Evaluation of diversity between different Spanish chicken breeds, a tester
line, and a White Leghorn population based on microsatellite markers
S. G. Dávila,1 M. G. Gil, P. Resino-Talaván, and J. L. Campo
Departamento de Mejora Genética Animal, Instituto Nacional de Investigación y Tecnología
Agraria y Alimentaria, Apartado 8111, 28080 Madrid, Spain
ABSTRACT The present study was conducted to evaluate the genetic variability and the genetic divergence
of 13 Spanish chicken breeds, a tester line, and a White
Leghorn population, using 24 microsatellite markers. A
total of 150 alleles were detected across all population.
The number of alleles by locus ranged from 2 to 13,
with the mean value being 6.25. The mean polymorphic
information content was 0.591, ranging from 0.847 to
0.172. The combined parentage exclusion probability of
excluding 1 parent or 2 parents was 99 and 100%, respectively. The observed heterozygosity was lower than
the expected heterozygosity for all loci, the mean values being 0.461 and 0.637. The observed and expected
heterozygosity ranged from 0.003 to 0.735 and 0.181 to
0.863, respectively. Mean deficit of heterozygotes within populations (FIS) was 0.056 and mean fixation index
of each population (FST) was 0.244. The mean global
deficit of heterozygotes across populations (FIT) was
0.286. A total of 15 private alleles in 10 microsatellites
were observed, and in some populations, fixed alleles
were found for 7 microsatellites. A total of 300 birds
(83%) were properly assigned to the source population.
The average observed heterozygosity for each population was 0.461, ranging from 0.328 (Quail Castellana)
to 0.538 (Red Villafranquina), and the average expected heterozygosity was 0.488, ranging from 0.320 (Quail
Castellana) to 0.550 (White-Faced Spanish). All of the
Spanish breeds except the Quail Castellana were more
polymorphic than the White Leghorn population. The
mean value of the deviation of heterozygote number
was 0.052. Nei’s genetic distance showed a range from
0.109 (between White-Faced Spanish and Black Menorca) to 0.437 (between Buff Prat and White Leghorn). A
phylogenetic tree constructed by the neighbor-joining
method, based on Nei’s genetic distance, showed a clear
separation between the White Leghorn and the remaining breeds. The results indicate that the panel of microsatellite markers was useful in studying the genetic
diversity of chicken breeds.
Key words: Spanish breed, chicken, microsatellite marker, genetic diversity, phylogenetic tree
2009 Poultry Science 88:2518–2525
doi:10.3382/ps.2009-00347
INTRODUCTION
Because of centuries of domestication and breeding,
a wide range of chicken breeds exists today. However,
an increasing number of local chicken breeds are under threat of extinction, and valuable genotypes and
traits are at risk of being lost (Blackburn, 2006). The
local breeds represent both a heritage and a reservoir
of variability that deserves to be explored and properly
managed. With industrial aviculture development in
the 1950s and the 1960s, and the creation of hybrids to
produce eggs and meat, the number of traditional hens
fell dramatically to the point of extinction. Fortunately,
due to conservation programs and a few traditional hen
breeders, a large number of these breeds were saved
©2009 Poultry Science Association Inc.
Received July 14, 2009.
Accepted September 11, 2009.
1
Corresponding author: [email protected]
from extinction. The evaluation of local breeds as genetic resources is of interest in efforts to maintain genetic variation and includes records of phenotypes and
breeding history as well as determination of genetic
variability (Hammond, 1994). Quantitative assessment
of genetic diversity within and between populations is
an important tool for decision making in genetic conservation plans.
Spain has a rich genetic diversity of native chickens.
A total of 13 Spanish chicken breeds have been reported, based on morphological characteristics (Campo,
1998). Among these native breeds, 6 are white egg layers, 2 are tinted egg layers, 1 is a dark brown egg layer,
1 is a dual-purpose breed (meat and brown egg layer),
2 are feather producers, and 1 is a synthetic egg layer.
