Theoretical and Applied Genetics

, Volume 123, Issue 3, pp 465–474 | Cite as

Tomato fruit weight 11.3 maps close to fasciated on the bottom of chromosome 11

Original Paper

Abstract

Fruit weight is an important character in many crops. In tomato (Solanum lycopersicum), fruit weight is controlled by many loci, some of which have a major effect on the trait. Fruit weight 11.3 (fw11.3) and fasciated (fas) have been mapped to the same region on chromosome 11. We sought to determine whether these loci represent alleles of the same or separate genes. We show that fas and fw11.3 are not allelic and instead represent separate genes. The fw11.3 locus was fine-mapped to a 149-kb region comprised of 22 predicted genes. Unlike most fruit weight loci, gene action at fw11.3 indicates that the mutant allele is partially dominant over the wild allele. We also investigate the nature of the genome rearrangement at the fas locus and demonstrate that the mutation is due to a 294-kb inversion disrupting the YABBY gene known to underlie the fas locus.

Introduction

Fruit weight is an important selection criterion in the breeding programs of many fruit and vegetable crops. Domestication and selection of tomato (Solanum lycopersicum) led to dramatically increased fruit mass compared with fruit found in its wild relatives (Bai and Lindhout 2007; Paran and van der Knaap 2007). Tomato is also an excellent system for studying fleshy fruit development (Giovannoni 2001), and therefore genes controlling fruit size offer insights into basic plant developmental processes. Fruit weight is a quantitatively inherited character that is controlled by up to 28 quantitative trait loci (QTLs) (Grandillo et al. 1999). The loci fw1.1, fw2.2, fw2.3, fw3.1, fw4.1 and fw9.1 each explain more than 20% phenotypic variance in at least one independent study. In addition, fw1.1, fw2.1, fw2.2, fw3.1, fw3.2, and fw11.3 have been identified in at least four independent studies (Grandillo et al. 1999). The aforementioned loci are referred to as major QTLs and account for the majority of fruit weight variation in the evolution of tomato from a small berry to a large fruit. Two loci, locule number (lc) and fasciated (fas), increase locule number and fruit width, often resulting in increased fruit weight (Grandillo et al. 1999; Lippman and Tanksley 2001; Causse et al. 2007).

To date, the genes for two tomato fruit size loci, fw2.2 and fas, have been cloned (Frary et al. 2000; Cong et al. 2008). The large-fruited allele of fw2.2 is thought to be fixed in cultivated tomato accessions, and thus it is believed the mutation arose early during domestication (Nesbitt and Tanksley 2002; Tanksley 2004). Histological studies suggest that FW2.2 protein is a regulator of cell division because of more cells in large compared with small fruits (Frary et al. 2000; Cong et al. 2002). Expression analyses have indicated that higher gene expression and different timing of expression correlate with fewer cells and smaller fruit (Cong et al. 2002). There are many FW2.2-related genes in other plant species. Some of the FW2.2-related genes are found to play similar roles in the regulation of organ cell number. In maize the overexpression of tomato FW2.2-like gene, Cell Number Regulator 1 (CNR1), reduces plant and organ size by reducing cell number (Guo et al. 2010). In avocado, small fruit has fewer cells compared with normal-sized fruit. Pafw2.2, an FW2.2-like gene in avocado, is expressed at higher levels in small fruit compared with normal-sized fruit (Dahan et al. 2010). Greatly reduced expression of GmFWL1, a soybean FW2.2-like gene, significantly decreased nodule number and nuclear size (Libault et al. 2010) suggesting a role for FW2.2-like genes in organ initiation as well as organ size. The molecular function of FW2.2 and CRN1-like proteins is not well understood. However, based on protein localization at the plasma membrane (Cong and Tanksley 2006; Libault et al. 2010) and structural modeling (Guo et al. 2010), it has been suggested that their role in cell division is indirect and that this family of proteins might regulate transport of metals such as cadmium across membranes (Guo et al. 2010).

The main function of fas is to control locule number which affects fruit mass (Lippman and Tanksley 2001; Cong et al. 2008). Wild-type tomatoes carry fruit with 2–4 locules. However, certain accessions carry fruit of many locules, some of which are controlled by the FAS gene mutation (Cong et al. 2008; Rodriguez et al. 2011). This mutation also leads to increased floral organ number and fruit size (Lippman and Tanksley 2001; Barrero et al. 2006). It has been shown that a mutation in the YABBY gene underlies the fas locus (Cong et al. 2008). Furthermore, it has been postulated that the mutation is due to a 6- to 8-kb insertion in the first intron of YABBY resulting in down regulation of the gene (Cong et al. 2008). YABBY family members are regulators of organ polarity and the establishment of proper boundaries in meristems (Golz and Hudson 1999; Bowman 2000).

