UCEs (ultraconserved elements:
Bejerano et al. 2004), the prevailing view is that these sets of sequences are largely overlappingin their genesis and functions, and that their evolutionary dynamics are largely unknown(Polychronopoulos et al. 2017). In this paper, we call all such sequences “conserved noncoding
sequences” or CNSs. CNSs tend to cluster in the vicinity of genes with regulatory roles in
multicellular development and differentiation (Sumiyama and Saitou 2012). In fact, CNS
mutations may result in vertebrate morphological changes, or may cause human genetic
diseases (Polychronopoulos et al. 2017).
The recent rapid growth of genome data has made it possible to identify CNSs
particularly among vertebrates. For the last ten years, we have been studying CNSs among
various taxonomic groups such as plants (Hecchiarachci et al. 2014), vertebrates (Matsunami
et al. 2010; Matsunami and Saitou 2013; Hecchiarachci and Saitou 2016), mammals(Babarinde et al. 2013), rodents (Takahashi and Saitou, 2012), and primates (Takahashi and
Saitou, 2012; Babarinde and Saitou 2016; Saber et al. 2016, 2017). Some of them examinedthe contribution of putative regulatory CNSs in defining clade-specific phenotypes (e.g.,Babarinde and Saitou 2013; Matsunami and Saitou 2013; Saber et al. 2017). Recently, CNSs
have been identified as evolutionarily conserved elements, based on genome alignments using
tools such as PhastCons (Siepel et al. 2005) and GERP (Davydov et al. 2010). However,preparation of genome alignments and analyses using such tools are computationally intensive.
As far as we know, there are only four CNS-related databases. The VISTA Browser(https://enhancer.lbl.gov) distributes CNSs identified in humans and mice that have been tested
in vivo for enhancer activity (Visel et al. 2007), and VISTA’s web tools(http://genome.lbl.gov/vista/index.shtml) allow inspection and comparison of sequenceconservation profiles across specified genomic regions in a user-customizable manner (Brudno
et al. 2007). ANCORA (http://ancora.genereg.net), developed by Engstrom et al. (2008),
distributes metazoan CNSs identified by scanning pairwise genome alignments (e.g., humans
vs chickens). This web resource can be used to discover developmental regulatory genes and2

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(pufferfish) revealed that a class of noncoding genomic sequences display an extra degree ofto distinguish their chromosomal regulatory domains by viewing CNS locations and densities
in the UCSC Genome Browser (Kuhn et al. 2007). Persampieri et al. (2008) developed
cneViewer (http://bioinformatics.bc.edu/chuanglab/cneViewer/) for noncoding DNA elements
specific gene regulation, based on known developmental expression patterns of nearby genes.
Dimitrieva and Bucher (2013) developed UCNEbase (https://ccg.epfl.ch/UCNEbase) that
identifies 4,351 CNSs shared among 18 vertebrates. UCNEbase features a consistent naming
scheme to identify elements across genomes, along with descriptive statistics of element
distributions and synteny maps. These databases, however, are not frequently updated and do
not accommodate demands to identify CNSs using user-provided sequences as queries in
specific taxonomic sampling. Moreover, no database exists to link causal single-nucleotide
polymorphisms (SNPs) to morphological changes and/or genetic diseases.
New Approaches
By integrating CNSs among vertebrates scattered among databases and journal articles, wecreated a new database called dbCNS (http://yamasati.nig.ac.jp/dbcns). dbCNS allows users
not only to extract published CNSs as regulatory candidates of interest, but also to search for
CNSs in user-selected genomes. For this purpose, dbCNS also contains some invertebrate
genomes. dbCNS automatically produces coordinates, multiple alignments, and phylogenetic
trees. Using these outputs, users can evaluate extracted sequences as CNSs within areas ofinterest and can detect potential CNSs with accelerated substitution rates. Users can also countidentical CNSs in a genome in dbCNS, something no other database has been able to do,
because of their reliance on genome alignments to identify CNSs.

Results and Discussion
Interface and two query search modes
Figure 1 shows the upper part of the top page of dbCNS version 1. dbCNS contains ~6.9 million
CNSs published in journals and in databases (see Table 1), and it also contains sequences of162

vertebrateand9invertebrate

genomesdownloaded

fromEnsembl(http://www.ensembl.org) and NCBI (https://www.ncbi.nlm.nih.gov) (Table S1). Phylogenetic
relationships of the genomic sequence datasets in dbCNS are shown in Figure 2. dbCNS holds
a list of gene coordinates for each species to identify the nearest genes (upstream and3

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in zebrafish. Its key feature is the ability to search for CNSs that may be relevant to tissue-

downstream) of BLAST hits. Two main functions are available in dbCNS: (A) Query searchand (B) BLAST and alignment. Flowcharts are shown in Supplementary Fig. S1A. The web
design of dbCNS follows that of ORTHOSCOPE, developed by Inoue and Satoh (2019)
There are two query search modes (A1 and A2) in dbCNS. When a keyword isprovided by the user, dbCNS collects CNSs near the gene of interest in “Keyword search”
mode. For this purpose, each record of the CNS database has a name line, including the nameof the nearest gene locus (See example in Supplementary Fig. S1B). By finding the keyword
in name lines, dbCNS lists search results as output. An example output of 195 hits for the
keyword “HoxA1” is shown in Supplementary Fig. S2. One can download a tab-separated filefrom the link shown after “Download tab-separated file” located at the top of this output.
dbCNS also allows users to link the potential target gene and CNSs with a user-specified
distance with the option “CNS distance from the gene of keyword.” When a coordinate isprovided by the user in “Sequence extraction” mode, dbCNS extracts the corresponding
sequence from the genome data of a selected model organism with BLASTDBCMD (Altschul
et al. 1990). An example of output for “7:27097212-27097599” as the coordinates of a 388-bp
sequence at chromosome 7 for the HoxA1-related CNS (Matsunami et al. 2010) from thehuman genome, build GRCh38/hg38, is shown in Supplementary Fig. S3A. Alternatively,
when an SNP is provided with its coordinates, dbCNS generates a sequence consisting of the
SNP with 100-bp fragments both 5’ upstream and 3’ downstream. Fragment lengths can beselected with the “Flank lengths to SNPs” option. Example output for “11:31664397>A” as
the coordinate at chromosome 11 for the human genome, build GRCh38/hg38, is shown in
Supplementary

Fig.

