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Investigation of Genetic Relationships
Among Taiwan Black Pigs and Other Pig
Breeds in Taiwan Based on Microsatellite
Markers
Y. C. Chen
a b c
a
d
a
a
a
, J. T. Hsu , C. C. Chien , Y. C. Leu , C. Y. L. Chyr
a
e
, D. Y. Lin , E.-C. Lin , C. H. Chen & P. H. Wang
a
a
Department of Animal Science and Technology , National Taiwan
University , Taipei , Taiwan
b
INRA, UMR 1313. Unité de Génétique Animate et Biologie
Intégrative , Jouy-en-Josas , France
c
AgroParisTech, UMR1313 Unité de Géńetique Animale et Biologie
Intégrative , Paris , France
d
Department of Animal Breeding, Livestock Research Institute ,
Council of Agriculture, Executive Yuan , Tainan , Taiwan
e
Livestock Industry Section, Department of Animal Industry ,
Council of Agriculture, Executive Yuan , Taipei , Taiwan
Published online: 07 Nov 2012.
To cite this article: Y. C. Chen , J. T. Hsu , C. C. Chien , Y. C. Leu , C. Y. L. Chyr , D. Y. Lin , E.-C.
Lin , C. H. Chen & P. H. Wang (2012) Investigation of Genetic Relationships Among Taiwan Black Pigs
and Other Pig Breeds in Taiwan Based on Microsatellite Markers, Animal Biotechnology, 23:4, 278-290,
DOI: 10.1080/10495398.2012.700667
To link to this article: http://dx.doi.org/10.1080/10495398.2012.700667
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Animal Biotechnology, 23: 278–290, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 1049-5398 print=1532-2378 online
DOI: 10.1080/10495398.2012.700667
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INVESTIGATION OF GENETIC RELATIONSHIPS
AMONG TAIWAN BLACK PIGS AND OTHER PIG
BREEDS IN TAIWAN BASED ON MICROSATELLITE
MARKERS
Y. C. Chen1,2,3, J. T. Hsu1, C. C. Chien1, Y. C. Leu1,
C. Y. L. Chyr1, D. Y. Lin4, E.-C. Lin1, C. H. Chen5, and
P. H. Wang1
1
Department of Animal Science and Technology, National Taiwan
University, Taipei, Taiwan
2
INRA, UMR 1313. Unité de Génétique Animate et Biologie Intégrative,
Jouy-en-Josas, France
3
AgroParisTech, UMR1313 Unité de Géńetique Animale et Biologie
Intégrative, Paris, France
4
Department of Animal Breeding, Livestock Research Institute, Council of
Agriculture, Executive Yuan, Tainan, Taiwan
5
Livestock Industry Section, Department of Animal Industry, Council of
Agriculture, Executive Yuan, Taipei, Taiwan
The aim of this study was to investigate the genetic relationships between Taiwan black pigs
(TBP) and other pig breeds by means of 15 fluorescent-labeled microsatellite markers. DNA
from a total of 299 TBP from eight private farms and 234 purebred pigs representing six
breeds and one synthetic line was used. Among the 15 microsatellite loci, polymorphism information content (PIC) values were all above 0.500; the numbers of observed alleles were all
greater than the numbers of effective alleles (10.1 vs. 4.3 in averages). But 13 of the 15 microsatellite markers significantly deviated from the Hardy-Weinberg equilibrium (HWE);
moreover, 13 of the 15 tested populations also deviated from the HWE. The inbreeding coefficient (FIS) indicated that two TBP populations (TBP-3 and TBP-4) had heterozygote
deficiency (P < 0.01). The pair-wise FST, representing the genetic diversity between the
two populations, ranged from 0.0332 to 0.3809. Meishan and Taoyuan breeds with black hair
were previously considered closely related to TBP; however, the result of genetic relationship
refuted this assumption. In conclusion, TBP is more similar to the European than Chinese
breeds, and further investigations will need to clarify it more accurately.
Keywords: Genetic relationship; Microsatellite markers; Pig; Taiwan black pigs
Y. C. Chen and J. T. Hsu contributed equally to this paper.
We thank C. L. Wang and H. M. Lin for providing blood samples from the Pig Performance Test
Station, National Animal Industry Foundation. Also, we thank Dr. M. C. Wu for providing DNA samples of Taoyuan and Meishan pig. The project was supported in part by Council of Agriculture in Taiwan
(Grant No. 98AM-04.05-AD-08(3) and 99AM-04.05-AD-01).
