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Isolation and characterization of novel microsatellite loci for Asian sea bass, Lates calcarifer from genome sequence survey database

Indian Academy of Sciences
Isolation and characterization of novel microsatellite loci for Asian sea
bass, Lates calcarifer from genome sequence survey database
School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
[Abdul Rahman Z., Choay-Hoong L., Mat Khairuddin R., Ab Razak S. and Othman A. S. 2012 Isolation and characterization of novel
microsatellite loci for Asian sea bass, Lates calcarifer from genome sequence survey database. J. Genet. 91, e82–e85. Online only: http://www.]
Asian sea bass, Lates calcarifer is also known as
Barramundi, is an economically important finfish cultured
and marketed in southern Asia. It has an extensive natural
range from India through the Indo–malaysian archipelago,
east to northern Australia and north to southern China
(Greenwood 1976; Moore and Reynolds 1982). In Malaysia,
domestication of Asian sea bass using cage nets has been
established by the Fisheries Department since the 1970s,
although locally produced fry only became available in the
mid 1980s (Awang 1986).
Microsatellite markers are valuable as genetic markers
since they are codominant, highly abundant and have high
levels of polymorphism (McCouch et al. 1997; Hayden and
Sharp 2001). Molecular markers can be divided into type I
(coding) markers which are associated with genes of known
functions and type II (noncoding) markers which are asso-
ciated with anonymous genomic sequences (O’Brien 1991).
In general, most microsatellites represent type II markers.
Nongene sequences are free to mutate, causing high levels
of polymorphism, whereas sequences within protein-coding
regions generally show low levels of polymorphism due to
functional selection pressure (Chistiakov et al. 2006)and
are more difficult to develop (Liu et al. 1999). Large-scale
sequencing projects have generated the genome sequence
survey (GSS) database, offering an alternative means to
develop microsatellites in less time at lower cost. GSS
sequence databases ( provide
a rich source for isolating thousands of sequences con-
taining putative microsatellites, and offer the possibility of
developing hundreds of polymorphic microsatellite markers
(Chistiakov et al. 2006). Microsatellites for this particular
species are extensively available (Yue et al. 2002;Simand
For correspondence. E-mail:
Othman 2005;Zhuet al. 2006), newly identified microsatel-
lites loci may aid in other studies e.g., genome mapping.
We have taken the approach of mining microsatellite mark-
ers from GSS sequences to increase the availability of
microsatellites for L. calcarifer.
Materials and methods
A total of 5607 GSS sequences present in the database
( were screened for micro-
satellites using Tandem Repeat Finder 2.2 (Benson 1999).
From 5607 GSS sequences, 263 were identified with
microsatellite motifs. Only dinucleotide, trinucleotide, tetra-
nucleotide and pentanucleotide repeats were targeted, with
a minimum length of 20 bp. PRIMER3 (Steve and Helen
2000) was used to design primer for flanking region of each
microsatellite locus, and only a set of 206 microsatellite
primer pairs were designed and synthesized. Microsatellite
motifs that we failed to design as primers were eliminated.
Thirty individuals of L. calcarifer were collected from
the Straits of Malacca (west coast of peninsular Malaysia).
Genomic DNA was isolated from muscle tissues follow-
ing the standard phenol–chloroform method (Taggart et al.
1992) with some modifications. Tissue was homogenized in
500 μL of extraction lyses buffer together with 0.5 μg/mL
proteinase K and incubated at 55C. Following phenol :
chloroform : isoamyl alcohol (25 : 24 : 1) extractions, the
supernatants were precipitated by the addition of 2 volumes
of absolute ethanol. DNA was washed with 70% ethanol,
dissolved in distilled water and stored at 20C. PCR
amplification was conducted in a total reaction volume
of 25 μL consisting of 1.5 μL of template DNA (100 ng),
1×magnesium free Green GoTaq R
Flexi buffer, (1.5–3.0)
mM MgCl2, 2 mM dNTPs mixtures, 10 μmol of each primer
setand1UTaq DNA polymerase (Promega, Wisconsin,
Keywords. genome sequence survey; microsatellite; Lates calcarifer.
Journal of Genetics Vol. 91, Online Resources e82
Isolation and characterization of microsatellite loci for Lates calcarifer
Table 1. Characteristics of 23 polymorphic GSS-derived microsatellites for Lates calcarifer.
