WO2023129057A2 - Multiplex pcr method for identifying the resistance gene tsw of tomato spotted wilt virus (tswv), and resistance 5 gene l4 of pepper mild mottle virus (pmmov) in pepper (capsicum annuum) - Google Patents

Multiplex pcr method for identifying the resistance gene tsw of tomato spotted wilt virus (tswv), and resistance 5 gene l4 of pepper mild mottle virus (pmmov) in pepper (capsicum annuum) Download PDF

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WO2023129057A2
WO2023129057A2 PCT/TR2022/051618 TR2022051618W WO2023129057A2 WO 2023129057 A2 WO2023129057 A2 WO 2023129057A2 TR 2022051618 W TR2022051618 W TR 2022051618W WO 2023129057 A2 WO2023129057 A2 WO 2023129057A2
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tswv
pepper
pmmov
resistance
resistant
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WO2023129057A3 (en
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Cansu BULBUL
Nedim MUTLU
Duran SIMSEK
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Akdeniz Universitesi
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

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  • the present invention relates to a multiplex PCR method that allows for determining resistance status against both diseases simultaneously by means of the fact that marker systems for two different diseases in pepper (Capsicum annuum) are used in a single polymerase chain reaction (PCR) amplification and by forming a single restriction digestion, for identifying Tsw, which is the Tomato Spotted Wilt Virus (TSWV) Resistance Gene, and L4, which is the Pepper Mild Mottle Virus (PMMoV) Resistance Gene.
  • Tsw Tomato Spotted Wilt Virus
  • PMMoV Pepper Mild Mottle Virus
  • TSWV Tumor Spotted Wilt Virus
  • PMMoV Pieris spp.
  • Molecular markers are DNA fragments associated with any gene region or gene region in the genome, and are markers that reveal the DNA sequence difference of different genotypes in various ways. Use of nucleic acid-based genetic markers in genome analysis, especially for breeders is of great importance in determining both cultivars and disease resistance thereof.
  • Tsw gene controlled by a single dominant gene located on chromosome 10 of pepper is known to confer genetic resistance against TSWV, which is included in the genus Tospovirus.
  • Seven virus species included in the genus Tobamovirus cause disease in Capsicum (pepper) species. These are called Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), Bell pepper mottle virus (BPeMV), Paprika mild mottle virus (PaMMV), Obuda pepper virus (ObPV), and Pepper mild mottle virus (PMMoV).
  • TMV Tobacco mosaic virus
  • ToMV Tomato mosaic virus
  • TGMV Tobacco mild green mosaic virus
  • BPeMV Bell pepper mottle virus
  • PaMMV Paprika mild mottle virus
  • ObPV Obuda pepper virus
  • PMMoV Pepper mild mottle virus
  • allelic status of the resistance can be determined in a reliable manner by means of CAPS (Cleavage Amplified Polymorphic Sequence) marker systems that produce codominant (Rr, RR, rr) information, which is one of the molecular methods based on PCR used to determine disease resistance in plants.
  • CAPS Cleavage Amplified Polymorphic Sequence
  • results obtained from restriction of the product amplified with specific primers of the SCAC568 CAPS marker, which is the CAPS molecular marker associated with the Tsw gene, with Xbal, Taql, or Haelll enzymes are used in the diagnosis of resistance to TSWV in pepper [1 ].
  • the newly designed primer pair amplifies a 568 bp band common to susceptible and resistant alleles, and produces codominant information (detecting homozygous resistant, homozygous susceptible, or heterozygous genotype) after restriction with Xbal, Taql, or Haelll restriction enzymes.
  • Xbal Xbal
  • Taql or Haelll restriction enzyme
  • the patent document numbered EP1804571 B1 in the prior art provides codominant information about L4 allele-dependent resistance against PMMoV- P1.2.3 pathotype as a result of restriction of the product amplified with the help of primers designed specifically for the region associated with the L locus on chromosome 11 in pepper, with the Trul l enzyme.
  • Tm3-DRS marker system developed from the TG36 marker, which they previously determined in another study to be 6 cM away from the L locus on the 11th chromosome of pepper is also used for this L locus.
  • the primer pair P118/P119 designed specifically for this region can amplify the L locus associated region common to resistant and susceptible genotypes, and produces a band approximately 350 bp long. Approximately 350 bp product L4, which is formed as a result of enzymatic restriction of the resulting PCR product with Trul l restriction enzyme.
  • Trul l restriction enzyme is used for the two CAPS marker systems (Tsw and L4) in the work of Moury et al. (2000) described above and the patent document numbered EP1804571 B1 , analyzes are also made by creating separate PCR reactions.
  • PCR reaction was established using DNA extracted from PMMoV-resistant (R) and PMMoV- susceptible (S) materials by primers of 516 randomly selected RAPD marker systems for determining PMMoV - L4 resistance by classical PCR method [2], Among the resistant and susceptible genotypes, only one of the 516 primers showed polymorphism, and produced bands at 1500 bp (base pairs) only for resistant genotypes. Based on this polymorphism, this RAPD marker was converted into the dominant 20 nucleotide SCAR (Sequence Characterized Amplified Region) marker system and became the AP-7/AP-8 primer pair.
  • SCAR Sequence Characterized Amplified Region
  • the AP-7 I AP-8 SCAR marker can distinguish between resistant and susceptible individuals, it is only capable of amplifying the 1500 bp band of the resistant genotype. As a result of PCR amplification, no bands are produced in the susceptible genotype. In other words, the marker here is a dominant SCAR marker.
  • Information is obtained about the presence/absence of the allele that provides resistance to the disease or only the presence/absence of the disease in the plant by means of the use of dominant markers found in the above-mentioned prior art documents used in disease testing. That is, unlike the CAPS marker system, which produces codominant information, in tests with the dominant marker, it is not possible to obtain a result about whether the allele that gives resistance to the plant is in the homozygous (RR) or heterozygous (Rr) state since the marker targets a single allele. From the point of view of plant breeding, this situation causes genotypic segregation in future generations due to failure to identify the susceptible allele (r) in a heterozygous (Rr) plant, not determined at an early stage.
  • the PCR product is enzymatically cleaved with the appropriate restriction enzyme after firstly amplified by region-specific (to include the SNP region between the resistant and susceptible allele) primers that are common to the susceptible and resistant allele.
  • region-specific to include the SNP region between the resistant and susceptible allele
  • the difference between resistant and susceptible alleles is interpreted according to whether the sequence of the restriction site is present in the amplified PCR product. Therefore, in testing two different loci with CAPS markers, in addition to the costs of the PCR device, PCR reaction components and restriction enzymes, the time and labor spent on analyzes are doubled.
