UDK = 636.5:636.084.5
GENETIC CHARACTERISATION OF ITALIAN CHICKEN BREEDS USING A
PANEL OF TWENTY MICROSATELLITE MARKERS
E. Zanetti, Chiara Dalvit, M. De Marchi, R. Dal Zotto, M. Cassandro
Original scientific paper
Genetic relationships among four Veneto native breeds of chickens were studied on the basis of
microsatellites polymorphisms. A total of 190 DNA samples (45 Robusta Lionata, 43 Robusta Maculata, 45
Ermellinata di Rovigo, 45 Pèpoi) and a commercial broiler line (12 Golden Comet) were genotyped at 20
microsatellite loci. The average number of alleles per locus was 5 and the expected heterozygosity resulted
lower for the local breeds than for the commercial broiler line used as reference. The inbreeding coefficient
showed a deficit of heterozygotes, highest for the Robusta Lionata breed. Nei’s standard genetic distances
corrected for bias due to sampling of individuals (Da), based on allele frequencies, and Reynolds distances
(DReynolds) were calculated among breeds. The Robusta Lionata and Robusta Maculata resulted very similar
approving the same genetic origin. A Neighbor-Joining tree drawn from DReynolds distances clustered three
groups, one including the Robusta Lionata and Robusta Maculata breeds, the second one formed by the
Ermellinata di Rovigo and the Golden Comet commercial line and the third by the Pèpoi. The results showed
the genetic differences occurring between the local chicken breeds.
Key-words: chicken, microsatellites, genetic distances, biodiversity, conservation.
The dramatic size contraction of local poultry breeds due to replacement with cosmopolite improved
breeds showed the need for native genetic resources conservation. Rare poultry breeds and population
characterised by a limited size strictly depend on the maintenance of genetic differences (Wimmers et
al., 2000). Conservation of genetic variability is of great importance in animal science; the analysis of
breeds genetic structure can supply the basis for effective conservation programs. Since 2000, in the
Veneto, region of Italy, a total of 12 local poultry breeds derived from four different species (chicken,
duck, helmeted guinea fowl and turkey) have been available as genetic resources and involved in an in
situ marker assisted conservation scheme (Cassandro et al., 2004).
In the present study four of these Veneto chicken breeds, Robusta Lionata (PRL), Robusta Maculata
(PRM), Ermellinata di Rovigo (PER) and Pèpoi (PPP), were analysed using the commercial broiler
line Golden Comet (PBR) as a reference. Robusta Lionata and Robusta Maculata are medium-heavy
dual purpose breeds, selected in 1965 in the “Stazione Sperimentale di Pollicoltura” of Rovigo, by
crossing Orpington with White America and probably other unspecified breeds. The Ermellinata di
Rovigo breed was developed in 1959 crossing Sussex and Rhode Islands to obtain a valuable meat
breed also useful for eggs production. The Pèpoi is a small size breed originated in the north-western
part of the Veneto and Friuli regions of Italy. It appears to have a good attitude to the extensive
production systems and is particularly appreciated for its meat (Veneto Agricoltura, 2004).
The application of molecular biology techniques helps avoiding the risk of compromising genetic
variability of conservation programs of small populations. Until 2005 the genotyping of the individual
animals for marker assisted conservation scheme was carried out using the AFLP technique (De
Marchi et al., 2006). Afterward, microsatellites have been applied because these molecular markers
Enrico Zanetti, PhD. Student, Chiara Dalvit, PhD. Student; PhD. Massimo De Marchi, Assistant Professor;
PhD. Riccardo Dal Zotto, PhD. Martino Cassandro, Professor - Department of Animal Science, University of
Padova – Agripolis, Viale dell'Università,16 35020 Legnaro (PD), Italy – Ph. +39 049 8272616 - Fax: +39 049
8272633 – e-mail: firstname.lastname@example.org
are well dispersed in the genome and highly polymorphic (Cheng et al., 1995); their application to
characterise chicken breeds is relatively recent but it has been used in many countries to study the
genetic relationships among native breeds (Takahashi et al., 1998; Hillel et al., 2003).Aim of this study
was to define the genetic relationships among four local chicken breeds: Robusta Lionata, Robusta
Maculata, Ermellinata di Rovigo, Pèpoi and a commercial broiler line, the Golden Comet, using
microsatellite DNA polymorphisms as markers.
