A consensus linkage map of the grass carp (Ctenopharyngodon idella) based on microsatellites and SNPs.

Abstract

Grass carp (Ctenopharyngodon idella) belongs to the family Cyprinidae which includes more than 2000 fish species. It is one of the most important freshwater food fish species in world aquaculture. A linkage map is an essential framework for mapping traits of interest and is often the first step towards understanding genome evolution. The aim of this study is to construct a first generation genetic map of grass carp using microsatellites and SNPs to generate a new resource for mapping QTL for economically important traits and to conduct a comparative mapping analysis to shed new insights into the evolution of fish genomes.
We constructed a first generation linkage map of grass carp with a mapping panel containing two F1 families including 192 progenies. Sixteen SNPs in genes and 263 microsatellite markers were mapped to twenty-four linkage groups (LGs). The number of LGs was corresponding to the haploid chromosome number of grass carp. The sex-specific map was 1149.4 and 888.8 cM long in females and males respectively whereas the sex-averaged map spanned 1176.1 cM. The average resolution of the map was 4.2 cM/locus. BLAST searches of sequences of mapped markers of grass carp against the whole genome sequence of zebrafish revealed substantial macrosynteny relationship and extensive colinearity of markers between grass carp and zebrafish.
The linkage map of grass carp presented here is the first linkage map of a food fish species based on co-dominant markers in the family Cyprinidae. This map provides a valuable resource for mapping phenotypic variations and serves as a reference to approach comparative genomics and understand the evolution of fish genomes and could be complementary to grass carp genome sequencing project.

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RESEARCH ARTICLEOpen Access
A consensus linkage map of the grass carp
(Ctenopharyngodon idella) based on
microsatellites and SNPs
Jun Hong Xia1, Feng Liu2, Ze Yuan Zhu1, Jianjun Fu2, Jianbin Feng2, Jiale Li2,3*, Gen Hua Yue1,3*
Abstract
Background: Grass carp (Ctenopharyngodon idella) belongs to the family Cyprinidae which includes more than
2000 fish species. It is one of the most important freshwater food fish species in world aquaculture. A linkage map
is an essential framework for mapping traits of interest and is often the first step towards understanding genome
evolution. The aim of this study is to construct a first generation genetic map of grass carp using microsatellites
and SNPs to generate a new resource for mapping QTL for economically important traits and to conduct a
comparative mapping analysis to shed new insights into the evolution of fish genomes.
Results: We constructed a first generation linkage map of grass carp with a mapping panel containing two F1
families including 192 progenies. Sixteen SNPs in genes and 263 microsatellite markers were mapped to twenty-
four linkage groups (LGs). The number of LGs was corresponding to the haploid chromosome number of grass
carp. The sex-specific map was 1149.4 and 888.8 cM long in females and males respectively whereas the sex-
averaged map spanned 1176.1 cM. The average resolution of the map was 4.2 cM/locus. BLAST searches of
sequences of mapped markers of grass carp against the whole genome sequence of zebrafish revealed substantial
macrosynteny relationship and extensive colinearity of markers between grass carp and zebrafish.
Conclusions: The linkage map of grass carp presented here is the first linkage map of a food fish species based
on co-dominant markers in the family Cyprinidae. This map provides a valuable resource for mapping phenotypic
variations and serves as a reference to approach comparative genomics and understand the evolution of fish
genomes and could be complementary to grass carp genome sequencing project.
Background
A linkage map is an essential framework for mapping
traits of interest and is often the first step towards
understanding genome evolution and aids genome
assembly [1-4]. With the advent of molecular genetic
techniques and sophisticated statistical tools for linkage
analysis, linkage maps have been constructed for some
economically important aquaculture species, such as
channel catfish, tilapia, Asian seabass, European sea
bass, shrimps, and oysters [4]. Until last decade, domi-
nant DNA markers such as AFLP and RAPD were used
to construct linkage maps that are now replaced by
co-dominant markers such as microsatellites and SNPs.
Microsatellites have been extensively used in construc-
tion of genetic maps for some aquaculture fishes
recently, e.g., brown trout, Salmo trutta [5], nile tilapia,
Oreochromis niloticus [6], channel catfish, Ictalurus
punctatus [7,8], Asian seabass, Lates calcarifer [9] and
blacklip abalone, Haliotis rubra [10]. With the rapid
development of DNA sequencing and genotyping tech-
nologies, single nucleotide polymorphism (SNP) [11],
the most frequent polymorphism in genome, became
the most favored DNA markers for linkage mapping
and whole genome association studies in humans [12]
and model organisms [13]. However, the application of
SNPs in linkage mapping of aquacultured fish is just in
its infancy [8].