  • CAPS markers of the state of the art are used to determine the resistance of plants to Tsw and L4, due to the reasons such as the need for two different reactions, primer and enzyme, to determine the allelic status (homozygous/heterozygous) of both Tsw and L4, requiring two separate processes, creating a disadvantage in terms of time, cost and labor, the fact that the markers are the dominant SCAR markers, that is, they have the ability to amplify only the band of the resistant genotype, it is necessitated to develop CAPS markers and methods using these markers, that offer a single reaction for the determination of the allelic status (homozygous/heterozygous) of both Tsw and L4, that do not require two separate PCR reactions, that enable the identification of both susceptible and resistant alleles for both loci, in order to determine the two different disease resistance status simultaneously.
  • a multiplex PCR method that allows for determining resistance status against both diseases simultaneously by means of the fact that marker systems for two different diseases in pepper (Capsicum annuum) are used in a single polymerase chain reaction (PCR) amplification and by forming a single restriction digestion, for identifying Tsw, which is the Tomato Spotted Wilt Virus (TSWV) Resistance Gene, and L4, which is the Pepper Mild Mottle Virus (PMMoV) Resistance Gene.
  • Tsw Tomato Spotted Wilt Virus
  • PMMoV Pepper Mild Mottle Virus
  • the first object of the present invention is to determine the status of Tsw and L4 genes, which determine resistance against two different diseases in pepper, and homozygous and heterozygous alleles, with a single reaction.
  • Existing codominant CAPS markers used to determine resistance to TSWV and PMMoV (P1 .2.3) in pepper are developed, combined, and made operable by the multiplex PCR method by means of the present invention.
  • a single restriction digestion is formed by using the marker systems developed for two different diseases in a single PCR amplification, and thus, analysis can be performed for two diseases simultaneously.
  • Another object of the present invention is to provide a method that is advantageous in terms of cost and labor compared to the prior art, that enables analysis in a short time and at low cost while both Tsw and L4 genes, which determine resistance to two different diseases in pepper, were analyzed together.
  • Tsw and L4 genes which determine resistance to two different diseases in pepper, were analyzed together.
  • TSWV Tomato Spotted Wilt Virus
  • PMMoV Pepper Mild Mottle Virus
  • Another object of the present invention is to provide the addition of a new alternative enzyme to the existing enzyme variety (Xbal, Taql or Haelll) and to optimize the reaction conditions, in relation to the SNPs in the target region of the primers used in the CAPS marker system, where resistance to TSWV is tested in pepper.
  • Tru11 (Msel) enzyme has been added to be used in the SCAC568 CAPS marker system as an alternative to Xbal, Taql, or Haelll enzymes.
  • CAPS markers and methods using these markers that offer a single reaction for the determination of the allelic status (homozygous/heterozygous) of both Tsw and L4, that do not require two separate PCR reactions, that enable the identification of both susceptible and resistant alleles for both loci, in order to determine the two different disease resistance status simultaneously.
  • Figure 1 Representative map of amplified regions for determination of TSWV (Tsw) and PMMoV (L4) resistances in C. annuum a) The region amplified by P118/P119 primer pair on the chromosome 11 of C. annuum - PMMoV (L4) Chromosome 11 P118/P119; b) The region amplified by SCAC568 F/R (forward/reverse) primer pair on C. annuum chromosome - 10 TSWV Chromosome 10 SCAC568 F-R.
  • Figure 2 Representative map of regions amplified with the SCAC568-F/R primer pair a) the target region of the TSWV-susceptible (rr) genotype amplified with the SCAC568 F/R primer pair; b) The target region of the TSWV-resistant (RR) genotype amplified with the SCAC568-F/R primer pair.
  • Figure 3 In silico restriction of susceptible and resistant PCR products amplified with primer pair SCAC568-F and SCAC-R' by Tru11 enzyme (susceptible allele - 209 bp and resistant allele - 141 bp (+68 bp), M: 100 bp DNA Marker, S: sample).
  • Figure 4 L1 -L3: Restriction of multiplex PCR products of Rr, RR and rr genotypes with Trul l enzyme, respectively; Restriction of PMMoV (L4) ⁇ P118/P119 PCR products of Rr, RR, and rr genotypes with Trul l enzyme, respectively; L7-L9: Restriction of TSWV-SCAC568-F/SCAC-R' PCR products of Rr, RR, and rr genotypes with Tru11 enzyme, respectively; L10-L12: Restriction of TSWV-SCAC568-F/R PCR products of Rr, RR, and rr genotypes, respectively, with Xbal enzyme (M: 100 bp DNA Marker).
  • Figure 5 A. S1 -S6: Restriction of multiplex PCR products of Efests F1 (Demre type), Erciyes Rene F1 (Demre type), Assia F1 (Demre type), Mustang F1 (California Wonder), Doganay F1 (Dolma type), Koray F1 (California Wonder) samples with Trul l enzyme, respectively; Restriction of B. PMMoV (L4)- P118/P119 PCR products with Tru11 enzyme; restriction of C. TSWV- SCAC568- F/SCAC-R’ PCR products with Trul l enzyme; Restriction of TSWV-SCAC568- F/R PCR products with Xbal enzyme (M: 100 bp DNA Marker).
  • Figure 6 A1-20: F2 progenitors from inbreeding of Abraham F1 (California Wonder), C: TSWV- PMMoV (L4) heterozygous (Rr) control and K1 - K21 : Restriction of TSWV-PMMoV (L4) multiplex PCR products obtained with F2 progenitors obtained from inbreeding of Kioto F1 (California Wonder) with Trul enzyme (M: 100 bp DNA Marker).
  • the present invention relates to a multiplex PCR method that allows for determining resistance status against both diseases simultaneously by means of the fact that marker systems for two different diseases in pepper (Capsicum annuum) are used in a single polymerase chain reaction (PCR) amplification and by forming a single restriction digestion, for identifying Tsw, which is the Tomato Spotted Wilt Virus (TSWV) Resistance Gene, and L4, which is the Pepper Mild Mottle Virus (PMMoV) Resistance Gene.
  • Tsw Tomato Spotted Wilt Virus
  • PMMoV Pepper Mild Mottle Virus
  • annuum Pepper Zunla 1 species can be used as the pepper mentioned in the present invention without any geographical restriction.
  • Position of 11th chromosome for Capsicum annuum Pepper Zunla 1 preferably used in the present invention 1227700-1228034 positive thread NCBI accession number Ref_v1.0, NC_029987.1 ; Capsicum annuum Pepper Zunla 1 10th chromosome Position: The NCBI accession number for 204196455-204197020 positive thread is Ref_v1.0, NC_029986.1 The Capsicum annuum pepper zunla 1 sequences used were retrieved from the NCBI database. There is no geographical region expression in the database.
  • gene resources of Areo Seeds company were used for sequencing of TSWV-resistant and susceptible samples as genetic resources. Breeding materials with the code BWS60-2 (California Wonder) for TSWV-resistant; and BWS60-4 (California Wonder) for TSWV- susceptible were used, in order to control the designed multiplex system, commercial F1 varieties of different seed companies were used.