Address correspondence to P. H. Wang, Department of Animal Science and Technology, National
Taiwan University, Taipei, 10673, Taiwan. E-mail: [email protected]
278
MICROSATELLITES ANALYSIS FOR TAIWAN BLACK PIGS
279
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INTRODUCTION
The pork industry is the largest animal industry in Taiwan. In 2009, there were more
than eight million pigs slaughtered (1) and the market value was about two billion
USD which contributed 46% of livestock production and about 16% of total agricultural production (1). To deal with the import threat on the Taiwan pork industry
after joining the World Trade Organization (WTO) in 2000, the breeders sought
to develop a special pig population with local characteristics, and they focused on
Taiwan black pigs. In November 2010, the population of Taiwan black pigs contributed 15.83% of total on-farm pig population (about 6.2 million pigs), and each farm
reared about 185 Taiwan black pigs (2).
Taiwan black pigs are similar to other Chinese breeds, such as Meishan, with
slower growth rate, lower feed efficiency, thicker backfat, and lower lean percentage, but have higher intramuscular fat content in comparison with the Western
breeds (e.g., Landrace and Duroc). Moreover, the local pig breed, Taoyuan, introduced into Taiwan from southern China around 1877 (3) is also considered as one
of the origins of Taiwan black pigs. Animal breeders attempt to improve animal
production, product quality, or the efficiency of production by the way of selection
within breeds or use of differences among breeds through crossbreeding,
grading-up to a superior breed by repeated backcrossing, or formation of a synthetic population (4). The Livestock Research Institute (LRI) generated a synthetic
line, ‘‘TLRI Black Pig No. 1,’’ by crossbreeding Taoyuan and Duroc breeds (5).
However, except for the TLRI Black Pig No. 1, the mating systems for Taiwan
black pigs at different farms are not well organized. The farmers subjectively
choose their sires and dams without performance records; they are
experience-dependent. Thus, high diversity in body conformation, carcass, and
meat quality traits of commercial Taiwan black pigs makes the quality of pork
production difficult to predict and control. In order to understand the performance
and production potential of Taiwan black pigs, it is necessary to know how they
are related to other pig breeds in Taiwan.
Microsatellites are PCR-based markers and are highly polymorphic, abundant,
and fairly evenly distributed throughout the euchromatic parts of the genome. These
characteristics make microsatellites suitable for mapping, paternity testing, and
population genetics. In pig species over 300 microsatellite loci had been linked with
seven restriction fragment length polymorphic loci in a backcross reference population, and this map provided the basis for genetic analysis or quantitative inheritance of phenotypic and physiologic traits (6, 7). In addition, microsatellites are
proven tools for investigating genetic relatedness, genetic diversity, and genetic
structure in pigs (8–13). Microsatellite loci were selected from the ‘‘panel of markers
for PigMap diversity studies’’ for parentage control, and a multiplex polymerization
chain reaction (PCR) was developed to reduce cost and time for genotyping in
porcine populations (14).
The relationships between Taiwan black pigs and other pig breeds in Taiwan
could be analyzed based on genetic distance estimated from polymorphic microsatellite markers. The aim of this study is to investigate the relationships and the genetic
diversity between Taiwan black pigs and other pig breeds by means of 15 pairs of
fluorescent-labeled microsatellite markers.
280
Y. C. CHEN ET AL.
MATERIALS AND METHODS
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Sample Collection and Genomic DNA Isolation
The study included a total of 299 Taiwan black pigs from eight private farms
(TBP-1 to TBP-8) and 234 purebred pigs including 65 TLRI Black Pig No. 1
(TLRIBP), 38 Meishan, 32 Taoyuan, 12 Berkshire, 38 Duroc, 27 Landrace, and
22 Yorkshire pigs. They were provided by the Taiwan Animal Germplasm Center
(TAGC) of LRI and=or the Pig Performance Test Station. These eight private farms
in the southwestern Taiwan were randomly chosen, and the TAGC and Pig Performance Test Station collected the DNA of unrelated pigs from different farms in Taiwan. Blood samples were collected from the jugular vein using a K3EDTA
vacutainer (BD Vacutainer, USA). After centrifuging at 2270 g for 15 minutes, the
leukocyte buffy coat was collected for genomic DNA extraction. Genomic DNA
was extracted using Genomic DNA Isolation Reagent (GenePure Technology
CO., LTD., Taiwan). The nucleic acid concentration and purity were determined
by absorbance at 260 and 280 nm and the ratio of 260=280.
