Farid F Bensalah

Asmaa A Labtar

Christine C Delorme

Pierre Renault

Occurrence, isolation and DNA identification of Streptococcus thermophilus involved in Algerian traditional butter 'Smen'

Keywords: Streptococcus thermophilus, traditional butter, PCR, Enterococcus, DNA, species-specific primer

HAL is

INTRODUCTION

The microbial composition of traditional dairy products and, particularly, of European artisanal cow's and ewe's fermented milk and cheeses, have been subjected to microbiological investigation at genus and species level (Tornadjio et al., 1995;[START_REF] Cogan | Characterisation of the lactic acid bacteria in artisanal dairy products[END_REF][START_REF] Mannu | Genotypic and phenotypic heterogeneity among lactococci isolated from traditional Pecorino Sardo cheese[END_REF][START_REF] Mannu | A Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewes' milk cheese[END_REF][START_REF] Alegría | Diversity and evolution of the microbial populations during manufacture and ripening of Casín, a traditional Spanish, starter-free cheese made from cow's milk[END_REF][START_REF] Secchi | Bioconversion of ovine scotta into lactic acid with pure and mixed cultures of lactic acid bacteria[END_REF]. Many indigenous fermented foods from Algeria are based on lactic fermentation from cow's and ewe's milk [START_REF] Bensalah | Characterisation of thermotolerant cocci from indigenous flora of 'leben' in Algerian arid area and DNA identification of atypical Lactococcus lactis strains[END_REF]. Among these, *Corresponding author. E-mail: bensalahfarid@yahoo.fr.

Abbreviation: PCR, Polymerase chain reaction; glcK, gene encoding glucose kinase; dnaE, gene encoding DNA polymerase III; thrS, gene encoding threonyl-tRNA synthetase.

'Smen' is a traditional butter, which, so far has not been investigated for microbiological genotypic strains characterization. The nature of this fermented product is different from one region to another and depends on the local indigenous microflora; which in turn reflects the climatic conditions of the area. However, there is very little information on strain composition within species, and on the specific contribution of each wild strain to the lactic fermentation. Moreover, the study of strain heterogeneity in African traditional butter [START_REF] El-Baradei | Bacterial biodiversity of traditional Zabady fermented milk[END_REF][START_REF] Ongol | Main microorganisms involved in the fermentation of Ugandan ghee[END_REF], and cheese starters is believed to be of great importance to the dairy industry (Girrafa et al., 1998).

Fermented milk 'leben' or 'rayeb' [START_REF] Elotmani | Characterisation of anti-Listeria monocytogenes bacteriocins from Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis strains isolated from Raib, a Moroccan traditional fermented milk[END_REF][START_REF] Hegazi | Proteolysis and electrophoretic pattern of casein of some fermented milks[END_REF][START_REF] Bensalah | Characterisation of thermotolerant cocci from indigenous flora of 'leben' in Algerian arid area and DNA identification of atypical Lactococcus lactis strains[END_REF] can be eaten fresh or suffers a churning and creaming in goatskin called 'Chekoua'. Skimming is usually done in the morning; gourd is half filled with milk fermented, then strained by swelling, and then the 'Chekoua' is tied and shaken vigorously for half an hour. The formation of fat globules is considered by the sound change that occurs within the skin. To assist the agglomeration of particles of butter, water is usually added, hot or cold depending on the temperature of milk. The fresh butter is removed manually in a single lump called 'Zebda' which is transformed into 'Smen' by washing with salt water. 'Smen' has a very strong smell, easily recognizable during ripening. Implication of Streptococcus thermophilus in Egyptian artisanal butter 'zabady' was reported recently [START_REF] El-Baradei | Bacterial biodiversity of traditional Zabady fermented milk[END_REF]. This strain is an essential microorganism in the manufacture of many types of fermented dairy products, occurring in natural as well as commercial starter cultures [START_REF] Coppola | The microflore of natural whey cultures utilized as strarters in the manufacture of Mazzarella cheese from water-buffalo milk[END_REF][START_REF] Delorme | Safety assessment of dairy microorganisms: Streptococcus thermophilus[END_REF], and the increasing number of commercial strains available used as starters requires rapid and reliable methods to accurately differentiate strains at both species and strain levels in pure and mixed cultures in order to protect patent rights and eliminate risks of use confusion [START_REF] Ramos | DNA fingerprinting of lactococci and streptococci used in dairy fermentations[END_REF][START_REF] Moschetti | Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism: powerful methods to differentiate Streptococcus thermophilus strains[END_REF]. Enterococcus strains can harbour specific biochemical traits that are essential in the manufacture of various fermented milk products, and some strains are technologically exploited as functional starters or probiotics [START_REF] Giraffa | Enterococci isolated from dairy products: a review of risks and potential technological use[END_REF][START_REF] Franz | Enterococci at the crossroads of food safety?[END_REF][START_REF] Bensalah | A rapid PCR based method to distinguish between Enterococcus species by using degenerate and species-specific sodA gene primers[END_REF]. Enterococci has also been implicated in the spoilage of processed meats [START_REF] Franz | Thermotolerance of meat spoilage lactic acid bacteria and their inactivation in vacuum-packaged vienna sausages[END_REF] and include strains that have been recognized as emerging human pathogens mostly in nosocomial urinary tract infections and bacteremias [START_REF] Giraffa | Isolation of vancomycinresistant Enterococcus faecium from Italian cheeses[END_REF][START_REF] Martinez | Interactions among strategies associated with bacterial infection: pathogenicity, epidemicity, and antibiotic resistance[END_REF].

In the last few years, the development of new methods involving various DNA-based typing techniques [START_REF] Farber | An introduction to the hows and whys of molecular typing[END_REF], widely used in epidemiological studies for the identification of medically important bacterial strains (Kuhn et al., 1995;[START_REF] Descheemaeker | Evaluation of arbitrarily primed PCR analysis and pulsed-field gel electrophoresis of large genomic DNA fragments for identification of enterococci important in human medicine[END_REF], has opened up new perspectives for typing strains from raw milk and from traditional cheeses [START_REF] Bouton | Preliminary characterization of microflora of Comté cheese[END_REF][START_REF] Desmasures | Characterization of lactococci isolated from milk produced in the Camembert region of Normandy[END_REF]. It was previously reported that a genus-specific PCR assay based on the use of degenerate primers enabled amplification of an internal fragment representing approximately 85% of the sodA gene encoding a manganese-dependent superoxide dismutase (Mn-SOD) in various gram-positive bacteria including genera of Streptococcus [START_REF] Poyart | Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase[END_REF] and Enterococcus [START_REF] Poyart | Characterization of superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers[END_REF]. The sodA gene has been identified as a target for the identification of mycobacteria [START_REF] Zolg | The superoxide dismutase gene, a target for detection and identification of mycobacteria by PCR[END_REF]) and staphylococci at the species level by PCR [START_REF] Poyart | Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target[END_REF]. Another Streptococcus species-specific, PCRbased identification method was reported recently that utilises the housekeeping gene encoding glucose kinase (glcK), gene encoding DNA polymerase III (dnaE) and gene encoding threonyl-tRNA synthetase (thrS) [START_REF] Delorme | Extent of horizontal gene transfer in evolution of Streptococci of the Salivarius group[END_REF], and the oligonucleotide primers from the intergenic sequences 16S-23S rRNA [START_REF] Tissala-Timisjarvi | Development of oligonucleotide primers from the 16S-23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR[END_REF].

Moreover, since little is known about the involvement of thermophilic cocci with Algerian artisanal foods, this study was aimed at characterizing phenotypically and genotypically, the streptococci associated with the traditional fermented cow's and ewe's raw milk transformed into home-production butter provided from southern west Algeria. We applied the developed methodology of genus and species-specific primers from sodA, glcK, dnaE and thrS housekeeping genes in order to distinguish among reference, and wild strains of S. thermophilus and for their differentiation from Enterococcus spp.

MATERIALS AND METHODS

Sampling and bacterial strains

Six samples of cow's and ewe's traditional butter 'Smen' were obtained from individual households in rural arid area. Samples were collected in sterile small bottles and stored in Laboratoire de Génétique Microbienne, Es-Sénia, Algeria (LGM) under refrigeration at 4°C until use. Reference strains representing four Gram-positive species were used in this study and were provided from INRA collection (Jouy-En-Josas, France). These included S. thermophilus CNRZ 1066, Enterococcus faecalis JH2-2, Enterococcus faecium 64/3 and Corynebacterium glutamicum ATCC 13032.

Isolation and phenotypic characterization of selected isolates

Serials dilutions of homogenized 10 g of 'Smen' samples in 90 ml sterile solution saline 0.85% NaCl were used for microbial isolation with M17 agar [START_REF] Terzaghi | Improved medium for lactic streptococci and their bacteriophages[END_REF] containing 1% lactose (LM17). Plates were incubated anaerobically at 37, 45 or 50°C for 48 to 72 h for isolation of thermophilic streptococci. Identification was carried out according to previously published characterization schemes [START_REF] Devriese | Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups[END_REF][START_REF] Moschetti | Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism: powerful methods to differentiate Streptococcus thermophilus strains[END_REF]Muller et al., 2001;[START_REF] Michaylova | Isolation and characterization of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus from plants in Bulgaria[END_REF]. Gram staining, catalase activity, ammonia production from arginine and gas production from glucose were determined. Tests for growth on Slanetz-Bartley (SB) medium, in M17 broth at 10°C, pH value adjusted to 9.6 and with NaCl 6.5% were performed for all the isolates. Colonies morphology were determined for overnight cultures by using phase-contrast microscopy and all isolates were stored in liquid cultures with 15% glycerol at -80°C.

Oligonucleotides

The first preliminary phenotypic investigation was followed by using the nucleotide sequences of internal fragment with PCR identification analysis. Streptococcus /Enterococcus genus-specific primers sodAd1/sodAd2 (encoding superoxide dismutase) as previously described [START_REF] Poyart | Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase[END_REF][START_REF] Poyart | Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci[END_REF] and housekeeping genes of Streptococcus species-specific primers from glcK, dnaE and thrS, [START_REF] Delorme | Extent of horizontal gene transfer in evolution of Streptococci of the Salivarius group[END_REF] were used in this study.

