The evolutionary origin and domestication history of goldfish (Carassius auratus)

Abstract

Significance
We assembled one of the most contiguous genomes for the common goldfish and unveiled the genetic architecture of many well-known and anatomically interesting traits, owing to the sequencing of a large collection of goldfish varieties. The datasets that we have generated for candidate genes or genomic regions based on population genomics may help to elucidate the evolution of goldfish and goldfish varieties and may provide a resource for a wide range of studies with important implications for the use of goldfish as a model for vertebrate and fish genetics.

Full text

The evolutionary origin and domestication history of
goldfish (Carassius auratus)
Duo Chen
a,b,1
, Qing Zhang
c,1
, Weiqi Tang
d,1
, Zhen Huang
a,b,1,2
, Gang Wang
c,1
, Yongjun Wang
c
, Jiaxian Shi
a,c
,
Huimin Xu
c
, Lianyu Lin
c
, Zhen Li
c
, Wenchao Chi
d
, Likun Huang
e
, Jing Xia
e
, Xingtan Zhang
c
, Lin Guo
c
, Yuanyuan Wang
c
,
Panpan Ma
c
, Juan Tang
f
, Gang Zhou
f
, Min Liu
f
, Fuyan Liu
f
, Xiuting Hua
c
, Baiyu Wang
c
, Qiaochu Shen
c
, Qing Jiang
c
,
Jingxian Lin
c
, Xuequn Chen
c
, Hongbo Wang
c
, Meijie Dou
c
, Lei Liu
c
, Haoran Pan
c
, Yiying Qi
c
, Bin Wu
g
, Jingping Fang
a
,
Yitao Zhou
a,b
, Wan Cen
a
, Wenjin He
a,h
, Qiujin Zhang
a
, Ting Xue
a,h,i
, Gang Lin
a,i
, Wenchun Zhang
j
, Zhongjian Liu
k
,
Liming Qu
l
, Aiming Wang
m
, Qichang Ye
j
, Jianming Chen
d
, Yanding Zhang
b
, Ray Ming
n
, Marc Van Montagu
o,p,2
,
Haibao Tang
c,2
, Yves Van de Peer
o,p,q,r,2
, Youqiang Chen
a,i,2
, and Jisen Zhang
c,2

a
Public Service Platform for Industrialization Development Technology of Marine Biological Medicine and Product of State Oceanic Administration, College
of Life Sciences, Fujian Normal University, 350117 Fuzhou, China;
b
Fujian Key Laboratory of Developmental and Neural Biology, College of Life Sciences,
Fujian Normal University, 350117 Fuzhou, China;
c
Center for Genomics and Biotechnology, Haixia Institute of Science and Technology, Fujian Provincial
Laboratory of Haixia Applied Plant Systems Biology, College of Life Sciences, Fujian Agriculture and Forestry University, 350002 Fuzhou, China;
d
Institute of
Oceanography, Marine Biotechnology Center, Minjiang University, 350108 Fuzhou, China;
e
Fujian Key Laboratory of Crop Breeding by Design, Fujian
Agriculture and Forestry University, Fuzhou, 350002 Fujian, China;
f
Technical Department, Biomarker Technologies Corporation, 101300 Beijing, China;
g
Laboratory Department, Fujian Fisheries Technology Extension Center, 350002 Fuzhou, China;
h
Center of Engineering Technology Research for Microalgae
Germplasm Improvement of Fujian, Southern Institute of Oceanography, Fujian Normal University, 350117 Fuzhou, China;
i
Fujian Key Laboratory of Special
Marine Bio-Resources Sustainable Utilization, College of Life Sciences, Fujian Normal University, 350117 Fuzhou, China;
j
Department of Technical Science,
Minhou County Nantong Chunyuanli Ecological Farm, 350001 Fuzhou, China;
k
Key Laboratory of National Forestry and Grassland Administration for Orchid
Conservation and Utilization at College of Landscape Architecture, Fujian Agriculture and Forestry University, 350001 Fuzhou, China;
l
Editorial Department,
The Straits Publishing House, 350001 Fuzhou, China;
m
Department of Breeding, Aimin Goldfish Farm, 350001 Fuzhou, China;
n
Department of Plant Biology,
University of Illinois at Urbana–Champaign, Urbana, IL 61801;
o
Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent,
Belgium;
p
Center for Plant Systems Biology, Vlaams Instituut voor Biotechnologie, 9052 Ghent, Belgium;
q
Department of Biochemistry, Genetics and
Microbiology, University of Pretoria, 0028 Pretoria, South Africa; and
r
College of Horticulture, Nanjing Agricultural University, 210095 Nanjing, China
Contributed by Marc Van Montagu, September 3, 2020 (sent for review March 26, 2020; reviewed by Ingo Braasch, Axel Meyer, and Manfred Schartl)
Goldfish have been subjected to over 1,000 y of intensive domes-
tication and selective breeding. In this report, we describe a high-
quality goldfish genome (2n =100), anchoring 95.75% of contigs
into 50 pseudochromosomes. Comparative genomics enabled us to
disentangle the two subgenomes that resulted from an ancient
hybridization event. Resequencing 185 representative goldfish
variants and 16 wild crucian carp revealed the origin of goldfish
and identified genomic regions that have been shaped by selective
sweeps linked to its domestication. Our comprehensive collection
of goldfish varieties enabled us to associate genetic variations
with a number of well-known anatomical features, including fea-
tures that distinguish traditional goldfish clades. Additionally, we
identified a tyrosine-protein kinase receptor as a candidate causal
gene for the first well-known case of Mendelian inheritance in
goldfish—the transparent mutant. The goldfish genome and diver-
sity data offer unique resources to make goldfish a promising
model for functional genomics, as well as domestication.
Carassius auratus
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goldfish
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domestication
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GWAS
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genome evolution
Goldfish (Carassius auratus) were domesticated in ancient
China from crucian carp (both are still considered the same
species) (1–3), which is one of the most important farmed fish,
with global aquaculture production of 3.096 million tons of
crucian carp in 2018 (4). The appearance of red scales on nor-
mally gray or silver crucian carp was first recorded during the
Chinese Jin Dynasty (AD 265 to 420) (3). During the Tang
Dynasty (AD 618 to 907), goldfish with preferred phenotypes
were selected to be raised in ornamental ponds and water gar-
dens (5). In the Song Dynasty (AD 960 to 1279), the gold (yel-
low) variety of goldfish was the symbol of the imperial family,
and goldfish became known as the “royal fish”while commoners
were forbidden to raise these yellow goldfish (5). The goldfish
was introduced into Japan (6) and Europe at the beginning of
the 17th century (7) and introduced to North America ∼1850
where it quickly became popular (8).