S3B.

This

SNP

C>A

at

rs606231388

in

dbSNP

(http://www.ncbi.nlm.nih.gov/SNP) causes the human ocular disease, aniridia (Bhatia et al.
2013; see “A case study” below).
BLAST and multiple alignment
In the “BLAST & alignment” mode of dbCNS, a CNS should be provided in FASTA format.
An example CNS (a 201-bp sequence in the human Simo enhancer region: GRCh38_1131664297-31664497)

is

shown

in

http://yamasati.nig.ac.jp/dbcns/examples/exampleQuerySeq.html. A BLAST search (Altschul
et al. 1990) is first conducted using that query sequence in dbCNS. BLAST hits are thenmultiply aligned using MAFFT (Katoh and Standley 2013) and TRIMAL (Capella-Gutierrez

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(https://www.orthoscope.jp).

et al. 2009), and the corresponding neighbor-joining tree (Saitou and New 1987) for these
multiply aligned sequences is generated using APE 3.0 (Popescu et al. 2012) automatically.
The most parameter-rich model in the program, the TN 93 model (Tamura and Nei 1993), is
Before starting an analysis, the user needs to set parameters in “BLAST options” forthe similarity search: “-tasks” sets parameters to typical values for a specific type of search.
“BLASTN” finds regions of local similarity between nucleotide sequences. For much longer
DNA sequences, “MEGABLAST” can be selected for intraspecific comparisons with large“word-size” (see below) and “DC-MEGABLAST” to find more distant (interspecific)
sequences. “-word_size” determines the length of an initial exact match. “-evalue” is a
threshold expect value for saving hits, and “-num_alignments” determines the number of
BLAST hits report per genome. “perc_identity” discards alignments that do not meet aminimum%

identity.

In“DC-MEGABLAST

option”

using

DC-MEGABLAST,“template_length” determines lengths of templates. In “BLASTDBCMD option”, using
BLASTDBCMD, “-range” provides lengths of 5’ upstream and 3’ downstream sequences for
extracting flanking sequences of BLAST hits. Taxonomic sampling is determined by selecting
species in “Genome taxon sampling” or by uploading a batch file (See Appendix for details ofbatch file description).
If we submit example file to dbCNS, the result file is created after ~33 seconds ofcomputation. Figure 3 shows the flow of information in this example. The summary output(Supplementary Fig. S4A) can be seen by clicking the link after “Status Finished”, just above
the “SUBMIT” button. This summary output shows the query sequence, numbers of BLAST
hits for each selected genome sequence, multiple alignment of BLAST hits, a phylogenetic
tree, and setting details. In addition to numbers of BLAST hits for each species, dbCNS
provides coordinates and nearest genes in name lines. These are linked to the Ensembl genome
browser to show their genomic positions. In the resultant alignment, poorly aligned sites are
identified using TRIMAL with the option “-gappyout.” Such sites are marked with “0” whereas
unambiguously aligned sites are identified with “1.” One can download the output (in zip
format) from the link shown after “Download”, located at the top of this summary output. Thisdetailed output folder contains files, including an analytical summary, a multiple alignment,
and a phylogenetic tree.
Three case studies related to PAX6

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applied with a gamma distributed rate for site heterogeneity (Yang 1994).

We demonstrate the utility of dbCNS using three case studies related to the PAX6 gene, withtaxonomic sampling relative to gnathostomes and teleosts. The multi-functional developmental
regulator, PAX6, is essential to development and maintenance of the central nervous system2013). This gene is best known for its critical role in eye development (Gehring and Ikeo 1999;
Cvekl and Callaerts 2017). In nearly every species that uses vision, development of the eyes iscritically dependent on the presence and dosage of PAX6 (Gehring 2005). Extensive effort hasgone into characterizing spatio-temporal regulation of PAX6 expression (Kleinjan et al. 2006).
A genomic regulatory block has been identified by finding long syntenic arrays of CNSs
clustered around this block (Kikuta et al. 2007).
Case study 1: Construction of CNS alignment, including an SNP that causes human disease
Based on coordinates of human SNP sites, dbCNS can construct multiple sequence alignments
to evaluate evolutionary conservation of genomic regions, including specified sites. Aniridia(OMIM ID 106210) is a panocular disease characterized by a variable degree of iris/foveal
hypoplasia, nystagmus, and ciliary body abnormalities. In a patient with aniridia and no exonic
mutations or chromosomal abnormalities, direct sequencing of cis-regulatory elements activein various eye tissues revealed a single nucleotide change in a conserved ocular enhancer,
SIMO, located 150 kb downstream from PAX6 (Bhatia et al. 2013). The SNP that causes
aniridia (Bhatia et al. 2013) is C>A at rs606231388 in dbSNP.
As we already showed in an example of “sequence extraction mode”, dbCNS
extracted a 201-bp sequence, including this SNP site, from the reference human genome
sequence (hg38) using “11:31664397>A” as a keyword (Supplementary Fig. S3B). Using thissequence, output of the BLAST & alignment mode was generated with 38 gnathostome
genomes (Supplementary Fig. S4A). In this analysis, the “-num_alignments” option was set at
two in order to count identified CNSs in each species. As a result, dbCNS identified at mostone BLAST hit for each species (shown in [# of blast hits]) and automatically aligned them.
The alignment showed that all BLAST hits of gnathostomes contain the PAX6 binding site andbelong to the SIMO region (Bhatia et al. 2013), except for the partial sequence of Erpetoichthys
calabaricus (reedfish). Then we confirmed that these BLAST hits are identical to the humanquery sequence and form a CNS as a highly conserved part of the SIMO enhancer (Antosova
et al. 2016).

