doi:10.1111/j.1365-2052.2009.02014.x
Construction of integrated genetic linkage maps of the tiger shrimp
(Penaeus monodon) using microsatellite and AFLP markers
E.-M. You*, K.-F. Liu†, S.-W. Huang*, M. Chen§, M. L. Groumellec¶, S.-J. Fann** and H.-T. Yu*
*Institute of Zoology and Department of Life Science, National Taiwan University, Taipei, 10617, Taiwan, Republic of China. †Tungkang
Biotechnology Research Center, Fisheries Research Institute, C. O. A., Pingtung, 92845, Taiwan, Republic of China. §Center for Medical
Genetics, Department of Genomic Medicine, Changhua Christian Hospital, Changhua city, Changhua County, 500, Taiwan, Republic of
China. ¶Aquaculture de la Mahajamba, Unima, Mahajanga 401, Madagascar. **Institute of Biomedical Science, Academia Sinica, Taipei,
115, Taiwan, Republic of China.
Summary
The linkage maps of male and female tiger shrimp (P. monodon) were constructed based
on 256 microsatellite and 85 amplified fragment length polymorphism (AFLP) markers.
Microsatellite markers obtained from clone sequences of partial genomic libraries, tandem
repeat sequences from databases and previous publications and fosmid end sequences were
employed. Of 670 microsatellite and 158 AFLP markers tested for polymorphism, 341 (256
microsatellite and 85 AFLP markers) were used for genotyping with three F1 mapping
panels, each comprising two parents and more than 100 progeny. Chi-square goodness-offit test (v2) revealed that only 19 microsatellite and 28 AFLP markers showed a highly
significant segregation distortion (P < 0.005). Linkage analysis with a LOD score of 4.5
revealed 43 and 46 linkage groups in male and female linkage maps respectively. The male
map consisted of 176 microsatellite and 49 AFLP markers spaced every 11.2 cM, with an
observed genome length of 2033.4 cM. The female map consisted of 171 microsatellite and
36 AFLP markers spaced every 13.8 cM, with an observed genome length of 2182 cM.
Both maps shared 136 microsatellite markers, and the alignment between them indicated
38 homologous pairs of linkage groups including the linkage group representing the sex
chromosome. The karyotype of P. monodon is also presented. The tentative assignment of
the 44 pairs of P. monodon haploid chromosomes showed the composition of forty metacentric, one submetacentric and three acrocentric chromosomes. Our maps provided a solid
foundation for gene and QTL mapping in the tiger shrimp.
Keywords amplified fragment length polymorphism, F1 mapping panel, karyotype, linkage
maps, microsatellite, Penaeus monodon.
Introduction
The tiger shrimp Penaeus monodon is a marine species of
high commercial value. The culture and production of the
tiger shrimp have been concentrated in the Asia and Pacific
regions where shrimp products are exported to the world
(Fao 2006). The aquaculture of tiger shrimp depends on
wild-caught broodstocks to a great extent because the
Address for correspondence
Dr A. H-T. Yu, Institute of Zoology, National Taiwan University, Taipei,
10617, Taiwan.
E-mail: [email protected]
Accepted for publication 15 November 2009
shrimps do not easily reach their reproductive potential
once kept in artificial conditions. This also keeps the tiger
shrimps from being domesticated (Wilson et al. 2002;
Fao 2006). In addition, the high dependence on wildcaught seed shrimps makes the tiger shrimp industry
vulnerable to deterioration of broodstock quality. Consequently, the production of tiger shrimp is often affected by
outbreaks of deadly infectious diseases caused either by
bacteria or by viruses (Fao 2006; Maneeruttanarungroj
et al. 2006).
To implement the manipulation for improvement of the
shrimp production, construction of a genetic map is usually
the first priority (Wilson et al. 2002). Once a linkage map
has been constructed, it can be used in combination with
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studies of breeding and assessment of quantitative traits of
interest, thus allowing the quantitative trait loci (QTL) to be
placed on the linkage map (Liu & Cordes 2004). Subsequently, coding regions can be located on the linkage
groups by mapping the microsatellite markers found in the
expressed sequence tags (ESTs) (Liu & Cordes 2004;
Maneeruttanarungroj et al. 2006). Eventually, the highresolution linkage map can be integrated with a physical
map (i.e. fosmid contigs from fosmid library) to facilitate a
whole genome sequencing project in the future.