  • TSWV For TSWV, scans were performed with primer pair SCAC568-F with SEQ ID NO: 1 and SCAC568-R with SEQ ID NO:2 on chromosome 10; and for PMMoV (L4), scans were performed with primer pair P118 with SEQ ID NO:3 and P119 with SEQ ID NO:4 on chromosome 11 .
  • the sequence of the target region determined by these primer pairs was converted to FASTA format and turned into representative maps with the SnapGene Viewer program.
  • Tru11 (Msel) restriction enzyme sequence (T'TAA) (only one restriction site in this region) used for the restriction of the 350 bp long region amplified by the Primer pair P118 with SEQ ID NO:3 and P119 with SEQ ID NO:4 in the CAPS marker system is located in the region where the primer pair SCAC568-F with SEQ ID NO:1 and SCAC568-R with SEQ ID NO:2 are also amplified (Fig. 1 a, b).
  • T'TAA Tru11 restriction enzyme sequence
  • Trul l (Msel) restriction enzyme recognition region in Figure 1 b is common to both the resistant and susceptible genotype, however, Trul l region located in another site within the same region is polymorphic between the two genotypes (Fig. 2a, b).
  • Trul l enzyme recognition region in Figure 1 b is common to both the resistant and susceptible genotype, however, Trul l region located in another site within the same region is polymorphic between the two genotypes (Fig. 2a, b).
  • silico restriction of sequences of 568 bp PCR product obtained from amplification of TSWV-resistant and susceptible genotype DNAs with the SCAC568-F/R primer pair with the sequences SEQ ID NO:1 and SEQ ID NO:2 with Trul l enzyme was analyzed in Molbiotools application.
  • the PCR amplification product obtained by using primer combination SCAC568-F with SEQ ID NO:1 and SCAC-R' with SEQ ID NO:5 is expected to be 209 bp long, regardless of whether they are resistant/susceptible.
  • silico restriction of PCR products obtained from the amplification of DNAs of TSWV-resistant and susceptible genotypes with this new primer combination with Tru11 enzyme are analyzed in the Molbiotools application, and two fragments of 141 and 68 bp lengths occurred for the resistant allele, while no cleavage (209 bp) was found for the susceptible allele (figure 3).
  • the method which is the subject of the present invention, is a detection method by multiplex polymerase chain reaction (PCR) based on the identification of TSWV resistance gene Tsw and PMMoV resistance gene L4, for the simultaneous detection of resistance status to disease caused by Tomato Spotted Wilt Virus (TSWV) and Pepper Mild Mottle Virus (PMMoV) and the polymorphism between susceptible and resistant genotype in pepper (Capsicum annuum), wherein said method comprises the process steps of; i.
  • PCR polymerase chain reaction
  • amplifying nucleic acids by using the primer with the sequence SEQ ID NO:1 and the primer pair with the sequence SEQ ID NO:5, and the primer with the sequence SEQ ID NO:3 and the primer pair with the sequence SEQ ID NO:4 together in a single polymerase chain reaction, ii. restricting of the amplified PCR products with Tru11 restriction enzyme, and iii. identifying genotypes by obtaining at least one fragment that determines Tsw-resistant and susceptible genotypes and at least one fragment that determines /.4-resistant and susceptible genotypes according to the size of the restricted products.
  • the conditions for the polymerase chain reaction in the step (i) mentioned in this method are as follows, respectively; a. 1 cycle at 95°C for 13 minutes b. 35 cycles at 95°C for 15 seconds, at 56°C for 45 seconds, at 72°C for 1 minute, respectively, c. 1 cycle for 7 minutes at 72°C
  • the fragment TSWV is the fragment with the length of
  • At least one of said fragments is obtained according to the disease resistance and phenotypes found in the pepper, and disease resistance is defined according to these fragments.
  • said fragment PMMoV (L4) is a fragment with the length of
  • At least one of said fragments is obtained according to the disease resistance and phenotypes found in the pepper, and disease resistance is defined according to these fragments.
  • annuum preferably Capsicum annuum Pepper Zunla 1, genotypes (Rr, RR and rr) SCAC568-F (SEQ ID NO:1 )/SCAC-R' (SEQ ID NO:5) that have been previously tested and resistance status of which is known.
  • Capsicum annuum Pepper Zunla 1 that is used to check the primers and the accuracy of the obtained fragments is a member of Capsicum annuum, it is assumed to be the same as the sequences of the samples used (Capsicum annuum).
  • confirmation of homozygous resistant and heterozygous resistant individuals is recommended as a result of restriction of the products amplified with the SCAC568-F/R primer pair with the Taql enzyme.
  • Table 1a TSVW and PMMoV (L4) multiplex PCR components.
  • the PCR reaction is carried out under the conditions in Table 1 b by using the components in Table 1 a in the amounts given.
  • Table 1b TSWV and PMMoV (L4) multiplex PCR conditions.
  • L1-L3 in Figure 4 Restriction of multiplex PCR products of Rr, RR and rr genotypes with Trul l enzyme, respectively; Restriction of PMMoV (L4) ⁇ P118/P119 PCR products of Rr, RR, and rr genotypes with Trul l enzyme, respectively; L7-L9: Restriction of TSWV-SCAC568-F/SCAC-R' PCR products of Rr, RR, and rr genotypes with Tru11 enzyme, respectively; L10-L12: Restriction of TSWV-SCAC568-F/R PCR products of Rr, RR and rr genotypes, respectively, with Xbal enzyme.
  • a second test was performed to reinforce the accuracy of the TSWV-PMMoV (L4) multiplex PCR method, which produces correct fragments as a result of the analysis.
  • three commercial F1 hybrids with TSWV-resistant and no PMMoV (L4) resistance BASF, Nunhems: Efests F1 , Erciyes Rene F1 , Assia F1
  • three commercial F1s with TSWV and PMMoV (L4) resistances BASF, Nunhems: Mustang F1 , Yuksel Seed: Doganay F1 , Koray F1 ) were tested by the TSWV-PMMoV (L4) multiplex PCR method.
  • S1 -S6 show restriction of multiplex PCR products of Efests F1 (Demre type), Erciyes Rene F1 (Demre type), Assia F1 (Demre type), Mustang F1 (California Wonder), Doganay F1 (Dolma type), Koray F1 (California Wonder) samples with Trul l enzyme, respectively; Restriction of B. PMMoV (L4)- P118/P119 PCR products with Trul l enzyme; restriction of C. TSWV- SCAC568-F/SCAC-R’ PCR products with Trul l enzyme; Restriction of TSWV-SCAC568-F/R PCR products with Xbal enzyme.
  • genotypic segregation occurred in F2 progenitors, which were compatible with the TSWV-Rr x PMMoV (L4)-Rr crossing profile.