MATERIAL AND METHODS
Individual blood samples 190 belonging to four local breeds of Veneto region, Robusta Lionata (PRL,
43 individuals), Robusta Maculata (PRM, 45 individuals), Ermellinata di Rovigo (PER, 45), Pèpoi
(PPP, 45) and the Golden Comet commercial broiler line (PBR, 12) were randomly collected within
breed in three different herds. Twenty sets of primers (Table 1), included in the lists of recommended
primers for chicken analysis suggested by the FAO organisation (FAO, 2004), were chosen on the
basis of their position in the chicken genome. The PCR primer pairs were synthesized and 5’ ends of
the forwards primers were fluorescently labelled. Chicken genomic DNA used as a template for PCR
reaction was isolated from blood using a modified DNA purification kit (Gentra System PUREGENE
DNA). The 20 microsatellites (STR) were individually analyzed by a PX2 Thermohybaid thermal
cycler at the following conditions, the X temperature being the annealing t° of each primer (NCBI):
initial denaturation step of 10 min at 94°C, 35 cycles of 45 s at 94°C, 1 min at X°C and 1.5 min at
72°C and a final extension of 10 min at 72°C. A reaction volume of 15 µl contained 25 ng of genomic
DNA, 1.5 mM MgCl2, 1.5 µl of Taq Buffer 1X, 0.04 U Taq Gold (Sigma), 3mM dNTPs and 10 µM of
each primer. Analysis of fragments was performed using an automated DNA sequencer (CEQ 8000
Genetic Analysis System, Beckman Coulter) and a computer software (CEQ 8000 Beckman Coulter).
Alleles were designated according to PCR product size whereas allelic frequencies were estimated.
Values of observed, non biased (i.e. observed heterozygosity corrected for bias due to sampling) and
expected heterozygosity, FIS values (Weir and Cockerham, 1984) and genetic distances among breeds,
calculated according to Nei (1978), were determined using the Genetix software (Belkhir, 1996-2002).
Reynolds distances (DReynolds) (Reynolds et al., 1983) were calculated using the Phylip 3.66 software
package (Felsenstein, 2005). A χ2 test was performed to evaluate significant differences between
observed and expected heterozygosity (H) values using the Genepop software (Raymond, 1995). A
factorial correspondence analysis was carried out using the software Genetix, in order to define latent
variables which explain the whole genetic similarity relation system existing among individuals.
RESULTS AND DISCUSSION
All twenty microsatellites examined approved to be polymorphic, a total of 100 alleles were detected
and the average number of alleles per locus was 5 (Table 1).
Table 1. Polymerase chain reaction primers for microsatellite markers, chromosomes involved (Chr.),
alleles detected and minimum and maximum fragments length
Loci Chr. Alleles Length Loci
MCW0295 4 5 86-98 MCW0222
MCW0078 5 6 134-146 MCW0037
MCW0104 13 7 190-216 MCW0098
MCW0123 14 6 112-134 ADL0278
MCW0081 5 6 143-155 LEI0166
MCW0014 6 6 166-181 ADL0268
MCW0248 1 4 215-223 MCW0016
LEI0094 4 6 259-283 MCW0165
MCW0111 1 4 98-106 MCW0020
MCW0216 13 4 141-145 MCW0103
Expected and observed H values are reported in Table 2. These parameters are important because the
conservation program aims to increase the genetic variability within and between breeds. The broiler
line showed the highest value of expected and observed heterozygosity (0.5580 and 0.6777,
respectively). The PRL, PER and PPP showed a significant deficit of heterozygotes, deviating from
Hardy-Weinberg equilibrium. The PBR, as expected from a commercial hybrid, showed a significant
excess of heterozygotes. All the local breeds showed evidenced low H values if compared to those
reported by other authors regarding other indigenous breeds (Zhang et al., 2002), but similar to those
reported by Hillel et al. (2003) about standardized breeds selected on morphology (European breeds).