Cell number, cell size, and endoreduplication play important roles in the control of plant organ size such as the fruit (Cheniclet et al. 2005). FW2.2 regulates fruit mass by altering cell number (Frary et al. 2000; Cong et al. 2002), and FAS regulates fruit mass by altering the boundaries within the floral meristem resulting in changes in locule number (Lippman and Tanksley 2001; Cong et al. 2008). To elucidate additional genes that were selected during domestication of tomato and to further understand the molecular mechanism that regulate tomato fruit size, it is important to isolate the genes underlying the other loci controlling fruit weight in this species. The fw11.3 locus overlaps with fas on tomato chromosome 11 (Grandillo et al. 1999; Lippman and Tanksley 2001; van der Knaap and Tanksley 2003). Therefore, we hypothesized that fw11.3 and fas were allelic and that fw11.3 was the weaker allele of fas. In this study, the fw11.3 locus was narrowed down to a ~149-kb region, which was 45 kb downstream of fas indicating that these loci were not allelic. Twenty-two predicted genes were found in the fw11.3 target region. Moreover, the mutation at fas was not due to a 6- to 8-kb insertion but instead to a ~294-kb inversion in the first intron of YABBY, thereby disrupting the gene.

Materials and methods

Plant materials

Tomato seeds were obtained from the TGRC (LA0767, LA0925, LA1215, LA1589, LA1786, LA2452); Tomato Growers Supply Co (Howard German, Orange Strawberry, Yellow Stuffer, Zapotec Pinked Ribbed); Tomato Bob (Costoluto Genovese); Sunseeds (Sun1642); IPK Gatersleben (LYC281, LYC439, LYC444, LYC1823, LYC1848, LYC1917, LYC2456, T1708); MJ Gonzalo (UPV7637); Heinz (Heinz1706).

S. lycopersicum cv Howard German bears large and on average five locules per fruit as a result of the lc but not the fas mutation (Gonzalo et al. 2009; Rodriguez et al. 2011). We predicted that Howard German carried the cultivated allele of fw11.3 because we found that QTLs associated with fruit shape coincided with the fw11.3 locus (Brewer et al. 2007; Gonzalo and van der Knaap 2008). The rationale is that fruit shape is more pronounced in the large-fruited background and hence the effect is enhanced by a fruit weight QTL. A recombinant inbred line population HGBC1F5 was derived from a cross between Howard German and the wild species S. pimpinellifolium accession LA1589. The F1 was backcrossed to Howard German and subsequent generations were self-pollinated in the field in Wooster, OH, USA, four times (Supplementary Fig. 1). One hundred forty-seven HGBC1F5 plants were evaluated in summer 2008 for the segregation of fw11.3. To confirm and map fw11.3 to a defined interval, two recombinant plants, 09S11-1 and 09S12-12, were selected from 187 HGBC1F3 seedlings and self-pollinated in the greenhouse. These recombinant parents were chosen because other fruit size QTLs (fw1.1, fw2.2, fw3.1, fw3.2, and fw4.1) were fixed for the Howard German allele. From each recombinant parent, 11–12 homozygous recombinant and 11–12 homozygous non-recombinant progeny seedlings were selected and grown in the field in summer 2009. From each plant, 20 fruits were evaluated for fruit mass. To identify additional recombinants around fw11.3, 188 seedling progeny of 09S11-1 and 376 seedling progeny of 09S106-2 (selected from 09S11-1 and heterozygous for fw11.3) were screened with marker YABBY and EP1654 (Supplementary Table 1). A subset of the recombinant plants was selected for progeny test in the field in 2010. To determine the gene action of fw11.3, two heterozygous plants, 09S225-47 and 09S225-185, were selected from the progeny of 09S106-2. From the progeny of each plant, a set of homozygous plants carrying the Howard German allele (EE), the LA1589 allele (PP), or heterozygous (EP) were selected for evaluation of fruit mass in the field in 2010.

To screen recombinants around the fas locus, we genotyped 1504 seedlings of an F2 population derived from a tomato cultivar Orange Strawberry carrying the fas mutation (Rodriguez et al. 2011) crossed with LA1589, with markers EP1312, YABBY, and EP1057. The two EP markers were derived from the sequence of the Heinz1706 BAC clone HBa0323E19 carrying the YABBY gene. The progeny tests of four recombinants were conducted in the field or the greenhouse. From each plant, 20–30 fruits were evaluated for locule number.