Comparative mapping contributes largely to solve
issues in the evolution of individual species, differences
* Correspondence: jlli@shou.edu.cn; genhua@tll.org.sg
1Molecular Population Genetics Group, Temasek Life Sciences Laboratory,
National University of Singapore, 117604 Republic of Singapore
2Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,
Shanghai Ocean University, Ministry of Education, Shanghai 201306, China
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© 2010 Xia et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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and similarities between various species and to charac-
terize functions of genomes by providing a detailed ana-
lysis of conservation among orthologous intervals in
different species [14]. This would not only help to
reduce cost and increase efficiency in genetic research
across species through offering the possibility to transfer
genomic information available from model species to
nonmodel organisms, but also aids whole genome
sequence assembly as well as molecular breeding
[15-17]. The availability of an increasing number of fish
whole genome sequences (e.g., zebrafish, medaka, fugu,
stickleback and freshwater pufferfish) offer opportunities
to accelerate fish genomic comparative studies, such as,
transferring genomic information from model species to
food fish. Genetic mapping for non-model species with
sequence-based molecular markers could provide a sui-
table support for the knowledge of genome organization
and evolutionary studies by comparative map analysis. It
represents a key towards integrating known genome
data from model species into identifying genes in the
species of interest [15,18,19].
The grass carp (Ctenopharyngodon idella) is one of
the largest members of the family Cyprinidae [20,21]. It
is an herbivorous freshwater fish of great commercial
value and is extensively cultivated in Eastern Asia for
food. Cultured grass carp may grow up fast reaching up
to 1 kg in the first year, growing approximately 2-3 kg/
year in temperate areas and 4.5 kg/year in tropical areas
[20]. Grass carp also has extensive ecological adaptabil-
ity, of the 115 countries in which grass carp have been
introduced, at least 58 (~50%) appear to have self-sus-
taining populations [22]. In addition, due to their ability
to be cultured easily, hardiness and effective biological
controls on wide variety of aquatic vegetation; this spe-
cies has been extensively used for aquatic weed control
purposes in rivers, fish ponds and reservoirs [21]. In
recent years, commercial harvesting of grass carp has
increased in many countries. According to the statistics
of Food and Agriculture Organization of the United
Nations [23], the global annual production of grass carp
summed up to 4,010,281 metric tons in 2006 with a
30% increase for past decades and was listed as the 3rd
biggest contributor to the world’s aquaculture produc-
tion. The revenue generated by aquaculture of this spe-
cies was over 4 billion US dollars in 2006 [23].
Therefore, the species represents one of the most pro-
mising species in world aquaculture [24]. However,
research on grass carp genomics remained at slow pace.
Although to date around 100 microsatellite sequences of
grass carp are available in GenBank database, and some
microsatellite markers have been applied in genetic
diversity and comparative analysis of grass carp [25-27],
no genetic map is available in grass carp.
The aim of this study is to construct a first generation
genetic map of grass carp using microsatellite and SNP
markers to supply a basis for genome-wide search of
QTL for economically important traits and to conduct a
comparative mapping analysis to provide new insights
into the evolution of fish genomes and to aid future
genome assembly.
Results
Markers
A total of 283 microsatellites and 24 SNP markers were
informative in the two mapping families and could be
used for linkage analysis. Four microsatellite markers
(CID131A/B, CID320A/B, CID532A/B, CID816A/B)
were scored as duplicates.
Linkage analysis
Two grass carp families containing 96 progenies in each
were scored for 307 informative loci. A total of 279 mar-
kers (263 microsatellites and 16 SNPs) were mapped to
24 linkage groups (Additional file 1: Table S1) whereas
28 remained (20 microsatellites and 8 SNPs) unmapped.
There is no significant deviation of Mendelian segrega-
tion for mapped markers after Bonferroni correction.
Using linkage analysis and LOD ≥ 3 threshold, for male
segregation data, 212 loci were finally assembled into 24
linkage groups (LGs) in both families, and for female
data, 219 loci were assembled into 24 linkage groups in
both families (Figures 1, 2 and 3 and Table 1). Among
them, 152 loci were located in both maps which could be
used to identify homologous pairs of linkage groups in
both sexes. The merged sex-specific maps in both
families showed different length. The map length for
female and male was 1149.4 cM and 888.8 cM respec-
tively. The average spacing between loci in male map (4.2
± 1.7 cM) was lower than that in the female map (5.2 ±
1.8 cM), indicating that sex-specific differences in recom-
bination rates existed in grass carp. Comparison of
recombination differences between the parents of two
mapping families revealed that the average recombina-
tion ratio (female: male) across all pairwise comparisons
was 2.03 [N = 287 comparisons; G-test value (1 d.f) =
1132.2] for family 1 and 2.00 [N = 307 comparisons;
G-test value (1 d.f) = 1226.9] for family 2. Therefore, a
significantly higher recombination rate was evident in the
female map but no significant family-specific difference
in overall recombination ratio for both sexes was
detected [Female (family1)/Female (family 2) = 1.02,
N = 243 comparisons, G-test value (1 d.f) = 0.91; Male
(family1)/Male (family2) = 1.01, N = 281 comparisons,
G-test value (1 d.f) = 0.29]. The distributions of recombi-
nation ratio between both parents in two mapping
families were given in Additional file 2: Figure S1.
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Figure 1 Linkage groups 1 to 8 of the consensus linkage map of grass carp based on microsatellite and SNP markers. The female
linkage group (left) is named as “CIDF_LG” and 1-2 numbers; the male linkage group (middle) is named as “CIDM_LG” and 1-2 numbers; the
sex-averaged linkage group (right) is named as “CID_LG” and 1-2 numbers. Estimates of map distances between markers are indicated in
Kosambi centimorgans.
