# Patent

## Title
Non-transgenic tomato varieties having increased shelf life post-harvest

## Abstract

A series of independent human-induced, non-transgenic mutations found in at least one non-ripening (NOR) gene of tomato; tomato plants having these mutations in at least one of their NOR genes; and a method of creating and identifying similar and/or additional mutations in the NOR gene by screening pooled and/or individual tomato plants. The tomato plants of the present invention exhibit fruit that ripen more slowly, rot more slowly, are firmer, and have a longer shelf life post-harvest as a result of non-transgenic mutations in at least one of their NOR genes.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 13/123,391 filed Apr. 8, 2011, which is a United States §371 National Phase application of PCT/US2009/060235, filed Oct. 9, 2009, both of which claim the benefit of U.S. Provisional Application No. 61/104,628, filed Oct. 10, 2008, all of which are hereby incorporated by reference in their entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Contract No. W911QY-07-C-0121 awarded by the United States Department of Defense. The Government has certain rights in the invention.


FIELD OF THE INVENTION
This invention relates to novel human-induced, non-transgenic mutations of the non-ripening (NOR) gene of tomatoes and tomato plants having such non-transgenic mutations in at least one of their NOR gene sequences. This invention further relates to tomatoes that ripen more slowly, rot more slowly, are firmer, and have a longer shelf life post-harvest as a result of human-induced, non-transgenic mutations in at least one of their NOR genes. This invention also relates to a method that utilizes non-transgenic means to create tomatoes having mutations in at least one of their NOR genes.
BACKGROUND
One of the main challenges facing today's tomato industry is how to deliver to a processing plant or to the marketplace tomato fruit that have been vine-ripened (and thus are desirable to consumers in terms of taste, texture, and color), but that remain firm without the usual post-harvest ripening-related softening that reduces shelf life of harvested fruit. Using traditional breeding methods, which are very labor intensive, it could take years to develop a novel tomato variety that ultimately may display only a modest increase in shelf life. Instead, recent studies have utilized genetic and biochemical techniques in an effort to identify the factors that regulate fruit ripening. By identifying and modifying the expression of specific genes, researchers and breeders hope to develop new tomato varieties that have the desirable qualities of vine-ripened fruit, but that are resistant to post-harvest softening and therefore display an extended shelf life.
Ripening is a complex process involving numerous physiological and biochemical changes including changes in color, firmness, sugar content, and pathogen resistance. Post-harvest ripening limits the shelf life of fresh produce, such as tomatoes. Several genes involved in the ripening process have been identified by analysis of single locus mutations that result in a non-ripening phenotype. One of these genes has been called NOR after a naturally occurring mutation at the nor (non-ripening) locus of tomato. The non-ripening phenotype results from a 2 base pair deletion in the NOR gene, which causes a frame shift that affects NOR protein synthesis (see U.S. Pat. No. 6,762,347). This NOR deletion mutation severely impairs tomato fruit ripening and causes a broad range of undesirable traits that have proven difficult to eliminate through traditional breeding. It is the only characterized mutation in the NOR gene of tomato. NOR is a member of the NAC protein family, a large family of plant-specific transcription factors involved in multiple developmental processes, including formation of shoot apical meristem, floral organs, lateral shoots, hormone control and defense mechanisms, and programmed cell death. The structure of the DNA-binding NAC domain has recently been determined (Ernst et al., EMBO Reports 5(3):297-303, 2004; Olsen et al., Trends in Plant Science, 10(2):79-97, 2005).
