WO2003057917A2

WO2003057917A2 - An isolated nucleotide sequence responsible for the tomato dark green (dg) mutation and uses thereof

Info

Publication number: WO2003057917A2
Application number: PCT/IL2003/000023
Authority: WO
Inventors: Ilan Levin; Avraham Lalazar; Nehama Gilboa
Original assignee: The State Of Israel, Ministry Of Agriculture, Agricultural Research Organization, The Volcani Center
Priority date: 2002-01-13
Filing date: 2003-01-08
Publication date: 2003-07-17
Also published as: AU2003207951A8; WO2003057917A3; AU2003207951A1

Abstract

The present invention describes an isolated nucleotide sequence responsible for the tomato dark green (dg) phenotype, wherein said sequence comprises an altered tomato DET1 gene sequence or fragment thereof. Further described is a method for detecting the presence of the dg mutation in a plant, comprising the steps of isolating the genomic DNA from said plant, amplifying a gene fragment containing said dg mutation from said genomic DNA by use of a PCR technique and determining the presence of said dg mutation in said genomic DNA. Further described is a method for preparing double-mutant lines of Lycopersicon esculentum having genotype dg/dg p/p, wherein p represents any recessive photomorphogenic mutation that is genetically unlinked to the dg mutation.

Description

An Isolated Nucleotide Sequence Responsible for the Tomato Dark Green (dg) Mutation and Uses thereof

Field of the Invention

The present invention relates to a modified nucleotide sequence that is responsible for producing the dark green ( dg) phenotype in tomatoes. More specifically, the present invention discloses a point mutation in the tomato homolog of the Arabidopsis DETl gene and the uses of said modified nucleotide sequence.

Background of the Invention

Plants respond to light intensity, direction, duration, and spectral quality by modulating their developmental processes in an array of interactions that are referred to as photomorphogenesis . Photomorphogenic mutants have been proven to be an excellent tool in research of the complex interactions between light and plant development and some of them have also been used in several agricultural crop breeding programs. Photomorphogenic mutants have been reported in a number of species, including Arabidopsis, Sorghum, Brassica , tobacco, tomato and pea. In general, these mutants may be classified either as defective in photoreceptors, or altered in some aspect of light signal transduction chain (Chory, 1993) .

Several light-hypersensitive mutants have been described in tomato [Lycopersicon esculentum) . Among these, mutants carrying the monogenic recessive high pigment (hp-1 and hp- 2) and dark green ( dg) mutations are characterized by their exaggerated light responsiveness. These mutants display higher anthocyanin levels, shorter hypocotyls, and greater fruit pigmentation in comparison to their semi-isogenic wild type plants (Mochizuki and Kamimura 1984; Wann et al . 1985) . The increased fruit pigmentation seen in these mutants is due to significantly elevated levels of carotenoids, primarily lycopene, and flavonoids in the mature ripe red fruit. As a consequence of their effect on fruit color, hp and dg mutations were introgressed into several commercial processing and fresh-market tomato cultivars that are currently marketed as Lycopene Rich Tomatoes (LRT) (Wann, 1997) .

The hp-1 mutant was originally discovered in 1917 as a spontaneous mutant at the Campbell Soup Company farms

(Riverton, NJ) (Reynard, 1956), the hp-2 mutant was reported in the Italian San Marzano variety in 1975

(Soressi, 1975) , and the dg mutant appeared in trellized planting of the Manapal variety (Konsler, 1973) . Despite some initial confusion, it is now clear that hp-1 and hp-2 mutations map the tomato chromosomes 2 and 1, respectively, and are therefore non-allelic (Van Tuinen et al . 1997; Yen et al. 1997). At each of these loci, two mutant alleles have been identified: hp-1 and hp-l^w, hp-2 and hp-2^

(Kerckhoff and Kendrick 1997; Van Tuinen et al. 1997) .

WO 99/29866 discloses the cloning and sequencing of the HP- 2 gene, said gene being found to encode the tomato homolog of the Arabidopsis nuclear protein DEETIOLATED1 ( DETl) . This publication further discloses that a point mutation and deletion mutation, both of which are located in exon 11 at the 3' end of the coding sequence of HP-2, give rise to the previously-identified hp-2^j and hp-2 mutants respectively. In the case of the hp-2 mutant, a point mutation directs alternative splicing of intron 10 that leads to a nine base pair deletion in exon 11.

The dg mutant is phenotypically similar to other hp mutants, but has a much darker mature-green fruit, resulting from higher total chlorophyll content (Wann et al . 1985; Konsler 1973) .Another significant difference between the phenotypes of the dg mutant and certain other photomorphogenic mutants such as the hp-2³ mutant is that red-ripe fruit of the hp-2³ mutants is sensitive to concentric cracking resulting in fruits with poor quality. Red-ripe fruit of the dg mutants are significantly more crack resistant and therefore of higher quality.

The map location of the dg mutation was unknown prior to the present invention.

It is a purpose of the present invention to provide an isolated nucleotide sequence that contains the mutation responsible for the dg photomorphogenic mutant of tomato plants .

It is a further purpose of the present invention to provide a polymorphic PCR-based DNA marker that may be used as a molecular diagnostic tool for the identification and selection of dg mutants.

It is a still further purpose of the present invention to provide a molecular diagnostic tool for use in the early selection of dg mutant plants at the seedling stage.

A yet further purpose of the present invention is to provide a molecular diagnostic tool that may be used for genotypic selection in the production of photomorphogenic double mutants.

Another purpose of the invention as claimed herein is to provide a diagnostic tool that may be used to determine the presence of the dg mutation in a post-control setting in seed production.

A further purpose of the invention is to provide an efficient method for preparing double mutant plants for two photomorphogenic genes, wherein said method obviates the need for laborious test crosses.

Other purposes and advantages of the present invention will become apparent as the description proceeds .