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The resulting sex-averaged map composed of 24 linkage
groups (Figures 1, 2 and 3 and Figures 4, 5 and 6) con-
tained 279 loci. Six (CID131A, CID320A, CID320B,
CID532B, CID816A and CID816B) of the duplicate loci
were assigned to 4 linkage groups (CID_LG2, 8, 15
and19) and the remaining two were unmapped
(CID131B and CID532A). The marker order in the sex-
averaged map was the same as in the sex-specific maps.
The map spanned 1176.1 cM with individual linkage
groups ranging from 23.4 to 95.2 cM (mean 49.0 ± 16.2,
Table 2). The average resolution of the map was 4.2 ±
1.6 cM ranging from 2.2 cM (CID_LG18) to 8.6 cM
(CID_LG20) for individual linkage groups. The average
number of loci per linkage group was 11.6 ± 4.1 with a
range varying from 4 (CID_LG20) to 19 loci (CID_LG2)
per linkage group. Only 2 (0.9%) out of the 221 intervals
Figure 2 Linkage groups 9 to 16 of the consensus linkage map of grass carp based on microsatellite and SNP markers. The female
linkage group (left) is named as “CIDF_LG” and 1-2 numbers; the male linkage group (middle) is named as “CIDM_LG” and 1-2 numbers; the
sex-averaged linkage group (right) is named as “CID_LG” and 1-2 numbers. Estimates of map distances between markers are indicated in
Kosambi centimorgans.
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were larger than 25 cM. Overall statistics including
number of markers, linkage length and map length for
the sex-specific and sex-averaged maps are given in
Table 1 and 2.
Comparative mapping
The sequences of 263 mapped microsatellites and 16
SNPs in ESTs and gene loci were used for similarity
searches against genome sequences of four model fish
species. Our results showed that 10.1% (medaka
sequences: 28 hits) to 59.2% (zebrafish: 164 hits) of the
blasted sequences were significantly conserved between
grass carp and model fish species (e < 10-7; Table 3).
Between grass carp and fugu/medaka, no microsatellites
with more than 5 or more repeat motif were conserved,
whereas between grass carp and zebrafish, 43.9% of
microsatellite loci were conserved. When compared to
microsatellite data, much higher conservation (ranging
from 43.8% for medaka to 100% for zebrafish) of homo-
logous ESTs and gene loci between grass carp and
Figure 3 Linkage groups 17 to 24 of the consensus linkage map of grass carp based on microsatellite and SNP markers. The female
linkage group (left) is named as “CIDF_LG” and 1-2 numbers; the male linkage group (middle) is named as “CIDM_LG” and 1-2 numbers; the
sex-averaged linkage group (right) is named as “CID_LG” and 1-2 numbers. Estimates of map distances between markers are indicated in
Kosambi centimorgans.
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model species was detected. Of the total 176 loci with
significant hits (e < 10-7), 42 loci (23.9%) matched at
least 2 model species and 13 (7.4%) matched all the 4
model species. By syntenic detection, some blocks with
very high conservation were also found. For example, a
syntenic block defined by markers SNP0010 and
CID0278 on CID_LG1 was conserved among at least 3
species. The detailed comparative mapping results were
presented in Additional files 3-4 Tables S2 and S3.
The grass carp genetic map was utilized in the estab-
lishment of gene orthology relationships based on con-
served syntenies among species. The zebrafish genome
with 25 chromosomes was fairly characterized. One
hundred and sixty-four (148 microsatellites and 16
SNPs) markers were common in grass carp linkage map
and zebrafish genome map. The marker density in the
comparative map ranged from 2 (CID_LG10) to 13
(CID_LG1 and CID_LG4) markers with an average of
6.8 ± 3.0 per LG (Table 4). These allow us to evaluate
large-scale synteny patterns between the two species.
All 24 grass carp LGs were clearly associated with
zebrafish chromosomes (Figures 4, 5 and 6 and Table
4). In most cases, the grass carp LG showed extensive
synteny towards a particular zebrafish chromosome,
sharing two or more loci with zebrafish chromosome.
The average number of syntenic loci in one pair of grass
carp LG and homologous zebrafish chromosome was
5.5 ± 2.7 with a range varying from 2 (CID_LG10-
DR_LG11, CID_LG20-DR_LG25, CID_LG22-DR_LG2
and CID_LG24-DR_LG22) to 11 (CID_LG1-DR_LG7)
per chromosome (Table 2). In total, 6 of the 24 grass
carp LGs (CID_LG10-DR_LG11, CID_LG15-DR_LG15,
CID_LG16-DR_LG9, CID_LG19-DR_LG24, CID_LG21-
DR_LG8, CID_LG23-DR_LG17) mapped each to a
single zebrafish chromosome, 12 yielded hits with
2 chromosomes, 5 with 3 chromosomes and one
(CID_LG4) mapped to 6 chromosomes (Figures 4, 5 and
6 and Table 4).
