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Isolation and characteristics of polymorphic microsatellite

markers inParabramis pekinensis

Abstract

The bleeker (Parabramis pekinensis) is an economically important freshwater fish

which are widely distributed in China. In order to obtain a set of genetic markers for

evaluating the genetic diversity of bleeker, we developed thirty-four microsatellites

from a (C) !"-enriched genomic library in "# individuals collected from $iang%i

$ake. &eventeen of those loci were polymorphic with allele numbers ranged from ' to

. The observed, epected hetero%ygosities and polymorphism information content

range from #.*### to !.####, #.+" to #.!! and #.*! to #., respectively. The

polymorphic microsatellite loci developed in this study will be of potential use in

understanding population genetic structure and conservation genetics ofP. pekinensis.

KeywordsParabramis pekinensis, microsatellite, polymorphism

Introduction

The bleeker (/arabramis pekinensis) is the most common and widespread species in the genus of/arabramis in China. It is characterised by an increased body height that is "'.'0 to '#.##0 of

standard length, a protracted diamond body and skinny body breadth (1u 21 et al., !+').

&everal species are considered endemic to the 3eilong 4iver and the $iaohe 4iver

includingP. pekinensis P. strenosomusandP. liaohonensis. 3owever, it has a great

commercial importance as food resource since it5s a crucial a6uaculture income for

the coastal communities. 7arious factors such as water pollution, overfishing,

construction of dams for power generation and so on affected the population

drastically which led to a decline in population of P. pekinensis.8espite decades of

study, there is not much information pertaining to geneticsof this species ($i et al.

##9 :iao et al. #!"). :icrosatellite markers are commonly used to assess genetic

population structure because of their high level of polymorphism and co-dominant

inheritance (;oldstein and &chlerguson et al. #!!), Siniperca chuatsi($iu et al. #!!) andPelteobagrus fulvidraco

(3u et al. ##). ?ut no :icrosatellite markers have been developed for P. pekinensis.

Therefore, in this study we focused isolate novel polymorphic microsatellite loci of P.


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pekinensisusing >I&C@ (>ast Isolation by >$/ of &e6uences Containing 4epeats)

for future population genetic studies.

Methodology

;enomic 8A ofP. pekinensiswas etracted from caudal fin by using proteinase-B

digestion and mmonium acetate chromatography with 4Aase treatment, and then an

enriched partial genomic library for the repeat motif (C)!"was constructed using the

>I&C@ protocol (ane et al. ##) with some modifications (hu et al. ##*).

Dnriched fragments were ligated into p:8!-T vector (TaBa4a) and then

transformed into Escherichia coli83*E (TaBa4a) cells. ;rown overnight in a &@C

solid medium with ampicillin, clones were confirmed by polymerase chain reaction

(/C4) amplifications using :!" forward and reverse primers. fter /C4

confirmation, * positive clones were se6uenced by &angon (&hanghai). &e6uences

were analy%ed for the repeat region using software &&43unter !.". The software

/rimer *.# was used to design * pairs of primers flanking the repeat regions of

interest.

Thirty individuals of adultP. pekinensiswere sampled from $iang%i $ake in 3ubei

province of China. ;enomic 8A of P. pekinensiswas etracted using ammonium

acetate high salt chromatography, and these 8A samples were used for the test and

characteri%ation of polymorphism of the isolated microsatellite loci.

The conditions for polymerase chain reaction were optimi%ed for each pair of

primers, and the tests of polymorphism were performed in "# individuals of P.

pekinensispopulation. The /C4 amplifications were carried out in !# F$ volume on a

7eriti + well Thermal Cycler (?I), the miture containing ! G /C4 buffer (!#m:

Tris-Cl, !.*m: :gCl, *#m: BCl), !#ng genomic 8A, #.!F: for each primer,

!##FmolH$ dAT/s and #. rTaq8A polymerase (TaBa4a). The amplification

system was set as a initial denaturation at * for * min9 "" cycles including

denaturation for '*s, annealing at annealed temperature (Table !) for '*s and

elongation at for "#s9 and a final etension at for !# min. mplified

fragments were si%e-fractionated on !#0 non-denaturing polyacrylamide gels running

at !"*7 for ## min and visuali%ed by silver nitrate staining, photographed, and

analy%ed using the ltraviolet ;el 8ocument &ystem (?iotech). The analyses of

polymorphism, including allele diversity, observed (H0), polymorphism informationcontent (/IC), epected hetero%ygosities (HE), and the eact test for 3ardy-1einberg


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e6uilibrium (31D) was performed using ;DAD/@/ online version

(httpJHHgenepop.curtin.edu.auH) (eng, #!!).