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Abstract

The present invention relates to a multiplex PCR method that allows for determining resistance status against both diseases simultaneously by means of the fact that marker systems for two different diseases in pepper (Capsicum annuum) are used in a single polymerase chain reaction (PCR) amplification and by forming a single restriction digestion, for identifying Tsw, which is the Tomato Spotted Wilt Virus (TSWV) Resistance Gene, and L4, which is the Pepper Mild Mottle Virus (PMMoV) Resistance Gene. By means of the present invention, it is provided CAPS markers and methods using these markers, that offer a single reaction for the determination of the allelic status (homozygous/heterozygous) of both Tsw and L4, that do not require two separate treatments, that enable the identification of both susceptible and resistant alleles for both loci, in order to determine the two different disease resistance states simultaneously.

Description

MULTIPLEX PCR METHOD FOR IDENTIFYING THE RESISTANCE GENE TSW OF TOMATO SPOTTED WILT VIRUS (TSWV), AND RESISTANCE GENE L4 OF PEPPER MILD MOTTLE VIRUS (PMMOV) IN PEPPER (CAPSICUM ANNUUM)
Technical Field of the Invention
The present invention relates to a multiplex PCR method that allows for determining resistance status against both diseases simultaneously by means of the fact that marker systems for two different diseases in pepper (Capsicum annuum) are used in a single polymerase chain reaction (PCR) amplification and by forming a single restriction digestion, for identifying Tsw, which is the Tomato Spotted Wilt Virus (TSWV) Resistance Gene, and L4, which is the Pepper Mild Mottle Virus (PMMoV) Resistance Gene.
State of the Art
Although many of the plant diseases that cause serious yield and economic losses are virus-based, diseases caused by especially "Tomato Spotted Wilt Virus (TSWV)” and "Pepper mild mottle virus (PMMoV)" viruses are very common in greenhouse and open field pepper (Capsicum spp.) cultivation worldwide. Molecular markers are widely used for identifying genetic resistance status against these diseases.
Molecular markers are DNA fragments associated with any gene region or gene region in the genome, and are markers that reveal the DNA sequence difference of different genotypes in various ways. Use of nucleic acid-based genetic markers in genome analysis, especially for breeders is of great importance in determining both cultivars and disease resistance thereof.
Tsw gene controlled by a single dominant gene located on chromosome 10 of pepper is known to confer genetic resistance against TSWV, which is included in the genus Tospovirus. Seven virus species included in the genus Tobamovirus cause disease in Capsicum (pepper) species. These are called Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), Bell pepper mottle virus (BPeMV), Paprika mild mottle virus (PaMMV), Obuda pepper virus (ObPV), and Pepper mild mottle virus (PMMoV).
It is known that different dominant alleles (L1 , L2, L3, L4) at the L locus located on chromosome 11 of pepper in PMMoV confer resistance against different pathotypes (P0, P1 , P1.2, P1.2.3). Among these, the L4 resistance gene also provides resistance against the P1 .2.3 pathotype, which other alleles (L1 , L2, L3) cannot provide resistance. The allelic status of the resistance (homozygous/heterozygous) can be determined in a reliable manner by means of CAPS (Cleavage Amplified Polymorphic Sequence) marker systems that produce codominant (Rr, RR, rr) information, which is one of the molecular methods based on PCR used to determine disease resistance in plants.
Within the scope of determining the allelic state of resistance with CAPS marker systems, according to the results obtained in a study by Moury et al. (2000) in the prior art, results obtained from restriction of the product amplified with specific primers of the SCAC568 CAPS marker, which is the CAPS molecular marker associated with the Tsw gene, with Xbal, Taql, or Haelll enzymes are used in the diagnosis of resistance to TSWV in pepper [1 ]. Bulk DNAs generated from 8 Tsw- resistant and Tsw-susceptible genotypes, determined to be homozygous by Moury et al. (2000) were tested with 250 10-mer RAPD markers and it was determined that 4 of them (OPAC10, OPAH13, OPAF16, OPB01 ) were polymorphic and associated with the Tsw locus. The result of PCR amplification with the OPAC10 primer (1 .69 ± 1 .23 cM) , which was determined to be relatively close to the Tsw locus from these 4 markers produces a product of 593 bp in the resistant genotype. This was made very close (0.9 ± 0.6 cM) to the Tsw locus by conversion of the RAPD marker to the CAPS marker (SCAC568). The newly designed primer pair amplifies a 568 bp band common to susceptible and resistant alleles, and produces codominant information (detecting homozygous resistant, homozygous susceptible, or heterozygous genotype) after restriction with Xbal, Taql, or Haelll restriction enzymes. As a result of the restriction of the 568 bp long PCR product with the Xbal enzyme, the TSWV-resistant allele is enzymatically cleaved, while the TSWV-susceptible allele is not. In the same way, the Taql (or Haelll) enzyme makes it possible to detect the susceptible allele, if any, when the 568 bp PCR product is enzymatically cleaved.
Similarly, the patent document numbered EP1804571 B1 in the prior art provides codominant information about L4 allele-dependent resistance against PMMoV- P1.2.3 pathotype as a result of restriction of the product amplified with the help of primers designed specifically for the region associated with the L locus on chromosome 11 in pepper, with the Trul l enzyme. As mentioned in the patent document numbered EP1804571 B1 , Tm3-DRS marker system developed from the TG36 marker, which they previously determined in another study to be 6 cM away from the L locus on the 11th chromosome of pepper is also used for this L locus. The primer pair P118/P119 designed specifically for this region can amplify the L locus associated region common to resistant and susceptible genotypes, and produces a band approximately 350 bp long. Approximately 350 bp product L4, which is formed as a result of enzymatic restriction of the resulting PCR product with Trul l restriction enzyme. However, although different enzymes are used for the two CAPS marker systems (Tsw and L4) in the work of Moury et al. (2000) described above and the patent document numbered EP1804571 B1 , analyzes are also made by creating separate PCR reactions.
In a study by Matsunaga et al. (2003) in the prior art, PCR reaction was established using DNA extracted from PMMoV-resistant (R) and PMMoV- susceptible (S) materials by primers of 516 randomly selected RAPD marker systems for determining PMMoV - L4 resistance by classical PCR method [2], Among the resistant and susceptible genotypes, only one of the 516 primers showed polymorphism, and produced bands at 1500 bp (base pairs) only for resistant genotypes. Based on this polymorphism, this RAPD marker was converted into the dominant 20 nucleotide SCAR (Sequence Characterized Amplified Region) marker system and became the AP-7/AP-8 primer pair. Although the AP-7 I AP-8 SCAR marker can distinguish between resistant and susceptible individuals, it is only capable of amplifying the 1500 bp band of the resistant genotype. As a result of PCR amplification, no bands are produced in the susceptible genotype. In other words, the marker here is a dominant SCAR marker.
