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Molecular Ecology Notes (2007)
7
, 422–424 doi: 10.1111/j.1471-8286.2006.01606.x
© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd
Blackwell Publishing Ltd
PRIMER NOTE
A set of polymorphic microsatellite loci for the bay scallop,
Argopecten irradians
HONGJUN LI ,*† XIAO LIU,* J INGJIE HU,‡ ZHENMIN BAO‡ and GUOFAN ZHANG**
Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Shandong 266071, China,
†
Graduate School, Chinese Academy of Sciences, Beijing 100039, China,
‡
Laboratory of Marine Genetics and Breeding, Division of Life Science and Biotechnology, Ocean University of China, Qingdao, Shandong 266003, China
Abstract
The method of creating enriched microsatellite libraries can supply an abundant source ofmicrosatellite sequences at a considerably reduced cost. Here we report the development of15 polymorphic microsatellite loci from the bay scallop,
Argopecten irradians,
using enrichmentprotocol. Polymorphism was assessed in a sample of hatchery population (
n
= 38) revealingthree to seven alleles per locus. The expected and observed heterozygosities ranged from0.198 to 0.813 and from 0.083 to 0.833, respectively. These markers will be useful for geneticvariation monitoring and parentage analysis.
Keywords
: bay scallop, polymorphic, simple sequence repeats
Received 3 August 2006; revision accepted 8 October 2006
Bay scallop,
Argopecten irradians
, is not native to China. Itwas introduced from the USA in 1982 and has become oneof the most important aquaculture species in China, owingto its high economic value, fast growth and adaptation todifferent geographical regions for aquaculture. In 2000,China produced about 200 000 tons of bay scallops (Zhang2003). We initiated a selective breeding program in2001. This program includes establishing broodstocks forselection, estimating heritability for economically importanttraits and developing molecular markers for populationgenetics. An inevitable problem in captive populationis loss of genetic variability resulting from increase ininbreeding, especially in a hermaphroditic species such asbay scallops. This will limit genetic gain from artificialselection. Microsatellite markers are sensitive and usefulfor studying genetic variation (Kenchington
et al
. 2006) andestimating relationship between individuals of unknownpedigree (Norris
et al
. 2000; Blouin 2003). As part of aproject to investigate the genetic diversity of differentstocks of bay scallops in China, we have isolated 15 poly-morphic microsatellite loci from
A. irradians
.Genomic DNA was extracted from adductor muscle
using a standard phenol-chloroform protocol (Sambrook
et al
. 1989). DNA was digested with
Hind
III and
Rsa
I
restriction enzyme. Selected fragments (300–800 bp) wererecovered after agarose electrophoresis. The productswere ligated to oligonucleotide adaptors (21-mer 5
′
-CTCTTGCTTGAATTCGGACTA-3
′
, and 25-mer 5
′
phos-phate-TAGTCCGAATTCAAGCAAGAGCACA-3
′
) withT4 DNA ligase at 16
°
C for 16 h. Ligation products wereamplified using the adaptor and then partially enrichedfor (CA)
n
and (GA)
n
containing sequences by hybridizationto a 1-cm
2
piece of Hybond-N membrane saturated withsynthetic CA and GA polymers. A polymerase chainreaction (PCR) was effectuated with the eluted DNA astemplate and amplified products were used to transforminto competent
Escherichia coli
DH5
α
cells. Recombinantclones were screened on nitro-cellulose transfer membranesand hybridized with (GA)
20
and (CA)
20
oligonucleotideprobes labelled with Gene Images 3
′
-oligolabelling (AmershamBiosciences). Positive clones were sequenced using BigDyechemistry with M13 forward and reverse primers on anApplied Biosystems ABI PRISM 310 Genetic Analyser.
PCR amplifications were carried out in 15
µ
L reactionscontaining 1
×
PCR buffer, 0.34
µ
m
of each primer, 200
µ
m
each dNTP, 1.5 m
m
MgCl
2
, 0.5 U
Taq
polymerase andabout 50 ng of template DNA. The thermal profile for PCRamplification was as follows: 5 min at 94
°
C, 35 cycles of30 s at 94
°
C, 30 s at a primer-specific annealing temperature(Table 1), 30 s at 72
°
C, and a final step at 72
°
C for 5 min.PCR products were separated by electrophoresis on a 6%
Correspondence: Xiao Liu, Fax: +86-532-82898509; E-mail: [email protected]
P R I M E R N O T E
423
© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd
denaturing polyacrylamide gel sized with the DNA ladder
Φ
X174/
Hin
fI (Fermentas Life Sciences) and visualized by silverstain. Allele sizes were determined using the original clone.