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(Osumi et al. 2008), the olfactory system (Nomura et al. 2007), and the pancreas (Hart et al .

The alignment (Fig. 4A) confirmed that in this aniridia-related site, most tetrapods
share the same nucleotide C and the mutation changed the human nucleotide from C>A. In
addition, the alignment showed that all five snakes share A at this site. In this case, dbCNS can(Fig. 4B) suggested that in the snake lineage, branches leading to the common ancestor of thefive snakes possessed an increased number of substitutions compared to peripheral branches.
These findings imply that characteristics of the snake SIMO region were fixed before
divergence of the major snake lineages. This CNS diversification in snake ancestors is
consistent with their possible subterranean lifestyle (Da Silva et al. 2018) and the loss of opsinsin the early stage of snake evolution (Simoes et al. 2015). In contrast, four subterraneanmammals (species names are shown in red) showing convergent eye degeneration shared thenucleotide C with most other tetrapods (Fig. 4A). In subterranean mammals, several CNSs near
PAX6 loci and other transcription factors important for eye development exhibit accelerated
substitution rates (Partha et al. 2017). In this analysis of the SIMO region, an accelerated
substitution rate was suggested for the lineage leading to the subterranean mammal,
Heterocephalus glaber (naked mole rat), compared to other eutherians (Fig. 4B). The naked
mole rat sequence is not placed next to related species probably due to its high sequence
divergence. For more sophisticated analyses of accelerated substitution rates with user-definedtree-topologies, users can employ state-of-the-art methods, such as RERconverge (Kowalczyk
et al. 2019), using dbCNS outputs.
dbCNS produces a link to dbSNP (build 153) using BLAST-hit coordinates derived
from the human genome (hg38). By clicking the link “11:31664297-31664497” located below“Human SNP in dbSNP:” in the output html file (Supplementary Fig. S4A), it was confirmedthat the aniridia-causing SNP site (rs606231388) is located in this human BLAST hit.
Moreover, dbCNS can analyze SNPs identified in genome-wide association studies (GWAS).
For example, nasopharyngeal carcinoma-related SNP (Madelaine et al. 2018) can be analyzed
using “3:169364845>A” (hg38) as a keyword (Supplementary Fig. S4B).
Case study 2: Detection of CNSs in gnathostome genomes
dbCNS can evaluate the existence or number of CNSs in genomes. In addition to the SIMO
region, several CNSs were annotated as cis-regulatory elements that control expression of
PAX6 in various tissues, including the eye. Bhatia et al. (2014) identified CNSs in the RCN1PAX6 intergenic region by employing a strategy that analyzes gnathostome sequence

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Downloaded from https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msaa296/5983335 by Idenkagaku Kenkyujo user on 19 November 2020be used to detect CNS candidates with accelerated substitution rates. The estimated CNS treeconservation and tests identified CNSs of the elephant shark for enhancer activity using a
combination of zebrafish and mouse transgenic studies.
Thus, we examined CNSs shared among other gnathostomes. Using 20 published CNS
from human genome data (Supplementary Table S2A). The existence of identical CNSs wasthen evaluated for 38 gnathostome genomes using extracted CNSs as queries in the “BLAST& alignment” mode. The option “-num_alignments” was set at two to detect duplicated CNSs
in each genome. A method for conducting multiple analyses and summarizing results is shownon the instruction page. BLAST hits were detected for all species analyzed only for agCNE2
and cre149, while all four teleost genomes lacked nine CNSs (Fig. 5A). Although most BLAST
hits were single, two hits were detected for several species, such as Podarcis muralis (common
wall lizard), Equus caballus (horse), and Aotus nancymaae (Nancy Ma's night monkey).
These BLAST hits were mapped onto genomic regions of eight gnathostome species
to determine the presence of CNSs around the PAX6 locus (Fig. 5B). Summary statistics fromthose 20 analyses were generated by using our customized command-line scripts available fromthe dbCNS instruction page. In addition to their genomic positions, we identified CNSs byevaluating sequence alignments and bit scores. As a result, a single CNS was identified in thisregion, although Podarcis muralis possessed six duplicated CNSs.
The six duplicated CNSs (agCNS9–13 and P2) of P. muralis formed a pair of blocks:
an 11-kbp region consisting of the six CNSs with the same order as in the human genome anda 37-kbp region, including additional three CNSs (agCNS6–8) with reversed order. The
arrangement of P. muralis CNSs appears to show vestiges of duplication and inversion, though
CNSs of other gnathostomes were aligned in the same order as in the human genome. Thisconserved architecture shared among gnathostomes is probably important for the PAX6
system.
To illustrate the novelty of dbCNS, identified CNSs were compared with thoseestimated using a pioneering web tool in this field, mVISTA (Frazer et al. 2004;
http://genome.lbl.gov/vista/mvista/submit.shtml), with special reference to the vestiges of
duplication and inversion in the intergenic RCN1–PAX6 region. As far as we know, except for
dbCNS, mVISTA is the only other web tool that can identify CNSs in response to user requests.
Although mVISTA has a feature to identify novel CNSs, users must prepare sequences ofinterest for all species. When the intergenic RCN1–PAX6 region was compared among eightspecies used in gnathostome analyses (Fig. 5B), mVISTA identified almost the same CNSs