Development of PCR-based markers

Putative SSR sequences in sequenced BACs or tomato whole genome scaffold sequences located on the bottom of chromosome 11 were identified by the microsatellite search tool, SSRHunter1.3 (Li and Wan 2005). PCR primers matching the flanking regions of these SSR sequences were designed using the PrimerSelect Lasergene program (DNASTAR Inc., Madison, WI, USA.). To develop the marker based on insertion/deletions (InDels) or single nucleotide polymorphism (SNP), PCR products from the parental lines were separately purified and sequenced. The sequence of PCR products were aligned using the software Sequencer 4.5 (Gene Codes Corporation, Ann Arbor, MI, USA). Large InDels (≥14 bp) and SNPs were used to develop primers flanking the InDel, cleaved amplified polymorphic sequences (CAPS) or derived cleaved amplified polymorphic sequence (dCAPS) (http://helix.wustl.edu/dcaps/dcaps.html; Neff et al. 2002) markers, respectively (Supplementary Table 1). The amplification of the markers was conducted using the following profile: 94°C 2 min; 35 cycles of 30 s at 94°C, 30 s at 52°C, and 30 s at 72°C; 5 min at 72°C. PCR products were separated on 3% agarose gel. All markers were mapped in silico to the tomato SL2.30ch11 (version 2.30).

RT-PCR experiment

Young flower buds (length <2 mm) were harvested from the progeny of 09S130-11 and were frozen in liquid nitrogen immediately. Total RNA was isolated using TRIzol Reagent (Invitrogen). Total RNA samples were treated with DNase I (New England Biolabs) to remove genomic DNA. The first strand of cDNA was synthesized from 5 µg of total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and then diluted to 60 µl. For PCR, 2 µl cDNA sample was taken into a reaction volume of 30 µl using gene-specific primers (Supplementary Table 1). Reactions were performed with Taq Polymerase on the DNA Engine Tetrad 2 Peltier Thermal Cycler (BIO-RAD), with the following profile: 94°C 2 min; different cycles (eIF4α 21 cycles, YABBY 34 cycles) of 30 s at 94°C, 30 s at 54°C, and 1 min at 72°C; 72°C 5 min.

Data analysis

One-way analysis of variance was performed to determine the correlation of markers and fruit weight from plants in the HGBC1F5 population grown in 2008. Significant difference at P < 0.01 was used as threshold for estimating the linkage of fw11.3 and the marker. To fine map fw11.3, the mean fruit weight of homozygous recombinants and homozygous non-recombinants from each progeny family were analyzed using Student’s t test. A probability of P < 0.01 was used as threshold for determining that the locus was segregating in the parent. The gene action of fw11.3 was estimated as the difference of mean fruit weight between the homozygous plants carrying the Howard German allele (EE), the LA1589 allele (PP), and heterozygous plants EP using the formula: d/a = [2EP − (EE + PP)]/(EE − PP). To fine map fas in Orange Strawberry, the mean locule number of homozygous recombinants and homozygous non-recombinants from each progeny family were analyzed using Student’s t test. A probability of P < 0.01 was used as threshold for determining that the locus was segregating in the parent.

Predicted genes in 149 kb fw11.3 region

The predicted genes in the region encompassing fw11.3 were identified by searching the available tomato genome annotation database, ITAG version 1 (SGN: http://solgenomics.net/genomes/Solanum_lycopersicum/genome_data.pl#annotation). Information about the expression of the predicted genes was found by BLASTN searching of the tomato full-length cDNA, Unigene, and ESTs databases, including Sol Genomics Network (SGN http://solgenomics.net) and The National Center for Biotechnology Information (NCBI http://www.ncbi.nlm.nih.gov).

Genome structure analysis at the fas locus in tomato

To determine the location of the breakpoint of the rearrangement in the first intron of the YABBY gene, fragments derived from the intron sequence were used as probe in Southern blot analysis according to standard protocols (Sambrook and Russell 2001) (Supplementary Table 1). To identify the sequence of the rearranged segment, inverse PCR was conducted as described previously (Rodriguez et al. 2011) and a fragment of approximately 4 kb was obtained. The sequence from the rearranged region was used as query to the tomato Heinz1706 genome sequence database using BLASTN at SGN (http://solgenomics.net/). To check the validity of the genome rearrangement, sets of primers were developed spanning the breakpoints on both ends of the inversion (Supplementary Table 1).

