Identication of sex-linked DNA markers by AFLP in

Portunus trituberculatus
XiaoFang Lai , Huan Gao, Binlun Yan, Wenhua Yan, Shanrui Shen
Ocean and Fisheries College, Huaihai Institute of Technology, Lianyungang 222005, China
a b s t r a c t a r t i c l e i n f o
Article history:
Received 14 January 2014
Received in revised form 13 April 2014
Accepted 17 April 2014
Available online 21 April 2014
Keywords:
Portunus trituberculatus
Sex-linked
AFLP
Cluster analysis
To investigate the mechanism of sex determination of crabs, amplied fragment-length polymorphism DNA
analysis was applied to identify sex-specic markers for Portunus trituberculatus. Eleven pairs of primers were
used to amplify DNA isolated from male and female crabs. A total of 481 and 499 fragments were amplied
for females and males, respectively, and the ratios of polymorphic fragments were 20.5965.52% and
27.2762.22%, respectively. Distribution difference values between female and male groups for 10 polymor-
phisms were greater than 40%; Cluster analysis revealed that Nei's genetic distances for 57 polymorphisms clus-
tered according to sex. The results indicate that a difference at the DNA level exists between female and male
crabs and provide data for further study on sex determination.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Sex determination and differentiation mechanisms are relevant for a
wide range of research topics within the life sciences, including heredi-
ty, development and evolution (Lou et al., 2004). Crustacean species are
of particular interest as they exhibit guttate chromosomes, no resolu-
tion centromere, and usually lack heteromorphic sex chromosomes
(Wang et al., 2002). DNA-based tests could potentially be used for re-
search on sex determination and differentiation mechanisms of the
crab Portunus trituberculatus; however, sex-specic markers have not
yet been identied.
Various analyses have been used to identify sex-specic DNA se-
quences in several species; for example, using expressed sequence
tags (EST) for Penaeus monodon (Leelatanawit et al., 2009), randomam-
plied polymorphic DNA (RAPD) for Eriocheir sinensis (Qiu and Lu,
1998), Dioscorea bulbifera (Wang et al., 2007), Populus tomentos (Hou
et al., 2008) and Fenneropenaeus chinensis (Liu and Wu, 1989) and
sequence-related amplied polymorphisms for Ficus pumila (Wu et al.,
2008). Amplied fragment-length polymorphism (AFLP) (Tang et al.,
2008) provides several advantages over other techniques: it combines
the efciency of PCR with the stability of restriction fragment-length
polymorphism (RFLP) analysis, requires small DNA quantities, has
been used to identify many polymorphisms, and can be used to selec-
tively examine genomic coding sequences. AFLPhas beenused to screen
sex-linked DNA markers in plants, such as Cannabis sativa (Lv et al.,
2010), Taxus cuspidate (Xiao et al., 2011), Ginkgo biloba (Wang et al.,
2001) and Vitis amurensis (Tang et al., 2008), and in marine animals,
such as Cyprinus carpio var. Jian (Si, 2006), Oreochromis niloticus (Yang
et al., 2007), Cynoglossus semilaevis (Ma H.Y. et al., 2009; Ma H. et al.,
2009), Oncorhynchus mykiss (Felip et al., 2005), Salmo salar (Artieri
et al., 2006), Gasterosteus aculeatus (Grifths et al., 2000), Plecoglossus
altivelis (Watanabe et al., 2004), Hucho taimen (Zhang et al., 2010),
Scylla serrata (Wang et al., 2004), Neophocaena phocaenoides
asiaeorientalis (Xia et al., 2005) and Marsupenaeus japonicus (Li et al.,
2003). Most of the relationships between the identied DNA markers
and sex determination require further verication.
The crustaceans play very important roles in evolutionary biology
(their sex determination and differentiation are more primitive), diver-
sity and plasticity than vertebrates. In addition, crustaceans exhibit sig-
nicant sex-specic differences in many biological characteristics, such
as growth rate and individual size. Accordingly, the study of the mech-
anisms of crustaceansex determination and differentiation will contrib-
ute to further understanding of evolutionary biology. P. trituberculatus is
a commercially important farmed species in China. The natural distribu-
tion of this species ranges from the Bohai Sea and the Yellow Sea to the
South China Sea and waters of Japan, Korea and Malaysia (Du, 1993).