Standard breeding methods have utilized the NOR deletion mutation in tomatoes (see U.S. Pat. No. 6,180,854). The usefulness of this deletion mutation is limited however since these mutant fruit fail to ripen normally. While the NOR deletion mutant fruit may have an increased shelf life, they have decreased sensory qualities (e.g., impaired flavor, aroma, and color) compared to wild type fruit which makes the nor deletion mutant fruit less appealing to consumers. Fruit that are homozygous for the NOR deletion mutation fail to ripen and remain hard and green. Breeders have attempted to use the NOR deletion mutation in the heterozygous state to develop firmer fruit. Even fruit that are heterozygous for the NOR deletion mutation fail to fully develop the red color and sensory qualities that consumers desire in ripened fruit.
To date, other useful characterized mutations in the NOR gene of tomato are not available. Because NOR exerts pleiotropic effects, it would be useful to have an allelic series of mutations in the NOR gene that provide a spectrum of firmness and color phenotypes that could be used to optimize the breeding of extended shelf life tomato varieties that retain many of the quality traits of vine-ripened tomatoes. Additional useful NOR mutations would include those that increase shelf life but do not affect flavor, aroma and color as adversely as the naturally occurring nor deletion mutation. Tomato lines with NOR mutations that have been genetically characterized could also be crossed with lines that carry mutations in other genes involved in ripening.
In addition to standard breeding methods utilizing the nor mutation, transgenic approaches that targeted the NOR gene (see U.S. Pat. No. 6,762,347; U.S. Patent Application No. 20050076410) have been proposed for tomato fruit development. However, public acceptance of genetically modified plants, particularly with respect to plants used for food, is not universal. Because a cultivated tomato that is resistant to post-harvest softening and has improved shelf life with quality traits acceptable to consumers would be useful, an allelic series of novel mutations in the NOR gene of tomato were created. A cultivated tomato with reduced fruit softening as a result of altered NOR that was not the result of genetic engineering would have tremendous value for the tomato industry, including fresh market tomatoes, processor tomatoes and tomato food products such as sliced tomatoes, canned tomatoes, ketchups, soups, sauces, juices and pastes.
SUMMARY OF THE INVENTION
In accordance with one exemplary embodiment, this invention includes a tomato plant having tomato fruit with increased firmness, reduced rot rate, and increased shelf life post-harvest compared to wild type tomato fruit due to a human-induced, non-transgenic mutation in the NOR gene, as well as fruit, seeds, pollen, plant parts and progeny of that plant.
In accordance with another exemplary embodiment, this invention includes a tomato plant having tomato fruit that ripen more slowly post-harvest compared to wild type tomato fruit due to a human-induced non-transgenic mutation in the NOR gene, as well as fruit, seeds, pollen, plant parts and progeny of that plant.
In accordance with another exemplary embodiment, this invention includes food and food products incorporating tomato fruit having increased firmness, reduced rot rate, and increased shelf life post-harvest caused by a human-induced non-transgenic mutation in the NOR gene.
In accordance with yet another exemplary embodiment, this invention includes a tomato plant having fruit with increased shelf life compared to wild type tomato fruit created by the steps of obtaining plant material from a parent tomato plant, inducing at least one mutation in at least one copy of a NOR gene of the plant material by treating the plant material with a mutagen to create mutagenized plant material, culturing the mutagenized plant material to produce progeny tomato plants, analyzing progeny tomato plants to detect at least one mutation in at least one copy of a NOR gene, selecting progeny tomato plants that have fruit with extended shelf life compared to the parent tomato plant; and repeating the cycle of culturing the progeny tomato plants to produce additional progeny plants having extended shelf life.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NO: 1 shows a Solanum lycopersicum NAC domain protein (NAC-NOR) gene, NAC-NOR-NOR allele, complete cds (NCBI Accession Number AY573803).
SEQ ID NO: 2 shows the protein encoded by SEQ ID NO: 1 (NCBI Accession Number AAU43922).
SEQ ID NOs: 3-8 show the DNA sequences for the Solanum lycopersicum NOR specific PCR primers used to detect the mutations of the present invention.
A substitute sequence listing is herein incorporated by reference to the material contained in the associated file, entitled, “substitutesequencelist.txt,” submitted herewith, created on Apr. 5, 2011, and comprising 8.72 kb in size.


DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In accordance with one exemplary embodiment, the present invention provides tomatoes that have an extended shelf life as compared to wild type tomatoes due to a mutation in at least one of their NOR genes and without the inclusion of foreign nucleic acids in the tomatoes' genomes. In accordance with other exemplary embodiments, the present invention provides a series of independent non-transgenic mutations in the NOR gene; tomatoes having these mutations in at least one of their NOR genes; and a method of creating and identifying similar and/or additional mutations in the NOR gene of tomatoes.
In order to create and identify the NOR mutations and tomatoes of the present invention, the present inventors utilized a method known as TILLING. See McCallum et al., Nature Biotechnology 18:455-457, 2000; McCallum et al., Plant Physiology 123:439-442, 2000; Colbert et al., Plant Physiology 126:480-484, and U.S. Pat. No. 5,994,075 and U.S. Publication No. 20040053236, all of which are incorporated herein by reference. In the basic TILLING methodology, plant material, such as seed, is subjected to chemical mutagenesis, which creates a series of mutations within the genomes of the seeds' cells. The mutagenized seeds are grown into adult M1 plants and self-pollinated. DNA samples from the resulting M2 plants are pooled and are then screened for mutations in a gene of interest. Once a mutation is identified in a gene of interest, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with the gene of interest.
Any cultivar of tomato having at least one NOR gene with substantial homology to SEQ ID NO: 1 may be used in accordance with the present invention. As used herein, “substantial homology” means that the DNA sequence of the gene is sufficiently similar to SEQ ID NO: 1 at the nucleotide level to code for the equivalent protein as SEQ ID NO: 1, allowing for allelic differences between cultivars. In accordance with one aspect of an exemplary embodiment of the invention, “substantial homology” may be present when the homology between the NOR gene and SEQ ID NO: 1 is as low as about 85%, provided that the homology in the conserved regions of the gene is higher (e.g., at least about 90%). Preferably, the percent identity in the coding region is 85-90%, more preferably 90-95%, and optimally, it is above 95%. One of skill in the art may prefer a tomato cultivar having commercial popularity or one having specific desired characteristics in which to create the NOR-mutated tomatoes. Alternatively, one of skill in the art may prefer a tomato cultivar having few polymorphisms, such as an in-bred cultivar, in order to facilitate screening for mutations within the NOR loci.
In accordance with one aspect of an exemplary embodiment of the present invention, seeds from tomatoes were mutagenized and then grown into M1 plants. The M1 plants were then allowed to self-pollinate and seeds from the M1 plant were grown into M2 plants, which were then screened for mutations in their NOR locus. While M1 plants may be screened for mutations, an advantage of screening the M2 plants is that all somatic mutations correspond to the germline mutations. One of skill in the art would recognize that a variety of tomato plant materials, including, but not limited to, seeds, pollen, plant tissue or plant cells, may be mutagenized in order to create the NOR-mutated tomatoes of the present invention. However, the type of plant material mutagenized may affect when the plant DNA is screened for mutations. For example, when pollen is subjected to mutagenesis prior to pollination of a non-mutagenized plant, the seeds resulting from that pollination are grown into M1 plants. Every cell of the M1 plants will contain mutations created in the pollen, thus these M1 plants may then be screened for NOR mutations instead of waiting until the M2 generation.
Mutagens that create primarily point mutations and short deletions, insertions, transversions, and or transitions (about 1 to about 5 nucleotides), such as chemical mutagens or radiation, may be used to create the mutations of the present invention. Mutagens conforming with the method of the present invention include, but are not limited to, ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N-nitrosurea (ENU), triethylmelamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N′-nitro-Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7, 12 dimethyl-benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), and the like), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride (ICR-170), and formaldehyde. Spontaneous mutations in NOR that may not have been directly caused by the mutagen can also be identified in accordance with various embodiments of the present invention.
Any suitable method of plant DNA preparation now known or hereafter devised may be used to prepare the tomato plant DNA for NOR mutation screening. For example, see Chen and Ronald, Plant Molecular Biology Reporter 17:53-57, 1999; Stewart and Via, Bio Techniques 14:748-749, 1993. Additionally, several commercial kits are available, including kits from Qiagen (Valencia, Calif.) and Qbiogene (Carlsbad, Calif.).
In accordance with one aspect of an exemplary embodiment of the invention, DNA samples from individual tomato plants are prepared and then pooled in order to expedite screening for mutations in NOR of the entire population of plants originating from the mutagenized plant tissue. The size of the pooled group may be dependent upon the sensitivity of the screening method used. In accordance with one aspect of an exemplary embodiment of the invention, groups of four or more individual tomato plants are pooled.
In accordance with another aspect of an exemplary embodiment, after the DNA samples are pooled, the pools are subjected to NOR sequence-specific amplification techniques, such as Polymerase Chain Reaction (PCR). For a general overview of PCR, see PCR Protocols: A Guide to Methods and Applications (Innis, Gelfand, Sninsky, J., and White, eds.), Academic Press, San Diego, 1990. Any primer specific to the NOR locus or the sequences immediately adjacent to the NOR locus may be utilized to amplify the NOR sequences within the pooled DNA sample. Preferably, the primer is designed to amplify the regions of the NOR locus where useful mutations are most likely to arise. Most preferably, the primer is designed to detect mutations in the coding region of the NOR gene. Additionally, it is preferable for the primer to avoid known polymorphic sites in order to ease screening for point mutations. To facilitate detection of PCR products on a gel, the PCR primer may be labeled using any conventional or hereafter devised labeling method.
Exemplary primers (SEQ ID NOs: 3-8) that have proven useful in identifying useful mutations within the NOR sequence are shown below in Table 1. These primers are also detailed in the Sequence Listing appended hereto.