Information is obtained about the presence/absence of the allele that provides resistance to the disease or only the presence/absence of the disease in the plant by means of the use of dominant markers found in the above-mentioned prior art documents used in disease testing. That is, unlike the CAPS marker system, which produces codominant information, in tests with the dominant marker, it is not possible to obtain a result about whether the allele that gives resistance to the plant is in the homozygous (RR) or heterozygous (Rr) state since the marker targets a single allele. From the point of view of plant breeding, this situation causes genotypic segregation in future generations due to failure to identify the susceptible allele (r) in a heterozygous (Rr) plant, not determined at an early stage. Especially in pure line development studies, which are important in terms of breeding, failure to predict heterozygous genotype-induced segregation (Mendel's principle of independent assortment) makes reaching the goal difficult. In the presence of a heterozygous inheritance, uniformity cannot be achieved between plants, and therefore, it causes the formation of different genotypes (RR, Rr, rr) and phenotypes in the next generation. This situation highlights the importance of using codominant markers compared to dominant markers in marker assisted selection (MAS) studies. As a result, it is important to use codominant markers such as CAPS markers instead of existing dominant marker systems in determining TSWV and PMMoV-L4 resistance.
In the tests performed with the classical PCR method by using existing systems, analysis methods that require individual amplification of each locus are used in order to detect the presence of alleles associated with the Tsw and L4 loci. In case of homozygous/heterozygosity testing for both genes; first of all, DNA samples are amplified in PCR with Tsw specific (SCAC568) primers, in the second step, these PCR products representing each genotype are enzymatically cleaved separately with Xbal and Taql restriction enzymes, and finally the restriction products are run separately in agarose gel. Similarly, when plant samples are to be tested for the L4 allele; this time, PCR products are obtained with L4 specific (P118-P119) primers, these PCR products are then enzymatically cleaved with the Msel restriction enzyme, and finally after running on an agarose gel, the presence of homozygous/heterozygous L4 allele can be determined. These steps are performed separately for Tsw and L4 loci, and separate enzymes are used for these different loci and analyzes are made by creating separate PCR reactions.
In tests using the CAPS marker, the PCR product is enzymatically cleaved with the appropriate restriction enzyme after firstly amplified by region-specific (to include the SNP region between the resistant and susceptible allele) primers that are common to the susceptible and resistant allele. The difference between resistant and susceptible alleles is interpreted according to whether the sequence of the restriction site is present in the amplified PCR product. Therefore, in testing two different loci with CAPS markers, in addition to the costs of the PCR device, PCR reaction components and restriction enzymes, the time and labor spent on analyzes are doubled.
In case the CAPS markers of the state of the art are used to determine the resistance of plants to Tsw and L4, due to the reasons such as the need for two different reactions, primer and enzyme, to determine the allelic status (homozygous/heterozygous) of both Tsw and L4, requiring two separate processes, creating a disadvantage in terms of time, cost and labor, the fact that the markers are the dominant SCAR markers, that is, they have the ability to amplify only the band of the resistant genotype, it is necessitated to develop CAPS markers and methods using these markers, that offer a single reaction for the determination of the allelic status (homozygous/heterozygous) of both Tsw and L4, that do not require two separate PCR reactions, that enable the identification of both susceptible and resistant alleles for both loci, in order to determine the two different disease resistance status simultaneously.
Brief Description of the Invention
In the present invention, it is disclosed a multiplex PCR method that allows for determining resistance status against both diseases simultaneously by means of the fact that marker systems for two different diseases in pepper (Capsicum annuum) are used in a single polymerase chain reaction (PCR) amplification and by forming a single restriction digestion, for identifying Tsw, which is the Tomato Spotted Wilt Virus (TSWV) Resistance Gene, and L4, which is the Pepper Mild Mottle Virus (PMMoV) Resistance Gene.
The first object of the present invention is to determine the status of Tsw and L4 genes, which determine resistance against two different diseases in pepper, and homozygous and heterozygous alleles, with a single reaction. Existing codominant CAPS markers used to determine resistance to TSWV and PMMoV (P1 .2.3) in pepper are developed, combined, and made operable by the multiplex PCR method by means of the present invention. By means of the present invention, a single restriction digestion is formed by using the marker systems developed for two different diseases in a single PCR amplification, and thus, analysis can be performed for two diseases simultaneously.
Another object of the present invention is to provide a method that is advantageous in terms of cost and labor compared to the prior art, that enables analysis in a short time and at low cost while both Tsw and L4 genes, which determine resistance to two different diseases in pepper, were analyzed together. Considering both the enzyme costs and the time and effort spent on two separate analyzes for the Tsw and L4 genes, by means of the present invention, it is possible to integrate the available amplification techniques developed as separate analyzes with the multiple PCR method. By means of the present invention that makes resistance statuses against diseases caused by Tomato Spotted Wilt Virus (TSWV) and Pepper Mild Mottle Virus (PMMoV) simultaneously detectable, the total cost and labor spent for analyzes are halved.
Another object of the present invention is to provide the addition of a new alternative enzyme to the existing enzyme variety (Xbal, Taql or Haelll) and to optimize the reaction conditions, in relation to the SNPs in the target region of the primers used in the CAPS marker system, where resistance to TSWV is tested in pepper. By means of the present invention, Tru11 (Msel) enzyme has been added to be used in the SCAC568 CAPS marker system as an alternative to Xbal, Taql, or Haelll enzymes. By optimizing the restriction digestion reaction within the scope of the present invention, it is ensured that the desired result is obtained in a shorter time compared to the prior art, by using the least amount of chemicals. By means of the present invention, it is provided CAPS markers and methods using these markers, that offer a single reaction for the determination of the allelic status (homozygous/heterozygous) of both Tsw and L4, that do not require two separate PCR reactions, that enable the identification of both susceptible and resistant alleles for both loci, in order to determine the two different disease resistance status simultaneously.
Description of the Figures
Figure 1 : Representative map of amplified regions for determination of TSWV (Tsw) and PMMoV (L4) resistances in C. annuum a) The region amplified by P118/P119 primer pair on the chromosome 11 of C. annuum - PMMoV (L4) Chromosome 11 P118/P119; b) The region amplified by SCAC568 F/R (forward/reverse) primer pair on C. annuum chromosome - 10 TSWV Chromosome 10 SCAC568 F-R.
Figure 2: Representative map of regions amplified with the SCAC568-F/R primer pair a) the target region of the TSWV-susceptible (rr) genotype amplified with the SCAC568 F/R primer pair; b) The target region of the TSWV-resistant (RR) genotype amplified with the SCAC568-F/R primer pair.
Figure 3: In silico restriction of susceptible and resistant PCR products amplified with primer pair SCAC568-F and SCAC-R' by Tru11 enzyme (susceptible allele - 209 bp and resistant allele - 141 bp (+68 bp), M: 100 bp DNA Marker, S: sample).
Figure 4: L1 -L3: Restriction of multiplex PCR products of Rr, RR and rr genotypes with Trul l enzyme, respectively; Restriction of PMMoV (L4)~ P118/P119 PCR products of Rr, RR, and rr genotypes with Trul l enzyme, respectively; L7-L9: Restriction of TSWV-SCAC568-F/SCAC-R' PCR products of Rr, RR, and rr genotypes with Tru11 enzyme, respectively; L10-L12: Restriction of TSWV-SCAC568-F/R PCR products of Rr, RR, and rr genotypes, respectively, with Xbal enzyme (M: 100 bp DNA Marker).