Locus Sequence Repeat motif Ta(C) Size range NAHOHEPvalue Accession
Lc02G003F04(A) F: GGTGACAGTGCTGCTGAGAA (ATAG)12 45.0 153–181 8 0.533 0.766 0.003 HQ713527
Lc02G003F04(B) F: TTTCCCCATTGTGGGACTAA (TTATC)657.7 151–176 5 0.467 0.592 0.148 HQ713526
Lc02G042G09 F: GGCCATGACTCACCAGTTTT (AC)12 60.0 178–200 12 0.759 0.883 0.001aHQ713522
Lc02G044F11 F: GGCTCTGACCTTCCTCACAG (AC)7A-A(AC)860.0 200–228 6 0.400 0.594 ****aHQ713528
Lc02G044H04 F: TGACAACCCCCTGCATTTAT (GT)4AT(G-T)20 62.7 122–146 13 0.929 0.894 ****aHQ713523
Lc02G045G12 F: CCCACACTATTGCAGCAGAA (GT)20 45.0 195–235 14 0.667 0.912 ****aHQ713533
Lc02G047E11 F: CCACCGTGATCTCAGCTTTT (CA)5TAA-A(CA)12 52.5 222–268 14 0.933 0.938 ****aHQ713524
Lc05G005B05 F: CACTATTCCCTGTGGTGTCG (GA)11 53.9 127–149 10 0.767 0.859 0.004 HG713511
Lc05G007G11 F: GAGCTGCTGGGTAACAGAGG (AC)16 59.7 172–190 10 0.767 0.866 ****aHQ713512
Lc05G010F10 F: ATGGGGAGTTATGGTTCCTT (AC)13 59.7 210–246 17 0.800 0.918 0.010 HQ713529
Lc05G017A04 F: GCGTGAAGGACAGCTACCTC (AC)12(TC-AC)3(AC)259.7 217–253 9 0.767 0.859 ****aHQ713514
Lc02G002H09 F: GAGGTGAGGCTCAAAACTGG (AC)14 57.7 191–217 8 0.741 0.829 0.001aHQ713525
Lc05G005D05 F: CGGTGCAGAGTGGATCAGTA (TG)19 56.0 137–181 19 0.767 0.918 ****aHQ713530
Lc05G010E09 F: AAGCACAATCACGCACTCAC (TG)15 60 120–138 3 0.667 0.560 ****aHQ713513
Lc05G017H11 F: CCATCTGTGGGCCTGTTTAT (TG)11 60.0 200–246 16 0.800 0.916 ****aHQ713515
Lc02G012G04 F: TCTTCCAAGCCTCTTCCAAA (CA)19 61.0 170–186 9 0.733 0.802 ****aHQ713516
Lc02G015H11 F: ATACAGGGGGCGTAGAAGGT (CA)21 54.2 177–221 19 0.767 0.917 ****aHQ713531
Lc02G016E12 F: CCGGGTAATTGTATCGGACA (CA)12 64.7 214–250 16 0.897 0.926 0.108 HQ713517
Lc02G018F03 F: ATTTCTGCCATGTTCGCTCT (AC)15 65.0 181–199 10 0.724 0.796 0.001aHQ713518
Journal of Genetics Vol. 91, Online Resources e83
Zulaiha Abdul Rahman et al.
Table 1 (contd)..(contd).
Locus Sequence Repeat motif Ta(C) Size range NAHoHEPvalue Accession
Lc02G019C11 F: ATTGGGCTGTGAGGTTTCAG (GT)2TT(GT)863.9 143–159 8 0.833 0.834 0.003 HQ713519
Lc02G020F03 F: TACGTCCCAGGCTGATCTGA (GT)11 64.7 196–276 24 1.000 0.951 ****aHQ713520
Lc02G022C11 F: TGGGAGGCAGAGTCATTTCT (TG)11 56.4 153–209 22 0.833 0.944 ****aHQ713532
Lc02G029G12 F: GCAGAAAGACCCTGAGCTTG (TG)9-TA(TG)362.7 234–292 16 0.800 0.908 ****aHQ713521
HO, observed heterozygosity; HE, expected heterozygosity; NA, number of alleles; Pvalue were calculated for 30 L.calcarifer individuals; Ta, actual annealing temperature.
Accession is GenBank ID.
**** for Pvalue <0.0001.
aDeviation from HWE.
USA). Amplification was performed using thermal cycler
DNA Engine (PTC-200) (MJ Research, Massachusetts,
USA). The PCR profile was: initial denaturation at 94Cfor
2 min; followed by 34 cycles of 94C for 1 min, annealing
(see table 1) for 1 min, and 72C for 1 min and finally 1 cycle
of 72C for 5 min. Amplification products were resolved
on 6.0% nondenaturing polyacrylamide gel and detected by
ethidium-bromide staining. Standard samples were included
across all gels to aid in accuracy and consistent sizing. Gel
images were captured and scoring was carried out against
20-bp Extended Range DNA Ladder (Cambrex, New Jersey,
USA) using Kodak Digital Science ID software. The number
of alleles (NA), expected heterozygosity (HE)and observed
heterozygosity (HO)were calculated using POPGENE v1.31
(Francis and Rong-Cai 1999). Tests for deviation from
Hardy–Weinberg equilibrium (HWE) and linkage disequilib-
rium (LD) were performed using GENEPOP v4.0
(Rousset 2008). Micro-Checker v2.2.3 was used to deter-
mine the most probable cause of departure from HWE
(Van Oosterhout et al. 2004). LD was determined by using
Arlequin v3.11 software (Excoffier et al. 2005).
