Sandrine Demane

Herve ´sanguin

John Pote

Elisabeth Navarro

Dominique Bernillon

Patrick Mavingui

Walter Wildi

Timothy M Vogel

Pascal Simonet
email: pascal.simonet@ec-lyon.fr.

James M Tiedje

P S Wrote

Antibiotic-resistant soil bacteria in transgenic plant fields

Understanding the prevalence and polymorphism of antibiotic resistance genes in soil bacteria and their potential to be transferred horizontally is required to evaluate the likelihood and ecological (and possibly clinical) consequences of the transfer of these genes from transgenic plants to soil bacteria. In this study, we combined culture-dependent and -independent approaches to study the prevalence and diversity of bla genes in soil bacteria and the potential impact that a 10-successive-year culture of the transgenic Bt176 corn, which has a blaTEM marker gene, could have had on the soil bacterial community. The bla gene encoding resistance to ampicillin belongs to the beta-lactam antibiotic family, which is widely used in medicine but is readily compromised by bacterial antibiotic resistance. Our results indicate that soil bacteria are naturally resistant to a broad spectrum of beta-lactam antibiotics, including the third cephalosporin generation, which has a slightly stronger discriminating effect on soil isolates than other cephalosporins. These high resistance levels for a wide range of antibiotics are partly due to the polymorphism of bla genes, which occur frequently among soil bacteria. The blaTEM116 gene of the transgenic corn Bt176 investigated here is among those frequently found, thus reducing any risk of introducing a new bacterial resistance trait from the transgenic material. In addition, no significant differences were observed in bacterial antibiotic-resistance levels between transgenic and nontransgenic corn fields, although the bacterial populations were different.

antibiotic resistance ͉ GMO ͉ HGT B ecause of their efficiency against susceptible Gram-positive and proteobacteria [START_REF] Thomson | The threat of antibiotic resistance in Gram-negative pathogenic bacteria: Beta-lactams in peril![END_REF], their specificity, and a general absence of toxicity for humans, beta-lactam antibiotics, including penicillin, ampicillin, cephalosporins, and monobactams, are the largest antibiotic family used in medicine. However, the use of these antibiotics is readily compromised by bacterial antibiotic resistance [START_REF] Livermore | Are all beta-lactams created equal?[END_REF][START_REF] Wilke | beta-Lactam antibiotic resistance: a current structural perspective[END_REF], partly because of the production of betalactamases. The most common beta-lactamases are the TEM beta-lactamases that are encoded by the blaTEM1 gene and its descendants. TEM1 beta-lactamase hydrolyzes only penicillins and some older cephalosporins, but descendants of TEM1 have evolved the ability to hydrolyze modern cephalosporins and monobactams [START_REF] Medeiros | Evolution and dissemination of beta-lactamases accelerated by generations of beta-lactam antibiotics[END_REF]. Indeed, the widespread use of beta-lactams in the past few decades has led to the evolution of a new generation of beta-lactamases, which have extended substrate spectra [i.e., extended spectrum beta-lactamases (ESBLs)]. ESBLs are generally plasmid-mediated enzymes that confer resistance to oxyimino-beta-lactams, such as cefotaxime, ceftazidime, and aztreonam [START_REF] Jacoby | More extended-spectrum beta-lactamases. Antimicrob[END_REF]. Numerous questions remain about the role that natural environments play in the maintenance and dispersion of antibiotic resistance genes and about the frequency with which these genes are exchanged among indigenous bacteria and whether they can spread from these commensal strains to clinical isolates. After many years of contradictory data about the actual production of antibiotics by indigenous microorganisms in soil (6), several reports were published confirming that antibiotics are produced in soils at sufficiently high concentrations to inhibit bacterial growth in the vicinity of the producers [START_REF] Li | Factors affecting co-inoculation with antibiotic-producing bacteria to enhance rhizobial colonization and nodulation[END_REF][START_REF] Thomashow | Production of the antibiotic phenazine-1-carboxylic acid by fluorescent Pseudomonas species in the rhizosphere of wheat[END_REF][START_REF] Hansen | Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining wholecell biosensors and flow cytometry[END_REF][START_REF] Anukool | In situ monitoring of streptothricin production by Streptomyces rochei F20 in soil and rhizosphere[END_REF]. These results suggest that in situ conditions are selective enough to drive the development of antibiotic resistance mechanisms as a necessary survival strategy to guard against antibiotic-producing strains. Moreover, the use of antibiotics in therapeutic treatments or as growth promoters and field cultivation of some genetically modified plants (GMPs) are suspected to increase the risk of antibiotic resistance gene dissemination. Several commercial GMPs contain antibiotic resistance genes that are still under the control of bacterial promoters as remnants of the bacterial vectors used to construct the GMPs. These former bacterial genes could be transferred more easily than other plant genes to soil bacteria because of a high degree of homology facilitating recombination in potential bacterial recipients [START_REF] Goldstein | Human safety and genetically modified plants: A review of antibiotic resistance markers and future transformation selection technologies[END_REF]. This might be the case for the insect-protected Bt176 corn that confers protection against attack by Lepidopteran insects because of the production of a Bt toxin. A bla gene under a bacterial promoter control that encodes a TEM116 betalactamase conferring resistance to ampicillin was cointroduced into corn during the Bt176 event.

The possible impact of GMPs in terms of gene transfer to bacteria and modification of the soil microbial community with antibiotic resistance genes cannot be evaluated without considering the initial ecology of these genes in this community. Indeed, the impact level would strongly depend upon the natural prevalence of these genes in soil bacteria and their polymorphism that reflects their actual evolutionary level susceptible of providing the community with the ability to hydrolyze a more or less wide range of antibiotic molecules.

We addressed the questions of antibiotic resistance gene evolution and GMPs impact by investigating the diversity of bla genes in soil bacteria from fields cultured for as many as 10 successive years with transgenic (Bt176 event) and traditional corn lines and from a prairie soil in the same area. The study was based on a combination of culture-dependent and -independent approaches that aimed to determine the actual state of evolution of bla genes in soils. Then, we addressed the potential impact of the transgenic plant on the prevalence of bla genes and the emergence of new resistant strains. In addition, the modifications of the bacterial community structure due to a potential (trans)gene transfer to bacteria and/or a direct effect of the GMPs was monitored by these methodological approaches and by the use of a 16S rRNA microarray (phylochip).

Results and Discussion

The study was conducted on soils sampled in Bazie `ges (Station Inter Institut Association Ge ´ne ´rale des Producteurs de Maı ¨s-AGPM-Technique, Haute-Garonne, France), where transgenic Bt176 corn (T1 and T2 soil samples) and traditional corn (C1 and C2 soil samples) have been cultured in separate fields for 10 successive years. Soil samples from a prairie (P1 and P2 soil samples), recovered in the same area, were also analyzed. The insect-protected Bt176 corn was obtained by microprojectile bombardment into immature embryos of inbred corn line CG00526 (Zea mays L.), using two different plasmids, including pCIB4431 and pCIB3064, both containing a copy of the blaTEM116 gene that confers resistance to ampicillin and carbenicillin under the control of a bacterial promoter.

Total and Beta-Lactam-Resistant Cultivable Bacteria in Soils. Soil suspensions plated on appropriate media led to the recovery of cultivable bacteria and among them ampicillin-resistant isolates potentially carrying a bla gene. The total number of cultivable bacteria was not significantly different (0.36 Ͻ P Ͻ 0.62) between the three soils (1.7 ϫ 10 4 in C1 to 2.5 ϫ 10 5 in T2) [supporting information (SI) Table 1], but the level of resistant isolates was significantly different (P Ͻ 0.01) between cultivated and prairie soils. In the corn fields, the prevalence of cultivable ampicillinresistant bacteria exhibited some heterogeneity that correlated not to the transgenic status of the plant but rather to field location. Percentages of resistant bacteria varied from 0.4% to 6.5% of total cultivable bacteria between samples T2 and T1, respectively, and from 5.5% to 8.0% in samples C2 and C1, respectively (SI Table 1). These results are consistent with other studies that found that the population levels of ampicillinresistant bacteria in corn field soils did not exhibited significant difference between Bt176 transgenic and nontransgenic corns but did significantly differ with field location, sampling stage, and year of study [START_REF] Badosa | Lack of detection of ampicillin resistance gene transfer from Bt176 transgenic corn to culturable bacteria under field conditions[END_REF]. In prairie soil, prevalence of ampicillinresistant bacteria is significantly higher, ranging from 54.4% to 69.6% in P2 and P1 samples, respectively (SI Table 1). This is a possible indication that bacterial communities not disturbed by agricultural practices may be more exposed to antibioticproducing strains or in better contact for gene exchange and, as a consequence present a higher degree of antibiotic resistance.

A library of 576 ampicillin-resistant isolates (96 isolates per soil sample) was constructed. Beta-lactam resistance pattern of these isolates was determined by testing growth on media supplemented with antibiotics of the penicillin group, including ampicillin, carbenicillin, amoxicillin, penicillin G, and oxacillin, and the cephalosporin group, including cephalothin, cefamandole, and ceftriaxone, which are first-, second-, and thirdgeneration cephalosporins, respectively.

