Gustavo R Rodríguez

Stéphane Muños

Claire Anderson

Sung-Chur Sim

Andrew Michel

Mathilde Causse

Brian B Mcspadden Gardener

David Francis

Esther Van Der Knaap
email: vanderknaap.1@osu.edu

Distribution of SUN, OVATE, LC and FAS in the Tomato Germplasm and the Relationship to Fruit Shape Diversity

ABSTRACT

Phenotypic diversity within cultivated tomato (Solanum lycopersicum) is particularly evident for fruit shape and size. Four genes that control tomato fruit shape have been cloned. SUN and OVATE control elongated shape whereas FASCIATED (FAS) and LOCULE NUMBER (LC) control fruit locule number and flat shape. We investigated the distribution of the fruit shape alleles in the tomato germplasm and evaluated their contribution to morphology in a diverse collection of 368 predominantly S. lycopersicum and S. lycopersicum var. cerasiforme accessions. Fruits were visually classified into eight shape categories that were supported by objective measurements obtained from image analysis using the Tomato Analyzer software. The allele distribution of SUN, OVATE, LC and FAS in all accessions was strongly associated with fruit shape classification. We also genotyped 116 representative accessions with additional 25 markers distributed evenly across the genome. Through a model-based clustering we demonstrated that shape categories, germplasm classes and the shape genes were non-randomly distributed among five genetic clusters (p <0.001), implying that selection for fruit shape genes was critical to subpopulation differentiation within cultivated tomato. Our data suggested that the LC, FAS and SUN mutations arose in the same ancestral population while the OVATE mutation arose in a separate lineage. Furthermore, LC, OVATE and FAS mutations may have arisen prior to domestication or early during the selection of cultivated tomato whereas the SUN mutation appeared to be a post-domestication event arising in Europe.

INTRODUCTION

Tomato (Solanum section Lycopersicon) is native to western South America, from Ecuador and Peru to Bolivia and northern Chile. Cultivated tomato (S. lycopersicum L.) is postulated to have been domesticated in Mexico (Jenkins, 1948) with Peru suggested as an alternative location (De Candolle, 1886). The fruit of S. lycopersicum var. cerasiforme, also known as "cherry tomato", are typically larger than fruit of the wild species but smaller than those of cultivated tomato. Wild cherry tomato is hypothesized to be a direct progenitor of cultivated tomato (Rick and Holle, 1990). However, others consider "var. cerasiforme" a revertant from cultivation (i.e.,feral plants) or a possible hybrid between wild and weedy taxa (Peralta et al., 2008). Indeed, previous studies have shown that most accessions of S. lycopersicum var. cerasiforme are more closely related to cultivated tomato than to wild relatives and others that are an admixture between S. lycopersicum and the wild relative S. pimpinellifolium possibly resulting from the frequent hybridizations between them (Nesbitt and Tanksley, 2002;Ranc et al., 2008). Some of the most important changes that occurred during the domestication and improvement of tomato were increased fruit weight and the emergence of variable fruit shapes and colors (Paran and van der Knaap, 2007). Tomato varieties have been classified based on fruit morphology into shape categories described by the International Union for the Protection of New Varieties of Plants (UPOV) and the International Plant Genetic Resources Institute (IPGRI) (IPGRI, 1996;UPOV, 2001). In addition to the fruit shape categories, tomatoes have also been categorized into germplasm classes based on geographic origin and/or age. However, definitions for germplasm classes are neither accepted by all nor clearly delineated, with categories partially overlapping. Tomatoes classified as regional or landraces are farmer or gardener-selected and are 5 adapted to the local environment typically in areas of local subsistence (Male, 1999). The term "vintage" and "contemporary" (modern) tomato refers to tomato accessions released before (vintage) or after (contemporary) a certain year (Williams and St Clair, 1993;Park et al., 2004;Sim et al., 2009). Breeding and elite breeding lines are used in current breeding programs that seek to develop commercially competitive varieties. U.S. heirloom tomatoes comprise a diverse and loosely defined group. Heirlooms have been referred to as accessions handed down from generation to generation, old commercial varieties, contemporary varieties created to fill niche markets, those of mysterious origin, or "treasured" (Male, 1999). Many heirloom varieties were brought to North America by European settlers and therefore could be considered as regional accessions or landraces in the country from where they originated. Fruit shape and locule number are quantitatively inherited characters with estimates of QTL number ranging from four to 17 with the major loci explaining 19 % to 79 % of the genetic variation (Barrero and Tanksley, 2004;Brewer et al., 2007;Gonzalo and van der Knaap, 2008). The sun and ovate loci control fruit elongation and the underlying genes are known. SUN encodes a protein that is a positive regulator of growth resulting in elongated fruit and is hypothesized to alter hormone or secondary metabolite levels (Xiao et al., 2008). The mutation is the result of a gene duplication event that was mediated by the retrotransposon Rider (Xiao et al., 2008;Jiang et al., 2009). OVATE encodes a negative regulator of growth, presumably by acting as a repressor of transcription and thereby reducing fruit length (Liu et , 2002;Hackbusch et al., 2005;Wang et al., 2007). The OVATE allele that conditions an elongated fruit carries a premature stop codon and is presumed to be a null allele (Liu et al., 2002). Locule number, which has a pleiotropic effect on fruit shape and size, is controlled by the fas and lc loci. FAS 6 encodes a YABBY transcription factor and down regulation of the gene is caused by a large insertion in the first intron (estimated to be 6 to 8 kb) resulting in fruits with high locule number (Cong et al., 2008). The molecular nature of LC was recently identified (Muños et al. unpublished data). Two single nucleotide polymorphisms (SNP) were found to be critical in controlling the locule number phenotype and were located ~1200 bp downstream of the stop codon of a gene encoding a WUSCHEL homeodomain protein, members of which regulate stem cell fate in plants (Mayer et al., 1998).

The goals of this study were to determine whether allelic distribution of SUN, OVATE, LC, and FAS was associated with fruit shape, genetic background as well as geographical and historical origin in a diverse collection of cultivated accessions. This information would offer important insights into the number of genes involved and their effect on fruit shape in the tomato germplasm. Moreover, the knowledge of the distribution of the fruit shape gene alleles would allow us to examine the molecular events that accompanied domestication and selection of this important crop. We showed that the diversity in tomato fruit morphology was explained to a large extent by mutations in the SUN, OVATE, LC, and FAS genes. We analyzed the genetic clustering of this dataset relative to fruit shape category and germplasm class, and demonstrated non-random distribution of the major fruit shape alleles. Moreover, our data suggested that FAS and SUN arose in the LC mutant background. OVATE on the other hand arose in a different ancestral population. Finally, our studies offered valuable insights into the evolution of tomato from a round berry to a fruit with diverse shapes.

RESULTS

Tomato germplasm and fruit shape categorization

We visually classified 368 tomato accessions according to eight fruit shape categories: "flat", "round", "rectangular", "ellipsoid", "heart", "long", "obovoid" and "oxheart" (Figure 1, Table S1). These accessions represented eight germplasm classes based on geographic origin and/or history (Table I). Some of the fruit shape categories were represented by accessions from all germplasm classes whereas other shape categories contained accessions from only a few classes (Table I). For example, fruit in the ellipsoid category was found in nearly all germplasm classes with the exception of the seven wild accessions, which produced only round fruits. In contrast, long tomatoes were commonly found among U.S. heirloom and regional Spanish accessions and rectangular shape was represented mostly by Italian accessions (Table I).

Phenotypic diversity analyzed by Tomato Analyzer

To analyze fruit shape objectively, and determine whether the visual categories were supported by quantitative shape measurements, we obtained the values for 36 fruit attributes using image analysis. A subset of 120 accessions (hereafter called the subcollection) that represented the diversity of the fruit shape and germplasm classes observed in the larger collection of 368 accessions was phenotypically evaluated using Tomato Analyzer (TA) (Brewer et al., 2006;Gonzalo et al., 2009). Iterations of Linear Discriminant Analysis (LDA) led to the identification of the seven most important attributes that define the fruit shape categories. The final set of attributes identified for their predictive value were: fruit shape index, distal end protrusion, widest width position, the proximal end blockiness value at 20% from the proximal end, rectangular, distal angle at 20% along the boundary from the tip of the fruit and proximal eccentricity. The range of these attributes for each shape category is listed in Table S2. Using the 8 measurements of these attributes, 83% of fruit could be accurately classified by a simple linear discriminant function according to the cross validation test (Table II). The relatively few discrepancies between the visual classification and the objective classification based on TA attributes arose with fruits classified as ellipsoid, obovoid or rectangular. Each of these three categories had some accessions classified as one or the other. Upon closer inspection, approximately one third of these were misclassified during the initial visual scoring of the collection. The remaining misclassified accessions were the result of transitioning and overlapping values for the seven attributes in the ellipsoid, obovoid and rectangular shape categories (Table S2). Regardless, the objective assessment using the seven attributes in TA provided a robust classification of fruit shape categories consistent with subjective international descriptors.