The female crab, which is more nutritious and has greater economic
value than the male, is commonly referred to as green crab and ma-
rine ginseng (Wang et al., 2004). As sexing this crab is of economic im-
portance, we aimed to identify sex-specic markers for P. trituberculatus
using AFLP and provided data of mechanism of sex determination and
differentiation.
Gene 544 (2014) 216219
Abbreviations: EST, expressed sequence tags; RAPD, random amplied polymorphic
DNA; AFLP, amplied fragment-length polymorphism; RFLP, restriction fragment-length
polymorphism; PTC, Peltier Thermal Cycler; MEGA, Molecular Evolution Genetics
Analyses; SCAR, sequence-characterized amplied regions.
Corresponding author.
E-mail address: lai.xiaofang@163.com (X. Lai).
http://dx.doi.org/10.1016/j.gene.2014.04.034
0378-1119/ 2014 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Gene
j our nal homepage: www. el sevi er . com/ l ocat e/ gene
2. Materials and methods
2.1. Samples
P. trituberculatus were collected from Haizhou Bay, Lianyungang,
Jiangsu, China. Collected crabs had an average body length of 13.25
1.746 cm and an average body weight of 135 19.02 g. The crabs
used in this study (12 females and 17 males) were chosen randomly.
Upon arriving in the laboratory, the claw of each crab was collected
and kept at 20 C immediately for DNA extraction.
2.2. DNA extraction
DNA was extracted from the claw muscle of each crab according to
the standard protocol described by Lai et al. (2013) with modication.
Approximately 100 mg of tissue was homogenized and transferred to
a tube containing 500 l DNA extraction buffer (100 mM NaCl, 10 mM
Tris, pH 8.0, 25 mM EDTA, 0.5% SDS and freshly added 0.1 mg/ml pro-
teinase K). Lysates were incubated at 55 C for 35 h and subsequently
extracted with phenol and chloroform. DNAwas precipitated by adding
1/25 volume of 5 MNaCl and two volumes of ethanol and incubating at
20 C for 3 h. DNA was collected briey by centrifugation, washed
twice with 70% ethanol, air-dried and dissolved in TE buffer. The concen-
tration was determined by absorption at 260 nmwith a GENEQUANT Pro
(Pharmacia Biotech) RNA/DNA spectrophotometer.
2.3. AFLP analysis
AFLP analysis was performed according to the standard protocol de-
scribed by Vos et al. (1995) with modication. Genomic DNA was
digested with EcoRI and MseI (Takara, Dalian, China) in 1 Tang (O)
buffer at 37 C for 3 handligated to EcoRI and MseI adapters ina reaction
mixture containing T4 DNA ligase and 1 T4 DNA ligase buffer (Takara,
Dalian, China) at 16 C overnight. A pre-amplication reaction was per-
formed using a Peltier Thermal Cycler (PTC-200) with a pair of primers
that contain adapter sequences plus one additional nucleotide (E00 and
M00, Table 1). The reaction was subjected to 25 amplication cycles at
an annealing temperature of 56 C for 30 s. The 20 l reaction was dilut-
ed tenfold with distilled water and was used as a template for the sub-
sequent selective PCR amplication using the selective amplication
primers listed in Table 1 (E1E8 and M1M8). Dilutions were subjected
to 10 cycles at a touchdown annealing temperature of 6555 C (1 C
per cycle) followed by 25 cycles at an annealing temperature of 55 C
for 30 s.
2.4. Gel electrophoresis and silver staining
PCR products were mixed with 10 l AFLP loading buffer (99% form-
amide, 10 mM EDTA, 0.05% bromophenol and 0.05% xylene cyanol),
denatured at 95 C for 25 min, and incubated on ice to cool. A 6% dena-
turing polyacrylamide gel (5.7% acrylamide, 0.3% bisacrylamide, 7 M
urea and1TBE) was pre-runat 50 Wfor 30 min. Five microliters of de-
natured sample was loaded onto the gel and electrophoresed for 1.5 h at
50 W and 50 C. The gel was stained according to Merril et al. (1979)
with modication. After electrophoresis, the gel was xed in 10% acetic
acid for at least 30 min. The gel was rinsed in distilled water and stained
with a mixture of 0.2%silver nitrate and 0.06% formaldehyde for 30 min.
The stained gel was rinsed again with distilled water and immersed in a
developing solution (3% sodium carbonate, 0.037% formaldehyde,
0.00084% sodium thiosulfate). Development was subsequently stopped
with 10% acetic acid when visible bands reached desired intensity. All
markers were visually scored independently by two persons to mini-
mize scoring and interpretation errors (Lai et al., 2010).