Initially, we determined the resistance pattern of Escherichia coli DH5␣ and found that the presence of pCIB4431 plasmid, which was used to construct transgenic corn Bt176 and carried the blaTEM116 gene, conferred a robust resistance to penicillins, but would not provide the resistance to second-and third-generation cephalosporins. Then, the resistance pattern was established for all soil isolates, which exhibited a broad beta-lactam resistance spectrum (SI Fig. 2). This led to the observation that none of the 576 isolates tested exhibited the same resistance pattern as E. coli containing pCIB4431 because of their natural extended antibiotic resistance pattern. Almost all of the isolates from the three soils were resistant to penicillins and cephalosporin antibiotics belonging to the first and second generation, except sample T2, where bacteria were slightly less resistant to the second cephalosporin generation (94.6%). The only discriminating antibiotic belonged to the third cephalosporin generation, with resistance percentages ranging from 38.7% in sample P2 to 96.4% in sample C1. No marked difference was observed in bacterial antibiotic resistance level and pattern between transgenic and nontransgenic corn fields (SI Fig. 2 and SI Table 1). These data indicate that the growth of transgenic plants for Ͼ10 successive years on the same field has not increased the measurable level and spectrum of antibiotic resistance in the cultivable soil bacterial fraction, thus excluding any cumulative effect related to the constant release of transgenic plant DNA. The observed multiantibiotic resistance patterns of soil communities has been reported with bacteria resisting up to eight antibiotics belonging to various other antibiotic families [START_REF] Dcosta | Sampling the antibiotic resistome[END_REF][START_REF] Nwosu | Antibiotic resistance with particular reference to soil microorganisms[END_REF].

Diversity of blaTEM Genes in Bacterial

Isolates and PCR-Amplified DNA from Soils. Presence and putative diversity of the blaTEM genes in the 576 ampicillin and carbenicillin-resistant soil isolates was studied after genomic DNA was amplified by PCR. The expected 828-bp PCR product, which generates 295-and 553-bp RsaI cut fragments according to the published sequence (GenBank accession no. AY425988), were obtained for 505 of these 576 genomic DNA (87.7%), confirming that the use of the two antibiotics efficiently selected blaTEM gene harboring strains or that there is a natural prevalence of this gene among ampicillincarbenicillin-resistant soil bacteria. For the 71 other sequences, each digestion pattern was sequenced and did not correspond to a bla sequence (results not shown).

For soil isolates, 80 PCR products were sequenced (15 from T1, 10 from T2, 16 from C1, 11 from C2, 12 from P1, and 16 from P2). Ten were identical to the blaTEM116 sequence of the transgenic corn (four T1, two C2, one P1, and three P2 sequences). This specific sequence was detected in bacteria isolated from the transgenic and nontransgenic corn fields and from the prairie soil. Some of these bacteria were resistant to ceftriaxone, indicating that these bacteria contained other antibiotic resistance mechanisms in addition to that encoded by the blaTEM116 gene or that its expression in soil bacteria increased the antibiotic resistance spectrum to third-generation cephalosporins. The 70 other PCR products had one nucleotide different from the blaTEM116 sequence (nucleotide 232, where G replaces A), which would substitute an isoleucin by a valin residue (neutral evolution) to provide a bla sequence used in cloning vectors (GenBank accession nos. X56095, L42764, AJ299425, and AJ431686) and encoding ampicillin resistance [START_REF] Smits | New alkane-responsive expression vectors for Escherichia coli and Pseudomonas[END_REF]. Several PCR products yielded both A and G at position 232. Their cloning and sequencing revealed that some soil isolates carried at least two blaTEM genes. This agrees with data demonstrating that clinical isolates can produce as many as five distinct betalactamases [START_REF] Bush | New beta-lactamases in gram-negative bacteria: Diversity and impact on the selection of antimicrobial therapy[END_REF]. The observation of only two types of blaTEM gene sequences in soil isolates might demonstrate the restricted soil diversity compared with clinical environments or that the high ampicillin and carbenicillin concentration and the culture medium used strongly selected genes and/or bacteria. To test these hypotheses, we first conducted the analysis of 16S rDNA sequences of 16 of these isolates (4 strains from T soil, 6 strains from C soil, and 6 strains for P soil). The 16S rDNA sequences classified isolates as belonging to only two genera, 15 as Pseudomonas genus (99% similarity) and 1 as Stenotrophomonas sp. (99% similarity).

Because cultivable bacteria represented Ͻ1% of total bacteria, diversity of blaTEM genes in soil bacterial community was further investigated by a culture-independent strategy. Bacterial metagenomic DNA was extracted from soil samples and submitted to PCR, using primers bla1 and bla2 (SI Table 2). PCR products were cloned, and 178 inserts were selected on the basis of RsaI digestion pattern were sequenced (GenBank accession nos. EF514028-EF514205). This included 54, 56, and 68 PCR products from transgenic corn (named Bt1 to Bt54), nontransgenic corn (named M1 to M56), and prairie (named S1 to S68) soils, respectively. A group of 10 DNA inserts that originated from transgenic (five Bt sequences) and prairie (five S sequences) soil DNA exhibited the blaTEM116 gene sequence. A khi2 test, performed to compare the distribution of the bla-TEM116 gene in the three soils demonstrated the absence of a transgenic plant effect (P Ͻ 0.10), thus confirming previous isolate-based data about its natural prevalence in environmental bacteria. In addition, this blaTEM116 gene was detected in DNA from another prairie soil (La Batie-Divisin, France) located 400 km away from the transgenic field, thus eliminating any possibility of contamination by the Bt176 plant DNA and reinforcing the potentially ubiquitous occurrence of this gene in prairie and agricultural soils. This result would justify a posteriori the choice of the blaTEM116 gene in the transgenic plant construction strategy, because it might have been already common in environmental bacteria and, thus, would limit the impact of a putative transfer from the transgenic plant to microbial biota. In addition to the blaTEM116 sequence, 153 different blaTEM nucleotide sequences were found in the PCR-amplified bacterial DNA, indicating a particularly high polymorphism level of this family of genes and confirming the interest in using a direct approach to avoid biases related to cultivation and selection methods. Polymorphism in the blaTEM genes was already noticed for clinical isolates with Ͼ150 variants [START_REF] Jacoby | Beta-lactamase nomenclature[END_REF]. A phylogenetic relationship was established by using environmental and National Center for Biotechnology Information database sequences aligned between nucleotides 28 and 845 of blaTEM116 gene, corresponding to nucleotides 237 to 1054 of blaTEM1 gene, using the amino acid residue numeration as established in ref. [START_REF] Ambler | A standard numbering scheme for the class A beta-lactamases[END_REF]. All of the blaTEM genes originating from soils grouped in a single cluster with only four sequences (Bt3, Bt8, Bt9, and Bt10) at the interface with sequences derived from medical facilities (Fig. 1) because of the presence of a G at position 458 (SI Fig. 3). Considering the low number of mismatches between blaTEM genes, the low bootstrap values, and the absence of a clear ancestor, the best representation was an unrooted phylogenic tree that demonstrates the separation between clinical and environmental strains except for the blaTEM116 and bla-TEM157 genes, which might have arisen from environmental origins. An alignment analysis clarified the differences between medical and environmental blaTEM sequences. Some bases are preferentially represented in medical genes and others are preferentially represented in environmental sequences, such as positions 442, 458, and 759 in blaTEM1 sequence (SI Fig. 3). Substitution of a G by a A at position 458 and substitution of a C by a T at position 759 are particularly specific of soil sequences, because no medical bla other than blaTEM116 and blaTEM157 possessed these substitutions. Such results cannot be due to PCR biases related to bla1 and bla2 primers that were designed to amplify all blaTEM and most bla genes but would confirm that beta-lactamases can easily evolve through point mutations. Furthermore, strains characterized by intermediate mutation frequencies have significantly higher levels of antibiotic resistance than strains with low and high mutation frequencies [START_REF] Denamur | Intermediate Mutation Frequencies Favor Evolution of Multidrug Resistance in Escherichia coli[END_REF]. Correlation between point mutation and polymorphism could explain why uncultured soil bacteria are a reservoir of antibiotic resistance genes [START_REF] Riesenfeld | Uncultured soil bacteria are a reservoir of new antibiotic resistance genes[END_REF].

BLA Mutation Analyses.

To complete the previous study on bla diversity, we studied the potential activity of the partial BLA sequences retrieved from soils by comparing them to known beta-lactamases. The TEM1 beta-lactamase, a plasmid-borne enzyme (GenBank accession no. AF309824), was the first en-zyme described in 1963 and is the most commonly detected in clinical bacteria [START_REF] Jacoby | Beta-lactamase nomenclature[END_REF][START_REF] Bastarrachea | On the origin of plasmid-borne, extended-spectrum, antibiotic resistance mutations in bacteria[END_REF]. Point mutations in plasmid bla genes expand the resistance spectrum of beta-lactamases even to the third cephalosporin generation, and most of the ESBLs from clinical isolates are mutants of TEM1 with one to four amino acid substitutions [START_REF] Jacoby | More extended-spectrum beta-lactamases. Antimicrob[END_REF][START_REF] Bastarrachea | On the origin of plasmid-borne, extended-spectrum, antibiotic resistance mutations in bacteria[END_REF].

In the present study, translation into a single protein was possible for only 139 of the 178 soil DNA PCR-amplified sequences. Other sequences contained internal stop codons or exhibited evidence that insertion or deletion events had modified the reading frame, and, therefore, these other sequences could be pseudogenes. A unique amino acid sequence was detected for 112 of the 178 analyzed fragments. Among the 139 sequences able to be translated, 35 sequences were 100% similar to BLA-TEM116 (15 Bt, 7 M, and 13 S sequences).