Each zebrafish chromosome was mainly syntenic to
one grass carp LG and shared ≥50% orthologous mar-
kers of the chromosome in common (Figures 4, 5 and
6). The zebrafish genome map covered by these syntenic
markers summed up to 809.5 Mb with a range of 1.1
Mb (DR_LG22) to 55.7 Mb (DR_LG6) per chromosome,
accounting for 63% of the displayed regions of zebrafish
reference genome (Table 2). A large amount of syntenic
Table 1 Number of markers and genetic length of sex-specific linkage groups of grass carp
LG Female Male
No. of loci Length (cM)cM/markerNo. of lociLength (cM)cM/marker
1
2
3
4
5
6
7
8
9
16
15
7
14
14
8
9
11
15
5
6
9
8
7
8
8
13
17
8
4
5
3
5
4
219
83.3
76.0
50.1
48.3
70.9
59.2
49.0
59.0
59.6
14.1
38.8
48.5
48.5
48.5
44.3
56.0
60.5
46.4
42.7
40.3
27.3
22.4
44.5
11.2
1149.4
47.9 (± 17.4)
5.2
5.1
7.2
3.5
5.1
7.4
5.4
5.4
4.0
2.8
6.5
5.4
6.1
6.9
5.5
7.0
4.7
2.7
5.3
10.1
5.5
7.5
8.9
2.8
-
11
15
8
11
12
8
10
10
9
8
9
8
5
8
10
10
14
14
6
2
4
6
4
10
212
75.5
63.9
50.5
62.6
43.2
47.2
40.4
46.7
50.8
57.4
54.3
45.0
23.1
19.8
36.0
27.8
19.7
27.7
16.8
12.5
14.6
21.1
3.5
28.7
888.8
6.7
4.3
6.3
5.7
3.6
5.9
4.0
4.7
5.6
7.2
6.0
5.6
4.6
2.5
3.6
2.8
1.4
2.0
2.8
6.3
3.7
3.5
0.9
2.9
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Total
Average (± SD) 9.1 (± 4.2)5.2 (± 1.8)8.8 (± 3.3)37.0 (± 18.9) 4.2 (± 1.7)
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Figure 4 Sex-averaged map (LG1 to 8) of grass carp (CID_LG) and synteny relationship to the assembled zebrafish genome sequence
(DR_LG). Each vertical line represents individual grass carp linkage group (left) or zebrafish chromosome (right). Marker names and genetic
distances (cM) of each linkage group were shown on left side, and the marker names and their physical distances (Mb) for zebrafish
chromosome were given on right side. For the marker on the zebrafish chromosome without assignment to the current linkage group, its
targeted LG on the grass carp linkage map was shown in italics on the right side of zebrafish chromosome.
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Figure 5 Sex-averaged map (LG9 to 18) of grass carp (CID_LG) and synteny relationship to the assembled zebrafish genome sequence
(DR_LG). Each vertical line represents individual grass carp linkage group (left) or zebrafish chromosome (right). Marker names and genetic
distances (cM) of each linkage group were shown on left side, and the marker names and their physical distances (Mb) for zebrafish
chromosome were given on right side. For the marker on the zebrafish chromosome without assignment to the current linkage group, its
targeted LG on the grass carp linkage map was shown in italics on the right side of zebrafish chromosome.
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blocks could be defined by two or more syntenic loci in
this study. This showed substantial macrosynteny rela-
tionship existed between the two species.
The extensive colinearity was very evident for some
syntenic chromosomes/linkage pairs e.g. CID_LG3-
DR_LG21, CID_LG5-DR_LG13, CID_LG8-DR_LG1,
CID_LG9-DR_LG12, CID_LG10-DR_LG11 but partial
collinearity was more frequently observed for other
chromosomes/linkage pairs, indicating large-scale chro-
mosomal rearrangements were rare but small inversions
and translocations were prevailing. However, there were
few exceptions e.g. both of the zebrafish chromosomes,
DR_LG10 and DR_LG22, were mainly syntenic to one
grass carp linkage group, CID_LG24 and shared ≥50%
orthologous markers of the chromosome. In addition,
16 SNP markers mapped to 10 grass carp LGs were
assigned to 10 chromosomes of the zebrafish genome
map of which, 14 (87.5%) were found in syntenic
regions (defined with ≥50% orthologous markers). How-
ever, for the 263 mapped microsatellite markers, only
47.5% were found in syntenic regions. This data indi-
cates a strong conservation of functional genes in the
two species.
Discussion
DNA markers and genetic maps
Genetic maps are essential tools for fish genomic studies
[28]. Development of a large number of sequence-based
genetic markers including microsatellites and SNPs is
necessary for mapping QTLs for traits of interest and
carrying out marker-assisted selection (MAS). Using 307
informative DNA makers, we have constructed a first
generation linkage map of grass carp including 263
microsatellites and 16 SNPs. Due to difficulty in scoring,
Figure 6 Sex-averaged map (LG19 to 24) of grass carp (CID_LG) and synteny relationship to the assembled zebrafish genome
sequence (DR_LG). Each vertical line represents individual grass carp linkage group (left) or zebrafish chromosome (right). Marker names and
genetic distances (cM) of each linkage group were shown on left side, and the marker names and their physical distances (Mb) for zebrafish
chromosome were given on right side. For the marker on the zebrafish chromosome without assignment to the current linkage group, its
targeted LG on the grass carp linkage map was shown in italics on the right side of zebrafish chromosome.