These native breeds are used for quality chicken production in free-range systems, and they are maintained
in a conservation program of genetic resources that was
started in 1975 (Campo and Orozco, 1982). The Span-
2518
SPANISH CHICKEN GENETIC DIVERSITY
ish population includes one of the oldest Mediterranean
breeds (its ancestor having been introduced into Spain
by the Arabians in the 8th century), the only breed
in the world with white face, and the original breed
carrying the blue gene. The Spanish breeds carry 6 different alleles in the main polyallelic locus determining
the plumage color in chickens (extended black, birchen,
dominant wheaten, wild type, brown, and buttercup),
and some of them can be used for chick sexing of egg
lines (Campo, 1991) or have low cholesterol content
(Campo, 1995).
Recent advances in molecular technology have opened
up new horizons for estimating genetic relatedness between and within animal populations, and molecular
markers may serve as an important initial guide to
develop conservation strategies. Studies in molecular
technology have found that local breeds have more genetic diversity when compared with recent commercial
breeds (Siegel et al., 1992; Plotsky et al., 1995; Ponsuksili et al., 1996; Zhou and Lamont, 1999; Lee et al.,
2000; Okumura et al., 2006). The microsallite markers
are extensively used for estimating genetic structure,
diversity, and relationships because of many advantages: they are numerous and ubiquitous throughout
the genome, show a higher degree of polymorphisms,
and have a codominant inheritance (Tautz, 1989). Information in literature has revealed that microsatellite
markers are the most accurate and efficient method for
estimating genetic diversity and relationships among
populations (Takezaki and Nei, 1996).
Several studies have been conducted to characterize
the diversity in traditional chicken breeds based on microsatellite markers. Many of them have been made
in Asian breeds (Takahashi et al., 1998; Zhang et al.,
2002; Osman et al., 2006; Ya-Bo et al., 2006; Tadano et
al., 2007). Additionally, Wimmers et al. (2000) evaluated genetic distinctness of African, Asian, and South
American chickens, and Romanov and Weigend (2001)
analyzed genetic relationships between various chicken
populations in Europe and jungle fowl. Genetic relationships between a wide range of chicken types from
Europe, Asia, and Africa have been analyzed in the
AVIANDIV project (Hillel et al., 2003), and Berthouly
et al. (2008) studied genetic diversity of French and
Asian breeds, comparing them with those of the AVIANDIV project. Finally, Twito et al. (2007) compared
the biodiversity of 20 chicken breeds with SNP and
microsatellite markers, noting that analysis based on
microsatellites resulted in significantly higher clustering success due to their multiallelic nature.
The objective of this study was to evaluate the genetic variability and the genetic divergence of 13 Spanish chicken breeds, a tester line, and a White Leghorn
population using microsatellite markers. Up to now,
only 1 study with Spanish breeds has been conducted
to evaluate genetic diversity based on DNA polymorphism (the AVIANDIV project included 2 Spanish
chicken breeds).
2519
MATERIALS AND METHODS
Chickens
A total of 360 hens, randomly selected, from the 15
populations were used (24 hens by population). All of
them came from the experimental station of El Encín
(Madrid, Spain), where the conservation program of
Spanish genetic resources is located. The number of
hens and cocks in each population ranged from 128 to
395 and from 25 to 80, respectively. The effective population size ranged from 66 to 310 (Campo et al., 2002),
higher than the minimum size (50) required to avoid
inbreeding depression in the short-term. Black Castellana, Black Menorca, White-Faced Spanish, BlackBarred Andaluza, Blue Andaluza, and Black-Breasted
Red Andaluza are white egg layers, whereas Buff Prat
and White Prat are tinted egg layers, and Red-Barred
Vasca and Red Villafranquina lay brown and dark
brown eggs, respectively. Birchen Leonesa and Blue Leonesa are used to produce hackles and saddles for fishermen. In the study are included a synthetic Spanish
breed (Quail Castellana or Melanotic Prat) originated
from an F2 cross between Black Castellana and Buff
Prat (Campo and Orozco, 1986), a tester line carrying
the recessive wheaten allele (Smyth, 1976), and a White
Leghorn population (Campo and Jurado, 1982).
