Genetic sequence in solanaceous plants having improved resistance to tospovirus.

Abstract

The present invention relates to a specific genetic sequence, whose presence in Solanaceae plant genome confers an improved resistance to infections caused by some viruses belonging to the "Tospovirus" family. In particular, the described genetic sequence confers an improved resistance to some viral strain, such as Groundnut RingSpot Virus (GRSV) and Tomato Chlorotic Spot Virus (TCSV).

Description

“GENETIC SEQUENCE IN SOLANACEOUS PLANTS HAVING IMPROVED RESISTANCE TO TOSPO VIRUS”.* * * *DESCRIPTIONSCOPE

[0001] The present invention relates to a specific genetic sequence, the presence of which in the genome of Solanaceae plants is able to confer an improved resistance to infections caused by some viruses of the Tospovirus family.

[0002] In particular, the genetic sequence described in the present invention confers on plants belonging to the Solanaceae family a better resistance against some viral strains, such as Groundnut RingSpot Virus (hereinafter referred to as GRSV) and Tomato Chlorotic Spot Virus (hereinafter referred to as TCSV).STATE OF THE ART

[0003] It is known that the generic term “Tospovirus” indicates a family of viruses strongly pathogenic to a large number of plants, such as to cause outbreaks of considerable geographical extent in plant species spread both in temperate regions and in subtropical regions.

[0004] The wide spread of these viruses seems to be due to the intercontinental transport of infected ornamental plants, while the level of extent of the epidemic in a specific territory depends very much on the presence of insects of the Thripidae family (thrips), which constitute the main vector for this family of viruses.

[0005] The Tospovirus genome consists of three RNA segments designated as L, M and S in order of decreasing size, which encode six proteins. The Tospovirus virions are quasi- spherical particles, comprising the above-mentioned three RNA genomic segments, each encapsulated by the nucleoprotein and associated with protein molecules L. The viral glycoproteins are incorporated within the lipid envelope.

[0006] Tospoviruses are transmitted from about ten species of thrips in a persistent manner. Replication of viral RNA and assembly of virions occur in the cytoplasm of both plant and insect cells. The ability to spread to new geographical areas thousands of kilometres apart in a few years has been demonstrated. Therefore, there are numerous Tospoviruses on all continents and the situation is extremely fluid.

[0007] Tospoviruses have an extremely wide range of possible hosts, since they can infect more than 500 species among ornamental plants, fruit plants and horticultural plants. Solanaceae, Compositae and leguminous plants are particularly sensitive to this family of viruses. Some plants of very different species which can be seriously affected by Tospoviruses are tomato, pepper, tobacco, peanuts, lettuce, pineapple, papaya, dahlia and gloxinia. If 19 Tospoviruses were known in 2009, after only 6 years ten new Tospoviruses were characterized.

[0008] The losses resulting from Tospovirus infections are proportional to the number of plants early infected by the virus, and infection rates reaching 70-90% are common.

[0009] The pathogenic symptoms vary greatly depending on the plant species affected, the specific organ of the plant involved, and the age of the plant at the time of infection. In general, the symptoms caused by infections due to the presence of these viruses appear in the following forms:- rings, lines or chlorotic spots on leaves, stems and fruits;- necrotic streaks on stems;- brown colouring, curling and wilting of the leaves;- rings, necrotic spots and malformations on the fruits;- growth arrest and necrosis of whole parts or plants.

[0010] With particular reference to tomato plants, the first symptoms of Tospovirus infection are usually small orange spots on some central or lower leaves, or on sepals. Upon the appearance of new spots,the older leaves become brown, die and decay.

[0011] Similar spots or stripes appear on stems and stalks, the entire plant becomes dwarfed, and with its falling leaves it resembles a plant hit by wilting. Yellowish spots of up to 10 mm in diameter appear on green fruits, usually with distinct concentric areas of yellow or brown tones, alternating with green and subsequently with pink or red.

[0012] As known in the field of plant genetics, resistance is the ability of a plant variety to limit the growth and development of a specific pathogen capable of attacking this plant and the ability to reduce the damage that such pathogen can cause, compared to plant varieties susceptible under similar environmental conditions and pathogen pressure.

[0013] In order to try to limit the spread of Tospoviruses there are substantially two strategies: the first one focuses on the attempt to control the vector represented by thrips, and the second one is based on the increase in the genetic resistance of the plant species that can be infected.

[0014] However, limiting the spread of these viruses through vector control is hindered by the rapid development of resistance to insecticides within the thrips populations. Recently, biological alternatives to chemical control, such as the use of thrips predators or thrip-proof mosquito nets, are gaining importance. However, the non-optimal climatic conditions resulting from reduced ventilation in thrip-proof greenhouses are an important disadvantage for this approach and cannot be implemented in the open field.

[0015] The alternative strategy of increasing the genetic resistance of the plant has important advantages: in fact, it is always active, is independent from the technical means and from the cultivation techniques. On the other hand, the continuous appearance of new viral strains can make progress vain after a few years or decades. Therefore, the continuous search for new resistant plants is an unquestionable advantage for theagricultural sector. Although forms of genetic resistance have been introduced into various cultures, they have almost always been overcome by the rapid emergence of new strains of the virus that break this resistance. It is believed that some Tospoviruses naturally exist as a complex heterogeneous mixture of distinct isolates, which can exchange genetic information through the reassortment of genome segments. This provides a readily available reservoir of genetic information facilitating the adaptation of these viruses. For the reasons set out above, the control of Tospovirus epidemics is still an open challenge.

[0016] For example, the Sw5 Tomato Spotted Wilt Virus (TSWV) resistance gene has been widely used in commercial varieties for about 20 years, but strains of TSWV breaking the resistance have been isolated from Sw5-bearing tomato varieties initially in Spain, Italy and later in many areas of the world.