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coordinates (Supplementary Table S1), the dbCNS “Sequence extraction” mode reported CNSs

sets (Supplementary Fig. S6A) as those identified by dbCNS (Supplementary Fig. S6B).
However, despite weak detection of two CNSs (agCNE7 in Lepisosteus and agCNE12 inDanio) not detected in dbCNS analyses, mVISTA could not identify eleven CNSs locatedfrom identifying these duplicated CNSs using multiple sequence alignments.
Case study 3: Detection of lineage-specific CNSs from teleost genomes
dbCNS can detect lineage-specific CNSs. Due to the additional whole genome duplication in
the teleost lineage (teleost genome duplication [TGD]) and its consequently increased rate ofevolutionary divergence, teleost genomes lack many CNSs identifiable in other vertebrates(Lee et al. 2011). In fact, only three CNSs (agCNE9, agCNE13, and cre149 in Fig. 5B) wereidentified around the PAX6b locus of the Danio rerio (zebrafish) genome in our gnathostome
analysis. Using the keyword “PAX6b” for an analysis in the “Keyword search” mode, 164
CNSs conserved between zebrafish (D. rerio) and sticklebacks (Gasterosteus aculeatus) werelisted. Among those, 30 zebrafish sequences (zs1–zs30 in Supplementary Table S2B) had morethan four BLAST hits when analyses were conducted in BLAST & alignment mode with our
teleost taxon sampling (Fig. 6A). Results of these 30 analyses are summarized on the right sideof Fig. 6A.
Single BLAST hits were detected in many cases (Fig. 6A). When mapping BLAST
hits of Oryzias latipes (medaka) chromosome 3 on the region around the PAX6b locus, 17 of
30 query CNSs of D. rerio had identical CNSs (blue letters in Fig. 6B). However, two BLAST
hits were detected when some of these 30 CNS queries were used, especially for D. rerio (Fig.
6A). When mapping BLAST hits of D. rerio around the PAX6a locus in chromosome 25, 10
out of 30 query CNSs (blue letters in Fig. 6B) had TGD-derived counterparts. The teleost
PAX6a gene is known as the counterpart of the PAX6b gene derived from the TGD (Feiner et
al. 2014). Given the preservation of the ancestral PAX6 synteny block around the teleost PAX6b
locus (Fig. 6C), and the singleton status of PAX6b-adjacent genes, RCN1 and ELP4, in the lastcommon ancestor of teleosts (Supplementary Fig. S5), counterparts of teleost RCN1 and ELP4
genes from TGD are considered lost from the region around the PAX6a locus (Fig. 6C). Thisdisappearance of adjacent genes, RCN1 and ELP4 counterparts, supports the hypothesis that in
the D. rerio genome, these 10 CNS counterparts function as regulatory elements of the PAX6agene, as suggested by Kikuta et al. (2007).

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within the 37-kbp block of Podarcis. Vestiges of duplication and inversion prevented mVISTA

Conclusion
dbCNS (http://yamasati.nig.ac.jp/dbcns), a dynamic web database, enables researchers in gene
regulation and human diseases to identify CNSs and their genomic properties. Recently, toapproaches based on assessing chromatin state (ChIP-seq) and accessibility (e.g. DNaseI-seq,
ATAC-seq) have been applied (Martinez-Morales 2015; Roscito et al. 2018). Researchers can
examine how such novel elements have changed during evolution of traits and species using
dbCNS. In addition, dbCNS can evaluate CNSs identified by other CNS-identification
programs using genome-wide data such as PHAST (Hubisz et al. 2011) and CNEr (Tan et al.
2019). Identified CNSs can be used to test their enhancer activity using suitable alternativemodel systems, such as transgenic reporter zebrafish (Bhatia et al. 2014). Moreover, dbCNS
can be used not only to evaluate clade-specific CNSs, but also to examine architectures ofnoncoding sequences. dbCNS currently has several limitations: (1) analyses are specialized forsingle-molecule data, not for genome-wide data. (2) Users should evaluate alignments,
coordinates, and bit scores of BLAST hits to confirm the presence of CNSs in genomic regionsof interest. (3) Lengths of query sequences should be less than 1,000 bp to avoid separation ofa target sequence into several BLAST hits. In addition to current vertebrate data, dbCNS will
include published CNSs and genome sequences from non-vertebrate metazoans, plants, fungi,
and prokaryotes in the near future. Moreover, as our future tasks, the CNS database can be
integrated

with

gene

regulatory

data

from

resources

such

as

ENCODE(http://genome.ucsc.edu/ENCODE) and FANTOM (https://fantom.gsc.riken.jp). Use of
dbCNS by researchers will facilitate our updates.

Materials and Methods
The dbCNS server runs on the Linux operating system. An Apache HTTP Server provides web
services. Python scripts process all data and requests from users. All these resources have been
extensively used and are well supported.
Acknowledgements
We thank Genesis Healthcare for financial support and all members of the Population
Genomics Laboratory for discussions about the database. We thank Steven D. Aird forediting the manuscript. Critical comments from three anonymous reviewers were useful for

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identify novel regulatory elements in the whole genome of a single species, high-throughputimproving the manuscript. This work was supported by the Japan Society for the Promotionof Science (JSPS) Grants-in-Aid for Scientific Research (C) (18K06396) to J.I.

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Figure Legends
Fig. 1
The front page of dbCNS.
Fig. 2Phylogenetic relationships of 180 genomes for which sequence data is included in dbCNS.
Fig. 3Results of dbCNS analyses for an SNP that causes aniridia. (A) Query sequence. The letterwith a red background indicates the SNP site. (B) Name line of alignment. The name line
includes the nearest gene of the BLAST hit identified by the transcription start site (TSS).
Links of coordinates and nearest genes lead to the Ensemble genome browser. (C) Genomic
position in Ensembl.

