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Reprod Dev Biol 37(4) : 205-211 (2013) ISSN : 1738-2432 (P r i n t)

http://dx.doi.org/10.12749/RDB.2013.37.4.205 ISSN : 2288-0151 (Online)

Individual Identification using The Multiplex PCR with


Microsatellite Markers in Swine

Lee-Kung Kima, Chang-Min Parka, Sun-Ae Park, Seung-Chang Kim, Hoyoung Chung,
Han-Ha Chai, Gyeong-Yong Jeong and Bong-Hwan Choi†
Division of Animal Genomics & Bioinformatics, National Institute of Animal Science, RDA, Suwon 441-706, Republic of Korea

ABSTRACT

The swine is one of the most widespread mammalian throughout the whole world. Presently, many studies concer-
ning microsatellites in swine, especially domestic pigs, have been carried out in order to investigate general diversity
patterns among either populations or breeds. Until now, a lot of time and effort spend into a single PCR method.
But simple and more rapid multiplex PCR methods have been developed. The purpose of this study is to develop
a robust set of microsatellites markers (MS marker) for traceability and individual identification. Using multiplex-PCR
method with 23 MS marker divided 2 set, various alleles occurring to 5 swine breed (Berkshire, Landrace, Yorkshire,
Duroc and Korea native pig) used markers to determine allele frequency and heterozygosity. MS marker found 4 alle-
les at SW403, S0227, SWR414, SW1041 and SW1377. The most were found 10 alleles at SW1920. Heterozygosity
represented the lowest value of 0.102 at SWR414 and highest value of 0.861 at SW1920. So, it was recognized appro-
priate allele frequency for individual identification in swine. Using multiplex-PCR method, MS markers used to
determine individual identification biomarker and breed-specific marker for faster, more accurate and lower analysis
cost. Based on this result, a scientific basis was established to the existing pedigree data by applying genetics additio-
nally. Swine traceability is expected to be very useful system and be conducted nationwide in future.
(Key words : Microsatellite marker, Swine, Multiplex PCR, Individual identification, traceability)

INTRODUCTION meat can be used. Traceability of cow was most resear-


ch activity done. However, studies related to the trace-
ability of swine in the country were insufficient. Addi-
The swine (Sus scrofa domestica) is one of the most wi- tionally, gene profiling studies utilizing of mitochondria
dely widespread domestic animal species in the world. DNA, blood protein, minisatellite, microsatellite, single
It is supposed that in the history of pig domestication, nucleotide polymorphism (SNP) and various genetic
complex factors such as men-deriving forces and natural markers by the recognition of the value of conservation
selection have influenced the actual diversity and po- and utilization in many traditional livestock as genetic re-
pulation structure of the species (Amaral et al., 2011). sources in various worlds carried out in the traditional
Its domestic pig is of economic importance and the pre- livestock (Chung et al., 2001; Girish et al., 2007; Ichika-
sent day varieties are the result of multiple domestica- wa et al., 2001; Kim et al., 2010; Oh M. Y. et al., 1992;
tion events that occurred in different regions. And ge- Signer and Jeffreys, 1997). In particular, the microsate-
netic diversity at swine breed is impossible to individual llite has a regular repeating sequence of 2∼6 base pairs
identify. One case changed foreign and domestic pigs of DNA, are distributed approximately 50,000 to 100,000
were frequently. In addition, these case scientific foren- across the entire mammalian genome and is non-co-
sic techniques are nonexistent situation to prevent me- ding DNA sequence of the wide range of high poly-
thod of currently disguised distributors. So, develop- morphism (Debrauwere et al., 1997). It is widely used
ment of DNA marker techniques based on swine gene- to analysis of the genetic diversity of livestock popu-
tic is required. Moreover, these characteristics are the lations because of convenience and polymorphism of
recent increasing interest for traceability system of the the experiment among the technology using DNA (Bar-

* This work was supported by the “High-throughput sequencing and variation for genetic markers development in Korean native animals”
project of the National Institute of Animal Science, RDA, Korea.
a
These authors contributed equally to this work.
†Corresponding author : Phone: +82-31-290-1592, E-mail: bhchoi@korea.kr
205
206 Kim et al.