Results and Discussion

@ut of the * pairs of primers designed, " failed to amplify /C4 products. In sum,

! of the "' successfully amplified microsatellite loci were polymorphic, with the

number of alleles per locus ranging from ' to . >or those ! polymorphic loci, H0,

HEand /IC ranged from #.*### to !.####, from #.+" to #.!! and from #.*! to

#., respectively (Table !). The remaining ! loci were monomorphic. The tests for

31D revealed that the maKority (" out of !) of these polymorphic microsatellites

were in 31D, and /pe*, /pe!#, /pe! of the ! loci showed significant departure

from 31D (/L#.##*), which may suggest population subdivision or occurrence of

null allele may be detected as homo%ygotes. These ! polymorphic microsatellite loci

ofP. pekinensiswill facilitate future studies on conservation genetics inP. pekinensis

populations.
http://genepop.curtin.edu.au/http://genepop.curtin.edu.au/


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Table ! Characteri%ation of ! polymorphic microsatellites ofP. pekinensisin $iang%i $ake

$ocus;en?ank

ccession Ao.4epeat motif /rimer se6uence (*M-"M)

T

()

si%e

range

(bp)

Aa 3@ 3D /IC

/pe! B>#*+(C)#N(C)*N(C)*N(C)!*N(C)*N(C)*N

(C)!N(C)"+

>J CTTTTTCTCTT;TCT

*# "'-"+ !.#### #.#*+ #.4J ;CTTT;TCTTCT;;;

/pe B>#*# (C)+N(C)*>J TTC;T;CCCTCTCC;

* '#-'*! #.""" #.!"" #.4J ;CTTCCCCTCCCC;

/pe" B>#*!(TTC)+N(C)#N(C)!#N

(C)*N(C)#

>J ;TTCT;;C;TC*" "+-"+ * #.### #.''' #.#*

4J ;CTCT;T;T;T;T;;T

/pe' B>#* (C)'>J TCTTTTC;TTCCCTCC

*' -*! * #.++ #.#*+ #.++4J T;;C;TCTCCTCCT

/pe* B>#*" (;T)'">J ;TCTCTTCCTCC;TCC

** !-"#" * #.### #.+' #."!O4J ;;;TTTT;;T;TTTT;T;

/pe+

B>#*'

(T;)"'N(T;)>J TCCCTCTCC;C;

** '#-'* + #.++ #.+ #.4J TCTTTCCCCTTCTCCC

/pe (;T)!+>J CTCCC;C;;;;T

* !*-* * #.*### #.+**# #.+#'4J ;C;;;CC;;CTTC

/pe B>#** (T;)!'>J T;T;;;;;;;;TC

*' !'-*# ' #.### #.! #.+++4J T;CTTT;TTTT;T;;C

/pe B>#*+ (;T)!N(;T)*>J C;TCT;T;CT;T;

*! !+-!+ ' #.+""" #.+'' #.+4J TCC;;;CTC

/pe!# B>#* (T;)N(T;)!"N(T;)N(T;)!#>J T;T;;T;TT;;;;

*. #"-! ' #.'""" #.+!! #.+O4J ;;C;T;TTTT;;;

/pe!! B>#* (T;)>J T;TTCT;CT;;;TC

*' "#-"" + #.+""" #.+ #.+**4J ;;CTCT;;C;;


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/pe! B>#* (C)'>J CT;;T;TTT;T;T;TT;T;

*+ "-""* #.+""" #.!! #.O4J ;C;;CTTCCTTCCTCT

/pe!" B>#*# (C)!N(C)!N(C)!'N(CT)*>J ;;T;;T;;;T;TT

*.* '!-'*! ' #.*### #.+" #.*!4J T;;TCT;;C;;T

/pe!' B>#*!(C)N(C)N(C)*N(C)!#N

(C)!#N(C)!"

>J T;;;;;T;T;;;T;** ""+-"+# * #.### #.! #.++

4J CCC;;T;;TTT;TTT

/pe!* B>#* (C)#N(C)! >J CCC;;CC;C ** !*-*! * #.### #.! #."+

4J TC;TTC;CCCTCTC

/pe!+ B>#*" (C)>J CCTCCCCCC;C

*+ #-' * #.+""" #.+" #."!4J ;;CTTCTTTTCCT;C;

/pe! B>#*' (T;)N(TC)+N(T;)>J TCCTTT;T;TTT;CCCT

*" '#-# * #.*""" #.## #."#4J ;TTCTTTTT;;TT;CTT;C

TJ annealing temperature ()9 AaJ number of alleles9 3@J observed hetero%ygosity9 3DJ epected hetero%ygosity9 /ICJ polymorphism

information content9 O indicated deviation from 3ardy-1einberg e6uilibrium (/L#.#*) after ?onferroni correction


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Acknowledgements

This study was supported by grants from the :odern groindustry Technology

4esearch &ystem, PP&taple >reshwater >ishery Industry Technology &ystem55 (Ao.

C4&-'+-#*), >undamental 4esearch >unds for the Central niversities (Ao.

#!!/Q#"#!!/Q#'"#!!&C, #!!C#!! and #!Q?#), and ;uangdong

3aid ;roup Co., $td.

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"J '-'!.

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