Allelic distribution of SUN, OVATE, LC and FAS according to fruit shape category and germplasm class.

We determined the alleles for SUN, OVATE, LC, and FAS in the 368 accessions comprising the entire collection. The data showed that all obovoid, and many of the ellipsoid (83%), rectangular (59%) and heart (48%) tomatoes carried the mutant allele of OVATE whereas most of the long (88%) and oxheart (83%) tomatoes carried the mutant allele of SUN (Table I). The most frequent mutation in flat tomatoes was LC (82%) followed by FAS (28%). Many of the long tomatoes also carried the mutation in the LC gene (63%), a finding that was supported by genetic evidence for the lc QTL in a population that segregated for elongated fruit shape (Gonzalo et al., 2009). All oxheart tomatoes carried the LC mutation in addition to SUN and/or FAS. Most round tomatoes carried the wild type allele at the four shape loci, with the LC mutation most prevalent 9 at 33%. The majority of the round tomatoes with the LC mutation were S. lycopersicum var cerasiforme lines (Table S1). To evaluate whether fruit shape category and shape gene mutations were correlated to one another, we conducted a Chisquare test. The test corroborated the lack of independence between fruit shape categories and alleles of SUN, OVATE, LC, and FAS (χ 2 = 790, df = 84, p < 0.0001) indicating that the tomato shape mutations have a major impact on fruit morphology. These results were further supported by a quantitative association of fruit shape alleles with specific shape attributes (Table S3).

The mutant alleles of LC and OVATE were well represented in all germplasm classes with the exception of the wild species (Table I). The FAS mutation was often present in U.S. heirloom and regional Italian collection whereas the SUN mutation was often present in U.S. heirloom and regional Spanish accessions. The most common mutant alleles in the Italian collection were for OVATE and LC. The FAS mutation was more common in the regional than in the contemporary Italian collection. There were very few Italian accessions that carried the mutation in the SUN gene (Table 1). We also investigated the distribution of alleles in the Latin American germplasm. As expected, only wild type alleles for the fruit shape genes were found in the wild species (Table I). Moreover, none of the Latin American regional and cerasiforme accessions carried the mutation in SUN, suggesting that the gene duplication resulting in elongated fruit shape did not occur in South or Central America. In contrast, the mutations in OVATE, LC, and FAS were found in Latin American regional and cerasiforme accessions. The most common mutant allele was for LC, followed by OVATE and FAS (Table I). Two cerasiforme accessions (LA1655 and VIR739) carried both the LC and FAS mutations and exhibited flat fruits and higher locule number (6.8 and 7.5, respectively) than the LC mutation alone (3.4 locules) in this background (Table S1).

Genetic analysis of the subcollection

To evaluate genetic structure in relation to germplasm and morphological categories, we genotyped 120 accessions with an additional 25 loci that were randomly chosen based on their distribution across the genome. With the exception of two cerasiforme accessions, these 118 represented cultivated tomato. After the genotypic evaluations, we removed four accessions because they were identical to another accession in the subcollection. The number of alleles per locus ranged from two to nine for ten SSR markers with a mean of 5.5 (Table S4). The remaining markers had two alleles per locus with the exception of the markers SP and LeOH16.2 which had three. The resulting dataset of 114 cultivated and two cerasiforme accessions and 29 markers (including the four fruit shape genes) was analyzed with the STRUCTURE 2.2 software (Pritchard et al., 2000). We tested population structure for K = 1-15 and determined that the best number of clusters is 5 (Evanno et al., 2005) (Figure S1 andS2). We tested the consistency among five different runs at K = 5 after which we determined the ranking of inferred ancestry among accessions within each cluster as well as the stability of accessions in the same cluster (Figure 2). This analysis indicated that none of the accessions changed from one to another cluster and that the grouping of the accessions was robust. The analysis was repeated with identical settings for burn-in and iterations but only including the 25 randomly chosen markers and omitting the four fruit shape genes. This analysis did not define a best K for the number of clusters (Figure S1). Therefore, the alleles of SUN, OVATE, LC, and FAS appeared to be essential in determining genetic structure for this dataset, suggesting that selection for fruit shape was responsible for differentiating statistically distinct subpopulations within the collection of 116 cultivated accessions.

Pairwise FST and Nei's genetic distances of the STRUCTURE clusters

To test the significance of the genetic clusters at K = 5, we conducted pairwise FST (θ) (Weir and Cockerham, 1984) analyses. We found that the STRUCTURE clusters were significantly different from one another, strongly supporting the genetic grouping (Table S5). Based on Nei's genetic distances (Nei, 1978), one of the most distinct STRUCTURE groups was represented by cluster 5, comprised of contemporary accessions, sharing the fewest common alleles with other clusters. This finding is not surprising because recent introgression of disease resistance genes from wild relatives have enhanced the diversity in this germplasm class (Ruiz et al., 2005;Sim et al., 2009). Clusters 1 and 2 as well as clusters 3 and 4 shared more alleles relative to other pairwise comparisons (Table S5). Germplasm classes including regional Italian, regional Spanish and U.S. heirloom lines and fruit shape categories including flat, ellipsoid and obovoid were associated with genetic clusters at p < 0.001 (Table S6A,B). Specifically, heirloom accessions were significantly over-represented in cluster 3, regional Spanish accessions were significantly over-represented in cluster 4 whereas regional Italian accessions were significantly over-represented in cluster 1 (p < 0.05) (Table S6A). In addition, three of four contemporary U.S. lines and four of nine contemporary Italian lines grouped together in cluster 5 (Figure 2). We did not statistically analyze these two germplasm classes because there were fewer than 10 in each group. Nevertheless, these results suggested that the contemporary accessions, whether American or 12 Italian, carried similar alleles implying that the accessions were related to one another or that selection through breeding favored the same allele at multiple loci. With respect to shape category, the flat category was significantly over-represented in cluster 3 and 4, and the ellipsoid category in cluster 1 and 2. The obovoid category was randomly distributed among the different genetic clusters (p > 0.05, Table S6B).

The accessions that carry the mutation in the LC gene were over-represented in cluster 3 and 4 (Table S6C), which was consistent with the over-representation of flat fruit in these clusters (Table S6B). OVATE was significantly over-represented in cluster 1 and 2 (Table S6C) which was consistent with the over-representation of ellipsoid fruit in these clusters (Table S6B).

The mutations in FAS and SUN were significantly over-represented in cluster 3, representing all the oxheart, and most of the flat and long fruit (Figure 2).

The occurrence of mutant alleles in certain genetic clusters might indicate separate origin of the mutations. The most wide-spread mutations in the tomato germplasm were for the OVATE and LC genes. These mutations were found in distinct genetic clusters, indicating separate origin. The data showed that accessions with both OVATE and LC mutations were indeed rare (Table III). It is possible that the lack of coinheritance was reinforced by repulsion phase linkage of OVATE and LC on chromosome 2. Accessions that carry both SUN and OVATE as well as FAS and OVATE were also found less often than would be expected by chance (Table III). This observation suggested an independent origin for the OVATE mutation and genetic isolation o lineages carrying the mutant allele. On the other hand, SUN, LC and FAS are found more often together in the same accession than expected by chance indicating that the mutations arose in the same ancestral population (Table III).

Presence of fruit shape genes in commercially grown fresh market tomato varieties

Nearly all tomato accessions evaluated in this study were heirloom, regional and contemporary accessions with a few exceptions (Table S1). As was evident from our analysis, selection for mutant alleles of the four fruit shape genes played a key role in defining population structure in the subpopulation dataset. To determine the relevance of these mutant alleles in commercial germplasm, we obtained varieties from local retail stores and evaluated them for shape and the alleles of the shape genes. We found that all the store-bought varieties carried one or more mutant alleles for the four shape genes. For example, ellipsoid shaped grape tomatoes, which occupy a distinct niche market, carried the null allele of OVATE, indicating that the introduction of this group was accompanied by selection for the mutation in this gene. Unexpectedly, while the heirloom Roma carried the OVATE mutation (Table S1), all three storebought ellipsoid shaped Roma-type tomatoes carried the mutation in SUN instead. Two of the three store bought Roma tomatoes also carried the LC mutation. Flat shaped and high loculed varieties carried the LC mutation and one of the three carried the FAS mutation as well. Similar to our findings with the older accessions, several round and low loculed varieties carried LC, indicative of modifiers of this mutation. It appears that the SUN, LC, FAS, and OVATE mutant alleles are not unique to heirloom, regional and contemporary accessions but are found in commercially grown varieties sold at retail stores and, thus, are highly relevant today.