ker et al., 1997; Bjornstad et al., 2003; Laval et al., 2000; MATERIALS AND METHODS
Li et al., 2000). Currently, multiplex PCR as well as
simple PCR technique using microsatellite marker (MS
marker) are also being developed to save time and costs Animals and Extraction of DNA from Blood
(Jamsari et al., 2011; Koskinen and Bredbacka, 1999; Wei- DNA was extracted from blood of total 96 by 5 swi-
ssenberger et al., 2010). ne at each breed (Berkshire, Landrace, Yorkshire, Duroc
Microsatellites can be used to investigate the popula- and Korea native pig,) using Wizard genomic DNA
tion structure, including the genetic relationships, am- purification kit (Promega, USA) and analyzed concen-
ong subpopulations. This study examined to determine tration and purity at absorbance of 260 nm and 280 nm
gene identification and individual identification through using ND-1000 spectrophotometer (Nanodrop, USA).
traits associated biomarkers and breed- specific marker
for faster, more accurate and ways to reduce the analy- MS Marker for Allele Analysis
sis cost using optimized multiplex-PCR method with MS markers utilized this study were firstly selected
known MS marker. 511 MS markers based on microsatellite genetic loci of

Table 1. List of microsatellite markers and primer

Set Primer Tempe- Product


CHR Name Dye
No. Foward(5’→3’) Reverse(3’→5’) rature size

16 SW403 GTGTATGTTCATGCATGGGTG GTCTCTGCTTTGCTTGCATG 100∼114 Fam

2 S0226 GGTTAAACTTTTNCCCCAATACA GCACTTTTAACTTTCATGATGCTCC 175∼206 Fam

14 SW210 TCATCACCATCATACCAAGATG AATTCTGCCAAGAAGAGAGCC 215∼250 Fam

8 SW2410 ATTTGCCCCCAAGGTATTTC CAGGGTGTGGAGGGTAGAAG 102∼124 VIC

4 S0107 CAAGGATGCCTGTAACTGGTGCAG TCCTTAAGGCCTCGTAGGATCTGT 165∼190 VIC


Set
4 S0227 GATCCATTTATAATTTTAGCACAAAGT GCATGGTGTGATGCTATGTCAAGC 60℃ 225∼256 VIC
01
18 SWR414 GATTTGACCCCATGCCTG AAGGCAAACCCCTTGAGTTC 138∼158 NED