Summary of the Invention

It has now been found that, despite the abovementioned phenotypic differences between the dg mutant and other photomorphogenic mutants, the mutation responsible for the dark green ( dg) mutant is located within the same gene as the mutations responsible for both the hp-2 and hp-2³ mutants, namely the tomato homolog of the DETl gene ( HP-2) . Surprisingly, however, despite the fact that both of the known photomorphogenic mutations of the DETl gene (i.e. the hp-2³ and hp-2 mutations) are close to the 3' end of the coding sequence, the dg mutation, which is disclosed for the first time herein, is located close to the 5' end of the coding region of said gene.

The present invention relates to an isolated nucleotide sequence responsible for the tomato dg phenotype, wherein said sequence comprises an altered tomato DETl gene sequence or fragment or homolog thereof, wherein the alteration in said altered sequence or fragment or homolog comprises an A-to-T transversion at nucleotide 29 of the second exon of said DETl gene sequence.

In one preferred embodiment of the invention, the isolated nucleotide sequence comprises the sequence shown in Fig. 2A.

The present invention is particularly directed to the use of the abovementioned isolated nucleotide sequence as a molecular diagnostic tool. Said tool may be used to aid the introgression of the dg mutation into various genetic backgrounds for the purpose of improving fruit quality and nutritional value.

Thus in one aspect, the present invention provides a method for detecting the presence of the dg mutation in a plant, comprising the steps of isolating the genomic DNA from said plant, amplifying a gene fragment containing said dg mutation from said genomic DNA by use of a PCR technique using primers designed to amplify the flanking regions of said dg mutation and detecting the presence of said dg mutation in said genomic DNA.

In one preferred embodiment of the above-disclosed method of the invention, the presence of the dg mutation is detected by the use of restriction site analysis, wherein the loss of an Acl I site indicates the presence of said mutation. In another preferred embodiment of the method of the invention, the presence of the dg mutation is determined by direct sequencing of the amplified fragment and comparison of the sequence obtained with that shown in Fig. 2A.

In a further preferred embodiment of the method of the invention, the forward and reverse primers used to amplify the flanking regions of the dg mutation are:

5'-TTC TTC GGA TTG TCC ATG GT-3 ' and

5'-CAC CAA TGC TAT GTG CCA AA-3', respectively.

In another preferred embodiment, the plant in which the presence of the dg mutant is being detected is of the species ^~Lycopersicon esculentum .

In another aspect, the invention also encompasses the use of the above-described method as a means of quality control, or post-control in seed production, for detecting the presence of the dg allele in cultivars and their parental lines. The term post-control is used herein to indicate quality control checks that are performed following seed production, in order to confirm the intended genotype of said seeds.

In yet another aspect, the invention is directed to a method for the determination of the presence of two different photomorphogenic mutations in a plant, wherein one of said mutations is the dg mutation, comprising detecting the presence of a photomorphogenic mutation other than the dg mutation by genotypic or phenotypic selection means, and detecting the presence of the dg mutation by means of the method described hereinabove.

In one preferred embodiment of the immediately-preceding method, the phenotypic selection means for determining the presence of the non- g mutation comprises germinating seeds obtained from the plant in which the presence of the mutations is being determined in a temperature controlled chamber, under a yellow plastic screen, said screen preventing transmittance of light having a wavelength less than 500nm, and selecting the non-etiolated seedlings eight days post-sowing.

The term non-etiolated refers to seedlings with significantly shorter hypocotyls and lower cotyledon mass, characterized by significantly higher anthocyanin and chlorophyll content. In other words: seedlings that appear as if they were grown in the presence of normal light levels, although in fact they were grown under suboptimal light conditions.

In another aspect, the present invention is also directed to a method for preparing double-mutant lines of Lycopersicon esculentum having genotype dg/dg p/p, wherein p represents any recessive photomorphogenic mutation that is genetically unlinked to the dg mutation, said method comprising the steps of:

a) cross-hybridization of a homozygous dg/dg +/+ line (or plant) with a homozygous +/+ p/p line (or plant) to yield double heterozygous dg/+ p/+ Fi plants; b) self-crossing of the Fi plants obtained in step (a) in order to yield F₂ seeds; c) light-sensitivity selection of light-hypersensitive seedlings germinated from F₂ seeds; d) selection of light-hypersensitive seedlings containing the dg/+ p/p mutations, by use of the above-described method for detecting dg/+ heterozygous plants; e) self-crossing of the selected plants obtained in step (d) to generate F₃ seeds, and germination of said seeds under normal conditions; f) selection of double homozygous dg/dg p/p seedlings from the seedlings obtained in step (e) , by use of the above- described method for detecting the presence of the dg mutation in a plant.

The term "genetically unlinked" as used herein refers to the situation wherein the second photomorphogenic mutation is located within a gene other than DETl .

In one preferred embodiment of the immediately preceding method for preparing double-mutant lines, the recessive photomorphogenic mutation p is a mutation that causes photomorphogenic phenotypic effects similar or identical to those caused by the hp-1 mutation.

In the present context the abovementioned "phenotypic effects similar or identical to those caused by the hp-1 mutation" are those already described hereinabove, i.e., exaggerated light responsiveness, higher anthocyanin levels, shorter hypocotyls, and greater fruit pigmentation (in comparison to their semi-isogenic wild type plants) .

In a more preferred embodiment, the abovementioned recessive photomorphogenic mutation p that causes phenotypic effects similar or identical to those caused by the hp-1 mutation is a lycopene-enhancing mutation. In a particularly preferred embodiment, the lycopene-enhancing mutation is hp-l^w. The term "lycopene-enhancing mutation" in this context is used to describe photomorphogenic mutations that cause a significant increase in the amount of lycopene produced by the host plant containing said mutation, as compared to a plant bearing the wild-type, non-mutated gene.