Results

Identification of the fw11.3 locus in tomato cultivar Howard German

To confirm the presence of the large-fruited allele of fw11.3 in the variety Howard German, new markers were developed spanning approximately 33 cM and covering the bottom half of chromosome 11. These markers were used to genotype HGBC1F5 population derived from a cross between Howard German and LA1589 that would segregate for fw11.3 if Howard German carried the large-fruited allele (Fig. 1a). The analysis of the fruit weight for each plant in the population showed that markers TG36 (P < 0.001), EP1055 (P < 0.0005) and EP1254 (P < 0.01) were significantly associated with the trait. This result indicated that the fw11.3 locus was segregating in the Howard German-derived population and located in the EP1141-EP1254 interval on chromosome 11 (Fig. 1a), as reported previously (Grandillo et al. 1999; Van der Knaap and Tanksley 2003).
Fig. 1

Fine-mapping of the fw11.3 locus afw11.3 locus was identified within EP1141-EP1254 interval on tomato chromosome 11 in the Howard German BC1F5 population. bfw11.3 locus was confirmed and narrowed down to EP1055- EP1254 interval. cfw11.3 was narrowed down to EP1057-EP1573 interval. d Annotated scaffold SL1.00sc06004 encompassing the fw11.3 region. Numbers indicate predicted genes (Table 3) and the arrows indicate the direction of transcription

To better define the fw11.3 region, a progeny test was performed on two plants that were recombinants between AC217002 and EP1254 (Table 1; Fig. 1b). The EP1055-EP1254 interval of 09S11-1 and the AC217002-EP1254 interval of 09S12-12 were heterozygous while many other regions were homozygous for the Howard German alleles, such as fw1.1, fw2.2, fw3.1, fw3.2, and fw4.1 loci (Grandillo et al. 1999). When comparing the fruit weight of the non-recombinants with the recombinants in each family, a significant (P < 0.00001) increase in mean fruit weight for 09S11-1 (37.7 g) and 09S12-12 (21.4 g) was observed. This represented an increase of 57.6 and 50.1% in fruit mass, respectively (Table 1). These results indicated that the fw11.3 locus was located in the EP1055-EP1254 interval on chromosome 11 (Fig. 1b).
Table 1

Progeny test to confirm the fw11.3 locus

BC1F3 Plant

Parental Pedigree

Filial Pedigree

Markersa

Nb

AFW(g)c

P-valued

FW△(g)e

FW△(%)f

AC217002

EP926

EP1141

TG36

EP1055

YABBY

EP1057

EP1254

09S11-1

05S113

09S106

1

1

1

1

1

1

1

1

12

103.2 ± 13.2

2.97E-7

37.7

57.6

1

1

1

1

1

3

3

1

12

65.5 ± 12.3

09S12-12

05S120

09S107

1

1

1

1

1

1

1

1

11

64.1 ± 8.6

3.09E-6

21.4

50.1

1

3

3

3

3

3

3

1

11

42.7 ± 6.5

aMarker score: 1 homozygous for S. lycopersicum alleles (E); 3 homozygous for S. pimpinellifolium alleles (P)

bThe number of tomato plants

cAFW: average fruit weight

dP values of t-tests for comparisons within each BC1F4 family

eThe mean fruit weight in grams (g) for the EE class subtracted by the mean fruit weight (g) for the PP class

fFruit weight △ (g) divided by the mean fruit weight (g) of the PP class

Fine mapping of fw11.3 locus

Based on the genomic sequence of tomato cv. Heinz 1706 (SL2.30ch11), the length of EP1055-EP1254 interval is 487 kb. To further delineate the position of fw11.3, five markers were developed to divide this interval in smaller segments (Fig. 1c). From the 564 seedlings that were genotyped with markers YABBY and EP1654, 49 recombinants were identified corresponding to an interval of 4.3 cM and a ratio of 76.4 kb/cM. Among the 49 recombinants, 12 recombinants located within YABBY-EP1057 interval; 16 recombinants located within EP1057-EP1573 interval; 7 recombinants located within EP1573-EP1449 interval; and 14 recombinants located within EP1449-EP1564 interval (Fig. 1c). From the total number of recombinant plants, 20 were selected for progeny testing based on the location of the crossover, sufficient number of seeds, maximum fruit weight, and regular fruit shape of the parental accession. The progeny test showed that fw11.3 was located between markers EP1057 and EP1573, a 149-kb region on chromosome 11 (Fig. 1c, Supplementary Table 2).