Panel of Microsatellite Markers Selected and Amplified
All animals were genotyped for the 15 fluorescence-labeled microsatellite markers amplified in three multiplex PCRs. Primer designs, fluorescent labeling, and
expected fragment sizes are listed in Suppl. 1 (14). The multiplex-PCRs were performed in a total of 15 mL volume containing 50 to 100 ng of porcine genomic
DNA as template, 1 X PCR buffer, 0.1 to 1.0 mM of forward and reverse primers
(dependent on different pairs of primers), 200 mM of each dNTP, 2.5 mM Mg2þ,
and 0.375 U of Taq polymerase (TAKARA Co., Japan). The reactions were performed on the ABI thermal cycler (GeneAmp PCR System 9700, Applied Biosystem,
USA) under the thermal cycle profile: denaturation at 95 C for 10 minutes in the first
cycle, 30 cycles of 95 C for 30 seconds, 55–60 C for 30 seconds, 72 C for 80 seconds,
and extension at 72 C for 30 minutes for the last cycle. Electrophoresis in 1% agarose
gel was used to make sure that PCR products were well amplified.
Genotyping of the Microsatellite Markers
The multiplex-PCR products were genotyped using capillary electrophoresis with
fluorescent detection (ABI 3730 DNA Analyzer, Applied Biosystems, USA). The PCR
products were diluted with deionized water and then mixed with Hi-Di formaldehyde
(Applied Biosystems, USA) and the internal size standard (GeneScan-500 LIZ Size
Standard, Applied Biosystems, USA). The mixtures were loaded into a 96-well plate
for detection of the fragment size of PCR products. The fragment size was calibrated
with Peak Scanner Software version 1.0 (ABI PRISM, Applied Biosystems, USA).
Statistical Analysis
Several computational and statistical software programs for population genetics were used in the present study, including the Excel Microsatellite Toolkit
(15), POPGENE software package (16), GENEPOP (17), FSTAT (18), PHYLIP
software package (19), and XLSTAT (20). Raw data for the fragment size was transformed using the EXCEL Microsatellite Toolkit and GENEPOP.
MICROSATELLITES ANALYSIS FOR TAIWAN BLACK PIGS
281
Genetic variability of microsatellite loci. For each locus, the average number of observed and effective alleles (Na and Ne) and the observed and expected
heterozygosities were obtained with POPGENE, whereas polymorphism information content (PIC) (21) was calculated with the EXCEL Toolkit, and an exact test
of the Hardy-Weinberg equilibrium (22) was carried out using GENEPOP.
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Within-population diversity. Genetic variations within the 15 microsatellite
markers in each population were estimated. Allele frequencies at each locus, the average number of observed and effective alleles (23) of the 15 microsatellite markers and
observed and expected heterozygosities were computed using POPGENE. The
inbreeding coefficient (FIS) of each population was estimated with the program
FSTAT (24). Exact tests of the Hardy-Weinberg equilibrium (22) and heterozygote
deficiency (25) were performed by GENEPOP for each locus in the populations.
Between-population diversity. The population differentiation was evaluated by Wright’s fixation indices (26, 27). The programs GENEPOP and FSTAT
were used to calculate the measure of the genetic differentiation over populations,
FST, pair-wise FST, and inbreeding parameters FIS (24). The unbiased Nei’s genetic
distances (28, 29) based on allele frequencies were estimated using POPGENE
between each pair of populations.
Clustering, phylogenetic tree reconstruction and measures of population diversity. Distances between each pair of populations were used to construct
the phylogenetic tree by the unweighted pair-group method with arithmetic mean
(UPGMA) (30), which was obtained using the PHYLIP software package. The bootstrapping procedure of PHYLIP software package was used to evaluate the significance of tree nodes and was extended to account for unequal sample size across
populations and loci (8). In the present study, the bootstrapping value was 1000.
Allele frequencies were also applied to a principal component analysis (PCA) to represent geometric relationships among the 15 populations in this study (13). The PCA
was performed using the XLSTAT program.