In another preferred embodiment, the recessive photomorphogenic mutation p is the hp-1 mutation.

The present invention is further directed to double-mutant hybrid plants of the species ^~Lycopersicon esculentum having genotype dg/dg p/p, wherein p is as defined hereinabove. In one particularly preferred embodiment, said double- mutant hybrid plants have genotype dg/dg hp-l/hp-1 . In another particularly preferred embodiment, the double- mutant hybrid plants have genotype dg/dg hp-l^w/hp-l^w .

According to a preferred embodiment of the invention, the abovementioned double-mutant hybrid plants are prepared by the method disclosed hereinabove.

All the above and other characteristics and advantages of the present invention will be further understood from the following illustrative and non-limitative examples of preferred embodiments thereof. Brief Description of the Drawings

Fig. 1 Visual phenotypic characterization of heterozygous hp-2 / dg Fi plants. A. A photograph showing a typical hp- 2³/dg Fi plant with mature-green fruits obtained from a cross between hp-2³ and dg mutants; B. A photograph showing developmental differences between heterozygous hp-2 / + and hp-2³ / dg Fi plants, two months after sowing; C. A photograph showing mature green and ripe-red fruits of heterozygous hp-2³ / + and 6 hp-2 / dg _\ plants.

Fig. 2 provides an alignment of nucleotide (A) and deduced amino acid sequence (B) of the first (I) and part of the second (II) exons of DETl gene in wild type (w.t.) and dg plants. The site of the mutation, in both the nucleotide and amino acid sequences, is indicated by an enlarged bold letter. The Acl I cleavage recognition sequence is underlined in the wild-type nucleotide sequence.

Fig. 3 demonstrates cleaved amplified polymorphic DNA marker that is used to identify dg mutant plants in cv Manapal background, in accordance with the method of the invention.

Fig. 4 depicts the segregation of the cleaved amplified polymorphic DNA marker for dg mutant plants in an F₂ population, showing the two homozygous genotypes (single bands) and the heterozygous genotype (two bands) .

Detailed Description of Preferred Embodiments

In one of its aspects, as described hereinabove, the present invention provides a method for detecting the presence of the dg mutation in a plant, comprising the steps of isolating the genomic DNA from said plant, amplifying a gene fragment containing said dg mutation from said genomic DNA by use of a PCR technique using primers designed to amplify the flanking regions of said dg mutation, digesting the PCR amplification product using the Acl I endonuclease, and detecting the presence of said dg mutation in said genomic DNA by means of electrophoresis in 1.0% agarose and staining with ethidium bromide.

The term "PCR" (or polymerase chain reaction) technique as used hereinabove and hereinbelow refers to a family of techniques that are based on the use of heat-stable polymerases for achieving the amplification (i.e. increase in number of copies) of specific DNA sequences by repeated polymerase reactions. This reaction can be used as a replacement for cloning: all that is required is knowledge of the nucleic acid sequence. In order to carry out PCR, primers are designed which are complementary to the sequence of interest. The primers are then generated by automated DNA synthesis.

PCR and other methods of amplifying DNA and/or RNA are well known in the art, and can be used according to the present invention without the need for undue experimentation, based on the teaching and guidance presented herein. Several PCR methods (as well as related techniques) are described, for example, in US patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, as well as in Innis et al . eds . , PCR Protocols : A guide to method and applications . Examples

Methods

Plant material , crosses, growth conditions and experimental design

The hp-2 and hp-2³ mutants and the corresponding semi- isogenic wild-type tomato seeds (Solanum Lycopersicon esculentum cv Money Maker) were kindly provided by R.E. Kendrick and M. Koornneef (Wageningen Agriculture University, the Netherlands) . The dg mutant and its corresponding isogenic wild type tomato seeds (Solanum Lycopersicon esculentum cv Manapal) were kindly provided by R.T. Chetelat (Tomato Genetics Cooperative, UC Davis, USA). Seeds from wild-type tomato (Solanum Lycopersicon esculentum cv Ailsa Craig) and a line nearly isogenic and homozygous for the hp-1 mutation were obtained from J.J Giovannoni (Texas A and M University, USA) . Lines n474 and n935, which were used to sequence the DETl gene, are dg mutant lines developed by the late R. Frankel, D. Lapushner and I. Levin at the Volcani Center, Bet Dagan, Israel. Several wild-type tomato cultivars used in this study, such as: Ailsa-Craig, M82, VF-36, 189, 124, 139, NC8288 and Florida were from seed stocks available at the Volcani center .

The initial cross was carried out between hp-2³ and dg during the summer of 2000 in a screen-house at the Volcani Center, Bet Dagan, Israel. In this cross, the indeterminate hp-2³ (cv Money Maker) plants served as a maternal line, whereas a mixture of pollen derived from 10 unrelated determinate parental breeding lines, represented the paternal dg genotype. Seven of the Fi plants resulting from the above cross were planted in an environmentally controlled greenhouse at the Volcani Center during the winter of 2000/2001. All of these plants displayed a delayed growth development, and set mature-green and ripe- red fruits with a characteristic dg phenotype, identical to homozygous dg/dg plants (Figure 1) . All of these i plants were found to be true hybrids, using markers complementary to the SELF-PRUNING locus (Pnuelli et al. 1998). F₂ Seeds were individually extracted from each one of these plants at the end of the season.

Sixty-four F₂ seeds, from each one of the 7 original F_lf plants were germinated under a yellow plastic screen that prevents transmittance of light having a wavelength less than 500nm (Mochizuki and Kamimura, 1984), in the Spring of 2001 in an environmentally controlled greenhouse at a commercial nursery (Hish-Shtill, Nehalim, Israel) . The photomorphogenic response of seedling hypocotyl length was visually compared to several wild-type genotypes germinated under the same conditions. Twenty-eight of the more etiolated F₂ seedlings, representing each one of the 7 original F₁ plants (A total of 196 plants) , were planted in an open field at the Hula valley research station located in northern Israel.