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28 markers were not assigned to the linkage map. Two
hundred and seventy-nine loci were arranged into 24
linkage groups. Grass carp is a diploid fish with 2n = 48
chromosomes [29,30], therefore the linkage group num-
ber is corresponding to the haploid chromosome num-
ber of grass carp. According to our best knowledge, this
map is the first linkage map of a food fish species in the
family Cyprinidae exclusively based on co-dominant
markers (i.e. microsatellites and SNPs).
The genome size of grass carp estimated with a hap-
loid C-value of 1-1.09 pg [31] and the conversion
formulas (1 pg = 978 Mb) [32] was 978-1066 Mb. The
length of the grass carp linkage map was 1176.1 cM.
Table 3 Number of the conserved orthologous markers
between grass carp and four model fish species
Typea
ZebrafishTetraodonFuguMedaka
1
2
3
4
Total (%)b
126
12
13
13
164 (59.2%)
3
8
12
13
36 (13.0%)
3
2
15
13
33 (11.9%)
2
2
6
13
28 (10.1%)
’a’, shows the conservation of orthologs, e.g., type 1 indicates unique
orthologs identified for each model species, type 2 shows orthologs identified
only in two model species (the given model species and another model
species), type 3 shows orthologs identified in three model species (the given
model species and other two model species), etc;’b’, ratio of all orthologous
markers to the total informative markers used for similarity searches.
Table 2 Summary of the sex-averaged linkage map of grass carp and the homologous zebrafish chromosomes that
share at least two markers in common
Grass carp linkage mapZebra fish genome map
LGLength of LG
(cM)
No. of lociALD (cM)Homologous
chromosome1
No. of
syntenic loci2
Length of syntenic
region (Mb)
Syntenic ALD
(Mb)
R
1
2
3
4
5
6
7
8
9
95.2
69.9
68.5
61.3
57.0
56.9
56.1
55.4
55.2
54.1
53.7
50.9
50.8
47.3
43.3
41.9
40.6
39.1
38.9
34.3
29.6
28.7
24.0
23.4
23.4
1176.1
18
19
10
16
15
11
13
13
15
9
10
12
10
10
11
10
17
18
10
4
7
6
5
10
10
279
5.3
3.7
6.9
3.8
3.8
5.2
4.3
4.3
3.7
6.0
5.4
4.2
5.1
4.7
3.9
4.2
2.4
2.2
3.9
8.6
4.2
7.8
4.8
2.3
2.3
–
7
3
11
7
3
8
8
4
9
7
3
2
5
8
3
7
8
4
8
9
5
2
6
2
4
3
2
138
54.6
38.1
39.6
42.0
39.0
36.4
38.2
35.3
24.9
41.6
31.4
37.8
15.7
51.6
44.3
34.4
53.6
55.7
23.6
11.9
30.4
3.4
22.8
2.1
1.1
809.5
5.0
5.4
13.2
5.3
4.9
9.1
4.2
5.0
8.3
20.8
6.3
4.7
5.2
7.4
5.5
8.6
6.7
6.2
4.7
6.0
5.1
1.7
5.7
0.7
0.6
–
78%
60%
86%
91%
72%
79%
78%
63%
52%
92%
73%
71%
28%
91%
94%
67%
77%
94%
59%
36%
54%
6%
44%
5%
3%
63%
21
19
13
23
18
1
12
11
4
16
14
20
15
9
5
6
24
25
8
2
17
10
22
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
24
Total
Average (± SD)49.0 (± 16.2)11.6 (± 4.1) 4.2 (± 1.6)– 5.5 (± 2.7)32.4 (± 14.6)6.3 (± 4.0)–
ALD, average inter-locus distance; ‘1’ the homologous zebrafish chromosome that share at least two markers in common with a given grass carp linkage group;
‘2’, the number of syntenic loci in one pair of grass carp LG and homologous zebrafish chromosome; R, the ratio of syntenic region to displayed region of
reference chromosome (Mb) in NIH genetic sequence database http://blast.ncbi.nlm.nih.gov/Blast.cgi; Length of syntenic region (Mb) was calculated based on the
distance between the longest syntenic pair of loci in each homologous zebrafish chromosome.
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Therefore, the ratio between physical and linkage dis-
tance was 832 - 906 kb/cM. This figure is similar to
some other fishes, such as rainbow trout [16], but higher
than that of turbot (530 kb/cM)[19] and tiger pufferfish
(420 kb/cM) [33]. The length of the grass carp map is
far less than the linkage map of the common carp
(Cyprinus carpio; 4111 cM)[34], but similar to the
length of silver carp (Hypophthalmichthys molitrix; 1150
cM) and bighead carp (Aristichthys nobilis; 1209 cM)
genetic maps [35].
Compared to density of first-generation linkage maps
of other species, such as the turbot (6.5 cM) [19], tiger
pufferfish (7.1 cM) [33], Barramundi (4.7-6.2 cM) [9]
and olive flounder (4.7 cM) [36], the average resolution
of our map was slightly better with an average distance
of 4.2 (± 1.6) cM between loci. Therefore, such a map
would be dense enough for the initial mapping of inter-
esting agriculture traits and facilitate future comparative
genomic analyses for understanding genome evolution.