[0017] In addition to TSWV (Tomato Spotted Wilt Virus), in recent years the Groundnut RingSpot Virus (GRSV) and Tomato Chlorotic Spot Virus (TCSV) have become important and widespread, two Tospoviruses identified in 1993 in Brazil and Africa. In the last 10 years these viruses have been frequently found in many countries of South and Central America and in different areas of the USA. They are transmitted by 3 species of thrips called: Frankliniella occidentalis, F. schultzei and F. intonsa.

[0018] GRSV, TCSV and TSWV can jointly infect the same plant, but currently only among the first two viruses the ability to accomplish recombination has been observed. This phenomenon makes viruses potentially able to evolve faster and become more infectious.

[0019] Symptoms of GRSV or TCSV infection in pepper and tomato resemble those induced by TSWV, and include chlorotic and necrotic spots on the leaves, leaf rolling and general plant growth arrest. The fruits of the infected plants are generally discoloured, often deformedand with chlorotic or necrotic rings. Such fruits are not marketable.

[0020] The Groundnut RingSpot Virus (GRSV) was recognized as a distinct species of Tospovirus based on serology (serogroup II) and phylogenesis of the nucleoprotein gene sequence of one isolate from peanuts in South Africa. In addition to numerous wild species, such virus is capable of infecting many cultivated plants such as tomato, pepper, eggplant, tomatillo, peanut, coriander and lettuce.

[0021] The Tomato Chlorotic Spot Virus (TCSV), another Tospovirus of serogroup (II), was identified as distinct from the GRSV based on the difference in nucleoprotein gene sequence of one isolate from tomato in Brazil. The most affected plants are tomato, pepper, lettuce and stramonium.

[0022] Various resistance genes have been identified and designated Swal, Swbl, Sw2, Sw3 e Sw4, Sw-5b, Sw-5b2, Sw6, Sw7 against some Tospoviruses. However, in many cases these genes have proved ineffective in stopping the spread of outbreaks generated by GRSV and TCSV in various plant species.

[0023] US 2002 / 062504 Al discloses a plant nucleic acid which confers resistance to Tospoviruses and the generation of plant resistant to Tospoviruses expressing said nucleic acid. The protein Sw-5 (SEQ ID NO: 2) encoded by such sequence is resistant to GRSV and TCSV.

[0024] WO 2023 / 165711 Al discloses Solatium lycopersicum plants resistant to Tospovirus comprising a Tomato Spotted Wilt Virus (TSWV) resistance gene. A plant comprising a Sw-5 resistance gene that encodes for the Sw-5 protein is described. The tomato Sw-5 gene is known to provide a dominant resistance to TSWV, GRSV e TCSV.

[0025] Kraide Heron Delgado et al: "Pre-infection of tomato plants carrying the Sw-5 gene with tomato chlorosis virus did not alter infection with groundnut ringspot virus in the field", PLANT PATHOLOGY, vol. 72, no. 4, 5 January 2023 (2023-01-05), pages 742-751, discloses studieson the pre-infection of tomato plants carrying the Sw-5 gene with tomato chlorosis virus. For these studies, plants comprising Sw-5 have been infected with, inter alia, GRSV and TCSV. Tomato plants comprising the Sw-5 gene (Viradoro) are resistant to infection with GRSV and TCSV.

[0026] Leastro Mikhail Oliveira et al: "The NSm proteins of phylogenetically related tospoviruses trigger Sw-5b-mediated resistance dissociated of their cell-to-cell movement function", VIRUS RESEARCH, Amsterdam, NL, vol. 240, 25 July 2017 (2017-07-25), pages 25-34, discloses NSm proteins of tospoviruses that trigger Sw-5- mediated resistance. The document states that Sw-5b has been shown to confer resistance against, inter alia, GRSV and TCSV.

[0027] Athos S. De Oliveira et al: "The Sw-5 Gene Cluster: Tomato Breeding and Research Toward Orthotospovirus Disease Control", FRONTIERS IN PLANT SCIENCE, vol. 9, 19 July 2018 (2018- 07-19), discloses the Sw-5 gene cluster and its implication in the disease control of orthotospoviruses. Said gene provides high levels of resistance to TSWV, TCSV and GRSV.

[0028] In the light of the above considerations, there is the need to develop plants belonging to the Solanaceae family, which satisfy the cultivation and marketability requirements of their berries, and which are endowed with resistance to Tospovirus-derived infections, in particular due to the viral strains "Groundnut RingSpot Virus" (GRSV) and "Tomato Chlorotic Spot Virus" (TCSV).

[0029] The Applicant has found that the above technical problem can be solved by the presence in the genome of Solanaceae plants of a specific genetic determinant, which has proved particularly useful for increasing the resistance of plants to the infections derived from Tospoviruses, in particular, the viral strains GRSV and TCSV.

[0030] In order to overcome the above-mentioned drawbacks of the prior art and to achieve these and further objects and advantages, theApplicant has devised, tested and embodied the present invention.SUMMARY OF THE INVENTION

[0031] The present invention is set forth and characterized in the independent claims, while the dependent claims describe other preferred and non-essential features of the present invention, or variants of the main solution idea.

[0032] A first object of the present invention relates to a genetic determinant TS-1, whose presence in the genome of Solanaceae plants confers resistance on some strains of Tospovirus, such as Groundnut RingSpot Virus (GRSV) and Tomato Chlorotic Spot Virus (TCSV).

[0033] The genetic determinant TS-1 according to the invention is capable of coding for the amino acid sequence SEQ ID No.2 described below.