DNA template preparation

Template DNA was prepared by a modification of the method of

Primer

Sequence Product size (bp) sodAd1 (universel-F) sodAd2 (universel-R) 5'-CITAYICITAYGAYGCIYTIGARCC-3' 5'-ARRTARTAIGCRTGYTCCCAIACRTC-3' 438; Enterococcus 480; Streptococcus Streptococcus thremophilus (glcK) 5'-TGGGCAGAAACTCAAGA-3' 5'-AACACCACCACCGATAAC-3' 470

Streptococcus thremophilus (dnaE) 5'-GGACTGGGAGCCTGGGAT-3' 5'-ACTCCCTGCAGCAGACCC-3' 480

Streptococcus thremophilus (thrS) 5'-ATCACTGAAGATGGAAGC-3' 5'-CCAAGTTTACGGTGGTCA-3' 497 [START_REF] Moschetti | Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism: powerful methods to differentiate Streptococcus thermophilus strains[END_REF]. Total DNA was extracted from 100 µl of overnight cultures which was picked from one colony and resuspended in M17 broth. Cells were pelleted in a microcentrifuge at 8000 × g for 5 min, resuspended in 20 µl of GeneReleaser TM (Bio Ventures, Inc.) and lysed using PCR program as follows: 65°C for 30 s, 8°C for 30 s, 65°C for 1 min 30 s, 97°C for 3 min, 8°C for 1 min, 65°C for 3 min, 97°C for 1 min, 65°C for 1 min and 80°C for 5 min.

I = A,T,G or C; Y = C or T; R = A or G.

Amplification of internal part from sodA gene by using degenerate primers

PCR procedure was performed in 25 µl reaction mixtures containing: 2.5 µl 10X PCR buffer, 2 µl dNTP, 0.5 µl of each primer, 0.25 µl of Taq DNA polymerase (Takara) and 5 µl of DNA from cellular lysates. Amplification was carried out in a Thermal cycler (Applied Biosystem), as follows: an initial denaturation step of 95°C for 3 min, 30 cycles of amplification (60 s of annealing at 37°C, 60 s of elongation at 72°C, and 30 s of denaturation at 95°C) and 7 min at 72°C for the last elongation. PCR primers pairs used in this study are shown in Table 1. An amount of 5 µl of the PCR product was electrophoresed at 100 V on a 1% agarose gel. The documented sizes of the amplicons were 480 and 438 bp from sodA gene in Streptococci and Enterococci respectively [START_REF] Poyart | Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase[END_REF][START_REF] Poyart | Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci[END_REF]. 200 bp DNA Smartladder (Eurogentec) was used as molecular size marker and DNA of references strains was included.

Streptococcus-specific PCR

DNA identification analysis by PCR was followed with Streptococcus species-specific primers from S. thermophilus CNRZ1066 used as the positive control. The cellular lysates were obtained with the genereleaser TM (bio Ventures, inc.) from overnight cultures as described above and the following three primers pairs were used for this study (Table 1). The amplification profile included a denaturation step of 94°C for 5 min, and then subjected to 30 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 1 min and a final soak at 4°C. DNA fragments were determined by comparing the positive control bands from reference strains with amplified fragments of wild members within indigenous flora isolates. The documented bands of the amplicons were 470, 480 and 497 from glcK, dnaE and thrS respectively [START_REF] Delorme | Extent of horizontal gene transfer in evolution of Streptococci of the Salivarius group[END_REF]. The presence of PCR products was determined by gel electrophoresis in 1% agarose gel containing ethidium bromide. Electrophoresis in Tris-borate-EDTA was performed at 100 V, and DNA Smartladder (Eurogentec) of 200 bp was used as the molecular size marker.

RESULTS

Biochemical and morphological characteristics

12 strains were selected and presumptively identified as cocci thermophilic lactic acid bacteria (LAB). All the strains were Gram-positive, catalase negative and produced no gas from glucose. Eight strains did not grow at all or poorly in 6.5% NaCl, three of them were able to grow at pH 9.6 and showed no or only weak growth on SB medium. Morphology cells were spherical, ovoid in short or means and long chain. Enterococci are reported to grow at 10°C, but six strains presumptively identified in these genera by classical methods failed to do so. The contradictions were established in strains which grew well with pink colonies in SB agar but failed to grow in broth with 6.5% NaCl and at pH 9.6. Therefore, another identification approach was necessary to clarify the taxonomic positions of these atypical isolates.

Analysis of DNA amplicons products from sodA gene

Identification by using the universal primers of sodA database of Gram-positive cocci is frequently reported particularly from genus and species of streptococci and enterococci. In this work, the specificity of the genus was determined by testing all isolates presumptively identified as thermophilic lactic acid bacteria in short and long chain. Only five strains reacted with sodAd1/sodAd2 genus universal primers, and occurred as a single product. One band of weak intensity at 438 bp in the references and three isolates (LGM5, LGM6 and LGM7) and another band of strong intensity located approximately between 438-480 bp was revealed in only the reference and two selected strains (LGM1 and LGM2). No significant common bands were found from other isolates, and from the control-negative strain C. glutamicum (Figure 1a). These results represent a similar profile and do not indicate the discriminating power of the universal primers of sodA gene PCR described here.

Amplification using streptococcal species

On the basis of the unclear results of phenotypic identification and the weak discriminating power of sodA gene between Streptococcus and Enterococcus genus, species-specific PCR of glcK, dnaE and thrS from streptococci were carried out to determine the taxonomic identification and to compare the isolates with reference strains. In one hand, the expected 470-pb band from glcK gene was revealed in the streptococci reference strain and two isolates (LGM1 and LGM2). However, a double product band was visualised also at approximately 600 bp in two reference enterococci strains and in three traditional butter isolates (LGM5, LGM6, and LGM7), while no significant bands were found from the other strains tested (Figure 1b). Two other strains suspected to be streptococci were confirmed by using primers of glcK,

LGM1 LGM2

LGM3

LGM4 LGM3 and LGM4, (results not shown).

On the other hand, the specific 480 and 497 bp streptococci bands from dnaE and thrS respectively were obtained in four isolates which reacted also with glcK from all isolates tested, (Figure 1c,d). Finally, in this study, we demonstrated that four isolates reacted with the three species-specific glcK, dnaE and thrS amplicons used in work, indicating that they are members of Streptococcus thermophilus which are provided from Algerian traditional butter 'Smen'. These results indicate the discriminating power of the glcK-up/glcK-dn, dnaEup/dnaE-dn and thrS-up/thrS-dn primers described in this work. This evidence allowed us to clearly differentiate Streptococcus thermophilus (Figure 2) from Enterococcus spp.

DISCUSSION

While the association of Streptococci and Enterococci with European fermented foods is well known [START_REF] Moschetti | Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism: powerful methods to differentiate Streptococcus thermophilus strains[END_REF][START_REF] Cogan | Characterisation of the lactic acid bacteria in artisanal dairy products[END_REF][START_REF] Sengun | Identification of lactic acid bacteria isolated from Tarhana, a traditional Turkish fermented food[END_REF][START_REF] Secchi | Bioconversion of ovine scotta into lactic acid with pure and mixed cultures of lactic acid bacteria[END_REF], little is known about the involvement of these bacteria in African fermented foods [START_REF] Youssif | Molecular characterization, technological properties and safety aspects of enterococci from 'Hussuwa', an African fermented sorghum product[END_REF]. Streptococci were shown in this study to form a predominantly inhabitants of the microflora of fermented milk product 'Smen', as these bacteria could be isolated throughout the fermentation process. Enterococci were also present in 'Smen', they appear to be often associated with fermented products from Africa, although at a high incidence compared with other cocci lactic acid bacteria (LAB) [START_REF] Mathara | Isolation, identification and characterisation of the dominant microorganisms of kule naoto: the Maasai traditional fermented milk in Kenya[END_REF][START_REF] Bensalah | A rapid PCR based method to distinguish between Enterococcus species by using degenerate and species-specific sodA gene primers[END_REF][START_REF] Bensalah | Characterisation of thermotolerant cocci from indigenous flora of 'leben' in Algerian arid area and DNA identification of atypical Lactococcus lactis strains[END_REF].

Streptococcus and Enterococcus are Gram-positive cocci, commonly recognized by their growth on Rogosa agar plates. These genera differ phenotypically; however, biochemical and physiological tests often give imprecise specifications. Due to complicated and uncertain identification, efforts have been made to develop simple, fast and discriminating methods for identification of Streptococcus from Enterococcus species (Moshetti et al., 1998). The result of this study demonstrates the necessity for use of molecular methods which is crucial for successful identification of unknown thermophilic cocci strains. PCR analysis is useful for clearly discriminating a multitude of species of LAB (like Streptococcus, Lactococcus and Enterococcus species) [START_REF] Mannu | A Genetic diversity of lactococci and enterococci isolated from home-made Pecorino Sardo ewes' milk cheese[END_REF][START_REF] Bensalah | A rapid PCR based method to distinguish between Enterococcus species by using degenerate and species-specific sodA gene primers[END_REF][START_REF] Bensalah | Characterisation of thermotolerant cocci from indigenous flora of 'leben' in Algerian arid area and DNA identification of atypical Lactococcus lactis strains[END_REF]. Species-specific PCR was previously used for the identification of clinical and food isolates of Enterococci and Streptococci [START_REF] Poyart | Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase[END_REF][START_REF] Delorme | Extent of horizontal gene transfer in evolution of Streptococci of the Salivarius group[END_REF].

The primers used for the sodA gene in this study gave a DNA profile almost similar to Streptococci and Enterococci; however, the use of three other primer pairs glcK, dnaE and thrE helped highlight different profiles and amplified the expected DNA bands. The loci chosen formed a suitable basis for DNA identification procedures and the housekeeping gene provided evidence and demonstrated that species-specific glcK, dnaE and thrE discriminate among streptococci and enterococci isolates. PCR methods in this work proved to be useful for species identification and to reveal different patterns throughout this genus. We also attempted to show that phenotypic characterisation and amplified DNA amplicons using PCR analyses were quick, reliable and convenient methods to differentiate Streptococci from Enterococci. PCR assays developed here are technically affordable for laboratories that do not have easy access to more sophisticated or expensive procedures. Also, the genotypic and phenotypic analysis carried out on wild Streptococci and Enterococci isolated from traditional fermented 'Smen' highlighted diversity among strains. This study enabled the dominance biotypes in 'Smen' to be defined at each period of maturation and, it was found that the strains that dominated the first stage of ripening were not necessarily predominant in the later periods [START_REF] Mannu | Genotypic and phenotypic heterogeneity among lactococci isolated from traditional Pecorino Sardo cheese[END_REF].

To our knowledge, this is the first report on the occurrence, isolation and molecular identification of S. thermophilus in 'Smen' in Algeria, and further investigation into strain variability within species should be extended not only to more old samples but also, to dairy artisanal product from spontaneously fermented milk. The analysis of bacterial properties as synthesis of exopolysaccharides [START_REF] Minic | Control of EpsE, the phosphoglycosyltransferase initiating exopolysaccharide synthesis in Streptococcus thermophilus, by EpsD tyrosine kinase[END_REF], proteolysis, aroma, acidifying power and antagonistic activity must be realised. These studies are of particular interest as they favour the isolation and selection of future cultures starters as S. thermophilus from traditional dairy products. It is, therefore, of great importance to preserve the genetic pool of strains of lactic acid bacteria in Africa.