Chinese scientists are planning to sequence the com-
plete genome of grass carp. Therefore, this linkage map
could be complementary to a genome sequencing
project.
Sex recombination ratio
It is important to know the relative rates of recombina-
tion for both sexes of any species [37]. Significant differ-
ences in sex recombination ratios have been reported
for many fishes such as rainbow trout (F:M, 1.68:1 and
3.25:1) [16,38], zebrafish (2.74:1.0) [37], Atlantic halibut
(1.89-2.53:1) [39], European sea bass, Dicentrarchus lab-
rax (1.5:1) [40], turbot (1.6:1) [19], Barramundi, Lates
calcarifer (2.06:1) [9]. Family-specific recombination
rates were also demonstrated in some salmon species
[41]. These studies have shown that reduced male
recombination rates might exist extensively in teleost
fishes although it varied greatly among chromosomes
and sub-chromosomal regions. The female:male recom-
bination rates (~ 2:1) for the parents of two families in
this study are similar to the ratios that were reported
previously. However, we observed some exceptions for
some marker intervals with higher recombination rates
Table 4 Macrosyteny relationship between grass carp and zebrafish genome
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 Total
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
171110
2
7
5
9
10
13
6
4
6
2
3
8
4
8
10
7
10
9
9
4
3
5
6
4
2
7
5
81
19
1111
6
4
1113
2
3
8
13
8
118
1114
91
18
117
31
12
1
1
1
4
5
12
Total
Chra
12
2
9
3
4
2
13
6
9
2
5
2
10
2
8
2
5
3
2
1
7
3
9
2
4
2
8
2
8
1
4
1
10
3
10
2
5
1
3
2
6
1
3
2
4
1
6
3
164
–
“D1-25” refer to the 25 zebrafish chromosomes and “C1-24” refer to the 24 grass carp linkage groups, respectively; ‘a’, the zebrafish chromosome number that
shows synteny to a given grass carp linkage group.
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in male, which have also been reported in some fishes,
such as zebrafish, trout and Barramundi [9,37,38].
Previous studies suggested many factors such as peri-
centromeric suppression, CpG islands, GC content,
polyA/polyT content, simple repeats, LINE, SINE ele-
ments and other sequence features would influence
recombination rate, however the molecular mechanisms
responsible for the difference between the two sexes are
still not well understood [3,37].
Comparative mapping
Both grass carp and zebrafish belong to the family
Cyprinidae with the divergence time of 63 ± 2 million
years (Mya) [26] while the time of divergence between
zebrafish and Takifugu was 290 ± 6 Mya [42]. The simi-
larity searches against 4 model fish species reference
genomes showed that 59.2% grass carp sequences used
were considered to be significantly similar to zebrafish
genome sequences. However, much lower significant
hits ranging from 10.1% to 13.0% in Tetraodon, fugu
and medaka was found. The result is in agreement with
phylogenetic data [42], showing zebrafish was more
closely related to grass carp than other model species.
Karyotype evolution in teleosts occurred mainly by
inversion and not by fusion or fission of chromosomes
[33,43]. Since grass carp (2n = 48) and zebrafish (2n =
50) have different karyotyes and genome sizes (C-value
of 1-1.09 pg for grass carp and 1.68-2.28 pg for zebra-
fish) [31], the strict pairwise synteny of chromosome
pairs between grass carp and zebrafish was not expected.
Our study demonstrated extensive colinearity for some
syntenic chromosome/linkage pairs. For example, each
zebrafish chromosome was mainly syntenic to one grass
carp LG and shared ≥50% othologous markers of the
chromosome in common. This study also indicated that
substantial macrosynteny relationship, inversions and
translocations existed between grass carp and zebrafish.
Therefore, our results provide further support to the
studies of karyotype evolution in teleosts by Jaillon et al.
[43] and Kai et al. [33]. However, due to the precision
in marker order on the current grass carp linkage map
was not high enough for some markers, and reference
sequences were not free of assembly error [44], it was
possible that some of the disagreements in marker order
between grass carp and zebrafish were not accurate,
which can be confirmed by further cytogenetic studies,
such as FISH experiments.
Comparative genomics had shown the existence of
several blocks of synteny between zebrafish and human
gene maps [45]. Our study showed a strong conserva-
tion of gene order between grass carp and zebrafish and
other model species. For example, 87.5% of the gene-
based SNP markers and 47.5% of the flank sequence-
based microsatellite markers were found in syntenic
regions of grass carp to zebrafish; and 23.9% of the loci
with significant hits matched at least 2 model species
and 7.4% matched 4 model species. However, the con-
servation was different for most of the compared chro-
mosomes between grass carp and zebrafish. This could
be reflected by differentiated homologous zebrafish
chromosome number that shows synteny to different
grass carp linkage group (ranging from 1 to 6 chromo-
somes for different grass carp LG). Some studies had
reported that chromosomes might be functionally parti-
tioned [46] and large-scale genomic rearrangements
were nonrandom with nucleotide variation [47]. Bour-
que et al. [48] also suggested that the total number of
microrearrangements was much higher for anonymous
sequence-based data than for gene-based data, as many
of the microrearrangements in sequence-based synteny
blocks lay outside exons. These studies demonstrated
that conservation of synteny might reflect important
functional relationships at chromosomal levels.