[0034] The genetic determinant TS-1 is a nuclear gene identified with the nucleotide sequence SEQ ID No.1 and its synonym sequences, which will be explained in the detailed description of the present invention. The genetic determinant TS-1 is provided with the start and end codons, is expressed and does not have introns.

[0035] The expression of the genetic determinant TS-1 is constitutive since it is present in the transcriptome, both before and after a possible Tospovirus infection.

[0036] Solanaceae plants are a family of dicotyledonous angiosperms of great importance for man, since they include some species used throughout the world as vegetables. Important plants for human nutrition such as tomatoes, potatoes, eggplants and peppers are part of this family. Tobacco, Goji berries and belladonna are also part of this family of plants.

[0037] For the sake of clarity, some terms used in the present description are defined as follows.Genotype: The set of all genes forming the DNA of an organism.Each gene, individually and / or cooperatively, contributes differently to the development, physiology and functional maintenance of an organism.Phenotype: A combination of all the characteristics manifested by a living organism, therefore its morphology, its development, its biochemical and physiological properties.Pure-line: The pure-lines are organisms whose sexual reproduction gives the same result as that of asexual reproduction; practically they are individuals who retain the same traits from one generation to the next. In an autogamous species such as tomato, a pure- line is obtained by repeated autogamy starting from a plant with a high degree of heterozygosity.Parental line: Defines a plant or group of plants used in the realization of a hybrid. Usually, in tomato, pure-lines are used as parental lines.Hybrid: Plant resulting from the crossing of two plants, called parental lines.

[0038] As said, the presence of the genetic determinant TS-1 of the present invention in the genome of Solanaceae plants makes this family of plants resistant to the viral strains Groundnut RingSpot Virus (GRSV) and Tomato Chlorotic Spot Virus (TCSV).

[0039] The presence of the genetic determinant TS-1 in the plant genome can be carried out by means of a non-essentially biological process, in the sense that the production of plants resistant to the viral strains GRSV and TCSV can also be carried out through methods that do not provide exclusively for the crossing of the plants and the selection. The presence of the genetic determinant TS-1 in a tomato genome can be obtained by mutagenesis induced by physical or chemical agents on tomato plants susceptible to Tospoviruses and subsequent plant selection by the PCR technique described in the present patent. Such plants will include the genetic determinant TS-1 in a stable manner, i.e. it will remainpresent throughout the life of the plant and will be transferred to the progeny either by autogamy, cross-pollination, or vegetative reproduction (e.g. by cutting), or cell culture. Therefore, the resistance traits are observable both in the original plants and in the progeny. The presence in the genome of the genetic determinant TS-1 can also be obtained through inserting external genetic elements such as plasmids, T-DNA or CRISPR- CAS modified to contain the sequence TS-1.

[0040] By virtue of the above, a second object of the present invention relates to Solanaceae plants comprising in their genome the genetic determinant TS-1 and obtained by a non-essentially biological process.

[0041] According to a preferred embodiment of the present invention, the Solanaceae plants comprising the genetic determinant TS-1 belong to the Solatium lycopersicum (tomato) subgroup. The experimentation of the advantages related to the present invention was carried out by developing the TS-1 genetic sequence in the tomato plant genome.

[0042] As will be apparent from the embodiments of the invention, tomato plants comprising the above-mentioned innovative genetic sequence have proved resistant to the Groundnut RingSpot Virus (GRSV) and the Tomato Chlorotic Spot Virus (TCSV) after being subjected to artificial infection.

[0043] In particular, the percentage of tomato plants with systemic infection was of the order of 7-8% for plants comprising in their genome the genetic determinant TS-1, whereas for tomato plants without this genetic sequence the systemic infection was of the order of 93-95% after artificial inoculation. This result refers to plants homozygous for the TS-1 sequence and phenotypically stable (pure-lines).

[0044] The present description also provides a method for evaluating the presence of the genetic determinant TS-1 in DNA extractedfrom plant cells belonging to Solanaceae plants.

[0045] Therefore, a further object of the present invention is a method for identifying and / or selecting a Solanaceae plant or parts of Solanaceae plants comprising in their genome the genetic determinant TS- 1, said method comprising:- extracting a DNA sample from cells or tissues of a Solanaceae plant using the cationic detergent CTAB (cetyltrimethylammonium bromide) as buffer;- subjecting said DNA sample to a polymerase chain reaction (PCR) by using primers Pom FW 1.1 and Pom RW 1 to obtain amplification of a specific DNA fragment;- identifying said genetic determinant TS-1 by agarose gel electrophoresis.

[0046] Further specific aspects and advantages of Solanaceae plants having the innovative features of the present invention will be more clearly highlighted in the following detailed description of the invention. FIGURES

[0047] Figure 1 shows the genetic determinant TS-1 nucleotide sequence (SEQ ID No.l) and amino acid sequence (SEQ ID No.2), the nucleotide sequence of the PCR amplification product obtained from the primers Pom Fwl. l and Pom RWL (SEQ ID No.3), the nucleotide sequence of primer Pom Fwl. l (SEQ ID No.4) and the nucleotide sequence of primer Pom Rwl (SEQ ID No.5).DETAILED DESCRIPTION OF THE INVENTION

[0048] As previously stated, the presence in the genome of Solenaceae plants of the genetic determinant TS-1 confers improved resistance on some strains of Tospoviruses, such as Groundnut RingSpot Virus (GRSV) and Tomato Chlorotic Spot Virus (TCSV).

[0049] In order to identify this specific genetic determinant, a sequencing of the entire genome of a tomato pure-line resistant to the twoabove-mentioned viruses was carried out. Sequencing has shown the presence of new, or non-deposited sequences in GenBank: such resistant plants do not have the sequences already known to confer resistance to Tospovirus, sequences such as Sw-5b, Sw-5b2 mentioned in the prior art.