Comparisons to TEM1 were done by using the amino acid residues numeration as established in ref. [START_REF] Ambler | A standard numbering scheme for the class A beta-lactamases[END_REF] and according to www.lahey.org/studies. The environmental sequences included 120 different amino acid substitution positions (SI Table 3) with up to four amino acid residues modified. Some of them corresponded to sequence patterns detected in clinical environment (SI Table 4), such as mutations F16L, L21F, A42V, L51P, M69V, E104K, S130G, R164H, N175I, M182T, R204Q, G218E, E240K, N276D, and A280V. In addition, two specific mutations present in TEM116 and TEM157 and not in other medical sequences (V84I and A184V) were also detected in soil sequences. Mutation V84I was generally observed in soil sequences but was not present in Bt3, Bt8, Bt9, and Bt10 sequences. These sequences were at the interface between soil and clinical sequences in the phylogenetic tree (Fig. 1). Mutation A184V was detected in all soil sequences except in clone M27, presenting instead mutation A184I, and can be thus considered as the signature of BLA-TEM sequences in soil bacteria. Considering these data, BLA-TEM116 and BLA-TEM157 could originate from soil. Amino acid substitutions at positions located in close proximity to the active-site cavity, including positions 104, 164, 237, 238, and 239, are expected to open up novel enzyme-substrate interactions [START_REF] Collatz | Molecular evolution of ubiquitous betalactamases towards extended-spectrum enzymes active against newer beta-lactam antibiotics[END_REF]. Indeed, incorporation of a lysine at position 104, a serine at position 164 or 238, or the doublet threoninelysine at positions 237 and 239 conferred an increased substrate range [START_REF] Mabilat | Development of ''oligotyping'' for characterization and molecular epidemiology of TEM beta-lactamases in members of the family Enterobacteriaceae[END_REF][START_REF] Sougakoff | Plasmid-mediated resistance to third-generation cephalosporins caused by point mutations in TEM-type penicillinase genes[END_REF]. These substitutions were detected in five TEM beta-lactamases, e.g., TEM12 and TEM17 [START_REF] Jacoby | More extended-spectrum beta-lactamases. Antimicrob[END_REF][START_REF] Sougakoff | Plasmid-mediated resistance to third-generation cephalosporins caused by point mutations in TEM-type penicillinase genes[END_REF][START_REF] Collatz | Substitution of serine for arginine in position 162 of tem-type beta-lactamases extends the substrate profile of mutant enzymes, tem-7 and tem-101, to ceftazidime and aztreonam[END_REF][START_REF] Sougakoff | Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in enterobacteriaceae[END_REF]. Accordingly, mutations E104K and R164H detected in clones Bt17 and Bt10, respectively, should produce an ESBL phenotype. Because serine 70 is a key catalytic residue, its substitution by another amino acid is expected to have a strong effect on enzyme efficiency, including resistance loss to beta-lactams [START_REF] Herzberg | Bacterial resistance to beta-lactam antibiotics: crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.5 Å resolution[END_REF]. Clone M34 exhibits mutation S70G, which was never observed in clinical isolates. However, the PCR-based selection of blaTEM genes among soil DNA did not exclude those that no longer confer the resistance phenotype or pseudogenes.

l a T E M - 1 2 b l a T E M - 6 0 b l a T E M - 1 2 1 b l a T E M - 1 0 1 b l a T E M - 4
These data indicate that the numbers of possible mutational combination pathways that increase or maintain blaTEM enzyme activity without decreasing its stability are not unlimited. For instance, resistance to cefotaxime conferred by a blaTEM1 gene to an E. coli hypermutator strain submitted to directed evolution was only observed for the E104K/M182T/G238S mutation combination [START_REF] Orencia | Predicting the emergence of antibiotic resistance by directed evolution and structural analysis[END_REF], and DNA shuffling approaches revealed that antibiotic resistance level increased for alleles containing A18V, A42G, G92S, and R241H mutations [START_REF] Stemmer | Rapid evolution of a protein in vitro by DNA shuffling[END_REF]. All but one of these blaTEM mutations, which increase enzyme activity (E104K, M182T, A18P, A42V, and G92S) or substrate range (R241H), were detected in PCR-amplified soil DNA and in several clinically referenced bacteria. Thus, soil bacteria apparently have access to a wide range of adaptive alleles including some that guard against novel and synthetic antimicrobial molecules.

Bacterial Diversity in Soils. Bacterial diversity in the different soils used was studied to determine whether the similarity observed between bla genes in the three soils was due to the presence of similar microbial structures. A microarray approach was developed to analyze and compare microbial community composition of soils recovered from a prairie and traditional and transgenic corn fields. For this purpose, a 16S rRNA-based taxonomic microarray composed of 575 probes targeting bacterial diversity [START_REF] Sanguin | Development and validation of a 16S taxonomic microarray for characterizing and monitoring the rhizosphere bacterial community[END_REF] at various taxonomic levels from phyla to species was used. Principal component analysis (PCA) and k-means clustering of total bacterial community hybridization results (SI Fig. 4) showed significant differences between the three soils. However, the major was between the soils from corn fields (traditional and transgenic) and the prairie soil (43% of the total variability).

Changes in microbial communities due to the Bt trait have been shown to be smaller than changes due to soil type, plant growth stage, different (non-Bt) corn cultivar, and different crops (such as grass) [START_REF] Griffiths | A comparison of soil microbial community structure, protozoa and nematodes in field plots of conventional and genetically modified maize expressing the Bacillus thuringiens is CryIAb toxin[END_REF]. Other studies demonstrated that bacterial diversity as measured by metabolic profiling and molecular analysis of 16S rRNAs in nontransgenic and transgenic corn rhizospheres was not significantly different [START_REF] Fang | Bacterial diversity in rhizospheres of nontransgenic and transgenic corn[END_REF][START_REF] Saxena | Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil[END_REF]. Indeed, rhizospheric bacterial diversity is affected more by soil texture or growing season than by cultivation of transgenic varieties [START_REF] Fang | Bacterial diversity in rhizospheres of nontransgenic and transgenic corn[END_REF][START_REF] Schmalenberger | Genetic profiling of noncultivated bacteria from the rhizospheres of sugar beet (Beta vulgaris) reveal field and annual variability but no effect of a transgenic herbicide resistance[END_REF]. In contrast, genetically modified potatoes expressing antibacterial compounds were observed to induce different microbial activity rates and structures [START_REF] Rasche | Rhizosphere bacteria affected by transgenic potatoes with antibacterial activities compared with the effects of soil, wild-type potatoes, vegetation stage and pathogen exposure[END_REF]. However, in that study, the impact of the genetic modification cannot be correlated to the transgenic trait, but rather to the plant genotype as the traditional corn was not the isogenic variety of the Bt corn.

In conclusion, the similarity observed between bla genes in the three soils was not correlated to the microbial structures, because they were significantly different.

Hypothesis for a Transgenic Contamination. The continuously increasing antibiotic resistance in pathogenic (and maybe commensal) bacteria is one of the most striking demonstrations that human activity might accelerate evolutionary changes in other species, especially in highly adaptable organisms, such as bacteria. Ampicillin is one of the numerous examples of an antibiotic, which has rapidly lost its efficacy. Ampicillin was deployed in 1961, and resistance was first observed 15 years later [START_REF] Palumbi | Humans as the world's greatest evolutionary force[END_REF]. Antibiotic resistance genes detected on numerous plasmids today were apparently lacking from bacterial replicons isolated in the preantibiotic era [START_REF] Hughes | Conjugative plasmids in bacteria of the ''pre-antibiotic'' era[END_REF][START_REF] Nwosu | Antibiotic resistance with particular reference to soil microorganisms[END_REF]. These dramatic changes confirm the fundamental role of horizontal gene transfer (HGT) in the dispersion of these genes between bacteria. However, several fundamental questions remain about whether HGT occurs in hospitals or in the environment and the role of commensal bacteria, including those from soils as donors or recipients of DNA, thus justifying studies to evaluate the possible impact that transgenes could have as templates for further gene evolution.

Transgene transfer from plants to bacteria has been detected under greenhouse conditions with specifically selected donor and recipient organisms [START_REF] Kay | In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria[END_REF]. Despite these experiments that simulated the environment, plant bacteria HGT events remain undetected under field conditions. These limitations justified carrying out studies on soil samples originating from fields cultivated with the Bt176 corn plants for 10 successive years. This period could increase the probability that the gene would be transferred to soil bacteria and that the soil bacterial community would demonstrate some effect. In addition, this plant was one of the first transgenic plants to have been developed and will certainly remain as one of the rare GMPs used in which a resistance gene has been cloned with its own promoter. The prokaryotic origin of the blaTEM116 gene and presence of a bacterial promoter are two factors susceptible to facilitate its transfer to bacteria and to improve the fitness of the recombinants that would be able to express this gene.

The potential that the diversity of the blaTEM gene in metagenomic DNA could have evolved from the transgene was evaluated by comparing the divergence of soil blaTEM sequences with blaTEM116 gene. To further test this hypothesis, synonymous (silent, dS) and nonsynonymous (amino acid altering, dN) nucleotide substitutions were estimated [START_REF] Nei | Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions[END_REF]. dN/dS ratios Ͻ1, Ͼ1, or equal to 1 correspond to negative, positive, or neutral evolutions (related to selective pressure), respectively. A coding sequence is considered to be positively selected if the nonsynonymous substitution rate is significantly higher than the synonymous substitution rate. Among the 153 different metagenomic sequences tested, only five sequences (3.3%) were positively selected (SI Fig. 5). Two sequences arose from the transgenic corn soil (Bt10 and Bt24), and the three others came from the prairie soil (S46, S54, and S63). These genes could have evolved through environmental pressure leading to more adapted sequences. Negatively selected sequences include 36.6% of the cloned sequences leading to less adapted genes, which might not even be functional. Their activity could not be tested, because PCR primers used were designed inside the bla gene. However, this relatively high percentage might illustrate the general lack of selection pressure in these soils. Finally, there are 60.1% of sequences with the dN/dS ratio equal to 1, indicating that these genes are in the same evolution state as the transgenic sequence without any apparent additional selective pressure and that these genes were either well adapted to their environment or perhaps unnecessary for bacterial survival. Thus, the envi-ronmental selective pressure cannot be shown to influence the diversity of the blaTEM genes.

Conclusion

After the first cultivation of GMPs in the fields, questions have soon arisen about the risk of transfer of the transgenes to soil bacteria with possible consequences on the bacterial community structure and on the spread of antibiotic resistance to soil and clinical strains.