Synteny among species or genera might also aid initial
QTL experiments with candidate gene approaches [49].
In this study, 16 SNPs derived from functional genes or
ESTs related to complex genetic basis for interesting
quantitative traits in productions such as muscle myosin
heavy chain (MHY; SNP0038), TNF-related apoptosis
inducing ligand (TRAIL; SNP0053) and c-Fos (c-fos;
SNP0067) were mapped on to the linkage map at high
confidence level. Most of them were also assigned to the
conserved synteny regions of grass carp linkage map to
zebrafish genome. The orthologous markers closely
linked to these genes on conserved chromosomes of
related model fish species might be considered to iden-
tify candidate genes and carry out MAS in further
breeding strategies of the grass carp.
Conclusions
We constructed a first generation linkage map contain-
ing 279 DNA markers (263 microsatellites and 16 SNPs
in ESTs). The marker density was 4.20 cM/locus.
Recombination rate was higher in females than in males.
This linkage map will provide important base for map-
ping QTL affecting economically important traits and
could be complementary to grass carp genome sequen-
cing project. Comparative mapping between the grass
carp and other model organisms revealed that substan-
tial macrosynteny relationship and extensive colinearity
existed between grass carp and zebrafish. Comparative
mapping will facilitate understanding genome evolution.
In near future the identification of QTL associated
traits, such as related to disease and parasite resistance
and growth rate, would be conducted with the markers
on the map. Our study also showed strong syntenic rela-
tionships and a number of conserved colineality existed
between grass carp and zebrafish genome. Therefore,
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gene prediction directly from zebrafish to grass carp was
possible. Further studies focusing on the transferring of
genomic information available from zebrafish to the
selection of grass carp had been put forward. A high
density map with gene-based markers was also planned,
which would provide a detailed analysis of conservation
among orthologous intervals in related species and a
basis for location of genes and MAS of grass carp or
other Cyprinidae.
Methods
Mapping families and DNA isolation
Prior to construction of reference mapping families, 150
candidate grass carp parents (75 males and 75 females)
derived from wild populations in Yangtze and Pearl
river systems were genotyped with 6 microsatellite mar-
kers (Ci02, Ci08, Ci09, Ci11, Ci12 and Ci15) as
described previously [50] and 3 microsatellite markers
(CID0025, CID0029 and CID0037) developed in this
study. Genetic relatedness was calculated on the basis of
microsatellite genotypes [51]. Two pairs of grass carps
with the most genetic difference were selected for artifi-
cial insemination to produce F1 offspring. From each
pair, Two millions of fertilized eggs were obtained and
96 progenies at the age of one month were randomly
collected and stored in 75% ethanol for subsequent link-
age analyses. Parental fin clips of 192 offspring from two
families were sampled for DNA extraction with the
method described as in Yue and Orban [52].
Development and genotyping of DNA markers
Two microsatellite-enriched partial genomic libraries
(CA and GA repeats) were constructed using DNA from
one adult grass carp according to method described by
Yue et al [53] with some minor modifications [50].
From each library, 2000 clones were sequenced in both
directions with M13 and M13 reverse primers and Big-
Dye chemicals on an ABI 3730×l Genetic Analyzer
(Applied Biosystems). The microsatellite loci containing
seven or more repeat units and enough flanking regions
were identified for primer design using the program Pri-
merSelect (DNASTAR, Wilmington, DE). In addition,
17 microsatellites from a previous publication [50] were
selected. Microsatellites were named as “CID” followed
by 4 numbers while duplicate markers were named as
“CID” and 3 numbers plus an “A” or “B”. In addition,
six EST-derived microsatellite loci were chosen by scan-
ning the grass carp EST database in GenBank and their
primers flanking repeats were developed. Three of them
were mapped and given names as “EST” followed by 4
numbers.
To optimize PCR for each microsatellite, PCR reaction
was performed in 25 μL volume containing 10 ng geno-
mic DNA, 1×PCR buffer, 100 μmol of each dNTPs, 0.2
μmol forward primer, 0.2 μmol reverse primer and 1 U
of Taq DNA polymerase (FINNZYMES, Espoo, Finland)
on a thermal cycler (MJ Research, CA, USA) with the
following cycling profile: one denaturation step for 2
min at 95°C was followed by 35 cycles of 30 sec at 94°C,
30 sec at 50°C, 55°C or 60°C and 45 sec at 72°C. The
final step was a prolonged extension of 5 min at 72°C.
PCR products were resolved on 2% agarose gel and
visualized by ethidium bromide staining. Five hundred
and ten primer that pairs amplified clear and strong
bands were selected for labeling with fluorescent dyes.
For each microsatellite, one primer was labeled either
FAM or HEX (1stBase, Singapore). These microsatellite
markers were selected for genotyping parents. Finally
263 microsatellite markers, being informative among
four parents, were selected for genotyping the two map-
ping families comprising 192 individuals. Microsatellites
were amplified on grass carp genomic DNA with fluor-
escent primers under the same conditions used for
amplification with unlabeled primers. PCR products
were resolved on ABI3730×l Genetic Analyzer (Applied
Biosystems, CA, USA) and were sized relative to an
internal size standard (GeneScan-500 ROX) using Gene-
Mapper 3.5 software package (Applied Biosystems, CA,
USA) as described previously [54].