TABLE 1







PCR primers specific for the NOR 


gene in tomato.











SEQ
PRIMER




ID
NAME
SEQUENCE







3
NORA-3193
tgaattcaggtcaactcaaaca





tcgtaaattg



 



4
NORA-3194
aattcactttttacacgttatc





gtggatatcttttg



 



5
NORB-3195
aaagtagtggacaaacataaag





tagtggacccataa



 



6
NORB-3196
tgaaagttgaatcaagtcatct





acaacaacaaca



 



7
NORC-3235
aatgaaaatcctgaatcggcca





ctaactttaac



 



8
NORC-3236
atgattgattgatcgattgatt





ttacagggcta










In accordance with one aspect of an exemplary embodiment of the invention, the PCR amplification products may be screened for NOR mutations using any method that identifies nucleotide differences between wild type and mutant sequences. These may include, without limitation, sequencing, denaturing high pressure liquid chromatography (dHPLC), constant denaturant capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (TGCE) (see Li et al., Electrophoresis 23(10):1499-1511, 2002), or by fragmentation using enzymatic cleavage, such as used in the high throughput method described by Colbert et al., Plant Physiology 126:480-484, 2001. Preferably, the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences. In accordance with another aspect of an exemplary embodiment, cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program.
The present inventors have determined that to achieve reduced post-harvest softening in tomatoes, mutations that alter NOR function are desirable. Preferred mutations include missense, nonsense and splice junction changes, including mutations that prematurely truncate the translation of the NOR protein from messenger RNA, such as those mutations that create a stop codon within the coding regions of the NOR gene. Such mutations include insertions, repeat sequences, modified open reading frames (ORFs) and, most preferably, point mutations.
In accordance with yet another aspect of an exemplary embodiment of the invention, once an M2 plant having a mutated NOR sequence is identified, the mutations are analyzed to determine its affect on the expression, translation, and/or activity of the protein. In accordance with one exemplary embodiment, the PCR fragment containing the mutation is sequenced, using standard sequencing techniques, in order to determine the exact location of the mutation in relation to the overall NOR sequence. Each mutation is evaluated in order to predict its impact on protein function (i.e., completely tolerated to loss-of-function) using bioinformatics tools such as SIFT (Sorting Intolerant from Tolerant; Ng et al., Nucleic Acids Research 31:3812-3814, 2003), PSSM (Position-Specific Scoring Matrix; Henikoff and Henikoff, Computer Applications in the Biosciences 12:135-143, 1996) and PARSESNP (Taylor and Greene, Nucleic Acids Resarch 31:3808-3811, 2003). For example, a SIFT score that is less than 0.05 and a large change in PSSM score (e.g., roughly 10 or above) indicate a mutation that is likely to have a deleterious effect on protein function.
In accordance with a further aspect of an exemplary embodiment, if the initial assessment of a mutation in an M2 plant indicates it to be of a useful nature and in a useful position within the NOR gene, then further phenotypic analysis of the tomato plant containing that mutation is pursued. First, the M2 plant is backcrossed or outcrossed twice to create a BC1 plant in order to eliminate background mutations. Then the backcrossed or outcrossed BC1 plant is self-pollinated in order to create a BC1F2 plant that is homozygous for the NOR mutation.
Several physical characteristics of these homozygous NOR mutant plants are assessed to determine if the mutation results in a useful phenotypic change in the tomato. Mutant NOR tomatoes are evaluated post-harvest for several traits including rate of ripening, firmness, rot rate and shelf life compared to normal (e.g., wild type) parental tomatoes or to wild type sibling control tomatoes. Evaluations can be performed during storage. Examples of standard storage conditions include room temperature storage (approximately 68° F./20° C.) or refrigerated storage (approximately 55° F./13° C.). Normal fruit ripens on the vine or during storage such that the color of the tomato changes from light green to red. As this change occurs, the fruit tends to become softer such that compression under a specified weight becomes greater and/or the force required to depress the surface of the fruit a specified distance becomes less. See Cantwell, Report to the California Tomato Commission: Tomato Variety Trials: Postharvest Evaluations for 2001; Edan et al., J. Food Science 62(4): 793-796, 1997; Errington et al., Postharvest Biology and Technology 11: 141-147, 1997; Lesage and Destain, Postharvest Biology and Technology 8: 45-55, 1996. For lycopene measurements, see Alba et al., Plant Physiology 123:363-370, 2000.
The following novel mutations identified in Table 2 are exemplary of the mutations created and identified according to various embodiments of the present invention. The only previously reported mutation in the NOR gene—the nor two base pair deletion mutation (U.S. Pat. No. 6,762,347)—results in a frameshift beginning at the glutamine at amino acid 183 according to SEQ ID NO: 2 that ends in a truncation (stop*) four amino acids later (QRSID to QVHR*).







TABLE 2







Examples of novel mutations created and identified in the NOR gene of tomato.

















Nucleotide
Amino Acid (a.a.)
Amino Acid (a.a.)






Mutation
Mutation
Mutation


Type of

Primer
EMS
According to
According to
According to


Mutation
Variety
SEQ IDs.
Treatment
SEQ ID NO: 1
SEQ ID NO: 2
SEQ ID NO: 2





Missense
Shady
B
0.8%
G995A
G68E
glycine to



Lady




glutamic acid








at a.a. 68


Missense
NC84173
B
1.2%
G1048A
A86T
alanine to








threonine at








a.a. 86


Truncation
NC84173
C
1.2%
C2277T
Q206*
glutamine to


(stop)





stop at a.a.








206


Missense
NC84173
C
1.2%
G2425A
G255D
glycine to








aspartic acid








at a.a. 255


Truncation
Shady
C
0.8%
G2434A
W258*
tryptophan to


(stop)
Lady




stop at a.a.