[0050] Sequencing of the transcriptome of the resistant tomato plants was also carried out, showing that among the new sequences there are 3 sequences which are expressed. Among these, a single sequence, called TS-1, was statistically present in resistant tomato plants and absent in tomato plants susceptible to infections caused by GRSV or TCSV.

[0051] The genetic determinant TS-1 thus identified, results to be a nuclear gene of 3738 nucleotides, is provided with the start and end codons, is expressed and is not provided with introns.

[0052] The genetic determinant TS-1 is a nuclear gene of the plant identified with a nucleotide sequence of DNA SEQ ID No. l. The nucleotide sequence SEQ ID No. l of nucleotides is reported below: atggctgaaaatgaaattgaggaaatgttagagcacctgagaaggatcaagagtggaggtgatctggattggt tcgacatattgcgaattaaggaacttgaaatggtgctaagagtttttagaacctttacaaagtatcatgatgttct tttgcctgattccttagtcgaactcacaaagagggccaaattgattggggaaatacttcaccgggtgttgggtag tattccacataaatgtaaaactaaccttaatctggaaaggctagaatcacatttgttggaattctttcaaggtaa tacggcaagtttaagtcacaattatgagttgaatgattttgatctgtcgaaatatatggattgtctggaaaatttt ctaaatgatttactgatgatgttcttgcaaaagggtaatttcatccattccagagaacaacttgcaaaacatcga tcaataaaggaactgaaaattgttcaaaagaaaataagatttttgaaatacatatatgccacagagataaatg gttatgtcgactatgagaagctggaatgtttggagactcgaattcagttcatgactaacactgtgggacaatatt gtttggccgtattagattatgtcactgagggtaaacttaatgaagaaaatgacaactttagtaaacttccttacc tattatcattgattgtgttagtggagctggaaatgaagaagatttttcatggtgaagtaaaggcttcaaagttta ctcaatcaaaaactttcaaggacaagaaattaccaaaaggattttcacatcatctccacaatctgttgatgtatc tcagaaacaaaaagctcgagaattttcctaataatatcgctgctcaaaatattgatgtggcaatagagttcttgt tggttttccttgatgctgatgtgtcaaatcatgttattaatggtaactggttgaaagaggtcttgttaaaggttgga gctatagcgggtgatattctatatgtaattcaaaagcttcttcctagatcaataaacaaagatgaaactagcaa cgtaagtctttgctcaatacagatattggagaagactaaagatctgaaggcacaagttgagacgtactacaaatccttaaaatttactccatctcagttccccacctttggtggattgagctttctggattctcttttaaggaaactgaat gagatgtcgacatctaagtccggattaggtttcctgatgaaacctcttttagggaatttggagaaagagctatca tctcttacatccattttagagaaggagctctcatccattttccgtgatgtcgtgcaccatgaacatagcattccta aagatcttcagagacgtaccatcaatttggcatatgaggctgaggttgctattgattctattcttgctcagtataa tgtttttttgcatattttttgctcacttcctacaattctaaaagagatcaagcaaattaatgcagaggtgactgag atgtggtcagcggatattcctcttaatcctcactatgtggctgctccattaaaacatctgctggatcgacatagca atcttgtaactgatgaggaggtagtgggttttgagaataaagcagaagaactaattgattatctgattagaggt acaaatgagctagacgttgtcccaattgtaggcatgggaggacaagggaaaacgacaattgctagaaagttg tacaataatgacattattgtttctcgctttgatgttcgagcatggtgcatcatttctcaaacgtataatcggagag agttattacaagatatttttagtcaagttacaggtgtcaacgacaatggagctacggttgacgatgttgccgaca tgttgaggagaaaattaatgggaaagagatatctcattgtattggatgatatgtgggattgtatggtatgggat gacttaaggctttcctttccagattccggaaatagaagcagaatagtcgtaacaactcgacttgaagaagtggg taagcaagtcaagtaccatactgatccttattctcttccattcctcacaacagaagagagttgccaattgttgca gaaaaaagtgtttcaaaaggacgattgcccgcctgaactacaagatgtgagtcaagcagtagcagaaaaatg caaaggactgcccctagtggttgtcttggtagctggaataatcaaaaaaaggaaaatggaagaatcttggtgg aatgaggtgaaagatgctttatttgactatattgacagtgagttcgaagaatatagtctggcgactatgcagttg agttttgataacttaccccactgtttaaagccttgtcttctttatatgggaatgttttcggaggacgcaagaattcc agcatctacattgataagtttatggattgctgaaggattcgtggagaacactgaatctgggatattaatggaag aggcagctgaaggttacttgatggatctcattagcagtaacttggtaatgctttcaaagagaagttataagggt aaagtcaaatactgtcaggttcatgatgttgtgcatcacttttgcttggaaaagagtagagaagcaaagtttatg cttgcagtgaagggtcaatgtatccagtttcaaccttcggattggaagggtactcgagtgagcttcagttttagt gaagagctttccaagtttgcatctctggtctccataacacagaagcctttccatcaacacttgaggtcattgata acgacaggtcgagaaaaatatattgatgtgattctcttctgtcagattagtgaattgcgacttctcaaagtcttgg atttgagttcttataatgtggaatttttgtcgttagctacattcaaaccactaaatcagctgaagtacctcgcagtt caggctgataaattctattttgatccaggatcacatcttccccatatagaaactttcattgtaaagaattttcctta taatatagggttaccagtgtctttttgggaaatgaaaaaattaaggcatgctcattttggtaaggctgaatttgat aagcaggggctctttgaaggatcctctaaattggaaaatttgaggatattaaagaatattattggatttgatagg gtggatgtgttatcaaggaggtgtcctaatcttcaacaacttaaaatcagatattttggaaataatgaagagcct ttttgtcccaaattggagaatcttacccagcttcaacaacttcgactttcctctgtgagtccccacactctatccgg gttacagttaccttcaaatttaaataagttggcacttgtaggaattcatataggaagtgttattcccttcattgcgggactaccaagcctggaatatctccaattacaggatgtgtgttttcctcaatcagaagagtggtgccttggagat attacgttccataaacttaagttgttgaaactggtgaagttaaatatatcaaggtgggatgcctcagaggaatc atttccgttgcttgaaacactcgttataaagaagtgcggtgacctagaggagattccacttagctttgctgatatt ccaacattggaacaaattaaattgattgggtcctggaaagtatctctggaggattcagctgtgagaatgaagga agaaatcaaagagactgaaggatgtgatcgtttacacctcgtcaaacaacgctcagattga