Currently, three genetic maps of Penaeus monodon (Wilson
et al. 2002; Maneeruttanarungroj et al. 2006; Staelens et al.
2008) have been constructed, primarily based on AFLP
markers. The major advantage of AFLPs is to generate a
large number of markers without any prior knowledge of
the genomic sequences, which is a suitable method to study
a non-model organism like P. monodon. However, as a result
of high sequence polymorphism of the restriction enzyme
cut sites and insertion/deletions in the AFLP fragments
itself, a good proportion of the AFLPs were not shared even
within closely related families [see for example (Staelens
et al. 2008)]. As a result, the AFLPs are not easily transferred between mapping families without further experimental procedures (such as acquiring the sequence
information of particular AFLP fragments). In contrast,
microsatellite markers are co-dominantly inherited, transferable between families but require substantial efforts to
develop.
Although obtaining the microsatellites is time-consuming
and expensive, they are abundant in tiger shrimp genomes
(Tassanakajon et al. 1998; Wuthisuthimethavee et al.
2003; Pan et al. 2004; Maneeruttanarungroj et al. 2006).
In addition, their characteristics of small amplification size
and high polymorphism make them an extremely popular
marker type in genetic linkage mapping (Liu & Cordes
2004). Microsatellite-based linkage maps have been generated for several economically significant aquaculture species
such as Nile tilapia (Kocher et al. 1998; Lee et al. 2005),
rainbow trout (Sakamoto et al. 2000), Japanese flounder
(Coimbra et al. 2003), Atlantic salmon (Gilbey et al. 2004),
Arctic char (Woram et al. 2004), ayu (Watanabe et al.
2004a), Pacific oyster (Hubert & Hedgecock 2004), tiger
pufferfish (Kai et al. 2005), sea bass (Chistiakov et al. 2005)
and Pacific whiteleg shrimp (Alcivar-Warren et al. 2007;
Zhang et al. 2007). Therefore, microsatellites have been one
of the priority choices for constructing linkage maps in
various applications.
In this study, we developed F1–pedigreed linkage maps
for both male and female P. monodon, comprising primarily microsatellites (170–176) plus a minor proportion
of AFLP (36–49) markers. We took a two-step mapping
strategy. First, we used one mapping family (Y3) to
construct basic maps, utilizing both microsatellite and
AFLP markers. Second, we chose microsatellite markers
from the basic maps as anchors and recruited more
polymorphic microsatellite markers to be placed onto the
basic maps, using two additional mapping families (EL4120 and EL41-28). The resulting linkage maps are a
powerful tool for selective breeding and mapping of
quantitative trait loci (QTL) in farmed tiger shrimp populations. A previous study reported the chromosome
number of P. monodon to be 2n = 88 for tiger shrimps
collected from the Indian Ocean (Kumar & Lakra 1996).
However, our study (You et al. 2008) has shown that the
tiger shrimp populations from the Australasian region are
apparently distinct from those in the Indian Ocean. As a
result of this observation, we extended our efforts to
further examine the karyotypes of our shrimp samples
from the Australasian region, to ensure that there was no
discrepancy of chromosome numbers of the shrimps
between the two regions. Both the linkage maps and the
karyotype will serve as a reference for complete sequence
analysis of the tiger shrimp genome as well as gene
mapping and comparative genomics.
Materials and methods
Production of family
Three first-generation pedigreed families were produced
either at the Tungkang Biotechnology Research Centre in
Taiwan (Y3) or at Unima Aqualma in Madagascar (EL4120 and EL41-28). The Y3 female parent was a gravid
female brooder captured from the southern coastal waters of
Taiwan. The EL41-20 and EL41-28 parents belong to the
breeding stock at UNIMA/AQUALMA generated from
domestication of nine generations. The tissues of male
parents were from paternal spermatophores acquired from
sperm receptacles of female brooders. The female brooders
spawned in captivity, and the resulting F1 generations were
raised in a nursery tank. The offspring of the Y3 family
(n = 311) were collected over 3 months (weight >1 g). As
for the EL41-20 and EL41-28 families, the offspring
(n = 102) were collected at the post-larvae stage 48 and 49
respectively. Therefore, all the offspring analysed were fullsibs. The parents were not related.