Figure 1 .

 1 Figure 1. Genus and species-specific PCR amplifications for sodA gene. (a) Genus PCR with primer pair d1/d2. Lane M, 200-pb DNA molecular mass marker; lane 1, S. thermophilus CNRZ 1066 as positive control; lane 2, LGM1 isolate; lane 3, LGM2 isolate; lane 4, C. glutamicum ATCC 13032 as negative control; lane 5, LGM5 isolate; lane 6, LGM6 isolate; lane 7, LGM7 isolate; lane 8, E. faecalis JH2-2 as positive control; lane 9, E. faecium 64/3 as positive control. (b) Species-specific PCR with primer pair glcKup/glcK-dn. Lane M, 200-pb DNA molecular mass marker; lane 1, LGM5 isolate; lane 2, isolate 6; lane 3, LGM6 isolate; lane 4,LGM7 isolate; lane 5, isolate 7; lane 6, isolate 8; lane 7, LGM1; lane 8, isolate 9; lane 9, LGM2; lane 10, E.. faecalis JH2-2 as negative control; lane 11, isolate 10; lane 12, E. faecium 64/3 as negative control; lane 13, isolate 11; lane 14, isolate 12; lane 15, St. thermophilus CNRZ 1066 as positive control. (c) Species-specific PCR with primer pair dnaE-up/dnaE-dn. Lane M, 200-pb DNA molecular mass marker; lane 1, LGM5 isolate; lane 2, LGM6 isolate; lane 3, LGM7 isolate; lane 4, isolate 6; lane 5, LGM1; lane 6, LGM2; lane 7, isolate 7; lane 8, isolate 8; lane 9, isolate 9; lane 10, LGM3; lane 11, LGM4; lane 12, isolate 10; lane 13, isolate 11; lane 14, isolate 12; lane 15, E.. faecalis JH2-2 as negative control. (d) PCR with primer pair thrSup/thrS-dn. Lane M, 200-pb DNA molecular mass marker; lane 1, Ent. faecium 64/3 as negative control; lane 2, E.. faecalis JH2-2 as negative control; lane 3, LGM1; lane 4, LGM2; lane 5, isolate 6; lane 6, LGM 3; lane 7, LGM5; lane 8, LGM6; lane 9, LGM4; lane 10, LGM7, lane 11, isolate 7, lane 12, isolate 8; lane 13, isolate 9; lane 14, isolate 10; lane 15, isolate 11; lane 16, isolate 12.

Figure 2 .

 2 Figure 2. Microscopic cell morphology of wild Streptococcus thremophilus LGM1, LGM2, LGM3 and LGM4 isolated from traditional butter 'Smen' (×1000).

Table 1 .

 1 PCR primers.


Nishtman N Abdi

Reza R Darvishzadeh
email: r.darvishzadeh@urmia.ac.ir

Hamid H Hatami Maleki

Haddadi Parham

Ahmad A Haddadi

Sarrafi

Parham Haddadi

Ahmad Sarrafi

Identification of quantitative trait loci for relative water content and chlorophyll concentration traits in recombinant inbred lines of sunflower ([i]Helianthus annuus[/i] L.) under well-watered and water-stressed conditions

Keywords: Helianthus annuus, linkage map, photosynthetic traits, plant water-status, QTL mapping, transgressive segregation

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Drought is the major factor limiting crop productivity worldwide [START_REF] Chimenti | Divergent selection for osmotic adjustment results in improved drought tolerance in maize (Zea mays L.) in both early growth and flowering phases[END_REF]. Crops with improved tolerance level to drought stress appear to be crucial in areas where dry seasons are common [START_REF] Andrew | Does maintaining green leaf area in sorghum improve yield under drought? II. Dry mater production and yield[END_REF]. Drought tolerance is not a unique and easily quantifiable plant attribute; it is a complex character that can be evaluated with relative water content (RWC), relative water loss, chlorophyll fluorescence, stomatal resistance, cell membrane stability, free prolin accumulation and many other characters [START_REF] Zarei | Evaluation of some indirect traits and indices to identify drought tolerance in bread wheat[END_REF]. Among the traits used for evaluating plant waterstatus, RWC gives best measure of the level of the water deficit in the plant at a specific time-point. As RWC is related to cell volume, it may closely reflect the balance between water supply to the leaf and transpiration rate [START_REF] Sinclair | Influence of soil water supply on the plant water balance of four tropical grain legumes[END_REF]. Decreased RWC and leaf water potential (ψw) inhibit the photosynthesis capacity in sunflower [START_REF] Tezara | Effects of water deficit and its interaction with CO 2 supply on the biochemistry and physiology of photosynthesis in sunflower[END_REF]. It was reported that among the photosynthetic traits, chlorophyll concentration is an index for estimating environmental stress effects on growth and yield, since this trait was closely correlated with the rate of carbon exchange [START_REF] Guo | Effects of high nocturnal temperature on photosynthetic organization in rice leaves[END_REF][START_REF] Fracheboud | Genetic analysis of cold-tolerance of photosynthesis in maize[END_REF]. Chlorophyll concentration as one of the major chloroplast components has a positive relationship with photosynthetic rate [START_REF] Guo | Studies on photosynthetic characteristics in rice hybrid progenies and their parents I. chlorophyll content, chlorophyll-protein complex and chlorophyll fluorescence kinetics[END_REF]. So, this parameter can be used as a reliable indicator to evaluate the energetic/metabolic imbalance of photosynthesis and yield performance across genotypes under water deficit [START_REF] Araus | Chlorophyll fluorescence as a selection criterion for grain yield in durum wheat under Mediterranean conditions[END_REF].

In the past, progress in improving drought tolerance via conventional breeding methods has been slow, due to genotype × environment interactions, poor inbred lines of sunflower (Helianthus annuus L.) under well-watered and water-stressed conditions definition of the target environment, the complexity and difficulty of drought-evaluation procedures, and the inconsistency of morpho-physiological traits as selection criteria for drought tolerance [START_REF] Ceccarelli | Breeding for drought resistance in a changing climate[END_REF]2007). In the last decade, molecular marker technologies have been successfully used to identify quantitative trait loci (QTLs) controlling important traits in various crop plants in responses to drought stress [START_REF] Verma | Mapping quantitative trait loci for flag leaf senescence as a yield determinant in winter wheat under optimal and drought-stressed environments[END_REF][START_REF] Abdi | Genetic analysis and QTL mapping of agromorphological traits in sunflower (Helianthus annuus L.) under two contrasting water treatment conditions[END_REF]. Marker assisted selection may solve problems associated with genotype × environment interactions, improve the selection efficiency and facilitate pyramiding different genes into a single genotype background. Although sunflower is moderately tolerant to water stress, its distribution and production are greatly influenced by drought stress. Several researches were conducted to identify putative QTLs related to osmotic adjustment [START_REF] Jamaux | Looking for molecular and physiological markers of osmotic adjustment in sunflower[END_REF]Poormohammad Kiani et al., 2007 a), agronomic traits (Poormohammad [START_REF] Kiani | QTL analysis of yield-related traits in sunflower under different water treatments[END_REF][START_REF] Haddadi | Genetic dissection of tocopherol and phytosterol in recombinant inbred lines of sunflower through quantitative trait locus analysis and the candidate gene approach[END_REF], photosynthesis parameters [START_REF] Herve | QTL analysis of photosynthesis and water status traits in sunflower (Helianthus annuus L.) under greenhouse conditions[END_REF][START_REF] Kiani | QTL analysis of chlorophyll fluorescence parameters in sunflower (Helianthus annuus L.) under well-watered and water-stressed conditions[END_REF], whereas, a few studies have been carried out to determine the QTLs linked to RWC [START_REF] Herve | QTL analysis of photosynthesis and water status traits in sunflower (Helianthus annuus L.) under greenhouse conditions[END_REF]Poormohammad Kiani et al., 2007 a) and chlorophyll concentration [START_REF] Herve | QTL analysis of photosynthesis and water status traits in sunflower (Helianthus annuus L.) under greenhouse conditions[END_REF] in sunflower. [START_REF] Herve | QTL analysis of photosynthesis and water status traits in sunflower (Helianthus annuus L.) under greenhouse conditions[END_REF] reported 4 QTLs for chlorophyll concentration and 1 QTL for RWC which explained 53% and 9.8% of their phenotypic variation, respectively. Poormohammad Kiani et al. (2007 a) have identified 6 QTLs for RWC whose phenotypic variation ranged from 7% to 25%.

In the present study, we aimed to identify QTLs involved in genetic variation of chlorophyll concentration and relative water content in RILs of sunflower under well-watered and water-stressed conditions using an integrated and high density genetic-linkage map based on SSR and SNP markers.

Material and methods

Plant materials and experimental methodology.

Recombinant inbred lines (RILs) used in this research were developed through single seed descent from F 2 plants, derived from the cross between PAC2 and RHA266 (Flores [START_REF] Berrios | AFLP mapping of QTLs for in vitro organogenesis traits using recombinant inbred lines in sunflower (Helianthus annuus L.)[END_REF]. Seeds of 70 RILs and their two parents kindly provided by National Institute for Agricultural Research (INRA, France) were evaluated in both well-watered and water-stressed conditions using a rectangular 8 × 9 lattice design with two replications. The experiment was arranged in research farm of Urmia University, Iran in 2010. Each plot comprised 1 line 8 m long, with a spacing of 75 × 25 cm between lines and plants, respectively. The distance between well-watered and water-stressed experiments was considered 5 m. From the beginning of planting time until the complete establishment of sunflower plants (eight-leaf (V8) stage), irrigation in both well-watered and water-stressed experiments was carried out when the amount of evaporated water (from class 'A pan' evaporation) reached to 60 mm [START_REF] Pourtaghi | Effect of irrigation water deficit on antioxidant activity and yield of some sunflower hybrids[END_REF]. After V8 stage, the irrigation was continued in the same manner (60 mm evaporation from class 'A pan') in well-watered experiment but it was carried out in the water-stressed experiment when the amount of evaporated water (from class 'A pan' evaporation) reached to 180 mm [START_REF] Pourtaghi | Effect of irrigation water deficit on antioxidant activity and yield of some sunflower hybrids[END_REF].

Traits measurement. Chlorophyll concentration. Leaf chlorophyll concentration was determined using a chlorophyll meter SPAD-502 ("Minolta", Japan). During filling grain stage, chlorophyll concentration on the youngest fully expanded leaves of five plants from each genotype per replicate were measured before irrigation in both well-watered and water-stressed experiments. Three measurements were made at random locations in the middle of the leaf for each plant and the average was used for the analysis.