Fine mapping and genome structure analysis of the fas locus

In an effort to develop near isogenic lines (NIL) for fas, we screened for recombinant plants in which the crossover occurred within 50 kb from YABBY gene. The source of the fas allele for NIL development was Orange Strawberry, a tomato cultivar bearing multilocular fruit that carries the mutant allele of fas (Rodriguez et al. 2011). From the recombinant screen using markers on BAC clone HBa0323E19 carrying the YABBY gene, 11 recombinants were identified that all fell within the 45-kb interval YABBY-EP1057 (for position of the markers, see Fig. 1b, c). Progeny testing of three recombinants, 08S610-18, 08S610-227, and 09S86-274, between YABBY and EP1057 showed that marker EP1057 is outside the fas locus (Supplementary Table 3). Progeny testing of an additional recombinant plant, with a crossover between EP1489 (derived from SGN marker T0302, approximately 375 kb upstream of YABBY) and YABBY, showed that fas is located in EP1489-EP1057 interval including YABBY (Supplementary Table 3), similar to what is published previously (Cong et al. 2008).

The dramatic change in recombination frequency on one compared with the other side of the YABBY gene (from 11 recombinants between YABBY and EP1057 to 0 recombinants between YABBY and EP1312) suggested that the fas mutation was due to an inversion and not to an insertion. To investigate the nature of the rearrangement, we sought to identify the sequence at the breakpoint of the rearrangement. As a first step, we conducted Southern blot analyses with DNA from tomato accessions that carried many loculed fruit and probes derived from the first intron where the rearrangement was located. These analyses suggested that the many loculed varieties exhibited a different genome structure compared with control accessions carrying 2–3 locules (Supplementary Fig. 2a and 2b). Moreover, only one band was observed in both many- and low-loculed varieties. Using inverse PCR, we obtained an approximately 4 kb fragment that covered the breakpoint of the rearrangement (Supplementary Fig. 3). Southern blot analysis using as probe a DNA fragment from the rearrangement, EP1067, showed also only one band in high- as well as low-loculed varieties (Supplementary Fig. 2c). These findings suggested that the rearrangement was not due to a duplication or deletion but instead might be the result of a translocation or an inversion. We genetically mapped EP1067 in an F2 derived from a cross between Yellow Stuffer and LA1589 (Van der Knaap and Tanksley 2003). Yellow Stuffer does not carry the fas mutation and therefore represented wild-type gene structure at the locus. The mapping showed that the rearrangement originated 3 cM below TG36 and 2 cM above YABBY (data not shown). Therefore, the rearrangement appeared to be small and intrachromosomal. After the public release of the tomato genome sequence, we used the 4-kb rearranged fragment sequence as query against the tomato Heinz1706 genome sequence (http://solgenomics.net). The first part of the sequence (1–3,561 bp) corresponding to the rearranged part matched the SL2.30ch11 (51,956,539–51,960,099, +strand), and the last part of the sequence (3,560–4,137 bp) corresponding to the intron of YABBY gene matched the SL2.30ch11 (52,253,523–52,254,100, −strand) which is approximately 294 kb apart in wild-type tomato (Supplementary Fig. 3). To confirm the presence of the rearrangement in additional high-locule-number varieties, mainly those that were studied previously (Barrero and Tanksley 2004; Cong et al. 2008), we designed four sets of primer pairs that were predicted to span the breakpoints of the rearrangement (Supplementary Table 1). Amplification results indeed confirmed the presence of a ~294-kb inversion in the fas mutants (Fig. 2b, c). This inversion clearly affected the expression of YABBY as indicated by RT-PCR of RNA isolated from young flower buds (< 2 mm) of plants carrying the mutant allele of fas compared with wild type (Fig. 2). These results indicated that YABBY is knocked out as a result of the ~294-kb inversion.
Fig. 2

The inversion of the fas locus in tomato a The fas locus harbored a 294-kb inversion. The left breakpoint of the inversion was between predicted gene PDE247 (pigment defective 247, SL1.00sc06004_200.1) and R3aL (Disease resistance protein R3a-like protein, SL1.00sc06004_201.1), the right breakpoint of the inversion was within the first intron of YABBY gene (YABBY2-like transcription factor YAB2, SL1.00sc06004_243.1). fas-WT-1 (primer pair EP1069 and EP1617) and fas-WT-2 (primer pair EP1070 and EP1071) were used to check the wild type genome structure; fas-I-1 (primer pair EP1069 and EP1071) and fas-I-2 (primer pair EP1070 and EP1617) were used to check inversion at fas. b PCR amplification using the primers mentioned in (a) resulted in the detection of the inversion on the left. c PCR amplification using the primers mentioned in (a) resulted in the detection of the inversion on the right. 1 LA1589, 2 Heinz 1706, 3 Howard German, 4 Yellow Stuffer, 5 LA0767, 6 LA0925, 7 LA1786, 8 LA2452, 9 Orange Strawberry. 1, 2, 3, 4 wild tomato or cultivars with low locule numbers; 5, 6, 7, 8, 9 tomato cultivars with high locule numbers (Barrero and Tanksley 2004; Cong et al. 2008; Rodriguez et al. 2011). d The expression of YABBY in wild-type and fas tomato. DNA marker, ΦX174 DNA-HaeIII Digest (NEB)