The pleopods and abdominal muscles of the female and its
offspring were sampled for DNA extraction. All the tissues
and spermatophores were preserved in RNAlater (Ambion,
Inc.) or 95% ethanol, and were stored at )20 C.
Genomic DNA extraction
The tissues of each individual (0.05–0.1 g) and the paternal
spermatophore (0.1 g) were carefully extracted and were
placed into individual sterile 1.5-ml microcentrifuge tubes.
DNA was extracted using the Classic Genomic DNA Isolation Kit (Lamda Biotech, Inc) following the manufacturerÕs
protocol. DNA was precipitated using absolute ethanol.
Pellets were washed in 70% ethanol, dried and resuspended
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Microsatellite-based linkage maps of P. monodon
in 100–250 ll of TE buffer. Finally, the DNA was checked
for quality in 1% agarose gel.
Sources of microsatellites
The microsatellite markers were recruited from three sources: (1) clone sequences from partial genomic libraries (see
for an example Pan et al. 2004); (2) public database and
previous publications, in which ESTs were included
(Tassanakajon et al. 1998; Xu et al. 1999; Brooker et al.
2000; Li et al. 2007) and (3) end sequences from a fosmid
genomic library (unpublished data). A total of 670 microsatellite markers were obtained for primer design. We
determined polymorphisms of the microsatellite markers
through the use of an eight-sample test for which we
amplified eight shrimp for each family, including two parents and six of their progeny. Only those that showed more
than two allele sizes were considered polymorphic and were
to be included for further genotyping.
Microsatellite genotyping by PCR
Amplification was carried out in a 10 ll PCR containing
1x Taq Plus Master (LAMDA BIOTECH), 2.5 mM of MgCl2,
5–50 ng of genomic DNA and 0.8 lM of each primer. Forward or reverse primers for each primer pair were used for
selective amplification and thus were end-labelled with a
fluorescent dye (FAM, HEX or TAMRA). The thermocycling
profile consisted of 2 min at 95 C, followed by 35 cycles of
30 s at 95 C, 30 s at 44–61.8 C (varied amongst markers), 40 s at 72 C, and a final extension of 5 min at 72 C.
PCR products were revealed on linear polyacrylamide (LPA)
gels with a MegaBACE 1000 automated sequencer (GE
Healthcare). Either ET-400 or ET-550 Size Standard (GE
Healthcare) was used to determine the allele sizes. Individuals with ambiguous genotypes were amplified and scored
at least twice.
AFLP analysis
Following the protocol of Vos et al. (1995), AFLP analysis
was performed with minor modifications. A total of 100 ng
of genomic DNA was used in the initial ligation step which
was performed on both EcoRI/MseI-cut DNA (Vos et al.
1995) and PstI/MseI-cut DNA (Thomas et al. 1995) using
adaptors and as described. Following the digestion and
ligation step in a 50 ll reaction volume, 0.5 ll was used in
a 25 ll preamplification reaction using (MseI + A) and
(EcoRI + A) primers. Then 0.5 ll of the preamplification
reaction was used directly in a 25 ll selective amplification
reaction with appropriate EcoRI/MseI or PstI/MseI primers
with three selective bases. For this step, we used 23 primer
pairs as described by Wilson et al. (2002) for scoring a
mapping population including 280 F1 offspring of the Y3
family. PCR reaction conditions were implemented as
described by Vos et al. (1995), and the EcoRI and PstI primer used for selective amplification was end-labelled with a
fluorescent dye (FAM or TAMRA). Amplified DNA of the 23
primer pairs (Wilson et al. 2002) was revealed on linear
polyacrylamide (LPA) gels with a MegaBACE 1000 automated sequencer (GE Healthcare). AFLP markers, ranging
from 100 to 550 base pairs, were sized by comparison with
the ET-550 Size Standard (GE Healthcare) and were scored
by using GeneMarker Version 1.6 (SoftGenetics LLC).
Linkage analysis
A two-step mapping strategy was executed as a mapbuilding procedure. First, a mapping population including
280 F1 progeny of the Y3 family was scored for informative
microsatellites and AFLP markers to produce first step ÔbasicÕ
male and female maps. Second, all microsatellite markers
from the basic male and female maps were screened using
two mapping populations, the EL41-20 and EL41-28 families, to obtain anchor markers (no less than two of each
linkage group) and additional polymorphic markers for the
two families respectively. These additional markers were
incorporated in the basic male and female maps to produce
the final maps.