2.5. Statistical analysis
All assays were performed in triplicate. For a dominant AFLP molec-
ular marker, two results from denaturing polyacrylamide gel electro-
phoresis are possible, presence or absence, indicated with 1 and 0,
respectively, for distinguishing female and male. Ratio of polymorphic
loci, observed number of alleles (Na), effective number of alleles (Ne),
Nei's gene diversity (H), Shannon's information index (I), and Nei's
genetic identity and genetic distance were obtain using POPGENE soft-
ware version 1.32 (http://www.ualberta.ca/~fyeh/fyeh/). Cluster analy-
sis of the data from 12 females and 17 males was conducted with the
UPGMA method using Molecular Evolution Genetics Analyses (MEGA)
software version 4.0 (http://www.megasoftware.net).
3. Results
3.1. Screening and isolation of polymorphic fragments
Atotal of 64 primer combinations were used in AFLP analysis of DNA
from male and female crabs. Eleven primer combinations, generating
more than seven polymorphic fragments, amplied a total of 481 and
499 fragments, with averages of 43.73 and 45.36 fragments per primer
pair fromfemales and males, respectively (Fig. 1, Table 2). The frequen-
cy of polymorphic fragments for each primer combination ranged from
7 (M7E2 in females) to 29 (M4E8 in females and males). A total of 206
and 211 polymorphic fragments were produced with averages of 18.73
and 19.18 for each primer pair from females and males, respectively.
The ratios of polymorphic fragments ranged from 20.59% (M7E2 in fe-
male) to 65.52% (M4E5 in female).
Table 1
Primer sequence used in AFLP.
EcoRI
primer
Sequence MseI primer Sequence
E00 GACTGCGTACCAATTCA M00 GATGAGTCCTGAGTAAC
E1 GACTGCGTACCAATTCAGG M1 GATGAGTCCTGAGTAACTT
E2 GACTGCGTACCAATTCAGC M2 GATGAGTCCTGAGTAACTG
E3 GACTGCGTACCAATTCACG M3 GATGAGTCCTGAGTAACTC
E4 GACTGCGTACCAATTCACC M4 GATGAGTCCTGAGTAACTA
E5 GACTGCGTACCAATTCACT M5 GATGAGTCCTGAGTAACAT
E6 GACTGCGTACCAATTCACA M6 GATGAGTCCTGAGTAACAG
E7 GACTGCGTACCAATTCAAG M7 GATGAGTCCTGAGTAACAC
E8 GATGAGTCCTGAGTAACAC M8 GATGAGTCCTGAGTAACAA
Note: EcoRI primers were named with E1E8; MseI primers were named with M1M8.
M 1 12 29 M
2000bp
1000bp
750bp
500bp
250bp
100bp
Fig. 1. AFLP electrophoresis result of M1E6 in 29 samples. M: DNA marker; samples 112:
, samples 1329: .
217 X. Lai et al. / Gene 544 (2014) 216219
3.2. Polymorphisms in female and male crabs
A total of 57 polymorphisms exhibited differential distribution in fe-
male and male crabs, and 10 of these had a distribution difference value
of greater than 40% (Table 3). The largest distribution difference value,
74.02%, was observed for the M4E3 primer pair and corresponded a
polymorphism which we refer to as M4E3-312, for which 11 females
(12 total) and 3 males (17 total) were positive. However none of the
10 polymorphic fragments was specic for males.
Observed number of alleles (Na), effective number of alleles (Ne),
Nei's gene diversity (H), Shannon's information index (I), and Nei's ge-
netic identity and genetic distance between female and male crabs ob-
tained using POPGENE are listed in Table 4. The genetic diversity was
slightly higher for males than for females. The Nei's genetic identity
and genetic distance between females and males obtained for the 57
polymorphic fragments were 0.8526 and 0.1595, respectively. The re-
sults indicate that genetic diversity between females and males was
low and the genetic differentiation was also low.
3.3. Cluster analysis
Nei's genetic distances for the 57 polymorphisms were subjected to
cluster analysis using UPGMAof MEGAsoftware (Fig. 2). Asingle cluster
was generated, in which female crabs clustered at the lower dendro-
gram and male crabs clustered at the upper dendrogram. These results
indicate that the Nei's genetic distance for crabs of same sex was smaller
than for crabs of different sex, suggesting that the distribution of the 57
polymorphisms is inuenced, in part, by sex.