DNA Isolation and Amplification
Blood samples were collected by brachial venipuncture aseptically into tubes using EDTA as anticoagulant. Blood samples were stored at −20°C. Deoxyribonucleic acid was extracted from 40 μL of blood using
resuspension buffer (0.1 M Tris-HCl, 0.01 M NaCl, 0.1
M EDTA, pH 8), lysis buffer (0.1 M Tris-HCl, 0.1 M
EDTA, 0.01 NaCl, 1% SDS, pH 8), 12.5 μL of proteinase K (20 mg/mL), 2 mL of 5 M NaCl, 6 mL of isopropanol, and resuspension in Tris-EDTA. The DNA was
quantified spectrophotometrically and the concentration was adjusted to 15 ng/μL.
Twenty-four microsatellite markers were chosen
based on their genomic location and their degree of
polymorphism. The PCR products were obtained in a
total volume of 10 µL of reaction, with 1 µL of DNA
(15 ng/µL), 0.5 µM of forward and reverse primers,
1.5 mM MgCl2, 200 µM deoxynucleoside triphosphate,
and 0.02 U/µL of Taq polymerase. The amplification
involved first denaturation at 94°C for 3 min, 30 cycles
of denaturation at 94°C for 1 min, annealing at primerspecific temperature (from 45 to 60°C) for 1 min, and
extension at 72°C for 1 min, followed by final extension
at 72°C for 10 min. Fluorescent end-labeled (carboxyfluorescein) PCR primers were used and size characterization of PCR product was performed by an ABI 370
DNA Genetic Analyzer (Applied Biosystems, Foster
City, CA).
2520
Dávila et al.
Statistical Analysis
ing in the White Leghorn population. The number of
alleles by locus ranged from 2 to 13, with the mean
value being 6.250 (SD = 2.770; residual sum of squares
= 2.009), whereas the effective number ranged from
1.220 to 7.219, with the mean value being 3.339 (SD =
1.506). The mean polymorphic information content was
0.591 (SD = 0.178), ranging between 0.847 and 0.172.
The parentage exclusion probabilities of excluding
first parent and parents pair ranged from 0.016 to 0.567
and from 0.166 to 0.889, respectively (Table 1). Combined parentage exclusion probabilities [1 − Π(1 − pi)]
across all loci were 99.999 and 100% (first parent and
parents pair, respectively).
The observed heterozygosity ranged from 0.003 to
0.735 (Table 2). The minimum observed heterozygosity was at the MCW0294 locus, and it was practically
zero. The lowest and greatest expected heterozygosity
was 0.181 and 0.863, respectively, with only 4 loci having the expected heterozygosity lower than 0.5. The
observed heterozygosity was lower than the expected
heterozygosity for all loci, the mean values being 0.461
(SD = 0.191) and 0.637 (SD = 0.175), respectively. The
F-statistics (FIS, FST, and FIT) for each locus are also indicated in Table 2. Mean deficit of heterozygotes within
populations (FIS) was 0.056 and mean fixation index of
each population (FST) was 0.244, ranging from 0.099 to
0.467. The global deficit of heterozygotes across populations (FIT) ranged from 0.097 to 0.996, the mean
value being 0.286.
Six of the Spanish populations (Blue Andaluza, RedBarred Vasca, White-Faced Spanish, Black Castellana,
Birchen Leonesa, and Red Villafranquina) and the
White Leghorn had a total of 15 private alleles at 10
Number of alleles, effective allele number, polymorphism information content, observed and expected
heterozygosity, Wright’s F-statistics, heterozygote
deficiency or excess, and test of the Hardy-Weinberg
equilibrium using Bonferroni correction were estimated
using POPGENE (Yeh et al., 1999), FSTAT (Goudet,
2001), and CERVUS (Kalinowski et al., 2007) computer packages. Neighbor-joining method (Saitou and
Nei, 1987) with arithmetic mean based on Nei’s genetic
distance (Nei et al., 1983) was used to construct the
phylogenetic trees. The robustness of the phylogenetic
trees was evaluated by resampling bootstrap of the loci
with a total of 1,000 replications. All calculations were
made using the DISPAN package (Ota, 1993). The
GENECLASS program (Cornuet et al., 1999) was used
for assigning individuals to populations using Nei’s genetic distance. The probability that each individual was
assigned or not to a population was calculated using a
direct estimation of frequencies, with 10,000 simulated
individuals per population and applying a rejection
threshold of 0.01. Parentage exclusion probabilities (pi)
of first parent and parents pair (Jamieson and Taylor,
1997) were calculated with CERVUS.