To distinguish the markers with significant segregation
distortion, genotype frequencies were tested against the
Mendelian expectations at the a = 0.005 level based on the
goodness-of-fit test (v2). Linkage analysis and map building
were performed by JoinMap 4 (Kyazma B. V.) (Van Ooijen
2006) with default parameters. The cross-pollination (CP)
coding scheme for outbreeding population with unknown
linkage phase was employed, and significance of linkage
between markers was determined at a LOD threshold of
4.5–5.
Mapping of sex-linked AFLP marker
As we are interested in locating sex-linked tag markers into
linkage maps, a sex-linked AFLP marker (E06M45M347.0)
(Staelens et al. 2008) was examined. E06M45M347.0 was
first determined to show complete linkage with female
individuals in three tiger shrimp families (Staelens et al.
2008). The sequence of the AFLP fragment was obtained
and primers were designed to incorporate the AFLP marker
into a PCR-based allele-specific assay (Staelens et al. 2008),
which could later be applied to other mapping populations.
Subsequently, we examined the mapping families by using
the amplification of the above AFLP fragment to identify the
sex-linked linkage group in the current map. The PCR
reaction was performed in a 10 ll PCR reaction containing
1 · Taq Plus Master (LAMDA BIOTECH), 2.5 mM of MgCl2,
5–50 ng of genomic DNA and 0.8 lM of locus-specific
primers (5¢-TCTAACAGTTCATAAGATC CTAT-3¢ and
5¢-TTAAGCATATACTAAGAATCCAT-3¢) (Staelens et al.
2008). One of each primer pair was end-labelled with a
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You et al.
fluorescent dye FAM. An initial of denaturation step of
2 min at 95 C was followed by 35 cycles of 30 s at 95 C,
30 s at 52 C, 40 s at 72 C, and a final extension for 5 min
at 72 C. PCR products were revealed with a MegaBACE
1000 automated sequencer (GE Healthcare).
Genome length
The genome length was estimated for the male and female
linkage maps separately by the following method: First, the
total lengths of all linkage groups were calculated by adding
the space between adjacent markers in all linkage groups.
Second, to calculate the terminal chromosome regions, the
average space (ÔsÕ) between adjacent markers was calculated
by dividing the total length of all linkage groups by the
number of intervals (number of markers minus number of
linkage groups) (Hubert & Hedgecock 2004). Third, the
genome length was estimated by adding 2 s to the total
lengths of all linkage groups (Fishman et al. 2001). As the
genome of P. monodon contains about 2.17*109 bp
(unpublished data), the relationship between physical and
genetic distance could also be estimated.
Karyotype
Karyotypes of marine decapods are rather difficult to obtain
for two reasons: (1) the mitotic index is low across various
tissues and (2) chromosome sizes are small, which hampers
microscopic observation. (Chow et al. 1990; Campos-Ramos
1997). We had tried to acquire dividing cells from various
preparations such as testes, ovaries, minced larvae, haemolymph and fertilized egg cleavages for karyotyping
analysis, but only a small number of mitotic cells from the
fertilized eggs were obtained according to a protocol (Campos-Ramos 1997) with some modifications.
Spawn from two freshly captured female P. monodon in
southern Taiwan were collected at 3 h after spawning.
The water temperature was about 27.5 C and the salinity
was about 35 ppt. The number of cleaving eggs (4–16 cell
stage) was counted in 400 eggs from one spawning to
confirm the fertilization rate to be at least 50%. Eggs were
washed with fresh sea water using 40 lm mesh and placed
in 20 ml plastic tubes to get about 2 cm of sample depth.
Concentration at 0.025%, 0.05% and 0.1% colchicine
(Sigma Chemical Co.) was used to incubate the eggs in fresh
sea water for 45 min. The eggs were then incubated in
0.9% sodium citrate in distiled water for 30 min. The
samples were then fixed in the freshly prepared CarnoyÕs
fixative (methanol to acetic acid 3:1) for five changes with
each lasting 10 min. The fixed eggs were then stored at 4 C
overnight. Two quick changes of freshly prepared CarnoyÕs
fixative were performed followed by five changes of 50%
acetic acid in distilled water at 40 C to macerate the eggs.