Relative water content (RWC). The youngest fully expanded leaf of four plants per genotype per replicate was sampled in both well-watered and waterstressed conditions. Leaf discs of 4 cm 2 area in rectangle shape per plant were prepared and the fresh weight (Wf) was measured. Leaf discs were then dipped in the glass vials containing 20 ml deionized water. These vials were left for four hours at room temperature (about 25ºC) with no illumination. After four hours, leaf discs were blotted and their turgid weight (Wt) was recorded. Leaf discs were oven dried in 80ºC for 24 h and weighed (Wd). RWC was calculated using the following formula:

. Map construction and quantitative trait loci (QTL) mapping. Simple sequence repeat (SSR) mapping. The genomic DNA of RILs and their parents (PAC2, RHA266) were extracted according to the method of extraction and purification presented by [START_REF] Porebski | Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components[END_REF]. We used Picogreen fluorescent stain (Quanti-iT TM Picogreen®, Invitrogen) to quantify DNA concentration with the BioTek FL600 Fluorescence Microplate Reader (BioTek Instruments Inc., USA). One hundred and fourteen SSRs were studied. All SSR markers are public and can be provided upon request. We used multiplex polymerase chain reaction (PCR) method, in which several SSR markers are simultaneously amplified in the same reaction (we used 4 SSR markers in the same reaction thanks to their size). PCR is done using a forward primer with a nucleotide extension at its 5'-end, identical to the sequence of an M13 sequencing primer and a standard length reverse primer and fluorescently (6-FAM, NED, VIC and PET) labelled M13 primer. During PCR, the SSR product is fluorescently labelled following participation of the M13 primer after the first few cycles of amplification. Therefore, instead of synthesizing one specific fluorescently labelled primer for each SSR marker, only a dye labelled M13 primer is needed. PCR products were diluted with ultrapure water (2 µl of each PCR product in 20 µl water) and 2 µl of diluted PCR products mixed with 7.94 µl Formamide HiDi TM and 0.06µl GeneScan TM 500 LIZ TM size standard (Applied Biosystems, USA). After denaturing at 94°C for 5 min, we used sequencer ABI3730 and fragments were sized using the GeneMapper® software, version 4 (Applied Biosystems). Chi-square-tests were performed for segregation distortion of each locus. All new SSRs are mapped to our previous map [START_REF] Haddadi | Genetic dissection of tocopherol and phytosterol in recombinant inbred lines of sunflower through quantitative trait locus analysis and the candidate gene approach[END_REF] by Carthagène (De [START_REF] Givry | Carthagene: multi-population integrated genetic and ISSN 1392-3196[END_REF] and Mapmaker [START_REF] Lander | Mapmaker: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations[END_REF].

Candidate genes (CGs) mapping. Some important tocopherol and phytosterol pathway-related genes, enzymatic antioxidant-related genes, droughtresponsive genes and Arabidopsis Sec14 homologue genes were selected to introduce in our map. Respective sequence data for CGs coding for these proteins were obtained from the Arabidopsis information tesource (www.arabidopsis.org). In order to seek the Helianthus homolog sequences to the Arabidopsis genes, we used the Compositae expressed sequence tag (EST) assembly clusters, available at the Helianthus-devoted bioinformatics portal HeliaGene (www.heliagene.org).

The Helianthus EST clusters presenting the reciprocal blast with the highest score and lowest E value with regarding to the original Arabidopsis genes were chosen for our studies. All primers were designed by MATLAB. Forward primers were tailed at 5' with M13-Fwd tail (5'CACGACGTTGTAAAACGAC3') and reverse primers were tailed at 5' with M13-Rev tail (5'ACAGGAAACAGCTATGAC3'). Between 2 to 4 various primer combinations per each candidate gene were tested on agarose gel. The PCR program was: 4 min at 94°C followed by 35 cycles; 30 s at 94°C, 30 s at 55°C or 58°C, 1 min at 72°C and final extension of 5 min at 72°C. One PCR fragment per gene was sequenced using M13-Fwd and M13-Rev primers. After sequencing, SNP-PHAGE (SNP discovery Pipeline with additional features for identification of common haplotypes within a sequence tagged site (Haplotype Analysis) and GenBank (-dbSNP) submissions), through the website (www. heliagene.org), was applied for analyzing sequence traces from both parents to identify SNPs. Several types of markers such as dominant, co-dominant, HRM: high resolution melting, InDel (short insertions and deletions), and SNP-based cleaved amplified polymorphic sequences (CAPS) markers are developed for genotyping of the studied candidate genes.

QTL mapping. QTL mapping of the studied traits was performed by composite interval mapping (CIM), using Win QTL Cartographer, version 2.5 [START_REF] Wang | Windows QTL cartographer 2.5[END_REF]. The genome was scanned at 2 cM intervals with a window size of 15 cM and up to 15 background markers were used as a cofactors in the CIM analysis identified by the SRmapqtl program (model 6). LOD (log 10 likelihood ratio: likelihood that the effect occurs by linkage/ likelihood that the effect occurs by chance) thresholds resulting from permutation tests were used for identifying significant QTLs. Additive effects of the detected QTLs were estimated with Zmapqtl program. The percentage of phenotypic variance (R 2 ) explained by the QTLs was estimated at the peak of curve by Win QTL Cartographer. MapChart 2.2 was used to draw a graphical presentation of linkage groups and map position of QTLs.

Statistical analysis. Analysis of variance was performed using PROC GLM procedure in the SAS software (SAS/STAT® 9.2, 2009). Phenotypic correlations between studied traits in water treatment states were determined using PROC CORR in the SAS software.

Results and discussion

Phenotypic variation among inbred lines.

Analysis of variance indicated the presence of genetic variation and possibility of mapping the genes involved in the inheritance of studied drought tolerance criteria (chlorophyll concentration and relative water content) in sunflower (Table 1). The effects of water treatment condition, genotype, and the interaction between genotype and water treatment were significant (Table 1). The presence of significant genotype × water treatment interaction on the studied traits means that the changes occur in relative performance of genotypes across water treatment conditions. So, a genotype showing promise in one water treatment condition may not be exceptional in another condition. Slicing significant interaction effects for relative water content trait revealed the significant difference among genotypes in each one of water treatment conditions, while concerning chlorophyll concentration there were significant difference among genotypes only in well-watered condition (Table 1). Phenotypic performance of RILs and their parents for chlorophyll concentration and relative water content under the two water treatment conditions are summarized in Table 2. Parental lines showed different chlorophyll concentration under water-stressed condition (Table 2). The differences between the mean of RILs ( RIL ) and the mean of their parents ( P ) were not significant for studied traits (Table 2). This nonsignificant difference shows that the RILs used in this study are representative of possible recombination of the cross PAC2 × RHA266. The genetic gain calculated as difference between the mean values of 10% selected best RILs ( 10%bestRILs ) and the mean of parents ( p ) was significant for the studied traits in both water treatment conditions except for chlorophyll concentration in waterstressed condition (Table 2). High standard deviation was observed for studied traits in both water treatment conditions. Transgressive segregation that would be the result of the accumulation of positive alleles from both parental lines was observed for both of the studied traits (Table 2). Transgressive segregation for osmotic adjustment, photosynthesis and plant water status traits under well-watered and water-stressed conditions as well as for physiological traits associated to low temperature growth under early sowing conditions has been also reported by [START_REF] Herve | QTL analysis of photosynthesis and water status traits in sunflower (Helianthus annuus L.) under greenhouse conditions[END_REF] Results revealed that under water-stressed condition, the mean value of chlorophyll concentration decreased and the mean value of relative water content increased compared with the well-watered condition.

In this study, chlorophyll concentration was significantly and positively correlated with relative water content under water-stressed condition (Table 3). Therefore, changes in water content are not directly related to the chlorophyll concentration in all types of environments. Furthermore, highly significant correlations between relative water content in two water treatments were observed that show the phenotypic value under well-watered condition explain a large proportion of the variation for performance under water-stressed condition (Table 3). This result suggests that selection under well-watered conditions could partly be effective to improve relative water content under water-stressed condition.

Identification of quantitative trait loci for relative water content and chlorophyll concentration traits in recombinant inbred lines of sunflower (Helianthus annuus L.) under well-watered and water-stressed conditions Notes. P -mean of parents, RILs -mean of all RILs, 10%bestRIL -mean of the 10% selected RILs for each measured characters. GG10% -genetic gain when the mean of 10% selected RILs is compared with the mean of parents. LSD-I 0.05 -least significant differences calculated using t 0.05 and error mean square of each experiment. LSD-C 0.05 -least significant differences calculated using t 0.05 and error mean square of combined analysis of experiments. Linkage map. Of 114 SSR primer pairs tested, 23 SSR primers (20.2%) produced clear polymorphisms between the parental lines which segregated in a Mendelian manner. These new SSR markers together with 5 new candidate genes identified with the code HuCL were assigned to the previously reported linkage map [START_REF] Haddadi | Genetic dissection of tocopherol and phytosterol in recombinant inbred lines of sunflower through quantitative trait locus analysis and the candidate gene approach[END_REF]. New SSR can be found in linkage groups 1,2,3,4,5,6,8,10,13,14, 15 and 17 and new 5 candidate genes (HuCLs) in linkage groups 2, 8 and 16. The updated map consisted of 210 SSR markers and 11 genes placed in 17 linkage groups. Linkage groups were named as 1 to 17 according to the reference linkage map of sunflower [START_REF] Tang | Simple sequence repeat map of the sunflower genome[END_REF]. The total map length is 1,653.1 cM with a mean density of 1 marker per 7.44 cM (Fig.). The number of markers per linkage group ranged from 5 to 26. Linkage group 14 is the largest in term of cM size (197.6 cM) while linkage group 4 (32 cM) is the smallest (Fig. ).

Quantitative trait loci (QTL) analysis. QTL mapping showed the presence of 16 QTLs on 9 linkage groups in the expression of chlorophyll concentration and relative water content under both water treatment conditions (Table 4). The number of detected QTLs for each trait varied from 2 to 7 depending on water treatments. The phenotypic variance explained by QTLs (R 2 ) ranged from 0.39% to 52.48%, and the sign of additive gene effects showed that both parental lines contributed to the expression of studied traits. For chlorophyll concentration, 3 QTLs were detected under well-watered condition on linkage groups 10 and 16 with the phenotypic variance ranging from 0.56% to 42.19% (Table 4,Fig 4.30 29.48 Notes. QTL names were constructed using the trait abbreviation name and suffixed with numbers presenting the linkage group and order of QTL on the linkage group. The QTLs names were also followed by either W or S presenting well-watered and water-stressed conditions, respectively. The positive additive effect shows that PAC2 allele increase the trait and negative value shows that RHA266 allele increases the trait. LGlinkage groups; cM -centimorgan; LOD -log 10 likelihood ratio: likelihood that the effect occurs by linkage/likelihood that the effect occurs by chance; % -percentage of phenotypic variance explained by the individual QTLs.