RESULTS
Genetic Variability of Microsatellite Loci
A total of 152 alleles were observed in the 15 microsatellites; polymorphisms
at all loci were observed in all of the examined populations. The observed fragment
sizes of PCR products were in line with those expected in the UniSTS database,
with the exception of S0228, S0005, and S0386 (Suppl. 1). The discrepancy of
fragment sizes suggests that some new alleles were present in the pig populations
examined in the present study. Table 1 presents the genetic variability of the 15
microsatellite loci. The average number of observed alleles per locus was 10.13,
ranging from 6 (SW951) to 22 (S0005). The average number of effective alleles
per locus ranged from 2.54 (S0355) to 9.09 (S0005), with a mean across loci of
4.32. The average expected heterozygosity was 0.741 among the 15 microsatellite
loci, ranging from 0.607 (S0355) to 0.891 (S0005). The polymorphism information
content (PIC) showed the level of polymorphism at the 15 microsatellite loci was
the least for S0386 (0.574) and the greatest for S0005 (0.880). All loci, except
282
Y. C. CHEN ET AL.
Table 1 Characterization of the 15 microsatellite loci analyzed in the 15 pig populations
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F-statistics3
Locus
Na=Ne1
HO=HE2
FIS
FST
PIC4
Exact test of HWE5
SW936
SW911
S0101
S0228
SW72
S0227
SW857
SW24
S0155
S0090
SW951
S0005
SW240
S0386
S0355
8=4.1
8=3.9
9=3.3
14=3.2
8=4.3
12=3.0
11=4.7
9=5.9
8=4.5
9=4.5
6=2.9
22=9.1
11=6.2
8=2.6
9=2.5
0.576=0.755
0.602=0.745
0.578=0.701
0.525=0.689
0.717=0.770
0.540=0.674
0.702=0.788
0.732=0.831
0.587=0.779
0.657=0.777
0.548=0.658
0.721=0.891
0.711=0.840
0.361=0.612
0.412=0.607
0.098
0.015
0.039
0.077
0.044
0.063
0.043
0.028
0.011
0.023
0.035
0.005
0.002
0.245
0.045
0.165
0.192
0.153
0.187
0.116
0.155
0.156
0.154
0.269
0.145
0.208
0.200
0.163
0.234
0.307
0.715
0.715
0.671
0.673
0.738
0.626
0.757
0.808
0.747
0.746
0.607
0.880
0.824
0.574
0.583
All loci
10.1=4.3
0.598=0.741
0.025
0.185
–
NS
NS
–
Abbreviations: NS, not significant.
1
Na ¼ Number of observed alleles; Ne ¼ Number of effective alleles.
2
HO ¼ Observed heterozygosity; HE ¼ Expected heterozygosity.
3
FIS is the measure of the deviation from Hardy-Weinberg proportions within subpopulations. FST is
the measure of the genetic differentiation over subpopulations. , FIS values with significance deviated
from 0 (P < 0.01).
4
Polymorphism information content.
5
Hardy-Weinberg equilibrium.
P < 0.05. P < 0.01.
for SW951 and SW240, were significantly deviated from the Hardy-Weinberg equilibrium (P < 0.05).
Within-Population Genetic Variation
Average number of observed and effective alleles of the 15 microsatellite loci
and observed and expected heterozygosities were calculated for each population
(Table 2). The average number of observed and effective alleles ranged from 4.07
(TLRIBP) to 6.50 (TBP-5) and from 2.45 (TBP-1 and Meishan) to 3.99 (TBP-7),
respectively. The observed and expected heterozygosities ranged from 0.474
(Meishan) to 0.802 (TBP-5) and ranged from 0.521 (Meishan) to 0.736 (TBP-7),
respectively. The FIS was used as the inbreeding coefficient and to indicate the test
of heterozygote deficiency. In the populations of Taiwan black pigs, only TBP-3
and TBP-4 had heterozygote deficiency; moreover, the six pure breeds were all significant in the test (P < 0.01). The number of loci showing a significant deviation
from the Hardy-Weinberg equilibrium within each population ranged from 0
(TBP-6) to 8 (TBP-7, Meishan, and Taoyaun). An exact test of the Hardy-Weinberg
equilibrium showed that all of the examined populations, except for TBP-6 and
TLRIBP were significantly deviated from the equilibrium (P < 0.05).