The suspensions were then centrifuged at 1000 rpm for
3 min after each change. The eggs now turned from
translucent to having a white crystalline appearance.
Ten millilitres of resuspended eggs was gently placed in a
55 C preheated Petri dish. We used a glass rod (10 cm in
length and 1 cm in diameter) to crush the eggs in the Petri
dish. The eggs were then placed in a 10-ml centrifuge tube
and observed against the light. Small fragments with crystalline splinters were suspended in the fluid with the
uncrushed eggs staying in the bottom. We discarded the
uncrushed eggs and resuspended the small fragments from
the crushed eggs in 50% acetic acid in distilled water. The
suspensions were centrifuged at 500 rpm for 5 min. The
supernatant was discarded, resuspended and centrifuged for
three changes. The final cell suspensions were placed in
50% acetic acid in distilled water.
Slide preparation was performed by dropping the cell
pellet from 10 cm height to a pre-cleaned slide placed on
the heating plate (70 C). Slides were air-dried for at least
48 h or baked at 95 C in an oven for 30 min. Slides
were then frozen at )20 C for future analysis. Metaphases were counted using the Cytovision system
equipped with a fluorescent microscopy coupled with
high-resolution camera and image capturing software
(Applied Imaging).
Results
Microsatellite markers
To obtain informative microsatellite markers for linkage
analysis, we examined the segregation types of 670
markers from eight individuals (including two parents and
six of their progeny) in each mapping family. In these
eight-sample tests, 494 of the 670 markers could be
successfully amplified by PCR. Amongst the 494 microsatellite markers, 256 (52%) were informative markers for
segregation analysis in at least one mapping family. These
included 128 microsatellite markers cloned from the
partial genomic libraries, 82 from public database and
previous publications (Brooker et al. 2000; Pan et al.
2004; Maneeruttanarungroj et al. 2006; Li et al. 2007)
and 46 from the fosmid end sequences (unpublished
data).
AFLP markers
The AFLP protocol with selected AFLP primers resolved
33–161 bands per primer pair in the size range
60–550 bp. A total of 205 eligible polymorphic fragments
from 23 primer pairs were scored in the Y3 family,
yielding an average of 9 informative markers per primer
pair. Finally, only 157 AFLP fragments ranging from 100
to 550 base pairs were used for the construction of
linkage maps (Table 1).
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Microsatellite-based linkage maps of P. monodon
Table 1 Number of polymorphic bands of 23 pair primer combinations for AFLP analysis.
MseI primer
PstI primer
AAG
ACT
CCT
CAC
CGA
CGT
0P
10 T
4R
8V
1S
3W
4Q
6U
CAC
CGA
MseI primer
CCT
EcoRI primer
AAG
AAC
ACG
ACC
AGC
CTA
CTG
9B
11 C
CTC
CTT
18 D
10 H
16 E
CAG
CAT
9F
15 J
20 G
4K
16 A
2L
6N
20 I
5M
8O
The 23 letters on the right side of the number represent the 23 pairs of primer combinations.
Segregation distortion
Segregation distortion from expectation under Mendelian
inheritance was found in 19 (7.4%) of 256 microsatellite
markers. Although these markers normally are excluded
from linkage analysis, the use of the independence LOD, one
of the grouping parameters provided by JoinMap 4, allows
these markers to be included. This test for independence is
not affected by segregation distortion and leads to less
spurious linkage. As a result, 17 of these 19 markers were
placed onto the linkage map after the linkage analysis. The
departures from 1:1 Mendelian ratios were also found in 28
of the 157 polymorphic AFLP markers (17.8%), but 11 of
these 28 AFLP markers can still be placed onto the final
linkage map after the linkage analysis.
Linkage mapping
Three F1 mapping panels representing three families, each
consisting of two parents and >100 progeny, were used for
genotyping. All informative markers from different families
were finally combined to form a single male map and a
single female map. We used the Y3 family to construct basic
male and female maps. From the basic maps, we chose
microsatellite anchor markers, which were included in map
construction for EL41-20 and EL41-28. The distances
between the additional microsatellite markers to the anchor
markers, resulting from EL41-20 and EL41-28 maps, were
incorporated into the basic maps of the Y3 family to produce
the merged maps. Characteristics of the microsatellite loci
mapped are given in Table S1.