The positive alleles for these QTLs come from PAC2. The major QTL for chlorophyll concentration (ChlW.10.1) is found on linkage group 10 (Table 4). This QTL was also identified for relative water content, with an R 2 value of 23.00% in water-stressed condition (Table 4,Fig.). The most important QTL detected for chlorophyll concentration under well-watered condition (ChlW.10.1) is also reported for leaf area duration under well-watered condition by Poormohammad [START_REF] Kiani | QTL analysis of chlorophyll fluorescence parameters in sunflower (Helianthus annuus L.) under well-watered and water-stressed conditions[END_REF]. Under water-stressed condition, 7 QTLs were identified for chlorophyll concentration on linkage groups 3, 8, 10, 14, 15 and 16 with the phenotypic variance ranging from 0.39% to 52.48% (Table 4,Fig Note. Bars represent intervals associated with the quantitative trait loci (QTLs) in two water treatment conditions. QTL names are constructed using the trait abbreviation name suffixed with numbers presenting the linkage group and order of QTL on the linkage group. The QTLs names were also followed by W or S presenting well-watered water-stressed conditions, respectively.

Figure.

Linkage map of sunflower based on 11 single nucleotide polymorphism (SNP) and 210 simple sequence repeat (SSR) markers using the cross between PAC2 × RHA266 inbred lines of sunflower (Helianthus annuus L.) under well-watered and water-stressed conditions contributed positive alleles for all detected QTLs. In this study, one identified QTL for chlorophyll concentration under water-stressed condition (ChlS.3.1) was also reported for leaf area duration under both water-treatment conditions by Poormohammad [START_REF] Kiani | QTL analysis of yield-related traits in sunflower under different water treatments[END_REF] and for 1000 kernel weight and palmitic acid content under water-stressed condition by [START_REF] Ebrahimi | QTL mapping of seed-quality traits in sunflower recombinant inbred lines under different water regimes[END_REF]2009). Four QTLs were detected for relative water content, on linkage groups 11, 12, 16 and 17 under well-watered condition, and 2 QTLs on linkage group 10 under waterstressed condition (Table 4,Fig.). These putative QTLs explained 0.93% to 21.18% and 23.00% to 29.48% of the total phenotypic variance of target trait in each one of the conditions, respectively (Table 4). The identified QTL on linkage group 10 (RWCS.10.2) under water-stressed condition, was also reported by [START_REF] Ebrahimi | QTL mapping of seed-quality traits in sunflower recombinant inbred lines under different water regimes[END_REF]2009) for percentage of protein, 1000 kernal weight, oil content and palmitic acid content under well-watered condition. Results revealed that PAC2 and RHA266 contributed equally to QTLs controlling relative water content.

QTLs overlapped among studied traits and water treatments. Results showed that some detected QTLs are associated with two traits and some others are specific for only one trait or a given water treatment (Table 4,Fig.). Co-localization of QTLs for different traits implies likely the presence of pleiotropic or closed linkage between QTLs controlling traits [START_REF] Tuberosa | Mapping QTLs regulating morphophysiological traits and yield in drought-stressed maize: case studies, shortcomings and perspectives[END_REF]. For instance, 2 QTLs including ChlW.16.1 and RWCW.16.1 for chlorophyll concentration and relative water content under well-watered condition were overlapped on linkage group 16 (Fig.). These QTLs were also reported for stomatal conductance and plant dry weight by [START_REF] Herve | QTL analysis of photosynthesis and water status traits in sunflower (Helianthus annuus L.) under greenhouse conditions[END_REF] and for linoleic acid content by [START_REF] Haddadi | Genetic control of protein, oil and fatty acids content under partial drought stress and late sowing conditions in sunflower (Helianthus annuus L.)[END_REF]. According to simple correlation (Table 3), negative correlation between relative water content and chlorophyll concentration is justified by opposite additive effects of their overlapped QTLs. These phenomena possess potential challenges to breeders for simultaneous improvement of both traits. However, independent segregation of QTLs for studied traits at genetic level provides opportunity for simultaneous improvement of these traits in sunflower breeding programs. QTLs common for both water treatments seem to be more important as it gives a constitutive performance for the traits without being affected by water treatment. For instance, overlapped QTLs between two water treatment regimes was observed in interval 17-18 cM on linkage group 10 near markers HA928 and ORS456 for chlorophyll concentration and relative water content (ChlW.10.1 and RWCS.10.1). However, majority of identified QTLs were specific for each or both water treatment conditions, which could be of interest for marker-assisted selection (MAS) when favourable alleles are selected. Overlapping QTLs involved in studied traits show that the plant water status and chlorophyll concentration are genetically related, and should be considered together when used as the selection criteria for drought tolerance improvement in sunflower. Therefore, this region may increase yield under stress through drought avoidance by increasing water uptake and mineral nutrients resulting in better growth and photosynthesis.

Conclusions

1. Quantitative trait loci (QTL) analysis for water status and photosynthetic traits using our improved map with 210 simple sequence repeats (SSRs) and 11 genes enabled us to investigate with greater precision the genetic basis of trait association by looking for colocation of corresponding QTLs on the genetic map.

2. The identification of genomic regions associated with water status and chlorophyll concentration under well-watered and water-stressed conditions will be useful for marker-based approaches to improve drought tolerance in sunflower.

3. In addition, our map complements the public SSR and SSR/SNP maps [START_REF] Tang | Simple sequence repeat map of the sunflower genome[END_REF][START_REF] Lai | Identification and mapping of SNPs from ESTs in sunflower[END_REF] as a genetic framework for quantitative and qualitative trait analysis for sunflower (Helianthus annuus L.) ISSN 1392-3196 / e-ISSN 2335-8947 Zemdirbyste-Agriculture, vol. 100, No. 2 (2013), p. 159-166 DOI 10.13080/z-a.2013.100.020 Kiekybinių požymių lokusų (KPL) nustatymas santykinio drėgmės kiekio ir chlorofilo koncentracijos požymiams tikrosios saulėgrąžos (Helianthus annus L.) rekombinantinėse inbredinėse linijose lietinant ir esant drėgmės stresui

Santrauka

Tyrimo tikslas -identifikuoti kiekybinių požymių lokusus (KPL), lemiančius tikrosios saulėgrąžos (Helianthus annuus L.) santykinio drėgmės kiekio ir chlorofilo koncentracijos įvairovę lietinant ir esant drėgmės sukeltam stresui. Septyniasdešimt rekombinantinių inbredinių linijų (RIL) iš 123, gautų sukryžminus PAC2 × RHA266, ir jų tėvinės linijos vertintos atsitiktine tvarka išdėstytoje 8 × 9 bandymo schemoje dviem pakartojimais lietinant ir drėgmės sukelto streso sąlygomis. Lyginant vertinamus požymius, didelis genetinis kintamumas ir transgresinė segregacija nustatyta tarp RIL esant abiem drėgmės sąlygoms. Kiekybiniai požymių lokusai nustatyti naudojant atnaujintą didelio tankio paprastųjų pasikartojančių sekų ir vieno nukleotido pakitimų genolapį. Genolapį sudarė 210 paprastųjų pasikartojančių sekų ir 11 genų, išsidėsčiusių 17 sankibos grupių. Bendras genolapio ilgis yra 1653,1 cM su vidutiniu tankiu 1 žymeklis per 7,44 cM. Lietinant 3 ir 6 KPL buvo identifikuoti atitinkamai chlorofilo koncentracijai ir santykiniam drėgmės kiekiui. Drėgmės sukelto streso sąlygomis buvo identifikuoti atitinkamai 7 ir 2 KPL. Fenotipinės variacijos (R 2 ) dalis, paaiškinama KPL, svyravo nuo 0,39 iki 52,48 %. KPL chlorofilo koncentracijai ir santykiniam drėgmės kiekiui 10 ir 16 sankibos grupėse buvo persidengusios. Bendri KPL įvairiems požymiams, esant abiem drėgmės sąlygoms, sankibos grupėse 10, atrodo, yra svarbesni, nes pasižymi pastovumu nepriklausomai nuo lietinimo sąlygų.

Reikšminiai žodžiai: augalų aprūpinimo drėgme būklė, fotosintezės požymiai, genolapis, Helianthus annuus, KPL lokalizavimas, transgresinė segregacija.

  , Poormohammad Kiani et al. (2007 a; b) and Allinne et al. (2009) in sunflower.

  .).

Table 1 .

 1 Mean squares (MS) of chlorophyll (Chl) concentration and relative water content (RWC%) in sunflower recombinant inbred lines (RILs) and their two parents under two water treatment conditions CV% -coefficient of variation, df -degree of freedom; *, **, *** -significant at 0.05, 0.01 and 0.001 probability level

	Source of variation	df	Chl	MS	RWC%
	Water treatment	1	516.66*** 857.59***
	Replication (water treatment)	2	465.86*** 586.05***
	Genotype	71	37.75**		92.25*
	Genotype × water treatment	71	33.83*	101.87**
	Block (water treatment × replication)	32	21.86 ns	170.24**
	Residual	110	21.47		58.46
	Genotype × water treatment
	effect sliced by water
	treatment for genotype
	Water-stressed	71	29.25*		70.80*
	Well-watered	71	31.11*		84.47 ns
	CV%		24.63		11.03

Table 2 .

 2 Genetic parameters and gain for chlorophyll (Chl) concentration and relative water (RWC%) content in sunflower recombinant inbred lines (RILs) and their two parents under two water treatment conditions

	Conditions	Well-watered	Water stressed
	Item	Chl RWC% Chl RWC%
	PAC2 (P1)	15.19 73.32	69.3	75.75
	RHA266 (P2)	19.10 63.86 67.30 80.21
	P1-P2	-3.91	9.46 -10.93 -4.46
	P	17.14 68.59	68.3	77.98
	Max	34.74 87.90 25.93 88.84
	Min	10.50 44.59	6.74	52.04
	RIL	18.86 67.40 15.97 70.98
	RIL -P	1.70	-1.17 -4.43 -6.90
	10%bestRIL	26.72 80.61 22.30 83.44
	GG10%= 10%bestRIL -P 17.60 * 19.31 * 5.46 ns 10.86 *
	STDEV	4.24	7.84	3.75	7.21
	LSD-I 0.05	6.51	12.56	5.72	8.87
	LSD-C 0.05	9.08	14.99

Table 3 .

 3 Correlation among traits in sunflower recombinant inbred lines (RILs) under well-watered and water-stressed conditions

	Trait	Chl	RWC%
		well-watered condition
	Chl	1
	RWC%	-0.25 ns	1
		water-stressed condition
	Chl	1
	RWC%	0.34*	1
		well-watered with water-stressed
		0.11 ns	0.32*
	Chl -chlorophyll content, RWC% -relative water content;

ns -non significant; * -significant at 0.05 probability level, respectively

Table 4 .