Because goldfish can produce thousands of eggs and dozens of
these small fish can be raised in the same pond, 1,000 y of do-
mestication have been characterized by strong artificial selection.
When describing goldfish, Charles Darwin once wrote, “Passing
over an almost infinite diversity of color, we meet with the most
extraordinary modifications of structure”(9), highlighting that
goldfish provide a rich resource for investigating the genetics of
diverse morphological features. The study of variation under
domestication in goldfish supplied Darwin with the idea of
Significance
We assembled one of the most contiguous genomes for the
common goldfish and unveiled the genetic architecture of
many well-known and anatomically interesting traits, owing to
the sequencing of a large collection of goldfish varieties. The
datasets that we have generated for candidate genes or ge-
nomic regions based on population genomics may help to
elucidate the evolution of goldfish and goldfish varieties and
may provide a resource for a wide range of studies with im-
portant implications for the use of goldfish as a model for
vertebrate and fish genetics.
Author contributions: Y.C. and J.Z. conceived this genome project and coordinated re-
search activities; W.T., Z.H., M.V.M., H.T., Y.V.d.P., Y.C., and J.Z. designed the experiments;
D.C., Z.H., J.S., Q.S., W.H., Qiujin Zhang, G.L., W.Z., L.Q., A.W., Q.Y., and J.Z. collected and
generated goldfish and crucian carp materials; D.C., Qing Zhang, Z.H., G.W., Yongjun
Wang, H.X., L. Lin , X.Z., F.L., and H. T. studied geno me evolution; D. C., Qing Zhang,
W.T., G.W., J.T., G.Z., M.L., and J.Z. contributed to the population genetic analysis; Qing
Zhang, X.Z., X.C., Y.Q., Y. Zhou, and T.X. assembled and annotated the genome; D.C.,
G.W., J.S., Yongjun Wang, H.X., Z.L., W.C., L.H., J.X., L.G., Yuanyuan Wang, P.M., X.H.,
B.W., Q.J., J.L., H.W., M.D., L. Liu, and H.P. manually checked the gene annotation; D.C.,
Qing Zhang, J.C., Y. Zhang, R.M., H.T., Y.V.d.P, Y.C., and J.Z. wrote the manuscript.
Reviewers: I.B., Michigan State University; A.M., University of Konstanz; and M.S.,
University of Würzburg.
The authors declare no competing interest.
Published under the PNAS license.
1
D.C., Qing Zhang, W.T., Z.H., and G.W. contributed equally to this work.
2
To whom correspondence may be addressed. Email: zhuang@fjnu.edu.cn, marc.
vanmontagu@ugent.be, tanghaibao@gmail.com, yves.van depeer@psb.ugent.be,
yqchen@fjnu.edu.cn, or zjisen@fafu.edu.cn.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.2005545117/-/DCSupplemental.
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selection, whether natural or artificial, as the driving force be-
hind evolution (9). Over a century ago, William Bateson also
considered goldfish promising study material for biological var-
iation (10). Using genetic stocks of goldfish populations, Shisan
C. Chen pioneered the study of modern genetics in China (11)
and presented the first examples of traits exhibiting Mendelian
inheritance in goldfish, including traits of transparency and
mottling (12).
After over 1,000 y of domestication and breeding, hundreds of
variants in body shape, fin configuration, eye style, and colora-
tion exist, making goldfish an excellent genetic model system for
fish physiology and evolution. Goldfish had also been used as a
model for Mendelian genetics and biological variations before
the development of the zebrafish system. In this study, to sys-
tematically understand these and other phenomena in goldfish,
we have generated a high-quality reference genome for the
common goldfish and have resequenced a large collection of 185
representative goldfish varieties covering major classification
groups and famous ornamental lines, as well as 16 wild crucian
carp individuals, to provide genomic insights into the evolution,
domestication, and genetic basis of artificial selection in goldfish.
Results
Genome Sequencing, Assembly, and Annotation. Through flow
cytometry, we estimated the genome size of a 12-generation in-
bred line of a female common goldfish (G-12) at 1.8 gigabases
(Gb). Approximately 140 Gb of sequence data generated using
the PacBio RS II platform (SI Appendix, Table S1) were as-
sembled using Canu (13) and polished with gigabases of Illumina
paired-end sequences, yielding an initial 1.657-Gb draft assembly
with contig N50 of 474 kilobases (kb) (SI Appendix, Table S2).
We generated another 1,050,985 BioNano DNA molecules >150
kb to further correct the above genome assembly (SI Appendix,
Table S3). The final assembly using the BioNano approach
spanned 1.73 Gb with scaffold N50 of 606 kb (Table 1 and SI
Appendix, Table S2 and Fig. S1).
In total, 480 million 150-base pair (bp) reads were generated
from three high-throughput chromatin conformation capture
(Hi-C) libraries and uniquely mapped onto the draft assembly
contigs using ALLHiC (14, 15), followed by manual correction
(SI Appendix, Table S4). Approximately 99.30% (1,727.09 meg-
abases [Mb]) of the assembled sequences were anchored onto
the 50 pseudochromosomes (scaffold N50 31.84 Mb), and
95.75% (1,653.62 Mb) were oriented and ordered (SI Appendix,
Table S5 and Fig. S2). To validate the Hi-C assembly, a genetic
map of crucian carp with 8,487 single nucleotide polymorphism
(SNP) markers and 50 linkage groups (16) was aligned to the
assembled goldfish genome, which showed that the ordered
scaffolds and the genetic map are highly concordant with one
another, with average Pearson’s correlation coefficients of 0.923
(Fig. 1 and SI Appendix, Fig. S3). This goldfish genome assembly,
based on an advanced generation inbred line, is more complete
and of higher quality than the recently published draft goldfish
genome “Wakin”(1) (SI Appendix, Fig. S4), but comparable to
another goldfish genome published recently (2). CEGMA (17)
and BUSCO (18) were used to recall 219 (88.7%) complete gene
models from among 248 ultraconserved core eukaryotic genes
and 4,344 (94.7%) complete gene models from among 4,584
conserved actinopterygian genes in our assembly (SI Appendix,
Tables S6 and S7).