Of the 341 (256 microsatellites + 85 AFLPs) markers
for segregation analysis informative in at least one mapping family, 170 microsatellites and 36 AFLPs were
mapped onto 46 linkage groups (LGs) of the female
framework map, and 176 microsatellites and 49 AFLPs
were assigned to 43 linkage groups of the male map
(Table 2 and Fig. 2). Both maps shared 136 microsatellite
markers, and the alignment between them indicated 38
homologous pairs of linkage groups, designed as LG1LG38 (Figs 1 and 2).
The female map spanned 2182 cM and the spaces between markers range from 0 to 60.5 cM, with an average of
13.8 cM (Table 2). Fifty-two per cent of the intervals between markers ranged from 0 to 10 cM, 25% ranged from
11 to 20 cM, 13% ranged from 21 to 30 cM, 6% ranged
from 31 to 40 cM and 4% ranged from 41 to 60 cM. The
sizes of linkage groups ranged from 5.4 to 143.2 cM with
an average of 48.1 cM, and the number of markers per
linkage group varied from 2 to 11, with an average of five
markers per group (Table 2).
The male map spanned 2033.4 cM and the spaces between markers ranged from 0 to 43.5 cM, with an average
of 11.2 cM (Table 2). Sixty-two per cent of the intervals
between markers ranged from 0 to 10 cM, 20.0% ranged
from 11 to 20 cM, 12% ranged from 21 to 30 cM, 6%
ranged from 31 to 40 cM and 1% ranged from 41 to 50 cM.
The sizes of linkage groups ranged from 1.4 to 107.9 cM
with an average of 47.3 cM, and the number of markers per
linkage group varied from 2 to 11, with an average of five
markers per group (Table 2).
Genome length and the relationship between the
genetic and physical distances
After adding the estimated lengths of telomeres (1251.2 and
960.6 cM) to the total lengths of all linkage groups, the
genome lengths for female and male were 3433.2 and
2994.0 cM respectively (Table 2).
The genome size of P. monodon is approximately
2.17 · 109 bp as estimated from the C value measured by
flow cytometry of hemocytes (unpublished data). The
combination of physical and genetic distances thus gives
an estimate of about 395 kb per cM, although the rela-
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You et al.
Table 2 Summary of male and female genetic linkage map of Penaeus monodon.
Male map
Previous1
Number of microsatellite
markers on the map
Number of AFLP markers
on the map
Number of linkage groups
Observed size of linkage
groups (cM)
Estimated genome size (cM)
Average space between
two markers (cM)
Size of linkage group
(average)
Number of markers per linkage
group (average)
1
2
Female map
Previous2
Current
Previous1
Previous2
Current
34
0
176
28
0
170
117
757
49
82
494
36
48
1101.0
44
2378
43
2033.4
36
891.4
43
2362
46
2182.0
–
7
–
2994.0
11.2
–
8
–
3433.2
13.8
0–84.2
(23.4)
2–8 (3.3)
3.3
10.7–111.3
(54.0)
3–52 (17.2)
1.4–107.9
(47.3)
2–10 (5.2)
0–100.5
(24.8)
2–10 (3.1)
5.2
20.9–132.7
(54.9)
4–28 (11)
5.4–143.2
(48.1)
2–11 (4.5)
Animal Genetics 37, 363–8, 2006.
Genetics 179, 917–25, 2008.
tionship between physical and genetic distances may vary
from one genomic region to another (Brooks & Marks
1986).
Differences in recombination rate between male and
female
To reveal the sex-specific pattern in recombination rates,
recombination fractions for pairs of adjacent markers that
were heterozygous for both parents (Y3 family) and were
significantly linked (LOD score >4.0) were selected. The
recombination rates between the 108 pairs of adjacent
markers, obtained from 29 linkage groups (Fig. 3), are an
average of 0.162 in females and 0.112 in males. Therefore,
the relative recombination ratio (female-to-male; F:M) in
these pairs was 1.45:1, significantly higher in female than
in male (t-test, P < 0.001, d.f. = 193).