RESULTS
Polymorphism of Markers
A total of 150 alleles were detected across all population for the 24 microsatellites examined (Table 1),
the Spanish breeds having 72 alleles which were miss-
Table 1. Microsatellite markers, allele size range, number of alleles (Na), effective number of alleles (Ne), polymorphism information
content (PIC), and parentage exclusion probabilities (pi) for all loci across breeds
Microsatellite
ADL0034
ABR0341
MCW0034
ADL0114
ADL0019
ABR0345
ADL0267
ADL0102
ADL0181
MCW0294
ADL0124
LEI0194
MCW0081
ADL0023
MCW0295
ADL0278
MCW0037
MCW0330
MCW0069
ADL0101
ADL0118
MCW0150
ADL0032
MCW0222
1
First parent.
Parents pair.
2
Chromosome
Allele size (bp)
Na
Ne
PIC
pi1
pi2
20
28
2
2
1
8
2
10
2
Z
1
1
5
5
4
8
3
17
26
1
14
3
18
3
124 to 166
90 to 124
247 to 261
173 to 189
161 to 181
172 to 182
99 to 117
221 to 249
152 to 158
219 to 223
223 to 229
86 to 102
165 to 180
111 to 133
111 to 127
109 to 125
93 to 113
158 to 176
254 to 286
102 to 114
108 to 140
156 to 170
220 to 248
300 to 316
13
12
11
6
8
8
7
7
6
5
5
7
6
5
7
4
4
7
6
3
5
2
3
3
7.219
5.863
5.781
4.771
4.469
4.527
4.025
3.777
3.752
3.249
2.795
3.067
3.075
2.930
2.717
2.816
2.453
2.296
2.300
2.307
1.817
1.510
1.401
1.220
0.847
0.809
0.805
0.760
0.751
0.746
0.717
0.698
0.693
0.645
0.623
0.617
0.616
0.589
0.578
0.570
0.527
0.514
0.510
0.470
0.392
0.281
0.259
0.172
0.567
0.497
0.489
0.414
0.407
0.394
0.358
0.334
0.330
0.274
0.262
0.260
0.252
0.221
0.217
0.210
0.177
0.168
0.172
0.160
0.103
0.057
0.041
0.016
0.889
0.846
0.838
0.776
0.782
0.757
0.727
0.704
0.696
0.632
0.632
0.601
0.586
0.527
0.553
0.505
0.477
0.491
0.490
0.401
0.355
0.224
0.236
0.166
2521
SPANISH CHICKEN GENETIC DIVERSITY
Table 2. Observed and expected heterozygosity (Ho and He, respectively) and F-statistics (FIS, FIT,
and FST1) for all loci across breeds
Microsatellite
ADL0034
ABR0341
ADL0114
ADL0102
MCW0034
ADL0181
ADL0023
ADL0267
ADL0019
ABR0345
MCW0295
ADL0278
MCW0069
LEI0194
MCW0081
MCW0037
MCW0330
ADL0118
ADL0101
MCW0150
ADL0124
ADL0032
MCW0222
MCW0294
Ho
He
FIS
FIT
FST
0.735
0.700
0.667
0.629
0.625
0.616
0.603
0.581
0.542
0.531
0.528
0.494
0.492
0.476
0.464
0.431
0.408
0.393
0.353
0.253
0.247
0.211
0.081
0.003
0.863
0.831
0.791
0.736
0.828
0.735
0.660
0.753
0.778
0.780
0.633
0.646
0.566
0.675
0.676
0.593
0.565
0.450
0.567
0.338
0.665
0.286
0.181
0.692
0.011
−0.071
0.019
−0.223
0.042
−0.008
−0.095
−0.010
0.054
0.109
−0.020
−0.022
−0.029
0.099
0.062
0.042
0.068
0.037
0.198
−0.031
0.334
−0.043
0.407
0.992
0.156
0.170
0.166
0.158
0.257
0.172
0.097
0.238
0.315
0.331
0.178
0.248
0.140
0.307
0.326
0.287
0.290
0.132
0.388
0.288
0.628
0.305
0.576
0.996
0.146
0.225
0.149
0.311
0.224
0.178
0.176
0.244
0.275
0.249
0.193
0.264
0.164
0.231
0.281
0.256
0.237
0.099
0.239
0.311
0.444
0.326
0.296
0.467
1
FIS = mean deficit of heterozygotes within populations; FIT = mean global deficit of heterozygotes across populations; FST = mean fixation index of each population.