[0053] It is important to point out that for the purposes of the protection conferred by the present patent application the term "genetic determinant TS-1" includes not only the specific DNA sequence SEQ ID No.1 above, but also all the possible synonym sequences of SEQ ID No.1, which encode the amino acid sequence SEQ ID No.2. To better understand the meaning of synonym sequences in molecular biology, the following explanation is provided.

[0054] The translation of a nucleotide sequence of the mRNA into the amino acid sequence of the protein is based on the pairing of the complementary bases between a triplet (codon) of the mRNA and the corresponding complementary triplet (anticodon) of the tRNA carrying a specific amino acid. For each of the three positions in a codon there are four possible nucleotide bases. The four nucleotides present in the DNA molecule, in groups of three, give rise to 64 different codons. Of these, three codons (UAG, UAA and UGA in RNA) do not encode any amino acids but signal the end of the genetic message and therefore stop codons or non-sense codons are defined. Thus, 61 codons specifying 20 amino acids (sense codons) remain: therefore, most amino acids correspond to more than one codon. This is known as degeneration or redundancy of the genetic code.

[0055] The degeneration of the genetic code implies that a single tRNA can correspond by base pairing to more than one codon and that each of the amino acids must correspond to more than one tRNA. In fact, both situations occur: for some amino acids there really exists more than one tRNA molecule, moreover the constitution of the anticodon of some tRNAmolecules is such to require the accurate pairing of the bases only in the first two positions of the codon, while a discrepancy in the third base is tolerated. Such a phenomenon, whereby a tRNA can recognize multiple codons, is called wobbling of the genetic code. The pairing between codon and anti-codon is regular as far as A-U and G-C are concerned for the bases in the first two positions of the codon, but admits the pairing G-U in the third position. In general, when the base at the third codon position is a pyrimidine (U or C), the same anticodon may pair with two alternative codons. This “uncertain” pairing explains why many of the alternative codons for the same amino acid differ only in the third nucleotide of the triplet.

[0056] Mutations occurring on the encoding DNA may be “synonym” or silent, when they do not modify the gene product sequence (protein), or “non-synonym” if they alter the gene product sequence. A synonym mutation results in a codon change, but not an amino acid change, due to the degeneration of the genetic code. Two DNA sequences are called synonym when coding for the same amino acid sequence.

[0057] From the above description it is clear that both the above DNA sequence SEQ ID No. l and all the relevant synonym sequences are capable of coding for the amino acid sequence SEQ ID No.2.

[0058] The amino acid sequence SEQ ID No.2 consists of 1245 amino acids and is reported below: MAENEIEEMLEHLRRIKSGGDLDWFDILRIKELEMVLRVFRTFTKYHDVLLPDSLVELTKRAKLIGEILHRVLGSIPHKCKTNLNLERLESHLLEFFQGNTASLSHNYELNDFD LSKYM DCLENFLNDLLMM FLQKGNFIHSREQLAKHRSIKELKIVQKKIRFLKYIYAT EINGYVDYEKLECLETRIQFMTNTVGQYCLAVLDYVTEGKLNEENDNFSKLPYLLSL IVLVELEMKKIFHGEVKASKFTQSKTFKDKKLPKGFSHHLHNLLMYLRNKKLENFP NNIAAQNIDVAIEFLLVFLDADVSNHVINGNWLKEVLLKVGAIAGDILYVIQKLLPR SINKDETSNVSLCSIQILEKTKDLKAQVETYYKSLKFTPSQFPTFGGLSFLDSLLRKLN EMSTSKSGLGFLM KPLLGNLEKELSSLTSILEKELSSIFRDVVHHEHSIPKDLQRRTINLAYEAEVAIDSILAQYNVFLHIFCSLPTILKEIKQINAEVTEMWSADIPLNPHYVAAP LKHLLDRHSNLVTDEEVVGFENKAEELIDYLIRGTNELDVVPIVGMGGQGKTTIAR KLYNNDIIVSRFDVRAWCIISQTYNRRELLQDIFSQVTGVNDNGATVDDVADM LR RKLMGKRYLIVLDDMWDCMVWDDLRLSFPDSGNRSRIVVTTRLEEVGKQVKYH TDPYSLPFLTTEESCQLLQKKVFQKDDCPPELQDVSQAVAEKCKGLPLVVVLVAGII KKRKM EESWWNEVKDALFDYIDSEFEEYSLATMQLSFDNLPHCLKPCLLYMGMF SEDARIPASTLISLWIAEGFVENTESGILMEEAAEGYLM DLISSNLVM LSKRSYKGK VKYCQVHDVVHHFCLEKSREAKFM LAVKGQCIQFQPSDWKGTRVSFSFSEELSKF ASLVSITQKPFHQHLRSLITTGREKYIDVILFCQISELRLLKVLDLSSYNVEFLSLATFK PLNQLKYLAVQADKFYFDPGSHLPHIETFIVKNFPYNIGLPVSFWEMKKLRHAHFG KAEFDKQGLFEGSSKLENLRILKNIIGFDRVDVLSRRCPNLQQLKIRYFGNNEEPFC PKLENLTQLQQLRLSSVSPHTLSGLQLPSNLNKLALVGIHIGSVIPFIAGLPSLEYLQL QDVCFPQSEEWCLGDITFHKLKLLKLVKLNISRWDASEESFPLLETLVIKKCGDLEEI PLSFADIPTLEQIKLIGSWKVSLEDSAVRMKEEIKETEGCDRLHLVKQRSD

[0059] As stated above, a further object of the invention consists of a method for identifying and / or selecting a plant or part of a Solanaceae plant comprising in its genome the genetic determinant TS-1.