258


Missense
Shady
C
0.6%
G2646C
E329Q
glutamic acid



Lady




to glutamine








at a.a. 329


Missense
NC84173
C
1.2%
G2673T
G338W
glycine to








tryptophan at








a.a. 338









The nomenclature used in the Table 2 indicates the wild type nucleotide or amino acid, followed by its position according to the referenced SEQ ID NO, followed by the changed nucleotide or amino acid at that position using standard genetic code terminology (see specific examples below).
The following Examples are offered by way of illustration only, and not limitation. It is to be understood that the mutations below are merely exemplary and that similar mutations are also contemplated.
EXAMPLE 1
Mutagenesis
In one embodiment of the present invention, tomato seeds of cultivars Shady Lady (hybrid) and NC84173 were vacuum infiltrated in H2O (approximately 1,000 seeds/100 ml H2O for approximately 4 minutes). The seeds were then placed on a shaker (45 rpm) in a fume hood at ambient temperature. The mutagen ethyl methanesulfonate (EMS) was added to the imbibing seeds to final concentrations ranging from about 0.1% to about 1.6% (v/v) in accordance with one aspect of an exemplary embodiment of the invention. Following a 6 to 24-hour incubation period, the EMS solution was replaced 4 times with fresh H2O. The seeds were then rinsed under running water for approximately 1 hour. Finally, the mutagenized seeds were planted (96/tray) in potting soil and allowed to germinate indoors. Plants that were four to six weeks old were transferred to the field to grow to fully mature M1 plants. The mature M1 plants were allowed to self-pollinate and then seeds from the M1 plant were collected and planted to produce M2 plants.
DNA Preparation
DNA from these M2 plants produced in accordance with the above description was extracted and prepared in order to identify which M2 plants carried a mutation at their NOR loci. The M2 plant DNA was prepared using the methods and reagents contained in the Qiagen (Valencia, Calif.) DNeasy 96 Plant Kit. Approximately 50 mg of frozen plant sample was placed in a sample tube with a tungsten bead, frozen in liquid nitrogen and ground 2 times for 1 minute each at 20 Hz using the Retsch Mixer Mill MM 300. Next, 400 μl of solution AP1 [Buffer AP1, solution DX and RNAse (100 mg/ml)] at 80° C. was added to the sample. The tube was sealed and shaken for 15 seconds. Following the addition of 130 μl. Buffer AP2, the tube was shaken for 15 seconds. The samples were placed in a freezer at minus 20° C. for at least 1 hour. The samples were then centrifuged for 20 minutes at 5,600×g. A 400 μl aliquot of supernatant was transferred to another sample tube. Following the addition of 600 μl of Buffer AP3/E, this sample tube was capped and shaken for 15 seconds. A filter plate was placed on a square well block and 1 ml of the sample solution was applied to each well and the plate was sealed. The plate and block were centrifuged for 4 minutes at 5,600×g. Next, 800 μl of Buffer AW was added to each well of the filter plate, sealed and spun for 15 minutes at 5,600×g in the square well block. The filter plate was then placed on a new set of sample tubes and 80 μl of Buffer AE was applied to the filter. It was capped and incubated at room temperature for 1 minute and then spun for 2 minutes at 5,600×g. This step was repeated with an additional 80 μl Buffer AE. The filter plate was removed and the tubes containing the pooled filtrates were capped. The individual samples were then normalized to a DNA concentration of 5 to 10 ng/μl.
Tilling
The M2 DNA was pooled into groups of four individuals. The DNA concentration for each individual within the pool was 0.25 ng/μl with a final concentration of 1 ng/μl for the entire pool. The pooled DNA samples were arrayed on microtiter plates and subjected to gene-specific PCR.
PCR amplification was performed in 15 μl volumes containing 2.5 ng pooled DNA, 0.75× ExTaq buffer (Panvera®, Madison, Wis.), 2.6 mM MgCl2, 0.3 mM dNTPs, 0.3 μM primers, and 0.05U Ex-Taq (Panvera) DNA polymerase. PCR amplification was performed using an MJ Research thermal cycler as follows: heat denaturation at 95° C. for 2 minutes; followed by 8 cycles of “touchdown PCR” (94° C. for 20 seconds, followed by an annealing step starting at 68-70° C. for 30 seconds and decreasing 1° C. per cycle, then a temperature ramp increasing 0.5° C. per second to 72° C., and followed by 72° C. for 1 minute); then 25-45 more cycles of PCR (94° C. for 20 seconds, 61-63° C. for 30 seconds, a ramp increasing 0.5° C. per second up to 72° C., 72° C. for 1 minute); and finally extension, denaturation and re-annealing steps (72° C. for 8 minutes; 98° C. for 8 minutes; 80° C. for 20 seconds; followed by 60 cycles of 80° C. for 7 seconds decreasing 0.3° C. per cycle).
The PCR primers (MWG Biotech, Inc., High Point, N.C.) were mixed as follows:
9 μl 100 μM IRD-700 labeled left primer
1 μl 100 μM left primer
10 μl 100 μM right primer