Figure 5: A. S1 -S6: Restriction of multiplex PCR products of Efests F1 (Demre type), Erciyes Rene F1 (Demre type), Assia F1 (Demre type), Mustang F1 (California Wonder), Doganay F1 (Dolma type), Koray F1 (California Wonder) samples with Trul l enzyme, respectively; Restriction of B. PMMoV (L4)- P118/P119 PCR products with Tru11 enzyme; restriction of C. TSWV- SCAC568- F/SCAC-R’ PCR products with Trul l enzyme; Restriction of TSWV-SCAC568- F/R PCR products with Xbal enzyme (M: 100 bp DNA Marker).
Figure 6: A1-20: F2 progenitors from inbreeding of Abraham F1 (California Wonder), C: TSWV- PMMoV (L4) heterozygous (Rr) control and K1 - K21 : Restriction of TSWV-PMMoV (L4) multiplex PCR products obtained with F2 progenitors obtained from inbreeding of Kioto F1 (California Wonder) with Trul enzyme (M: 100 bp DNA Marker).
Detailed Description of the Invention
The present invention relates to a multiplex PCR method that allows for determining resistance status against both diseases simultaneously by means of the fact that marker systems for two different diseases in pepper (Capsicum annuum) are used in a single polymerase chain reaction (PCR) amplification and by forming a single restriction digestion, for identifying Tsw, which is the Tomato Spotted Wilt Virus (TSWV) Resistance Gene, and L4, which is the Pepper Mild Mottle Virus (PMMoV) Resistance Gene.
First of all, within the scope of the present invention, in the target region of the TSWV / SCAC568 F (forward)-R (reverse) and PMMoV (L4) / P118-P119 primer pairs, which belong to the CAPS markers that are widely used today for the determination of TSWV and PMMoV (L4) resistance, it was determined whether there was the same restriction enzyme restriction site. In an embodiment of the present invention, since DNA sequence information of primers and their PCR products is known, these sequences were searched by the NCBI/Genome (National Center for Biotechnology Information, U.S.) database in the C. annuum genome and corresponding chromosomes where the resistances were found. All C. annuum species, especially C. annuum Pepper Zunla 1 species, can be used as the pepper mentioned in the present invention without any geographical restriction. Position of 11th chromosome for Capsicum annuum Pepper Zunla 1 preferably used in the present invention: 1227700-1228034 positive thread NCBI accession number Ref_v1.0, NC_029987.1 ; Capsicum annuum Pepper Zunla 1 10th chromosome Position: The NCBI accession number for 204196455-204197020 positive thread is Ref_v1.0, NC_029986.1 The Capsicum annuum pepper zunla 1 sequences used were retrieved from the NCBI database. There is no geographical region expression in the database.
In order to avoid possible sequence differences in the resistance source, in the determination of TSWV resistance, confirmation of homozygous resistant and heterozygous resistant individuals is recommended as a result of enzymatic restriction of the products amplified with the SCAC568-F/R primer pair with the Taql enzyme.
Within the scope of the present invention, gene resources of Areo Seeds company were used for sequencing of TSWV-resistant and susceptible samples as genetic resources. Breeding materials with the code BWS60-2 (California Wonder) for TSWV-resistant; and BWS60-4 (California Wonder) for TSWV- susceptible were used, in order to control the designed multiplex system, commercial F1 varieties of different seed companies were used.
For TSWV, scans were performed with primer pair SCAC568-F with SEQ ID NO: 1 and SCAC568-R with SEQ ID NO:2 on chromosome 10; and for PMMoV (L4), scans were performed with primer pair P118 with SEQ ID NO:3 and P119 with SEQ ID NO:4 on chromosome 11 . The sequence of the target region determined by these primer pairs was converted to FASTA format and turned into representative maps with the SnapGene Viewer program.
By interpreting sequence information on these maps, it was determined that Tru11 (Msel) restriction enzyme sequence (T'TAA) (only one restriction site in this region) used for the restriction of the 350 bp long region amplified by the Primer pair P118 with SEQ ID NO:3 and P119 with SEQ ID NO:4 in the CAPS marker system is located in the region where the primer pair SCAC568-F with SEQ ID NO:1 and SCAC568-R with SEQ ID NO:2 are also amplified (Fig. 1 a, b). However, since the CAPS-SCAC568 and Trul l enzyme combination is a new method, Sequencing of resistant and susceptible alleles was performed to observe if this enzyme restriction site is present in both the resistant and susceptible allele. Because in codominant information producing marker systems, to obtain the polymorphism between the susceptible and resistant genotype, the enzyme restriction site should be present in only one of the susceptible or resistant allele. Therefore, in accordance with the protocol specified in the prior art, products of 568 bp obtained as a result of PCR amplification of DNA extracted with the 2% CTAB method belonging to susceptible and resistant genotypes, resistance of which was determined previously, were sequenced. TSWV-resistant and TSWV-susceptible chromatograms obtained by sequencing were compared by turning them into representative maps in SnapGene Viewer program.
It was determined that the Trul l (Msel) restriction enzyme recognition region in Figure 1 b is common to both the resistant and susceptible genotype, however, Trul l region located in another site within the same region is polymorphic between the two genotypes (Fig. 2a, b). In silico restriction of sequences of 568 bp PCR product obtained from amplification of TSWV-resistant and susceptible genotype DNAs with the SCAC568-F/R primer pair with the sequences SEQ ID NO:1 and SEQ ID NO:2 with Trul l enzyme was analyzed in Molbiotools application.
A result of the restriction of the TSWV-resistant allele with the Trul l enzyme, fragments of 218, 208 and 141 bp lengths are obtained, while fragments of 359 and 208 bp lengths are obtained for the susceptible allele. Due to the 208 bp fragment occurring for both allelic cleavages, it is provided to amplify the current sequence by shortening it and to eliminate the fragment that is not related to this resistance. In this context, a reverse (R) primer (SCAC-R1) was designed with the Primer3Plus program to exclude the common Trul l restriction site by using the available sequence information. The PCR amplification product obtained by using primer combination SCAC568-F with SEQ ID NO:1 and SCAC-R' with SEQ ID NO:5 is expected to be 209 bp long, regardless of whether they are resistant/susceptible. In silico restriction of PCR products obtained from the amplification of DNAs of TSWV-resistant and susceptible genotypes with this new primer combination with Tru11 enzyme are analyzed in the Molbiotools application, and two fragments of 141 and 68 bp lengths occurred for the resistant allele, while no cleavage (209 bp) was found for the susceptible allele (figure 3). Likewise, as a result of restriction of PCR products (335 bp) obtained with PMMoV (L4) - P118/P119 primers with Trul l enzyme, fragments of 335 bp (no cleavage occurs) for the resistant allele and 299 bp for the susceptible allele are known to occur. Due to the fact that the products obtained with the TSWV-SCAC568-F (SEQ ID NO:1 )/SCAC-R' (SEQ ID NO:5), and PMMoV (L4)-P118 (SEQ ID NO:3)/P119(SEQ ID NO:4) primer combinations (209 bp and 335 bp, respectively) do not overlap in size with the fragments obtained from cleavage with the Tru11 enzyme; these four primer combinations are used for the multiplex PCR method in order to simultaneously determine two different disease resistance status by means of the present invention.