Comment

DISCUSSION

In contrast to wild relatives that carry round and two-loculed fruit, cultivated tomato fruit is highly diverse in shape. In this study, we demonstrate that the diversity in fruit morphology in the cultivated germplasm is explained to a large extent by mutations in the SUN, OVATE, LC, and/or FAS genes. Individually, the alleles of these genes explain as much as 71% of the observed variation for specific fruit shape attributes (Table S3). At the same time, it is evident that interactions between genes and uncharacterized modifiers also affect fruit shape. For example, store-bought Roma types and Sun1642 carry the duplication of the SUN gene and exhibit an ellipsoid instead of a long shaped fruit. Sun1642 is a contemporary U.S. accession that clusters genetically with similar accessions such as M82 and Rio Grande demonstrating that the mutant SUN allele was introgressed from another accession, most likely an heirloom. Moreover, differences in fruit shape of varieties carrying the OVATE, FAS and LC mutations also suggest that suppressors and enhancers of these genes are present within the cultivated germplasm. For example, accessions that carry the OVATE mutation display a range of fruit shapes from long (e.g. LYC1340) and obovoid (e.g. Yellow Pear) to round (e.g. Gold Ball Livingston) whereas accessions carrying LC mutation produce long (e.g. Howard German), oxheart (e.g. Cuore de Toro), round (e.g LA1215), or flat (e.g. Druzba) fruit. Although Howard German fruit has on average five locules controlled by LC (Gonzalo et al., 2009), the effect of SUN is dominant over LC in controlling overall fruit shape (i.e. long), even though locule number is impacted. When adding the FAS mutation to the SUN-LC mutant background, the fruit is less elongated and, instead, oxheart in shape. Fruit shape in rectangular, ellipsoid and heart shaped varieties that do not carry the OVATE or SUN mutation might be controlled by genes such as those underlying the shape QTL fs8.1 and/or tri2.1/dblk2.1 controlling fruit elongation (Grandillo et al., 1996;Ku et al., 2000;Brewer et al., 2007;Gonzalo and van der Knaap, 2008). However, without the knowledge of the underlying genes at fs8.1 and tri2.1/dblk2.1 and the mutations that gave rise to the altered fruit shape phenotype, it is not possible to survey the alleles at these QTL throughout the germplasm. Alternative explanations to describe variation in fruit shape are also plausible. While it is likely there are more than two alleles for each fruit shape gene with the exception of SUN, it is not clear whether any of the other alleles would result in the fruit shape changes described for the known mutant alleles. accessions also showed there are more than 2 alleles. However, only one SNP was associated with elongated fruit shape (Rodriguez, unpublished data). The SUN mutation was likely to have occurred recently based on findings presented herein. In fact, there are no nucleotide differences between the ancestral and derived locus with the exception of the template switch that accompanied the transposition event (Xiao et al., 2008). Thus, the existence of other alleles of SUN that feature elongated fruit shape is extremely unlikely. A third allele has been reported for FAS (Cong et al., 2008), although we did not find this allele in any of our accessions. Because we did not search for additional alleles of FAS, the existence of more than two alleles that would result in highly loculed fruit is possible albeit unlikely.

The population structure analysis resulted in the identification of five genetic clusters, some exhibiting significant associations with fruit shape category and germplasm class. The fruit shape alleles of SUN, OVATE, FAS and LC are responsible for the observed clustering indicating that selection for fruit shape is responsible for the underlying genetic structure in tomato. Genetic groupings according to tomato fruit morphology have been reported in other studies (Mazzucato et al., 2008), supporting the notion that selection of diverse fruit shapes played a critical role in tomato domestication. We demonstrate that the fruit shape controlled by SUN and FAS is significantly over-represented in STRUCTURE cluster 3, LC in clusters 3 and 4, and OVATE in clusters 1 and 2 (Figure 2, Table S6C). In addition, the analysis of Nei's genetic distances shows that clusters 1 and 2 share a large number of alleles as do clusters 3 and 4. This observation and the distribution of fruit shape alleles suggest a separate origin of the OVATE mutation (Table S5). The OVATE lineage remained separate from the lineages carrying mutations in the other fruit shape genes during the domestication and selection of tomato, otherwise random interbreeding would have resulted in more accessions carrying mutations in OVATE and one of the other genes. However, it is also conceivable that the combination of these two mutations results in seed sterility and/or reduced plant viability which would preclude the formation of lineages that carry both OVATE and LC mutations.

Evidence about the consumption of tomato before and immediately after the discovery of America by Christopher Columbus is extremely limited. As described in the Florentine Codex writings by de Sahagún who lived between 1499 and 1590, tomatoes were eaten with salt and chili pepper (De Sahagún, 1959). Historical evidence demonstrated that tomato arrived from Mexico to Spain and Italy following Columbus' exploration of the Americas. The first written record of tomato in Europe was in 1544 where it was described as flat and segmented fruit (Matthiolus, 1544). Other descriptions of fasciated fruit followed soon thereafter (Oellinger, 1941). The fasciated phenotype of those tomatoes suggests that the earliest tomatoes that arrived in Europe carried both LC and FAS mutations. It was not until 1813 that Dunal documented in "Solanum" the pear-shaped fruit and named it Lycopersicum piriforme, the earliest mention of a different shape of tomato (Dunal, 1813). Therefore, it is plausible that tomatoes carrying the OVATE mutation traveled to Europe centuries after the first wave of tomatoes that carried the LC and FAS mutations. The mutant allele subsequently spread in Italy where, at present time, 71 out of 109 elongated accessions carry the OVATE mutation (Table I). The latter finding demonstrates that the OVATE mutation underlying the classical Italian paste tomato is in fact very wide-spread in its germplasm. All four tomato fruit shape gene mutations are wide-spread in the cultivated tomato germplasm, including commercial fresh market varieties sold at present time in grocery stores. This finding clearly shows that the fruit shape gene mutations, whether maintained for curiosity's sake, for cultural and culinary purposes, or to develop high yielding and uniquely shaped varieties, played key roles in the selection of tomato and that these mutations are still highly relevant today.

Although speculative, it has not escaped our notion that the distribution of the four fruit shape genes in the tomato germplasm enables us to develop a model for the domestication and selection of tomato. Based on the data presented in this work, we hypothesize how tomato evolved from a spherical to a variably shaped fruit, and where and when the fruit shape mutations arose (Figure 3). Accessions of S. lycopersicum var. cerasiforme represent an admixture of the remnant ancestral gene pool from which tomato may have been domesticated as well as feral derivatives of cultivated varieties (Nesbitt and Tanksley, 2002;Peralta et al., 2008;Ranc et al., 2008). If we were to assume that S. lycopersicum var. cerasiforme is the direct progenitor of cultivated tomato, the FAS, LC, and OVATE mutations would have arisen prior to domestication since the mutant alleles were found in the progenitor's gene pool. On the other hand, if we were to assume that the S. lycopersicum var. cerasiforme genetic pool is a transition between the wild and the cultivated germplasm, selection of the LC, FAS and OVATE mutations would be directly involved in the domestication of tomato. Based on the distribution of LC in the cerasiforme germplasm, where over half of them carry the mutant allele, the mutation probably arose prior to FAS and OVATE (Figure 3). The notion that LC is an old mutation is supported by data presented by Muños and colleagues (Muños et al., unpublished data). However, increased selection for the LC mutation and genetic drift could also explain the broad presence of the mutant allele in the progenitor's germplasm. Approximately 12% of the cerasiforme accessions carry the mutant alleles of OVATE or FAS, suggesting that they arose at roughly the same time but in different lineages. It is likely that FAS arose in the LC background because many accessions that carry FAS (80%) also carry LC mutations. However, some accessions including two cerasiforme lines only carry FAS, thus the mutation could have also arisen in independently in the wild type LC background.

The SUN mutation arose much later than the OVATE and FAS mutations. None of the cerasiforme or regional Latin American accessions carry this mutation and therefore, SUN arose most likely post-domestication and in Europe (Figure 3). The SUN mutation is found in approximately half of the U.S. heirloom and Spanish regional accessions with elongated fruit. Many of the U.S. heirloom accessions carrying SUN are of Northern European origin such as Opalka, Spitz, German Red Strawberry and Tyboroski Giant Plum (Table S1). This result supports the notion that SUN arose in Europe and came to North America as an heirloom. It is highly unlikely however, that the SUN mutation arose in Italy since only six out of 109 elongated fruit accessions from the Italian collection carry SUN.

It is likely that SUN arose in the LC background in cultivated tomato. In general, the older accessions representing the heirloom and regional accessions carry both SUN and LC mutations. The exceptions are Sun1642, UPV18983, Long John, Banana Legs, Orange Banana, Tegucigalpa, LYC1903, LYC1744, and T923 (Table S1). Sun1642 and UPV18983 are considered contemporary accessions and the result of recent breeding efforts (Figure 2). Banana Legs is a created heirloom that results from a deliberate cross (Male, 1999), and represents an admixture genotype in our cluster analysis. We also assume the admixture genotype and deliberate crosses that generated Long John, Orange Banana, LYC1903, LYC1744, and T923 accessions (Figure 2). The latter three are Italian accessions and since the SUN mutation is quite rare in this germplasm, it is likely that the mutation was bred from an accession that originated elsewhere. In all, these findings suggest that like FAS, SUN most likely arose in the LC mutant background.