Gene action of fw11.3

To determine gene action of fw11.3 locus, fruit weight was measured in the homozygous and heterozygous plants in two families that were segregating for the EP1057-EP1573 interval (Table 2). Based on the mean fruit weights of EE, PP, and EP plants of the progeny of 09S225-49 and 09S225-185, the degree of dominance of Howard German allele was found to be 0.46 and 0.75, respectively. These results suggested that the fw11.3 allele from Howard German was partially dominant over the allele from S. pimpinellifolium supporting earlier findings (Van der Knaap and Tanksley 2003).
Table 2

Gene action of fw11.3

BC1F5 plant

Parental pedigree

Filial pedigree

Markersa

Nb

AFW(g)c

d/ad

TG36

EP1055

YABBY

EP1057

EP1573

EP1449

EP1564

EP1254

EP1550

09S225-49

09S106-2

10S93

1

1

1

1

1

1

1

1

1

12

83.5 ± 8.7

0.46

1

1

2

2

2

2

2

1

1

11

78.4 ± 7.0

1

1

3

3

3

3

3

1

1

12

64.5 ± 4.4

09S225-185

09S106-2

10S83

1

1

1

1

1

1

1

1

1

12

72.9 ± 7.7

0.75

1

1

1

2

2

2

2

1

1

11

71.0 ± 9.5

1

1

1

3

3

3

3

1

1

12

57.5 ± 5.9

aMarker score: 1 homozygous for S. lycopersicum alleles (E); 2 heterozygous (EP); 3 homozygous for S. pimpinellifolium alleles (P)

bThe number of tomato plants

cAFW: average fruit weight

dd/a = degree of dominance of the E allele [(2EP-EE-PP)/(EE-PP)]

Gene annotation of the 149-kb fw11.3 region

Twenty-two predicted genes were found in the 149-kb region corresponding to fw11.3 by searching the tomato genome annotation database (Table 3). With the exception of SL1.00sc06004_256.1 and SL1.00sc06004_257.1, all are predicted to encode functional proteins. Most of the predicted genes, with the exception of SL1.00sc06004_250.1, SL1.00sc06004_262.1, and SL1.00sc06004_264.1, corresponded to a full-length cDNA or a unigene. Twelve genes corresponded to ESTs expressed in tomato reproductive tissues such as flower, ovary and/or fruit (Table 3).
Table 3

Predicted genes in the fw11.3 region

ID

Predicted CDSa

Putative protein function

Full-length cDNA

Unigeneb

ESTs source tissues

1

SL1.00sc06004_250.1

CLAVATA 1, Serine/threonine kinase

   

2

SL1.00sc06004_251.1

Outer envelope protein of 80 kDa

 

SGN-U588538

Leaf

3

SL1.00sc06004_252.1

Large subunit GTPase 1 homolog

AK319274

SGN-U568726, SGN-U569667

Crown gall, flower, fruit, leaf, root, seedlings, shoot

4

SL1.00sc06004_253.1

Serine/threonine protein phosphatase

 

SGN-U572554

Flower

5

SL1.00sc06004_254.1

Serine/threonine protein phosphatase

 

SGN-U572555

Fruit, leaf

6

SL1.00sc06004_255.1

DnaJ homolog subfamily C member 8

AK328152, BT013084

SGN-U569691, SGN-U589611

Crown gall, flower, fruit, leaf, root, seed, suspension cultures

7

SL1.00sc06004_256.1

Unknown protein

AK247611

SGN-U584550

Leaf

8

SL1.00sc06004_257.1

Unknown protein

AK328791

SGN-U586284

Fruit, leaf, root, seedling

9

SL1.00sc06004_258.1

FAD-linked sulfhydryl oxidase ALR

 

SGN-U598120

Leaf

10

SL1.00sc06004_259.1

Serine/threonine-protein kinase Nek5

 

SGN-U599760

Callus

11

SL1.00sc06004_260.1

Pyridine nucleotide-disulphide oxidoreductase

 

SGN-U571091, SGN-U596521

Root

12

SL1.00sc06004_261.1

ACTIN 4 structural constituent of cytoskeleton

 

