In tomato fruit, the number of locules (cavities containing seeds that are derived from carpels) varies from two to up to 10 or more. Locule number affects fruit shape and size, and is controlled by several Quantitative Trait Loci (QTLs). The large majority of the phenotypic variation is explained by two of these QTLs, fasciated and locule number (lc) that interact epistatically with one another. FASCIATED has been cloned, and mutations in the gene are described as key factors leading to the increase in fruit size in modern varieties. Here, we report the map-based cloning of lc. The lc QTL includes a 1600 bp region that is located 1080 bp from the 3' end of WUSCHEL, which encodes a homeodomain protein that regulates stem cell fate in plants. The molecular evolution of lc showed a reduction in diversity in cultivated accessions with the exception of two SNPs. These two SNPs were shown to be responsible for the increase in locule number. An evolutionary model of locule number is proposed herein, suggesting that the fas mutation appeared after the mutation in the lc locus to confer the extreme high locule number phenotype.

INTRODUCTION

The domestication of wild plant species has altered particular sets of traits, such as the size and the diversity of the consumed parts (Frary and Doganlar, 2003). During this process, genetic diversity decreased, not only in those genes controlling domestication traits as a result of artificial selection, but also at the whole genome level as a result of genetic bottlenecks.

Improvements of cultivars and production systems have increased crops yield. At the genetic level, the yield of domesticated plants has increased considerably compared to their wild ancestors (Buckler et al., 2001), particularly over the last 40 years (http://faostat.fao.org).

However, this increase will probably reach its limit due to the increase of the world human population and the decrease in arable lands. The loss of genetic diversity at important loci that accompanied the yield increase could limit improvements in the future. The identification of new alleles or new allelic combinations from wild species or tomato accessions that did not experience an erosion of their genetic diversity is essential but requires an understanding of crop domestication. Linking the evolution of genotypes and phenotypes to understand domestication is one of the main challenges of modern genetics.

The tomato clade, Solanum section Lycopersicum, is composed of several wild species and the cultivated tomato (Solanum lycopersicum). The wild species are distributed along the Andean coast from Ecuador to Chile, with two species (S. cheesmaniae and S. galapagense) endemic to the Galapagos Islands. The cultivated tomato is a self-pollinating species that is thought to be derived from its closest wild ancestor S. pimpinellifolium (Nesbitt and Tanksley, 2002). Cherry tomato accessions (S. lycopersicum var. cerasiforme) have an intermediate position between these two species, as their genomes are mosaics of those from S. lycopersicum and S. pimpinellifolium (Ranc et al., 2008).

Compared to wild species, domesticated accessions show a range of morphological diversity, particularly with respect to fruit shape and size (Paran and van der Knaap, 2007) (Supplemental Photo 1), despite the strong loss of molecular diversity throughout the whole genome (Van Deynze et al., 2007).

A number of genes controlling tomato fruit morphology have been cloned: FW2.2 (Frary et al., 2000) controls fruit weight; SUN (Xiao et al., 2008) and OVATE (Liu et al., 2002) control elongated fruit shape; and FASCIATED (Cong et al., 2008) controls locule number, fruit size and flat fruit shape. fw2.2 controls up to 30% of fruit weight variation, and was the first gene underlying a QTL that was identified by a positional cloning approach (Frary et al., 2000).

The gene is differentially expressed in the carpel resulting in increased cell number in large fruit. FASCIATED encodes a YABBY-like transcription factor that is expressed early in the development of stamen and carpels. The characterization of both FW2.2 and FASCIATED illustrate the link between fruit size and genes involved in developmental processes.

In tomato, locule number influences fruit shape and size. The locules are directly derived from the carpels in the flower. In addition to fasciated, several QTLs controlling locule number have been mapped and a candidate gene approach has been used to map genes regulating floral meristem development which might colocalize with known QTLs for locule number (Barrero et al., 2006). Although none of the genes responsible for these QTLs was successfully identified, the results of this study were used to develop several interesting hypotheses.

Two QTLs, fasciated and locule number (lc, also named lcn2.1) have a major effect on the phenotype. FASCIATED, which could be considered a major gene, has the strongest effect by increasing the number of locules from two to more than six. lc has a weaker effect by increasing the number of locules from two to three or four. An epistatic interaction between the two QTLs influences the phenotype with both lc and fasciated synergistically contributing to extremely high locule number (Barrero and Tanksley, 2004;Lippman and Tanksley, 2001).

WUSCHEL, a gene controlling stem cell fate in the apical meristem, was mapped to the same region as lc (Barrero et al., 2006). The lack of polymorphisms in the gene and its promoter and the similar expression level of the gene in both wild type and mutant accessions led the authors to conclude that the DNA region which has been analyzed did not correspond to lc.

The gene responsible for lc and its role during domestication thus remains unknown.

We report herein the map-based cloning and the identification of two SNPs immediately downstream of WUSCHEL that control the trait. The characterization of the lc locus shows a remarkable pattern of diversity. In addition, we demonstrate that the selection of this locus has been necessary to increase locule number during the domestication of tomato.

RESULTS

Physical mapping of lc

The lc locus, which was first described in 1937 (Yeager, 1937), was mapped close to the ovate region (Butler, 1952). This QTL is a major locus known to modify the number of locules in tomato fruits, with a phenotypic effect that varies according to the genetic background. The QTL location on chromosome 2 was more accurately defined using molecular mapping with polymorphic markers (Lippman and Tanksley, 2001;van der Knaap and Tanksley, 2003).

Our study exploited a recombinant inbred line population obtained from an intraspecific cross between two cultivated tomato accessions (Causse et al., 2002), Cervil, a cherry tomato line, and Levovil, a classical French fresh market line carrying round fruits with many locules weighing approximately 100g. The QTL was initially mapped to the TG463 region (Lecomte et al., 2004) using two near-isogenic lines (CF12-C and CF13-L) that were genetically identical except for a 30 cM region containing lc. CF12-C contains the low locule number allele from Cervil (C), and CF13-L contains the high locule number allele from Levovil (L) (Fig. 1A).

An F2 segregating population (2688 plants) derived from the cross between CF12-C and CF13-L was used to identify 215 plants with a recombination between the markers T1555 and TG191 surrounding lc in a 4.8 cM region. The segregation of the phenotype was tested in the progeny of each recombinant line (Supplemental Table S1), allowing the mendelization of the QTL (Fig. 1B). Heterozygous plants have an intermediate phenotype (2.7 locules ± 0.50 SD)

that was between homozygous plants with low locule number (2.4 locules ± 0.51 SD) and homozygous plants with high locule number (3.5 locules ± 0.75 SD). This indicated that the mutant allele was partially recessive to the wild type allele. Homozygous plants carrying both the Cervil wild type alleles produced fruits with mostly two locules. In contrast, homozygous plants carrying both the Levovil mutant alleles produced fruits mostly containing three or more locules. Given the semidominant nature of the QTL and the limited phenotypic difference between the two extreme values of the two alleles, heterozygous plants could not be differentiated from either homozygous plant unless the recombinant plant was subjected to progeny testing.

The sequence analysis of the region around the closest linked marker TG463, indicated that the lc region was partially syntenic to a region in A. thaliana surrounding At2g18000 (Fig. 2A). Sequence analysis of TG463 showed homology with the BAC end of the tomato clone Le_HBa0139K19. The mapping of an insertion-deletion marker found at the other BAC end confirmed that this clone originated from the lc region. The ctof-13-f10 marker sequence was present on this BAC as confirmed by PCR amplification. Two recombinant plants, PA205-2 and JB149-9, showed a recombination event in the Le_HBa0139K19 BAC. The PA205-2 line produced fruits with few locules (2.3). The JB149-9 line produced fruits with a higher number of locules (3.8). Taken together, these observations indicated that the Le_HBa0139K19 BAC covered the lc QTL.

Sequence analysis of the BAC clone Le_HBa0139K19 (109.5kb) showed that it contained 11 putative Open Reading Frames (ORF). Polymorphisms found in the BAC sequence were used to identify the recombinant breakpoints in PA205-2 and JB149-9 which allowed us to narrow down the locus to 26.6 kb (Fig. 2A). This region contained three ORFs encoding a transducin WD40 repeat regulatory protein (similar to At5g66240), an unknown protein and LeWUSCHEL.

WUSCHEL (Laux et al., 1996;Reinhardt et al., 2003) was a likely candidate for the lc QTL because its function is associated with floral organ number (Mayer et al., 1998) and meristem size via the regulation of another key regulator of apical meristem development, Clavata3 (Brand et al., 2000;Schoof et al., 2000). The WD40 repeat protein was also a likely candidate because these proteins are linked to several important functions in eukaryotes (Smith et al., 1999), such as cell division (Feldman et al., 1997) and chromatin remodeling (Vitaliano-Prunier et al., 2008).