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Fig. 4(A)

Alignment of the main part of the SIMO region (Fig. S4). In the query sequence,

YOURSEQ1, the SNP site is highlighted with a red background.
Phylogenetic tree based on sequences of the SIMO region (121 sites).

Fig. 5Results of gnathostome analyses.
(A) BLAST hits for CNS queries around human PAX6 locus. An arrowhead indicates the rowof humans, sequences of which were used as queries. Phylogenetic positions of wholegenome duplications (VGD, vertebrate genome duplication; TGD, teleost genome
duplication) follow Braasch and Postlethwait (2012). The heatmap was summarized by usinga script available from the dbCNS instruction page.
(B) Overview of CNS positions around PAX6 loci. The black line represents DNA. Red
letters indicate CNS queries in humans and blue letters indicate CNS BLAST hits in nonhuman gnathostomes. Rectangles indicate the PAX6 locus (red) and adjacent RCN1 (green)and ELP4 loci (yellow). In P. muralis, thin horizontal arrows indicate putative duplicatedregions. Arrows within gene loci indicate TSS.
Fig. 6
Results of teleost analyses.
(A) BLAST hits for CNS queries around the zebrafish PAX6b locus. All 30 zsCNSs wereidentified by comparing zebrafish and stickleback genomes in the ANCORA database(Engstrom et al. 2008). An arrowhead indicates the row of D. rerio, sequences of which wereused as queries.
(B) Overview of CNS positions around zebrafish and medaka PAX6b and zebrafish PAX6aloci. Red letters indicate CNSs queries around zebrafish PAX6b loci and blue letters indicate
CNS blast hits in other regions.
(C) Ancestral PAX6 synteny blocks of teleosts. PAX6 synteny blocks were compared amongbony vertebrate genomes using a conserved synteny browser, Genomicus ver. 98.01 (Muffato
et al., 2010). Based on the PAX6 gene tree (Feiner et al. 2014), hypothetical ancestral statesaround the PAX6 locus were reconstructed using parsimony.

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(B)

Appendix
Batch file example of the taxon sampling list

Downloaded from https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msaa296/5983335 by Idenkagaku Kenkyujo user on 19 November 2020

Callorhinchus-milii_Black
Rhincodon-typus-N_Black
Erpetoichthys-calabaricus_Green
Lepisosteus-oculatus_Green
Danio-rerio_Green
Gasterosteus-aculeatus_Green
Tetraodon-nigroviridis_Green
Oryzias-latipes_Green
Latimeria-chalumnae_Purple
Microcaecilia-unicolor-N_Purple
Xenopus-tropicalis_Purple
Sphenodon-punctatus_Orange
Podarcis-muralis-N_Orange
Anolis-carolinensis_Orange
Python-bivittatus-N_Orange
Protobothrops-mucrosquamatus-N_Orange
Thamnophis-sirtalis-N_Orange
Pseudonaja-textilis-N_Orange
Notechis-scutatus_Orange
Chelonoidis-abingdonii_Magenta
Alligator-mississippiensis-N_Magenta
Nothoprocta-perdicaria_Magenta
Gallus-gallus_Magenta
Calidris-pugnax_Magenta
Ornithorhynchus-anatinus_Blue
Monodelphis-domestica_Blue
Choloepus-hoffmanni_Blue
Chrysochloris-asiatica-N_Red
Condylura-cristata-N_Red
Myotis-lucifugus_Blue
Equus-caballus_Blue
Bos-taurus_Blue
Heterocephalus-glaber-male_Red
Nannospalax-galili_Red
Mus-musculus_Blue
Propithecus-coquereli_Blue
Aotus-nancymaae_Blue
Homo-sapiens_Blue15c

http://ancora.genereg.net/downloads/danRer10/vs_stickleback/HCNE_danRer10_gasAcu1_70pc_30col.bed.gzdhttp://ancora.genereg.net/downloads/hg38/vs_chicken/HCNE_hg38_galGal4_100pc_50col.bedehttps://genome.ucsc.edu/cgi-bin/hgTablesf
http://ancora.genereg.net/downloads/hg38/vs_dog/HCNE_hg38_canFam3_100pc_50col.bed.gzg
http://ancora.genereg.net/downloads/canFam3/vs_horse/HCNE_canFam3_equCab2_80pc_50col.bed.gzhhttp://ancora.genereg.net/downloads/canFam3/vs_horse/HCNE_canFam3_equCab2_100pc_50col.bed.gzi
http://ancora.genereg.net/downloads/hg38/vs_mouse/HCNE_hg38_mm10_80pc_50col.bed.gz

16

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Table 1. CNSs stored in dbCNS————————————————————————————————————————
Clade
Comparison
Sequence# of CNS Data source————————————————————————————————————————
Vertebrata
18 vertebrates
Human4351 https://ccg.epfl.ch//UCNEbase(hg19)
Gnathostomata
19 gnathostomes
Human
208 Matsunami et al (2010)a(hg19)
Bony vertebrates Human, Zebrafish Human
18,852 ANCORAb(hg38)(70% identity over 50 columns)
Actinopterygii
Clupeocephala
Zebrafish,
Zebrafish
200,099 ANCORAc
Stickleback(danRer10)(70% identity over 30 columns)
Zebrafish,
Stickleback
175,168 ANCORAc
Stickleback(BROADS1)(70% identity over 30 columns)
Sarcopterygii
8 tetrapods
Human ortho
7650 Matsunami and Saitou (2013)(GRCh37)
8 tetrapods
Human para
309 Matsunami and Saitou (2013)(GRCh37)
Amniota
Human, Chicken
Human
12,041 ANCORAd(hg38)(100% identity over 50 columns)
Mammalia
20 mammals
Human2752 UCSC Genome Browser e(hg38)(phastCons100way , <1000 bp)
Boreoeutheria
Human, Dog
Human95,462 ANCORAf(hg38)(100% identity over 50 columns)
Laurasiatheria
Dog, Horse
Dog
5,284,452 ANCORAg(80% identity over 50 columns)
Dog, Horse
Dog
126,218 ANCORAh(canFam3)(100% identity over 50 columns)
Euarchontoglires Human, Mouse
Human
946,151 ANCORAi(hg38)(80% identity over 50 columns)
Human, Rat, Mouse Human
481 Bejerano et al (2004)(hg19)
Rodentia Mouse,
Rat Mouse
21,128 Takahashi and Saitou (2012)(mm10)
Primates
Simiiformes
Human, Marmoset Human
8,198 Takahashi and Saitou (2012)(hg38)
Hominoidea
5 hominoids
Human
679 Saber and Saitou (2017)(GRCh37)
Hominidae
4 hominids
Human
1,658 Saber et al (2016)(GRCh37)————————————————————————————————————————a
Hox clusters onlyb
http://ancora.genereg.net/downloads/hg38/vs_zebrafish/HCNE_hg38_danRer7_70pc_50col.bed.gz