The goldfish genome comprises 56,251 coding genes and
10,098 long noncoding transcripts (Table 1 and SI Appendix,
Tables S5 and S8). Comparison with Cyprinus carpio,Cteno-
pharyngodon idellus,Danio rerio, and humans revealed 937 (4%)
gene families unique to goldfish (SI Appendix, Fig. S5). Trans-
posable elements (TEs) spanned 33.04% of the goldfish genome
assembly, which was lower than that of common carp (39.20%)
(19), “Wakin”(39.60%) (1), and zebrafish (54.30%) (20), but
higher than the 33.00% for cave fish (21) and 30.68% for Oryzias
latipes (22) (SI Appendix, Table S9). Potential centromeric re-
gions were predicted for 38 of the 50 chromosomes, again
highlighting the near completeness of the assembled goldfish
genome (SI Appendix, Figs. S6 and S7 and Table S10).
Disentangling the Subgenomes in Goldfish. For more than 50 y, it
has been speculated that goldfish is a tetraploid species (23, 24).
A recent study of cyprinids in the Barbinae (2n =50) subfamily
suggested that species in this subfamily might be the closest
diploid ancestors of goldfish (25) and led to the hypothesis that
Barbinae may be the progenitors of the diploid lineage leading to
goldfish. We performed whole-genome shotgun sequencing of
six representative diploid Barbinae species, including Puntius
semifasciolatus,Hypsibarbus vernayi,Mystacoleucus marginatus,
Balantiochelos melanopterus,Barbonymus schwanenfdi, and
Hampala macrolepidota (SI Appendix, Fig. S8), and aligned an
average of ∼14 million reads from these six Barbinae species to
the goldfish genome assembly.
The 50 goldfish chromosomes can be clearly separated into
two sets (subgenomes), as evidenced by the alignment of a dis-
proportionate number of reads (average of 80.4%) to one of the
homeologous genomes, despite overall similar sizes between
pairs of homeologous chromosomes (Fig. 1 Aand Band SI
Appendix, Figs. S9 and S10 and Table S5). The biased proportion
of mapped reads toward one homeologous chromosome (be-
tween 70.11% and 84.52%) is highly consistent across different
chromosomes, suggesting that C. auratus originated from two
ancestral lineages, one of which was common to Barbinae. Thus,
we defined the set of chromosomes with the highest proportion
of reads aligned between goldfish and Barbinae as subgenome A
(ChrA01∼A25) and designated the remaining set as subgenome
B (ChrB01∼B25). For comparison, SI Appendix, Table S11
shows how these different subgenomes relate to other (sub)ge-
nomes discussed in other studies. The repeat family was con-
sidered enriched in a subgenome by the criterion A/(A + B) −B/
(A + B) ≥0.8 or B/(A + B) −A/(A + B) ≥0.8, resulting in one
A-specific and six B-specific repeat families, which are the hAT-
Ac and TcMar-Tc1 elements, respectively (26) (SI Appendix,
Figs. S11–S13). Moreover, a phylogenetic tree was constructed
based on a nuclear gene: i.e., the connective tissue growth factor-
like gene, which has only one copy in diploid cyprinids but two
copies in tetraploid cyprinids (SI Appendix, Fig. S14). The results
showed that the inferred homolog from Barbinae species (P.
semifasciolatus) was clustered with the gene from subgenome A
Table 1. Statistics of C. auratus genome assembly
Assembly feature C. auratus
Estimated genome size, Gb 1.80
No. of contigs 5,888
Contig N50, bp 606,731
Contig N90, bp 137,868
Longest contig, bp 7,117,495
No. of scaffolds 1,770
Scaffold N50, bp 31,841,898
Scaffold N90, bp 24,975,486
Longest scaffold, bp 60,771,278
Assembly length, bp 1,739,655,870
Assembled portion of genome, % 96.11
Repeat portion of assembly, % 33.04
Predicted gene models 56,251
Average coding sequence length, bp 1,394
Average exons per gene 8.59
Chromosomal assembled portion, % 99.30
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Fig. 1. Goldfish genome features and their evolution. (A) The rings from outermost to innermost represent complete genomes in Mbp. Subgenome A is
colored with purple, and subgenome B is colored with orange (ring A). Shown are gene density in goldfish (ring B), SNP density in the population of 201
goldfish (ring C), InDel density in the population of 201 goldfish (ring D), GC (guanine-cytosine) content of the whole goldfish genome (ring E), TEs inthe
whole goldfish genome (ring F), gene expression (ring G), chromosome collinearity between the subgenomes (ring H), gene expression (ring I), and chro-
mosome collinearity between the subgenomes (ring J). Each signal was calculated in 500-kb sliding windows with 100-kb steps. (B) The proportion of P.
semifasciolatus reads against mapped reads of goldfish. For patterns of the reads mapping for the other five Barbinae species, see SI Appendix, Figs. S9 and
S10.(C) The distribution of nonsynonymous substitutions between selected pairs among taxa C. auratus (goldfish), C. carpio, C. idellus,D. rerio, illustrating the
genetic distance between orthologous gene pairs (between two different taxa) or paralogous gene pairs (within the same taxon). (D) Genome duplications
and chromosome rearrangements in C. auratus,C. carpio,C. idellus,P. semifasciolatus, and D. rerio.
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in goldfish, with zebrafish and glass carp as outgroups. The re-
sults indicated that subgenome A may have originated from a
progenitor species within the Barbinae subfamily whereas the
diploid progenitor of subgenome B probably went extinct or de-
rived from a yet unknown Cyprininae lineage (25, 27). We spec-
ulate that the origin of goldfish is due to an allotetraploidy event
although other evolutionary events, such as autopolyploidy, cannot
be ruled out, although much less likely. Chromosome numbers
were designated in accordance with their collinearity to those of
zebrafish (20) (SI Appendix,Fig.S15). The goldfish A and B
subgenomes contain 28,133 and 26,141 genes, respectively (SI
Appendix,Fig.S16andSupplementary Text).