The successful transfer of transgene-borne antibiotic resistance genes to bacteria might be unavoidable according to a plethora of scientific data. This includes the long-term DNA persistence in soil, the heterogeneous soil structure favoring contact between DNA and bacteria, the prokaryotic origin of the plant transgene sequences that represent a specific risk for a facilitated integration in a bacterial genome by HGT as demonstrated under laboratory [START_REF] Gebhard | Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA[END_REF], and greenhouse conditions [START_REF] Kay | In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria[END_REF]. In addition, in the Bt176 event that we investigated here, the bacterial promoter was introduced concomitantly with the antibiotic resistance gene that would facilitate its expression in a potential recipient. Finally, these GMPs were cultured in the same field for 10 successive years, making this GMP-field combination particularly suitable to address gene transfer questions and impact on soil bacteria.

However, the detection of such events remains very difficult, and, in this study, we, like others before, did not detect any cellular or molecular evidence that the blaTEM116 gene from the Bt176 transgenic plant was transferred to bacteria. If such transfer events ever happened (although undetectable), they apparently remain without consequences on the soil bacterial community structure. In addition, the use of the sensitive microarray-based hybridization technique failed to detect any significant changes in soil bacteria that could be specifically related to the presence of the transgenic plants or to the expression of the transgene, including cry genes.

These results are probably partly due to the low frequency at which these transfer events happen and at a limited efficiency of the investigation protocols. However, they are also and mainly because these genes are already present in soil. Bacteria that would have acquired a blaTEM116 gene from the plant would not have a specific selective advantage relative to other resistant bacteria. Our results indicate that indigenous bacteria are already involved in evolutionary processes by point mutations and HGT as evidenced by the polymorphism of the bla gene in the various soils tested. This could indicate that soil is certainly a reservoir in which all bacteria, including clinical pathogens, can acquire the genetic determinants that could permit them to adapt rapidly to present and future antibiotics. These results confirm the interest of considering the evolutionary potential of bacteria when evaluating the impact of GMPs. Our data are sufficiently informative to conclude that the risk that antibioticresistant genes in GMPs can pose to commensal and clinical bacteria should be considered as almost null. This risk has to be neglected not because these genes cannot be transferred but because the plethora of genes already present in soil bacteria and the constant evolution to which they are subjected limit the impact that a newly acquired, yet identical, gene from a plant can have.

Experimental Procedures

Bacteria, Media, and Plasmids. Bacteria, media, and plasmids are described in SI Text. Soil Sampling. Soil was sampled (SI Text) by using geostatistical methods as described by Atteia et al. [START_REF] Atteia | Geostatistical analysis of soil contamination in the Swiss Jura[END_REF]. The soil was sieved at 2 mm. Some soils characteristics are showed in SI Table 5. Trace elements in soil were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS HP 4500; Agilent), using external calibration and internal standard correction (SI Table 6).

Isolation and Enumeration of Bacteria. Recovery of bacteria was performed by using a Nycodenz gradient separation method (SI Text) as described in ref. 43. To enumerate total and resistant soil cultivable bacteria, appropriate dilutions of bacterial suspension were spread onto tryptic soil agar (TSA) 1/10 medium alone or supplemented with ampicillin and carbenicillin. Colonies were counted after a 2-day incubation at 28°C. Colonies grown on selective plates were used to constitute a library of 96 ampicillin and carbenicillin-resistant isolates for two samples of each of the three soils. PCR Assays. The different primer sets, the targeted genes, and the sizes of the generated PCR products are described in SI Table 2. Detection of blaTEM-like genes was performed by amplifying an Ϸ828-bp fragment with primers bla1 and bla2, whose sequences are complementary to a conserved portion of the blaTEM gene sequences. Amplifications of blaTEM genes from soil isolates were first performed in a final volume of 20 l (SI Text). Samples presenting expected sizes were selected for a new PCR in a final volume of 100 l for sequencing.

Antibiotics

Amplifications of blaTEM sequences from metagenomic DNA (SI Text) were performed on 5 l of extracted DNA in a final volume of 200 l in presence of 1 g of T4 gene 32 protein (Roche) and 1 unit of titanium Taq polymerase (Clontech). After purification on 1% (wt/vol) agarose gel, amplified DNA was cloned into vector TOPO-TA and transformed into E. coli TOP10. Cloned metagenomic blaTEM sequences were checked by PCR with primers M13F and M13R (provided in the cloning kit) on 2 l of liquid culture of transformed E. coli strains with the Taq polymerase from Invitrogen in a final volume of 20 l.

The 16S rDNA genes of ampicillin-resistant isolates and of metagenomic DNA were amplified by using the primers pA and pHЈ (SI Table 2 and SI Text). Amplified DNA was then loaded on a 0.8% (wt/vol) agarose gel, and excised bands were purified by using the QIAquick Gel MiniElute Extraction kit (Qiagen).

Microarray Assays. Fluorescence labeling of 16S rRNA PCR products from bacterial communities, manufacturing and processing of the 16S rRNA-based microarrays, hybridization, scanning, and data analysis of microarrays were performed as described in refs. 30 and 44. Comparisons of microarray data were carried out by PCA and k-means clustering, using the package ade4 and stats for R (www.r-project.org), respectively. PCA was performed by using the covariance matrix and k-means clustering the algorithm of Hartigan and Wong [START_REF] Hartigan | A K-means clustering algorithm[END_REF]. For each soil sample, two hybridization replicates were done. DNA Sequencing. The nucleotide sequences of blaTEM and 16S rDNA were determined from soil isolates by direct sequencing of the PCR products after column purification performed by the sequencing society (Genoscreen, Lille, France or Genome Express, Meylan, France). The nucleotide sequence of blaTEM was also obtained from soil metagenomic DNA by sequencing cloned PCR products with primers M13F/M13R.

Sequence and Phylogenetic Analyses. Sequences were submitted to the National Center for Biotechnology Information data bank and compared by using the BLASTN and BLASTX algorithm [START_REF] Altschul | Basic local alignment search tool[END_REF]. On the basis of the results of the database analyses, bla sequences were aligned in between and with the bla sequence of plasmid pCIB4431, using ClustalX software, Version 1.64b. Phylogenetic reconstructions were generated with three different analytical methods, maximum parsimony, maximum likelihood, and neighbor-joining, using the Phylowin software package [START_REF] Galtier | SEAVIEW and PHYLOWIN: Two graphic tools for sequence alignment and molecular phylogeny[END_REF], and visualized with NjPLOT [START_REF] Perriere | WWW-query: An on-line retrieval system for biological sequence banks[END_REF]. The dN/dS ratio was calculated after the clustalX alignment (49) between DNA sequences with the SNAP software [START_REF] Nei | Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions[END_REF].

Chimeric 16S rRNA gene sequences were checked by using the chimera detection program Pintail version 0.33 (available at www.cardiff.ac.uk/biosi/ research/biosoft/pintail/pintail.html). Data management and computation were performed with Microsoft Excel software.

Fig. 1 .

 1 Fig.1. Phylogenic relationship of blaTEM sequences isolated from medical origin and amplified from a transgenic corn field (Bt), a traditional corn field (M), and a prairie soil (S). Green shading identifies blaTEM sequences from the transgenic corn field. Black shading identifies blaTEM sequences from other origins. Red shading identifies the two blaTEM medical sequences that might originate from the environment.

  -Resistance Assays. The following beta-lactam antibiotics were used in this study: ampicillin, carbenicillin, amoxicillin, penicillin G, oxacillin, cephalothin, cefamandole, and ceftriaxone. Details are provided in SI Text. DNA Manipulations. DNA extraction and cloning step are described in SI Text.

͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800072105 Demane `che et al.

͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800072105 Demane `che et al.

͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800072105 Demane `che et al.

ACKNOWLEDGMENTS.

We thank G. Grundmann for fruitful discussions with the microarray approach. This work was supported by the Programme National de Recherches sur les Organismes Ge ´ne ´tiquement Modifie ´s from the Agence Nationale de la Recherche project Ploben Grant ANR-05-POGM-004-01 and Agence Franc ¸aise de Se ´curite ´Sanitaire de l'Environnement et du Travail project AntiReSol Grant EST-2006/1/44.


Whole rosette

Rosetteleaf blades

. Hydrostatic hydraulic conductivity (K) of whole rosettes (whole rosette) or of rosettes with all leaf blades excised (rosette -leaf blades). K was measured in rosettes excised during day time from plants grown under a normal photoperiodic regime. K was successively measured on whole rosettes and after excising all blades at their basis. Pooled data from two independent cultures (n = 8 plants). Values are mean ± SE.


I A K Youssao

P C Tobada

B G Koutinhouin

M Dahouda

N D Idrissou

G A Bonou

U P Tougan

S Ahounou

V Yapi-Gnaoré

B Kayang

X Rognon

M Tixier- Boichard

Phenotypic characterisation and molecular polymorphism of indigenous poultry populations of the species Gallus gallus of Savannah and Forest ecotypes of Benin

Keywords: Body weight, plumage colour, molecular polymorphism, local chickens, Benin

Phenotypic characterisation and molecular polymorphism of indigenous poultry populations of the species Gallus gallus of Savannah and Forest ecotypes of Benin

I.

INTRODUCTION

The genetic resources of poultry in West Africa are mainly represented by domestic local chickens (Gallus *Corresponding author. E-mail: issaka.youssao@epac.uac.bj. Tél: 00 229 97 91 20 74 / 95 28 59 88.

gallus domesticus), guinea fowls (Numida meleagris) and ducks (Cairina sp.). These local avian species are bred under traditional breeding system. The animals are bred for meat as well as for eggs; the divagation system is the rule and different species can be found intermingled. This type of breeding is described as family poultry farming and still constitutes the main part of poultry rearing in sub-Saharan Africa. The size of the national poultry livestock of Benin was estimated in 2005 to be 16 690 000 heads of which 81.29% were local chickens (Bebay, 2006) for 6 000 000 inhabitants. In spite of this numerical importance, the local production of poultry meat is below the needs expressed by the consumers and this deficit has led to increased imports as well as to programmes of introduction of exotic breeds in Benin.