Result and discussion
Only 23 out of 206 loci showed polymorphism. The rest were
monomorphic, or had presence of stutter bands, while some
loci were not easily amplified using a standard protocol. This
may be because either the flanking regions were too short or
failed to meet minimum amplification criteria. Data obtained
are summarized in table 1. All 23 loci were novel microsatel-
lites. The number of alleles per locus ranged from 3 to 24
with an average of 12.30 alleles per locus and values of the
observed and expected heterozygosities ranged from 0.400 to
1.000 and from 0.560 to 0.951, respectively. Six loci, namely
Lc05G010F10,Lc02G016E12 and Lc02G019C11, con-
formed to HWE, while the remaining 17 loci showed
significant deviation from HWE after Benferroni correction
at 5% significance level (P>0.002). No significant LD
was detected in any loci. Micro-Checker analysis suggested
that there was no evidence for scoring error due to stuttering
and no evidence for large allele dropout. It also suggested
that loci Lc02G003F04(A),Lc02G044F11,Lc02G045G12,
Lc05G010F10,Lc05G005D05,Lc02G015H11 and
Lc02G022C11, showed signs of the presence of null alleles.
In addition to the presence of null allele, the other factor that
may contribute to deviation from HWE may be nonrandom
sampling. These loci are valuable as molecular markers as
they show high levels of polymorphism and heterozygosity.
This project was funded by the Malaysian Genome Institute
(MGI) under grant UKM-MGI-NBD0004-2007/(304/PBIOLOGY/
650389/M128). The authors would like to thank P. C. Boyce
Journal of Genetics Vol. 91, Online Resources e84
Isolation and characterization of microsatellite loci for Lates calcarifer
(PPSKH-USM) for constructive critical reading of this manuscript.
The fourth author is grateful to USM fellowship for providing
financial support.
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Received 24 August 2011, in revised form 2 January 2012; accepted 23 May 2012
Published on the Web: 8 August 2012
Journal of Genetics Vol. 91, Online Resources e85
... They were analyzed with polymorphic microsatellites isolated earlier in our laboratory (Yue et al., 2001;Yue et al., 2002) that according to our knowledge were the first such set of markers for the species. Later, others have also contributed to the collection (Sim and Othman, 2005;Abdul Rahman et al., 2012). As the results of a phylogeographic analysis based on microsatellites have shown lack of clear separation among the above three major groups (Yue et al., 2009), the geographic origin of broodstock individuals was not taken into consideration during the selection process. ...
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An efficient total DNA isolation protocol, suitable for routine population genetic screening purposes is described. This phenol based extraction can utilize fresh, frozen or ethanol preserved tissues.
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Polymerase chain reaction (PCR)-based isolation of microsatellite arrays (PIMA) technique was used to isolate seven polymorphic microsatellite loci in sea bass, Lates calcarifer Bloch. A total of 62 samples of wild and cultivated sea bass collected from a few populations within Peninsular Malaysia were used in the study. For seven polymorphic loci, the number of alleles ranged from four to nine and locus heterozygosities ranged from 0.710 to 1.000. The loci will be useful for studying population structure, genetic variability of wild and hatchery stocks of L. calcarifer and broodstock management purposes.
Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1–4.
DNA degradation, low DNA concentrations and primer-site mutations may result in the incorrect assignment of microsatellite genotypes, potentially biasing population genetic analyses. MICRO-CHECKER is WINDOWS(R)-based software that tests the genotyping of microsatellites from diploid populations. The program aids identification of genotyping errors due to nonamplified alleles (null alleles), short allele dominance (large allele dropout) and the scoring of stutter peaks, and also detects typographic errors. MICRO-CHECKER estimates the frequency of null alleles and, importantly, can adjust the allele and genotype frequencies of the amplified alleles, permitting their use in further population genetic analysis.
Microsatellites represent codominant molecular genetic markers, which are ubiquitously distributed within genomes. Due to their high level of polymorphism, relatively small size and rapid detection protocols, these markers are widely used in a variety of fundamental and applied fields of life and medical sciences. In the field of aquaculture, microsatellites represent workhorse markers, which are useful for the characterization of genetic stocks, broodstock selection, constructing dense linkage maps, mapping economically important quantitative traits, identifying genes responsible for these traits and application to marker-assisted breeding programmes. In this review, genomic distribution, function, evolution and practical applications of microsatellites are considered, with special emphasis on fish genetics and aquaculture.