13 S0068 AGTGGTCTCTCTCCCTCTTGCT CCTTCAACCTTTGAGCAAGAAC 211∼260 NED

17 SW1920 GATCCGTATCTATAGCCACCTG ATGAAAGCTACCAACCCTTCC 90∼135 PET

2 SWR2516 GTGCATTATCGGGAGGTATG ACCCTGTATGATACTGTAACTCTGG 154∼178 PET

7 S0101 GAATGCAAAGAGTTCAGTGTAGG GTCTCCCTCACACTTACCGCAG 197∼216 PET

5 S0018 GCACAGTTGATGCTTCATGC GATCAAAAGTCCCCAATTCC 248∼277 NED

10 SW1041 ATCAGAAAATGGTCAACAGTTCA GGAGAATTCCCAAAGTTAATAGG 93∼103 PET

11 SW1377 TTCAAGGTTGGAAAGACAGTCC ATGAGGAGTTTGAACTATTGGG 205∼228 NED

14 SW1557 TTCAAGGTTGGAAAGACAGTCC ATGAGGAGTTTGAACTATTGGG 84∼100 NED

15 SW1989 TGCTCTAATCTACCCGGGTC CCACCCCACTCCCTTCTG 228∼243 Fam

Set 9 SW2401 TGAACAAGTCCAACCAAGAGC CCCAACTAACGGGCTTGTG 148∼170 Fam


61℃
02 X SW2456 GAGCAACCTTGAGCTGGAAC AATGTGATTGATGCTGTGAAGC 189∼211 PET

14 SW2515 CCATCTCATCCAGAAACATCC AGGATGCTGAGGTGTTAGGC 90∼108 Fam

6 SW316 TTCTCCAGCCATCATGAGTG AATGACCATTCCTGAGGCTG 133∼159 PET

5 SWR1526 CGGTGGCTACAGATAACAATAC ATCCGATTCAACCCCTAGC 114∼146 VIC

10 SWR1849 CCTGTTCTGCCTCTAGCCTG CTGAGAAGCCTGTGCATCAG 115∼160 NED

13 SWR1941 AGAAAGCAATTTGATTTGCATAATC ACAAGGACCTACTGTATAGCACAGG 202∼222 VIC


Identification with Microsatellite Markers in Swine 207

swine reported Mapviewer database of NCBI (National Conditions of Thermal Cycler PTC-0240 (MJ Research,
Center for Biotechnology Information) and were se- Inc., MA, USA) were as follows: 15 min at 95℃ for ini-
lected 23 MS markers considered annealing tempera- tial denaturation, followed by 5 cycles with denatura-
ture of 61℃, product size and type of dye for Gradient tion at 94℃ for 40 sec, annealing at 61℃ for 40 sec
PCR thereafter. The selected MS markers divided into and elongation at 72℃ for 40 sec, 5 cycles with dena-
2 set of each 12 and were composed of the final set of turation at 94℃ for 40 sec, annealing at 60℃ for 40 sec
11 and 12 that satisfied condition of multiplex PCR. and elongation at 72℃ for 40 sec, 25 cycles with dena-
Total 23 primers of 2 set are shown in Table 1. turation at 94℃ for 40 sec, annealing at 59℃ for 40 sec
and elongation at 72℃ for 40 sec. The final had a ex-
Multiplex PCR and MS Analysis tension temperature of 72℃ 20 min. PCR products we-
Multiplex PCR was set up in 25 ul reaction volume re analyzed using the ABI-3730XL genetic analyzer (App-
consisting 6 ul (20ng/ul) of genomic DNA, 0.4 ul (10 lied Biosystems, USA) and GeneMapper version 4.0 (App-
pmole) each of fluorescence dye primer set (forward lied Biosystems, USA).
and reverse), 1 ul (Unit/ul) of Hot Start Taq DNA poly-
merase, 4 ul of 10× buffer and 3 ul of 2.5 mM dNTP. Statistic Analysis

Table 2. Allele and heterozygosity at microsatellite marker

Set No. MS marker Allele size Allele (Total) Allele (K) Allele (B) Allele (L) Allele (Y) Allele (D)

SW403 100∼114 4 2 3 3 4 2

S0226 175∼206 5 4 5 5 3 2

SW210 215∼250 8 8 6 5 3 3

SW2410 102∼124 6 1 1 4 3 3

S0107 165∼190 9 3 4 5 5 3

Set 01 S0227 225∼256 4 2 2 2 1 3

SWR414 138∼158 4 1 3 2 2 3

S0068 211∼260 9 3 6 5 3 6

SW1920 90∼135 10 3 6 5 3 6

SWR2516 154∼178 7 2 6 4 4 2

S0101 197∼216 7 2 4 4 4 3

S0018 248∼277 6 2 3 4 3 4

SW1041 93∼103 4 3 2 2 4 2

SW1377 205∼228 4 3 3 2 2 2

SW1557 84∼100 6 2 3 6 5 4

SW1989 228∼243 8 3 5 4 3 4

SW2401 148∼170 7 3 5 5 5 4
Set 02
SW2456 189∼211 5 3 4 5 3 5

SW2515 90∼108 9 2 7 6 5 2

SW316 133∼159 9 3 5 4 4 3

SWR1526 114∼146 5 1 4 3 4 5

SWR1849 115∼160 8 3 5 3 4 4

SWR1941 202∼222 9 2 4 5 6 4

K: Korea native pig, B: Berkshire, L: Landrace, Y: Yorkshire, D: Duroc


208 Kim et al.

Alleles of MS marker from Genotyper Software were Specific allele appearing to comparing different spe-
organized individual and group by analyzing using Ver- cies-specific alleles can be used as a measure of genetic
sion 3.0 program (version 3.0, The University of Edin- distinction within species and between species, therefo-
burgh). The Heterozygosity of entire population, allele re, we were calculated the number of allele of locus and
frequency and number of allele at each locus and at br- breed group about each MS marker (Table 2).
eed group were calculated. Also, it showed up variety The first set consists of 11 MS marker comes out 73
of allele in marker about each breed through calculated alleles. Especially, SW1920 have the highest of 10 al-
the value of expected heterozygosity and observed he- leles and SW403, S0227 and SWR414 has the smallest
terozygosity about 5 swine breed. of 4 alleles. The second set consists of 12 MS marker
comes out 80 alleles. Among them SW2515, SW316 and
SWR1941 have the highest of 9 alleles and lowest SW-
RESULTS AND DISCUSSION 1041 and SW1377 emerged as having 4 alleles. As a re-

Table 3. Expected and observed heterozygosities and PIC value at 23 microsatellite in 5 swine breed