Table 2. Average values of expected (H exp), non biased (H nb), observed (H obs) heterozygosity and
inbreeding coefficient (FIS)
Genetic type H exp H nb H obs
Robusta Lionata 0.3666 0.3712 0.3223
Robusta Maculata 0.3062 0.3098 0.3074
Ermellinata di Rovigo 0.4143 0.4202 0.3836
Pèpoi 0.2304 0.2334 0.2294
Broiler 0.5580 0.5830 0.6777
*** = P<0.001; * = P<0.05; n.s. = not significant
FIS value, which indicates the degree of departure from random mating, was particularly high in
Robusta Lionata (0.1233) compared to other breeds, indicating heterozygosity deficiency. It might be
a result of a bottleneck effect, since its population size decreased drastically (few hundreds) before the
beginning of the conservation project.
Nei’s standard genetic distance (Da), corrected for bias, due to sampling of individuals, and Reynolds
distance estimates (DReynolds) are reported in Table 3. Reynolds distance (Reynolds, 1983) were used to
estimate pairwise genetic distances between the breed. This measure is recommended by Eding and
Laval (1999) for populations with short divergence time. Calculating both distances, PRL and PRM
breeds were closer (0.388 and 0.392) than the other breeds and the broiler line individuals. This result
is in agreement with the known genetic origin of these two breeds, approving that the use of
microsatellite markers for the study of genetic biodiversity is accurate and reliable.
Table 3. Distance matrices estimated by Da (above diagonal) and DReynolds (below diagonal) distances
PBR PRL PRM PER PPP
PBR 0.442 0.385 0.457 0.565
PRL 0.311 0.388 0.646 0.623
PRM 0.319 0.392 0.697 0.728
PER 0.298 0.434 0.479 0.852
PPP 0.428 0.534 0.596 0.561
The Neighbor-Joining tree in Figure 1 was drawn from DReynolds distance matrix obtained analysing the
molecular markers. For the validation of the tree topology, 1000 bootstraps resampling were
performed. The dendrogram clearly clustered two groups (supported by a bootstrap value of 63%): one
includes PRL and PRM, the other one the remaining three breeds. In this second group PPP forms a
separate cluster but with lower bootstraps scores (43%).
Figure 1. Neighbor-Joining tree drawn from DReynolds distance estimated by microsatellite markers (1000
The factorial correspondence analysis defined three main factors (Figure 2). The first one explained
the 34% of total variance, the second one 24% while the third one 20%. On the whole, this analysis
reported a clear breed grouping trend and a good distinction among breeds.
Figure 2. Distribution of individual factorial weights for factor 1, factor 2 and factor 3 of broiler (PBR),
Robusta Lionata (PRL), Robusta Maculata (PRM), Ermellinata di Rovigo (PER) and Pèpoi (PPP) chicken
Microsatellite markers permitted the genetic characterisation of the four indigenous breeds of
chickens. The optimum use of such information can help to preserve allelic diversity and the existing
genetic variation. The obtained results seem to be promising to define and control the ongoing animal
genetic resources conservation program. The microsatellites panel adopted for this study could also be
useful for genetic traceability purposes. Tracing the breed of origin of animal products represents an
opportunity for the promotion of local genetic resources with benefits for local economy, breed
valorisation and sustainable conservation of biodiversity.
The authors wish to thank Martina Gervaso and Maristella Baruchello for their support. This
research was funded by the “Veneto Agricoltura” regional agency.