The IRD-700 label can be attached to either the right or left primer. Preferably, the labeled to unlabeled primer ratio is 9:1. Alternatively, Cy5.5 modified primers or IRD-800 modified primers could be used. Additionally, both primers could be labeled simultaneously with distinguishable labels such as IRD-700 and IRF-800. In the present invention, the IRD-700 label was coupled to the oligonucleotide using conventional phosphoramidite chemistry.
PCR products (15 μl) were digested in 96-well plates. Next, 30 μl of a solution containing 10 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (pH 7.5), 10 mM MgSO4, 0.002% (w/v) Triton X-100, 20 ng/ml of bovine serum albumin, and CEL 1 (Transgenomic, Inc.; 1:100,000 dilution) was added with mixing on ice, and the plate was incubated at 45° C. for 15 min. The specific activity of the CEL 1 was 800 units/μl, where a unit was defined by the manufacturer as the amount of enzyme required to produce 1 ng of acid-soluble material from sheared, heat denatured calf thymus DNA at pH 8.5 in one minute at 37° C. Reactions were stopped by addition of 10 μl of a 2.5 M NaCl solution with 0.5 mg/ml blue dextran and 75 mM EDTA, followed by the addition of 80 μl isopropanol. The reactions were precipitated at 80° C., spun at 4,000 rpm for 30 minutes in an Eppendorf Centrifuge 5810. Pellets were resuspended in 8 μl of 33% formamide with 0.017% bromophenol blue dye, heated at 80° C. for 7 minutes and then at 95° C. for 2 minutes. Samples were transferred to a membrane comb using a comb-loading robot (MWG Biotech). The comb was inserted into a slab acrylamide gel (6.5%), electrophoresed for 10 min, and removed. Electrophoresis was continued for 4 hours at 1,500-V, 40-W, and 40-mA limits at 50° C.
During electrophoresis, the gel was imaged using a LI-COR (Lincoln, Nebr.) scanner, which was set at a channel capable of detecting the IR Dye 700 label. The gel image showed sequence-specific pattern of background bands common to all 96 lanes. Rare events, such as mutations, create new bands that stand out above the background pattern. Plants with bands indicative of mutations of interest were evaluated by TILLING individual members of a pool mixed with wild type DNA and then sequencing individual PCR products. Plants carrying mutations confirmed by sequencing were grown up as described above (e.g., the M2 plant was backcrossed or outcrossed twice in order to eliminate background mutations and self-pollinated in order to create a plant that was homozygous for the mutation).
Physical and Biochemical Measurements
Tomatoes Selected for Study
Individual tomatoes selected for study were picked from plants derived from siblings of the same cross to preserve background phenotypes as much as possible. The plants and fruit were genotyped as homozygous for the mutation, heterozygous for the mutation, or wild type. Genotyping was performed using Taqman SNP Genotyping Assays (Applied Biosystems) to discriminate the three different alleles of the NOR locus.
Evaluation of Sensory Qualities
In general, tomato fruit that had one or more mutant alleles in NOR were similar in sensory qualities to wild type control tomatoes, though the attainment of full flavor, color and aroma was delayed in tomatoes with NOR mutations due to their delayed ripening. This is an improvement over fruit with the original NOR deletion mutation described in U.S. Pat. No. 6,762,347, which fail to fully develop the sensory qualities associated with ripe fruit. For example, fruit that were heterozygous for the W258* mutation were compared to wild type control fruit in a blind taste test. Tomatoes were stored at approximately 40° F. (4° C.) and approximately 55° F. (13° C.) and evaluated every two weeks. Results showed that W258* heterozygous tomatoes were as acceptable in odor, quality of flavor-point balance, texture attributes and overall quality as wild type control fruit. W258* heterozygous tomatoes were found to be superior to wild type tomatoes at 40° F. (4° C.). At 55° F. (13° C.), taste testers ranked W258* heterozygous tomatoes as under ripe at 2 weeks, fully ripe at 4 weeks, and slightly over ripe at 6 weeks post-harvest, whereas they ranked wild type control tomatoes as fully ripe at 2 weeks and over ripe at 4 and 6 weeks. Thus, W258* heterozygous tomatoes developed the full sensory profile and retained more quality over time than wild type control tomatoes.
Measurement of Fruit Firmness
Fruit (homozygous, heterozygous, and/or wild type siblings) were harvested at breaker stage and allowed to ripen at room temperature to light red stage. After the light red stage, tomatoes were stored at 55° F. (13° C.). Firmness was measured using a model TA-XT Texture Analyzer (Texture Technologies, Scarsdale, N.Y). The amount of force required to depress the tomato fruit surface 5 mm was recorded for each sample. Fruit firmness was measured twice for each fruit, equatorially, at two time points. The first two measurement locations were marked on the fruit, and subsequent measurements were taken at least 7 days later at different equatorial locations. Thus, each fruit was depressed four times. In general, time points were 7 days or increments of 7 days apart. In general, tomato fruit that had one or more mutant alleles in NOR were more firm than wild type control fruit. Exemplary data from measurements at 21 or 28 days post-harvest at shown in Table 3.







