GENETIC CHARACTERISATION OF ITALIAN CHICKEN BREEDS USING A PANEL OF TWENTY MICROSATELLITE MARKERS
ABSTRACT 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.
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ABSTRACT: In common domestic species for which varieties, strains or breeds are in danger of extinction, the population levels at which action needs to be taken are object of research in many countries. Different approaches have been developed and exploited to understand the different aspects that contribute to breed differentiation and to study the typical products that originate from them. The thesis is made up of three contributes. The objectives of the first one were to determine genetic variation and to analyze population structure in six Italian local chicken breeds involved in a conservation program. Twenty microsatellite markers were investigated in 337 animals belonging to six breeds: Ermellinata di Rovigo, Robusta Maculata, Robusta Lionata, Pepoi, Padovana and Polverara; a commercial layer cross was used as reference. One-hundred-twelve alleles were detected in the overall population, with a mean number of 5.6 ± 2.1 alleles per locus. For the local breeds, the observed and expected heterozigosity ranged from a minimum of 0.240 to a maximum of 0.413 and from 0.243 to 0.463 for the Pépoi and Polverara breeds, respectively. Deviation from Hardy-Weinberg equilibrium has been observed in five breeds and in the commercial cross. The overall population heterozygote deficiency FIT, resulted 0.427, the average FIS 0.097, while FST was 0.437, indicating a high heterozygote deficiency mainly due to breed subdivisions. Reynolds distances were used to draw an unrooted Neighbor-Joining tree, which topology gave information on the genetic origin of these breeds and confirmed their known history. The estimated molecular kinship within breed ranged from 0.559 to 0.769, evidencing high coancestry. Structure analysis was performed to detect the presence of population substructures. Inferred clusters corresponded to the different breeds, without presence of admixture. Exception was the Polverara, for which a more complex genetic structure was found. Obtained results confirmed the usefulness of molecular markers, as microsatellites, to characterize local breeds and to monitor genetic diversity in livestock conservation schemes. The objective of the second contribute was to describe carcass characteristics and qualitative meat traits of three local chicken breeds showing, at maturity, light, medium-light, and medium live weights. By the fact, those breeds could permit to extend and diversify consumer’s offer to fit all the local demands in typical diversified poultry products. The experiment involved 60 male chickens reared in an organic production system where housing was an indoor pen with access to a grass paddock was carried out in order to investigate carcass characteristics and qualitative meat traits of three slow-growing Italian local breeds of chicken (Ermellinata, Padovana, and Pépoi). Chicks were randomly selected at hatch, raised together under the same conditions, slaughtered at 190 days of age, dissected for carcass traits and meat was stored for subsequent analysis of breast and thigh meat quality. Ermellinata chickens were consistently heavier than Padovana and Pépoi chickens for live, carcass, thigh weight and there were differences among breeds for protein percentage (Ermellinata > Pépoi and Padovana), shear force (Padovana < Ermellinata and Pépoi), and cooking loss (Pépoi > Padovana and Ermellinata). The CIE system values of lightness (L*), redness (a*), and yellowness (b*) evidenced a distinctive darker and lighter colour of Padovana for meat and skin, respectively. Fatty acid composition of breast was similar among the studied breeds, while saturated and monounsaturated fatty acids contents of Ermellinata were higher and lower, respectively than the other breeds. Aim of the third study was to apply a proteomic approach for characterization of local chicken breeds. The experiment involved a total of 29 males of Pépoi, Padovana, and Ermellinata local chicken breeds. Samples were taken from breast muscle (Pectoralis superficialis). Sarcoplasmic protein fractions of breast muscle were analysed by bidimensional electrophoresis. Image analysis followed by statistical analysis enabled to differentiate groups of individuals on the similarities of protein expression. Individuals were distinguished into clusters and groups, corresponding to the breed of origin. SAM analysis enabled identification of the most relevant spots; 10 of these were identified by Mass Spectrometry revealing preliminary evidences on the mechanics of the breed differentiation process. Results evidenced a possible utilisation of proteomic approach in the field of breed characterization studies as an alternative to genomic analyses performed using molecular markers, both for breed and product traceability purposes.
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ABSTRACT: Microsatellite markers have been a useful genetic tool in determining diversity, relationships and individual discrimination studies of livestock. The level of genetic diversity, relationships among two Korean indigenous chicken brand populations (Woorimatdag: WR, Hanhyup3: HH) as well as two pure populations (White Leghorn: WL, Rhode Island Red: RIR) were analyzed, based on 26 MS markers. A total of 191 distinct alleles were observed across the four chicken populations, and 47 (24.6%) of these alleles were unique to only one population. The mean and PIC were estimated as 0.667 and 0.630. Nei's genetic distance and factorial correspondence analysis (FCA) showed that the four populations represented four distinct groups. However, the genetic distance between each Korean indigenous chicken brand (WR, HH) and the pure population (WL, RIR) were threefold that among the WR and HH. For the STRUCTURE analyses, the most appropriate number of clusters for modeling the data was determined to be three. The expected probabilities of identity among genotypes of random individuals (PI) were calculated as (All 26 markers) and , (9, 12 with the highest PI value, respectively). The results indicated that the brand chicken breed traceability system employing the own highest PI value 9 to 12 markers, and might be applicable to individual identification of Korean indigenous chicken brand.Journal of Animal Science and Technology. 01/2013; 55(3).
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ABSTRACT: Domestication of livestock species and a long history of migrations, selection and adaptation have created an enormous variety of breeds. Conservation of these genetic resources relies on demographic characterization, recording of production environments and effective data management. In addition, molecular genetic studies allow a comparison of genetic diversity within and across breeds and a reconstruction of the history of breeds and ancestral populations. This has been summarized for cattle, yak, water buffalo, sheep, goats, camelids, pigs, horses, and chickens. Further progress is expected to benefit from advances in molecular technology.Animal Genetics 05/2010; 41 Suppl 1:6-31. · 2.58 Impact Factor
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: email@example.com
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.
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(Received on 16 June 2007; accepted on 3 July 2007)