Karyotype analysis
Amongst the 122 slides we prepared, only one contained
enough metaphase cell spreads (n = 22) for definite analysis, illustrating the difficulties when anyone attempts to
karyotype marine decapods. The diploid number was
determined to be 88, corresponding to previous studies
(Xiang et al. 1993; Kumar & Lakra 1996). The P. monodon
karyotype (Fig. 4) contained 40 pairs of metacentrics, three
pairs of acrocentrics/telocentrics (arm ratio more than seven) and one pair of submetacentrics (the arm ratio is 4.0,
which is classified as submetacentrics with the range of arm
ratio 1.77.0). As a result of the extremely small sizes of the
chromosomes, it was very hard to discriminate heteromorphic sex chromosomes.
Sex-specific AFLP marker
The sex-linked AFLP marker E06M45M347.0 (Staelens
et al. 2008) was applied to map into the present maps from
our mapping panel. Using a PCR-based allele-specific assay
converted from E06M45M347.0, we placed this locus on to
our female map LG26, which was also covered by four
microsatellite markers (c5425, PmMS4, PmMS17, and
cb1866; Fig. 2). Therefore, our LG 26 should correspond to
the sex chromosome (Staelens et al. 2008).
Discussion
Markers, linkage groups and genome size
Maneeruttanarungroj et al. (2006) have reported linkage
maps for P. monodon with 58 microsatellite markers and
193 other types of markers (largely AFLP markers). In
contrast, we constructed the linkage maps primarily with
microsatellite markers (n = 211) that complement previous
mapping efforts. Moreover, our linkage maps are more
feasible for transferring additional markers and for merging
linkage maps amongst different families of distinct geographical/genetic origins because our linkage groups contain, on average, more than three microsatelllite markers
(3.7 in female and 4.1 in male).
The male and female maps contain 43 and 46 linkage
groups (Table 2) respectively, very close to the number of
chromosome sets (n = 44). Furthermore, more than 52%
of the intervals between adjacent markers ranged from 0
to 10 cM, revealing high resolution for short genetic
distances between two markers in the present maps. Still,
there are three small linkage groups with their lengths
2010 The Authors, Journal compilation 2010 Stichting International Foundation for Animal Genetics, Animal Genetics, 41, 365–376
Figure 1 The male linkage map of Penaeus monodon based on 176 microsatellite and 49 amplified fragment length polymorphism (AFLP) markers. Linkage analysis was performed using JoinMap 4 with a
LOD score of 4.5. The names of microsatellite markers are to the right of each linkage group, and Kosambi recombination distances (cM) between markers are on the left of each group. c-, PM-:
microsatellites developed in this study. BTPm-, PmMS-, Pmo-: microsatellite obtained from databases and previous publications. f-: microsatellite from fosmid end sequences. A box with the locus name:
AFLP marker.
Microsatellite-based linkage maps of P. monodon
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371
Figure 2 Genetic linkage maps of female Penaeus monodon. The female map encompasses 170 microsatellite loci and 36 amplified fragment length polymorphism (AFLP) loci. Linkage analysis is performed
using JoinMap 4 with a LOD score of 4.5. The names of microsatellite markers are to the right of each linkage group, and Kosambi recombination distances (cM) between markers are on the left of each
group. c-, PM-: microsatellites developed in this study. BTPm-, PmMS-, Pmo-: microsatellite obtained from databases and previous publications. f-: microsatellite from fosmid end sequences. A box with the
locus name: AFLP marker.
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2010 The Authors, Journal compilation 2010 Stichting International Foundation for Animal Genetics, Animal Genetics, 41, 365–376
Microsatellite-based linkage maps of P. monodon
Figure 4 The karyotype of P. monodon. T, telocentrics; SM, submetacentrics; All the remaining unmarked chromosomes are metacentric.
shorter than 10 cM (Figs 1 and 2), and 27 microsatellite
markers are unassigned to any of the linkage groups in
the present maps. The above observation suggests that
the gaps exist and still remain to be filled amongst linkage
groups.
The tiger shrimp genome size was estimated to be
2400 cM from an AFLP-based linkage map in a previous
study (Staelens et al. 2008). In this study, we estimate the
genetic genome size to be 3433.2 and 2994 cM for female
and male maps respectively (Table 2), a substantial increase
in coverage.