microsatellites. The Black Castellana, Birchen Leonesa,
and Red Villafranquina breeds had 1 private allele at
3 different loci, whereas the White-Faced Spanish had
2 private alleles at 1 locus. The Red-Barred Vasca had
1 private allele at 2 loci, and the Blue Andaluza had 1
private allele at 1 locus and 2 private alleles at another
locus. Finally, the White Leghorn had 1 private allele
at 2 loci and 3 private alleles at another locus. A total
Table 3. Number of birds assigned to the population (n1), to the
population and to another population (n2), to another population (n3), and number assigned to the population or to another
population (n4)
1
Population
n1
AA
AF
AP
B
CB
22
20
22
22
15
CC
19
CN
ey
IN
MN
LEG
P
PA
PW
VF
21
18
19
16
20
21
20
22
23
1
n2
1 (MN)
1 (PA)
1 (ey)
1
4
1
1
(MN, AA)
(MN)
(CN, MN)
(CN)
2 (AP)
6 (CB)
2 (PW)
2 (CB)
n3
n4
1
1
3
1
1
3
2
3
2
5
2
4
1
4
1
AA = Blue Andaluza; AF = Black-Barred Andaluza; AP = BlackBreasted Red Andaluza; B = Red-Barred Vasca; CB = White-Faced
Spanish; CC = Quail Castellana; CN = Black Castellana; ey = tester
line; IN = Blue Leonesa; MN = Black Menorca; LEG = White Leghorn;
P = Buff Prat; PA = Birchen Leonesa; PW = White Prat; VF = Red
Villafranquina.
of 9 private alleles had frequencies higher than 10%.
Fixed alleles were observed in 7 loci.
The assignment of the individual to the different
populations is presented in Table 3. A total of 300 birds
(83%) were properly assigned to the source population,
some additional birds being assigned to the source population and to another population (20 birds) or to 2
different populations (2 birds). Thirty-five birds were
neither assigned to the source population nor to another population, and only 3 birds were assigned to
another population.
Diversity of Population
Observed and expected heterozygosities for each
population are indicated in Table 4. Mean expected heterozygosity was higher than mean observed
heterozygosity in most populations except in the Red
Villafranquina, White Prat, Blue Andaluza, and Quail
Castellana breeds. The average observed heterozygosity
was 0.461 (SD = 0.041), ranging from 0.328 to 0.538,
whereas the average expected heterozygosity was 0.488
(SD = 0.060), ranging from 0.320 to 0.550. All of the
Spanish breeds except the Quail Castellana had greater observed heterozygosity than the White Leghorn
population. The number of alleles per locus and breed
ranged from 3 to 4, the average mean value being 3.405
± 0.393. The 4 breeds indicated above showed deficit of
heterozygotes, whereas the 11 remaining breeds showed
excess of heterozygotes, the average mean value being
0.052.
The number of loci that deviated significantly (P <
0.05) from the Hardy-Weinberg equilibrium ranged from
2522
Dávila et al.