MATERIALS AND METHODS

Plant material, DNA extraction and fruit scanning

A total of 368 tomato accessions were grown in the field in Wooster, Ohio USA in the summers of 2005 to 2007 (Table S1). The collection includes U.S. heirloom (47 accessions), contemporary U.S. (13), regional Spanish (22), regional Latin American (20), S. lycopersicum var. cerasiforme (46) and wild (7) accessions. The Italian germplasm was obtained from two sources (Table S1) and was divided into "regional" (162) and "contemporary" (38) based on the presence of the uniform ripening locus u located on chromosome 10 (Kinzer et al., 1990;Philouze). Older tomato accessions such as those found in the heirloom and regional categories often carry fruit with green shoulders when unripe, whereas accessions in the contemporary category lack green shoulders and ripen evenly. The seeds were obtained from a variety of sources (Table S1 and in the Tomato Cultivars and Heirlooms section found at the Sol Genomics Network -http://solgenomics.net/). Approximately eight fruit from each plant were cut longitudinally through the center, placed cut side-down on a scanner and digitalized at 100 dots per inch (dpi) as previously described (Brewer et al., 2006). Total genomic DNA was isolated from young leaves as described previously (Bernatzky and Tanksley, 1986;Fulton et al., 1995).

Fruit shape categories

The fruit shape terms and the number of categories in UPOV (UPOV, 2001) and IPGRI (IPGRI, 1996) classification systems are not completely consistent. Moreover, terms from an older version of UPOV (1992) are not the same as those in the most recent version (2001).

The UPOV and IPGRI fruit shape terms are also inconsistent with the prevailing ontology terms (http://solgenomics.net/tools/onto/) (SP:0001000, Solanaceae phenotype ontology). Therefore, to maintain consistency with terms present in the trait ontology database, we renamed categories and merged ones for which varieties were often classified in both (Table S7). We merged the "flattened" and "slightly flattened" categories into just one category entitled "flat". The term "round" is used for spherical shaped fruit. The category "ellipsoid" represented oval shaped fruit. The "heart-shaped" category in UPOV and IPGRI was renamed "oxheart". Fruit categorized as "oxheart" tended to be large and tapered with prominent shoulders. The term "heart" represented fruit that were larger toward the proximal end than the distal end, had less prominent shoulders than oxheart, and had a distinct tip at the distal end. Instead of "pear-shaped" or "pyriform", we adopt the term "obovoid". The category "long" included varieties that produced very elongated, cylindrical, tapered and often slightly curved fruit. The term "rectangular" remained the same. Thus the eight categories are "flat", "round", "rectangular", "ellipsoid", "heart", "long", "obovoid" and "oxheart". After this modification of the fruit shape categories, we classified our germplasm accordingly (Table S1).

Comment

Phenotypic analysis of the tomato subcollection of 120 accessions

We selected 120 representative cultivated tomato accessions from the larger set of 368 examined. This selection was balanced to equally represent the eight shape categories as well as regional representation. Two S. lycopersicum var. cerasiforme accessions were also included whereas more distant wild relatives were not included. Phenotypic data were collected using Tomato Analyzer software program (TA) (Brewer et al., 2006;Gonzalo et al., 2009). The TA attribute values were subjected to multiple iterations of Linear Discriminant Analysis (LDA) to determine which attributes were most important for defining shape. First, all 36 attributes were subjected to LDA after which seven were removed because they showed high correlations with one or more attributes that were kept in the analysis. Then, several combinations of three attributes were subjected to LDA. The highest value of the proportion of correct assignments in the cross validation test was obtained when fruit shape index, distal end protrusion and width at the widest position were included. Additional attributes were added one by one and kept only if they led to an increase in the correct proportion of assignments in the cross validation test. This process led to the selection of seven TA-defined attributes for an objective classification scheme (Table S2). The predictive accuracy of an objective image-based classification scheme using a fixed linear discriminant function based on these seven attributes was then assessed. The analyses were carried out with MINITAB 15.1.0.0 Software.

Marker development for the fruit shape genes

Two alleles are known for the SUN, OVATE and LC genes (Gonzalo and van der Knaap, 2008;Muños et al., unpublished data; LC sequence accession numbers: JF284938 and JF284939). FAS is represented by more than two alleles. For this study however, we focused on the allelic variant that carried the proposed 6-8 kb insertion in the first intron which is underlying the FAS gene mutation in the cultivated germplasm. Another FAS allelic variant (Cong et al.,2008) appears to be unique since the allele has not been found in other accessions.

For OVATE, a dCAPS marker was developed using a fluorescently labeled M13 F primer (5'-CACGACGTTGTAAAACGAC-3') in combination with primers EP158 (5'-CACGACGTTGTAAAACGACAATGCTTTCCGTTCAACGAC-3') and EP159 (5'-CGTCGGTTTCTACGTCATCA-3'). After amplification, the products were digested with DdeI and separated on a LI-COR IR2 4200 (LI-COR Biosciences, Lincoln, Nebraska, USA). The null allele of the OVATE gene, resulting in an elongated fruit, yielded a fragment of 125 bp whereas the wild type allele yielded a fragment of 135 bp.

For FAS, we first determined part of the sequence of the large insertion present in the first intron of the YABBY gene. This large insertion is indicative of the allele that causes the increased locule number phenotype (Cong et al., 2008). Genomic DNA was digested with HindIII and separated on a 0.8% agarose gel. After transfer of the DNA to Hybond N+, the blots were hybridized to a DNA fragment amplified with EP1016 (5'- S4.

Genetic cluster analysis

Clusters of similar genotypes were delineated using STRUCTURE version 2.2 (Pritchard et al., 2000). To avoid redundancy and bias in our subcollection, we removed four accessions which had the same genotype based on the alleles for the 25 randomly distributed markers and four fruit shape genes. The accessions that were removed were Jersey Devil (identical to Howard German), PI513088 and PI513036 (identical to Opalka) and UPV11936 (identical to Yellow Plum). A model assuming admixture and independent allele frequencies was selected.

We used a burn-in period of 500,000 Markov Chain Monte Carlo (MCMC) iterations and then 1,000,000 iterations after burn-in to estimate the parameters. The selected run length was much longer than suggested by Pritchard and colleagues (Pritchard et al., 2007) in order to minimize the effect of the starting configuration as well as to obtain the most accurate parameter estimates. Twenty independent runs were done for K (= number of clusters) varying from 1 to 15. The K optimum was defined according to the method proposed by Evanno et al. (2005). The strong modal signal at the true K = 5 (Figure S1) was also supported by the plateau observed for parameter P(X|K) at K = 5 (Figure S2), the rate of change of the likelihood distribution, and the absolute values of the second order. The assignment of individuals to clusters was quite robust when compared to pre-defined classes. Some shape categories, genotype classes and fruit shape genes are over represented in some particular cluster (Table S6). For verification of the STRUCTURE groupings, we estimated pairwise FST (θ) (Weir and Cockerham, 1984) and Nei's standard genetic distance (Nei, 1978) using the Microsatellite analyzer (MSA) V4.05 (Dieringer and Schlotterer, 2003).

The P-value for the FST was calculated based on 10,000 permutations and a Bonferroni correction was applied. S1). Attributes included in LDA: Fruit shape index, distal end protrusion, widest width position, the proximal end blockiness at 20% of the height from the top of the fruit, rectangular, distal angle at 20% along the boundaries from the tip of the fruit and proximal eccentricity. A description of the attributes is given in (Brewer et al., 2006;Gonzalo et al., 2009). a Refers to number of accessions that carried the mutant alleles of the gene listed in the first column.

Chi-square analyses were conducted to determine whether the observed combination of alleles (number) were higher or lower than expected (number in parenthesis). The Chi-square values obtained in each paired analysis of the mutant alleles appears in the second row at ** p < 0.01, **** p < 0.0001. The Chi-square values obtained in the analysis of combinations of three or more fruit shape genes were all highly significant.

  Comment citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285. Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript

  citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285. Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript

  Comment citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285. Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript

  Comment citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285. Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript

  citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285. Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript

  Comment citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285.

Figure 1 .Figure 2 .

 12 FIGURE LEGENDS

  Comment citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285.

	Manuscrit d'auteur / Author manuscript
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random distribution of shape categories, germplasm class and alleles of the SUN, OVATE, LC and FAS genes.

	Non-
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Comment citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285.

  one marker. CAPS markers were scored on 2 to 4% agarose gels whereas the InDel and SSR markers were scored on the LI-COR IR2 4200 (LI-COR Biosciences, Lincoln, Nebraska, USA). Details on the markers used are given in Table

	only genotyped with
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Comment citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285.

  Number of accessions in each category is given, including the percentage from the total (in parenthesis). a Accessions that carry the SUN gene duplication, b Accessions that carry the premature stop codon mutation in the OVATE gene, c Accessions that carry the large insertion mutation in the FAS gene. d Accessions that carry the mutations near the LC gene, e Accessions that carry the wild types alleles at four loci.