4. Discussion
AFLP analysis is an effective technique for characterizing polymor-
phisms between organisms. Generally, a large number of loci can be
scored for each primer combination used (Vos et al., 1995), providing
tenfold greater efciency than simple sequence repeat and RAPDanaly-
sis (Pejic et al., 1998). In our study, primer combinations generated av-
erages of 18.73 and 19.18 polymorphic fragments from females and
males, respectively, which are slightly less than observed for Chlamys
farreri (20.8, Li et al., 2005), Pacic oyster (22.6, Li and Guo, 2004) and
eastern oyster (23.3, Yu and Guo, 2005), but muchhigher thanobserved
for kuruma prawn (5.6, Li et al., 2003), tilapia (9.3, Kocher et al., 1998)
andPseudosciaena crocea (10, Ning et al., 2007). The number of the poly-
morphisms for each primer pair varied notably for the eleven selected
primers: the M3E3, M4E3 and M4E8 primer pairs produced over twenty
polymorphic fragments, whereas M7E2 produced less than ten. Similar
results were observed for P. crocea (Ning et al., 2007). However, none of
the polymorphisms were male-specic.
Conversion of AFLP bands into single locus markers (sequence-
characterized amplied regions, SCARs) is relatively fast and simple
(Xu et al., 2001), and has been performed successfully for Cucurbita
pepo (Li et al., 2011), Carassius auratus gibelio (Wang et al., 2009),
O. niloticus (Yang et al., 2007) and V. amurensis (Tang et al., 2008).
The sequence of the M4E3-312 polymorphism was obtained and
primers were designed to amplify the corresponding sequences
from female and male crabs. However, polymorphic markers could
not be converted into SCARs (data not shown).
Our AFLP approach with a sex-type pool strategy quickly identi-
ed sex-specic polymorphisms in P. trituberculatus. Further char-
acterization of these genetic markers may ultimately aid detection
and mapping of sex genes in this species and further the under-
standing of sex-chromosome evolution and sequence variability
among populations.
Conict of interest
No conict of interest.
Table 2
The number of fragment and ratio of polymorphic fragment by 11 primer combinations.
Primer combinations Amplied fragment Polymorphic fragment Ratio of polymorphic fragment

M1E4 39 40 13 14 33.33% 35.00%
M1E6 52 55 14 15 26.92% 27.27%
M2E4 49 47 21 18 42.86% 38.30%
M3E3 43 45 28 28 65.12% 62.22%
M3E4 41 49 17 17 41.46% 34.69%
M4E3 40 40 22 21 55.00% 52.50%
M4E5 29 35 19 20 65.52% 57.14%
M4E8 55 55 29 29 52.73% 52.73%
M7E2 34 34 7 10 20.59% 29.41%
M7E3 56 56 18 22 32.14% 39.29%
M8E4 43 43 18 17 41.86% 39.53%
average 43.73 45.36 18.73 19.18 43.41% 42.55%
total 481 499 206 211 42.83% 42.28%
Table 3
Differences of distribution in 10 fragments between female and male groups of Portunus trituberculatus.
Primer combinations Fragment molecular weight (bp) Presence/total sample Distribution difference value

M1E4 275 11/12 4/17 68.14%
M1E6 450 9/12 5/17 45.59%
M2E4 200 11/12 8/17 44.60%
M4E3 480 7/12 3/17 40.69%
M4E3 228 7/12 3/17 40.69%
M4E3 312 11/12 3/17 74.02%
M4E8 181 12/12 9/17 47.06%
M4E8 218 8/12 2/17 54.90%
M7E2 174 11/11 10/17 41.18%
M7E2 480 9/11 7/17 40.64%
218 X. Lai et al. / Gene 544 (2014) 216219
Acknowledgments
This work was nancially supported by Special Funds from the Cen-
tral Finance to support the development of local universities (CXTD31),
Lianyungang Science and Technology Projects (CN1306), and by Jiangsu
Key Laboratory of Marine Biotechnology programof China (2012HS002).
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Table 4
Parameters of genetic diversity by M1E4 combination.
Sex Na Ne H I
1.8667 1.4417 0.2750 0.4239
1.9333 1.4775 0.2971 0.4581
Fig. 2. The dendrogram for 12 females and 17 males of P. trituberculatus based on Nei's
genetic distance. 112 were female individuals; 1329 were male individuals.
219 X. Lai et al. / Gene 544 (2014) 216219