MICROSATELLITES ANALYSIS FOR TAIWAN BLACK PIGS
283
Table 2 Average within-population genetic variations from 15 microsatellite loci
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Average number of alleles
Heterozygosity
Population
Sample
size
Observed
(SD)
Effective
(SD)
Observed
(SD)
Expected
(SD)
FIS3
TBP – 11
TBP – 2
TBP – 3
TBP – 4
TBP – 5
TBP – 6
TBP – 7
TBP – 8
TLRIBP2
Meishan
Taoyuan
Berkshire
Duroc
Landrace
Yorkshire
59
32
41
60
30
19
38
20
65
38
32
12
38
27
22
4.90 (1.78)
5.13 (1.46)
6.40 (2.10)
5.47 (1.51)
6.60 (1.35)
4.53 (1.64)
6.40 (1.80)
4.73 (1.71)
4.07 (1.22)
4.53 (1.19)
5.40 (1.40)
5.07 (1.39)
5.60 (2.41)
5.80 (2.31)
5.47 (2.80)
2.45 (0.86)
2.61 (0.92)
2.88 (1.04)
2.86 (1.07)
3.55 (0.79)
2.79 (1.17)
3.99 (1.17)
3.17 (1.32)
2.56 (0.70)
2.45 (0.95)
2.87 (0.80)
3.57 (0.96)
2.91 (1.18)
2.81 (1.21)
3.54 (2.01)
0.580 (0.221)
0.583 (0.176)
0.581 (0.192)
0.599 (0.140)
0.802 (0.117)
0.614 (0.216)
0.746 (0.188)
0.643 (0.233)
0.601 (0.162)
0.474 (0.237)
0.544 (0.172)
0.567 (0.192)
0.526 (0.139)
0.551 (0.200)
0.590 (0.191)
0.541 (0.191)
0.577 (0.162)
0.606 (0.178)
0.615 (0.126)
0.718 (0.063)
0.596 (0.181)
0.736 (0.091)
0.652 (0.150)
0.582 (0.131)
0.521 (0.233)
0.629 (0.131)
0.730 (0.094)
0.620 (0.132)
0.600 (0.151)
0.658 (0.185)
0.073 (2)
0.011 (3)
0.042 (3)
0.027 (2)
0.119 (2)
0.031 (0)
0.015 (8)
0.014 (4)
0.033 (2)
0.092 (8)
0.138 (8)
0.231 (5)
0.154 (5)
0.084 (4)
0.106 (3)
Exact test
of HWE4
NS
NS
Abbreviation: NS, not significant.
1
Taiwan black pigs from 8 private farms: Farm 1 to 8.
2
Taiwan Livestock Research Institute Black Pig No. 1.
3
Probability from multi-locus test that there is no heterozygote deficiency. Number in parentheses indicates the number of loci showing a significant deviation from Hardy-Weinberg equilibrium after correction
from multiple tests (P < 0.05).
4
Hardy-Weinberg equilibrium.
P < 0.05. P < 0.01.
Between-Population Genetic Diversity
The divergence between observed and expected heterozygosities for all individuals, subpopulations, and the total population was reflected in the F-statistics parameters, FIS (evaluation of with-population deficit) and FST (genetic differentiation
among populations). The result of F-statistics at each locus was summarized in
Table 1. The FIS values for SW936 (0.098) and S0386 (0.245) were positive and significantly deviated from zero (P < 0.01) indicating inbreeding. FST, representing genetic differentiation among populations, ranged from 0.116 (SW72) to 0.307 (S0355).