DNA was extracted from 5 plants chosen at random from each one of the 7 F₂ populations (A total of 35 plants) . These DNA samples were genotyped using a DNA marker developed based on a mutation discovered, in this research, in the tomato DETl homolog of dg mutants. Lycopene was extracted and its content determined in ripe-red fruits sampled from 21 of these genotyped plants. Fourteen plants that set an insufficient amount of fruits were discarded from further analysis . F₃ Seeds were extracted individually from each one of the above F₂ plants. A sample of 20 F₃ seeds, derived from 6 heterozygous F₂ plants ( dg/hp-2³ ) , representing 6 of the 7 original Fi plants (A total of 120 seeds) were sampled. These seeds were germinated under yellow plastic screen in an environmentally controlled growth chamber (25°C day/18°C night) during the autumn of 2001. Hypocotyl-length of the resulting seedlings was individually measured after 7 days, and compared to the length of wild type seedlings.

A linkage analysis study between the tomato DETl locus and the aggregated photomorphogenic response characterizing dg mutants was carried out using F₂ seeds of a cross between determinate dg mutant plants and wild type plants (cv PETO4303) . These seeds were allowed to germinate under yellow plastic screen in an environmentally controlled growth chamber during the autumn of 2001 (25°C day/18°C night) . Hypocotyl-length of individual F₂ seedlings was measured 9 days after sowing and their genotype determined using the DNA marker developed in this study.

Genomic DNA extraction and cDNA preparation

Genomic DNA was extracted from individual plants. The extraction procedure was according to Fulton et al . 1995.

PCR primers

Sequence analysis and locus-specific primer design were carried out with the DNAMAN, Sequence Analysis Software version 4.1 (Lynnon BioSoft, Quebec, Canada). All DNA primers used during the course of this study were purchased from M.B.C Molecular Biology Center Ltd., Ness-Ziona, Israel. PCR primers used to clone overlapping fragments spanning the entire coding sequence of the tomato DETl gene were:

TDR1: 5'-GTA CAC CTT AGT TGC TCG AGG GCG TG-3' CRISR: 5'-GTG ATT TCT AGG TTG ATT TCA ATC TAG AG-3 ' TDF2: 5' -GAT CCT AAT TCG AGC CCT CCT T-3' TDF1: 5'-GGA TGG AGC TAT ACT TGA CGA AAG GG-3' TDR: 5'-GCC GTT GCT TTA TAC CGC TCA GGA AA-3 ' TDR2: 5' -CAC TAG CAT CTA CGG GTC TGT TAT G-3'

PCR primers used to amplify the tomato DETl genomic DNA fragment flanking the dg mutation locus were:

TD-M2 FI: 5 ' -TTC TTC GGA TTG TCC ATG GT-3' TD-M2 Rl: 5 ' -CAC CAA TGC TAT GTG CCA AA-3'

The abovementioned TD-M2 FI primer anneals to the first intron of the tomato DETl sequence (bases 2171-2190 in NCBI accession AJ224356) , while the TD-M2 Rl primer anneals to exon 3 of the tomato DETl sequence (bases 2848-2867 in NCBI accession AJ224356) .

PCR reaction

The amplification reactions (25 μl final volume) were performed with 10 ng template DNA, 25 mM TAPS (pH=9.3 at 25°C) , 50 mM KC1, 2mM MgCl₂, ImM β-mercaptoethanol, 0.2 mM of each of the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP and dTTP) , 10 ng of each of two primers and 1 unit of thermostable Taq DNA polymerase (SuperNova Tag polymerase, Madi Ltd., Rishon Le Zion, Israel). Reactions were carried out in an automated thermocycler (MJ Research Inc., Watertown, MA, USA). Initial incubation was at 94°C for 1 min, followed by 34 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and polymerization at 72°C for 1.5 min. Final polymerization at 72°C was carried out, for 3 min, after the above cycles have been completed. The PCR amplification products were visualized by electrophoresis in 1.0% agarose gels and detected by staining with ethidium bromide.

Cloning and sequencing of the toma to DETl gene from dg mutant plants

Total RNA was extracted from breaker fruits (200 mg fresh weight) of individual dg mutant plants representing 2 independent lycopene-rich tomato breeding lines, n474 and n935 and the open pollinated wild-type cultivar Ailsa- Craig. The RNA extraction was carried out using the TRIzol reagent system (GibcoBRL Life Technologies, Gaithersburg, MD, USA) . The total RNA was used as template for first strand cDNA synthesis using the Superscript pre- amplification system (GibcoBRL Life Technologies, U.K.). The cDNA prepared was used as template, in a PCR reaction, to amplify three overlapping fragments of the gene encoding the tomato DETl from the two dg mutant plants. A fragment, 1783 bp in length, was amplified in two sequential PCR reactions. The first reaction was primed with TDF2-TDR2 primer combination. The resulting DNA product, 1816 bp in length, was excised from an agarose gel, purified using the GENECLEAN II kit (BIO 101 Inc., La Jolla CA, USA), and used for a nested PCR reaction primed with TDF2-CRISR. The two other fragments were amplified directly from the cDNA template using the primer combinations TDF2-TDR and TDF1- CRISR. The three PCR products were then cloned into pGEM-T Easy vector using the pGEM-T Easy Vector Systems according to the manufacturer recommendations (Promega Corporation, Madison, WI, USA) . Three independent clones of each of the three amplified fragments were sequenced, based on the vector T7, SP6, and the tomato DETl complementary primers (TDF2, TDF1 and TDR1) , using an ABI PRISM 377 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) . Equivalent fragments were cloned and sequenced from the wild type cv Ailsa Craig.