To explore any divergence in the transcriptional patterns be-
tween the two subgenomes, by means of RNA-seq analysis, we
analyzed the expression patterns of 16,362 homeologous gene
pairs in 10 tissues, including spleen, scale, muscle, midkidney,
head kidney, gut, gill, dorsal fin, atrium, and testis (but see also
ref. 2). In all of the examined tissues, 4,123 and 2,941 homeologs
were inferred to be dominantly expressed in subgenomes A and
B, respectively (Fig. 1Band SI Appendix, Figs. S16–S22). Ap-
parently, the homeologous expression exhibited asymmetric ex-
pression patterns between the two subgenomes, and the genome-
wide expression level dominance was overall more biased toward
subgenome A in the goldfish genome (2).
Genome Evolution of Goldfish. Large segmental inversions were
observed for the homeologous chromosome pairs ChrA01/
ChrB01, ChrA04/ChrB04, ChrA05/ChrB05, ChrA09/ChrB09,
ChrA11/ChrB11, and ChrA15/ChrB15, despite a strong overall
collinearity (Fig. 1B). These large segmental inversions might be
one of the reasons for the allotetraploidy formation (28).
According to the syntenic blocks detected in C. auratus, C. car-
pio,C. idellus,P. semifasciolatus, and D. rerio, the two sub-
genomes of goldfish (Ks [substitutions per synonymous site] =
∼0.17) and common carp (Ks =∼0.21) diverged 13.28 to 16.67
million years ago (MYA) (Fig. 1 Cand D), which was earlier
than the divergence between goldfish and common carp that
took place 7.81∼8.77 MYA (Ks =∼0.10). We hypothesize that
the whole-genome duplication (WGD) event occurred in the
shared lineage of goldfish and common carp and, thus, that they
may have the same number of chromosomes (2n =100), which is
twice the basic chromosome number (2n =50) of diploid
members of the Cyprininae subfamily, indicating these two spe-
cies are tetraploid (Fig. 1D). Moreover, zebrafish, which is dis-
tantly and equally related to all carps, diverged from the last
common ancestor (LCA) of C. carpio,C. auratus, and C. idellus
∼32.8 to 36.8 MYA (Fig. 1 Cand D). Segmental inversions in
ChrA04/ChrA14/ChrA19 occurred after the divergence of
zebrafish and the lineage leading to the Cyprininae subfamily
because the orientation of this chromosomal fragment was
conserved among the extant Cyprininae genomes examined to
date (SI Appendix, Figs. S15 and S23–S25).
Domestication and Population Structure of Goldfish. To examine the
evolutionary history and population structure of goldfish, we
generated 4.3 terabases of sequence data with an average se-
quencing depth of ∼12.5×for a total of 201 samples, including 16
wild crucian carps and 185 representative goldfish variants
(Datasets S1 and S2 and SI Appendix, Fig. S26, Table S1, and
Supplementary Text). The wild crucian carps used were collected
from 16 different locations in eastern China, including Zhejiang
and Jiangsu (5) (SI Appendix, Fig. S26). We aligned 33 previously
reported sequences of common carp (19) to our goldfish genome
and constructed a distance-based phylogenetic tree by using ∼6.5
million biallelic SNPs genotyped in 234 samples, including
common carp, crucian carp, and goldfish. The group containing
common carp was clearly separated from those containing
crucian carp and goldfish (SI Appendix, Fig. S27A), which was in
accordance with the principal component analysis (PCA) based
on the same SNP dataset (SI Appendix, Fig. S27B).
Phylogenetic reconstruction of the 201 crucian carp and goldfish
samples showed that common goldfish was more closely related to
crucian carp than to the other goldfish and divided the latter into
two lineages (Fig. 2 A–C). Both principal component 1 (PC1)
(11.29%) and PC2 (7.87%) generally distinguished common gold-
fish from Egg goldfish (dorsal fin absence) and Wen goldfish (dorsal
fin presence) (SI Appendix,Figs.S28–S30), respectively. The phy-
logenetic tree also indicated that Wen goldfish could be further
classified into three distinct subgroups, including Wenyu–Oranda,
telescope, and Wen–Pompon, whereas the Egg goldfish could be
classified into four subgroups, namely Eggfish–Lionhead, Egg–
Pompon, and Chinese Ranchu, consistent with the PCA based on
total SNPs (Fig. 2Band SI Appendix,Figs.S31–S37).
We further calculated drift parameters in light of the allelic
variants across the 201 sampled genomes and constructed trees
showing the phylogenetic relationships between goldfish and
crucian carp (Fig. 2C). The direction of the gene flow for
Wen–Pompon from Egg–Pompon (weight =0.142) (Fig. 2D)
indicated that it, including the Japanese Hanafusa, is the coun-
terpart of the Chinese Pompon (Egg–Pompon) (27).
Domestication and Selective Sweeps. To elucidate the genomics of
domestication in goldfish, we analyzed the rate of decay of
linkage disequilibrium (LD) (indicated by r2), Tajima’s D, and
genetic diversity (π) for the four subpopulations (crucian carp
[CC], common goldfish [CG], Wen, and Egg) (Fig. 3 Aand Band
SI Appendix, Figs. S38–S41). The decay of LD was dramatically
faster in the crucian carp than in the goldfish (Fig. 3A), sug-
gesting a considerably higher frequency of genetic recombination
in the crucian carp while inbreeding is more common in goldfish.
Moreover, LD decay in the Egg group was faster than in the Wen
group, indicating stronger artificial selection in the Egg goldfish
group, consistent with the estimates of the fixation index Fst,
which quantifies the population differentiation (Fig. 3Aand SI
Appendix, Fig. S41). Compared with crucian carp (1.133), Taji-
ma’s D values for the Egg group (1.770) and the Wen group
(2.315) were considerably higher (Fig. 3B), which supports the
hypothesized existence of a population genetic bottleneck during
the domestication and strong artificial selection in goldfish. The
different subgroups also displayed variations in genetic diversity
(π)(SI Appendix, Fig. S38). The value of πincreased from cru-
cian carp (0.00059) to common goldfish (0.00124) and increased
from common goldfish (0.00134) to both Wen goldfish and Egg
goldfish (0.00297). These observations suggest that the process
of domestication from crucian carp and common goldfish in-
creased genetic diversity as a result of artificial selection (Fig. 3B
and SI Appendix, Fig. S32), indicating the accumulation of sig-
nificant genetic variation in goldfish after their domestication
from crucian carp.