Genetic distance (DN) and mean FST estimates were used to evaluate the divergence between populations examined in the present study (Table 3). According to
Nei’s unbiased genetic distance between TBP-1 to 8 and the Meishan and Taoyuan
breeds, the genetic distance was about 1.000 (DN ¼ 0.8201 to 1.444), except for the
genetic distances between Meishan and TBP-8 (DN ¼ 0.4701). Duroc breed showed
shorter genetic distances of less than 0.2500 (DN ¼ 0.0680 to 0.2393) from TBP
populations, except for the genetic distance between Duroc and TBP-6
(DN ¼ 0.3243). For TBP-6, the pure breed with the least genetic distance was Berkshire breed (DN ¼ 0.2170). A higher pair-wise FST value indicates higher diversity
between the two populations. The result of pair-wise FST was coincident with the
284
b
a
–
0.2114
0.0880
0.1995
0.2226
0.3333
0.2766
0.2515
0.2656
1.0661
1.1295
0.7083
0.0934
0.3269
0.6567
1
0.1293
–
0.2136
0.2797
0.2615
0.4805
0.3444
0.3104
0.1682
1.1550
1.0776
0.7994
0.2082
0.4442
0.9028
2
0.0574
0.1125
–
0.1948
0.1639
0.3123
0.2037
0.2593
0.2192
1.1355
1.2516
0.6295
0.0680
0.3302
0.5156
3
0.1159
0.1375
0.0974
–
0.1914
0.3704
0.2658
0.2696
0.3549
1.2840
1.1003
0.6512
0.1027
0.3230
0.5355
4
5
0.1181
0.1143
0.0727
0.0822
–
0.4098
0.1894
0.2300
0.2056
0.8863
0.6720
0.4951
0.1047
0.3356
0.5264
Taiwan black pigs from 8 private farms: Farm 1 to 8.
Taiwan Livestock Research Institute Black Pig No. 1.
TBP – 1 (1)a
TBP – 2 (2)
TBP – 3 (3)
TBP – 4 (4)
TBP – 5 (5)
TBP – 6 (6)
TBP – 7 (7)
TBP – 8 (8)
TLRIBPb (9)
Meishan (M)
Taoyuan (T)
Berkshire (B)
Duroc (D)
Landrace (L)
Yorkshire (Y)
Population
0.1786
0.2091
0.1452
0.1621
0.1466
–
0.2050
0.5017
0.4759
1.4444
1.2827
0.2170
0.3243
0.3013
0.4276
6
0.1339
0.1348
0.0849
0.1044
0.0563
0.0828
–
0.3255
0.3607
0.8201
0.9844
0.3473
0.1917
0.2670
0.4312
7
0.1364
0.1414
0.1146
0.1169
0.0813
0.1873
0.1040
–
0.3076
0.4701
0.8927
0.5210
0.2393
0.4929
0.6829
8
0.1525
0.0971
0.1162
0.1653
0.0980
0.2072
0.1438
0.1423
–
1.1312
1.0572
0.7641
0.2281
0.5960
0.7802
9
0.3653
0.3600
0.3429
0.3497
0.2722
0.3809
0.2527
0.2142
0.3510
–
0.6712
0.7365
1.2479
1.3655
1.1299
M
0.3412
0.3009
0.3051
0.2874
0.1906
0.3095
0.2236
0.2458
0.3000
0.2653
–
0.7532
1.1808
1.1238
1.0432
T
0.2488
0.2353
0.1940
0.1962
0.1238
0.0847
0.0874
0.1471
0.2342
0.2569
0.1997
–
0.6611
0.5277
0.5657
B
0.0595
0.1066
0.0332
0.0519
0.0451
0.1439
0.0764
0.1018
0.1170
0.3476
0.2914
0.1904
–
0.2581
0.4998
D
0.1744
0.1969
0.1510
0.1465
0.1276
0.1418
0.1036
0.1846
0.2360
0.3700
0.2940
0.1717
0.1202
–
0.4022
L
Y
0.2530
0.2700
0.1883
0.1907
0.1526
0.1650
0.1270
0.2014
0.2541
0.3264
0.2613
0.1527
0.1775
0.1582
–
Table 3 Nei’s unbiased genetic distance (below the diagonal) and mean FST estimates (above the diagonal) between each pair of the 15 pig populations
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MICROSATELLITES ANALYSIS FOR TAIWAN BLACK PIGS
285
Figure 1 Dendrogram of UPGMA showing genetic similarity of Taiwan black pigs from Farm 1 to 8, the
synthetic breed TLRI Black Pig No. 1, and the pure breeds including Meishan, Taoyuan, Berkshire,
Duroc, Landrace, and Yorkshire. TLRIBP, Taiwan Livestock Research Institute Black Pig No. 1. #
Bootstrap value.
Nei’s genetic distances: higher FST values were between Meishan and Taoyuan
breeds and the TBP populations (FST ¼ 0.1906 to 0.3809), whereas lower FST values
were between Duroc breed and the TBP populations (FST ¼ 0.0332 to 0.1439).