Population

Locus Korean native pig Berkshire Landrace Yorkshire Duroc

Ex H Ob H PIC Ex H Ob H PIC Ex H Ob H PIC Ex H Ob H PIC Ex H Ob H PIC

SW403 0.262 0.3 0.222 0.511 0.316 0.397 0.582 0.368 0.473 0.65 0.632 0.571 0.462 0.263 0.349

S0226 0.699 0.7 0.621 0.642 0.579 0.568 0.617 0.526 0.542 0.397 0.421 0.35 0.514 0.579 0.375

SW210 0.645 0.7 0.555 0.804 0.789 0.751 0.707 0.789 0.645 0.496 0.368 0.389 0.364 0.316 0.327

SW2410 0 0 0 0 0 0 0.289 0.158 0.267 0.59 0.526 0.511 0.536 0.263 0.411

S0107 0.437 0.316 0.354 0.671 0.474 0.594 0.651 0.333 0.565 0.788 0.895 0.728 0.323 0.316 0.288

S0227 0.185 0.2 0.164 0.309 0.263 0.255 0.193 0.105 0.171 0 0 0 0.104 0.053 0.099

SWR414 0 0 0 0.323 0.263 0.288 0.193 0.211 0.171 0.102 0.105 0.095 0.4 0.474 0.356

S0068 0.56 0.444 0.481 0.818 0.789 0.765 0.543 0.4 0.496 0.16 0.167 0.149 0.79 0.556 0.735

SW1920 0.383 0.35 0.343 0.477 0.474 0.432 0.861 0.789 0.817 0.791 0.474 0.733 0.832 0.526 0.783

SWR2516 0.501 0.55 0.369 0.643 0.632 0.562 0.616 0.579 0.554 0.639 0.526 0.548 0.422 0.474 0.327

S0101 0.185 0.2 0.164 0.368 0.368 0.336 0.661 0.632 0.576 0.512 0.579 0.467 0.391 0.368 0.338

S0018 0.097 0.1 0.09 0.546 0.211 0.474 0.579 0.263 0.51 0.626 0.368 0.54 0.694 0.556 0.612

SW1041 0.574 0.65 0.499 0.491 0.684 0.364 0.501 0.526 0.369 0.468 0.421 0.415 0.508 0.684 0.372

SW1377 0.504 0.5 0.441 0.563 0.579 0.445 0.371 0.474 0.296 0.053 0.053 0.05 0.512 0.526 0.374

SW1557 0.185 0.2 0.164 0.599 0.421 0.496 0.643 0.474 0.562 0.248 0.211 0.234 0.677 0.316 0.597

SW1989 0.344 0.4 0.303 0.61 0.579 0.511 0.73 0.789 0.662 0.465 0.368 0.409 0.599 0.579 0.513

SW2401 0.681 0.6 0.59 0.741 0.632 0.68 0.573 0.474 0.523 0.775 0.842 0.714 0.246 0.211 0.23

SW2456 0.44 0.25 0.38 0.627 0.316 0.534 0.616 0.316 0.53 0.671 0.263 0.58 0.762 0 0.7

SW2515 0.224 0.25 0.195 0.669 0.526 0.609 0.706 0.579 0.632 0.657 0.895 0.604 0.508 0.684 0.372

SW316 0.617 0.6 0.516 0.73 0.579 0.66 0.677 0.421 0.6 0.755 0.789 0.687 0.586 0.368 0.504

SWR1526 0 0 0 0.596 0.526 0.539 0.665 0.722 0.571 0.613 0.263 0.549 0.7 0.444 0.643

SWR1849 0.645 0.7 0.555 0.72 0.737 0.646 0.585 0.579 0.474 0.599 0.632 0.513 0.57 0.474 0.469

SWR1941 0.45 0.45 0.342 0.529 0.632 0.469 0.603 0.737 0.52 0.748 0.895 0.696 0.653 0.789 0.584

Ex H: Expected Heterozygosity, Ob H: Objectived Heterozygosity, PIC: polymorphic information content.


Identification with Microsatellite Markers in Swine 209

sult, total 153 alleles were genotyped to determine. Ea- tating a new design for programmes of conservation of
ch of these breed, heterozygosity by various alleles also these genetic resources (Fabuel E, 2004).
derived the value of a relatively wide range, but are So allele number and heterozygosity will improve dis-
distributed for each set. crimination of each set because of various distribution
Many studies carried out in cattle and pig, were kno- of each set. Concretely, comparison using the frequency
wn that discrimination of the breed over 96% shown of allele expression by genetic marker of analysis target
by marker of a similar level (Fan et al., 2005; Oh et al., was able to detect specificity of breed group. When
2008). compared with graph of allele frequency of MS marker
Also, the old breed structure, with differentiated va- having relatively many alleles, it increased confidence
rieties locally distributed, has been replaced by a pyr- of genetic discrimination because they represent sig-
amidal structure based on crossbreeding with Duroc, and nificant differences (Fig. 1). At representative allele at
a strong dependence on a small number of breeding nu- each set, alleles of 5 swine breed at each locus had ma-
clei supplying purebred all the production tier. ny difference, therefore specificity of breeding was de-
In these circumstances, some ancestral varieties have cided easily by combination of various alleles. The ob-
disappeared, others are endangered or blended, necessi- served and expected heterozygosity, and Polymorphic

(A) (B)

(C) (D)
Fig. 1. Allele frequency distribution of microsatellite markers (MS marker) in 5 swine breed. K: Korea native pig, B: Berk-
shire, L: Landrace, Y: Yorkshire, D: Duroc.
210 Kim et al.

Informative Content (PIC) were calculated to determine 35-44.


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