TABLE 3







Exemplary data from measurement of fruit firmness. Data are


expressed in Newtons.














Firmness After
Firmness After 28




Sample
21 Days in Storage
Days in Storage


Mutation
Genotype
Size
(X ± SEM)
(X ± SEM)





G68E
HOM
n = 14
11.6 ± 0.95
—



WT
n = 8
 9.7 ± 0.92
—


Q206*
HOM
n = 3
—
38.7 ± 1.58



HET
n = 3
—
12.0 ± 0.42



WT
n = 3
—
10.3 ± 1.95


W258*
HOM
n = 11
20.3 ± 1.63
—



HET
n = 33
11.6 ± 0.38
—



WT
n = 22
 8.8 ± 0.30
—









Measurement of Rot Rate
Fruit for each genotype were harvested at the breaker stage of fruit development and ripened to red prior to commencing the study to ensure that tomatoes of each type were at the same physiological age. Tomatoes were stored at approximately 55° F. (13° C.) and evaluated on a weekly basis for signs of rot. The rot rate was then calculated over time as the percent of tomatoes exhibiting rot, and the number of days in storage until all fruit had rotted was recorded. The rot rate was then used to extrapolate the number of days at which 50% of the fruit showed signs of rot. Using both data points eliminates the effect of outliers and illustrates the progression of rot. Fruit homozygous for NOR mutations were compared to fruit that were heterozygous for the NOR mutations and/or wild type sibling controls.
In general, tomato fruit that had one or more mutant alleles in NOR showed a reduced rate of rot and increased shelf life compared to wild type control fruit. Exemplary data for rot rate in three mutant lines are shown in Table 4.







TABLE 4







Exemplary data of measurement of rot rate.














Days in Storage
Days in Storage




Sample
Until 50% of
Until 100% of


Mutation
Genotype
Size
Fruit Rot
Fruit Rot














G68E
HOM
n = 14
56
75



WT
n = 8
34
61


Q206*
HOM
n = 3
>90
>90



HET
n = 3
31
75



WT
n = 3
23
50


W258*
HOM
n = 11
72
75



HET
n = 33
58
75



WT
n = 22
31
43









Evaluation of Color
Tomato fruit color was measured analytically using a Minolta CR-400 Chromameter. The a* values from the CIE L*a*b* color space measurements generated by the instrument were used to provide quantitative values for degree of ripening from green to red. The a* spectrum is the part of the CIE L*a*b* color space that defines the green (negative a*) to red (positive a*) color of any sample. For our purposes, the a* values specifically define the developmental stage of the fruit of red-fruited tomato cultivars. Tomatoes are considered to be ripe at the light red stage of development when the a* values are >20. Tomatoes at the pink stage of development have values between 10 and 20. Turning tomatoes have values between 0 and 10 and breaker and mature green tomatoes have negative a* values.
Of the seven mutations examined, all except but one (Q206*) achieved a maximum color of light pink to red. One particularly useful mutation is G68E. Tomatoes that are homozygous for the G68E allele are firmer than wild type tomatoes (11.6 versus 9.7) and have a reduced rate of rot compared to wild type controls (21 and 14 days longer in storage before reaching rot rates of 50% and 100%, respectively). Surprisingly, the G68E homozygous tomatoes attained an equivalent red color under 55° F. (13° C.) storage. Exemplary data for measurement of color are shown in Table 5.







TABLE 5







Exemplary data for measurement of color.














Maximum Color
Maximum Color




Sample
Stored at 55° F.
Stored at 68° F.


Mutation
Genotype
Size
(13° C.)
(20° C.)