Figure 2 Scatter diagram showing the relative position of the 15 populations defined by principal component analysis (PCA) based on correlation matrix from allele frequencies of microsatellite loci. The first
principal component accounted for 19.37% and the second one accounted for 16.09% of the total variation. TLRIBP, Taiwan Livestock Research Institute Black Pig No. 1. (Color figure available online.)
286
Y. C. CHEN ET AL.
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Clustering Based on the Genetic Distances
A phylogenetic tree of UPGMA with bootstrap resampling (n ¼ 1,000) was
performed with PHYLIP software. There were two major branches (Figure 1) separating Meishan and Taoyuan breeds from the other populations. The Yorkshire breed
was relatively separated from the other populations. The Berkshire breed and TBP-6
were grouped in the same branch, and the other seven TBP populations were closely
grouped with the Duroc breed and TLRIBP. Principal component analysis (PCA)
was used to cluster possibly correlated populations and represent the relative positions among the examined pig populations. The first and second principal components accounted for 19.37 and 16.09% of the total variation (Figure 2). Similar to
the phylogenetic tree of UPGMA, PCA separated the 15 pig populations into three
groups: Group I of Meishan and Taoyuan breeds, Group II of Yorkshire breed, and
Group III of Duroc, TLRIBP, and TBP populations except for TBP-6. Landrace,
Berkshire, and TBP-6 were distributed in the area surrounded by the three groups.
DISCUSSION
The pig industry is the most important part of the agricultural economy in
Taiwan. The local Taoyuan breed was introduced into Taiwan from southern China
in 1877 (3); later in 1898, during Japanese occupation era of Taiwan, Berkshire breed
was introduced from Japan to improve the performance of Taoyuan breed by crossbreeding. In the 1960s, Duroc, Landrace, Yorkshire, and Hampshire breeds
were introduced by the Taiwan Sugar Corporation for crossbreeding. Hereafter,
the two-way crossbred (Duroc Landrace, DL) and three-way crossbred
(Duroc Yorkshire Landrace, DYL) pigs are the major commercial lines in the
Taiwan pork market, and the population of Taoyuan has declined due to its poor
performance (disadvantages to growth rate, feed efficiency, and lean content). However, in response to consumer interest in pork flavor and for use in religious celebration, a niche market of Taiwan black pigs has taken root in strong
competition with DL and DYL crossbred pigs and imported pork products. The
characteristics of Taiwan black pigs have not been scientifically determined; therefore, the relationships and genetic diversity between Taiwan black pigs and other
pig breeds are the primary concern of this research.
The average number of observed alleles over all loci was 10.1, which was greater than that in five types of Taihu pigs in China (31), other Chinese pig breeds (9, 10),
the Iberian pig populations (11), and PiGMaP (32), and was less than in the European pig breeds (8) and the 18 Chinese indigenous pig breeds with different morphological types (33). Among the 15 microsatellite loci, PIC values were above
0.5, and the numbers of observed alleles were all greater than their effective allele
numbers, which indicates that the 15 microsatellite loci recommended by Nechtelberger et al. (14) were suitable for detection of relationships and genetic diversity
between Taiwan black pigs and other pig breeds in Taiwan. Although the expected
heterozygosity and the number of effective alleles were lowest at S0355, the FST at
S0355 was greater than at the other loci, indicating that, even though of limited
use for within-breed analyses, these markers might be interesting for between-breed
differentiation (34).
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287
The larger proportion of Taiwan black pig farms are distributed in the middle
and the southern areas of Taiwan. In the present study, the samples of Taiwan black
pigs were collected from eight different private farms located in different areas of
Taiwan, representing the genetic diversity between Taiwan black pigs from different
farms and between Taiwan black pigs and other pig breeds in Taiwan. For the Western pig breeds (i.e., Duroc, Landrace, and Yorkshire) and Meishan, the expected heterozygosities were similar to those reported by Luetkemeier et al. (35). However,
Meishan, Taoyuan, Berkshire, Duroc, Landrace, and Yorkshire breeds were significantly deviated from Hardy-Weinberg equilibrium (P < 0.05) and were heterozygously deficient (P < 0.01). Deviation from the Hardy-Weinberg equilibrium in
these purebred pigs could result from smaller original populations leading to
inbreeding, from nonrandom sampling and from small sample size at the TAGC
and Pig Performance Test Station with limited sample sizes ranging from 12 (Berkshire) to 38 (Meishan and Duroc). According to the within-population genetic variations in the eight Taiwan black pig populations, Taiwan black pigs from different
farms were closely related to each other in comparison to the other purebred pigs
(DN ¼ 0.0880 to 0.5017). The pork market in Taiwan is mainly composed of DL
and DYL crossbred pigs, and Taiwan black pigs represent only 15.83% of the total
on farm pig population. Therefore, the breeding stock of Taiwan black pigs are
usually shared or purchased from other breeders. This might be the major reason
why Taiwan black pigs from different farms were genetically close and why these
populations deviated from the Hardy-Weinberg equilibrium.