To identify potential selective signals during goldfish domes-
tication, we scanned genomic regions based on genome-wide
calculations for selective sweeps by estimating Fst and the
composite likelihood ratio (CLR) (28), and performed entropy
analysis (Materials and Methods). To avoid the overrepresenta-
tion of goldfish data, we selected 33 individual goldfish and 16
individual wild crucian carp for this analysis. The top 1% of the
–log (P) value covered 50 genomic regions with a total of 25.2
Mb and harboring 946 genes (Fig. 3Cand Dataset S3). The
overall polymorphism level (minor allele frequency) within the
selected regions is 0.065 across all goldfish varieties, compared to
0.236 across the entire genome, representing a fourfold reduc-
tion of diversity, possibly due to selective breeding and domes-
tication. The ∼25.2-Mb sequences displayed nonlinearity to each
other (between the two subgenomes), which would indicate
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Fig. 2. Population structure in goldfish. (A) Principal components of SNP variation. Samples from subpopulations of crucian carp (CC), common goldfish (CG),
Wen goldfish (WG), and Egg goldfish (EG) and (B) Clusters of CG, Wenyu–Oranda (WO), Telescope eye (TE), Wen–Pompon (WP), Eggfish–Lionhead (EL),
Egg–Pompon (EP), and Chinese–Ranchu (CR) are shown, with all of the samples in A, but excluding CC. The plots show the first two principal components. (C)
Neighbor-joining clustering of CC, CG, WO, TE, WP, EL, EP, and CR based on genetic distance calculated from SNPs. Branch color indicates membership in one
of the eight classified goldfish populations. The scale bar shows number of substitutions per site. (D) TreeMix analysis of 185 goldfish divided into seven
clusters, with crucian carp samples serving as the outgroup. The arrows correspond to the direction of gene flow. (E) STRUCTURE plot for CC and goldfish. The
distribution of the K =6 genetic clusters is shown. The eight different populations and the sample number for each population are indicated after the
abbreviated population names.
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Fig. 3. Genome-wide screening for domestication-associated selective sweeps in goldfish. (A) Decay of LD, (B) Tajima’s D value, and (C) whole-genome
analysis of the domestication with selective sweeps inferred from the comparisons among common goldfish, Wen goldfish, Egg goldfish, and crucian carp.
The genome-wide threshold of 2.5 was defined by the top 1% of Fst values. We calculated CLR scores to confirm selective sweeps based on domestication
features; the highest CLR score was 5%. The arrows indicate the sweeps that occurred in goldfish during the domestication process, and the chromosome number of
subgenome A and B is colored with purple and orange, respectively. Representative candidate genes in this region include: eif3f (Cau.A01G0002820), dmd
(Cau.A01G0002960), nav3 (Cau.A04G0007000), scgn (Cau.A04G0010160), mat2aa (Cau.A04G0010480), aurka (Cau.A06G0010390), agrp (Cau.A07G0008060), slc4a3
(Cau.A09G0011550), grk5 (Cau.A10G0011480), rarga (Cau.A11G0000470), thsd7aa (Cau.A19G0007320), macf1a (Cau.A19G0007840), ndufs5 (Cau.A19G0007860), ak2
(Cau.A19G0007880), extl3 (Cau.A20G0012960), zap70 (Cau.B08G0005260), pxnb (Cau.B09G0001240), pcdh15a (Cau.B13G0010680), and nhsl1b (Cau.B20G0006630). (D
and E) Haplotype bifurcation diagram in the goldfish population, with two illustrative examples shown with the extended haplotypes at the eif3f-allele of ChrA01 (D)
and entpd5a-allele of ChrA17 (E). The haplotype bifurcation diagram visualizes the breakdown of LD at progressively longer distances from the core allelefrom the
focal SNP, which is identified by a vertical dashed line. The thickness of the lines corresponds to the frequency of the haplotype. (Fand G) EHH (extended
haplotype homozygosity) for SNP “ChrA01:7730883/A/T”(diagnostic for eif3f) in the goldfish and crucian carp populations is shown with a dark and light blue
line, respectively (F), and for SNP “ChrA17:229 30930/A/T”(diagnostic for entpd5a) in the goldfish and crucian carp populations is marked with a deep and light
red line, respectively (G).
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potential functional complementation of homeologous chromo-
somes in goldfish. Gene Ontology (GO) and Kyoto Encyclopedia
of Genes and Genomes (KEGG) analysis of the 940 genes in-
dicated that a significant portion of the candidate genes were
related to morphogenesis, pigmentation, behavior, immune re-
sponse/infectious diseases, energy metabolism, and response to
hormones (Dataset S3). We also examined genes associated with
phenotypes that have been observed in zebrafish mutants
(https://zfin.org/) and found that 173 of the genes corresponding
to 132 orthologs displayed mutant phenotypes in gene knockout
lines in zebrafish, including phenotypes related to pigmentation,
morphogenesis, and behavior that have undergone positive se-
lection for over 1,000 y in goldfish (Dataset S3).
These selective sweeps were further scrutinized in the above
201 samples, and 393 genes indicated regions of completed se-
lective sweep (Fig. 3 Cand Dand Dataset S4). Of the 393 genes,
21 representative candidate genes with a high degree of pop-
ulation differentiation (Fst), and highly negative Tajima’s D are
indicated (Fig. 3C). Among these 21 genes, 13 are orthologous to
genes for which there are knockout lines in zebrafish that display
mutant phenotypes related to behavior (pcdh15a and agrp), de-
creased eye size (ndufs5), cell migration (nav3 and zap70), or de-
creased brain size (aurka)(Dataset S3). Haplotype bifurcation
diagrams are shown for two examples, the eif3f-allele (Fig. 3 Dand
F)andtheentpd5a-allele (Fig. 3 Eand G). In the whole goldfish
population, the average level of genetic diversity (π)ofthese393
genesis1.14E−4, compared to 1.16E−3 for all genes in the entire
genome. These low-diversity genes likely contributed to the phe-
notypes associated with major domestication traits in goldfish.