Lycopene extraction and analytical determination

Lycopene was extracted from pericarp tissue of fresh ripe- red fruits. Pericarp tissue of a sample of 20 fruits per individual plant was minced to puree in a blender. An aliquot of 2 g puree was taken from each sample and stirred with 40 ml extraction buffer for 30 min in the dark. The extraction buffer consisted of n-hexane : isopropanol : acetone (2:1:1) and contained 0.05% BHT . Phase separation was achieved by the addition of 20 ml 0.1M NaCl. The organic upper-phase was collected for analysis. Spectrophotometric absorbance at 472 nm was used to calculate lycopene concentration using El%=3450 (Davis, 1965) .

Statistical analyses

Statistical analyses were carried out with the JMP Statistical Discovery software (SAS Institute Inc., Gary, NC, USA) . Example 1 Allele test of dg and hp-2³ mutants

Seven Fi hybrid hp-2³ / dg plants initially indicated that dg and hp-2³ are allelic. All of these Fi plants set fruit that turn extremely green at the mature-green fruit developmental stage (Fig. 1) . Such extreme greening is a characteristic phenotype of dg tomato mutants and appears much darker than hp-2³ mutants (not shown) . These seven plants were confirmed as true hybrids using a PCR genotyping protocol based on tomato SELF-PRUNING ( SP) gene sequence (Pnueli et al . 1998). All 7 Fi plants were found to be heterozygous at the SP locus as would be expected in this cross where an indeterminate hp-2³ /hp-2³ maternal line was crossed with a determinate dg/dg paternal line.

To extend the analysis to F₂ generation, seeds were individually extracted from each of the 7 hybrid plants and a sample of 64 seeds, from each plant, was allowed to germinate under yellow plastic screen that eliminates spectral transmittance of light wave-length below 500 nm. Although variable, all of these F₂ seedlings displayed a divergent hypersensitive photomorphogenic phenotype, visualized as much shorter hypocotyl lengths, in comparison to wild type lines germinated under the same conditions (i.e. cv Money Maker and M82; data not shown) .

To validate the allelic relationship between dg and hp-2³, adult F₂ plants were also analyzed. Twenty-eight of the more etiolated F₂ seedlings, representing each one of the 7 original Fi plants (A total of 196 plants), were planted in an open field. Five plants from each of these 7 F₂ populations were chosen at random for genotyping and fruit lycopene analysis (a total of 35 plants) . Genotyping the DETl locus in these plants (described hereinbelow) was carried out using DNA samples extracted from true leaves of each individual plant. Red-ripe fruits of 21 plants were analyzed for lycopene content. The fruit lycopene content of these F₂ plants exceeded those of their wild type controls (Table 1) . No hp-2³ /hp-2³ genotype was found among the plants that participated in this analysis, because of prior selection of the more etiolated plants for further analysis (see above) . This genotype most probably failed to enter this selection because of the extremely slow seedling development, short hypocotyls and the severe deetiolated phenotypes it usually displays (Table 2) .

Table 1. Lycopene content in mature red fruit of parental lines and randomly chosen F₂ plants. Different superscript letters represent statistically significant differences between genotypic means (P<0.05), based on Tukey-Kramer HSD test (Kramer, 1956) .

Genotype Line N Lycopene±S . E . Lycopene (ppm) range (ppm)

+ /+ M82 6 72^B±2 64-78

+ / + Money maker 2 83^B±5 79-88 hp-2³ /hp-2³ Money maker 2 171^A +25 146-197 dg/dg LRT915 1 245^A - dg/dg F₂ 6 224^A±13 182-263 hp-2³ / dg F₂ 15 193^A±10 121-262 Table 2. Hypocotyl-length photomorphogenic response of tomato wild-type and mutant seedlings at the DETl and HP-1 loci. Seedlings were grown under yellow plastic screen for 7 days after sowing. Different superscript letters represent statistically significant differences between means (P<0.05), within each genetic background, based on the Tukey-Kramer HSD test (Kramer, 1956) .

Genotype Genetic N Hypocotyl background length (mean±S . E . )

+/+ Money maker 39 6.4 ^A ±0.2 hp-2/hp-2 Money maker 38 5.7 ^B ±0.1 hp-2³ /hp-2³ Money maker 14 2.5 ^c ±0.3

+/+ Ailsa Craig 40 7.5 ^A ±0.1 hp-l/hp-1 Ailsa Craig 40 4.7 ^B ±0.1

+/+ M82 73 7.5 ^A ±0.1 hp-l/hp-1 M82 40 5.0 ^B ±0.1 dg/dg M82 40 4.9 ^B ±0.2

The allelic test between dg and hp-2³ was completed by hypocotyl-length photomorphogenic response analysis of F₃ seedlings. F₃ seeds representing heterozygous hp-2³ / dg plants from 6 of the 7 F₂ populations were collected separately and were germinated in a temperature controlled growth chamber under yellow plastic screen. Hypocotyl length of single plants was measured 7 days after sowing. Results demonstrate that all of the segregating F₃ hp-2³ / dg seedlings were hypersensitive-photomorphogenic as compared to the wild type controls (Table 3) .