 4 Map position and effect of quantitative trait loci (QTL) detected for chlorophyll (Chl) concentration and relative water content (RWC) in sunflower recombinant inbred lines (RILs) in well-watered and water-stressed conditions

	Trait	QTL	LG	Position cM	LOD	Additive effects	R 2 %
			Well-watered condition
		ChlW.10.1	10 18.31 2.81	2.69	42.19
	Chl	ChlW.10.2	10 25.41 3.57	2.79	0.56
		ChlW.16.1	16 43.01 3.29	2.47	30.29
		RWCW.11.1 11 40.51 3.36	-3.00 21.18
	RWC	RWCW.12.1 12 66.01 3.51 RWCW.16.1 16 47.01 3.37	0.19 -1.14	0.93 1.99
		RWCW.17.1 17 12.91 3.26	1.58	6.79
			Water-stressed condition
		ChlS.3.1	3	29.01 4.90	-1.41 21.10
		ChlS.8.1	8	35.21 4.19	-1.95 52.48
		ChlS.10.1	10 95.61 3.77	-0.09	0.39
	Chl	ChlS.14.1	14 195.31 4.30	-1.05 15.38
		ChlS.15.1	15 29.01 4.20	-0.62	2.18
		ChlS.16.1	16 109.01 3.73	-1.06 14.51
		ChlS.16.2	16 143.01 4.32	-0.73	7.01
	RWC	RWCS.10.1 10 17.31 2.51 RWCS.10.2 10 119.61 2.62	-2.88 23.00

  N. Abdi 1 , R. Darvishzadeh 1 , H. Hatami Maleki 2 , P. Haddadi 3 , A. Sarrafi 4 1 Urmijos universitetas, Iranas 2 Maragheh universitetas, Iranas 3 Prancūzijos nacionalinis žemės ūkio mokslų tyrimo Jean-Pierre Bourgin institutas 4 Ecologie Fonctionnelle laboratorija, Prancūzija

Acknowledgments

We would like to thank the Institute of Biotechnology, Urmia University, Urmia, Iran, for financial support of this work.

Received 31 08 2012 Accepted 08 01 2013


Jacques Teyssier

Martine Migaud

Nathalie Debus

Celine Maton

Emmanuel Tillard

Benoit Malpaux

Philippe Chemineau

Loys Bodin

Expression of seasonality in Merinos d'Arles ewes of different genotypes at the MT1 melatonin receptor gene

Keywords: sheep, seasonality, ovulatory activity, melatonin receptor gene, feeding level Implications

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Seasonality of reproduction remains an important constraint in temperate sheep breeds in which ovulatory activity generally is inhibited in spring. Measurements of spontaneous ovulatory activity (SOA) in early spring, before the introduction of males into the flock, has been used to study out-of-season breeding ability in several low seasonal sheep breeds such as Merinos d'Arles (MA; [START_REF] Hanocq | Genetics parameters of spontaneous spring ovulatory activity in Merinos d'Arles sheep[END_REF], Latxa (Beltran de [START_REF] Beltran De Heredia | Genetic aspects of out of season breeding ability in Latxa sheep[END_REF] and Chios [START_REF] Avdi | Sources of variation and genetic profile of spontaneous, out-of-season ovulatory activity in the Chios sheep[END_REF]. Very similar results with regard to SOA have been found in the above studies; this trait is repeatable and its heritability ranges from 0.12 to 0.22. In the MA breed, a strong association has been observed between breeding value for SOA in April and restriction polymorphism at position 605 in Exon II of the MT1 melatonin -E-mail: Jacques.Teyssier@supagro.inra.fr; Loys.Bodin@toulouse.inra.fr receptor gene, corresponding to the presence (allele 1) or absence (allele 2) of a Mnl I restriction site [START_REF] Pelletier | Association between expression of reproductive seasonality and alleles of the gene for Mel 1a receptor in the ewe[END_REF]. More intense seasonality was associated with genotype 2/2. The discovery of this association extended previous results of [START_REF] Messer | Mapping of the melatonin receptor 1a (MTNR1A) gene in pigs, sheep, and cattle[END_REF] and could point to a specific role of the MT1 genotype in seasonal anoestrus; this study was designed to test this hypothesis. After genotyping MA ewes in a single flock in the first year of the study, groups of ewes from each homozygous genotype (1/1 and 2/2) were identified and their SOA was assessed between the end of drying-off (January) and the beginning of spring mating (April) over two consecutive years. In the first year of this observation period (year two of the study), groups of ewes were managed together on rangelands (experiment 1). In the following year, they were kept in a sheepfold where half of the ewes of each genotype were fed at a higher level than the other half to also test the effect of feeding levels on SOA (experiment 2). This study provides results on genotype frequencies in MA, on SOA in early April for the genotyped ewes, and on the follow-up of SOA of 1/1 ewes and 2/2 ewes between January and mid-April (before spring mating) under different experimental conditions.

Material and methods

Association study between genotype at the MnlI locus and SOA Following genotyping of 314 MA ewes for the Mnl I site early in 2003, their SOA was assessed in April of two consecutive years (2003 and 2004) by measuring peripheral progesterone levels twice in a 9-day period. These ewes from one flock belonging to an experimental farm, Le Merle (south-eastern France, latitude 43.58N), were born in autumn lambing from 1998 to 2001, that is, aged 1 to 4 years at the beginning of 2003. The flock was established in the 1920s and at the time of the study included approximately 1100 pure MA ewes and 30 rams. Renewal was about 20% per year and the foreign breeding material introduced, consisting only of rams or semen, came from a regional breeding centre where male lambs from different sheep flocks were raised to provide breeding rams. These rams were the offspring of ewes qualified as 'ram dams' whose selection criteria were unrelated to seasonality with the exception that the male lambs had to be born in autumn to enter the breeding centre. Over the last 14 years, the percentage of ewes from identified sires has remained above 50%. Genotyping was performed on 314 DNA samples prepared from ewes using the procedure described in detail by [START_REF] Pelletier | Association between expression of reproductive seasonality and alleles of the gene for Mel 1a receptor in the ewe[END_REF]. The resulting genotypes were represented as 1/1, 1/2 and 2/2 according to the presence (allele 1) or absence (allele 2) of the Mnl I restriction site. In addition, the physiological status for SOA of the genotyped ewes (anovulatory or ovulatory) was determined in early April for 2 years (2003 and 2004). For this, we used the criteria established by [START_REF] Thimonier | Progesterone level analysis for the determination of the physiological status of female farm animals (in French)[END_REF], which hold that plasma progesterone concentrations in two blood samples taken 7 to 11 days apart are adequate for the detection of ovulatory activity at a given period in ewes with regular ovarian cycles of 17 days on average and to discriminate the two physiological statuses. The most accurate results are obtained at an interval of 9 days between blood samples. Out of the 314 genotyped ewes, 312 were sampled twice in April 2003; however, only 289 were sampled in April 2004 because some had died or had been culled over the intervening year. Ewes with a progesterone level above the threshold of 1 ng/ml in at least one of the two consecutive blood samples were considered to be ovulatory or cycling. Progesterone concentrations were measured by radioimmunoassay using the technique described by [START_REF] Terqui | Nouvelle me ´thode radioimmunologique rapide pour l'estimation du niveau de progeste ´rone plasmatique. Application pour le diagnostic pre ´coce de gestation chez la brebis et la che `vre[END_REF].

As a rule, all of the MA ewes involved in the study were mated for the first time at 18 months of age and were joined with rams between April and June in subsequent years. They were kept completely separate from males during the months between the annual spring mating.

Assessment of SOA over a long period in 1/1 and 2/2 ewes Experiment 1. In early 2004, two groups of 30 1/1 and 30 2/2 ewes of similar age distribution (2, 3 and 5 years old) were identified from the population of genotyped ewes. The other sampling criteria for adults (3 and 5 years old) were fertility and the number of lambs suckled during the previous autumn lambing. The paternity of all of the ewes was not known; however, when possible, we tried to include ewes from different sires. From 15 January to 14 April, the SOA of ewes was assessed by measuring progesterone concentrations in blood samples taken 7 days apart. As the period of observation was long, the 7-day interval was chosen for logistical reasons, that is, to have a fixed day each week to take blood samples in the ewe groups. With this collection interval, the detection of ovulation is similar to that obtained with an interval of 9 days, especially when one can examine the progesterone levels in more than two consecutive blood samples to determine the status of SOA at a given period [START_REF] Thimonier | Progesterone level analysis for the determination of the physiological status of female farm animals (in French)[END_REF]. The ewes were kept on separate rangelands to avoid repeated sorting of animals during the experiment, and their shearing was carried out over a single day at the end of March. At the end of blood sampling, the two groups of ewes were reunited on 15 April and three rams were introduced into this combined group for 1 month. They then joined the rest of the spring mating MA flock from mid-May to mid-June.

Experiment 2. In January of the following year (2005), 60 ewes from the population of genotyped ewes were divided into two groups of 30 ewes each; in each group, 15 ewes were 1/1 and 15 ewes were 2/2. These groups of ewes of similar age distribution (3, 4 and 6 years old) were balanced in terms of live weight (LW), body condition score (BCS), fertility and the number of lambs suckled during the previous autumn lambing. Several ewes involved in this experiment (15 1/1 and 7 2/2) were not the same as in the previous year due to the sampling procedure and the replacement of dead or culled animals. The two groups of ewes were Teyssier, Migaud, Debus, Maton, Tillard, Malpaux, Chemineau and Bodin transferred to a sheepfold with natural light and, after a 15-day period of acclimatization together, placed in two adjacent pens with different feeding levels from 3 February to 14 April. One group was fed at a low level (L): 70% of maintenance energy requirements, with a diet composed of straw and pelleted alfalfa. The other one was fed at a high level (H): about 170% of maintenance energy through the addition of pelleted alfalfa and pelleted beet pulp. Daily rations for the H and L diets were formulated to increase the BCS by about 1 point in 60 days (H diet) or to maintain the LW and BCS at the level at which they were at the end of drying-off (L diet). The H diet was reduced to maintenance requirements beginning in late March to prevent excessive fattening at mating. Daily rations were established on the basis of standard feeding values for the ingredients [START_REF] Jarrige | Alimentation des bovins, ovins et caprins, 1st edition[END_REF]. They were distributed, between midday and 1400 h, in racks allowing simultaneous access to all ewes. In each pen, the ewes had free access to water, salt blocks and a mineral-vitamin pre-mix.