SGN-U576083

Fruit, ovary

13

SL1.00sc06004_262.1

Homoserine dehydrogenase family protein

   

14

SL1.00sc06004_263.1

Homoserine dehydrogenase family protein

 

SGN-U601085

Fruit

15

SL1.00sc06004_264.1

Polyubiquitin 9

   

16

SL1.00sc06004_265.1

LTP family protein

 

SGN-U579533

Flower, leaf, trichomes

17

SL1.00sc06004_266.1

GDSL esterase/lipase

AK325531

SGN-U571716

Flower, fruit, leaf, root

18

SL1.00sc06004_267.1

F-box/WD-40 repeat-containing protein

AK320336

SGN-U569064, SGN-U579177

Flower, leaf, seed, trichomes

19

SL1.00sc06004_268.1

MYB family transcription factor-like

 

SGN-U584149

Trichomes

20

SL1.00sc06004_269.1

Speckle-type POZ protein

AK320634

SGN-U582672, SGN-U587095

Fruit, leaf, root

21

SL1.00sc06004_270.1

Kelch repeat-containing F-box family protein

 

SGN-U597645

Leaf, root

22

SL1.00sc06004_271.1

Translation initiation factor eIF-2B subunit gamma

 

SGN-U562684, SGN-U598746

Callus, crown gall, flower, fruit, leaf, root, shoot/meristem

Discussion

The fw11.3 is not an allele of fasciated

The locus fw11.3 is an important fruit weight locus explaining from 8 to 13% of the variation in F2 and BC1/BC2 populations derived from crosses with wild relatives (Grandillo et al. 1999; Van der Knaap and Tanksley 2003). fas is also a major locus resulting in larger fruit weight by up to 37% through increasing the number of locules (Lippman and Tanksley 2001). fw11.3 is found in a similar region on chromosome 11 as fas in low-loculed tomato cultivars (Grandillo et al. 1999; Van der Knaap and Tanksley 2003). Therefore, we hypothesized that fw11.3 and fas were allelic and that fw11.3 was the weaker allele of fas. The FAS gene has been cloned and has been found to encode a YABBY-like transcription factor (Cong et al. 2008). Our results show that the fas mutant allele is present in Orange Strawberry, a tomato cultivar bearing multilocular fruit. We confirmed the map location of fas in the EP1489-EP1057 interval including YABBY (Supplementary Table 3) similar to what was found previously (Cong et al. 2008). However, the fw11.3 locus in Howard German was fine-mapped to EP1057-EP1573 interval located 45 kb downstream of YABBY gene (Fig. 1c; Supplementary Table 2). Therefore, the fine mapping result of this study indicates that FW11.3 and YABBY are two genes that map close to each other and are not alleles of the same gene.

Chromosomal rearrangements in tomato evolution

Chromosome rearrangements are mutations that alter the structure of chromosomes. Duplications, deletions, inversions, and translocations are the basic types of rearrangements (Pierce 2002). Chromosome rearrangements play important role in the plant evolution (Levin 2002). In tomato, 24.7 kb DNA fragment duplication mediated by retrotransposon Rider results in the increase of SUN gene expression and the elongation of fruit in several tomato cultivars (Xiao et al. 2008; Rodriguez et al. 2011). A 2.6-kb DNA fragment deletion in Ripening-Inhibitor (Rin) gene leads to tomato fruit that fails to ripen (Vrebalov et al. 2002). The emergence and selection of fas is a very important step in the tomato domestication (Lippman and Tanksley 2001; Cong et al. 2008). In this study, we found that the disruption of FAS resulted from a 294-kb DNA fragment inversion and its breakpoint was within the first intron of the gene (Fig. 2). Therefore, chromosome rearrangements might play more important roles in the evolution of phenotypic novelty within tomato than previously thought.

The large fruit fw11.3 allele is partially dominant

Most QTL alleles are not completely dominant or recessive (Tanksley 1993). The genetic analyses of fw2.2 and fas have shown that the large fruit alleles are partially recessive (Alpert et al. 1995; Barrero and Tanksley 2004). Molecular studies have shown that they are negative regulators of cell division and increase in carpel number, respectively. Moreover, in the case of fas, the large fruit phenotype resulted from a loss-of-function mutation (Cong et al. 2008). Unlike fw2.2 and fas, the large fruit allele of fw11.3 is partially dominant (Table 2). Therefore, the future cloning of FW11.3 would shed novel insights into the molecular mechanism of increased fruit weight mediated by a partially dominant instead of a partially recessive allele.