To further refine the recombination map, an additional 6768 F2 plants from the same cross between CF12-C and CF13-L were genotyped with the z1416 and z1420 markers that flanked the lc locus. This experiment led to an additional 52 recombinant lines. Newly designed markers, located every 2 kb within this region, restricted lc to a 3 kb region (Fig. 2B). Final sequence analysis of the region restricted lc to a 1608-bp region which showed 14 polymorphic sites (13 SNPs and a 1 bp indel) between the two alleles (Fig. 2C).

Functional analysis of lc

The 1608-bp region to which lc was fine mapped, corresponded to a non-coding region located 1080 bp downstream of the stop codon of WUSCHEL. None of the in silico analyses of the region provided any information about its putative function. In addition, this region did not show any homology with known miRNAs nor did its sequence have a clear secondary structure that would predict a novel miRNA. Using northern blots and RT-PCR did not reveal any possible expression of this locus (data not shown). We also examined the effect of the lc locus on the expression of the two adjacent genes (Fig. 3). WUSCHEL expression was restricted to flower buds, whereas the WD40 repeat protein was expressed in all tissues examined. There was no significant expression difference between the wild type and mutant alleles of lc. Therefore, these results could not discriminate conclusively which of the two candidate genes underlies lc.

Given that WUSCHEL expression was restricted to floral buds, the effect of lc on floral development was evaluated by determining floral organ number in several representative near-isogenic lines (Fig. 4). The increase in locule number was positively correlated with an increase in petal number but was not associated with an increase in flower size.

Sequence polymorphisms among different tomato accessions validated lc

To further characterize lc, the 1608 bp region was sequenced in a set of 88 accessions composed of 16 S. lycopersicum, 62 S. lycopersicum var. cerasiforme and 10 S.

pimpinellifolium (Supplemental File 1). This panel of varieties was chosen to represent a large spectrum of tomato diversity (Ranc et al., 2008). Sequence analysis revealed 21 new polymorphic sites, with the majority present in the wild species. Two SNPs were found to be associated with locule number (p-value < 1 x 10 -6 ), and these two SNPs were in almost complete linkage disequilibrium, as shown by the existence of only one accession (LA2402) that showed a recombination event between the two SNPs. Together, the two SNPs were thus considered a unique haplotype. The correlation between the number of locules and the lc haplotypes was consistent except for three lines. The Pescio, Muchamiel and Stupicke Polni Rane cultivars produced fruits with high locules (3.8, 5.5 and 4.2, respectively) and contained the low locule number haplotypes, but the mutant genotype of these three lines at the fasciated locus explained their phenotype.

To validate the functional effect of the two SNPs linked to lc by association genetics, a 235 bp region containing the two SNPs was sequenced in 92 additional lines (Supplemental Table S2). The statistical association between the genotypes at the two SNPs and the number of locules was highly significant (p-value < 1.30 x 10 -12 ), demonstrating that the two SNPs located in the non-coding sequence were responsible for lc and could thus be considered Quantitative Trait Nucleotides (QTNs) (Fridman et al., 2004). None of the other SNPs in the region was associated with fruit locule number. The potential effect of lc on fruit weight was also examined. The two SNPs explained more than 12 % of fruit weight variation (p-value of association < 3.5 x 10 -6 ) in the core collection of 88 accessions. In the same set, one SNP in the 5'-UTR of fw2.2 explained the same range of variation in fruit weight but with a weaker significance level (p-value < 3 x 10 -4 ) and no association with locule number. The mutations responsible for fw2.2 remain unknown, and thus another SNP may be more significantly associated with fruit weight at this locus.

Molecular evolution of lc during domestication

Detailed sequence analysis revealed that the diversity of the locus was drastically reduced in the cultivated species with the exception of the two SNPs responsible for lc (Fig. 5A and5C).

This observation was particularly evident for a 311 bp window surrounding the two SNPs in which no sequence differences were observed in the cultivated accessions. These results indicate that lc is under high selective pressure, probably due to the functional importance of the locus. An analysis of 8 additional loci located on the Le_HBa0139K19 BAC and 16 molecular markers located along the entire length of chromosome 2 indicated that the two The tomato accessions studied for association mapping were not selected based on fruit locule number but rather their molecular diversity at neutral markers. They were mostly composed of S. lycopersicum var cerasiforme accessions producing fruits with low locules. The fasciated phenotype was under represented in all tested accessions (5.6%). Because lc and fasciated are known to interact epistatically, another collection of accessions was used to determine whether lc was necessary for the phenotypic expression of the fasciated phenotype.

Thus, a complementary set of 87 modern cultivars producing fruits with more than six locules and considered fasciated were analyzed. Among these 87 cultivars, only three carried the low locule number allele of lc (Supplemental Table S3). Approximately 97% of the cultivars carried the allele of lc producing a high locule number. These results suggested that lc is necessary for the expression of a clear fasciated phenotype. Surprisingly, only 39 cultivars (44%) among the 87 had the mutant allele of fasciated, confirming that fas is not the only locus that produces fasciated fruits.

DISCUSSION

An intraspecific cross between two cultivated tomato lines allowed a map-based cloning of the lc QTL

Almost all wild tomato species produce bi-locular fruits. During domestication, the number of locules in fruits has increased together with fruit weight, which ranges from a few grams in wild species to up to 1000 grams in some cultivated varieties. Compared to other plant species, tomato fruit size is an excellent model for understanding the molecular basis underlying domestication of fruit bearing crops. The molecular characterization of the loci that control fruit size also provides insight into fleshy fruit development. fw2.2, the first QTL controlling fruit weight to be cloned, was identified via a map-based cloning strategy using an interspecific cross. The same strategy was employed to clone the gene responsible for the fasciated QTL, which determines the multi-locular phenotype in beefsteak type tomatoes. In both examples, the wild type allele came from S. pennellii. This distant wild relative was used because it had high polymorphism levels compared to S. lycopersicum varieties. The population of introgression lines from S. pennellii (Eshed et al., 1992) could not be used in this study because both parents have the same haplotype at the two SNPs and the same low locule number phenotype. Although lc is described as a major QTL, its effect on the number of fruit locules is weak, compared to that of fasciated. Thus, all genetic background effects needed to be overcome to determine which phenotypic variations could be attributed to lc alone, without any segregation of other minor QTLs. The best method to ensure this requirement was the use of near isogenic lines. An intraspecific cross between two cultivated accessions was selected to clone the lc QTL, which had been previously mapped in our segregating population (Lecomte et al., 2004). The two near isogenic lines only differed from each other in the region containing lc. A 1608 bp region responsible for the phenotype was identified based on the genotyping of 9456 F2 plants from this cross and the phenotyping of studied by self-pollinating the F2 recombinant plants and measuring the phenotype in the resulting homozygous F3 plants.

Association mapping permitted the identification of two SNPs responsible for lc but alone would not have identified the QTL

Linkage disequilibrium mapping allows the identification of the genes responsible for complex traits (Long and Langley, 1999). This approach was first developed to overcome the impossibility of obtaining the large segregating populations necessary for QTL mapping in humans (Spielman et al., 1993). The principle is simple, involving the comparison of two groups of genetically unlinked individuals that differ for a particular trait. The two groups are then genotyped with molecular markers, and statistical associations between the phenotype and the genotype at the markers are determined.

Association mapping is based on linkage disequilibrium (LD), which varies significantly between species. In maize, linkage disequilibrium is low within distances ranging from 200 to 1500 bp (Remington et al., 2001). In tomato, it remains high within distances of 20 cM (van Berloo et al., 2008). Because high LD increases the risk of detecting false positive associations, this study used a combination of map-based cloning to identify the locus region and association mapping to refine its molecular characterization. The map-based cloning step identified a 1608 bp region. For the association mapping step, this region was sequenced in a core collection composed of 88 accessions selected from a larger population that was designed to maximize the molecular diversity. In addition to the 14 polymorphic sites found between both parental lines, 21 polymorphisms were identified from this core collection. As expected, more than 81% of the new polymorphic sites were found in S. pimpinellifolium wild accessions. Association mapping identified two SNPs that had an almost perfect association with the locule number phenotype. This association was then validated using a larger set of tomato accessions. The same result was obtained, confirming that the two SNPs were responsible for the phenotype.