	Wild type (%) e	16 (13)	25 (58)	11 (32)	8 (11)	10 (44)	0	0	0	70 (19)

Comment citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285.

Table II . Summary of classification with cross-validation from Linear Discriminant Analysis (LDA) for fruit shape categories and seven fruit shape attributes measured by Tomato Analyzer (TA).

 II Fruit shape category for each accession was visually defined (see Material and Methods and Table

					Fruit shape Category a				Total
		Ellipsoid	Flat	Heart	Long	Obovoid Oxheart Rectangular Round
	N	25	36	9	11	13	5	9	12	120
	N correct	19	32	8	9	10	4	6	12	99
	Proportion	0.76	0.89	0.89	0.82	0.77	0.800	0.67	1.00	0.83

a

Table III . The expected and observed combinations of fruit shape alleles in the 368 tomato accessions.

 III Comment citer ce document : Rodriguez, G. R., Munos, S., Anderson, C., Sim, S.-C., Michel, A., Causse, M., McSpadden Gardener, B. B., David, F., van der Knaap, E. (2011). Distribution of SUN OVATE, LC and FAS in the tomato germplasm and the relationship to fruit shape diversity. Plant Physiology, 156 (1), 275-285.

		N a	OVATE	LC	FAS	SUN
	OVATE	136	-	2 (55) 137.9 ****	5 (19) 18.7 ****	5 (14) 9.7 **
	LC	150	-	-	39 (21) 31.3 ****	27 (15) 17.3 ****
	FAS	51	-	-	-	11 (6.7) 8.7 **
	SUN	37	-	-	-	-
						33

ACKNOWLEDGMENTS

We thank TGRC, GRIN, IPK Gatersleben, and Drs. Silvana Grandillo and Maria Jose Gonzalo for providing the seeds of the accessions used in this study. We thank Dr. Maria Jose Gonzalo, Ms. Jenny Moyseenko and Ms. Na Zhang for fruit and image collections.

This research was supported by the National Science Foundation DBI 0227541 to EvdK, IOS 0922661 to EvdK and BMG, and by the United States Department of Agriculture grant no: 2004-35300-14651 to EvdK and DF.

(5'-TTTCGATTTTATGGAACTTTTTGA-3') corresponding to the 3' region of intron 1 in the FAS gene. The presence of a band of approximately 4.2 kb coincided with the high locule phenotype whereas a band of approximately 4.0 kb coincided with the low locule phenotype. Digested DNA samples from tomato accessions Zapotec Pinked Ribbed, Costoluto Genovese and LYC281 carrying the fas insertion allele were separated on a 0.8% low melt agarose gel overnight and the 4.2 kb fragments were extracted from the gel using ®-agarase (NEB, Ipswich, MA, USA). The HindIII DNA fragments were self-ligated overnight. Amplification across the HindIII site was conducted using primers EP1031 (5'-AGCATCAATTAGCACTCTTCG-3') and EP1032 (5'-GCTGCAAAGGCAACAGTACA-3') resulting in a 4 kb band that was sequenced in its entirety. This analysis permitted the identification of the breakpoint of the 3' end of the insertion as well as part of the insertion sequence. Southern blot analysis using the insertion fragment as probe revealed that the inserted region was unique in the tomato genome and that it had rearranged (moved, not duplicated) from a region that mapped very close to fas (data not shown). Based on the sequence analysis of the insert with that of the genomic DNA, we developed three primers: EP1069

(5'-CCAATGATAATTAAGATATTGTGACG-3'), EP1070

(5'-ATGGTGGGGTTTTCTGTTCA -3') and EP1071 (5'-CAGAAATCAGAGTCCAATTCCA -3'). When the insertion is present, EP1069 and EP1071 will amplify a band of 466 bp; when the insertion is absent (wild type), EP1070 and EP1071 will amplify a band of 335 bp. The amplification products were separated on 2% agarose gels. For SUN, we were unable to develop a reliable PCR-based marker. Instead, we used Southern blot analysis to detect the alleles at this locus. DNA digested with EcoRV was hybridized with a probe amplified with primers EP45 (5'-TTTACCCGATGTGAAAACGA-3') and EP46 (5'-CATCAATAGTCCAAGGGGAAA-3'). An extra 4.3 kb fragment signifies the presence of the gene duplication that leads to an elongated fruit shape whereas the absence signifies the wild type allele at sun. For LC, we developed four primers: lcn-SNP695-F (5'-GTCTCTTGGATGATGACTATTGCACTTT-3'), lcn-SNP695-R (5'-TCAGCGCCTCATTTTCTATAGTATTTGT-3'), lcn-SNP695-F-cer (5'-CTTTTCCTAAAAGATTTGGCATGAGGT-3'), and lcn-SNP695-R-lev (5'-AAAGTAGTACGAATTGTCCAATCAGTCAG-3') that are included in the same PCR master mix. When the cultivated allele is present, lcn-SNP695-F and lcn-SNP695-R-lev will amplify a band of 533 bp; when the wild type allele is present lcn-SNP695-F-cer and lcn-SNP695-R will amplify a band of 395 bp. A common band of 872 bp always is present resulted of lcn-SNP695-F and lcn-SNP695-R amplification. The amplification products were separated on 2% agarose gels.

Marker selection

To genotype the 120 accessions, we assessed alleles for 10 SSR, 14 CAPS and one InDel marker. These markers were selected from the Tomato Mapping Resource Database (http://www.tomatomap.net/) and chosen based on their polymorphisms within cultivated tomato as well as their random distribution across the genome. Consequently, two or three markers per chromosome were employed with the exception of chromosome 10 which was


Sophie Prache

Joel Ballet

Karine Meteau

Frédéric Guy

Marie-Angèle Musset

Eugenie Vannier

Comparison of meat and carcass sensory quality in organically and conventionnally pasture-fed lambs at two levels of herbage availability

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


Winai W Kaewlamun

Marcel M Okouyi

Patrice P Humblot

Dominique D Remy

Mongkol M Techakumphu

Christine Duvaux-Ponter

Andrew A A Ponter

Effects of a dietary supplement of β-carotene given during the dry period on milk production and circulating hormones and metabolites in dairy cows

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

Introduction

In the dairy cow, the peripartum period is a stressful time due to the dramatic physiological and metabolic adaptations required during the change from pregnancy to lactation. Energy demand is increased due to the need to meet the requirements for rapid foetal growth and milk production. During this period, cow immunity is suppressed leading to an increase in susceptibility to a number of diseases. It has been reported that neutrophil function before parturition is impaired [START_REF] Kimura | Decreased neutrophil function as a cause of retained placenta in dairy cattle[END_REF] and this has been linked to mammary gland and uterine infection. The changes in metabolism associated with rapid foetal growth, parturition and initiation of lactation results in increased production of free radicals. When the production of free radicals exceeds the antioxidant defence mechanisms present in the body, the cow is in oxidative stress. Oxidative stress in

SUMMARY

The objective of this study was to investigate whether a supplement of β-carotene given during the dry period is able to 1) improve milk production and milk composition and 2) modify hormone and metabolic status in dairy cows during the dry and postpartum periods. This study was conducted using 40 Holstein, primiparous and multiparous cows. On the day of drying-off, cows were allocated to one of two dietary treatments: control diet (n=20) or control diet plus 1g/cow/d β-carotene (n=20). The β-carotene supplement was given individually to the cows until calving. Blood samples were obtained regularly and the concentrations of β-carotene in blood and circulating metabolites and hormones in plasma were measured. Live weight and body condition score (BCS) were monitored once a month. Daily milk production was recorded starting 5 days after calving. Milk composition was measured every 15 days. The dietary supplement of β-carotene increased blood concentrations of βcarotene during the dry period (P<0.05) and although the difference decreased postpartum β-carotene concentrations remained higher compared to the control group until the end of experiment. Live weight, BCS, milk production and composition: milk protein, milk fat, milk urea and somatic cell count, were unaffected by treatment (P>0.05). Plasma concentrations of insulin, insulin-like growth factor-1, glucose, non-esterified fatty acids and urea were unaffected by dietary supplementation with β-carotene (P>0.05). In conclusion, supplementation with β-carotene during the dry period increased blood concentrations of β-carotene but had no effect on milk production and milk quality nor on hormone and metabolic status.

Keywords: β-carotene, supplement, dairy cow.