Analysis of possible candidate genes

By searching the tomato genome annotation database we found twenty-two predicted genes in the 149-kb region corresponding to fw11.3. Nine of them are considered likely candidate genes of FW11.3 based on their predicted role in cell division and/or cell expansion. SL1.00sc06004_250.1 and SL1.00sc06004_259.1 are putative serine/threonine kinase genes, SL1.00sc06004_253.1 and SL1.00sc06004_254.1 are putative serine/threonine phosphatase genes (Table 3). Serine/threonine kinases and serine/threonine phosphatases regulate many biological processes, such as cell cycle progression, growth factor response and hormone stimuli (Luan 2003; Inze and De Veylder 2006; Farkas et al. 2007; Krizek 2009). SL1.00sc06004_250.1 is a homolog of Arabidopsis CLAVATA 1 gene. The loss of function of CLAVATA 1 gene enlarges shoot and floral meristem size and increases floral organ number, mainly of the carpel (Clark et al. 1997). SL1.00sc06004_259.1 encodes a putative serine/threonine-protein kinase Nek5. The members of Nek family are involved in cell cycle control (O’Regan et al. 2007).

SL1.00sc06004_267.1 and SL1.00sc06004_270.1 are putative F-box genes. F-box protein is a component of Skp1/Cullin/F-box (SCF) complex, which is the largest and best characterized type of E3 enzyme. E3 enzyme is a very important component of ubiquitin–proteasome system (Smalle and Vierstra 2004). The ubiquitin–proteasome pathway plays an important role in the regulation of plant organ size (Disch et al. 2006; Song et al. 2007; Li et al. 2008; Kurepa et al. 2009; Sonoda et al. 2009).

SL1.00sc06004_252.1 is a putative large subunit GTPase 1 homolog gene. GTPases are involved in the regulation of many signaling pathways, including cell cycle procession, ubiquitin–proteasome pathway, and the auxin signaling pathway. The gene affects cell expansion and cell division (Nibau et al. 2006; Fu et al. 2009; Chen 2010).

SL1.00sc06004_255.1 is a putative DnaJ homolog subfamily C member gene. DnaJ/Hsp40 (heat shock protein 40) is a family of proteins containing a J-domain. They mostly play a role as chaperone and interact with Hsp70 to regulate protein translocation, assembly, and disassembly (Qiu et al. 2006; Kampinga and Craig 2010). Human Mrj protein is involved in the regulation of cell cycle (Dey et al. 2009). In addition to their function as chaperon proteins interacting with Hsp70, several plant DnaJ proteins also interact with other proteins and regulate abiotic stress responses (Ham et al. 2006; Yang et al. 2010), carotenoid accumulation (Lu et al. 2006), and formation of endosomes (Tamura et al. 2007).

SL1.00sc06004_268.1 encodes a putative MYB transcription factor-like protein. The MYB proteins are a large transcription factor family and have diverse function in all eukaryotes. Some MYB proteins are involved in the regulatory networks controlling cell cycle and cell differentiation (Berckmans and De Veylder 2009; Dubos et al. 2010).

In addition to the aforementioned candidate genes of FW11.3, SL1.00sc06004_257.1, SL1.00sc06004_261.1, SL1.00sc06004_263.1, SL1.00sc06004_265.1, SL1.00sc06004_266.1, and SL1.00sc06004_271.1 are expressed in tomato reproductive tissues (flower, ovary, and/or fruit) (Table 3). Therefore, these genes might also play a role in tomato fruit size.

To conclude, our results show that fw11.3 is not an allele of fas. The fw11.3 is found to be a major QTL controlling fruit weight in tomato. We fine-mapped the locus to a 149-kb region located 45 kb downstream of the FAS-YABBY gene. Unlike fw2.2 and fas, the large fruit fw11.3 allele is partially dominant over the wild-type allele from S. pimpinellifolium LA1589. Moreover, the mutation at fas was the result of a 294-kb inversion event that created a null allele of YABBY underlying the locus. The results from this study will not only contribute to the cloning of FW11.3 gene, but also to further our understanding of the molecular mechanism of increased fruit weight in tomato and other crops.

Notes

Acknowledgments

We thank Claire Anderson for her help with the analysis of the rearrangement at the fas locus. We thank all members of the Van der Knaap laboratory, in particular Jenny Moyseenko for their help with greenhouse and field experiments. The work was supported by National Science Foundation grant IOS 0922661 to EvdK.

Supplementary material

122_2011_1599_MOESM1_ESM.pdf (380 kb)
Supplementary material 1 (PDF 80 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Department of Horticulture and Crop ScienceThe Ohio State University, OARDCWoosterUSA

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