An additional 24 DNA fragments located on chromosome 2 were sequenced in the core collection (Ranc, unpublished data). The two SNPs responsible for lc, however, were not in linkage disequilibrium with any other polymorphic site adjacent to the locus or distantly located on chromosome 2. Given this observation, the identification of the molecular region corresponding to lc using association mapping alone would have been impossible except by sequencing the region by chance. New high throughput sequencing tools allow genomes to be an approach involving the comparison of a large number of full genomes could be used in the near future to identify an increasing number of genes for complex traits.

A model for locule number evolution in tomato fruits

Using SSR markers, the genome of cherry tomato accessions (S. lycopersicum var.

cerasiforme) has been shown to be a mosaic of the genomes from S. pimpinellifolium and S.

lycopersicum (Ranc et al., 2008). Cherry tomatoes are thus a useful tool for association mapping because of their intermediate molecular diversity level and admixture genome structure. The 180 accessions used for association mapping were mostly composed of S. lycopersicum var. cerasiforme tomatoes. Wild tomato species, however, mainly produce fruits with a low locule number. Among the 180 accessions, 81.1% produced fruits with two to four locules, and 18.9% produced fruits with more than four locules. This collection allowed the identification of the two SNPs responsible for lc but could not be used to determine the evolutionary history of locule number during tomato domestication. The mechanism by which fasciated could have evolved compared to lc was studied using a third set of tomato accessions that only included accessions producing fruits with more than six locules.

Interestingly, only 44% of the accessions carried the fasciated high locule number allele, but 97% carried the high locule number allele of lc. This result indicates that the high locule number allele of lc is necessary to express the fasciated phenotype in combination with the fas locus and that it is also required to produce the beefsteak tomato phenotype in combination with one or more other loci in addition to fasciated.

Additionally, all wild species tested contained the lc haplotypes that produces fruits with a low number of locules, suggesting that the two SNPs responsible for the increase of locule number appeared during tomato domestication in a S. lycopersicum var. cerasiforme cultivar and then spread among these cultivars. fasciated appeared later, and the combination of the two loci produced the fasciated phenotype exhibited by the first cultivars introduced in Europe ( Daunay et al., 2007). A model is thus proposed to explain the evolution of locule number during tomato domestication and breeding (Fig. 6).

The average value of Tajima's D for the entire chromosome 2 is -0.633, suggesting a recent demographic expansion that followed the bottleneck of domestication (Fig. 5B). The two In the proposed model, lc existed as a polymorphic locus in the wild species, and all alleles resulted in the production of fruits with two locules. Then, the two SNPs responsible for the increase in the number of locules appeared in a particular allele of lc, which has subsequently expanded in S. lycopersicum accessions because it conferred larger fruits. Later the fas mutation allowed a further increase in locule number when expressed in the lc background. In our hypothesis, we proposed that lc appeared prior to other loci responsible for the increase of locule number. The wild type allele of lc has been recently reintroduced in S. lycopersicum by modern breeding to diversify fruit shape.

How to characterize the molecular function of lc?

Interestingly, the genomic region of lc is located in a noncoding region between two putative candidate genes, WUSCHEL and a gene encoding a WD40 repeat protein. WUSCHEL, which was previously proposed as a candidate gene for lc (Barrero et al., 2006), has a central role in apical meristem development, being responsible for stem cell fate and affecting meristem size. No differences in the expression of the two genes could be detected in a set of sub-NILs.

To determine whether the lc region could be expressed, quantitative RT-PCR assays using several primer pairs inside and surrounding the locus were performed. These assays did not reveal any expression of this region. Instead of using transformation to try to validate the QTL, we preferred to use a combination of fine mapping and association mapping. This method successfully identified the two SNPs responsible for lc. Although transgenic plants are used to help understand the function of a gene, this method would have been difficult for lc due to the unclear limits of the functional region necessary to express the phenotype.

Indeed, the locus did not show any homology with a known coding sequence. Even though we concluded that the 1608 bp region contains the polymorphic sites responsible for the phenotype, we cannot exclude that the functional region could be larger than 1608 bp. et al., 2008). Interestingly, a YABBY transcription factor acts on the partitioning of shoot apical meristem in Arabidopsis (Goldshmidt et al., 2008). This observation could favor WUSCHEL as the best candidate interacting with fas. WUSCHEL expression is regulated by several factors (Dodsworth, 2009). Given that the lc SNPs are located 1080 bp from the stop codon of WUSCHEL, they could act as post-transcriptional regulators.

WD40 repeat proteins play important roles in eukaryotes. Interestingly, the WD40 protein near lc is orthologous to At5g66240 in Arabidopsis. At5g66240 is itself homologous to the At5g66430 gene that encodes the FAS2 protein (Kaya et al., 2001). FAS1 and FAS2 are two subunits of the CAF1 complex (Chromatin remodeling factor-1). The FAS genes are described as regulating the maintenance of the expression state of WUSCHEL in the shoot apical meristem. Mutations in fas1 and fas2 were also described as causing stem fasciation together with altered floral development (Leyser and Furner, 1992). Stem fasciation is often linked to fruit fasciation in tomato, which is due to the high increase of locule number.

All together, these observations do not allow discrimination between WUSCHEL and FAS2like as the gene that is modified by the two SNPs and thus is responsible for the phenotype. lc could act on shoot apical meristem development by regulating both or either gene in a complex way. The two SNPs could also act on the regulation of one or several genes elsewhere in the genome.

Version postprint

Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. ( 2011

CONCLUSION

Although we were unable to identify the function of the two polymorphic nucleotides responsible for lc, we can assume that they play an important role in meristem development.

Although the lc region is not expressed, the two SNPs have a pleiotropic effect on locule number, floral organ number and fruit weight and might act by regulating meristem size.

These QTN are flanked by two genes that could affect tomato fruit development. Thus, these two SNPs may regulate either or both of these two genes, but at a very specific stage.

MATERIALS AND METHODS

Plant material and phenotyping

All accessions are maintained in the GAFL research unit (INRA Avignon, France) and are available upon request (helene.burck@avignon.inra.fr, mathilde.causse@avignon.inra.fr).

All plants were grown in greenhouses in Montfavet (south of France) between 2004 and 2008.

Fine mapping and ultra-high resolution mapping were performed on a F2 population derived from a cross between the two near isogenic lines CF12-C and CF13-L (described as F8-V-C and F8-V-L, respectively (Lecomte et al., 2004)). Plants were sown in 96-well plates and transferred to 3-L containers after genotype selection. The locule number of each plant was determined by phenotyping 20 mature fruits (10 from the second truss and 5 each from the third and fourth trusses). The petal number and flower diameter of each plant was determined by analyzing 20 flowers.

For the diversity analysis, 10 fruits per accession were phenotyped.

Genotyping

The z100_CAPS and z274 primers were used to amplify the T1555 and TG191 markers, and the resulting PCR products were digested with BamHI and NdeI, respectively, and mapped on the F6 RIL population derived from the cross between Cervil and Levovil (Causse et al. 2002). Taqman markers developed from the associated SNPs were used to screen 2688 F2 plants for the fine mapping analysis. Those plants with different genotypes at T1555 and TG191 (i.e. recombination between the two markers) were selected for fine mapping. The same markers were also used to screen 8 F3 plants from the self-progeny of each F2 recombinant plant. Two plants homozygous for the segregating markers were then selected for phenotyping.

Polymorphisms in the z1416 and z1420 markers were used to develop Taqman markers that were used to screen 6768 F2 plants for the ultra-high resolution mapping analysis. As above, these markers were also used to genotype 8 F3 plants from the self-progeny of each recombinant F2 plant.

Alleles and BAC sequencing

All sequencing reactions were performed either manually on the ABI3730XL (Philippe et al. plasmid. All other sequencing reactions were performed using PCR products. All primers used for PCR amplification are described in Supplemental Table S4.

The LeHBa0139K19 BAC sequencing was performed by Genome Express.

Sequence and polymorphism analysis

Sequence alignment and SNP detection were performed manually using the Genalys software (Takahashi et al., 2003), which is available at http://software.cng.fr. S5.

Accession numbers

All

Expression analysis

Samples from leaves, flower buds or fruits at 7 or 14 days after anthesis were randomly harvested (4 plants/line) and immediately frozen in liquid nitrogen. Total RNA were extracted from 3 different pools with TRI Reagent Solution (Ambion, Austin,TX,USA) following the procedure described by the manufacturer.

RT-qPCR were performed and analyzed has described by Prudent et al. (2010). The genes of interest coding for WUSCHEL (AJ538329), the WD40 protein (SGN-U585584) and the eIF-4A-2 gene used as an internal control (SGN-U593757) were amplified; a specific PCR product was obtained with the corresponding efficiency: 100.8%, 108.1%, 110.8% respectively.

RT-PCR were performed in the same conditions with the same samples but amplified without SYBR Green on a classic thermal cycler.