The method, which is the subject of the present invention, is a detection method by multiplex polymerase chain reaction (PCR) based on the identification of TSWV resistance gene Tsw and PMMoV resistance gene L4, for the simultaneous detection of resistance status to disease caused by Tomato Spotted Wilt Virus (TSWV) and Pepper Mild Mottle Virus (PMMoV) and the polymorphism between susceptible and resistant genotype in pepper (Capsicum annuum), wherein said method comprises the process steps of; i. amplifying nucleic acids by using the primer with the sequence SEQ ID NO:1 and the primer pair with the sequence SEQ ID NO:5, and the primer with the sequence SEQ ID NO:3 and the primer pair with the sequence SEQ ID NO:4 together in a single polymerase chain reaction, ii. restricting of the amplified PCR products with Tru11 restriction enzyme, and iii. identifying genotypes by obtaining at least one fragment that determines Tsw-resistant and susceptible genotypes and at least one fragment that determines /.4-resistant and susceptible genotypes according to the size of the restricted products. The conditions for the polymerase chain reaction in the step (i) mentioned in this method are as follows, respectively; a. 1 cycle at 95°C for 13 minutes b. 35 cycles at 95°C for 15 seconds, at 56°C for 45 seconds, at 72°C for 1 minute, respectively, c. 1 cycle for 7 minutes at 72°C
In the method of the present invention, the fragment TSWV is the fragment with the length of
• 209 bp for the susceptible allele (rr), or
• 141 bp and 68 bp for the resistant allele (RR), or
• 209 bp, 141 bp, and 68 bp for the heterozygous allele (Rr).
At least one of said fragments is obtained according to the disease resistance and phenotypes found in the pepper, and disease resistance is defined according to these fragments.
In the method of the invention, said fragment PMMoV (L4) is a fragment with the length of
• 299 bp for the susceptible allele (rr), or
• 335 bp for the resistant allele (RR), or
• 299 bp and 335 bp for the heterozygous allele (Rr)
At least one of said fragments is obtained according to the disease resistance and phenotypes found in the pepper, and disease resistance is defined according to these fragments.
The utility of the method (TSWV-SCAC568-F (SEQ ID NO:1 )/SCAC-R' (SEQ ID NO:5) and PMMoV (L4)-P118 (SEQ ID NO:3)/P119(SEQ ID NO:4) + Trul l enzyme restriction), which is the subject of the present invention, integrated into multiplex PCR method for simultaneous testing of TSWV and PMMoV (L4) resistance status was analyzed together with the Trul l enzyme restriction of formed products, P118 (SEQ ID NO:3)/P119(SEQ ID NO:4), and Trul l enzyme restriction of formed products, and SCAC568-F (SEQ ID NO:1 )/SCAC568-R (SEQ ID NO:2), and Xbal enzyme restriction results of formed products in three different C. annuum, preferably Capsicum annuum Pepper Zunla 1, genotypes (Rr, RR and rr) SCAC568-F (SEQ ID NO:1 )/SCAC-R' (SEQ ID NO:5) that have been previously tested and resistance status of which is known.
In one sample of the present invention since Capsicum annuum Pepper Zunla 1 that is used to check the primers and the accuracy of the obtained fragments is a member of Capsicum annuum, it is assumed to be the same as the sequences of the samples used (Capsicum annuum). In order to avoid possible sequence differences in the resistance source, to avoid a possible difference, in the determination of TSWV resistance, confirmation of homozygous resistant and heterozygous resistant individuals is recommended as a result of restriction of the products amplified with the SCAC568-F/R primer pair with the Taql enzyme.
As a result of restriction of the product obtained with SCAC568-F/R with the Xbal enzyme (T'CTAGA) by the method determined in the prior art, it has been reported that the presence of the 568 bp long un-restricted fragment indicates the TSWV-susceptible allele (rr), the 280 bp fragment indicates the TSWV-resistant (RR) allele, and the presence of both indicates TSWV-heterozygosity (Rr). As an additional advantage of the method of the present invention and the primers designed, by optimizing the reaction conditions used in the analysis, the amount of chemicals used with the method in the previous art was also reduced. All contents and values in Tables 1 a, 1 b and 2a refer to the optimal conditions optimized within the scope of the present invention. Here, DNA from Capsicum annuum is used as a sample.
Table 1a. TSVW and PMMoV (L4) multiplex PCR components.
Figure imgf000014_0001
The PCR reaction is carried out under the conditions in Table 1 b by using the components in Table 1 a in the amounts given.
Table 1b. TSWV and PMMoV (L4) multiplex PCR conditions.
Figure imgf000015_0001
Table 2. Restriction components of multiplex PCR (PCR) products with Tru11 (Msel) enzyme.
Figure imgf000015_0002
As a result of restriction of the products obtained by multiplex PCR amplification with the PCR reaction content and conditions in Table 1 with Tru11 enzyme under the conditions in Table 2 (16 hours at 65°C) and electrophoresis of the products with 2.5% agarose gel for 1 hour (100 V), the accuracy of the method, which is the subject of the present invention, has been proven by obtaining the fragments, images of which are shown in Figure 4 and which were previously determined as in silico. L1-L3 in Figure 4: Restriction of multiplex PCR products of Rr, RR and rr genotypes with Trul l enzyme, respectively; Restriction of PMMoV (L4)~ P118/P119 PCR products of Rr, RR, and rr genotypes with Trul l enzyme, respectively; L7-L9: Restriction of TSWV-SCAC568-F/SCAC-R' PCR products of Rr, RR, and rr genotypes with Tru11 enzyme, respectively; L10-L12: Restriction of TSWV-SCAC568-F/R PCR products of Rr, RR and rr genotypes, respectively, with Xbal enzyme. As a result of the restriction performed with the Tru11 enzyme, 299 bp for PMMoV (L4)-susceptible allele (rr) and 335 bp (un- cleaved) fragments for resistant allele (RR) were obtained, while fragments of both lengths were obtained together for the heterozygous allele (Rr). 209 bp (un-cleaved) for the TSWV susceptible allele (rr), 141 and 68 bp for the resistant allele (RR), and for the heterozygous allele (Rr), these three fragments occurred together.