Genome-Wide Association Study of Dorsal Fin in Domesticated
Goldfish. Darwin noted that goldfish exhibited “the most ex-
traordinary modifications of structure”and recorded goldfish
variants lacking a dorsal fin, with double anal fins, and with triple
tails (29). The dorsal fin is a remarkable feature that distinguishes
Wen goldfish from Egg goldfish, and the diminution of the dorsal
finisthekeystepintheevolutionofEgggoldfish.Tostudythe
genetic segregation of the dorsal fin form, we crossed Chinese
Ranchu (Egg goldfish) with Lionhead (Wen goldfish). The F1
population from the female Chinese Ranchu and male Lionhead
contained 31% (62) normal, 4.5% (9) absent, and 64.5% (129)
abnormal dorsal fins. In contrast, the reciprocal cross F1 population
contained 25.5% (51) normal and 74.5% (149) abnormal dorsal fins
(SI Appendix,Fig.S42), suggesting that this dorsal fin trait is con-
trolled by multiple gene loci probably with maternal genetic effects.
Analysis of the homeolog retention of the associated genes for
dorsal fin revealed that, of 222 genes located in the subgenome
A, only 72 homeologs were present in subgenome B. These genes
were retained less often (retention rates are ∼53% and ∼46% for
genome-wide subgenomes A and B, respectively) than the av-
erage retained as two copies (retention rates are ∼89% and
∼83% for genome-wide subgenomes A and B, respectively) al-
though the differences are not statistically significant based on a
ttest (Pvalue =0.1702). We further performed a genome-wide
association study (GWAS) of the dorsal fin trait based on 96
controls (Wen goldfish) and 87 cases (Egg goldfish, excluding 2
fishes with ambiguous dorsal fins) (Fig. 4A). A total of 378 genes
associated with the dorsal fin phenotype were detected in 8.96
Mb of genomic regions spread across 13 chromosomes. A total
of 85.2% (322) of these genes were found on subgenome A, with
significant portions being located on five chromosomes, includ-
ing ChrA09 (84), ChrA07 (64), ChrA22 (56), ChrA16 (45), and
ChrA05 (41) (Fig. 4B,SI Appendix, Fig. S43, and Dataset S5), a
considerably higher proportion than the number of dorsal fin-
associated genes (56) located on subgenome B (Fisher’s exact
test, Pvalue <2.2e−16), suggesting an uneven subgenomic dis-
tribution of the genes associated with the dorsal fin. GO and
KEGG analyses indicated that the candidate genes for this trait
are putatively involved in the “cell surface receptor signaling”
pathway, “signal transduction,”“transmembrane transport,”
“skeletal system development,”and “primary metabolic process
and organ nitrogen compound metabolic process”(Dataset S5).
It is noteworthy that 57 genes of these 378 genes correspond to
50 zebrafish orthologs for which gene knockout lines display
mutant phenotypes (Dataset S5). The mutant phenotypes for 13
of these zebrafish genes (26%) are related to the development of
the dorsal fin, including decreased occurrence of the dorsal
longitudinal anastomotic vessel (itgb1a,rab13, and e2f8), dorsal
abnormal (kif5ba), dorsal aorta abnormal (fev,cxcr4b,gpr183a,
tal1,glrx2, and uchl5), ventralized (zic2a), and curved dorsal fin
(atp1a2a and dhfr)(Dataset S5). A haplotype bifurcation dia-
gram is shown for one example, dhfr (Fig. 4 C–F). We further
scrutinized the genotypes of our 183 goldfish samples (excluding
2 samples with ambiguous dorsal fin) (Fig. 4Eand Dataset S6)
and found that 24 of the corresponding regions in goldfish do not
contain annotated protein-coding exons while 246 genes in
goldfish display genotypes that generally coincide with the
presence/absence of the dorsal fin (Fig. 4Eand Dataset S6).
Eight representative genes are shown (Fig. 4E), including 7
genes related to dorsal development in zebrafish (dhfr,cxcr4b,
zic2a, gpr183a,tal1,kif5ba,andatp1a2a), while nrxn1a has been
associated with vertebral abnormalities in humans (30). Dihy-
drofolate reductase (dhfr) is a key enzyme in folate-mediated me-
tabolism and is involved in the de novo mitochondrial thymidylate
biosynthesis pathway. Recently, the enzymes regulating folate me-
tabolism have been reported to be up-regulated in zebrafish fins 4 d
postamputation (31). We speculate that dhfr may contribute, to
some extent, to the presence/absence of a fin. These genes probably
played key roles in the artificial selection of the Egg goldfish from
the Wen goldfish, as well as in dorsal fin development (31).
Candidate Genes for a Classic Case of Mendelian Inheritance in
Goldfish. The transparent goldfish mutant was first recorded in
1579 (5) and is a classic case of Mendelian genetics in fish and
even vertebrates with incomplete dominance at the T/t locus
(12). However, the gene located at the T/t locus had not been
identified after its first reporting in 1928. Goldfish with transpar-
ent scales are distributed across each of the subgroups in the panel
of 185 fish phenotypes because the single gene controlling this
transparent mutant has become widespread during goldfish
breeding over the past 400 y. Using 48 cases and 127 controls, we
detected a single strong association peak (−log10 (P) =17.3) with
five SNPs on one arm of ChrB15 (Fig. 5 A–E) that coincides with
the incomplete dominant genetic pattern of the T/t locus (Fig. 5F).