Table 3. Hypocotyl-length photomorphogenic response of wild type and segregating F₃ hp-2³ / dg seedlings. Seedlings were grown under yellow plastic screen for 7 days after sowing. Different superscript letters represent statistically significant differences between means (P<0.05) based on Tukey-Kramer HSD test (Kramer, 1956) . Hypocotyl length (cm)

Genotype N line Mean± S.E Range

+/+ 18 Money maker 5.6^A±0.1 4.5-6.6 Segregating 62 F₃ 2.8^B±0.1 1.2-4.4 hp-2³ /dg

Example 2

Allelic interaction between the dg and hp-2³ alleles of the

DETl gene significantly increases fruit lycopene content

A homozygous paternal dg line, selected for by the primer combination presented herein, was crossed with 3 isogenic homozygous maternal lines harboring the wild-type DETl allele (+) and the mutant ^'alleles : hp-2 and the hp-2³ . The parental lines and the F_x hybrids were planted in randomized block design- 3 blocks, each containing a plot of 10 plants of each of the parental and Fi hybrids. Ripe- red tomato fruits were harvested from each plot, weighted and analyzed for lycopene content. Results show that the mutant hybrid dg/hp-2³ produces, on average, a statistically significant higher level of lycopene which exceeds those of the dg/dg and hp-2³ /hp-2³ genotypes by 25.8 and 37.6 %, respectively (Table 4). These effects remained statistically significant after adjusting for fruit weight: slightly decreased compared to the hp-2³/hp- 2³ genotype (35.7%), but significantly increased compared to the dg/dg genotype (37.1%). These results show that specifically combining the dg and the hp-2³ alleles of the DETl gene can significantly increase lycopene concentration in the red-ripe tomato fruit and that this increase remains statistically significant after adjusting for fruit weight. Table . Weight adjusted and non adjusted average lycopene concentrations in wild-type, DETl mutant lines and their Fi hybrids. Different superscript letters represent statistically significant differences between means (P<0.05) based on Tukey-Kramer HSD test (Kramer, 1956).

Lycopene (ppm)

Genotype Genetic Non Weight background adj usted adj usted

+/+ Money maker 83^D 72^D hp-2/hp-2 Money maker 151^c 135^c hp-2³ /hp-2³ Money maker 182^B 207^B dg/dg M82 198^B 205^B dg/+ Fi 103^D 123^c dg/hp-2 Fi 202^B 212^B dg/hp-2³ Fi 249^A 281^A

Example 3 Sequence characterization of the tomato DETl in dg mutants

Two independently developed lycopene-rich breeding lines, homozygous for the dg mutation, were used for sequence analysis. The entire coding region of the tomato DETl cDNA was thoroughly sequenced. A single transversion of Thymine- to-Adenine was observed when dg mutant sequence was compared to wild type. This single base transversion was found to be located at nucleotide 29 of the second exon of the DETl gene sequence (Fig. 2A) , and thus its deduced amino-acid substitution would be Asparagine³⁴ to Isoleucine³⁴ (Fig. 2B) . This mutation also renders the dg mutant allele resistant to cleavage by the Acl I restriction endonuclease. This enzyme (whose recognition sequence is AACGTT, indicated by underlining in the nucleotide sequence given in Fig. 2A) cleaves the wild-type sequence into two fragments of size 578 and 119 bp respectively.

Example 4 Diagnostic tool for identifying the dg mutation

A DNA marker for use as a molecular diagnostic tool for identifying dg mutant plants based on the sequence results

(Fig. 2) was developed. The PCR primers (TD-M2 FI and TD-M2 Rl, sequence given hereinabove) were designed to amplify the genomic DNA sequences flanking the dg mutation. These genomic sequences were amplified, incubated with Acl I

(recognition sequence: AACGTT, indicated by underlining in the nucleotide sequence shown in Fig. 2A) and a clear polymorphism between dg and several wild-type plants representing 9 lines or cultivars (Money Maker, Ailsa Craig, M82, VF-36, 189, 124, 139, NC8288 and Florida; Data not shown) was seen. In the case of the homozygous dg mutant plants, a single amplified band of 679 bp was seen, while in wild-type plants, cleavage by Acl I led to the appearance of one 578 bp fragment and one 119 bp fragment. As expected, plants heterozygous for the dg mutation yielded the following three bands following Acl I cleavage: 679 bp, 578 bp and 119 bp. In order to substantiate these results, the DETl locus in dg mutant plants of the Manapal cultivar was genotyped and compared to that of the fully isogenic wild type plants. The polymorphism obtained (Fig. 3) , confirms that dg is a mutation at the tomato homolog of DETl gene. The segregation of individual plants using the DNA marker is presented in Fig 4. Example 5

Linkage analysis between the DETl locus and the photomorphogenic response

A linkage analysis study was carried out to test the association between the DETl locus and the characteristic hypersensitive-photomorphogenic response displayed by dg mutant plants (i.e. hypocotyl-length phenotype). For this purpose, F₂ • seeds of a cross between determinate dg mutant plants and wild type plants (cultivar PETO4303) were germinated under a yellow plastic screen in a controlled growth chamber. Nine days after sowing, the hypocotyl- lengths of individual seedlings were recorded and their DETl locus genotyped using the DNA marker described above. The results demonstrate a clear association between the DETl locus and hypocotyl-length (Table 5) . Homozygous recessive dg/dg seedlings displayed a highly statistically significant shorter average hypocotyl-length, indicative of a more hypersensitive-photomorphogenic response, in comparison to the two other genotypic groups (12<LOD Score<13, R²=51.1%). These results confirm that the mutation identified in the DETl locus of dg mutant plants is associated with one of its main characteristic phenotypes, i.e. shorter hypocotyl-length of seedlings.

Table 5. Linkage analysis between the tomato DETl locus and the hypocotyl-length photomorphogenic response. Seedlings were grown under yellow plastic screen for 9 days after sowing. Different superscript letters represent statistically significant differences between means (P<0.05) based on Tukey-Kramer HSD test (Kramer, 1956).

Genotype N Hypocotyl length LOD score R² ± S.E. (cm)

+/+ 18 7.7^A ±0.3 12<LOD<13 51.1% dg/ + 35 7.7^A ±0.2 dg/dg 33 5.4^B ±0.2

Example 6

Incorporation of two genetically unlinked lycopene enhancing mutations in a single tomato hybrid: Experimental

Approach

A common practice among breeders is to combine or incorporate two or more mutations positively affecting the same trait. Such procedure can be verified by laborious and time consuming test crosses. The diagnostic tool produced herein can facilitate the incorporation of two light hypersensitive lycopene-enhancing mutations in a single plant or breeding line.