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Fig. 1

Molecular Biology and Evolution

Deuterostomia

VertebrataTetrapoda

Mammalia
Eutheria

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Protostomia# ofspecies2
Ecdysozoa
1
Hemichordata1
Echinodermata
Cephalochordata 2
2
Urochordata
2
Cyclostomata
2
Chondrichthyes
49
Actinopterygii
Coelacanthiformes 1
5
Amphibia
11
Lepidosauria
4
Testudines
4
Crocodylia
Aves
18
Monotremata
1
Metatheria
1
Xenarthra
2
Afrotheria
6
Laurasiatheria
22
Glires9
Primates
16

Page 22 of 26

Fig. 2

Page 23 of 26

Molecular Biology and Evolution

Downloaded from https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msaa296/5983335 by Idenkagaku Kenkyujo user on 19 November 2020(A) Query sequence
11:31664397>A (hg38)>Homo-sapiens_GRCh38_11-31664297-31664497
AGATAAACTTTCCCATTGTCATTAATGAGGATGGATAATACTGAGTGACAAGTGCATTAGGAATGACACAGAAAGAGCTGTTAGATTTGTCAGAGCGTATaACTTCAGGCATTGACACTTGAGGACATTCTTTTTG
TGGATATTTGGTTNNNNNNNNAGTCTGATTTAAAGATACCAAAGTTTGTGTTTGCTAATAGAATA(B) Name line

Bit score

Homo-sapiens_GRCh38_11-31664297-31664497_PAX6_330
Assemblyversion

Coordinate

Nearestgene(C) Genomic position in Ensembl

Fig. 3

Molecular Biology and Evolution

PAX6 binding site

1: Unambiguously aligned site
Callorhinchus milii
Rhincodon typus
Erpetoichthys calabaricus
Lepisosteus oculatus
Danio rerio
Latimeria chalumnae
Microcaecilia unicolor
Xenopus tropicalis
Sphenodon punctatus
Podarcis muralis
Anolis carolinensis Snakes
Python bivittatus
Protobothrops mucrosquamatus
Thamnophis sirtalis
Pseudonaja textilis
Notechis scutatus
Chelonoidis abingdonii
Alligator mississippiensis
Nothoprocta perdicaria
Gallus gallus
Calidris pugnax
Ornithorhynchus anatinus
Monodelphis domestica
Chrysochloris asiatica
Condylura cristata
Myotis lucifugus
Equus caballus
Bos taurus
Heterocephalus glaber
Nannospalax galili
Mus musculus
Propithecus coquereli
Aotus nancymaae
Homo sapiens
YOURSEQ1
Subterraneanmammals

SIMO DSIMO CSIMO B
SNP for Aniridia(B) CNS tree
Callorhinchus milii
Rhincodon typus

Latimeria chalumnae
Erpetoichthys calabaricus

Danio rerio
Lepisosteus oculatus
Xenopus tropicalis
Heterocephalus glaber male
Monodelphis domestica

Myotis lucifugus
Mus musculus
Equus caballus
Chrysochloris asiatica
Nannospalax galili
Aotus nancymaae
YOURSEQ1_Homo sapiens_GRCh38_11-31664297-31664497
Homo sapiens
Propithecus coquereli
Bos taurus
Condylura cristata
Microcaecilia unicolor
Ornithorhynchus anatinus

Subterraneanmammals

Gallus gallus

0.05(substitutions/site)

Nothoprocta perdicaria
Chelonoidis abingdonii
Sphenodon punctatus
Calidris pugnax
Alligator mississippiensis
Anolis carolinensis
Podarcis muralis
Protobothrops mucrosquamatus
Notechis scutatus
Pseudonaja textilis
Thamnophis sirtalis
Python bivittatus

SnakesFig. 4

Downloaded from https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msaa296/5983335 by Idenkagaku Kenkyujo user on 19 November 2020(A) Alignment