[0060] This is a method based on Polymerase Chain Reaction (PCR) technology, which provides for DNA extraction from plant tissues, organs and cells. The polymerase chain reaction is a molecular biology technique that allows multiplication (amplification) of nucleic acid fragments of which the initial and terminal nucleotide sequences are known. The PCR multiplication allows to obtain in vitro very rapidly the amount of genetic material necessary for the subsequent applications. The DNA can be extracted using the cationic detergent CTAB (cetyltrimethylammonium bromide) as a buffer.

[0061] The main organ for DNA extraction is the leaves of the plant but also stems, cotyledons, fruits, seeds, roots or parts thereof and cell cultures can be used. The sequence TS-1 is identified by the use of the primers Pom FWl. l and Pom RWl reported below in Table A.

[0062] A primer is a short nucleic acid sequence, used to initiate DNA replication: the primers used in polymerase techniques are commonly DNA fragments of 18-20 bases length. These primers are constructed in the laboratory, through a chemical synthesis.

[0063] PCR amplification conditions are those reported in TableB; Promega GoTaq polymerase was used on a volume of 20 total microliters. Small deviations in such parameters are acceptable and depend on the quality of the DNA and the use of different polymerases.Table ATable B

[0064] The PCR amplification product is a 509 bp DNA fragment, the sequence of which is given below. Such fragment can be visualized on 2% agarose gel and Gelred staining, or alternative methods such as HRM, fluorescence, etc. can be used. This fragment is present only in plantscontaining the sequence TS-1.

[0065] The sequence of nucleotide bases (SEQ ID No.3) of the PCR amplification product obtained from the primers Pom Fwl. l and Pom RWL is reported below: tcaccgggtgttgggtagtattccacataaatgtaaaactaaccttaatctggaaaggctagaatcacatttgttggaattctttcaa ggtaatacggcaagtttaagtcacaattatgagttgaatgattttgatctgtcgaaatatatggattgtctggaaaattttctaaatg atttactgatgatgttcttgcaaaagggtaatttcatccattccagagaacaacttgcaaaacatcgatcaataaaggaactgaaa attgttcaaaagaaaataagatttttgaaatacatatatgccacagagataaatggttatgtcgactatgagaagctggaatgttt ggagactcgaattcagttcatgactaacactgtgggacaatattgtttggccgtattagattatgtcactgagggtaaacttaatga agaaaatgacaactttagtaaacttccttacctattatcattgattgtgttagtggagctggaaatgaagaaga

[0066] Further advantages and characteristics of the present invention will be apparent from the following embodiments, which are to be understood in a descriptive and non-limiting sense of the scope of the appended claims.EXAMPLES

[0067] The following examples relate to experimental tests to demonstrate the effectiveness of the presence of the genetic determinant TS-1 in increasing the resistance to the GRSV and TCSV Tospoviruses in tomato plants.Conditions for the growth of tomato plants

[0068] Tomato plants are grown in a mixture of peat and vermiculite (5: 1 ratio) in a growth chamber up to the first leaf stages (20 days after sowing) at 25-20°C with 70% humidity, light / dark cycle 16 / 8 hours.

[0069] For each genotype 12 plants are inoculated for each repetition. In total at least 3 repetitions are carried out. To control the quality of inoculation and screening, six sensitive controls are included every 90 plants. Three genotype plants are used as mock, i.e. plants treated with a virus-free solution, and are a reference in assessing normal plantdevelopment in the absence of infection.

[0070] One way to obtain tomato plants containing the genetic determinant TS-1 is the treatment with ethyl methanesulfonate (EMS): in detail, tomato seeds susceptible to the GRSV and TCSV viruses are immersed in an aqueous solution containing EMS; the seeds are then germinated. A leaf sample is taken from the seedlings and the DNA is extracted from it and processed by the PCR method reported. Only plants that have the mutations indicated in the TS-1 sequence are manually selfpollinated in a protected environment to avoid cross pollinations. From these fruits the seed (called M2) is obtained. The plants germinating from the M2 seed are analysed by the PCR technique reported. Only the plants homozygous for the genetic determinant TS-1 are manually self-pollinated in a protected environment. The seed obtained from these plants and all subsequent progeny are stably provided with the TS-1 gene. These plants are subjected to artificial infection with the GRSV and TCSV viruses to verify their acquired resistance.Preparation of the Groundnut RingSpot Virus (GRSV) inoculum

[0071] The preparation of the GRSV inoculum is carried out starting from tomato plants infected with GRSV. Symptomatic leaves are ground with mortar and pestle in the presence of a solution called "phosphate buffer" to which 0.1% of sodium sulphite is added. The "phosphate buffer" is prepared by combining 20.214 grams of disodium phosphate heptahydrate and 3.394 grams of sodium dihydrogen phosphate monohydrate, bringing everything to the volume of 1 litre with distilled water.