RÉSUMÉ

Effets d'une supplémentation en β-carotène pendant le tarissement sur la production laitière ultérieure et les concentrations circulantes d'hormones et de métabolites chez la vache laitière

Les objectifs étaient d'étudier, chez des vaches laitières, l'influence d'une supplémentation en β-carotène pendant le tarissement sur 1) la production laitière et la qualité du lait et 2) le statut hormonal et métabolique pendant le tarissement et après la mise-bas. Quarante vaches Holstein primipares et multipares ont été réparties en deux lots le jour du tarissement. Le groupe contrôle (n=20) a reçu un aliment témoin et le groupe β-carotène (n=20) a reçu le même aliment supplémenté d'1g/vache/j de β-carotène distribué individuellement jusqu'à la mise-bas. Des échantillons de sang ont été prélevés régulièrement afin de mesurer la concentration sanguine en β-carotène et la concentration plasmatique d'hormones et de métabolites. Le poids vif et la note d'état corporel ont été mesurés une fois par mois. La production laitière journalière a été enregistrée à partir de 5 jours après la mise-bas et la composition du lait a été mesurée tous les 15 jours. La supplémentation en β-carotène a augmenté les concentrations sanguines de β-carotène pendant le tarissement (P<0,05). Bien que la différence ait diminué après la mise-bas, les concentrations en β-carotène sont restées plus élevées que chez les témoins jusqu'à la fin des mesures. Aucun effet de la supplémentation en β-carotène n'a été observé sur le poids vif, la note d'état corporel, la production de lait ou sa composition (TP, TB, urée et taux de cellules) (P>0,05). Les concentrations plasmatiques d'insuline, d'IGF-1, de glucose, d'AGNE et d'urée n'ont pas été affectées par le traitement (P>0,05). En conclusion, une supplémentation en β-carotène pendant le tarissement augmente les concentrations sanguines de β-carotène mais n'a pas d'effet sur la production laitière et la composition du lait ni sur le statut hormonal et métabolique.

Mots clés : β-carotène, supplémentation, vache laitière.

Effects of a dietary supplement of β-carotene given during the dry period on milk production and circulating hormones and metabolites in dairy cows

W. KAEWLAMUN 1,2,4 , M. OKOUYI1,2 , P. HUMBLOT 3,7 , D. REMY 1,2 , M. TECHAKUMPHU 4 , C. DUVAUX-PONTER 5,6 , A.A. PONTER 1,2 * periparturient cows may be a contributory factor for disease susceptibility. Independent of its role as provitamin A, β-carotene may affect immune function. β-carotene has been found to enhance immune function through its ability to regulate membrane fluidity, gap junction communication and as an antioxidant [START_REF] Chew | Carotenoid action on the immune response[END_REF][START_REF] Tjoelker | Bovine vitamin A and β-carotene intake and lactational status. 1. Responsiveness of peripheral blood polymorphonuclear leukocytes to vitamin A and β-carotene challenge in vitro[END_REF][START_REF] Tjoelker | Effect of dietary vitamin A and β-carotene on polymorphonuclear leukocytes and lymphocytes function in dairy cow during the early dry period[END_REF]. In vitro studies showed that β-carotene enhanced bovine blood and mammary gland phagocytic cell killing ability and enhanced lymphocyte proliferation induced by a mitogen [START_REF] Daniel | β-carotene and vitamin A on bovine phagocyte function in vitro during the peripartum period[END_REF][START_REF] Daniel | In vitro effects of β-carotene and vitamin A on peripartum bovine peripheral blood mononuclear cell proliferation[END_REF][START_REF] Heirman | Modulatory effects of dietary β-carotene on blood mammary leukocytes function in periparturient dairy cows[END_REF]. β-carotene is a crucial member of a group of molecules which are free radical scavengers. βcarotene and other carotenoids are especially effective at quenching singlet oxygen and can prevent the subsequent formation of secondary reactive oxygen species (ROS) [START_REF] Sordillo | Impact of oxidative stress on the health and immune function of dairy cattle[END_REF]. CHAWLA and KAUR [START_REF] Chawla R | Plasma antioxidant vitamin status of periparturient cows supplemented with α-tocopherol and β-carotene[END_REF] reported a positive correlation between plasma β-carotene concentrations and antioxidant power measured by a ferric reducing antioxidant power assay in cows supplemented with β-carotene during the dry period. They also suggested that there is a need to give cows a β-carotene supplement during the dry period in order to improve their plasma antioxidant status and health after parturition and to improve milk production and quality due to reduced mammary gland infection. Negative energy balance commonly occurs in the early postpartum period and reaches a maximum during the first or second week postpartum [START_REF] Butler | Interrelationship between energy balance and post partum reproductive function in dairy cattle[END_REF][START_REF] Mcnamara | Effects of different transition diets on energy balance, blood metabolites and reproductive performance in dairy cows[END_REF]. Similarly, plasma concentrations of β-carotene and vitamin E in nonsupplemented cows decrease throughout the dry period and reach their lowest levels during the first week postpartum [START_REF] Heirman | Modulatory effects of dietary β-carotene on blood mammary leukocytes function in periparturient dairy cows[END_REF][START_REF] Calderón | Variation in carotenoids, vitamin A and E and color in cow's plasma and milk during late pregnancy and the first three months of lactation[END_REF]. Thus, the decrease in plasma concentrations of β-carotene could be associated with the occurrence of metabolic and infectious diseases during the peripartum in dairy cows. Additionally, the concentrations of several metabolites and metabolic hormones are modified by the adaptations which occur around calving. However, there have been few reports on the effect of supplemental β-carotene on milk production and quality and on blood metabolites in peripartum cows. Therefore, this study aimed to investigate whether a supplement of β-carotene given during the dry period is able to 1) improve milk production and milk composition and 2) modify hormone and metabolic status in dairy cows during the postpartum period.

Material and Methods

ANIMALS AND MANAGEMENT

Forty high-producing Holstein, primiparous and multiparous, cows were used in the experiment (average annual milk production 10 000kg). Cows were dried off 60 days before presumed calving date. On the day of drying-off cows were paired according to live weight, BCS, age, milk production level of previous lactation, expected calving date, and blood β-carotene concentrations and randomly allocated to one of two dietary treatments: control diet (C group: n=20) or control diet plus 1 g/cow/d β-carotene (BC group: n=20, DSM Nutritional Products Ltd., Paris, France). Treated and control cows were housed together in the same barn on straw bedding where they received a total mixed ration (TMR), formulated to meet average requirements for maintenance and production [START_REF] Hoden | Alimentation des vaches laitières[END_REF]. Three different diets were formulated (Table 1) and fed to the cows depending on their requirements (1 st or 2 nd month of the dry-period or lactation). The cows were given fixed quantities (limited compared to appetite) of diet during the dry-period and the lactation diet was given ad libitum. The diets contained a vitamin and mineral mix which covered vitamin A requirements. Cows had free access to water and salt licks. Before giving the meal in the morning the cows were restrained with head-lock stanchions and the β-carotene supplement was given individually to the cows by top-dressing on 500 g rapeseed meal. Cows in the control group only received the rapeseed meal. Once the treatments had been eaten the diet was distributed. The basal concentrations of β-carotene in the 1 st and 2 nd month dry-cow diet, lactating-cow diet and in the rapeseed meal were 2.35, 4.32, 3.72 and 0.63 mg/kg dry matter, respectively.

BLOOD SAMPLING

Blood samples were obtained at -8, -6, -4, -2 weeks before calving, at calving and at 1, 2, 3, 4, 6, 8 and 10 weeks after calving by caudal venipuncture before the morning feed. Blood was collected into 9 mL heparin-coated tube and placed immediately in ice-cooled water. Blood concentrations of βcarotene were measured and the sample was then centrifuged for 10 min at 4ºC and 2 500 rpm. Plasma was collected and frozen (-20ºC) until required for metabolite and hormone assay.

BLOOD CONCENTRATIONS OF β-CAROTENE

On-farm measurement of blood concentrations of β-carotene was performed on the day of sampling. The concentrations of β-carotene in blood were determined as previously described [START_REF] Schweigert | Cooperative European study for the validation of a novel cow-side β-carotene assay in serum and blood[END_REF] after extraction of β-carotene (iExTM) and measurement with a carotene photometer (iCheck™; BioAnalyt GmbH, Germany).

MILK YIELD AND MILK COMPOSITIONS

The individual daily milk yields (milk collected in the evening and the following morning) were measured with milk meters (MM15, DeLaval Inc., Elancourt, France) connected to Alpro (Alpro, DeLaval Inc., France). Daily milk production was recorded starting on the 5 th day postpartum. Milk yield per cow per day was average during the first 100 DIM.

Milk composition was measured individually once every two weeks on a composite mixture of milk from successive evening and morning milkings (mixture 50:50). The samples were kept at room temperature with a preservative (Bronopol, Lanxess, Langenfeld, Germany) until analysis. Samples were sent to the laboratory of the Milk Recording Organisation (Syndicat Interdépartemental de l'Elevage, Le Mée, France) to determine milk fat, protein and urea concentrations by infrared spectrophotometry (MilkoScan 6000, Foss Electric, Nanterre, France). Somatic cell counts (SCC) were evaluated by flow cytometric measurement (Fossomatic 5000, Foss Electric, Nanterre, France). In the present experiment there were no clinical cases of mastitis.

LIVE WEIGHT AND BODY CONDITION SCORE

Live weight and BCS were recorded on the day of dryingoff, 1 month after drying-off, just after calving, 1 and 2 months postpartum. Body condition score was evaluated by the same person on a scale of 1-5 with a 0.25 increment where 1 represents extremely thin or emaciated cows and 5 represents extremely fat or obese cows [START_REF] Ferguson | Principal descriptors of body condition score in Holstein cows[END_REF].