In 10 years, the quantum amount of poultry meat imported passed from 19361 tons metric in 1995 to 49634 tons metric in 2005 (FAO, 2008). Apart from this meat dependence toward foreign countries, certain diseases, which did not exist on the African continent, are present today in the majority of the West African countries in an endemic or epidemic form. Indeed, the Gumboro disease, the Marek's disease and the Newcastle disease are now present in Benin. Recently, the highly pathogenic avian influenza (HPIA) appeared in Africa in 2006(Bebay, 2006) and in 2007 in Benin (Direction de l'Elevage, 2007) after having caused significant economic losses in several countries of Asia and Europe.

In addition, the crossings between large format cockerels of exotic breeds and local hens (small size) were realized in the majority of the countries of West Africa to increase body weight of progeny (FAO, 2004). These anarchistic crossings failed most often because of the lack of follow-up or the lack of adapted husbandry techniques. In the majority of cases, the females resulting from these crossings do not have, unfortunately, a good maternal ability (incubation, hatching, chicks' follow-up) (FAO, 2004).

From all that proceeds, it comes out that the local production must be encouraged to reduce imports and limit the spreading of new diseases. The improvement of this production must start with a better knowledge of the endogenous genetic resources, in order to utilize them for the development of the poultry sector in Africa. The purpose of this study was to characterise two ecotypes of Gallus gallus (the indigenous poultry populations of the savannah and the forest zones of Benin) in terms of morphobiometric, phaneroptic and genetic diversity.

MATERIALS AND METHODS

Areas of study

The study on the phenotypic characterisation of local chickens of the species Gallus gallus was carried out from October 2005 to May 2006 in the Forest and Savannah areas of Benin. The savannah area covers the departments of Borgou and Donga, while the forest zone includes the departments of Atlantic, Littoral, Mono and Couffo. In the department of Borgou, the survey was carried out in the Communes of Parakou, N'Dali, Kalale and Bembereke. In the Department of Donga, the investigations were done in the Communes of Djougou and Copargo. In the Atlantic and Littotal, the area of study was the Communes of Abomey-Calavi, Allada and Cotonou. In the Departments of Mono-Couffo, the area of study was the Communes of Lokossa and Aplahoue. The farmers identified for these investigations had a herd of at least 10 chickens during the survey. These Communes were retained in order to have a good spatial distribution of the local chicken populations in each area. Figure 1 locates the surveyed Communes.

The two departments of Savannah are characterized by a climate of Sudanese type. This climate is marked by one dry season (November to May) and one rainy season (June to October) with a yearly rainfall varying from 900 to 1000 mm and an average monthly temperature which varies between 26 and 28°C [START_REF] Asecna | Première évaluation de la structure et de l'importance du secteur avicole commercial et familial en Afrique de l'Ouest. Synthèse des rapports nationaux[END_REF].

The Forest area of Benin has a subequatorial climate with 2 dry seasons and 2 rainy seasons. The rainfall mode is bimodal with a large and a small season of rains separated by dry seasons. The seasons of rains go from April to July and October to November. Average rainfall was 1200 mm per year, from 2000 to 2006 [START_REF] Asecna | Première évaluation de la structure et de l'importance du secteur avicole commercial et familial en Afrique de l'Ouest. Synthèse des rapports nationaux[END_REF]. The average monthly temperature varies between 27 and 31°C and the relative humidity fluctuates between 65% from January to March and 97% from June to July [START_REF] Asecna | Première évaluation de la structure et de l'importance du secteur avicole commercial et familial en Afrique de l'Ouest. Synthèse des rapports nationaux[END_REF].

Animals

The study on the phenotypic characterisation of the local chicken populations of the species G. gallus was carried out on 642 adult chickens, of which 326 were from the Forest ecotype and 316 from the Savannah ecotype. All the birds (both sex) used in the study were seven months of age at least. Females should have laid eggs once at least. According to the farmer's declarations, the experimental birds came from farms that had no prior contact with exotic breeds. The breeding system can be characterized as extensive with random mating.

The blood collection for the molecular typing was performed on 121 indigenous chickens raised at the Experimental Farm of the "Ecole Polytechnique d'Abomey-Calavi", of which 61 samples originate from the Savannah and 60 from the Forest area. The blood was sampled from birds that were used for the phenotypic study.

Data-collection

Production system

A survey was carried out by direct discussion with the farmer. Questions were organized in 4 sections: I. description of the farmer and the flock: farmer's education, age and sex, motivations for rearing local chickens, species raised and main productions; II. origin of the flock: number of founder animals, how where they obtained (purchase, gift, inheritance ); III. management of the flock: individual identification, flock size, feeding system, mating plan, choice of breeding animals, incubation system, final use of the animals; IV. health issues: mortality, time of occurrence and main causes of death, treatments.

Answers were collected using a pre-formatted Excel file, answer frequencies were calculated for each question and area of study. Possible differences between Savannah and Forest areas were analyzed with a Chi-square test.

Morphological and phenotypic traits

Body weight of each subject described was measured using a mechanical balance of 2.5 kg of capacity with a precision of 20 g. The following body measures were then recorded using one ribbon meter: breast circumference, body length, shank length. The phaneroptic characters were determined by direct observation on each animal according to the nomenclature described by [START_REF] Coquerelle | Les poules: diversité génétique visible. INRA, p. 181. Direction de l'élevage[END_REF]. These criteria were: plumage colour and feather patterns, feathers' structure, feather distribution, shank colour and eyes colour.

Colour variants are controlled by major genes, where generally, one mutant allele is known as compared to the wild type. Only the E locus (black extension) includes several alleles which are associated to a various proportion of black pigments on the body. Since interactions between colour mutations are frequent, it is not always possible to identify the genotype pertaining to one locus for all individuals. Thus, the analysis was done on phenotypic frequency, which does not strictly correspond to an allelic frequency.

Data collection was completed by taking pictures for all animals under study. These photographs were brought to laboratory and were submitted to a second description in order to check the accuracy of the phaneroptic characters recorded on the site of study. The identification of major mutations with visible effects on the phaneroptic characters of local chickens was made according to [START_REF] Coquerelle | Les poules: diversité génétique visible. INRA, p. 181. Direction de l'élevage[END_REF] and Bantam Club Français (2006).

DNA extraction and genotyping

Blood sampling was carried out at the level of the wing vein using a needle of 19G and preserved in a dry tube with heparin and of a capacity of 2 ml. Once conditioned, these tubes were preserved at -20°C in a refrigerated container of dry ice and were transported to the University of Legon in Ghana for the DNA extraction.

The DNA was obtained from 10 µl of the sampled blood, by an extraction with the Qiagen QIAamp kit in the laboratory of the Department of Animal Sciences of the Agriculture and Food Faculty of the University of Legon in Ghana. The genotyping was realized by the GIE LABOGENA (Jouy-en-Josas, France), using 22 microsatellite markers previously selected for the AvianDiv European project [START_REF] Hillel | Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools[END_REF]. Briefly, the genotyping procedure consisted in PCR amplification with fluorescent primers followed by migration on a capillary sequencer (ABI PRISM 3100 Genetic Analyzer, Applied Biosystems) as described by [START_REF] Berthouly | Using molecular and multivariate methods to study the genetic diversity on local European and Asian chickens breeds[END_REF].

Statistical analysis

Morphological and phenotypic traits analysis

The statistical analysis was carried out with SAS software (Statistical Analysis System, 1989) and several procedures were used. The procedure Proc freq was used to determine the phaneroptic frequencies. These frequencies were calculated by ecotype (Savannah vs Forest) and were compared by the test of Chisquare.

A linear model was adjusted to the data of body measures and live weight. This model included the fixed effects of the ecotype and the sex. The interaction between the ecotype and the sex was taken into account in the model of variance analysis, as shown below:

Yijk = µ + Ei + Sj + ESij + eijk
With -Yijk: the weight or a body measure of the k th hen of ecotype i and sex j; -µ: general mean; -Ei: fixed effect of the ecotype i (Savannah and Forest); -Sj: fixed effect of the sex j (female and male). -ESij: interaction between ecotype i and sex j; -eijk: residual error.

The procedure of the generalized linear models (Proc GLM) was used for the analysis of the variance and the means were then calculated and compared by a t-test.

Molecular analysis

Allelic frequencies, observed heterozygoty (Ho) and mean unbiased estimates of gene diversity (He) [START_REF] Nei | Estimation of average heterozygosity and genetic distance from a small number of individuals[END_REF], the average number of alleles (ANA) per locus and the average number of efficient alleles (Ae) per locus were calculated using the software GENETIX 4.4 [START_REF] Belkhir | GENETIX, Software under Windows TM for the Genetics of the Populations[END_REF]. These parameters describe the importance of polymorphism within the populations. The GENEPOP 4.0 software was used to determine departure from Hardy-Weinberg equilibrium [START_REF] Rousset | GENEPOP'007: a complete re-implementation of the GENEPOP software for Windows and Linux[END_REF], F-Statistics and pairwise genetic differentiation [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF][START_REF] Goudet | FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9[END_REF]. The multiple significance test was corrected using sequential Bonferroni procedure [START_REF] Rice | Analysing tables of statistical tests[END_REF]. The assignment of an individual to its supposed population, individuals were clustered according to the DAS [START_REF] Jin | Estimation of genetic distance and coefficient of gene diversity from single-probe multilocus DNA fingerprinting data[END_REF] genetic distance matrix, using the neighbor-joining [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF] method. Distance matrix and phenogram were computed using POPULATION 1.2.28 and TREEPLOT 0.7

RESULTS

Production system

The farmer's motivation for local chickens appeared to focus rather on meat quality and robustness of chickens in the Savannah, whereas tradition was the most frequent motivation declared in the Forest area. The owner of the animals was generally a farmer in the Savannah, but it could be craftsmen in the Forest. The surface of cultivated land per farmer was also greater on average in the Savannah. Women appeared more often to be in charge of poultry keeping in the Forest than in the Savannah.