G68E
HOM
n = 14
29.1
—



WT
n = 8
27.5
—


Q206*
HOM
n = 3
0.5
—



HET
n = 3
22.9
—



WT
n = 3
29.6
—


W258*
HOM
n = 11
17.1
 4.3



HET
n = 33
29
30.6



WT
n = 22
29.3
—









Identification and Evaluation of Mutation G68E
DNA from a tomato originating from seeds of cultivar Shady Lady that were incubated in 0.8% EMS was amplified using primers NORB-3195 and NORB-3196 (SEQ ID NOs: 5 and 6). The PCR amplification products were then incubated with CEL 1 and electrophoresed. The electrophoresis gel image showed a fragment that stood out above the background pattern for the PCR amplification products. Therefore, it was likely that this fragment contained a heteroduplex created by a mutation in a NOR sequence. Sequence analysis of this fragment showed the mutation was a G to A change at nucleotide 995 of SEQ ID NO: 1. This mutation correlates with a change from glycine to glutamic acid at amino acid 68 of the NOR protein [SEQ ID NO: 2].
Tomatoes homozygous for the G68E mutation in their NOR gene ripen more slowly, rot more slowly, are firmer and display a longer shelf life post-harvest than wild type sibling control tomatoes and have improved color compared to the original NOR deletion mutant described in U.S. Pat. No. 6,762,347. Tomatoes heterozygous for the G68E mutation in their NOR gene display a ripening rate that is intermediate between the homozygous and wild type controls.
Identification and Evaluation of Mutation Q206*
DNA from a tomato originating from seeds of cultivar NC84173 that were incubated in 1.2% EMS was amplified using primers NORC-3235 and NORC-3236 (SEQ ID NOs: 7 and 8). The PCR amplification products were then incubated with CEL 1 and electrophoresed. The electrophoresis gel image showed a fragment that stood out above the background pattern for the PCR amplification products. Therefore, it was likely that this fragment contained a heteroduplex created by a mutation in a NOR sequence. Sequence analysis of this fragment showed the mutation was a C to T change at nucleotide 2277 of SEQ ID NO: 1. This mutation correlates with a change from glutamine at amino acid 206 of the NOR protein [SEQ ID NO: 2] to a stop codon.
Tomatoes homozygous for the Q206* mutation in their NOR gene reach the Breaker stage of fruit development, which indicates an initiation of the ripening process. This observation differs from the original NOR deletion mutation described in U.S. Pat. No. 6,762,347 where fruit do not initiate ripening at all. In the heterozygous state, this mutation ripens to the pink stage of development and fruit rot more slowly, are firmer and display a longer shelf life post-harvest than wild type sibling control tomatoes.
Identification and Evaluation of Mutation W258*
DNA from a tomato originating from seeds of cultivar Shady Lady that were incubated in 0.8% EMS was amplified using primers NORC-3235 and NORC-3236 (SEQ ID NOs: 7 and 8). The PCR amplification products were then incubated with CEL 1 and electrophoresed. The electrophoresis gel image showed a fragment that stood out above the background pattern for the PCR amplification products. Therefore, it was likely that this fragment contained a heteroduplex created by a mutation in a NOR sequence. Sequence analysis of this fragment showed the mutation was a G to A change at nucleotide 2434 of SEQ ID NO: 1. This mutation correlates with a change from tryptophan at amino acid 258 of the NOR protein [SEQ ID NO: 2] to a stop codon.
Tomatoes homozygous for the W258* mutation in their NOR gene ripen more slowly, rot more slowly, are firmer and display a longer shelf life post-harvest than wild type sibling control tomatoes and have improved color when allowed to ripen at 55° F. (13° C.) compared to the original NOR deletion mutant described in U.S. Pat. No. 6,762,347. Tomatoes that are heterozygous for the W258* mutation in their NOR gene display an intermediate phenotype between homozygous and wild type tomatoes and thus ripen more slowly, rot more slowly, are firmer and display a longer shelf life post-harvest than wild type sibling control tomatoes, but unlike the homozygous tomatoes, these tomatoes ripen fully.
The above examples are provided to illustrate exemplary embodiments of the present invention but not limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims and all their equivalents. All publications, patents, and patent applications cited herein are hereby incorporated by reference.

## Claims

1. A non-ripening (NOR) gene from tomato comprising a human-induced, non-transgenic C2277T mutation; wherein said human-induced, non-transgenic C2277T mutation comprises a nucleotide change within the NOR gene; wherein said nucleotide change is identified according to SEQ NO: 1; and wherein said NOR gene codes for a protein having the amino acid sequence of SEQ ID NO: 2 with a truncation at amino acid 206.

2. A tomato plant containing a human-induced, non-transgenic mutation within its NOR gene; wherein said human-induced, non-transgenic mutation is C2277T; wherein said human-induced, non-transgenic mutation comprises a nucleotide change within the NOR gene; wherein said nucleotide change is identified according to SEQ ID NO: 1; and wherein said NOR gene codes for a protein having the amino acid sequence of SEQ ID NO: 2 with a truncation at amino acid 206.

3. A fruit, a seed, a pollen grain, a plant part, or the progeny of the tomato plant of claim 2; wherein the fruit, the seed, the pollen grain, the plant part, or the progeny comprises the mutation.

4. A food or a food product comprising the fruit of claim 3.

5. The tomato plant of claim 2, wherein expression of said mutation in the heterozygous state results in fruit that are firmer than wild type fruit and turn pink when ripened at standard storage conditions.

6. A NOR protein from tomato comprising a Q206* amino acid change; wherein said amino acid change results from a human-induced non-transgenic mutation in the NOR gene; wherein said amino acid change is identified according to SEQ ID NO: 2; and wherein said NOR protein has the amino acid sequence of SEQ ID NO: 2 with a truncation at amino acid 206.

7. A tomato fruit comprising the NOR protein of claim 6.

8. A food or a food product comprising the tomato fruit of claim 7.

9. The tomato fruit of claim 7, wherein expression of said amino acid change in its NOR protein in the heterozygous state results in fruit that are firmer than mild type fruit and turn pink when ripened at standard storage conditions.