INSULIN AND INSULIN-LIKE GROWTH FACTOR-1 (IGF-1)

Insulin and IGF-1 were analysed by radioimmunoassays respectively based on porcine insulin (Insulin-CT ® , CIS Bio International, Gif-sur-Yvette, France) and human recombinant IGF-1 (IGF-1-RIACT ® , CIS Bio International, Gif-sur-Yvette, France) in the samples collected at -8, -4, 0, 2, 4, 6 and 10 weeks in relation to calving. IGF-BPs were removed following the manufacturer's instructions. Intra-assay coefficients of variation were 4.8% at 153.8 pmol/l and 3.8% at 57.45 ng/ml for insulin and IGF-1, respectively.

MEASUREMENT OF BLOOD METABOLITES

Plasma samples were analysed by photometric methods for glucose (Glucose-RTU ® , BioMérieux, Lyon, France), NEFA (NEFA C ® , Wako Chemicals, Neuss, Germany), urea (Urea-kit S ® , BioMérieux, Lyon, France) in the samples collected at -8, -6, -4, -2, 0, 1, 2, 3, 4, 6, 8 and 10 weeks in relation to calving. Inter-assay coefficients of variation were 4.3% at 3.47 mmol/l, 12.65% at 0.31 mmol/l, 8.3% at 2.99 mmol/l, for glucose, NEFA, and urea, respectively.

STATISTICAL ANALYSIS

Statistical analysis was performed using SAS (Version 9.1; SAS Institute, Cary, NC, USA). The parameters measured in this study were repeated measures over time on individual cows. The MIXED procedure was used to determine the difference between treatment groups including a random female effect and contrast statement was used in the model [START_REF] Littell R | Statistical analysis of repeated measures data using SAS procedures[END_REF]. The somatic cell counts (SCC) were log transformed before analysis

Results

BLOOD β-CAROTENE CONCENTRATIONS

There was no significant difference in blood concentrations of β-carotene between the two treatment groups before the start of the dietary supplement (Figure 1). There was a significant effect of treatment before (BC > C, P<0.001) and after calving (BC > C, P<0.001) on blood concentrations of β-carotene.

LIVE WEIGHT AND BODY CONDITION SCORE

Neither live weight (P=0.95) nor BCS (P=0.92) were modified by dietary treatment (Figure 2). However, both parameters increased to a peak 1 month prior to calving and then decreased during the rest of the study (P<0.001).

MILK PRODUCTION

Average milk yield per day during the first 100 DIM was not affected by treatment (BC= 40.0 ± 6.1 kg/d, C=39.0 ± 7.2 kg/d; P=0.24). Milk production increased with time after calving to reach a plateau (P<0.0001) but there was no interaction between treatment and time (P=0.83).

INSULIN AND INSULIN-LIKE GROWTH FACTOR-1

Neither insulin (P=0.78) nor IGF-1 (P=0.87) (Figure 3) differed between treatments. Plasma concentrations of insulin increased slightly during the first month of the dry period and then drastically decreased at calving. The lowest blood concentrations of insulin were observed at calving in the BC group and at 2 weeks postpartum in the control group. Similarly, plasma concentrations of IGF-1 increased slightly during the first month after drying-off and then decreased and reached a minimum at 2 weeks postpartum. Thereafter IGF-1 increased gradually throughout the rest of the study.

BLOOD METABOLITES

The dietary supplement of β-carotene did not affect blood concentrations of glucose (P=0.20), NEFA (P=0.11), and urea (P=0.59) (Figure 4). Plasma concentration of these metabolites were affected by time postpartum (P<0.0001) but there was no interaction between time and treatment (P>0.05).

MILK COMPOSITION

Milk composition (protein (P=0.71), fat (P=0.85) and urea (P=0.61); Figure 5) and SCC (log transformed, (P=0.26) Figure 6) were not affected by treatment. Milk protein and fat were influenced by time postpartum (P<0.0001) while urea and SCC were unaffected. There was no interaction between treatment and time on milk composition (P>0.05).

Discussion

A dietary supplement of β-carotene given to dairy cows during the dry period increased blood concentrations of β-carotene. This is in agreement with several studies [START_REF] Bindas | Reproductive and metabolic characteristic of dairy cattle supplemented with beta-carotene[END_REF][START_REF] Akordor | Reproductive performance of lactating Holstein cows fed supplemental β-carotene[END_REF][START_REF] Wang | Effect of Beta-carotene supplementation on reproductive performance of lactating Holstein cows[END_REF][START_REF] Kawashima | Effect of βcarotene supply during the close-up dry period on ovulation at the first follicular wave postpartum in dairy cows[END_REF].

In the present experiment a β-carotene supplement had no effect on milk production in agreement with other studies [START_REF] Bindas | Reproductive and metabolic characteristic of dairy cattle supplemented with beta-carotene[END_REF][START_REF] Wang | Effect of Beta-carotene supplementation on reproductive performance of lactating Holstein cows[END_REF][START_REF] Rakes | Effects of adding beta-carotene to rations of lactating cows consuming different forages[END_REF]. In contrast, a study conducted with heat stressed cows showed that cumulative milk yield increased by 6 to 11% in βcarotene supplemented compared to non-supplemented cows [START_REF] Aréchiga | Effects of timed insemination and supplemental β-carotene on reproduction and milk yield of dairy cows under heat stress[END_REF]. The heat stressed cows were supplemented with β-carotene starting at approximately 15 to 20 days postpartum and for variable periods, the longest being 170 days. Moreover, OLDHAM et al. [START_REF] Oldham | Effects of supplemental vitamin A or beta-carotene during the dry period and early lactation on udder health[END_REF] found that a supplement of β-carotene during the dry period and early lactation could increase milk yield in non-heat stressed dairy cows. They concluded that the effect of β-carotene on milk production warranted additional research. Differences in β-carotene concentrations in the control diet, the initial blood concentration of β-carotene, the level of supplementation, the timing of supplementation, the duration of supplementation may have contributed to the lack of consistency in responses to β-carotene supplementation previously reported.

In this study, the evolution in plasma glucose was in agreement with a previous report [START_REF] Ingvartsen | On the relationship between lactational performance and health: is it yield or metabolic imbalance that cause production disease in dairy cattle? A position paper[END_REF]. What is commonly seen is that glucose concentrations remain stable or increase slightly during the prepartum period, rise at calving, and then decrease immediately after calving [START_REF] Santos | Effect of prepartum dietary protein level on performance of primigravid and multiparous Holstein dairy cows[END_REF][START_REF] Ingvartsen | Feeding-and management-related diseases in the transition cow: Physiology adaptations around calving and strategies to reduce feeding-related diseases[END_REF]. The increase in plasma glucose at calving reflects an increase in gluconeogenesis which is in response to calving stress. The decrease immediately after calving may be associated with the modest increase in dry matter intake concomitant with the very high uptake of circulating glucose by the mammary gland for lactose synthesis [START_REF] Wathes | Differences between primiparous and multiparous dairy cows in the inter-ralationships between metabolic traits, milk yield and body condition score in the periparturient period[END_REF].

The changes in plasma; NEFA, IGF-1 and insulin, together with the changes in live weight and BCS, reflect the nutritional status of the cows. These variables confirm the very large energy demands of cows during the last month of pregnancy. The energy supply from feed intake could not meet the requirements for maintenance and rapid foetal growth. Therefore, body energy reserves, mainly in the form of body fat, are mobilized to provide the energy and to cover requirements. This mechanism results in an increase in NEFA, and a decrease in insulin and IGF-1 and the loss of body condition.