Northern Blots were performed using lc locus as a probe on the same RNA samples as described by Muños et al. (2004). identified by positional cloning and 16 on the rest of the chromosome 2) were used to sequence the plants in this core collection. A mixed linear model (Yu et al., 2006) implemented using Tassel software was used for the association tests of the core collection.

Diversity analysis

A
The genetic structure of each sample was taken into account and calculated using Structure2.0 software (http://pritch.bsd.uchicago.edu/structure.html) based on microsatellite genotyping information (Ranc et al., 2008). The genetic background interaction was also taken into account using an estimation of kinship matrix following the recommendations of Yu et al. C. Sequence alignment of the two alleles of lc from Cervil and Levovil. The 14 polymorphic sites are highlighted in gray. The two underlined SNPs are responsible for lc (Fig. 5).

Figure 3. Expression analysis of the two candidate genes

RT-PCRs were performed on total RNA extracted from either floral buds, leaves or fruits.

Primers were designed on WUSCHEL (AJ538329 accession number), the WD40 repeat protein (SGN-U585584 tomato unigene). eIF4A2 (SGN-U593757 tomato unigene) was used as a reference gene. Cervil and Levovil are the parental lines. L2 and L4 have been obtained by marker assisted selection from Levovil; they are both identical to Levovil except for a region of chromosome 4 from Cervil (L4) and the region of the chromosome 2 from Cervil which contains lc (L2).

L2 and L4 must be compared to Levovil. CF12-C and CF13-L are a pair of identical isogenic lines except for a 30 cM region which contains lc. JB1538 and JB1546 are identical to CF12-C and CF13-L respectively but differ for less than 30cM. PA205-2 and JB149-9, on the same principle, differ from the one another by a 28kb region surrounding lc.

The lines are all homozygous at the lc locus and contain either the alleles from Cervil (C) which produce fruits with low locule number or from Levovil (L) which produce fruits with high locule number. The genetic background is either Cervil (C) or Levovil (L); it is recombinant (R) between Cervil and Levovil for the isogenic lines.

The mean of each line has been compared to Cervil. Statistical significances of the t-test are indicated as ns (not significant) or *** (P<0.001).

A B

Tomato accessions producing

A B

Tomato accessions producing fruits with more than 3 locules less than 3 locules more than 3 locules less than 3 locules

S. lycopersicum

Figure 6. Model of locule number evolution in tomato fruits during domestication

Solanum pimpinellifolium is considered the wild ancestor of the cultivated tomato Solanum lycopersicum. Based on the analysis of 267 tomato accessions, we propose a model that could explain the history of locule number evolution during tomato domestication. In our study, only 4.6% of the high locule number accessions (i.e., those with >3 locules) had the low locule allele of lc and 96.9% of the fasciated accessions (i.e., those with >6 locules) had the high locule allele of lc, but only 49.4% of them had the fas allele. These results indicate that lc was required for the increase in locule number in tomato fruits during domestication. The lc locus could have appeared before the fasciated locus. These two QTLs are the major loci controlling locule number. Modern breeding has used other loci to expand phenotypic diversity.

  Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997

  SNPs responsible for lc (positive Tajima's D) evolved differently from other loci on the chromosome (negative Tajima's D for the 23 other loci). The negative Tajima's D values for the 23 loci located along the entire chromosome 2 indicated an increase of the population size in contrast to lc with a positive value. The two SNPs were under balancing selection (Fig 5B, Tajima's D = 2.227, p-value < 0.05), indicating that both alleles were selected during the domestication process, in contrast to other loci selected during domestication such as tb1 in maize (Wang et al., 1999) or fw2.2 in tomato (Nesbitt and Tanksley, 2002) that were under positive selection.

  Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997

  267 recombinant sub-NILs lines. The success of a positional cloning approach is based on reliable phenotyping. Because lc is semidominant, heterozygous plants have an intermediate phenotype between that associated with the two homozygous alleles. The difference between the two extreme values of the two alleles was too close to distinguish the heterozygous plants from either homozygous plants. Thus, the segregation of the phenotype was systematically Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997

  SNPs responsible for lc exhibit a significantly different pattern. The evolutionary history of lc Version postprint Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997is more likely explained by a particular pattern of evolution rather than by the demographic evolution of the sample used to validate the QTL. The positive Tajima's D value for the two SNPs indicates a balancing selection. This type of selection is explained by the maintenance of the two alleles at the locus, with an equilibrated frequency.

lc

  had previously been shown to be necessary to obtain a fasciated phenotype(Lippman and Tanksley, 2001). The results presented herein further the understanding of the interaction between lc and fasciated. First, fasciated was not the only locus producing tomato fruits with a high number of locules. Second, high locule number allele at lc was necessary for the Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997 fasciated phenotype. At the molecular level, fasciated encodes a YABBY-like transcription factor (Cong

  The two QTLs fasciated and lc make tomato an excellent model to study floral meristem and fleshy fruit development. Herein, two SNPs responsible for the increase of locule number were identified and used to develop a model of the evolutionary history of tomato domestication. An understanding of the mechanism by which this non-coding region affects locule number in the fruits and floral organ number in the flowers still remains of great interest and will require additional experiments. Although this study did not reveal any evidence for the expression of the lc region, it might be expressed in specific cells or at a precise time of development. In situ hybridization experiments should be performed to determine whether the lc region is expressed at the cellular level in meristems, where it should act. Similar experiments using WUSCHEL and the FAS2-like protein as probes may also reveal differential expression between lines not identified by RT-PCR on floral buds.Version postprintComment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997

  , 2010) or by Genome Express (Meylan, France), GATC (http://www.gatc-biotech.com) or MWG (http://www.eurofinsdna.com). The TG463 sequence was obtained by sequencing the Version postprint Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997

  total of 180 accessions (37 S. lycopersicum, 128 S. lycopersicum var. cerasiforme, 15 S. pimpinellifolium) were phenotyped during the summers of 2007 and 2008. Part of this sample represents a core collection of 88 individuals selected to maximize the diversity of the whole collection (Ranc et al., 2008). A total of 24 genomic 500 bp fragments (8 on the BAC Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997

(Figure 1 .

 1 FIGURE LEGENDS

Figure

  Figure 2. Fine mapping (A), ultra-high resolution mapping (B) and polymorphism (C) of lc QTL.A. All sequences highlighted in green represent tomato genes syntenic with the At2g18000 region in Arabidopsis thaliana. Markers highlighted in yellow were used to screen for recombinant plants. The PA205-2 and JB149-9 recombinant lines allowed the identification of the BAC clone containing the QTL. This BAC, LeHBa139K19, contains 11 putative ORFs, with some showing homology to known genes or expressed sequences. lc is located in the region between z1416 and z1420.B. The z1416 and z1420 markers were used to screen for recombinant plants. Newly designed markers finally localized lc between z1497 and z1499. Seven plants with recombination events between these two markers were identified. The molecular characterization of these 7 lines restricted lc to a 1608 bp non-coding DNA region. The orientation (from ATG to stop codon) and the exons (in blue) of the cDNAs coding for WUSCHEL and the WD40 protein are shown.

Figure 4 .

 4 Figure 4. Effect of lc on locule number (A), flower size (B) and petal number (C).

Figure 5 .Figure 1 .

 51 Figure 5. Molecular diversity of the lc locus A. Tomato haplotypic structure of lc and other loci on chromosome 2 Each block corresponds to one of the 24 amplicons (average length of 500 bp). Columns and rows represent individuals and SNPs, respectively. With Heinz1706 used as reference, polymorphisms are indicated either in gray (identical allele) or in black (different allele).

Figure

  Figure 2. Fine mapping (A),

  The orientation (from ATG to stop codon) and the exons (in blue) of the cDNAs coding for WUSCHEL and for WUSCHEL and the WD40 protein are shown.

  lc from Cervil and Levovil. The 14 polymorphic sites are highlighted in gray. The two underlined SNPs are two underlined SNPs are responsible for lc (Fig.5).

Figure 5 .

 5 Figure 5. Molecular diversity of the lc locus lc QTN Figure 5. Molecular diversity of the lc locus A. Tomato haplotypic structure of lc and other loci on chromosome 2Each block corresponds to one of the 24 amplicons (average length of 500 bp). Columns and rows represent Each block corresponds to one of the 24 amplicons (average length of 500 bp). Columns and rows represent individuals and SNPs, respectively. With Heinz1706 used as reference, polymorphisms are indicated either in gray (identical allele) or in black (different allele). (identical allele) or in black (different allele).B. Tajima test over the whole chromosome and within lc.Stars indicate a significant departure from the neutrality hypothesis. A significant positive value of Tajima's D for lc Stars indicate a significant departure from the neutrality hypothesis. A significant positive value of Tajima's D for lc indicated either a balancing selection of the locus or a population decrease. An analysis of the entire chromosome showed that lc evolved differently from the entire chromosome: lc underwent a balancing selection, whereas showed that lc evolved differently from the entire chromosome: lc underwent a balancing selection, whereas chromosome 2 evolved following a population expansion. Dashed lines represent the mean and standard error for the whole chromosome. whole chromosome. C. Molecular diversity of lc in wild and cultivated lines. The diversity (π) of the locus was drastically reduced in cultivated lines (black lines) except for two SNPs (arrow) in comparison with wild accessions (gray lines). A total of 17 cultivated and 11 wild lines were used in this analysis.

  Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located

	lc_allele_from_Cervil C	TGACTAATATTTTAAGATGTTTGTATTTTTTTCATTTCAAATTTAAATTAATGATTATTCTAATTCAAATTGGTTTAATTAAATTTCGTAAAACGAAACA
	lc_allele_from_Levovil	TGACTAATATTTTAAGATGTTTGTATTTTTTTCATTTCAAATTTAAATTAATAATTATTTTAATTCAAATTGGTTTAATTAAATTTCGTAAAACGAAACA
	lc_allele_from_Cervil lc_allele_from_Cervil	TGACAAATCAGAATGAACCAAAGGAAATAATATGCGTGTAGTAGTTAGTAGTCTATATTTAGGGTTTGATCATGGTCTTGGTCTCTTGGATGATGACTAT TGACAAATCAGAATGAACCAAAGGAAATAATATGCGTGTAGTAGTTAGTAGTCTATATTTAGGGTTTGATCATGGTCTTGGTCTCTTGGATGATGACTAT
	lc_allele_from_Levovil	TGACAAATCAGAATGAACCAAAGGAAATAATATGCGTGTAGTAGTTAGTAGTCTATATTTAGGGTTTGATCATGGTCTTGGTCTCTTGGATGATGACTAT
	lc_allele_from_Cervil	TGCACTTTGAAAGTGGACAATTTTTCCTATTATAACTTTTTCTTGTTTAATTAATATCACCTTATAGTGATAGAGATTTGGCATAGACCTTTTACCTTGT
	lc_allele_from_Levovil	TGCACTTTGAAAGTGGACAATTTTTCCTATTATAACTTTTTCTTGTTTAATTAATATCACCTTATAGTGATAGAGATTTGGCATAGACCTTTTACCTTGT
	lc_allele_from_Cervil	AAATTAAATCCAAGTACAAATTATGTTAACCAGAAAGATGTATATTAGGACATCTCTCCAATTTGACCATCACTTTAGGTACTTCAACTAGTTTAGTAGA
	lc_allele_from_Levovil	AAATTAAATCCAAGTACAAATTACGTTAACCAGAAAGATGTATATTAGGACATCTCTCCAATTTGACCATCACTTTAGGTACTTCAACTAGTTTAGTAGA
	lc_allele_from_Cervil lc_allele_from_Cervil	TTTGAAGCAAGAAAAAAGAGCCTGTTTTTTTTTTGTTTTAAAAATCACTCTCTAATAATCTGAAAGTGAAGAGTGTTTATCTTCTGAACCACACGTCTCG TTTGAAGCAAGAAAAAAGAGCCTGTTTTTTTTTTGTTTTAAAAATCACTCTCTAATAATCTGAAAGTGAAGAGTGTTTATCTTCTGAACCACACGTCTCG
	lc_allele_from_Levovil	TTTGAAGCAAGAAAAAAGAGCCTGTTTTTTTTTTGTTTTAAAAATCACTCTCTAATAATCTGAAAGTGAAGAGTGTTTATCTTCTGAACCACACGTCTCG
	lc_allele_from_Cervil	TCAGAATGGCATGTTTACAAGACCACATGTTCATACAGAAGTATTATTTGAATGACATGAATTAGGATTGTGTTTGAGATGGAGATTTAGACGACATGAG
	lc_allele_from_Levovil	TCAGAATGGCATGTTTACAAGACCACATGTTCATACAGAAGTATTATTTGAATGACATGAATTAGGATTGTGTTTGAGATGGAGATTTAGACGACATGAG
	lc_allele_from_Cervil	AGACTTCGAAATGACTTTTCCAAACTTTATCTTACCAATAAGTTGTCTTGCCTGATATCCTTTTCCTAAAAGATTTGGCATGATGTTTACTAATTGGACA
	lc_allele_from_Levovil	AGACTTCGAAATGACTTTTCCAAACTTTATCTTACCAATAAGTTGTCTTGCCTGATATCCTTTTCCTAAAAGATTTGGCATGATGCTTACTGATTGGACA
	lc_allele_from_Cervil	ATTCGTACTACTTTATACATTTCATAGCATTCCATTTTTTTTTTGGACATATTTTACATTATATTCTATAAAATATTCAAGATCAAAACACTATTTTTAT
	lc_allele_from_Levovil lc_allele_from_Levovil	ATTCGTACTACTTTATACATTTCATAGCATTCCATTTTTTTTTTGGACATATTTTACATTATATTCTATAAAATATTCAAGATCAAAACACTATTTTTAT ATTCGTACTACTTTATACATTTCATAGCATTCCATTTTTTTTTTGGACATATTTTACATTATATTCTATAAAATATTCAAGATCAAAACACTATTTTTAT
	lc_allele_from_Cervil	ATACAAAACTACTTAAATTATGGTTATTGTTTTGTCCTTAATTTTAAGAATGGACACGAGGCATTATTTTATTTTTAACTAAAAGGGATCATTTGGTTTG
	lc_allele_from_Levovil	ATACAAAACTACTTAAATTATGGTTATTGTTTTGTCCTTAATTTTAAGAATGGACACGAGGCATTATTTTATTTTTAACTAAAAGGGATCATTTGGTTTG
	lc_allele_from_Cervil lc_allele_from_Cervil	AAATATTGATGTGTTCGGCCGGTTGGGAAATGTTTTTCAAAATTTTTTTTCGTCTTCCTTGCTTATTTTATGTGTTTGATATATAAATAAAACATATTAC AAATATTGATGTGTTCGGCCGGTTGGGAAATGTTTTTCAAAATTTTTTTTCGTCTTCCTTGCTTATTTTATGTGTTTGATATATAAATAAAACATATTAC
	lc_allele_from_Levovil	AAATGTTGATGTGTTCGGCCGGTTGGGAAATGTTTTTCAGAATTTTTTTTCGTCTTCCTTGCTTGTTTTATGTGTTTGATATATAAATAAAACATATTAC
	lc_allele_from_Cervil	TCTAATTTTTCATACGTATAATTTAGACAAATACTATAGAAAATGAGGCGCTGATAGAAAGATAGTGTGATGGTGATAACGGATAGAGGGATGAGTATGA
	lc_allele_from_Levovil	TCTAATTTTTCATACGTATAATCTAGACAAATACTATAGAAAATGAGGCGCTGATAGAAAGATAGTGTGATGGAGATAACGGATAGAGGGATGAGTATGA
	lc_allele_from_Cervil	TGAAGGTTATCTATATTAAAAACAAAGCATAATTAATATAAAATGATAATGTGATGAAAATGACGGATGGAGGGATGAGCATGAT-GAACGTTATTTATA
	lc_allele_from_Levovil	TGAAGGTTATCTATATTAAAAACAAAGCATAATTAATATAAAATGATAATGTGATGAAAATGACGGATGGAGGGATGAGCATGATTGAACGTTATTTATA
	lc_allele_from_Cervil	TTAAAAACAAAACATAATTAATGTAAAATATCATTATTCAACTTTATTTTCATTGATGAGCCTATTGGCCAAGGTAAAATACATAAATGTAGTGCGTGTA
	lc_allele_from_Levovil lc_allele_from_Levovil	TTAAAAACAAAACATAATTAATGTAAAATATCATTATTCAACTTTATTTTCATTGATGAGCCTATTGGCCAAGGTAAAATACATAAATGTAGTGCGTGTA TTAAAAACAAAACATAATTAATGTAAAATATCATTATTCAACTTTATTTTCATTGATGAGCCTATTGGCCAAGGTAAAATACATAAATGTAGTGCGTGTA
	lc_allele_from_Cervil	TAACCTCATAACATCTAAGAGAATCGTAAACTTGTACAGTCTCAACTTTCCGCAGCCTCATAATTAAATTACAAAATGGAATATGTATCAGAATATTTTA
	lc_allele_from_Levovil	TAACCTCATAACATCTAAGAGAATCGTAAACTTGTACAGTCTCAACTTTCCGCAGCCTCATAATTAAATTACAAAATGGAATATGTATCAGAATATTTTA
	lc_allele_from_Cervil	ATCATTAAATTAGACGATTCATCCTATACTCTCTCCATTTCAAAATAAAAATTAGTTTTTCCTTTTATTAAATTCAAATTGTGTTTAATGTATCAAAATA
	lc_allele_from_Levovil	ATCATTAAATTAGACGATTCATCCTATACTCTCTCCATTTCAAAATAAAAATTAGTTTTTCCTTTTATTAAATTCAAATTATGTTTAATGTATCAAAATA
	lc_allele_from_Cervil	TTTTTAAAAATTTTTTTAGACTTAAACATATCACGAGAAATATCAGCTTAAAATAATTAATTTCTATCAATTTCAATTTATTTGTCTTTAAAATCTATTT
	lc_allele_from_Levovil lc_allele_from_Levovil	TTTTTAAAAATTTTTTTAGACTTAAACATATCACGAGAAATATCAGCTTAAAATAGTTAATTTCTCTCAATTTCAATTTATTTGTCTTTAAAATCTATTT TTTTTAAAAATTTTTTTAGACTTAAACATATCACGAGAAATATCAGCTTAAAATAGTTAATTTCTCTCAATTTCAATTTATTTGTCTTTAAAATCTATTT
	lc_allele_from_Cervil	AAAAAGAAC
	lc_allele_from_Levovil	AAAAAGAAC