The local chicken production system was familial and extensive in both areas. Only one parent was known (the hen) and rarely both (the male and the female parent).

The incubation was natural according to all poultry breeders and less than 1% practiced occasionally the artificial incubation. The reproduction was not seasonal in the two areas and there was no evidence of a peak period for laying throughout. The average number of reproductive cocks was 3.42 in the Savannah and 2.95 in the Forest. The number of reproductive hens was more important (P < 0.001) in the Savannah (9.18) than in the Forest (5.45). The choice of the breeding animals (male and female) was made essentially at random in the two regions; it was very difficult for farmers to express any breeding objective, which could be considered in a breeding program involving selection within local popuations or crossing with exotic breeds or commercial lines.

The divagation was practiced by 92.11% of breeders in the Savannah of Benin and 94.12% of those of the Forest. The hen houses with frontage were rare and only used by 7.89% of breeders in the Savannah and 5.88 in the Forest; any permanent building was not found. The majority of the habitats were rudimentary and limited to a shelter used at night or during the rainy season; it did not comply with standard husbandry. The majority of the chicken breeders fed their animals by giving some cereals; the proportion of those giving some kitchen residues was more important (P < 0.001) in the Forest (76.47%) than the Savannah (9.65%) and the same tendency were observed on the use of termite in the poultry feeding.

In the Savannah as in the Forest, 85 to 88% of the breeders declared that majority of mortalities were observed between hatching and weaning. The causes of mortality varied with the age of animals and the region and could be grouped in four classes: accident, predator, disease and season. According to the interviewed breeders, the illnesses constituted the main reason of mortalities before the weaning, followed by the season, the predators and the accidents. It comes out from this study that the death rate was higher in the dry season than in the rainy season in the Savannah region of Benin whatever the age of the birds, whereas in the Forest region of the country, mortalities were noticed more often during the major rainy season. Veterinary treatments or vaccinations were more frequent in the Savannah than in the Forest. The reason for selling an animal was mainly the need for meat in the Savannah, whereas it was the need of money in the Forest. Males are sold in priority, according to the market price in the Forest, whereas price was generally negotiated on an individual basis in the Savannah.

Live weight and body measures

The live weight and body measures of the animals of Savannah ecotype were significantly higher (P < 0.001) than those of the animals from the Forest area (Table 1). The difference between the average live weight of chickens of Savannah ecotype and those of the Forest was 251 g, in favour of chickens of Savannah ecotype (P < 0.001). The live weight and body measures varied according to the sex (P < 0.001) with higher values in males.

The interaction between sex and area was significant (P < 0.01). The most important differences were obtained on live weight and tarsus length. Sexual dimorphism represented 20.6% of the female weight in the Savannah and 23.7% in the Forest, thus the dimorphism was slightly more noticeable in the Forest. For the tarsus, the difference between sexes was 26.9% of the value of the females in the Savannah and 16.8% in the Forest; thus, a more important dimorphism was observed for the tarsus length in the Savannah than in the Forest. In general, the females of the Savannah ecotype had live weights and body measures higher than those of the Forest (P < 0.01) and the same difference was observed in males. No significant difference was observed between females from the Savannah and males from the Forest for live weight, breast circumference and body length (Table 1).

Study of the phenotype

Plumage colour

A total of 22 colours of plumage was identified in the Forest and 18 in the Savannah. The identification was not possible for 6.96% of the animals in Savannah vs 6.24% in the Forest. Somen colours of feather were found in both sexes (self-coloured white, dirty white, ermined white, red ermined, fawn, black and grey), others only in males (golden black, coppered black, silver black, red with black tail and white with black tail) or in females (golden salmon, silver salmon, golden wheaten, wheaten and partridge colour). Table 2 shows the frequencies of observation of these various colours of feather per area. In the two ecotypes, the most frequent colours were the black and the white. The self-coloured white, black, coppered black and the golden wheaten dominated in the Savannah ecotype whereas the ermined, fawn, grey, white were found in the Forest ecotype (P < 0.05). The white with black tail, the golden salmon, the golden partridge with salmon breast and the silver partridge with salmon breast were exclusively met in the Forest ecotype. Feather colour and the corresponding mutations are described in Table 2 and the frequencies of the observed phenotypes are given in Table 3. The mutations I and E were the most frequent, as the wild phenotype for the shank colour (grey leg) with at least 45% of the chickens of the Savannah ecotype, while mutations E (black extended), EB ## : have nether been identified before; NS: not significant; *: P < 0.05; *: P < 0.01; **: P < 0.001.

(brown) and ID (white leg) were the most observed in chickens of the Forestern ecotype, with a respective frequency of 31, 33 and 38%.

Feather pattern

Feather pattern of 386 chickens of which 192 chicken of the forest ecotype and 194 of the savannah ecotype were recorded. The frequency of the feather patterns of local chicken population are shown in Table 4. The lack of feather patterns was higher (P < 0.001) in the Forest than in Savannah (79.17% vs 42.27). On the whole, 7 drawings of plumage were identified: the cuckoo with black foundation, the golden cuckoo, the mottled salmon, mottled black, the silver autosomal barring, the multiple lacing and the thousand-flowers. Some feathers exhibited several drawings at the same time, with a low frequency for both ecotypes. The mottling phenotype, either black or salmon or thousand-flowers, was more frequent in the Savannah than in the Forest, whereas the cuckoo pattern was slightly more frequent in the Forest than in Savannah (6.25 vs 1.03%). Apart from these drawings of plumage, the autosomal barring was rarely observed in the two areas and the multiple lacing was not observed in the Savannah, while it was rare in the Forest.

Shank colour

The colour of the legs was identified on 326 birds from the Savannah and 316 of the Forest. Table 5 presents the various colours encountered with the corresponding mutation and their frequency per area. Among these colours, the white legs (mutation at the locus ID) presented the highest frequency of occurrence and were significantly more numerous (P < 0.001) in the Forest (49.1%) than in Savannah (30.9%) area. This colour was followed in term of frequency, by the grey (wild type on the locus ID) and the black (locus E) which were more frequent in the Savannah than in the Forest (P < 0.05) with a difference of almost 10% for each of the two colours. Finally, yellow legs (locus W) were found in a small proportion in the two areas without any significant difference.

Eyes colour

The orange (wild-type colour) was met with a higher frequency in the Forest than in Savannah (72.7 vs 37.6%). It was followed respectively by the Brown (locus Br) and the red, largely more represented in the Savannah (37.6 and 24.2%) than in the Forest (17.5 and 9.1%). Finally the pearl eyes were rare in the Forest (0.65%) and the grey eyes (locus FM) were rare in Savannah (0.61%).

Earlobe colour

Six earlobe colours were observed. The white earlobe was the most represented with a proportion of 60.8% in the Forest ecotype against 45.1% in the Savannah ecotype. It was followed by the bluish white earlobe, more represented in the Savannah, with a difference of 10% as compared to the Forest. The same tendency was observed between the two areas for the sandy earlobes which presented a difference in percentage of 8.8. The red earlobes came then with 12.7% in the Forest against 5.5% in the Savannah. The grey earlobes were found in both areas at a low frequency (7.3 in the Savannah and 3.8 in the Forest). Finally the yellow earlobes were only observed in the Savannah with a very low proportion of 0.61%.

Rare mutations

H locus) were found at a frequency of 1.2% in the Savannah and 1.86% in the Forest. The pea comb mutation (P locus) was rare in both areas (0.61% in the Savannah and 1.3% in the Forest).

The Single comb without micro crest was also found in the Forest (0.63%) area. No significant difference was observed for the spur proportion between males of the Savannah ecotype (35.63 %) and males of the Forest ecotype (27.37%). Of the 316 animals of Savannah ecotype chicken, 7 cases of polydactyl were recorded whereas no polydactyl chickens were found within the 326 chicken of the forest ecotype recorded.

Molecular polymorphism

The average allele number per population and per locus was 5.73 for the Savannah and 5.91 for the Forest (Table 6). The average efficient allele number was 2.18 and 2.24, respectively for chickens of the Savannah and those of the Forest. The average effective allele number was definitely smaller than the total number of alleles, which reveals a heterogeneous distribution of the allelic frequencies.

The heterozygoty rate (observed and expected) was rather high (Table 6) and the FIS values ranged from -0.024 (Forest) to 0.012 (Savannah). After Bonferroni correction, only one locus exhibited significant deficit (locus MCW330, P < 0.01) for the Forest sample and no heterozygote excess was found. Although a low F STvalue (0.009), both samples were found significantly differentiated (P < 0.001). Figure 2 showed the neighborjoining tree constructed from the pairwise distances between all the individuals. The forest and Savannah samples exhibited a partial overlapping pattern.

DISCUSSION

Live weight and body measures

The weight and body measures of Savannah chickens were significantly higher than those of Forest chickens. This difference could be due to the breeding system, the climate or the genetic background. The results of the survey suggested that farmers from the Savannah were more involved in agriculture and breeding than those in the Forest, for whom poultry keeping was more a cultural tradition. For instance, farmers from the Savannah used veterinary treatments more often and paid attention to the body weight of males in order to sell them better. Nevertheless, both areas showed the same divagation system and the lack of a declared selection objective.

Similar differences between Savannah and Forest have been found for barymetric measures of other animal species. Regarding ruminants, the zebus, located in the zone of Malanville in Benin, have weights and body measures higher than the Borgou breed, located in the Departments of Borgou, Donga and the "Collines" in Benin [START_REF] Domingo | Contribution à l'étude de la population bovine des Etats du Golfe du Bénin[END_REF][START_REF] Youssao | Ethnologie des animaux de production[END_REF]. In the same way, the Borgou breed has higher weight and body measures than the "lagunaire" breed located in the Forest zones of Benin. The same tendency is observed with other animal species (sheep, goat and others) [START_REF] Youssao | Ethnologie des animaux de production[END_REF].