Map difference between sexes
In our study, the average space between two adjacent
markers is slightly less for the male map than for the female
map (11.2 vs. 13.8 cM, see Table 2), suggesting that the
recombination rate is slightly lower in males than in females.
This result is consistent with the AFLP-based map of P.
monodon (Wilson et al. 2002). The F:M recombination rate
ratio is 1.45 in our study, close to the F:M ratio in higher
vertebrates lying between 1.0 and 2.0 (Dib et al. 1996; Dietrich et al. 1996; Mellersh et al. 1997; Mikawa et al. 1999).
Karyotype
Figure 3 Male vs. female recombination rate, for 108 pairs of adjacent
loci segregating from both parents of Y3 family.
Few reports on the chromosome complement of P. monodon
are available in the literature. The only published karyotype
2010 The Authors, Journal compilation 2010 Stichting International Foundation for Animal Genetics, Animal Genetics, 41, 365–376
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You et al.
of this species was determined from individuals belonging to
the West Bengal population in the Indian Ocean (Xiang
et al. 1993; Kumar & Lakra 1996). The authors determined
8 metacentric, 10 submetacentric, 5 subtelocentric, and 21
acrocentric pairs in their study, with a diploid number of
88. However, the authors also reported that the chromosome numbers in their study range from 74 to 92. This
notable variation was believed to be because of the poor
quality of the chromosome preparation. It is partly because
of the cell source they chose (the minced post-larvae) and
partly because of the small sizes of the shrimp chromosomes. This renders the result dubious and affects the
accuracy in estimating the arm ratios.
We obtained a better quality of chromosome preparation
in this study and found that most of the shrimp chromosomes are metacentric (40 pairs, with the arm ratio ranged
between 1 and 1.7) with the exception of only three pairs of
acrocentrics/telocentrics and one pair of submetacentrics,
which was different from the result by Kumar and Lakra
(Kumar & Lakra 1996). On the other hand, our data conform to the observation that the diploid numbers (2n) of the
Penaeidae range from 68 to 90 (Murofushi & Deguchi
1990). The discrepancy in the number of different chromosome types between these two reports requires further
investigation to determine whether the two P. monodon
populations from the Indian and Pacific Oceans are differentiated in their genetic complements.
Sex-linked marker
The sex-specific AFLP marker, E06M45M347.0, was proven
to be highly associated with female gender and was assigned
to W chromosome (Staelens et al. 2008). We were able to
map E06M45M347.0 onto our LG26. Both male and female
maps share the homologous region of LG26 containing the
same order of four microsatellite markers (c5425, PmMS4,
PmMS17 and cb1866), which implies that the segment of
LG26 is homologous between females and males and is an
indication of a pseudoautosomal region of the W chromosome. Further comparison mapping of the W and Z
chromosomes should be carried out with our linkage
groups.
Future uses
The microsatellite-based linkage maps of P. monodon with
the addition of AFLP markers in this study provide a
foundation for future high-density linkage maps and a
thorough exploration of the entire genome. With the
advantages of being transferable between different strains
within a species, additional codominant markers such as
microsatellites, SNPs and expressed sequence tags will be
the priority to construct a more comprehensive linkage
map.
The present linkage maps also serve as a bridging
framework to line up the large insert library (BAC, fosmid,
etc.) clones leading to eventual physical maps resulting from
assembled sequence contigs (Somridhivej et al. 2008; Hill
et al. 2009; Yu et al. 2009). Our unpublished data for the
tiger shrimp genome sequences have shown that the species
contain much more repetitive genomic sequence than
human. This result points to the difficulty in assembling
sequence data even with high throughput shotgun
sequencing technology. Genetic maps like the one presented
in this study, along with a large-insert genome library, will
prove to be crucial for a tiger shrimp genome sequencing
project.
Acknowledgements
Financial support was granted to Hon-Tsen Yu by the
National Science Council of Taiwan (932317B002016,
942317B002009, 952317B002008 and 962317B
002012), and the National Taiwan University [97R006628
(BM0404)], ROC. We thank them all.
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Supporting information
Additional supporting information may be found in the
online version of this article.
Table S1 Characteristics of 211 microsatellite loci mapped
to the linkage maps of Penaeus monodon.
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