Several mutations in tomato positively affect lycopene content in the mature tomato fruit. Of these, at least 5 show a significant hypersensitive light response. These include:

1 . High pigment-1 (hp-1 )

2 . High pigment- l^w (hp-l^w)

3 . High pigment-2 (hp-2) 4 . High pigment-2^3' (hp-2³ )

5 . Dark green ( dg)

The hp-1 and hp-l^w mutations map to the HP-1 locus on the tomato chromosome 2 (Yen et al., 1997) . The hp-2, hp-2³ and, in accordance with the present invention, dg mutations map to the HP-2 locus on the tomato chromosome 1 (Mustilli et . al, 1999) . Incorporation of lycopene enhancing dg and either one of the two mutations that map to the HP-1 locus (hp-1 and hp-l^w) can be more efficiently achieved through the following procedure:

1. Cross homozygous dg with homozygous hp-1 mutants to generate double heterozygous Fi plants:

dg/dg +/+ X +/+ hp-l/hp-1

dg/+ hp-l/+

2 . Self-cross the Fi double heterozygous plants to generate F₂ seeds. These F₂ seeds will segregate into 9 genotypes that can be further categorized into two groups according to their light hypersensitivity (Table 6) .

3. Germinate the F₂ seeds in a temperature controlled chamber, under yellow plastic screen that prevents transmittance of light having a wavelength of less than 500nm (Mochizuki and Kamimura, 1984) . Discard the etiolated seedlings representing the non-hypersensitive genotypes eight days post-sowing (see Table 6) . Use the diagnostic tool (see Example 4 hereinabove) on DNA extracted from the remaining, light hypersensitive seedlings, in order to detect the presence of the dg mutation. Select heterozygous dg+ seedlings that comprise a single genotypic group of plants that are already hp-1 homozygous (see Table 6).

5. Self-cross the selected F₂ dg/+ hp-l/hp-1 plants to generate F₃ seeds. These seeds will segregate into 3 genotypic groups: dg/dg hp-l/hp-1, 2. dg/+ hp-l/hp-1 , and 3. +/+ hp-l/hp-1.

6. Germinate the F₃ seeds under normal conditions.

7. Use the diagnostic tool (Example 4, hereinabove) on DNA extracted from the F₃ seedlings. Select homozygous dg/dg plants. These plants will necessarily be double mutant plants, combining two lycopene-enhancing mutations .

_

Table 6

Light hypersensitive

Yes no dg/dg hp-l/hp-1 dg/+ hp-l/f dg/dg hp~l/+ dg/+ +/+ dg/dg +/+ ++ hp-l+ dg/+ hp-l/hp-1 +/+ +/+ +/+ hpl/hpl

Example 7

Incorporation of two genetically unlinked lycopene enhancing mutations in a single tomato hybrid significantly increases lycopene yield: Working Example

Two semi-isogenic hybrids, one homozygous for the hp-1 mutation, hp-l/hp-1 , and the other for the dg mutation, dg/dg, were crossed hybridized to yield Fi plants (hp-l/+ dg/+) . These Fi plants were self-hybridized to yield F₂ seedlings. These F₂ seedlings were screened using a yellow plastic screen, genotyped and self-hybridized to yield F₃ plants, as outlined in Example 6. These F₃ plants were genotyped again using the primer combination presented herein and self-hybridized to yield F₄ plants as also outlined in Example 6. These plants are, as outlined in Example 6, double mutant plants (hp-l/hp-1 dg/dg) . Two horticulturally acceptable plants were selected and allowed to self hybridize to yield two F₅ lines. These F₅ lines were cross hybridized to yield a double mutant hybrid. This hybrid was tested, together with the semi-isogenic single mutant hybrids used in the initial cross (see above) , in 4 locations in northern Israel during the spring season under open field conditions. Results presented in Table 7 show that, unexpectedly, the lycopene yield of the double mutant hybrid is statistically higher compared to its isogenic single mutant hybrids. The increase in lycopene yield of the double mutant hybrid was 19 and 61% compared to the lycopene yield of the dg/dg and hp-l/hp-1 single mutant hybrids, respectively.

Table 7. Lycopene yield of single and double mutant hybrid cultivars carrying hypersensitive lycopene-enhancing mutations. Different superscript letters represent statistically significant differences between means (P<0.05) based on Tukey-Kramer HSD test (Kramer, 1956).

Cultivar genotype Lycopene yield (gr/dunam*)

+/+ hp-l/hp-1 1136^c dg/dg +/+ 1538^B dg/dg hp-l/hp-1 1824^A

* 1 dunam= 1000 square meters

Example 8 Use of the diagnostic tool for post control analysis of parental lines and hybrid seeds .

Seed companies often use a battery of molecular markers for post- or quality- control of parental seed stocks and hybrid-seeds . Several commercial lycopene-rich tomato cultivars carry the dg mutation either at a homozygous or heterozygous state. Up until now, detection of the dg trait within a particular stock could only be performed by the lengthy procedure of germinating samples of the seeds, and performing complicated phenotypic analyses on the parental cultivars and subsequent generations.

The diagnostic tool developed in this study (see Example 3, hereinabove) can be used to positively detect the dg allele in such cultivars and their parental lines, and thus enable post-production quality control to be carried out over a time scale of 1-2 days instead of weeks or months.

References

Chory J (1993) Out of darkness: mutants reveal pathways controlling light-regulated development in plants. Trends in Genet 9:167-172

Davis BH (1965) Analysis of carotenoid pigments. In: Chemistry and Biochemistry of Plant Pigments. Goodwin TW ed. Academic Press. London and New York p.530.