Immediately before the different diets were introduced, SOA was determined by the progesterone levels in the two blood samples taken 9 days apart, using the rules of interpretation already defined for the two samples. SOA was then assessed through blood samples collected at 9-to 10-day intervals from 25 February to 14 April. Ewes were weighed at this interval and their condition scored at a double interval on a 5-point graded scale [START_REF] Russel | Subjective assessment of body fat in live sheep[END_REF] with 0.25 subdivisions. After an additional 12-day period of re-feeding (or feeding) at the maintenance requirement level in the sheepfold, the ewes were exposed to rams on pastures from 27 April to mid-June. For this experiment, shearing was delayed until the beginning of May, that is, during the mating period.

Periods of SOA For the long-term experiments, determination of status (cycled or non-cycled) of a ewe at each blood sampling point of time was based on progesterone level at the time of sampling and additional information obtained from other progesterone measurements on the same ewe during the sampling period. Unlike the analysis of two consecutive blood samplings, interpretation of progesterone levels in sequential blood samples required taking into account the timing of observations. A measurement below the threshold of 1 ng/ml of progesterone (coded 0, non-cyclic or follicular phase) that followed a measurement above the threshold (coded 1, luteal phase) was therefore included systematically in the definition of periods of SOA. With the 7-day interval retained in experiment 1, the infrequent occurrence of two consecutive measurements below the threshold that both followed and preceded observations above the threshold also were included in periods of continuous activity. For example, in experiment 1, the vector of n observations for a ewe [1 0 1 1 0 0 1 0 0 0 0 1 1 0] was interpreted as [ 1 1 1 1 1 1 1 0 0 0 1 1 1]. In experiment 2, in which blood samples were taken 9 or 10 days apart, the vector [0 1 0 1 0 0] was interpreted as [ 1 1 1 1 0]. The first observation was used to determine the SOA of ewes at the following date and analysis of modified data was carried out on n21 variables.

Statistical procedures

The repeatability of SOA for the 2 years of measurements in April on all genotyped ewes was estimated by the coefficient f, which is a measure of the degree of association between all-or-nothing traits as two binary variables [START_REF] Gu | On the methods of measuring association between two attributes, (with discussion)[END_REF]. The effects of genotype and feeding level on SOA variables (0/1 modified data) were investigated by a logistic regression of longitudinal data sets from experiments 1 and 2 using a marginal model, generalized estimating equations (GEE; [START_REF] Diggle | Analysis of longitudinal data[END_REF]. GEE can be regarded as an extension of GLM for the analysis of correlated data. In the general framework of longitudinal analysis, GEE takes into account the dependency of repeated observations within subjects by specifying a 'working correlation structure'. In this case, the correction for within-ewe correlation was carried out by assuming an auto-regressive correlation structure for the repeated-in-time measurements. This option was retained in the model to obtain efficient estimates of the standard error (robust s.e.) of coefficients and hence efficient inference tests (Wald test), the Wald statistic being used for assessing significance of coefficients [START_REF] Hosmer | Applied logistic regression[END_REF]. Under the null hypothesis, this statistic, also called z, will follow a standard normal distribution.

In these analyses, the date of observation was introduced as a linear continuous variable (coded from 0 for the first date to n for the last date) and genotype, age and diet as categorical variables. Interactions that are important for interpreting the effect of genotype, for example, genotype by date and genotype by diet, were introduced in a first step and were excluded from the final model if they had no significant effect (Wald test with a P-value .0.05). The genotype 2/2, the first class of age (2 or 3 years old according to the studied data set) and the L diet were used as reference categories. Analyses were performed using R software: GEE function, binomial family, logit link, AR-M (M 5 1) correlation structure (R Development Core Team, 2007).

Other comparisons between genotypes (and feeding levels) on SOA percentage were carried out using x 2 test and odds ratio (OR, and CI for its confidence interval). Onetailed t-test was used for the comparisons between feeding levels on ewe LW and ewe BCS.

Results

Genotypic frequencies and SOA in April

Frequencies of genotypes 1/1, 1/2 and 2/2 in the population of 314 genotyped ewes were 43.0% (n 5 135), 44.9% (n 5 141) and 12.1% (n 5 38), respectively; this agrees with the Hardy-Weinberg equilibrium (p 2 , 2pq, q 2 with p 5 0.65 and q 5 12p) and suggests that the Mnl I locus is not under selection in the MA population under investigation. With regard to this large sample of MA ewes (Table 1), there was no significant difference between genotypes in the percentage of ovulatory ewes in early April in Expression of seasonality in MA ewes of different MT1 genotypes either year of measurement (x 2 test: P 5 0.83 and P 5 0.35 in 2003 and 2004, respectively). Only 30 ewes were ovulating in early April in both of the 2 years, and none was of genotype 2/2 (17 1/1, 13 1/2). The repeatability of SOA for the two consecutive years was 0.28. Time changes in SOA of homozygous ewes Experiment 1. Two ewes of genotype 2/2 were eliminated during the experiment: one at the beginning, the other died before the last blood sampling. The representation of changes in the percentage of cyclic ewes from January to mid-April (Figure 1) displays a similar change in SOA over time in both groups of ewes (30 1/1 and 29 to 28 2/2). This change was marked by the occurrence of a temporary rise in ovulatory activity triggered prior to shearing on 29 March. In early April, which is the usual period of SOA measurement before rams are introduced, no difference was observed between genotypes. The overall percentage of cyclic ewes on 14 April was 25.9%.

Patterns of SOA for individual ewes (Figure 2) show that many ewes of both genotypes exhibited discontinuities in their ovulatory activity. In several of these ewes, no ovulatory activity was detected for periods of 2 to 3 weeks, suggesting the failure of a single cycle. The proportion of ewes continuously cycling at least until the apparent end of their SOA (11/30 and 6/29 for 11 and 2/2 ewes, respectively) did not differ significantly between genotypes (OR 5 2.22 with a 95% CI of 0.69, 7.12).

The results of the GEE analysis of this data set showed in the first place that the interaction between genotype and date was not significant. Therefore, the final fitted model was reduced to the main effects of genotype, age and date variables (Table 2). The effect of genotype on SOA was not significant (P 5 0.68). For the age effect, the 5-year-old ewes were much more likely to be cyclic than the 2-year-olds (P , 0.001), while no significant difference was observed between 3-and 2-year-old ewes (P 5 0.38). The effect of date was highly significant (P , 0.001) with a negative estimate of the coefficient; this linear variable indicates an overall decrease of SOA among the ewes from January to mid-April.

Experiment 2. Changes in ewe LW and ewe BCS during this experiment are shown in Table 3 for the groups of ewes fed at high and low levels (15 or 14 1/1 and 15 2/2 ewes per group; one 1/1 ewe was eliminated early after the Teyssier, Migaud, Debus, Maton, Tillard, Malpaux, Chemineau and Bodin constitution of the ewe groups). Average values for LW and BCS, which were equal between groups when they were constituted, differed significantly after 1 month of differential diets. From early February until the end of the experiment, the H diet induced an increase of 8.2 kg for LW and 0.8 point for BCS, while these traits remained approximately constant until the end of experiment for the ewes on the L diet.

For this controlled group feeding experiment, results of the GEE analysis of the SOA data set from 7 March to mid-April, when the ewes had been fed at different levels since early February, indicated similar trends as in experiment 1. The interaction between genotype and date as well as between genotype and diet were not significant; they consequently were excluded from the final model whose results are given in Table 4. These results confirmed the absence of effect of genotype on SOA (P 5 0.35); the presence of effect of age, with the 6-year-old ewes significantly more cyclic than the 3-year-olds (P , 0.01); and the significant decrease in SOA among ewes during the period of study (P , 0.001). In addition, the result for the diet variable (P 5 0.32) showed that the feeding level had no significant effect on the cyclicity of ewes within this period. The corresponding changes in SOA according to genotype (irrespective of diet) or to diet (irrespective of genotype) are presented in Figures 3 and4, respectively. At the end of this experiment on 14 April, the average percentage of cyclic ewes was 16.9%.

Fertility in spring mating

In experiment 1, in which ewes were joined with rams the day after the last blood sampling, there was no significant difference between genotypes in fertility in spring mating: 90% of 1/1 ewes and 89.3% of 2/2 ewes later lambed the following autumn. There also was no difference in prolificacy (1.32 and 1.28, respectively). During the first 15 days after joining, on the basis of autumn lambing, there were a few more 1/1 ewes fertilized or conceiving than 2/2 ewes (6 and 3 ewes, respectively), but this difference was not significant. Furthermore, the cumulative distribution of lambing dates did not differ significantly between genotypes (P . 0.05, non-parametric Kolmogorov-Smirnov two-sample test). In experiment 2, the comparison between genotypes of the fertility of ewes after the spring mating was not an objective because of the 12-day period of re-feeding of underfed ewes Expression of seasonality in MA ewes of different MT1 genotypes before mating and the delayed shearing time, which had been postponed until after mating began. [START_REF] Pelletier | Association between expression of reproductive seasonality and alleles of the gene for Mel 1a receptor in the ewe[END_REF] have shown that a mutation at position 612 in Exon II of the ovine MT1 gene leads to the absence of a cleavage site for the MnlI enzyme at position 605 (allele 2). They showed that this mutation was always found to be associated with a single allelic isoform among the different isoforms described for this exon. This haplotype is characterised by the specific and constant presence of four mutations, including the one (612) responsible for MnlI polymorphism, two other silent mutations at positions 453 and 891, and a mutation at position 706 that results in one base change in the amino acid sequence of the predicted MT1 receptor protein (substitution of a valine by an isoleucine). Several independent results point out a potential functional role of this locus on the control of seasonality:

Discussion

1. Within breeds, [START_REF] Pelletier | Association between expression of reproductive seasonality and alleles of the gene for Mel 1a receptor in the ewe[END_REF] found a highly significant difference in the frequency of homozygotes 2/2 between two groups of MA ewes screened for their SOA in spring. Ewes homozygous for the absence of the Mnl I site were not observed in the group of ewes cycling in April over three consecutive years (1995 to 1997). This study conducted on the same flock showed that 2/2 ewes also were not found in the group of 30 ewes that cycled in April over two consecutive years (2003 and 2004). Furthermore, a significant effect of Mnl I polymorphism on fertility in spring mating has been reported [START_REF] Notter | Evaluation of melatonin receptor 1a as a candidate gene influencing reproduction in an autumn-lambing sheep flock[END_REF] for adult ewes from a composite line (50% Dorset, 25% Rambouillet, 25% Finnsheep). Adult ewes with the genotype 2/2 had lower spring fertility. Significant differences between 1/1 or 1/2, and 2/2 genotypes in lambing frequency also were reported among Dorset ewes managed by an accelerated lambing system [START_REF] Mateescu | Association between melatonin receptor 1A gene polymorphism and reproductive performance in Dorset ewes[END_REF]. 2. Comparisons between breeds also support the functional role of the MT1 genotype. Substantial changes in allelic frequencies at the MnlI site with the degree of seasonality of breeds have been described by several different authors [START_REF] Messer | Mapping of the melatonin receptor 1a (MTNR1A) gene in pigs, sheep, and cattle[END_REF][START_REF] Pelletier | Association between expression of reproductive seasonality and alleles of the gene for Mel 1a receptor in the ewe[END_REF][START_REF] Notter | Opportunities for detection and use of QTL influencing seasonal reproduction in sheep: a review[END_REF]. For example, ewes of seasonal breeds had relatively higher frequencies of genotype 2/2 than ewes of low seasonal breeds. Thus, genotype 2/2 was not found in a large sample of ewes from the non-seasonal small-tailed Han breed in China (Chu et al., 2003) and the following gradient of the percentage of 2/2 ewes was encountered in samples of several breeds: 9% in Blackbelly; 7% in Barbarine; 10% in MA; 31% in Ile-de-France; 35% in Suffolk; 100% in wild Mouflon [START_REF] Trecherel | Relationship between MT 1 melatonin receptor polymorphism and seasonality of reproduction in sheep through genotyping and functional studies[END_REF]. 3. Various physiological studies reinforce the assumption of a close relationship between the expression of seasonality and polymorphism of the MT1 receptor gene. In MA 2/2 ewes, a higher density of melatonin receptors has been found in the pars tuberalis [START_REF] Pelletier | Association between expression of reproductive seasonality and alleles of the gene for Mel 1a receptor in the ewe[END_REF] as well as in four other brain areas [START_REF] Trecherel | Relationship between MT 1 melatonin receptor polymorphism and seasonality of reproduction in sheep through genotyping and functional studies[END_REF]. One of these areas belongs to the pre-mammillary hypothalamus, which is an important site of melatonin action regulating the reproductive function in sheep [START_REF] Malpaux | Biology of mammalian photoperiodism and the critical role of the pineal gland and melatonin[END_REF]. 4. Moreover, the contrast between the unique allelic isoform of Exon II associated with higher seasonality in several breeds and large variability of other haplotypes suggests a common origin of this particular haplotype, which had not been lost through selection and dissemination due to the selective advantage it confers.

Despite these findings, the relationship between the studied part of Exon II of the MT1 gene and the phenotype of seasonality remains unclear. First, this study indicates that in MA, no difference was observed between ewes of genotype 1/1 and 2/2 in the rate of SOA in early spring or on fertility in spring mating. Second, various attempts failed to find a difference by various criteria (beginning, end or length of the breeding season) within the genotyped progeny (1/1 and 2/2) of heterozygous sires in Ile-de-France [START_REF] Hernandez | Relationship between MT1 melatonin receptor gene polymorphism and seasonal physiological responses in Ile-de-France ewes[END_REF] and Latxa breeds [START_REF] Ugarte | Exploring the relationship between the polymorphism of the melatonin receptor gene and the variability of sheep seasonality[END_REF].

How may these two contradictory sets of findings be reconciled? It is unlikely that the relationship between the Teyssier, Migaud, Debus, Maton, Tillard, Malpaux, Chemineau and Bodin expression of seasonality and polymorphism of the MT1 gene found in MA [START_REF] Pelletier | Association between expression of reproductive seasonality and alleles of the gene for Mel 1a receptor in the ewe[END_REF], OOS [START_REF] Notter | Evaluation of melatonin receptor 1a as a candidate gene influencing reproduction in an autumn-lambing sheep flock[END_REF] and Dorset [START_REF] Mateescu | Association between melatonin receptor 1A gene polymorphism and reproductive performance in Dorset ewes[END_REF] ewes is casual. On the other hand, the mutated haplotype has been observed in enough animals of different breeds to exclude a sampling artefact. One hypothesis explaining this discrepancy would be the existence of another non-conservative mutation on an independent gene. Selection for spring ovulation would increase the frequency of favourable alleles at both sites, including the MnlI site. This would explain the results observed by [START_REF] Pelletier | Association between expression of reproductive seasonality and alleles of the gene for Mel 1a receptor in the ewe[END_REF]. In contrast, the screening of animals only for the presence or absence of the Mnl I site would not have any influence on the proportions of alleles at the other site, and this therefore would be insufficient to induce a change in the expression of seasonality. Support for this hypothesis would come when such a non-conservative mutation has been identified; however, it has not yet been found. With regard to environmental effects, it is important to note that the beginning of the unexpected rise of SOA in the first experiment (experiment 1) was observed on 25 March, that is, 4 days before shearing. No other change in management, handling or treatment may be associated with the increase of SOA on this date. The amplitude of the peak of SOA was probably related to shearing; however, its start before shearing might reflect spontaneous changes in the Mediterranean environment of grazing ewes with an increase in pasture availability and forage intake on rangelands after the colder months (January and February). Such events (shearing, re-growth on rangelands) could partially explain the inter-annual variations of the cyclicity rate observed in early April for MA ewes from first generations of selection lines for SOA at this time [START_REF] Teyssier | Preliminary results on selection lines for spontaneous spring ovulatory activity in Merinos d'Arles sheep[END_REF]. The introduction of controlled feeding levels in experiment 2 was also justified by different studies that have shown relationships between ovulatory activity and LW and BCS of ewes from Merino flocks [START_REF] Hanocq | Genetics parameters of spontaneous spring ovulatory activity in Merinos d'Arles sheep[END_REF][START_REF] Kleemann | Fertility in South Australian commercial Merino flocks: relationships between reproductive traits and environmental cues[END_REF]. In this study, the occurrence of a small divergence between diets at the end of the study period might suggest a delayed effect of feeding level on SOA [START_REF] Teyssier | Expression of seasonality in the Merinos d'Arles ewe according to the genotype of the melatonin receptor (MT1) gene and the feeding level[END_REF], but such a hypothesis was clearly challenged by the final analysis of results using a model suited to this type of data. The substantial differences in LW and BCS induced by differential feeding levels from February thus had no significant effect on SOA at the beginning of April. This agrees with results on Barbarine ewes [START_REF] Khaldi | Variations saisonnie `res de l'activite ´ovarienne, du comportement d'oestrus et de la dure ´e de l'anoestrus post-partum des femelles ovines de race Barbarine: influences du niveau alimentaire et de la pre ´sence du ma ˆle[END_REF], which have shown that significant variations of ewe LW between drying-off and the spring introduction of rams had no significant effect on the percentage of cyclic ewes before the beginning of mating, whereas initial differences in LW established during a preexperimental period before drying-off significantly affected this percentage.

In conclusion, results of this study show that genotype at locus MnlI of the MT1 receptor gene cannot be used alone as a genetic selection marker for spring (out-of-season) breeding in sheep, at least not in the MA breed. Further studies are required to define the role of this genomic region in the expression of seasonality. In addition, while the results illustrate the responsiveness of SOA to events such as shearing and spontaneous variations in the Mediterranean environment of grazing ewes, they do not show a significant effect of differential feeding of sheepfold ewes after dryingoff on their SOA before spring mating.

Figure 1 Figure 2

 12 Figure1Changes in the percentage of cyclic ewes between January and mid-April for 1/1 ewes (n 5 30) and 2/2 ewes (n 5 29 to 28) in experiment 1.

Figure 3

 3 Figure3Changes in the percentage of cyclic ewes between February and mid-April for 1/1 ewes (n 5 29) and 2/2 ewes (n 5 30) in experiment 2 (irrespective of diet).

Figure 4

 4 Figure 4 Changes in the percentage of cyclic ewes between February and mid-April for H level (n 5 30) and L level (n 5 29) ewes in experiment 2 (irrespective of genotype). Ewes were fed at different levels from 3 February.

Table 1

 1 Percentage of ovulatory ewes in early April in the MA genotyped for the MnlI site, according to genotype and year of measurement

		2003		2004
	Genotype	Number of ewes 1	Ovulatory ewes (%)	Number of ewes 1	Ovulatory ewes (%)
	1/1	133	30.8	129	20.2
	1/2	141	28.4	129	15.5
	2/2	38	26.3	31	25.8
	MA 5 Merinos d'Arles. 1 Number of ewes sampled on 2 and 11 April 2003 (n 5 312); 5 and 14 April 2004 (n 5 289).

Table 2

 2 Estimated coefficients, s.e., Wald statistics and P-values for the GEE model fitted to SOA data set from experiment 1, including genotype and age categorical variables and date continuous variable Variable Category 1 Coefficient s.e. 2 Wald statistic (z ) P . z

	Genotype 1/1	0.133 0.327	0.41	0.684
	Age	3 years old	0.472 0.537	0.88	0.380
		5 years old	1.264 0.335	3.77	,0.001
	Date		20.096 0.024	23.92	,0.001

GEE 5 generalized estimating equations; SOA 5 spontaneous ovulatory activity. 1 Reference categories for categorical variables are not included. 2 Robust s.e. of the coefficients provided by the GEE method with the option of an auto-regressive structure of correlation within each ewe. z 5 coefficient/s.e., with P-value calculated from a normal table.

Table 3

 3 Evolution of LW and BCS in the groups of ewes fed at H and L levels (170% and 70% of maintenance energy requirements, respectively) from early February: mean value (s.d.)

	Date

LW 5 live weight; BCS 5 body condition score; H 5 high level; L 5 low level. 1 Constitution of the groups of ewes. 2 Start of differential feeding levels.

3 Unilateral t-test by the hypothesis that the mean value for H diet was greater than the mean value for L diet.

Table 4

 4 Estimated coefficients, s.e., Wald statistics and P-values for the GEE model fitted to SOA data set from experiment 2 (7 March to 14 April), including genotype, age and diet categorical variables and date continuous variable Variable Category 1 Coefficient s.e. 2 Wald statistic (z) P . z

	Genotype 1/1	2 0.478 0.506	20.94	0.345
	Age	4 years old	0.977 0.686	1.43	0.154
		6 years old	1.711 0.635	2.70	0.007
	Diet	H level	0.522 0.522	1.00	0.318
	Date		20.368 0.091	24.04	,0.001
	GEE 5 generalized estimating equations; SOA 5 spontaneous ovulatory
	activity; H 5 high level.

1

Reference categories for categorical variables are not included. 2 Robust s.e. of the coefficients provided by the GEE method with the option of an auto-regressive structure of correlation within each ewe. z 5 Coefficient/s.e. with P-value calculated from a normal table.

Acknowledgements

We wish to thank especially P.M. Bouquet in charge of the experimental farm 'Domaine du Merle', J.D. Guyonneau, D. Montier and J.M. Capron for their contribution to the experiments and the staff of the INRA assay laboratory of Nouzilly for the progesterone assays. We are also grateful to A. Collet who performed genotyping of ewes. This work was supported by Institut National de la Recherche Agronomique (INRA): Departement PHASE (Grants CI 2004 and CI 2005), Departement GA.