Genetic relationships among breeds or populations were quite consistent in the
diagram of UPGMA based on Nei’s genetic distance and in the result of PCA based
on allele frequencies (Figure 1 and 2). Taiwan black pigs were considered breeding
from Meishan and Taoyuan breeds. However, Meishan and Taoyuan breeds were
in a separate branch from the eight populations of Taiwan black pigs and the Western pig breeds, even though Taoyuan was traditionally called Taiwan black pigs. The
Yorkshire breed was also distant from Taiwan black pigs, and the Berkshire, Duroc,
Landrace, and TLRIBP were mixed with Taiwan black pigs. Indeed, this was compatible with the common principle of choosing the Duroc breed as the terminal sire
in the breeding program of Taiwan black pigs. The vast majority of breeding stock
used by Taiwan pig industry includes Landrace, Yorkshire (as maternal lines), and
Duroc (as paternal line) since the crossbreeds DL and DYL pigs are the major commercial lines in the market. Thus, purebred Western breeds and the crossbreeds DL
and DYL pigs are widely involved in the breeding schemes either for meat production or for improvement of performances, even in Taiwan black pig farms.
The Berkshire breed is famous to its high fat content and meat with juiciness and
tenderness; therefore, some Taiwan black pig farmers used the Berkshire breed in
the breeding scheme. The genetic relationship analyzed by random amplification
of polymorphic DNA (RAPD) and mitochondrial D-loop sequence showed that
Taiwan black pigs from Guanmiao (in the southern Taiwan) were closely related
to Duroc breed, and the Hampshire, Berkshire, and Duroc breeds were highly correlated to Asian pig breeds (i.e., Meishan and Taoyuan breeds). Bootstrap values in
the diagram of UPGMA did not clearly establish the topology among the Taiwan
black pigs, TLRIBP, and other pig breeds in Taiwan. However, it was still useful
to make a note of the possible breed of breeding stock of Taiwan black pigs.
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The genetic distances between Taiwan black pigs from different farms and
TLRIBP ranged from 0.1682 to 0.3607 (Table 3). TLRIBP originated in 2001, and
the tests of production performance, such as genetic variation, growth rate, feed
efficiency, and crossbreeding experiments, were performed. Yen et al. (36) used
sequences of D-loop region, cytochrome c oxidase subunit 1, and cytochrome b of
mitochondrial DNA to analyze the genetic relationship between TLRIBP and eight
other pig breeds in Taiwan. The result showed that TLRIBP, Taoyuan, Berkshire,
Meishan, and Yorkshire breeds were clustered together, and Landrace and Duroc
were in the other branch. Mitochondrial DNA sequence reflects the genetic composition of maternal lines; thus, Taoyuan breed used as maternal breed for TLRIBP
was the most closely related. The discrepancy in the relationship between Taoyuan
and TLRIBP in the previous study (36) and the present study might be by the result
of sampling herds in different generations.
In conclusion, the present study clearly verified that using microsatellites as
polymorphic genomic markers was suitable to investigate the relationships and genetic diversity in different breeds or populations. Meishan and Taoyuan breeds with
black hair were previously considered more closely related to Taiwan black pigs. However, the genetic distance and the UPGMA tree showed that Taiwan black pigs are
more similar to the European than Chinese breeds. For the characteristics of
Taiwan black pigs, such as black hair and slower growth rate, the genetic origins of
these phenotype remain unknown. According to the history of Taiwanese pig
industry, there were some unofficial imported pig breeds and some unknown
Taiwanese local pig breeds in breeding Taiwan black pigs. In the future investigation
of parental breeds of Taiwan black pigs, it might need to increase the input from other
breeds, such as British Large Black and other local Taiwanese pig breeds, and also to
increase the number of microsatellite markers to make the estimation more accurate.
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