Page 24 of 26

Molecular Biology and Evolution

Species tree

Chondrichthyes
Actinopterygii

PAX6 ELP4

6
,69

8

43
11
,2

,31
67
11
,1

,61
1

73

91
9,6

9,6

65

11

7,9

E+ ,500
1
SI 20
M
cre O
2
9, 1

24

91

4,3

5,6

16

15

,03

,90

06
0,6
,90
15

66
11
60
81

E+
SI 120
creMO
21

04
36

66
,27
6,6
35

5,3
03
,92
1

E+
SI 120
creMO
21

44
,15
1
5,4

10
5,9
04
,56
4

E+
SI 120
creMO
21

10
5,7
01
,02
7

64
10
5,6
97
,3

9
14
cre

10
5,6
6

P2
P0

65
6,7
98
,7
,50
9,7
55
31

79
0,3
31
,78 24
4,7
79

14
(N 9
31 RE
)
,

cre

31
,8

P0 17,961

PAX6

ENSG00000007372

ELP4

ENSANAG00000025916
E+
1
SI 20
M
cre O
21

PAX6

E+
SI 120
creMO
21

7,0
46
,99
4

7,0
53
,08
1

cre

14

9

74
,66
5
7,0

P2
P0

ELP4

ENSMUSG00000027167

ENSANAG00000033670

P2

SI
creMO
21

6
,42
11
,1

57

14
14
14
9
14
9
14
cre

5,4

8,9

00

ENSGALG00000012127

ENSMUSG00000027168

6)

32
,0

ENSG00000049449

cre8

P061,44

ag ,496C
ag NE
agCNE6
C
ag NE7
agCNE8agCNE9agCNE10agCNE11
CN 12
E1
3

5,4
77

7,3
6

E- 0,38
ag250 0agCNE
agCNE1
agCN 2E3
agCNE
agCN 4agCNE5EC
ag N 6
agCNE7
agCNE8agCNE9agCNE10agCNE11CNE1
E12
3

E- 90,9ag250 04
agCN
E
agCNE1
agCN 2
3agCNE
E
agCNE4agCNE5agCNE6
agCNE7
agCNE8agCNE9agCNE10C
ag NE11CN 12
E1 (cr
3 e8

7,3
73
,27
7
5
5,7
5

RCN1

PAX6
ELP4
PAX6

ENSANAG00000027955

Homo sapiens
chr 11

cre

82

66
,4
5,5

E- 68,46
ag250 5
agCNE
agCNE1
agCNE2
agCNE3
CN 4
E5

10
5

RCN1

RCN1

ELP4
114597275

PAX6
114597346

ENSGALG00000012123

E- ,399,
ag250 319
C
ag NE
agCNE1
agCNE2
agCNE3
agCN 45
agCNE
agCNE6
7agCNE
E
C
ag N 8E
agCNE9agCNE10agCNE11
CN 12
E1
3

10
5,3
86
,29
1

11 kbp

ENSMUSG00000005973

Aotus nancymaae
KZ197012.1

9

1,1
,87
15

E- 63,3ag250 27
C
ag NE
C 1
ag NE
C
ag NE2C
P2 NE34ag
agCNE
agCNE13
agCNE12agCNE11C
ag NE10C 9
ag NE
Cag NE8agCNE7
agCNE6
agCNE9agCNE10agCNE11C
P2 NE1213
66,31
P0 9,3

7,8
04
,47
66

RCN1

ENSDARG00000008852

ENSDARG00000045936

37 kbp

ENSGALG00000012121

Mus musculus
chr 2

cre
56

3
E1
CN
ag

ag

CN

E9

51
,83
15

15

,81

8,7

0,2

78

Gallus gallus
chr 5

RCN1
114597410

ELP4

PAX6b

RCN1

5,5
75
,65
4

Podarcis muralis
chr1

ELP4

ENSLOCG00000004617

ENSLOCG00000004578

ENSDARG00000070006

32
,10

TGD

ELP4

ENSCMIG00000018323

PAX6

RCN1

ENSLOCG00000004562

Danio rerio
chr 7

9

,36
94
9,5

9,5

P2

5

,69
30
9,5

8

ag 8,58
agCNE2
agCNE5agCNE6
agCNE9agCNE10agCNE11
CN 12
E1
3

ag
C
ag NE
CN 1
E2

5

VGDs

cre

PAX6

ENSCMIG00000018190

66
26

RCN1

ENSCMIG00000018083

Lepisosteus oculatus
LG27

9

,40
34
11
,1

,93

6

8

ag
agCNE
agCNE1
agCNE2
agCN 3agCNE5agCNE6
7agCNE
E
agCNE8agCNE9C
ag NE11CN 12E13KI635862.1

11
,0

11
,0

25

PAX6 gene tree Callorhinchus milii

37

,64

4

(B) Genomic positions

ELP4

ENSG00000109911

Fig. 5

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VGDs

Callorhinchus milii
Rhincodon typus
Erpetoichthys calabaricus
Lepisosteus oculatus
TGD
Danio rerio
Gasterosteus aculeatus
Teleostei
Tetraodon nigroviridis
latipes
Coelacanthiformes Oryzias
Latimeria chalumnae
Amphibia
Microcaecilia unicolor
Xenopus tropicalis
Lepidosauria
Sphenodon punctatus
Podarcis muralis
Anolis carolinensis
Python bivittatus
P. mucrosquamatus
Thamnophis sirtalis
Pseudonaja textilis
scutatus
Archelosauria Notechis
Chelonoidis abingdonii
Alligator mississippiensis
Nothoprocta perdicaria
Gallus gallus
Calidris pugnax
Mammalia
Ornithorhynchus anatinus
Monodelphis domestica
Choloepus hoffmanni
Chrysochloris asiatica
Condylura cristata
Myotis lucifugus
Equus caballus
Bos taurus
Heterocephalus glaber
Nannospalax galili
Mus musculus
Propithecus coquereli
Aotus nancymaae
Homo sapiens
Homo sapiens chr 11
RCN1

cr
e1
E+ 49
12
SI 0
M
O
cr
e2
1

(A) Blast hits

E25
ag 0
CN
ag E1
CN
ag E2
CN
ag E3
CN
ag E4
CN
ag E5
CN
ag E6
CN
ag E7
CN
ag E8
CN
ag E9
CN
ag E1
CN 0
ag E1
CN 1
ag E1
CN 2
P2 E1
3
P0