[0072] Silicon carbide (400 mesh) is added to this inoculum to perform the mechanical infection of the first two true leaves of the plant. The leaves are then gently washed to remove the inoculum. After two days, infection on the same leaves is repeated to avoid false negatives.

[0073] The plants are kept in a controlled environment for 4 weeksprior to evaluation of the degree of resistance or susceptibility.

[0074] The upper leaves, which had not been infected in the initial phase, are tested with the ELISA method to verify the presence of the virus and confirm that the visible symptoms of susceptibility are due to the specific virus, that has therefore multiplied and spread in the plant from the point of infection: this phenomenon is indicated as systemic infection. Preparation of Tomato Chlorotic Spot Virus (TCSV) inoculum

[0075] The preparation of the TCSV inoculum is carried out starting from tomato plants infected with TCSV. Symptomatic leaves are ground with mortar and pestle in the presence of the cold "phosphate buffer" solution to which 0.1 % of sodium sulphite is added.

[0076] Silicon carbide (400 mesh) is added to this inoculum to perform the mechanical infection of the first two true leaves of the plant. The leaves are then gently washed to remove the inoculum. After 2 days, infection on the same leaves is repeated to avoid false negatives.

[0077] The plants are raised in a controlled environment for 4 weeks prior to evaluation of the degree of resistance or susceptibility.

[0078] The upper leaves, which had not been infected in the initial phase, are tested with the ELISA method to check for the presence of the virus and confirm that the visible symptoms of susceptibility are due to the virus, which then multiplied and spread in the plant from the point of infection.Definition of the degree of resistance

[0079] By the term "RESISTANCE" is conventionally meant the ability of a plant to limit the growth and development of a specific pathogen and to reduce the damage that such pathogen can cause to the plant.

[0080] Following the artificial infection of tomato seedlings with an aqueous solution containing the GRSV or TCSV viruses, symptoms variable as a function of the resistance of the plant can be noted. Suchsymptoms can be classified into five levels of plant strength based on their increasing severity: maximum resistance, good resistance, light susceptibility, average susceptibility and maximum susceptibility. The definitions of these resistance levels are given below:Maximum resistance: Well-developed plants, which have not been affected by viral infection, leaves and stems appear healthy and free of symptoms.Good strength: Plants with healthy leaves and stems but having a reduced development, organs appear intact and a smaller size only can be observed when compared to plants with maximum strength.Light susceptibility: Plants with even smaller size than those with good resistance. There are also slight deformations of the leaves or of the stem.Medium susceptibility: Plants with evident stem deformations and presence of very large leaf mosaic. Such symptoms are also present in the artificially uninoculated leaves.Maximum susceptibility: Plants with systemic necrosis and completely compromised in their development. The symptoms are also present in the initially uninoculated leaves.

[0081] The values reported in the subsequent tables relating to Examples 1-7 may be subject to slight variations depending on the growth conditions of the seedlings and the degree of virulence of the virus. Such variations do not alter the significance of the observed results.Example 1 (comparative)

[0082] Tomato plants, the cultivation of which is carried out in the manner indicated above and whose genome does not comprise the genetic determinant TS-1, are subjected to artificial infection by inoculation of GRSV Tospovirus.

[0083] A sample containing a number of tomato plants equal to 48 is tested. The percentage of plants with systemic infection is 95% and theresults relating to the resistance of the plants are shown in Table 1.TABLE 1Example 2

[0084] Tomato plants, cultivated in the manner indicated above and the genome of which comprises the genetic determinant TS-1 according to the present invention, are subjected to artificial infection by means of inoculation of GRSV Tospovirus.

[0085] A sample containing a number of tomato plants equal to 48 is tested. The percentage of plants with systemic infection is 8%.

[0086] The results relating to the resistance of the plants are indicated in Table 2, which refers to homozygous plants for TS-1 and phenotypically stable (pure-lines).TABLE 2Example 3 (comparative)

[0087] Tomato plants, the cultivation of which is carried out in the manner indicated above and whose genome does not comprise the genetic determinant TS-1, are subjected to artificial infection by the inoculation of TCSV Tospovirus.

[0088] A sample containing a number of tomato plants equal to 48 is tested. The percentage of plants with systemic infection is 93% and the results relating to the resistance of the plants are shown in Table 3.TABLE 3Example 4

[0089] Tomato plants, cultivated in the manner indicated above and the genome of which comprises the genetic determinant TS-1 according to the present invention, are subjected to artificial infection with TCSV Tospovirus.

[0090] A sample containing a number of tomato plants equal to 48 is tested. The percentage of plants with systemic infection is 7%.

[0091] The results relating to the resistance of the plants are indicated in Table 4, which refers to homozygous plants for TS-1 and phenotypically stable (pure-lines).TABLE 4Example 5

[0092] This example refers to hybrid plants obtained by crossing pure-lines of tomato plants containing the genetic determinant TS-1 and lines also lacking the genetic determinant TS-1 Such plants are subjected to artificial infection by inoculation of GRSV Tospovirus.

[0093] A sample containing a number of tomato plants equal to 48 is tested. The degree of systemic infection is 16%. The resistance results of these hybrid plants are shown in Table 5.TABLE 5Example 6

[0094] This example refers to hybrid plants obtained by crossing pure-lines of tomato plants containing the genetic determinant TS-1 andpure-lines lacking the genetic determinant TS-1. Such plants are subjected to artificial infection by TCSV Tospovirus inoculation.

[0095] A sample containing a number of tomato plants equal to 48 is tested. The degree of systemic infection is 14%. The resistance results of these hybrid plants are shown in Table 6.TABLE 6Example 7

[0096] In order to verify the correlation between genetic determinant TS-1 and the resistance phenotype to GRSV, the Applicant has recourse to the autogamy of hybrid tomato plants obtained by crossing pure-lines of plants containing the genetic determinant TS-1 and lines also lacking the genetic determinant TS-1.