Table 4. Observed and expected heterozygosity (Ho and He,
respectively), observed number of alleles per locus (Na), number
of loci that deviated from Hardy-Weinberg equilibrium (dHWE),
and deviation of heterozygote number (FIS)
Population1
VF
CB
AF
CN
MN
AP
B
PW
ey
PA
AA
IN
P
LEG
CC
Ho
He
Na
dHWE
FIS
0.538
0.517
0.495
0.493
0.478
0.474
0.471
0.464
0.462
0.460
0.460
0.434
0.425
0.416
0.328
0.528
0.550
0.518
0.540
0.535
0.502
0.529
0.454
0.481
0.535
0.448
0.469
0.427
0.486
0.320
3.250
3.958
3.500
3.958
3.708
3.292
3.708
3.042
3.583
3.791
3.458
3.208
2.875
3.125
2.625
2
2
2
3
3
4
4
2
3
6
4
2
1
1
1
−0.020
0.062
0.046
0.088
0.109
0.057
0.113
−0.023
0.040
0.142
−0.024
0.076
0.001
0.146
−0.026
1
AA = Blue Andaluza; AF = Black-Barred Andaluza; AP = BlackBreasted Red Andaluza; B = Red-Barred Vasca; CB = White-Faced
Spanish; CC = Quail Castellana; CN = Black Castellana; ey = tester
line; IN = Blue Leonesa; MN = Black Menorca; LEG = White Leghorn;
P = Buff Prat; PA = Birchen Leonesa; PW = White Prat; VF = Red
Villafranquina.
1 to 6 (Table 4), the average value being 2.666. A total of 330 Hardy-Weinberg equilibrium tests were made
(360 theoretical tests without taking in account the sexlinked locus and those corresponding to fixed alleles). A
total of 40 (12%) significant tests were observed, 11 of
them (28%) having an excess of heterozygotes and 29 of
them (72%) having a deficit of heterozygotes.
Relationships Between Populations
Ne’s genetic distance (Table 5) showed a range from
0.109 (between White-Faced Spanish and Black Menorca) to 0.437 (between Buff Prat and White Leghorn).
The genetic distance between the Spanish breeds laying
white eggs and the White Leghorn ranged from 0.246
(Black Menorca) and 0.352 (Black-Red Andaluza),
whereas the genetic distance between all of the Spanish
breeds and the White Leghorn ranged from 0.246 to
0.437. The 2 Spanish breeds included in the AVIANDIV
project (Black Castellana and Red Villafranquina) had
a genetic distance between them of 0.252, being 0.349
and 0.359 with the White Leghorn. A phylogenetic tree
showed a clear separation between the White Leghorn
and the remaining breeds (Figure 1). The most evident
clusters were those found with the Blue Andaluza and
Blue Leonesa, the 3 breeds with black plumage (Black
Castellana, Black Menorca, and White-Faced Spanish),
and the 3 varieties of the Prat breed (Buff, White, and
Quail).
DISCUSSION
Most of the microsatellite markers used in this study
showed a high degree of polymorphism. Barker (1994)
suggested that the average number of alleles per locus
in studies of genetic distances must be greater than
4 to reduce the SE in the estimation of genetic distances. In the present study, 4 microsatellites markers
had a lower value, whereas the mean number of alleles per locus was bigger than 4. Results in the current
study were similar to those found by Takahashi et al.
(1998), Wimmers et al. (2000), Ya-Bo et al. (2006),
Tadano et al. (2007), and Berthouly et al. (2008). On
the contrary, Romanov and Weigend (2001), Zhang et
al. (2002), Hillel et al. (2003), and Osman et al. (2006)
observed higher values of the average number of alleles.
The expected heterozygosity was generally higher than
0.5 and was especially high in the markers ADL0034,
ABR0341, and MCW0034, suggesting the usefulness of
these markers for this type of study. The panel of microsatellites showed a high power of parental exclusion,
higher than 99.999%, showing that this group of markers has a great capacity to discriminate paternities.