Page 25 of 26

(A) Blast hits
Species tree
Chondrichthyes

TGD
Teleostei

Sarcopterygii

Callorhinchus milii
Rhincodon typus
Erpetoichthys calabaricus
Lepisosteus oculatus
Paramormyrops kingsleyae
Scleropages formosus
Denticeps clupeoides
Danio rerio
Esox lucius
Gadus morhua
Labrus bergylta
Gasterosteus aculeatus
Larimichthys crocea
Tetraodon nigroviridis
Cynoglossus semilaevis
Lates calcarifer
Seriola lalandi dorsalis
Stegastes partitus
Oreochromis niloticus
Hippocampus comes
P. magnuspinnatus
Monopterus albus
Cyprinodon variegatus
Oryzias latipes
Latimeria chalumnae
Xenopus tropicalis
Anolis carolinensis
Gallus gallus
Mus musculus
Homo sapiens
Danio rerio chr 7
RCN1

PAX6b

ELP4

,90
5,6
zs
91
C
zs NS
CN 24
zs S2
C
5
zs NS
C 2
zs NS26
CN 7
zs S2
8
C
zs NS
CN 29
16 S30
,03
4,3
24
3

82
3,6

43
0,0

9,1

,87

,89

14

zs
C
zs NS
CN 26
S2
7
zs
CN
zs S
CN 29
S3
0

PAX6a

ENSDARG00000031420

09

1
CN
zs

zs
CN
15 S2
,02 2
9,0
4

4
S1

9,7

WT1a

ENSORLG00000009918

zsC
zs NS1C
7
zs NS1
CN 8
S1
9

81
zs

,04

S4
CN

,17
15

15

25
8,2

61
4,1
,21
15

ELP4

ENSORLG00000009913

14

zs

zs

PAX6b

ENSORLG00000029091

Danio rerio
chr 25

,36

CN

zs

RCN1

ENSDARG00000008852

zsC
zs NS1
CN 8
S1
9
zs
CN
S2
1
23
,35
8,5
17
zs
CN
S2
3

S2

CN

zs
C
zs NS
CN 12
S1
3
zs
CN
S1
5

78

CN
zs S8
CN
S
23 10
,34
6,4
05

ENSDARG00000045936

S
CN 4
zs S5
CN
S6
23
,34
0,0

19
8,4
,31

zs

23

Oryzias latipes
chr 3

ELP4

PAX6b

ENSDARG00000070006

zsC
zs NS2
CN 5
S2
6
zs
CN
zs S2
CN 8
S2
9
23
,41
6,9
5

RCN1

15

15

15

Danio rerio
chr 7

23

,81

8,7

78

,83
0,2
51
zs
CN
zs S1
CN
S
zs
C 2
zs NS3CN (zs S4 agCCN(
zs NS5 agC E9)
CN
NE
10
zs S6
)
C
(
zs NS7 agCNEC
zs NS8
13
CN
)
S9
15
,87
zs 1,1C
5
zs NS 6C 1
zs NS 0
CN 11
zs S
C 1
zs NS 2C 1
zs NS13C
4
zs NS1
C
5
zs NS
CN 16
zs S1
CN 7
zs S1
C
8
zs NS1 (creC914
zs NS29)C0
zs NS2
CN 1
15 S2
,90 2
0,6
06
zs
CN
S2
3

(B) Genomic positions

DNAJC24ENSDARG00000103379

ENSDARG00000023927(C) Ancestral PAX6 synteny blocks
PAX6 gene tree
ELF3M

WT1

RCN1

RCN1

Bony
vertebrates

TGD

PAX6

PAX6b

ELP4

Lepisosteus LG27
oculatus

ELF3M

Danio rerio chr 7
Oryzias latipes chr 3
Danio rerio chr 25

WT1

RCN1

PAX6

ELP4

IMMP1LDNAJC24

MEF2a

MCTP2b RCN1

PAX6b

ELP4

IMMP1L si:ch73-367j5.3

ELF3M

WT1b

PAX6b

ELP4

IMMP1L

MYOM2b

TCP11L1 CCDC73 WT1a

PAX6a DNACJ24 MPPED2NAV2bOryzias latipes chr 6

PDCD2L NFATC3b DUS2

PAX6a DNAJC24 SLC2A13b

KIF21aGallus gallus chr 5

ELF3M

WT1

RCN1

PAX6

ELP4

IMMP1LDNACJ24

Mus musculus chr 2

WT1

THEM7

RCN1

PAX6

ELP4

IMMP1LDNACJ24

Homo sapiens chr 11

ELF3M

WT1

RCN1

PAX6

ELP4

IMMP1LDNACJ24

ELP4 IMMP1L

RCN1 and ELP4
counterparts lost

PAX6a DNACJ24

Tetrapods

PAX6 synteny block

IMMP1L DNAJC24

RCN1

Teleosts

Downloaded from https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msaa296/5983335 by Idenkagaku Kenkyujo user on 19 November 2020

VGD

Page 26 of 26

zs
C
zs NS
C 1
zs NS
C 2
zs NS
C 3
zs NS (aC 4 g
zs NS (a CNE
C 5 gC 9
zs NS
NE )
CN 6
10
zs S (a
)
g
C 7 C
zs NS
NE
CN 8
13
zs S
)
C 9
zs NS
CN 10
zs S
C 1
zs NS 1C 1
zs NS 2C 1
zs NS 3C 1
zs NS 4C 1
zs NS 5C 1
zs NS 6
CN 17
zs S
C 1
zs NS 8 (C 1 crzs NS 9 e14
CN 20
9)
zs S
C 2
zs NS 1C 2
zs NS 2
CN 23
zs S
C 2
zs NS 4C 2
zs NS 5C 2
zs NS 6
CN 27
zs S
C 2
zs NS 8CN 29
S3
0

Molecular Biology and Evolution

Fig. 6