[0097] Autogamy generates a population of segregating tomato plants (called F2) for the genetic determinant TS-1. In this population, a reassortment of the chromosomes present in the initial pure-lines is observed, so that each plant has a different genetic combination.

[0098] The F2 population was artificially infected by GRSV inoculation, showing that there is a net segregation of resistance in plants. In fact, most plants were in the two most extreme classes: maximum resistance or maximum susceptibility. The results relating to the strength of these F2 population plants are shown in Table 7. This behaviour isconsistent with the presence of a single genetic determinant.TABLE 7

[0099] Sequencing of the genome and transcriptome of plants having maximum resistance was performed, showing that there were some new sequences in the genome since they were not present in the DNA database Genebank, and among them, 3 sequences were expressed.

[0100] By analysing the presence of these 3 sequences in the plants of the F2 population with maximum resistance and in plants with maximum susceptibility, it was observed that only the genetic sequence called TS-1 according to the present invention is statistically present in the resistant tomato plants and absent in the susceptible tomato plants. The significance of the result is demonstrated by the statistical chi-square test.Example 8

[0101] IPSP (Istituzione per la Protezione Sostenibile delle Piante - Institution for the Sustainable Protection of Plants) of CNR (Consiglio Nazionale delle Ricerche - National Research Council) headquartered in Turin performed tests on an artificial infection with GRSV virus (strain VE295), following the above-described methodology (Conditions for the growth of tomato plants and Preparation of inoculum). Homozygous lines were evaluated for the following genetic determinants: TS-1 and Sw5b. For the genetic determinant Sw5b plants having the resistant allele or thesusceptible allele were evaluated, in the following indicated with ‘ Sw5b resistant line’ and ‘Sw5b susceptible line’, respectively. These lines are not provided with the genetic determinant TS-1.

[0102] With “TS-1 line” the line containing the resistant allele of the genetic determinant TS-1 is meant.

[0103] The test classified the plants in ‘infected plants’ and ‘noninfected plants’ according to visible symptoms and ELISA tests. Table 8 contains the results of these tests.

[0104] The test shows outcomes different according to the analysed genotype. The plants of Sw5b susceptible line are completely infected, the plants of the Sw5b resistant line are infected with an infection rate of 67%, while the plants of the TS-1 line are completely non-infected, differing both from the Sw5b susceptible line and from Sw5b resistant line.TABLE 8Example 9

[0105] IPSP (Istituzione per la Protezione Sostenibile delle Piante - Institution for the Sustainable Protection of Plants) of CNR (Consiglio Nazionale delle Ricerche - National Research Council) headquartered in Turin performed tests on an artificial infection with TCSV virus (strain VE275), following the above-described methodology (Conditions for the growth of tomato plants and Preparation of inoculum). Homozygous lines were evaluated for the following genetic determinants: TS-1 and Sw5b.For the genetic determinant Sw5b plants having the resistant allele or the susceptible allele were evaluated, in the following indicated with ‘Sw5b resistant line’ and ‘Sw5b susceptible line’, respectively. These lines are not provided with the genetic determinant TS-1.

[0106] With “TS-lline” the line containing the resistant allele of the genetic determinant TS-1 is meant.

[0107] The test classified the plants in ‘infected plants’ and ‘noninfected plants’ according to visible symptoms and ELISA tests. Table 9 contains the results of these tests.

[0108] The test shows different outcomes for the different genotypes analysed. The Sw5b susceptible line is infected with a rate of 67%, An analogous behaviour was observed for the plants of the Sw5b resistant line, which are infected with an infection rate of 67%, too. On the contrary, the plants of the TS-1 line are completely non-infected, differing both from the Sw5b susceptible line and from Sw5b resistant line.TABLE 9

Claims

CLAIMS1. Genetic determinant TS-1 being present within the genome of Solanaceae plants to confer resistance to Groundnut RingSpot Virus (GRSV) and Tomato Chlorotic Spot Virus (TCSV) characterized by encoding for the amino acid sequence SEQ ID No.2.

2. Genetic determinant TS-1 according to claim 1, wherein said amino acid sequence SEQ ID No.2 comprises 1245 amino acids.

3. Genetic determinant TS-1 according to any one of the preceding claims, characterized in that it comprises the nucleotide sequence of DNA SEQ ID No. 1 and all the relative synonymous sequences translatable into said amino acid sequence SEQ ID No.2 comprising 1245 amino acids.

4. Genetic determinant TS-1 according to any one of the preceding claims, characterized in that said genetic determinant TS-1 has start and stop codons, is expressed and does not have introns.

5. Genetic determinant TS-1 according to any one of the preceding claims, characterized in that said nucleotide sequence of DNA SEQ ID No. 1 comprises 3738 nucleotides.

6. Solanaceae plants comprising the genetic determinant TS-1 according to claims 1-5 within their genome and obtained by a non-essentially biological process.

7. Solanaceae plants according to claim 6, wherein said Solanaceae plants belong to the subgroup Solanum lycopersicum (tomato).

8. A method for identifying and / or selecting a Solanaceae plant or parts of Solanaceae plants comprising in their genome the genetic determinant TS-1 according to claims 1-5, said method comprising:- extraction of a DNA sample from cells or tissues of a Solanaceae plant by using the cationic detergent CTAB(cetyltrimethylammonium bromide) as a buffer;- subjecting said DNA sample to a polymerase chain reaction (PCR) using the primers Pom Fwl. l SEQ ID No.4 and Pom Rwl SEQ ID No.5 to obtain amplification of specific DNA fragment;- identify said genetic determinant TS-1 by using agarose gel electrophoresis.

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