A genomic inversion was observed in this region in comparison to
homeologous chromosome 15. Furthermore, we scrutinized the
regions based on a larger variant dataset of the SNPs and inser-
tions/deletions (InDels) of the population and found that segre-
gation of 14 SNPs and two InDels coincides with the T/t locus, as
suggested (12). In the candidate region ChrB15:28,558−153,105,
we identified a gene, Cau.B15G0000010 (ChrB15:48,888−76,549),
encoding a tyrosine-protein kinase receptor UFO-like protein
containing four SNPs in predicted transcription factor-binding
sites in its 5′untranslated region (UTR) (Fig. 5G). A leukocyte
tyrosine kinase has been related to pigment cell development in
zebrafish (31), and members of the receptor tyrosine kinase family
play roles in melanoma development in humans (32) although the
roles of this gene family are not currently known in goldfish. In
goldfish, a tyrosine-protein kinase receptor might regulate tyrosine
kinase and thus result in pigmentation variation. In this region,
under strong association, the neighboring gene Cau.B15G0000020
encoding a beta-secretase 1-like protein (bace1) with a non-
synonymous single-nucleotide mutation has been related to me-
lanocyte migration in zebrafish (33) and in humans (34), which
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Fig. 4. GWAS for dorsal fin-related traits in goldfish. (A) Pictures of Wen goldfish (WG, with dorsal fin) and Egg goldfish (EG, without dorsal fin). (B) GWAS
for genes associated with dorsal fin in a Wen/Egg-goldfish population. Genes surrounding or within association peaks are indicated. Gene names are
highlighted in bold black/red in candidate regions potentially related to dorsal fin development in goldfish. Representative candidate genes for dorsal fin-related
traits include: dhfr (Cau.A05G0015640), cxcr4b (Cau.A09G0004970), zic2a (Cau.A09G0008030), gpr183a (Cau.A09G0007940), nrxn1a (Cau.A13G0000420), tal1
(Cau.A22G0009870), kif5ba (Cau.B02G0011950), and atp1a2a (Cau.B02G0012070). The chromosome number of subgenome A and B is colored with cyan and
orange. The dashed lines show different genome-wide significance thresholds, respectively. (Cand E) Haplotype bifurcation diagram in EG and WG subpopu-
lations, starting from the two alleles at one of the representative significant GWAS SNP sites. The haplotype bifurcation diagram visualizes the breakdown of LD
at progressively longer distances from the core allele from the focal SNP, which is identified by a vertical dashed line. The thickness of the lines corresponds to the
frequency of the haplotype. We show the extended haplotype at the dhfr allele of EG subpopulation in C, relative to the shorter haplotypes at dhfr allele of WG
subpopulation in E, which is in accordance with a selective sweep around the dhfr allele in the EG subpopulation. (Dand F) EHH for SNPs “ChrA05 :3858891 5”
(diagnostic for dhfr) in EG subpopulation (D) and WG subpopulation (F) is shown with maroon line (for G) and blue line (for C), respectively.
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Fig. 5. GWAS for transparent scale-related traits in goldfish population. (A) Goldfish with transparent scales or translucent scales served as case and control,
respectively. (B) PCA for the samples from case (transparent scale), control (translucent scale), and unknown populations based on whole-genome sequence data. PC1
and PC2 indicate scores of principal components 1 and 2, respectively. (C) Manhattan plot for transparent scale GWAS. The two dashed horizontal lines represent the
thresholds for very high significance [top 0.1%, –log10 (P) =3.38] and significance [top 1%, –log10 (P) =2.03]. The arrow indicates the highly significant peaks. (D)
Quantile–quantile plot for GWAS under a general linear model. (E) Local Manhattan plot surrounding the association peak on ChrB15. The double dashed horizontal
lines represent the thresholds for high significance [top 0.1%, –log10 (P) =3.38] and significance [top 1%, –log10 (P) =2.03]. The boxed areas in Dand Eindicate the
high-confidence SNPs. (F) Genotype analysis for the candidate region (Top) and gene order (Bottom) in the candidate region; green, yellow, orange, and gray blocks
represent reference allele homozygous (0/0), heterozygous (0/1), variant allele homozygous (1/1), and missing genotype (./.), respectively. The vertical orange bar
consists of orange blocks that represent variant allele homozygous (1/1). (G) Candidate genes and 5′UTR regulatory elements are shown. Exons, introns, and 5′UTR are
represented with blue boxes, broken lines, and cyan boxes, respectively. The variant sites are marked with red arrows.
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might explain the frequent development of black smudges or dots
on goldfish with transparent scales (Fig. 5A). Although functional
validation is still pending, we believe that the tyrosine-protein ki-
nase receptor is a strong candidate for the T/t locus in goldfish.
Discussion
Teleosts have undergone at least three rounds of WGD during
their evolutionary past, of which the most recent is teleost-
specific (Ts3R) and has been dated at 320 to 350 MYA (35,
36). Goldfish comprise one of the few teleost species that have
undergone an additional, considerably more recent, WGD.
Comparative genomics revealed that goldfish originated from a
merger of two progenitor species, one of which is closely related
to Barbinae species. The WGD in goldfish is assumed to origi-
nate from a hybridization event (allotetraploidy) between its two
progenitor species before the divergence of common carp and
goldfish, which dates to ∼13 to 16 MYA (Fig. 1C). The WGD in
goldfish might have coincided with the end of the rapid elevation
of the Tibetan Plateau (37) and accompanying global climate
change and could have given the allotetraploid goldfish a se-
lective advantage to survive in a quickly changed environment
(38, 39). As is generally assumed for allopolyploid plants (40),
goldfish also express genes predominantly residing on different
subgenomes (SI Appendix, Fig. S20), suggesting that the two
subgenomes probably diverged functionally. A recent study on
common carp (41) also revealed subgenome dominance and the
different expression of genes on homeologous chromosomes, gen-
erally supporting asymmetrical genome evolution in Cyprinidae.In
addition, the WGD in goldfish seems primarily to have resulted in
gene redundancy, increasing mutational robustness and likely
shielding goldfish from the deleterious effects of mutations (42),
thereby enabling artificial selection for mutations in key functional
genes in goldfish. With the availability of this high-quality genome
assembly, goldfish could become an excellent model system for
genetic studies of dominant deleterious mutations in vertebrates.
Artificial selection for the dorsal fin was shown to have pre-
ferred variants from goldfish subgenome A, which was derived
from the progenitor genome that is closely related to P. semi-
fasciolatus. Both subgenomes may have differentially contributed
to artificial selection for particular traits during the domestication
of goldfish. A similar phenomenon has been observed in allote-
traploid cotton, which displayed an asymmetric subgenome con-
tribution to the long fiber trait (43). Although our GWAS analysis
identified some genes previously known to be associated with the
development of related traits in teleosts, we also identified many
loci of unknown function. Very recently, the potentially associated
genes relative to the dorsal fin loss phenotype were also identified
in goldfish, and lrp6S was found to be located in subgenome S
(corresponding to subgenome B in our notation) (SI Appendix,
Table S11) (44). Based on classical genetics (crossing experi-
ments), GWAS, and population history analyses in this study, we
consider that the dorsal fin trait is likely controlled by multiple
loci, as supported by our much wider sampling of goldfish; thus,
the discrepancy between the current study and Kon et al. (44) is
possibly caused by the differences of the two GWAS pupations.