Fulton TM, Chunwongse J, Tansley SD (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Rep 13:207-209

Kerckhoff LHJ, Kendrick RE (1997) Photocontrol of antocyanin biosynthesis in tomato. J Plant Res 110:141-149

Konsler TR (1973) Three mutants appearing in 'Manapal' tomato. HortSci 8:331-333

Kramer CY (1956) Extension of multiple range tests to group means with unequal number of replications. Biometrics 12: 309-310

Mochizuki T, and Kamimura S (1984) Inheritance of vitamin C content and its relation to other characters in crosses between hp and og varieties of tomatoes . Eucarpia Tomato working group. Synopsis IX meeting 22-24 May, Wageningen, The Netherlands, pp 8-13

Mustilli AC, Fenzi F, Ciliento R, Alfano F Bowler C (1999) Phenotype of the tomato high pigment-2 is caused by a mutation in the tomato homolog of DEETIOLATEDl . Plant Cell 11:145-157 _ _ _

Pnuelli L, Carmel-Goren L, Hareven D, Gutfinger T, Alvarez J, Ganal M, Zamir D, and Lifschitz E (1998) The SELF- PRUNING gene of tomato regulates the reproductive switching of sympodial meristems and is the ortholog of CEN and TFL1 . Development 125:1979-1989

Reynard GB (1956) Origin of Webb Special (Black Queen) in tomato. Rep Tomato Genet Coop 40: 44-64

Soressi GP (1975) New spontaneous or chemically-induced fruit ripening tomato mutants. Rep. Tomato Genet Coop 25:21-22

Van Tuinen A, Cordonnier-Prat M-M, Pratt LH, Verkerk R,

Zabel P, and Koornneef M (1997) . The mapping of phytochrome genes and photomorphogenic mutants of tomato. Theor Appl Genet 94:115-122

Wann EV, Jourdain EL, Pressey R Lyon BG (1985) Effect of mutant genotypes hp og° and dg og^c on tomato fruit quality. J Amer Hort Sci 110:212-215

Wann, E.V. (1997) Tomato germplas lines T4065, T4099, T5019, and T5020 with unique genotypes that enhance fruit quality. HortSci 32: 747-748

Yen H, Shelton A, Howard L, Vrebalov J Giovanonni J (1997) The tomato high-pigment (hp) locus maps to chromosome 2 and influences plastome copy number and fruit quality. Theor And Appl Genet 95: 1069-1079.

Claims

1. An isolated nucleotide sequence responsible for the tomato dark green ( dg) phenotype, wherein said sequence comprises an altered tomato DETl gene sequence or fragment thereof, wherein said the alteration in said altered sequence or¹ fragment comprises an A-to-T transversion at nucleotide 29 of the second exon of said DETl gene sequence .

2. The isolated nucleotide sequence according to claim 1, wherein said sequence comprises the sequence shown in Fig. 2A.

3. A method for detecting the presence of the dg mutation in a plant, comprising the steps of isolating the genomic DNA from said plant, amplifying a gene fragment containing said dg mutation from said genomic DNA by use of a PCR technique and determining the presence of said dg mutation in said genomic DNA.

4. The method according to claim 3, wherein the presence of the dg mutation is determined by the use of restriction site analysis, wherein the loss of an Acl I site indicates the presence of said mutation.

5. The method according to claim 3, wherein the presence of the dg mutation is determined by direct sequencing of the amplified fragment and comparison of the sequence obtained with that shown in Fig. 2A.

6. The method according to claim 3, wherein the plant in which the presence of the dg mutant is being detected is of the species Lycopersicon esculentum .

7. Use of the method according to claim 3 as a means of post-control in seed production.

8. A method for the determination of the presence of two different photomorphogenic mutations in a plant, wherein one of said mutations is the dg mutation, comprising detecting the presence of a photomorphogenic mutation other than the dg mutation by either genotypic or phenotypic selection means, and detecting the presence of the dg mutation by means of the method according to claim 3.

9. The method according to claim 8, wherein the phenotypic selection means for determining the presence of the non-dg photomorphogenic mutation comprises germinating seeds obtained from the plant in which the presence of the mutations is being determined in a temperature controlled chamber, under a yellow plastic screen that is opaque to light having a wavelength less than 500nm, and selecting non-etiolated seedlings.

10. A method for preparing double-mutant lines of Lycopersicon esculentum having genotype dg/dg p/p, wherein p represents any recessive photomorphogenic mutation that is genetically unlinked to the dg mutation, said method comprising the steps of:

a) cross-hybridization of a homozygous dg/dg line or plant with a homozygous p/p line or plant to yield double heterozygous dg/+ p/+ F_x plants; b) self-crossing of the Fi plants obtained in step (a) in order to yield F₂ seeds; c) light-sensitivity selection of light-hypersensitive seedlings germinated from F₂ seeds; d) selection of light-hypersensitive seedlings containing the dg/+ p/p mutations, by use of the method of claim 3; e) self-crossing of the selected plants obtained in step (d) to generate F₃ seeds, and germination of said seeds under normal conditions; f) selection of double homozygous dg/dg p/p seedlings from the seedlings obtained in step (e) , by use of the method of claim 3.

11. A method according to claim 10, wherein recessive photomorphogenic mutation p is a mutation that causes photomorphogenic phenotypic effects similar or identical to those caused by the hp-1 mutation.

12. A method according to claim 11, wherein the recessive photomorphogenic mutation p is a lycopene-enhancing mutation.

13. A method according to claim 12, wherein the lycopene- enhancing mutation is hp-l^w.

14. A method according to claim 11, wherein p is the hp~l mutation.

15. Double-mutant hybrid plants of the species Lycopersicon esculentum having genotype dg/dg p/p.

16. Double-mutant hybrid plants according to claim 15, wherein said plants have genotype dg/dg hp-l/hp-1 .

17. Double-mutant hybrid plants according to claim 15, wherein said plants have genotype dg/dg hp-l^w/hp-l^w .