SNPs in ESTs were detected by PCR amplification of
DNA of four parents and sequencing of PCR products.
Briefly, 63 ESTs from grass carp were downloaded from
GenBank and were aligned with genomic sequence data
from zebrafish on GenBank. Primer sites in conserved
exon regions were identified and a total of 63 primer
pairs allowing PCR amplification of an intron-spanning
fragment were developed. The primer sets were given
names as “SNP” followed by 4 numbers in the genetic
map. Fifty-five primer pairs amplified intron-spanning
fragments which were sequenced as described above to
detect SNP in the four parents. SNPs were detected in
16 ESTs. The genotyping for the 16 polymorphic SNP
markers were performed based on ABI PRISM® snap-
shot™ Multiplex Kit (Applied Biosystems, CA, USA)
according to the manufacturer’s protocol. Two to six
SNP primers were combined in multiplex PCR reaction
and the resultant snapshot products were resolved on
ABI 3730×l Genetic Analyzer (Applied Biosystems) after
post-extension treatment with Shrimp Alkaline Phos-
phatase (Promega, Madison, WI). Genotyping of PCR
products were carried out against the internal size stan-
dard GeneScan-120 LIZ using GeneMapper 3.5 software
package (Applied Biosystems, CA, USA).
Details of polymorphic markers in parents used
to construct the grass carp genetic map were summar-
ized in Additional file 1: Table S1 and the sequences
were submitted to GenBank with accession nos
FJ883175-FJ883463.
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Map construction
The sex-specific linkage maps for two mapping families
were constructed independently using the program
LINKMFEX (ver. 1.5) [55]. Genotype data for both
families were checked for inconsistencies with Mende-
lian inheritance and manually corrected for error. Those
markers with LOD ≥ 3 for segregation data were
assigned to the same linkage group for both mapping
families respectively using a two-point analysis. Map dis-
tances were estimated for each best likely order linkage
group using the Kosambi function with the module
MAPDIS. Integration of the sex-specific linkage maps
and construction of a sex-averaged map for two map-
ping families was performed using the module MERGE.
Finally, the sex-averaged linkage groups were numbered
based on their group length (from large to small,
namely, CID_LG1 to CID_LG24). The maps were visua-
lized using MapChart software (ver. 2.1) [56]. For asses-
sing the differences in recombination rate between
sexes, comparisons of recombination differences
between both parents in two mapping families were per-
formed by analyzing all pairwise marker combinations
using a two-way contingency G-test as implemented in
the module RECOMDIF of the program LINKMFEX.
Comparative mapping
The flanking sequences of polymorphic microsatellite
loci and SNPs in ESTs and gene loci among parents
were used for similarity searches against the zebrafish
genomic sequences via the NIH genetic sequence data-
base and against medaka, fugu and tetraodon genomic
sequences via the Ensembl Genome Browser under
default settings. Hits with e < 10-7were considered as
significant. In cases where the searches hit two or more
scaffolds or loci with less than 100 fold difference in the
E-value, the genes were considered to be duplicated
within a genome, in a conservative way, no orthology
were assigned. The comparative map was drawn using
MapDisto software (ver. 1.7) [57].
Additional file 1: Table S1. Primer sequences of markers used in the
construction of a linkage map of grass carp.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-2164-11-
135-S1.XLS]
Additional file 2: Figure S1. Distribution of recombination ratio
between both parents in two grass carp mapping families.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-2164-11-
135-S2.DOC]
Additional file 3: Table S2. Putative othologous locus between the
grass carp and four model fish species genomes.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-2164-11-
135-S3.DOC]
Additional file 4: Table S3. Putative othologous locus between the
grass carp and zebrafish genomes.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-2164-11-
135-S4.XLS]
Abbreviations
LG: linkage group; AFLP: amplified fragment length polymorphism; RAPD:
random amplification of polymorphic DNA; SNP: single-nucleotide
polymorphism; QTL: quantitative trait loci; EST: expressed sequence tag; MAS:
marker-assisted selection; FISH: fluorescent in situ hybridization.
Acknowledgements
This study is funded by the internal funds of the Temasek Life Sciences
Laboratory, Singapore and by the earmarked fund for Modern Agro-industry
Technology Research System, China. We thank our colleague Dr Mamta
Chuahan for English editing.
Author details
1Molecular Population Genetics Group, Temasek Life Sciences Laboratory,
National University of Singapore, 117604 Republic of Singapore.2Key
Laboratory of Exploration and Utilization of Aquatic Genetic Resources,
Shanghai Ocean University, Ministry of Education, Shanghai 201306, China.
3Aquaculture Division, E-Institute of Shanghai Universities, Shanghai 201306,
China.
Authors’ contributions
GHY and JLL initiated the study. JHX, FL, ZYZ, JJF, JBF and GHY carried out
the experimental and molecular work. JHX conducted the data analysis and
drafted the bulk of the manuscript. All authors read and approved the final
manuscript.
Received: 6 September 2009
Accepted: 24 February 2010 Published: 24 February 2010
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