near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997

ACKNOWLEDGMENTS

We thank the master students who helped in the experiments: Abir Youssef, David Tricon, Xavier Titeca, Claire-Emmanuelle Modin.

Aurélie Chauveau and Rémi Bounon helped in the allele sequencing experiments.

Florent Leydet and Laure David participated in the fine mapping experiment and in the association mapping, respectively.

Hélène Burck manages and characterizes all the accessions of the tomato collection at INRA in Avignon and provided the seeds.

We thank the greenhouse experimental team of the GAFL research unit for growing all plants.

Jean-Paul Bouchet helped on primer design.

We gratefully thank Esther Van Der Knaap for critical review of the manuscript and for providing one marker to confirm some data for fas genotyping.

We thank Rebecca Stevens for editing the manuscript.

Version postprint

Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S., Duffe, P., Carretero, Y., Le Paslier, M.-C., Delalande, C., Bouzayen, M., Brunel, D., Causse, M. (2011). Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL. Plant Physiology, 156 (4), 2244-2254. , DOI : 10.1104/pp.111.173997

Implications for the evolution of fruit size in the domestication of cultivated tomatoes. decrease. An analysis of the entire chromosome showed that lc evolved differently from the entire chromosome: lc underwent a balancing selection, whereas chromosome 2 evolved following a population expansion. Dashed lines represent the mean and standard error for the whole chromosome.

C. Molecular diversity of lc in wild and cultivated lines.

The diversity (π) of the locus was drastically reduced in cultivated lines (black lines) except for two SNPs (arrow) in comparison with wild accessions (gray lines). A total of 17 cultivated and 11 wild lines were used in this analysis. Supplemental Photo 1. Photo of fruits from some varieties used in the study.

Fruits illustrate morphological diversity. Supplemental Table S1. Genotype and phenotype of the 215 recombinant F2 plants and their F3 progeny used for lc fine mapping.

Supplemental

These data show genotypes for T1555 and TG191 markers and phenotypes for locule number.

For each F2 recombinant plant, 8 F3 plants from the selfed progeny were genotyped in order to remove heterozygosity (H). Two F3 plants were selected: one with the two alleles from Cervil (C), the other with the two alleles from Levovil (L). Locule number corresponds to the average of 20 measured fruits. Data highlighted in purple are those of the two lines which allowed the identification of the BAC clone containing lc. For each pair of F3 lines, locule number averages have been compared by a Student's t-test, statistical significance are noted ns (not significant), *** (P<0.001), ** (P<0.01) and * (P<0.05). S2. Validation of the association by genotyping the two SNPs in 92 tomato accessions.

Supplemental Table

The data were obtained using 92 tomato accessions composed of 21 S. lycopersicum, 66 S. lycopersicum var. cerasiforme and 5 Solanum pimpinellifolium. These accessions have been grown and phenotyped for locule number in 2007 and 2008. 10 fruits have been measured for each accession. All accessions were genotyped by sequencing the 235 bp region containing the two SNPs responsible for lc. Genotype is noted 0 if homozygous for the low locule allele, 1 if homozygous for the high locule allele and 2 if heterozygous. S3. Analysis of lc and fas loci in tomato accessions producing fasciated fruits with more than 6 locules.

Supplemental Table

The data were obtained using 87 tomato accessions producing fasciated fruits with more than 6 locules. These accessions are a part of the collection maintained in the GAFL research unit (INRA, France). All accessions have been genotyped for the two SNPs responsible for lc and for fas. The genotype at the lc locus is noted 0 if homozygous for the low locule allele, 1 if homozygous for the high locule allele and 2 if heterozygous. The genotype at the fasciated locus is noted fas for the alleles producing fasciated fruits and wt for the alleles producing unfasciated fruits. S4. Primer sequences used in the study.

Supplemental Table

Version postprint

Comment citer ce document : Munos, S. (Auteur de correspondance), Ranc, N., Botton, E., Berard, A., Rolland, S.


H Archimède
email: harry.archimede@antilles.inra.fr

M Eugène

C Marie Magdeleine

M Boval

C Martin

D P Morgavi

P Lecomte

M Doreau

T A Mcallister

K A Beauchemin

X Hao

S Mcginn

H Robinson

Comparison of methane production between C3 and C4 grasses and legumes

Keywords: Methane production Ruminants Tropical forages Temperate forages Meta-analysis forage neutral detergent fibre, OM, organic matter, OMd, OM apparent digestibility

A meta-analysis was conducted to compare effects of C4 and C3 grasses as well as warm and cold climate legumes on CH 4 production of ruminants. For this purpose, a database was built using 22 in vivo studies containing 112 observations with 58 C3 grasses, 28 C4 grasses, 26 cold legumes and 12 warm legumes. Neutral detergent fibre (NDF), crude protein (CP) and total tract organic matter (OM) digestibility ranged from 415 to 753 g/kg DM versus 361 to 754 g/kg DM, from 24 to 254 g/kg DM versus 44 to 314 g/kg DM and from 0.51 to 0.71 versus 0.56 to 0.83 for C4 and C3 grasses, respectively. The NDF, CP and total tract OM digestibility ranged from 441 to 690 g/kg DM versus 252 to 684 g/kg DM, from 93 to 236 g/kg DM versus 141 to 269 g/kg DM and from 0.42 to 0.57 versus 0.38 to 0.79 for warm and cold legumes, respectively. Relationships between CH 4 production and forage characteristics were analysed by analysis of covariance. For grasses, the main factors tested as fixed effects were NDF and CP content of the diet, total tract OM digestibility, intake, animal species, forage family and random trial effect. For legumes, tannin level was included in the model. Results indicate that ruminants fed C4 grass produced 17% more CH 4 as L/kg OM intake (P<0.05) compared to those fed C3 grass. Animals fed warm legumes produced 20% less CH 4 (P<0.05) than those fed C4 grasses. In contrast, no difference in CH 4 production between C3 grasses and cold legumes. Use of some legumes in warm climates could be a strategy to reduce CH 4 emissions by ruminants.

Introduction

Diet composition and intake are main factors affecting CH 4 production by ruminants. Ruminants fed forages rich in structural carbohydrate produce more CH 4 than those fed mixed diets containing higher levels of non-structural carbohydrates [START_REF] Sauvant | Modélisation des interactions digestives et de la production de méthane[END_REF]. Forages are often the main ingredient in ruminant diets in the tropics. In these regions, feed resources (i.e., grasses, legumes, tree foliage) differ from those of temperate regions due to their chemical and structural composition and digestibility [START_REF] Leng | Factors affecting the utilization of poor-quality forages by ruminants particularly under tropical conditions[END_REF][START_REF] Assoumaya | Etude comparative de l'ingestion et de la digestion des fourrages tropicaux et tempérés[END_REF]. Tropical grasses generally use C4 metabolic pathways for photorespiration whereas most temperate grasses use C3 carbon fixation. Often, the C4 metabolic pathway leads to a higher rate and degree of deposition of lignin in plant tissues, a factor which can alter voluntary intake and digestion [START_REF] Wilson | Cell wall characteristics in relation to forage digestion by ruminants[END_REF], although there are exceptions. Some tropical forages, such as legumes, shrubs and tree foliages may contain secondary metabolites such as condensed tannins and saponins that alter rumen methanogenesis [START_REF] Jouany | Use of 'natural' products as alternatives to antibiotic feed additives in ruminant production[END_REF].