The live weight and body measures of males were significantly higher than those of females. The same tendency was observed by [START_REF] Godonou | Le système de production du poulet local des élevages suivis par le Programme d'Appui au Développement de[END_REF] and [START_REF] Dossou | Caractérisation phénotypique des types génétiques de populations locales de volailles de l'espèce Gallus gallus dans la Commune d'Abomey-Calavi[END_REF] in local chickens. The sexual dimorphism of the chicken becomes in general more noticeable with advanced age. Thus, the result of a larger dimorphism for shank length in the Savannah is logic but the slightly lower dimorphism on live body weight in the Savannah is more surprising. However, body weight can be modified by variable nutritional conditions at the time of weighing, whereas the tarsus length is a more stable indicator of format of adult animals.

In the Wet Dense Forest with Bimodal Pluviometry of Cameroun, on the whole of the studied Provinces, the average body weight was 1535 g for cocks and 1220 g for hens [START_REF] Fotsa | Caractérisation Phénotypique des Populations de Poules Locales (Gallus Gallus) de la Zone Forestière Dense Humide à Pluviométrie Bimodale du Cameroun[END_REF]. Local chickens of the "High lands" of the West Cameroun, where the climate is of sudano-guinean type, exhibited an adult weight of 1676 g for males against 1278 g for the female (Keambou et al., 2008). However, the body measures carried out on the animals of the West by [START_REF] Keambou | Caractères morphologiques, mensurations corporelles et diversité phylogénétique de la poule locale (Gallus gallus) des hautes terres de l'Ouest Cameroun[END_REF] showed that the tarsus was shorter but thicker than those of the animals of the wet forest. Thus, local populations of Cameroun exhibited higher body weight and body measures than those of Forest Benin, where pluviometry is also bimodal or of Savannah Benin where the climate is of sudanese type. Thus, climatic differences cannot be the only reason for a lower body weight of local chickens in Forest Benin. Consequently, it would be interesting to compare on station the growth performance of chickens from each ecotype in the same conditions, in order to test whether the difference in live weight could be due to climate, to the breeding system or to the genetic background.

Morphological mutations

An important phenotypic diversity was observed for both ecotypes. It must be recalled that some phenotypes cannot be observed in the presence of another one; for example, it is not possible to know if a self-coloured white animal carries the CO mutation or a mutation of feather drawing because the white can be due to the C mutation which inhibits any colour. Generally, the high variability of plumage colour is considered as a consequence of domestication which increases the survival of mutant phenotypes. The maintenance of this high variability indicates the absence of selection on a preferred phenotype, showing that the population is not standardized [START_REF] Lauvergne | Indices de primarité de chèvres au Nord Cameroun et au Tchad. Revue[END_REF]. Furthermore, the cultural value of various plumage colours has already been reported for village chickens in Africa [START_REF] Gueye | Village egg and fowl meat production in Africa[END_REF][START_REF] Gueye | Employment an income generation through family poultry in low income food-deficit countries[END_REF][START_REF] Benabdeljelil | Prise en compte des préférences des éleveurs pour la caractérisation des populations locales de poulets au Maroc[END_REF].

The present data showed differences in frequency of some plumage colours and shank colours between the Savannah and the Forest ecotypes, but the corresponding mutations are not usually associated with a significant effect on body weight in temperate climate. The relationship between such frequency differences and geographical areas is not easily explained, but it seems to occur in other African countries [START_REF] Fotsa | Caractérisation des populations de poules locales (gallus gallus) au Cameroun[END_REF].

Indeed, this difference in distribution of the feather colours was also observed in two different agro-ecological zones in Senegal [START_REF] Missohou | Caractéristique morphologique des poulets du Sénégal[END_REF]. Thus, in Dahra (agro-ecological zone, with weak pluviometry of Senegal), the fawn (26%), the red (9%), the white and fawn (7.4%) and the golden red (7.2%) plumage colours were observed, while in Kolda (ecological zone with large pluviometry), the colours observed were the white (16.4 %), the ermined fawn (9.6%), the thousand-flowers (9.5 %) and the white and fawn (8.9%). By comparing the two studies, it comes out that the fawn and the white were most frequent respectively in the agro-ecological zones with strong and weak pluviometry of Senegal, whereas in Benin, the frequently encountered plumage in the Savannah ecotype were the self-coloured white and the black plumage and the fawn and black colours in the Forest ecotype. The high frequency of the white colour in the savannah area is attributable to their frequent use at the time of religious sacrifices. Following the declarations of the villagers, the high frequency of white chickens in the savannah area is mainly attributable to their preference for this colour for religious purposes. The price of white hens in the savannah zones is accordingly higher than that of multicolored hens. In Benin, the frequency of hens with uniform black plumage colour or those with black plumage background (mottled, coppered and golden), is higher as that reported by [START_REF] Missohou | Caractéristique morphologique des poulets du Sénégal[END_REF] in Senegal. On the other hand, the partridge colour and partridge with black background (silver, golden, silver with salmon breast, golden with salmon breast) presented the same proportions in Benin and in Senegal, regarding the agro ecological zones with weak pluviometry (Savannah Benin and Dahra with respective proportions of 10.13 and 8%) as well as those with large pluviometry (Forest Benin and Kolda with respective proportions of 13.67 and 13%). According to [START_REF] Pedersen | Traditional chicken in a communal area of Zimbabwe. A baseline survey on production and management[END_REF], in Zimbabwe, the colours fawn (26%), black (23%) and grey (23%) were found in the area of Sanyati (ecological zone with weak pluviometry).

According to [START_REF] Dossou | Caractérisation phénotypique des types génétiques de populations locales de volailles de l'espèce Gallus gallus dans la Commune d'Abomey-Calavi[END_REF] and [START_REF] Youssao | Genetic diversity of poultry the local populations of the species Gallus gallus in the South and the North of the Benin. 1st Conference of the UAC of Sciences, Cultures and Technologies, from the 25[END_REF], the ermined fawn, the fawn, the black and the dirty white were the most common colours observed within the 311 chickens recorded in the Commune of Abomey-Calavi with frequencies ranging from 8.31 to 10.30%. These results are consistent with the observations made in the Forest area Youssao et al 389 of the present study. According to [START_REF] Akouango | Phénotypes et performances d'élevage chez les populations locales de volailles de genre Gallus gallus au Congo Brazzaville[END_REF], the various phenotypes met in Congo are the ermined fawn (37.25%), the negro black (16.7%), the golden (15.16%), the fawn (10.16%), the mottled (7.84%), the silver wheaten (7.6%) and the Pile (5.3%). By comparing the results of [START_REF] Akouango | Phénotypes et performances d'élevage chez les populations locales de volailles de genre Gallus gallus au Congo Brazzaville[END_REF] with those of this study, it comes out that the fawn and the Black (self-coloured or with black foundation) are the most dominant. Apart from a mixed type colour, all the plumage colours observed by [START_REF] Akouango | Phénotypes et performances d'élevage chez les populations locales de volailles de genre Gallus gallus au Congo Brazzaville[END_REF] were present in Benin. On the other hand, some colours such as: the silver, the dirty white, the silver or golden black and the salmon were not described by [START_REF] Akouango | Phénotypes et performances d'élevage chez les populations locales de volailles de genre Gallus gallus au Congo Brazzaville[END_REF].

Report by [START_REF] Dossou | Caractérisation phénotypique des types génétiques de populations locales de volailles de l'espèce Gallus gallus dans la Commune d'Abomey-Calavi[END_REF] in the Commune regarding shank colour, a high proportion of white legs (44.8%) were previously observed of Abomey-Calavi. In Senegal, the white was also dominating with a proportion of 56.5% [START_REF] Missohou | Caractéristique morphologique des poulets du Sénégal[END_REF]. Although yellow legs were found in a small proportion in Benin, it was more or less popular in Cameroun with proportions varying from 31.38 to 37.89% according to the area (Center, South and East of Cameroun) [START_REF] Fotsa | Caractérisation Phénotypique des Populations de Poules Locales (Gallus Gallus) de la Zone Forestière Dense Humide à Pluviométrie Bimodale du Cameroun[END_REF] and from 39 to 46% according to the zones (rural and urban) within area of Cameroun (Keambou et al., 2008). The strongest proportion of yellow legs observed in urban zone could be due to the introgression of exotic breeds, such as Rhode Island Red or White Leghorns, which have yellow legs. In the present study, the animals used are all chosen in farms where no crossing was declared. In Senegal, according to [START_REF] Missohou | Caractéristique morphologique des poulets du Sénégal[END_REF], the white earlobes were also frequent (78%). On the other hand, in Cameroun, the dominant colour of the earlobes were red (40.3%) and white (35.6%) (Keambou et al., 2008).

Molecular polymorphism

The indigenous chicken populations of the ecological areas analyzed in this study showed a higher level of genetic diversity than what is generally described for commercial lines [START_REF] Hillel | Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools[END_REF][START_REF] Granevitze | Genetic diversity within chicken populations from different continents and management histories[END_REF]. This result is consistent with what is generally observed in populations raised without a selection programme in contrast to what can be observed in commercial lines or standardised breeds [START_REF] Wimmers | Genetic distinctness of African, Asian and South American local chickens[END_REF][START_REF] Berthouly | Using molecular and multivariate methods to study the genetic diversity on local European and Asian chickens breeds[END_REF][START_REF] Muchadeyi | Absence of population substructuring in Zimbabwe chicken ecotypes inferred using microsatellites analysis[END_REF]. Only one case of a deficiency in heterozygotes was observed. This situation is relatively different from what can be observed in many local populations [START_REF] Berthouly | Using molecular and multivariate methods to study the genetic diversity on local European and Asian chickens breeds[END_REF]Granewitz et al., 2007;[START_REF] Muchadeyi | Absence of population substructuring in Zimbabwe chicken ecotypes inferred using microsatellites analysis[END_REF] where a Wahlund effect takes place, due to a fragmentation of the population in small sub-units having few or no exchanges of reproducers between them. With regard to morphometric and phaneroptics traits, the results from this study reveal some significant differences between the Savannah and the Forest chicken popula-tions in Benin, suggesting that their genetic background was partly different. Similar results were also reported by [START_REF] Bonou | Genetic diversity of indigenous chicken population of the species Gallus gallus in the North and the South of the Benin[END_REF] on phenotypic characteristics of local chicken of Benin.