Twenty-seven primer sets were designed by
primer
3(Rozen & Skaletsky 2000), of which 7 were not easilyamplified, 5 were monomorphic and 15 were polymorphic.Thirty-eight individuals were used to analyse these micro-satellite loci. The number of alleles ranged from three toseven. The expected and observed heterozygosities rangedfrom 0.1975 to 0.8125 and from 0.0833 to 0.8333, respectively.Departures from Hardy–Weinberg equilibrium (HWE)and linkage disequilibrium (LD) were tested using thesoftware
popgene
version 1.44 (Yeh
et al
. 2000). There wereonly five loci (BSAD117; BSAD124; BSAD168; BSAD179and BSAD190) conformed to HWE probably due to nullalleles and strong inbreeding in hatchery population. LDwas detected for two pairs of loci, i.e. BSDD003 & BSAD009and BSAD117 & BSAD105 (
P <
0.05). These loci, togetherwith those previously described (Roberts
et al
. 2005) havebeen shown useful for population genetics of
A. irradians
.
Acknowledgements
We would like to express our great thanks to Ai Bin Zhan, fromOcean University of China, for his generous help on this study.This work was supported by National High-Technology R & DPlan (2004AA626070 to X.L.), National Science Foundation ofChina (30671622 to G.Z.).
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Table 1 Characterization of 15 polymorphic microsatellite loci from Argopecten irradians
Locus (Accession no.) Primer sequence (5′−3′)
Ta (°C) Repeat motif
Size (bp) Na HO HE
HWAD003 F: AGTGGAGGTAACAGAACAGG 54 (TC)23 220 3 0.1842 0.5432(DQ779939) R: TAAAGGGTCTCCTCACAACGHWAD004 F: AACCTCATCTTAGGCATTC 55 (TC)9 … (TC)8 219 4 0.3684 0.6007(DQ779940) R: GGTCCTTGTGCTTATCTTGHWAD007 F: CCCGTGTATGATTTAACGTAG 54 (CA)37 195 4 0.2667 0.7407(DQ779941) R: ACTTTGTAGGCATTTATTGGACHWAD009 F: CATTCCCCATTTCCTCAGC 58 (TC)21 197 5 0.5676 0.7320(DQ779942) R: TATACGAGTTTGTGACTTGHWAD105 F: CACCATAACGAAAAGTCAC 58 (AG)30 191 7 0.2500 0.7979(DQ779943) R: GTTATTACCAGAAACCACG(HWAD117) F: TCTTACTGAAACAAACCGC 54 (AG)20GG 353 6 0.4848 0.6471(DQ779944) R: TGCCACTCTTAATACCAAC (AG)8 … (AG)12HWAD123 F: TTGTTGCTGCTGCAATAGC 60 (AG)16 173 5 0.5946 0.7257(DQ779945) R: ACCCTCAAGGTAAGGAAATHWAD124 F: GCTGGGGATGACGAGGATA 54 (AG)17 245 4 0.2105 0.1975(DQ779946) R: TTGGGCAGTTTAGTAGATGHWAD145 F: CGTCTTGACTGGTATTTTGG 53 (AG)9 … (AG)10 248 3 0.4595 0.6327(DQ779947) R: CCCGTAAGTTGCGAGGTTC GG(AG)13HWAD152 F: TTAGACTGGTATCAGGGTTAG 50 (AG)13 … (AG)24 275 5 0.083 0.7344(DQ779948) R: TGAGGACGAAATGAGGTAGHWAD153 F: CCGTAGTATGTGAAAAGTG 50 (TC)28 264 4 0.1905 0.5528(DQ779949) R: TCTCTCATAAGAAGGTAGCHWAD156 F: GTCAGCATCGGGAGTGATTAC 60 (TC)20 229 6 0.5625 0.8125(DQ779950) R: GGATTTGGCACAAGGTTTCHWAD168 F: GGTCCTTGTGCTTATCTTG 52 (AG)9 … (AG)5 217 4 0.6111 0.6545(DQ779951) R: AACCTCATTTAGGCATTCGHWAD179 F: CGAAGACAACATACTGATACC 51 (AG)20 290 4 0.8333 0.6910(DQ779952) R: GTGCTAAATGAAAGACCGAGHWAD190 F: CACTCGTGGGCGTAAATAG 50 (TC)6TT(TC)21 241 3 0.4000 0.5203(DQ779953) R: ACAAATAAAGGAAGCAACCG
Ta, optimal annealing temperature; Na, number of alleles; HO, observed heterozygosity; HE, expected heterozygosity.
424 P R I M E R N O T E
© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd
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