Further work will be necessary to functionally validate the specific
genes governing these artificially selected traits.
The population genomic datasets that we have gathered related
to candidate genes and genomic regions may help to elucidate the
genetic basis of various traits in goldfish, may provide a resource for
a wide range of studies, and may have important implications for
the use of goldfish as a model for vertebrate genetic studies. A fully
comprehensive understanding of goldfish genetics may also support
the use of goldfish as promising material for the study of natural
mutations and artificial selection and thus may provide connections
with genome duplication, morphological evolution, and domesti-
cation in animals and plants in the future (45).
Materials and Methods
Additional materials and methods are described at length in SI Appendix,
Supplementary Materials and Methods.
PacBio Sequencing. Genomic DNA was extracted from goldfish blood. Con-
centrated and sheared DNA (6 μg) was size-selected using the BluePippin
Automated DNA Size Selection System (Sage Science Inc.). SMRTbell libraries
(∼20 kb) were prepared according to the protocol described by PacBio. The
SMRT data were generated using the PacBio RSII system with P6-C4 chem-
istry (SI Appendix, Table S12).
Irys Optical Genome Maps. Genome mapping was performed using the
Bionano Genomics Saphyr System with NanoChannel array technology. Optical
maps were assembled by scaffolding using Irys with default parameters.
Hi-C Library Construction and Sequencing. Fresh blood was collected from
goldfish to prepare genomic DNA for construction of Hi-C libraries at the Bio-
Marker Technologies Company as follows (46). The paired-end 150-bp reads were
generated by sequencing the libraries on the Illumina HiSeq X Ten platform.
Raw Data Preprocessing. Reads with quality <0.75 or length <500 bp were
then filtered out. Next, SMRT reads were corrected using the parameter
corOutCoverage =100, which enables correction of all of the input PacBio
reads. The BioNano raw data were processed using the IrysView package
(Bionano Genomics, San Diego, CA). Molecules with label signal-to-noise
ratio (SNR) ≥3.0, average molecule intensity <0.6, and length >100 kb were
retained for use in genome assembly.
Genome Assembly and Hi-C–Based Pseudochromosome Construction. The ini-
tial de novo genome assembly of goldfish was performed with PacBio reads
(80×sequence coverage) using Canu (13) (v1.6). Subsequently, the draft
assembly was polished with 50×Illumina short reads using Pilon (47). ALLHiC
(15) was used as previously described to scaffold the allotetraploid goldfish
genome on chromosome-level scales.
Genetic Maps and Validation of Assembly. To validate the accuracy of the
chromosome-level assembly, a high-resolution genetic linkage map for crucian
carp (16) with 8,487 SNP markers assigned to 50 linkage groups wa s appl ied to
the 50 pseudochromosomes anchored by Hi-C using ALLMAPs (48).
Genome Annotation. Consensus TE sequences for the goldfish genome were
generated using RepeatModeler with a combination of de novo and ho-
mology strategies including two de novo repeat-finding programs, RECON
(49) and RepeatScout (50), which we imported into RepeatMasker (www.
repeatmasker.org/) to identify and cluster repetitive elements. Ab initio gene
models were evaluated using transcript and protein evidence to select the most
consistent model for each gene based on an Annotation Edit Distance (AED) value
by performing first-pass gene annotation using MAKER (51).
Estimation of Species Divergence Time. We used MCScanX (52) to identify
syntenic blocks (regions with at least five colinear genes) between species
and calculate Ks rates for syntenic genes using the Nei–Gojobori method,
and the median Ks value was used to estimate the divergence time based on
the C. idella evolution coefficient (53).
Population Structure Analysis. We used PLINK (54) (v1.9) to calculate the
proportions of heterozygotes, missing genotypes, and inbreeding coefficients for
each sample. A genetic relatedness matrix (GRM) of all pairwise comparisons of
samples was calculated using Genome-wide Complex Trait Analysis (GCTA)
(v1.26.0) software (55) and visualized using the R package pheatmap (56). Pop-
ulation structure inference was performed using ADMIXTURE (57) (v1.23).
Population Parameter Estimation. The LD decay analysis for genotypes in the
variantcallformat(VCF)filewascarriedout using popLDdecay (58). We estimated
the population parameters genetic diversity (π) and genetic distance (Tajima’sD)
for each group and calculated the statistic (Fst) to measure population differen-
tiation by comparing all samples to the crucian carp subpopulation using VCFtools
(59) for each nonoverlapping 50-kb genomic block across the whole genome.
Selective Sweep Detection. A set of SNP marker-specific crucian carp (C16) and
33 representative goldfish (G33) were filtered using PLINK (54) with the
parameters –mac 10 –geno 0.2. We used this marker set to perform pop-
ulation structure analysis (PCA and TreeMix for four groups), and the results
were confirmed by the previous results in this study.
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Case-Control GWAS of Morphologic Traits. PLINK (54) was used to perform case-control
GWAS. λGC is defined as the ratio between the 1-degree-of-freedom χ
2
value of
the median Pvalue and 0.455—the 1-degree-of-freedom χ
2
value for a Pvalue of
0.5. The top 0.1% of genetic corrected Pvalue (PGC) was used as a threshold.
Data Availability. We have submitted the goldfish genome and annotation to
National Center for Biotechnology Information (NCBI). The goldfish genome is
available from SAMN12618612. Goldfish RNA-Seq data and quality-filtered Illu-
mina reads for the 185 resequenced goldfish genomes, 16 crucian carp, and 6
Barbinae species were deposited into the NCBI BioProject database under
accession number PRJNA561458.
ACKNOWLEDGMENTS. We thank Tianshan Xue for collecting some goldfish
samples. This study was supported by the 13th Five-Year Plan for the Marine
Innovation and Economic Development Demonstration Projects (FZHJ14) and
Science and Technology Program of Fuzhou (2018-N-27). Y.V.d.P. acknowledges
funding from the European Research Council under the European Union’sHo-
rizon 2020 research and innovation program (Grant Agreement 833522).
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