A second test was performed to reinforce the accuracy of the TSWV-PMMoV (L4) multiplex PCR method, which produces correct fragments as a result of the analysis. For this purpose, three commercial F1 hybrids with TSWV-resistant and no PMMoV (L4) resistance (BASF, Nunhems: Efests F1 , Erciyes Rene F1 , Assia F1 ), and three commercial F1s with TSWV and PMMoV (L4) resistances (BASF, Nunhems: Mustang F1 , Yuksel Seed: Doganay F1 , Koray F1 ) were tested by the TSWV-PMMoV (L4) multiplex PCR method. As a control, all samples were analyzed to determine TSWV resistance with the SCAC568-F (SEQ ID NO:1 )/SCAC568-R (SEQ ID NO:2) + Xbal restriction combination. The results were consistent with each other, while 209+141 bp (heterozygous - Rr) fragments were formed for all samples with a TSWV resistance profile, only 299 bp fragments were formed in resistance samples (rr) without PMMoV (L4) resistance, and in PMMoV (L4)-resistant samples, fragments of 335+299 bp (Rr) were formed (Fig. 5). It was determined that all of the samples with TSWV- resistance profile were heterozygous, as a result of the restriction of the SCAC568-F (SEQ ID NO:1 )/SCAC568-R (SEQ ID NO:2) amplified PCR products with the Xbal enzyme, resulting in fragments of 568+280 bp, that it gives the same result as the new SCAC568-F (SEQ ID NO:1 )/SCAC-R' (SEQ ID NO:5) combination, and the optimized TSWV-PMMoV (L4) multiplex PCR method works with accuracy (Fig. 5). In Figure 5, S1 -S6 show restriction of multiplex PCR products of Efests F1 (Demre type), Erciyes Rene F1 (Demre type), Assia F1 (Demre type), Mustang F1 (California Wonder), Doganay F1 (Dolma type), Koray F1 (California Wonder) samples with Trul l enzyme, respectively; Restriction of B. PMMoV (L4)- P118/P119 PCR products with Trul l enzyme; restriction of C. TSWV- SCAC568-F/SCAC-R’ PCR products with Trul l enzyme; Restriction of TSWV-SCAC568-F/R PCR products with Xbal enzyme. In order to test the TSWV-PMMoV (L4) multiplex PCR method in F2 populations, DNAs extracted from 20 progeny (F2) obtained by inbreeding of Abraham F1 (HM. Clause: TSVW-IR/PMMoV (L4)-HR), and DNAs extracted from 21 progeny (F2) obtained by inbreeding of Kioto F1 (Syngenta: TSWV-IR I PMMoV (L4)-HR) are analyzed according to the conditions in Tables 1 and 2. As a result of electrophoresis with 2.5% agarose gel, genotypic segregation (dihybrid crossing) occurred in F2 progenitors, which were compatible with the TSWV-Rr x PMMoV (L4)-Rr crossing profile. In Figure 6, results obtained from the restriction of TSWV-PMMoV (L4) multiplex PCR products obtained with A1 -20: F2 progeny from inbreeding of Abraham F1 , C: TSWV-PMMoV (L4) heterozygous (Rr) control, and K1 - K21 : F2 progeny from inbreeding of Kioto F1 , with Trul enzyme, and results obtained from the restriction of multiplex PCR products amplified with SCAC568-F (SEQ ID NO:1 ), SCAC-R' (SEQ ID NO:5), P118 (SEQ ID NO:3), P119 (SEQ ID NO:4) primers combined with TSWV-PMMoV (L4) multiplex PCR method, with Tru11 (Msel) restriction enzyme proved that these primers and the restriction site are suitable for determining different TSWV and PMMoV (L4) resistances in pepper. By combining two different CAPS marker systems by means of the present invention, it is possible to produce codominant information about TSWV and PMMoV (L4) resistances.
REFERENCES
1. Moury, B., Pflieger, S., Blattes, A., Lefebvre, V., & Palloix, A. (2000). A CAPS marker to assist selection of tomato spotted wilt virus (TSWV) resistance in pepper. Genome, 43(1 ), 137-142. 2: Matsunaga, H., Saito, T., Hirai, M., Nunome, T., & Yoshida, T. (2003). DNA markers linked to Pepper mild mottle virus (PMMoV) resistant locus (L4) in Capsicum. Journal of the Japanese Society for Horticultural Science, 72(3), 218- 220.

Claims
CLAIMS Primer pairs to be used together in multiple polymerase chain reaction (PCR) method based on the identification of TSWV resistance gene Tsw and PMMoV resistance gene L4 for the simultaneous detection of resistance status to disease caused by Tomato Spotted Wilt Virus (TSWV) and Pepper Mild Mottle Virus (PMMoV) and the polymorphism between susceptible and resistant genotype in pepper (Capsicum annuum), characterized in that, said primer pairs are the primer with the sequence SEQ ID NO:1 and the primer pair with the sequence SEQ ID NO:5, and are the primer with the sequence SEQ ID NO:3 and the primer pair with the sequence SEQ ID NO:4. Detection method by multiplex polymerase chain reaction (PCR) based on the identification of TSWV resistance gene Tsw and PMMoV resistance gene L4, for the simultaneous detection of resistance status to disease caused by Tomato Spotted Wilt Virus (TSWV) and Pepper Mild Mottle Virus (PMMoV) and the polymorphism between susceptible and resistant genotype in pepper (Capsicum annuum), characterized by comprising the process steps of; i. amplifying nucleic acids by using the primer with the sequence SEQ ID NO:1 and the primer pair with the sequence SEQ ID NO:5, and the primer with the sequence SEQ ID NO:3 and the primer pair with the sequence SEQ ID NO:4 together in a single polymerase chain reaction, ii. cleaving the amplified PCR products with Tru11 restriction enzyme, and iii. identifying genotypes by obtaining at least one fragment that determines Tsw-resistant and susceptible genotypes and at least one fragment that determines /.4-resistant and susceptible genotypes according to the size of the cleaved products.
3. A method according to Claim 2, characterized in that, in said step (i), the polymerase chain reaction conditions are; a. 1 cycle at 95°C for 13 minutes, b. 35 cycles at 95°C for 15 seconds, at 56°C for 45 seconds, at 72°C for 1 minute, c. 1 cycle for 7 minutes at 72°C, respectively.
4. A method according to Claim 2, characterized in that, said fragment TSWV is the fragment with the length of
• 209 bp for the susceptible allele (rr), or
• 141 bp and 68 bp for the resistant allele (RR), or
• 209 bp, 141 bp, and 68 bp for the heterozygous allele (Rr).
5. A method according to Claim 2, characterized in that, said fragment PMMoV (L4) is the fragment with the length of
• 299 bp for the susceptible allele (rr), or
• 335 bp for the resistant allele (RR), or
• 299 bp and 335 bp for the heterozygous allele (Rr).
6. A primer pair according to Claim 1 , characterized in that, said pepper species is Capsicum annuum Pepper Zunla 1 .
PCT/TR2022/051618 2021-12-31 2022-12-27 Multiplex pcr method for identifying the resistance gene tsw of tomato spotted wilt virus (tswv), and resistance 5 gene l4 of pepper mild mottle virus (pmmov) in pepper (capsicum annuum) WO2023129057A2 (en)

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