1. Belkhir, K., Borsa, P., Chikhi, L., Raufaste, N., Bonhomme, F. (1996-2002): GENETIX v. 4.04.
Logiciel sous Windows TM pour la génétique des populations. Laboratoire Genome, Populations,
interactions CNRS UMR 5000, Université de Montpellier. France.
2. Cassandro, M., De Marchi, M., Targhetta, C., Dalvit, C., Ramanzin, M., Baruchello, M. (2004):
An in situ marker-assisted conservation scheme of 11 Italian avian breeds. 55th Anual meeting of
5 Download full-text
the European Association for Animal Production (EAAP), Bled, 5-9 September. Wageningen
Academic Publishers. Book of abstracts 10:30.
3. Cheng, H.H., Levin, I., Vallejo, R., Khatib, H., Dodgson, J.B., Crittenden, L.B., Hillel, J. (1995):
Development of a genetic map of the chicken with markers of high utility. Poultry Science,
4. De Marchi, M., Dalvit, C., Targhetta, C., Cassandro, M. (2006): Assessing genetic diversity in
indigenous Veneto chicken breeds using AFLP markers. Animal Genetics, 37:101-105.
5. Ending, J.H., Laval, G. (1999): Measuring genetic uniqueness in livestock. In: J.K. Oldenbroeck
(ed.), Genebanks and the Conservation of Farm Animal Genetic Resources. DLO Institute for
Animal Science and Health, Lelystad, The Netherlands, 33–58.
6. FAO. (2004): Secondary Guidelines: Measurement of Domestic Animal Diversity (MoDAD):
New Recommended Microsatellite Markers. Home page address: http://dad.fao.org/.
7. Felsenstein, J. (2005): PHYLIP v. 3.6. Department of Genome Sciences, Washington University,
8. Hillel, J., Groenen, M.A.M, Tixier Boichard, M., Korol, A.B., David, L., Kirzhner, V.M., Burke,
T., Barre Dirie, A., Crooijmans, R.P.M.A., Elo, K., Feldman, M.W., Freidlin, P.J., Mäki-Tanila,
A., Oortwijn, M., Thomson, P., Vignal, A., Wimmers, K., Weigend, S. (2003): Biodiversity of 52
chicken populations assessed by microsatellite typing of DNA pools. Genetics Selection
9. NCBI, Home page address http://www.ncbi.nlm.nih.gov.
10. Nei, M. (1978): Estimation of average heterozygosity and genetic distance from a small number of
individuals. Genetics, 89:583-590.
11. Raymond, M., Rousset, F. (1995): GENEPOP v. 3.1d. Journal of Heredity, 86:248-249.
12. Reynolds, J, Weir, B.S., Cockerham, C.C. (1983): Estimation of the coancestry coefficient: basis
for a short-term genetic distance. Genetics, 105:767-769.
13. Takahashi, H., Nirasawa, K., Nagamine, Y., Tsudzuki, M., Yamamoto, Y. (1998): Genetic
relationships among Japanese native breeds of chicken based on microsatellite DNA
polymorphisms. Journal of Heredity, 89(6):543-546.
14. Veneto Agricoltura. (2004). Home page address: http://www.venetoagricoltura.org/
15. Weir, B.S., Cockerham, C.C. (1984). Estimating F-statistics for the analysis of population
structure. Evolution, 38:1358-1370.
16. Wimmers, K., Ponsuksili, S., Hardge, T., Valle-Zerate, A., Mathur, P.K., Horst, P. (2000): Genetic
distinctness of African Asian and South American local chickens. Animal Genetics, 31:159-165.
17. Zhang, X., Leung, F.C., Chan, D.K.O., Yang, G., Wu, C. (2002): Genetic Diversity of Chinese
Native breeds Based on Protein Polymorphism, Randomly Amplified Polymorphic DNA, and
Microsatellite Polymorphism. Poultry Science, 81:1463-1472.
(Received on 16 June 2007; accepted on 3 July 2007)