In our study, there was no effect of a dietary supplement of β-carotene on milk protein content which is consistent with previous work [START_REF] Rakes | Effects of adding beta-carotene to rations of lactating cows consuming different forages[END_REF]. Recently, de ONDARZA et al. [START_REF] De | Case study: Effect of supplemental β-carotene on yield of milk and milk components and reproduction of dairy cows[END_REF] also reported that a supplement of β-carotene given after calving had no effect on milk protein and SCC but did increase milk fat compared to non-supplemented cows. HINO et al. [START_REF] Hino | Effect of β-carotene and α-tocopherol on rumen bacteria in the utilization of long chain fatty acids and cellulose[END_REF] found, in an in vitro study, that β-carotene plus α-tocopherol increased the growth of cellulolytic bacteria cultured in fatsupplemented media and increased cellulose digestion. The increase in fibre digestion in the rumen may explain why milk fat increased after β-carotene supplementation. Another possibility is that β-carotene supplementation is associated with altered rumen bio-hydrogenation as has been observed for another antioxidant, vitamin E [START_REF] Bell | Effect of safflower oil, flaxseed oil, monensin and vitamin E on concentration of conjugated linoleic acid in bovine milk fat[END_REF]. A supplement of vitamin E when given with a linseed supplement (rich in C18:3n-3) altered rumen bio-hydrogenation and resulted in an increase in the production of vaccenic acid (trans-11 C18:1) and a decrease in the production of trans-10 C18:1 in the rumen [START_REF] Pottier | Effect of dietary vitamin E on rumen biohydrogenetion pathways and milk fat depression in dairy cows fed high-fat diets[END_REF]. Since trans-10 C18:1 has been associated with milk fat depression [START_REF] Bauman | Regulation and nutritional manipulation of milk fat: low-fat milk syndrome[END_REF] this may explain why a dietary antioxidant supplement can in some situations increase milk fat level. In the present study, although the blood concentrations of β-carotene post partum were different between treatments, the BC cows were no longer receiving the dietary β-carotene supplement therefore it is unlikely that ruminal microbe growth and cellulolytic bacteria function would have been affected. In the studies where the supplement of β-carotene increased milk production, cows also received β-carotene during the post partum period [START_REF] Aréchiga | Effects of timed insemination and supplemental β-carotene on reproduction and milk yield of dairy cows under heat stress[END_REF][START_REF] Oldham | Effects of supplemental vitamin A or beta-carotene during the dry period and early lactation on udder health[END_REF][START_REF] De | Case study: Effect of supplemental β-carotene on yield of milk and milk components and reproduction of dairy cows[END_REF]. In the present study we wished to investigate whether there was a carry-over effect of β-carotene supplementation during the dry-period into the lactation period.

β-carotene functions as an antioxidant and may enhance immunity. This role is of interest in the fight against infection. Several studies have been conducted to determine the effect of β-carotene on cow immunity or udder health [START_REF] Chawla R | Plasma antioxidant vitamin status of periparturient cows supplemented with α-tocopherol and β-carotene[END_REF][START_REF] Oldham | Effects of supplemental vitamin A or beta-carotene during the dry period and early lactation on udder health[END_REF][START_REF] Leblanc | Peripartum serum vitamin E, retinal and beta-carotene in dairy cattle and their associations with disease[END_REF][START_REF] Spears | Role of antioxidants and trace elements in health and immunity of transition dairy cows[END_REF]. Some studies showed positive effects on udder health while others did not. Somatic cell count in cow milk has been used as an indicator of udder health and milk quality and can be related to the response of cellular immunity to pathogens. RAKES et al. [START_REF] Rakes | Effects of adding beta-carotene to rations of lactating cows consuming different forages[END_REF], for example, showed that a daily supplement of 300 mg β-carotene lowered SCC in milk. Others reported that the incidence of clinical mastitis was lower in β-carotene supplemented compared to non-supplemented cows [START_REF] Wang | Effect of Beta-carotene supplementation on reproductive performance of lactating Holstein cows[END_REF]. We, however, found no effect of a supplement of β-carotene on SCC. Although in the present experiment β-carotene remained higher in BC cows compared to C cows postpartum, even though the supplementation was stopped at calving, the difference was probably not large enough to affect SCC. Other studies have shown that β-carotene was ineffective in lowering somatic cell counts and protecting against mastitis [START_REF] Bindas | Reproductive and metabolic characteristic of dairy cattle supplemented with beta-carotene[END_REF][START_REF] Oldham | Effects of supplemental vitamin A or beta-carotene during the dry period and early lactation on udder health[END_REF][START_REF] Leblanc | Peripartum serum vitamin E, retinal and beta-carotene in dairy cattle and their associations with disease[END_REF].

The present study showed that a dietary supplement of βcarotene in a purified form given to dairy cows can escape complete degradation in the rumen and increase circulating concentrations in dairy cows. Although in the literature β-carotene has been shown to have specific effects on metabolism and production in dairy cows, the parameters that we measured in the present experiment were unable to shed light on the physiological functions that require β-carotene. Although the β-carotene supplement was stopped the day of calving, blood concentrations remained higher in the treated cows compared to the controls during the post partum period. There was no effect of β-carotene on milk production and metabolism after the supplement was stopped.

FIGURE 1 :FIGURE 2 :

 12 FIGURE 1: Blood β-carotene concentrations in Holstein cows given either: a control diet (n=20) or a control diet plus 1g/d β-carotene (n=20) starting 8 wks before calving until the day of calving. LSmean ± SEM. Significant difference between dietary treatments, *** P<0.001

FIGURE 3 :

 3 FIGURE 3: Plasma concentrations of a) insulin and b) insulin-like growth factor-1 (IGF-1) in dairy cows given either: a control diet (n=20) or a control diet plus 1g/d β-carotene (n=20) starting -8 wks before calving until the day of calving. LSmean ± SEM. C=calving.

FIGURE 4 :

 4 FIGURE 4: Plasma concentrations of glucose, non-esterified fatty acids (NEFA), and urea in dairy cows given either: a control diet (n=20) or a control diet plus 1g/d β-carotene (n=20) starting -8 wks before calving until the day of calving. LSmean ± SEM. C=calving.

FIGURE 5 :

 5 FIGURE 5: Milk composition (milk fat, protein and urea) in dairy cows given either: a control diet (n=20) or a control diet plus 1g/d β-carotene (n=20) starting -8 wks before calving until the day of calving. The samples were taken every 15 days post partum for 10 weeks. LSmean ± SEM.

FIGURE 6 :

 6 FIGURE 6: Somatic cell count (SCC, log transformed) in dairy cows given either: a control diet (n=20) or a control diet plus 1g/d β-carotene (n=20) starting 8 wks before calving until the day of calving. The samples were taken every 15 days post partum for 10 weeks. LSmean ± SEM.

TABLE I :

 I Composition and nutritional value of the diets given to dairy cows during the dry period and during lactation. Results are presented as LSmeans ± standard error of the mean (SEM). Significant differences are reported at P<0.05.

		Dry period		Lactation
		1 st month	2 nd month
	Diet composition	(kg DM/cow/d)	(kg DM/cow/d)	(% DM)
	Maize silage	5.2	5.9	38.74
	Brewers grains	-	-	6.44
	Sugar beet pulp	-	-	9.26
	Molasses	-	-	0.84
	Orange peel	0.6	-	-
	Rapeseed meal	1.4	0.9	18.04
	Grass hay	2.6	2.6	14.33
	Barley	-	-	6.92
	Mineral and vitamin mix*	0.2	0.2	1
	Urea	-	-	3.62
	Salt licks	ad lib	ad lib	ad lib
	CaCO 3	0.02	-	0.12
	MgCl	-	0.05	-
	Sodium bicarbonate	-	-	0.4
	Nutritional values	(/cow/day)	(/cow/day)	(/kg DM)
	NE L (Mcal)	14.11	13.50	1.632
	PDIN (g)	703	577	106
	PDIE (g)	755	673	101
	Calcium (g)	52.3	40.0	7.6
	Phosphorus (g)	38.8	33.4	4
	Starch (%)	-	-	16.8
	Crude fiber (%)	-	-	19.3
	β-carotene (mg)	42.3	41.8	2.35

*Mineral and vitamin mix contained 240g Ca, 35g P, 40g Na, 50g Mg, 1g Cu, 3.6g Zn, 3.6g Mn, 66mg I, 22mg Co, 20mg Se, 400,000 IU vitamin A, 66,700 IU vitamin D3, and 1200 IU vitamin E per kg as fed (Centralys, Trappes, France). NE L = Net energy for maintenance and lactation PDIN = true intestinal digestible proteins, when degraded N is the limiting factor PDIE = true intestinal digestible proteins, when energy is the limiting factor since the results did not have a normal distribution.

INRA, UMR 1198 Biologie du Développement et Reproduction, Domaine de Vilvert, 78350 Jouy-en-Josas, FRANCE.

Université Paris-Est, Ecole Nationale Vétérinaire d'Alfort, UMR 1198, 7 avenue du Général-de-Gaulle, 94704 Maisons-Alfort Cedex, FRANCE.

UNCEIA R&D, 13 rue Jouët, 94704 Maisons-Alfort, FRANCE.

Faculty of Veterinary Science, Chulalongkorn University, Henri Dunant Rd., Bangkok, 10330, THAILAND.

AgroParisTech, UMR 791 Modélisation Systémique Appliquée aux Ruminants, 16 rue Claude Bernard, 75005 Paris, FRANCE.

INRA, UMR

Modélisation Systémique Appliquée aux Ruminants, 16 rue Claude Bernard, 75005 Paris, FRANCE. 7 Present address: Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, SLU, Po Box 7054, SE 750 07 Uppsala, SWEDEN. *Corresponding author: aponter@vet-alfort.fr

PONTER (A.A.) AND COLLABORATORS

Acknowledgement

The authors thank DSM Nutritional Products for partial financial support. Winai Kaewlamun was supported by the Royal Golden Jubilee PhD Program of the Thailand Research Fund and PHC Franco-Thai project year 2007-2008. Marcel Okouyi was supported by the Gabonese government. Dominique Tristant and the farm workers at the experimental farm of Agro-ParisTech, Thiverval-Grignon and the technical assistance of Christine Ficheux are also acknowledged.