Conclusion

The present study showed that the local chicken populations of the species G. gallus of the two great climatic areas of Benin have a remarkable heterogeneity of their phenotypic characters. The distribution of plumage colour showed some differences between the Savannah and the Forest Benin, which probably reveal effects of climate as well as cultural preferences of the breeders. Although the Savannah and the Forest populations did not differ greatly on the basis of molecular polymorphism, they exhibited marked differences in body weight. They are theoretically many causes of variation of body weight of chickens, which could be addressed in further complementary investigations. Thus, it would be interesting to compare the growth performance of the Savannah ecotype chickens to those of the Forest ecotype in a controlled environment in station in order to test whether the difference of the live weight between those two ecotypes chickens is due to the climate, the production system or the genetic background. Furthermore, since the growth performance of local chicken was quite low, performance testing will be necessary to assess the possibility of further improvement of local chickens. A global strategy should also take into account the improvement of the production system in village conditions.

Figure 1 .

 1 Figure 1. Areas of study.

Figure 2 .

 2 Figure 2. Neighbour-joining tree derived from DAS distances, depicting the genetic relationships of 121 local chickens belonging to local populations from forest (red line) and savannah (green line) samples. Savannah and Forest genetic distances were based on 22 microsatellite markers. § Green lines: Savannah ecotype ; § Red lines: Forest ecotype.

Table 1 .

 1 Effect of ecotype, sex and the interaction between ecotype and sex on weight and body measurements of local chicken populations of the species G. gallus in Benin.

		Area		Sex		Interaction between area and sex			Significativity
	Parameters	Savannah Forest Female	Male	Savannah Male Female	Forest Male Female	RSD	Area Sex Interaction
	Weight (g)	1197a	946b	965a	1177b 1309a	1085b	1046b	846c	233	***	***	NS
	Breast circumference (cm)	34.2a	31.2b	31.3a	34.1b	35.9a	32.6b	32.4b	30.1c	2.4	***	***	*
	shank length (cm)	13.4a	12.5b	12.0a	13.9b	14.7a	12.2c	13.3b	11.8d	1.1	***	***	*
	Tarsus length (cm)	9.7a	8.5b	8.2a	10.0b	10.9a	8.6c	9.1b	7.8d	1.1	***	***	**
	Body length (cm)	20.6a	19.2b	19.1a	20.7b	21.6a	19.6b	19.8b	18.7c	1.5	***	***	*

* : P < 0.05 ; ** : P < 0.01 ; *** : P < 0.001 ; R S D: residual standard deviation. Within rows, means followed by different letters, differ significantly with the threshold of 5%.

Table 2 .

 2 Diversity of feather colours of local chicken (Gallus gallus) populations in Benin.

	Sex		Feather colour	Mutations pertaining to the wild type	Benin Savannah (N = 316) Male (%) Female (%) Total (%)	Benin Forest (N=326) Male (%) Female (%) Total (%)	Benin (%)	Test of Significance between areas
		Self-coloured White		I E	2.53	17.09	19.62	1.23	5.52	6.75	13.08	***
		Dirty white		I (heterozygous) and E 1.27	3.16	4.43	0.92	2.76	3.68	4.05	NS
	Phenotype found in the two sexes without feather drawing	Ermined white Ermined red Fawn		S CO EY CO EY CO EB	0.63 0.95 0.95	1.27 2.53 3.48	1.9 3.48 4.43	1.53 1.23 5.21	5.21 3.68 10.12	6.75 4.91 15.34	4.36 4.21 9.97	** NS **
		Black			E	4.75	17.41	22.15	3.07	7.36	10.43	16.20	***
		Grey			E BL	0.32	0.32	0.64	1.84	3.07	4.91	2.81	**
		Golden § black			4.11	0	4.11	5.52	0	5.52	4.83	NS
	Phenotype found in the cocks group without feather drawing	Coppery black Silver black Red with black tail		ER ER S DB	7.59 3.16 4.43	0 0 0	7.59 3.16 4.43	3.07 5.52 3.07	0 0 0	3.07 5.52 3.07	5.29 4.36 3.74	* NS NS
		White with black tail		DB S	0	0	0	1.23	0	1.23	0.62	*
		Golden salmon			0	0	0	0	2.45	2.45	1.24	**
		Silver salmon		S	0	3.8	3.8	0	1.84	1.84	2.80	NS
		Golden wheaten		EY	0	1.27	1.27	0	0	0	0.63	*
	Phenotype found in the hens group without feather drawing	Wheaten (light without black) Partridge Silver Partridge	EY I EB EB S	0 0 0	1.9 1.27 1.27	1.9 1.27 1.27	0 0 0	1.84 1.23 3.68	1.84 1.23 3.68	1.87 1.25 2.49	NS NS NS
		Golden Partridge		EB	0	7.59	7.59	0	6.75	6.75	7.16	NS
		Golden partridge with salmon breast # EB	0	0	0	0	3.07	3.07	1.56	**
		Silver partridge with salmon breast	EB S	0	0	0	0	2.45	2.45	1.24	**
		Unknown	##			2.85	4.11	6.96	2.15	3.37	5.52	6.23	NS
	§ golden stands for the wild-type allele at the S locus ;	# salmon breast indicates the wild-type allele for CO locus in females;

Table 3 .

 3 Phenotype and corresponding allele frequency per area.

	Allele	Frequency of phenotypes
		Savannah (%)	Forest (%)	Benin (%)
	E	59.57a	30.68b	44.90
	ID wild type	48.48a	38.36b	43.34
	I	45.57a	19.02b	32.09
	EB	14.56a	32.52b	23.68
	CO	9.81a	27b	18.54
	S	10.13a	21.47b	15.89
	wild type allele at CO locus	11.55a	18.02b	14.84
	EY	13.4a	15.34a	14.39
	wild type allele at E locus	12.15a	12.5a	12.33
	MO	20a	3.06b	11.40
	ER	10.75a	8.59a	9.65
	W	9.9a	0.00b	4.87
	DB	5.46a	4.3a	4.87
	B	1.82a	4.3a	3.08
	BL	0.64a	4.91b	2.81
	PG	1.03a	2.08a	1.56

Within rows, means followed by different letters differ significantly (P < 0.05).

Table 4 .

 4 Frequency of feather patterns occurrence of local chickens in Benin according to the sex and ecological areas

	Feather drawings	Mutations	Savannah Benin (N = 194) Male (%) Female (%) Total (%) Male (%) Female (%) Total (%) Forest Benin (N=192)	Benin (%)	Test of significativity per area
	Cuckoo (black foundation)	B E	0.52	0.52	1.03	1.04	5.21	6.25	3.63	**
	Golden cuckoo	B EY	2.06	0.00	2.06	2.08	0.00	2.08	2.07	NS
	Thousand flowers	MO wild type E	3.61	3.61	7.22	1.04	0.00	1.04	4.15	**
	Mottled salmon	MO EY	0.00	6.19	6.19	0.00	1.04	1.04	3.63	**
	Mottled black	MO E	9.79	27.32	37.11	1.56	4.17	6.25	21.76	***
	Silver autosomal barrure	PG DB S	1.03	0.00	1.03	0.00	0.00	0.00	0.52	NS
	Multiple lacing	PG	0.00	0.00	0.00	0.00	2.08	2.08	1.03	*
	Mixed pattern	-	1.03	2.06	3.09	0.52	1.56	2.08	2.59	NS
	Whithout drawing	-	10.31	38.14	42.27	20.83	60.42	79.17	60.62	***

NS : not significant; * : P < 0.05 ; * : P < 0.01 ; ** : P < 0.001.

Table 5 .

 5 Shank Colour of local chickens according to the area and the sex.

	colour	Allele	Savannah Benin (N = 316) Male (%) Female (%) Total (%) Male (%) Female (%) Forest Benin (N=326) Total (%)	Benin (%)	Test of significativity per area
	White	ID	7.58	23.33	30.91	12.89	36.16	49.06	40.13	***
	Yellow	W	0.91	3.33	4.24	1.26	4.40	5.66	4.96	NS
	Grey-blue	ID Wild type	12.42	36.06	48.48	10.06	28.30	38.36	43.34	**

NS: not significant; * : P<0.05 ; **: P<0.01 ; *** : P<0.001.

Table 6 .

 6 Summary of measurement parameters of within or between populations diversity and individual assignation of the 121 chicken.

	Ecotype	He	Ho	ANA	Ae	FIS	HWED
	Savannah	0.542 ± 0.152 0.536 ± 0.151	5.73	2.18	0.012	0
	Forest	0.554 ± 0.167 0.568 ± 0.181	5.91	2.24	-0.024	1 d
	He, expected heterozygosity; Ho, observed heterozygosity; ANA, average number of present allele per locus ; Ae : average number of effective alleles per locus; HWED, Hardy-Weinberg equilibrium departure. d Number
	of loci with heterozygote deficiency (after Bonferroni correction).

ACKNOWLEDGEMENT

The authors thank the Promoting Sustainable Development in Agricultural Research Systems Project (Project DURAS) for financial contribution.

Abreviation

HPIA, Highly Pathogenic Avian Influenza; FAO, Food and Agriculture Organization; ASECNA, Agence pour la Sécurité de la Navigation Aérienne; SAS, Statistical Analysis System.