It has been reported that ruminants fed tropical forages produce more CH 4 than ruminants fed temperate forages [START_REF] Mccrabb | Prediction of methane emissions from beef cattle in tropical production systems[END_REF]. Also, the presence of condensed tannins in legume, tree and shrub foliage has been found to lower CH 4 emissions [START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: progress and challenges[END_REF] mainly due to inhibition of methanogens in the rumen [START_REF] Martin | Methane mitigation in ruminants: from microbe to the farm scale[END_REF]. However, studies based on direct in vivo comparison of C4 versus C3 grass, or of legumes grown in warm ('warm' legume) versus cold environments ('cold' legume) have been rare due to geographical constraints.

Our objective was to compare CH 4 emissions of ruminants fed C4 versus C3 grass, and warm versus cold legumes using a meta-analysis approach with consideration for explanatory factors such as dietary fibre content, total tract digestibility and intake.

Materials and methods

Publications selection and codification for the database

The database used contained 112 treatments extracted from 22 scientific publications. To be included, a publication had to contain data on forage composition, feed intake, digestibility and CH 4 emission measurements. Only all forage diets were included and cereal forages, such as corn and sorghum, were not included. Forages were classified into grasses and legumes. A grass was assigned as C4 when it had a C4 metabolic pathway, which is characteristic of tropical grasses, and it was harvested in the tropics. A grass was assigned as C3 when it had a C3 metabolic pathway, which is characteristic of temperate grasses, and it was harvested in temperate environments. As all legumes have C3 metabolic pathways, they were classified into 'warm' and 'cold' types. A warm type was defined as being grown mainly in a tropical environment, and occasionally in a temperate environment during the warm season. A cold type was defined as being grown in a temperate environment. C4 grasses and warm legumes were defined as tropical forages, and C3 grasses and cold legumes were defined as temperate forages. This classification resulted in a distribution of studies among forage types of C3 grass (n = 58), cold legume (n = 14), C4 grass (n = 28) and warm legume (n = 12). As CH 4 production has been shown to be affected by tannins [START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: progress and challenges[END_REF], legumes were coded according to their tannin concentration. Three levels, being high, medium and low were defined on the basis of literature data. High concentrations corresponded to more than 300 g tannin/kg DM, medium concentration included legumes with 100-300 g tannin/kg DM and low included those legumes containing less than 100 g tannin/kg DM. Animal species were coded as large (i.e., cows, heifers, calves; n = 62) and small (i.e., sheep, goats; n = 50) ruminants. The methods used to measure CH 4 were coded as: calorimetric chamber, SF 6 gas tracer technique and gas bag technique.

Calculations

Some estimates were made of organic matter (OM) level of the diet and OM digestibility when data were lacking in the publications. The OM content of diet and OM digestibility were estimated as DM multiplied by 0.9 and DM digestibility multiplied by 1.05, respectively, according to tables of feed values in temperate and tropical countries. The CH 4 production was expressed in litres or per unit of DM intake (kg), (L/kg DM intake), OM intake (kg) (L/kg OM intake), or digested OM (L/g DOM).

Statistical analyses

The meta-analysis followed recommendations of [START_REF] Sauvant | Meta-analysis of experimental data in animal nutrition[END_REF]) using Minitab (2007). Variation in predictability of CH 4 production attributable to a number of variables including OM intake, neutral detergent fibre (NDF) intake, digestible OM intake, forage NDF (NDFf) and OM digestibility (OMd) was examined. Level of intake was considered using the OM intake/liveweight (LW) 0.9 [START_REF] Poppi | Studies of cattle and sheep eating leaf and stem fractions of grasses. 1. The voluntary intake, digestibility and retention time in the reticulorumen[END_REF] as covariates. Predictions of CH 4 production were similar to those obtained with LW as a covariate. Several qualitative factors were accounted for, such as type of forage (C3 grass, cold legume, C4 grass, warm legume), animal species (large and small ruminants), CH 4 measurement method (calorimetric chamber, SF 6 gas tracer technique, gas bag technique) and tannin level (low, medium and high). Moreover a random effect of study (n = 22) was also included in the model. Those variables that accounted for the majority of the predictability of CH 4 production, and were adequately represented among studies, were selected for the final model and included the factors: random study effect, forage, level of intake (OM intake/LW 0.9 ), OMd and NDFf. The statistical analysis model of the database was:

y ijk = + a i + b j + ˛OM intake + ˇOM digestibility + ıNDF content + ε ijk
where y ijk is the CH 4 production, a i is the ith random trial effect (i = 1, . . ., 22), b j is the jth forage type (j = 1, . . ., 4), ˛ is the coefficient associated with the covariable OM intake, ˇ is the coefficient associated with the covariable OM digestibility, ı is the coefficient associated with the covariable NDF content and ε ijk is the residual error.

When a treatment was an outlier (i.e., beyond 3 standard deviations from the adjusted mean) for a character or a specific relationship, it was removed from the statistical analysis.

An analysis of variance was completed on legumes to estimate tannin level effect on CH 4 production. Studies and tannins levels were considered as fixed effects. MOI/LW 0.9 , OMd and NDFf were used as covariates in the model:

y ijk = + a i + b j + ˛OM intake + ˇOM digestibility + ıNDF content + ε ijk
where y ijk is the CH 4 production of legumes, a i is the ith random trial effect (i = 1, . . ., 7), b j is the jth tannin level (j = 1, . . ., 3), įs the coefficient associated with the covariable OM intake, ˇ is the coefficient associated with the covariable OM digestibility, ı is the coefficient associated with the covariable NDF content and ε ijk is the residual error.

Results

Database characteristics

Studies included in the model are in Table 1, with at least 3 studies contributing to the mean values. Data included 50 studies in small ruminants (8 goats and 42 sheep) and 62 studies in large animals (34 cows, 21 heifers, 7 calves). Tropical forages are underrepresented relative to temperate forages, and grasses have been studied more than legumes. Whatever the origin of forage, the variation in chemical composition and digestibility is broad and representative of forage diversity.

Methane production and diet

Those measured components that had the closest relationship to aspects of methane production included: CH 4 L/kg DMintake = -4.2(±11.3) + 39.2(±9.2) × OMd + 21.1(±11.4) × NDFf -0.15(±0.06) × OM intake/LW 0.9 (n = 115, R 2 = 0.77, rsd = 4.1, P < 0.0001) CH 4 L/kg OMintake = 0.9(±11.4) + 39.8(±3.1) × OMd + 18.9(±11.49) × NDFf -0.2(±0.06) × OM intake/LW 0.9

(n = 115, R 2 = 0.84, rsd = 4.1, P < 0.0001) CH 4 L/kg OMD = 34.1(±9.6) + 40.4(±15.2) × NDFf -0.3(±0.09) × OM intake/LW 0.9

(n = 115, R 2 = 0.76, rsd = 6.6, P < 0.0001)

However, the accuracy of the models differed depending on how CH 4 production was expressed, with accuracy higher when CH 4 production was expressed as L/kg OM intake. Whatever the mode of expression of CH 4 , there were no interactions between class of forage and NDFf or between class of forage and OMd. As expected, CH 4 production increased with increasing NDF content and OMd of the diet, and decreased with declining intake.

Methane production was higher for animals fed C4 grasses (Table 2) compared to those fed C3 grasses regardless of how CH 4 production was expressed. Methane production was lower for animals fed warm legumes compared to those fed cold legumes.

A specific statistical analysis of legumes (n = 25, R 2 = 0.92, rsd = 2.6, P=0.01), indicated that CH 4 production (L/OM intake) was lower for animals fed high tannin legumes (21.8 L/kg MOI versus 27.8 L/kg MOI) compared to those fed low tannin legumes. The same model (n = 25, R 2 = 0.80, rsd = 5.6, P=0.01) resulted in estimates of 37.2 L/kg DOM intake versus 52.2 ± 2.7 L/kg DOM intake for high tannin legumes versus low tannin legumes.