Mean fixation index (FST) was 0.244, the global deficit of heterozygotes across populations being 0.286,
suggesting a high degree of population differentiation.
Typically, a fixation index of about 0.15 is considered
to be an indication of significant differentiation among
Table 5. Genetic distances between Spanish chicken breeds, a tester line (ey), and White Leghorn population
Population1
AA
AF
AP
B
CN
CB
CC
ey
IN
LEG
MN
P
PA
PW
AF
AP
B
CN
CB
CC
ey
IN
LEG
MN
P
PA
PW
VF
0.286
0.253
0.294
0.193
0.227
0.256
0.254
0.163
0.321
0.189
0.324
0.221
0.272
0.320
0.215
0.233
0.220
0.256
0.262
0.244
0.306
0.352
0.286
0.291
0.231
0.257
0.263
0.225
0.202
0.223
0.274
0.125
0.301
0.352
0.219
0.286
0.283
0.262
0.252
0.296
0.302
0.322
0.222
0.319
0.382
0.290
0.294
0.227
0.282
0.235
0.151
0.232
0.234
0.247
0.349
0.158
0.251
0.240
0.234
0.252
0.260
0.209
0.218
0.309
0.109
0.242
0.215
0.172
0.282
0.260
0.272
0.423
0.229
0.249
0.310
0.218
0.279
0.265
0.358
0.186
0.296
0.275
0.233
0.199
0.357
0.165
0.386
0.263
0.278
0.347
0.246
0.437
0.307
0.395
0.359
0.274
0.216
0.233
0.240
0.236
0.148
0.282
0.226
0.235
0.267
1
AA = Blue Andaluza; AF = Black-Barred Andaluza; AP = Black-Breasted Red Andaluza; B = Red-Barred Vasca; CB = White-Faced Spanish;
CC = Quail Castellana; CN = Black Castellana; ey = tester line; IN = Blue Leonesa; MN = Black Menorca; LEG = White Leghorn; P = Buff Prat;
PA = Birchen Leonesa; PW = White Prat; VF = Red Villafranquina.
SPANISH CHICKEN GENETIC DIVERSITY
populations (Frankham et al., 2002); this value was
found for all loci except for the ADL0118 locus. The
mean fixation index was similar to those indicated previously by Tadano et al. (2007) in 12 Japanese breeds
and 2 commercial lines, and Berthouly et al. (2008) in
14 French breeds and 6 different Japanese and Chinese
breeds (0.303 and 0.240, respectively). As an indirect
way to measure quantitative genetic diversity, a fixation index of about 0.25 means that 40% of total genetic variance could be explained by the among-breed
genetic variance [2FST/(1 + FST)], in agreement with
the range of values (30 to 50%) indicated in the literature for this parameter (Frankham et al., 2002).
The markers used in the current study may be of
great interest for the genetic identification of animals
because 15 private alleles were observed in the populations. A high percentage of birds were properly assigned to the source population, the birds assigned to
the source population and to another population being
explained most of the time by a common origin. For example, White-Faced Spanish, Black Menorca, and Blue
Andaluza come from the Black Castellana breed, and
Buff Prat and White Prat are 2 varieties of the same
breed.
All of the Spanish breeds except the synthetic Quail
Castellana were more polymorphic than the White Leghorn population. The highest value of heterozygosity
was observed in the Red Villafranquina breed (0.54);
although this breed has been selected for a very dark
2523
brown shell color for years, it shows a great variability
of shell color ranging from light to very dark brown.
Hillel et al. (2003) indicated a value for this breed of
0.46. The lowest heterozygosity was observed in the
Quail Castellana, which originated from an F2 cross
between the Black Castellana and Buff Prat breeds followed by 4 generations of selection to uniform the chick
down color until a completely black with brown face
type (Campo, 1991), the low values of heterozygosity
reflecting this selection. Similarly, the White Leghorn
population also showed a low value of heterozygosity
(0.42) and originated from the crossing of 3 commercial
stocks selected for egg production and egg weight (Babcock, Creighton, and Mount Hope; Campo and Jurado,
1982). Hillel et al. (2003) indicated a value for the white
egg layer line of 0.34. The mean observed heterozygosity in the 13 Spanish breeds was 0.46, in agreement with
the values reported by Hillel et al. (2003) for 23 standardized breeds selected for morphological traits (0.46)
and Wimmers et al. (2000), Tadano et al. (2007), and
Berthouly et al. (2008) for other native breeds (0.58,
0.40, and 0.49, respectively).