Discussion

The main objective was to compare CH 4 production in ruminants fed tropical or temperate forages. Some publications support the hypothesis that tropical C4 grasses are more methanogenic than temperate C3 grasses [START_REF] Kurihara | Methane production and energy partition of cattle in the tropics[END_REF][START_REF] Mccrabb | Prediction of methane emissions from beef cattle in tropical production systems[END_REF][START_REF] Ulyatt | Methane emission from dairy cows and wether sheep fed subtropical grass-dominant pastures in midsummer in New Zealand[END_REF]. To our knowledge, only [START_REF] Margan | Energy and protein values of four forages including comparison between tropical and temperate species[END_REF] directly compared of CH 4 production between two tropical forages and a temperate forage in the same study. Most comparisons are based on data obtained under different situations. Other conclusions come from inference of the positive correlation between forage fibre level and CH 4 production [START_REF] Blaxter | The utilization of the energy of different rations by sheep and cattle for fattening[END_REF][START_REF] Moe | Methane production in dairy cows[END_REF]. As tropical forages are usually higher in fibre than temperate forages, many models assign higher estimates of CH 4 production to them. However, this criterion does not consider the wide variability in fibre contents that exist within tropical and temperate forage groups with broad overlaps between these forage classes.

Our meta-analysis compared tropical and temperate forages at the same level of fibre, digestibility and level of intake. It demonstrated that CH 4 production for C4 grasses was 10-17% higher, depending on the unit, compared to C3 grasses. In contrast, CH 4 production from legumes grown in warm climates was 7-22% lower than for legumes grown in cold climates. These values can be compared with those of [START_REF] Margan | Energy and protein values of four forages including comparison between tropical and temperate species[END_REF] who found that sheep fed C4 grasses produced 23% more CH 4 (MJ/MJ intake) than sheep consuming a C3 grass, but only two C4 grasses were studied. Differences between C4 and C3 grasses occurred when CH 4 production was expressed as L/OM intake and L/DOM intake. These differences were not due to a lower digestibility for C4 grasses because the forages were adjusted for digestibility by inclusion of this variable as a covariate in the model. While intake and NDF content were included as covariables in the model, differences in CH 4 production between C4 and C3 grasses persisted. However, the nature of the fibre differs between tropical and temperate forages [START_REF] Wilson | Cell wall characteristics in relation to forage digestion by ruminants[END_REF] with C4 grass fibres tending to be more lignified and more resistant to physical and microbial digestion than those of C3 grasses. At the same level of digestibility, tropical grasses also have a longer retention time in the rumen [START_REF] Assoumaya | Etude des facteurs limitant l'ingestion chez les petits ruminants valorisant des fourrages tropicaux[END_REF]. This difference in rumen retention between C4 and C3 grasses may account for a portion of the difference in CH 4 production. Others have shown that increasing retention time of forages in the rumen can lead to increased CH 4 production [START_REF] Pinares-Patino | Rumen function and digestion parameters associated with differences between sheep in methane emissions when fed chaffed lucerne hay[END_REF].

The VFA profile could help to explain differences in CH 4 production between C3 and C4 grasses. Unfortunately, to our knowledge, neither in vivo nor in vitro experiments have been completed to compare VFA profiles between C3 and C4 grasses. Data from the comparison of old and young re-growth of C4 grass, which approximates digestion of C3 grass [START_REF] Assoumaya | Etude des facteurs limitant l'ingestion chez les petits ruminants valorisant des fourrages tropicaux[END_REF], could be of interest. [START_REF] Assoumaya | Etude des facteurs limitant l'ingestion chez les petits ruminants valorisant des fourrages tropicaux[END_REF] compared digestion of the C4 plant Panicum maximum between 14 and 56 days of re-growth and found a decrease in propionate relative to total VFA, while acetate increased with advancing maturity. Rumen fermentation profiles could therefore partially explain differences between temperate and tropical grasses. However, variation of CH 4 production among the 5 types of forage is lower than that in mixed diets where the proportion of forage and concentrate varies [START_REF] Johnson | Methane emissions from cattle[END_REF]. This likely reflects the small change in the ratio of propionate/acetate ratios in forages fed to ruminants in contrast to mixed diets [START_REF] Johnson | Methane emissions from cattle[END_REF].

Compared to grasses, legumes have at least two characteristics which could influence OM digestion in the rumen and, consequently, CH 4 production. The highly lignified cell walls and the presence of secondary metabolites such as tannins decrease cell wall digestion in legumes. Notwithstanding, there is high variability among legumes, in particular regarding the presence of secondary metabolites such as tannins which are more common in tropical versus temperate legumes [START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: progress and challenges[END_REF]. In our database, the legumes highest in tannin were those of tropical origin, a factor that could account for the lower CH 4 production in warm versus cold legumes. Studies conducted in vitro [START_REF] Bekele | Efficiency of Sesbania sesban and Acacia angustissima in limiting methanogenesis and increasing ruminally available nitrogen in a tropical grass-based diet depends on accession[END_REF][START_REF] Soliva | In vitro screening of various tropical foliages, seeds, fruits and medicinal plants for low methane and high ammonia generating potentials in the rumen[END_REF][START_REF] Tiemann | The ruminal degradability of fibre explains part of the low nutritional value and reduced methanogenesis in highly tanniniferous tropical legumes[END_REF] have shown that a large portion of the variability of CH 4 production in legumes can be accounted for by the presence or absence of secondary metabolites. In temperate conditions, fresh lucerne has been shown to produce less CH 4 than grasses [START_REF] Mccaughey | Impact of pasture type on methane production by lactating beef cows[END_REF], whereas clover did not [START_REF] Van Dorland | Effect of supplementation of fresh and ensiled clovers to ryegrass on nitrogen loss and methane emissions in dairy cows[END_REF]. Similarly, studies with temperate legumes found high variability in CH 4 production in ruminants depending on the presence or absence of secondary metabolites [START_REF] Waghorn | Methanogenesis from forages fed to sheep[END_REF][START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production: progress and challenges[END_REF][START_REF] Beauchemin | Dietary mitigation of enteric methane from cattle[END_REF][START_REF] Dewhurst | Nutritive value of forage legumes used for grazing and silage[END_REF].

Conclusions

When fed to ruminants, it is clear that C4 grasses produce more CH 4 than C3 grasses, that legumes from warm climates produce less CH 4 than legumes from cold climates, and that legumes produce less CH 4 than grasses.

The relative scarcity of data on CH 4 production from ruminants fed tropical forages (i.e., C4 grasses and warm legumes) suggests a need for additional studies. Results of this meta-analysis should be confirmed by coordinated experiments in temperate and tropical environments with the same animal genotype (i.e., tropical versus temperate) fed both tropical and temperate forages.

Table 1

 1 Statistical description of the chemical composition for each type of forage or animals in the database.

		Parameter	Grasses		Legumes
			C3 type	C4 type	Cold	Warm
		n	58	28	14	12
	NDF	Mean	557.3	646.4	447.2	568.2
	(g/kg DM)	SD	84.81	97.89	114.67	98.94
		Minimum	361.0	415.0	252.0	441.0
		Maximum	754.0	753.0	684.0	690.0
	CP	Mean	169.5	110.3	198.6	156.6
	(g/kg DM)	SD	55.94	62.80	38.40	56.91
		Minimum	44.0	24.0	141.0	93.0
		Maximum	314.0	254.0	269.0	236.0
	OM intake	Mean	19.8	17.7	17.8	18.6
	(g/kg LW)	SD	5.48	6.98	6.00	4.57
		Minimum	10.6	7.4	9.3	7.3
		Maximum	33.9	31	28.8	25.2
	OM	Mean	34.4	27.4	31.4	25.6
	intake	SD	8.27	11.81	9.30	6.44
	(g/kg LW 0.9 )	Minimum	15.7	10.7	13.8	10.7
		Maximum	48.9	56.9	51.1	36.3
	OM apparent total	Mean	0.70	0.62	0.63	0.48
	tract digestibility	SD	0.075	0.057	0.010	0.053
		Minimum	0.56	0.51	0.38	0.42
		Maximum	0.83	0.71	0.79	0.57

Table 2

 2 Effect of forage type on CH 4 production, expressed as L/kg dry matter (DM) intake, L/kg organic matter (OM) intake, L/kg digested OM (DOM) or L/100 kg live weight (LW).

		CH 4 (L/kg DM intake)	CH 4 (L/kg OM intake)	CH 4 (L/kg DOM)
	Grasses
	C3 type	30.0b	33.1b	52.1b
	C4 type	33.7c	38.8c	57.7b
	Legumes
	Cold	30.1b	33.7bc	52.4b
	Warm	25.9a	27.2a	40.7a
	SEM	1.77	1.79	2.89
	R 2	0.77	0.84	0.76
	P	0.001	0.001	0.001
	Mean values within column with no common letter (a-c) differ (P<0.05). Effects used in the model were: experiment, animal species, diet, forage NDF
	content, OM apparent total tract digestibility and OM intake/LW 0.9 .

Acknowledgements

This study was completed with the financial support of the "ANR -The French National Research Agency", project "ANR-09-STRA-01 EPAD". References included in the database can be obtained by request from the corresponding author.

Conflict of interest statement

None.