All populations showed significant deviations from
the Hardy-Weinberg equilibrium, suggesting that some
Spanish chicken breeds have been selected for years for
morphological traits such as plumage, shank and egg
colors, and comb and earlobe sizes, although the presence of null alleles or genotyping error could also be
the reason.
Figure 1. Neighbor-joining dendrogram based on Nei’s genetic distance. AA = Blue Andaluza; AF = Black-Barred Andaluza; AP = BlackBreasted Red Andaluza; B = Red-Barred Vasca; CB = White-Faced Spanish; CC = Quail Castellana; CN = Black Castellana; ey = tester line;
IN = Blue Leonesa; MN = Black Menorca; LEG = White Leghorn; P = Buff Prat; PA = Birchen Leonesa; PW = White Prat; VF = Red Villafranquina.
2524
Dávila et al.
In the current study, the 2 Spanish breeds (Black
Castellana and Red Villafranquina) included in the
AVIANDIV project had a Nei’s genetic distance between them and the White Leghorn population lower
than those indicated by Hillel et al. (2003), which were
0.426, 0.644, and 0.454, respectively. This fact suggests
that the values of genetic distances depend on the group
of populations studied (52 in the AVIANDIV project);
the white egg layer line used in the AVIANDIV project
had been selected for more generations, and the white
egg layer lines that were used in both studies had a different origin. Although Hillel et al. (2003) also calculated the Cavalli-Sforza and Reynolds genetic distances,
Takezaki and Nei (1996) suggested the use of Nei’s genetic distance in the analysis with microsatellite markers, when the main objective of the study is focused on
the correct distribution of the topology, rather than to
studies of evolutionary times.
A phylogenetic tree showed a clear separation between the White Leghorn and the Spanish breeds, suggesting that the latter do not come from the former,
although most of them are included in the Mediterranean group of breeds. Within the Spanish breeds, one
of the most evident clusters was found for the 2 breeds
carrying the blue gene, which produces a dilution of the
eumelanins coming from the extended black (Blue Andaluza) or the birchen (Blue Leonesa) alleles. Another
evident cluster was observed for the 3 breeds with black
plumage carrying the extended black allele (Black Castellana, Black Menorca, and White-Faced Spanish),
with the Black Castellana separated from the other 2
breeds. This fact is in agreement with the origin of
the Black Menorca and White-Faced Spanish from the
Black Castellana, due to the selection for a large size
of the earlobes (Black Menorca), which are so greatly
enlarged that they fall below the wattles covering all
of the face (White-Faced Spanish). The last evident
cluster was found for the 2 varieties of the Prat breed
(Buff and White), and the synthetic breed originated
from crossing this breed and the Black Castellana. This
fact suggests that the synthetic breed is more similar
to the Buff Prat than to the Black Castellana, in agreement with its melanotic columbian genetic background:
(eWh/eWh Co/Co Ml/Ml), the Buff Prat being columbian (Campo, 1991).
In conclusion, the panel of microsatellite markers
was of great usefulness in studying the genetic diversity
of chicken breeds, showing a high degree of polymorphism, a great capacity to discriminate paternities, and
a high degree of population differentiation. The Spanish
breeds had a high number of private alleles and were
more polymorphic than the White Leghorn population,
suggesting their potential to be selected for use in alternative production systems. Although Spanish chickens have been traditionally selected for morphological
traits, they have not been selected yet for productive
traits. A clear separation between the White Leghorn
and the Spanish breeds was found, indicating the im-
portance of including these populations in conservation
programs.
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