CN113943826B - Molecular marker closely linked with tomato neck rot resistance gene Frl and application thereof - Google Patents
Molecular marker closely linked with tomato neck rot resistance gene Frl and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker closely linked with a tomato neck rot resistance gene Frl and application thereof. The invention provides an application of a substance for detecting whether 165bp fragments are contained in a positioning interval of Frl genes in a genome of a tomato variety in identification or auxiliary identification of tomato neck rot resistance of tomatoes; the invention discovers that a 165bp large fragment inserted in a gene interval region in a Frl gene positioning region (in a region of 4.2-5.1Mb of a No. 9 chromosome of a tomato) in a genome of a tomato variety is related to resistance to tomato neck rot and root rot, and according to the fragment, a PCR-based molecular marker is designed, so that whether a tomato material to be detected contains the Frl gene and whether the Frl gene is homozygous or not can be detected, the breeding period can be shortened, and the accuracy of breeding selection can be improved.
Description
Technical Field
The invention belongs to the field of plant molecular breeding, and particularly relates to a molecular marker closely linked with a tomato neck rot resistance gene Frl and application thereof.
Background
The tomato neck rot root rot is a very serious soil-borne disease, and the pathogenic bacteria of the tomato neck rot root rot are fusarium oxysporum tomato specialization. Typical symptoms of the disease are that plant roots decay downwards from soil line positions, the overground parts wilt and lose water, vascular bundles brown, and plant growth stagnates until death after pathogen infection. When diseases occur, the yield of tomatoes can be reduced by more than 65 percent, even the tomatoes are in harvest, and the death rate of plants reaches 100 percent. At present, two main methods for preventing and treating the neck rot and root rot of tomatoes exist, namely physical and chemical prevention and treatment, and development and utilization of resistant resources to cultivate resistant varieties. The physical and chemical control can not only increase the production cost and influence the quality of tomato fruits, but also destroy soil and environment. Practice proves that the cultivation of the resistant varieties is the most economical, safe and effective way, and how to efficiently and quickly select the resistant tomato varieties is also a focus of attention of breeders.
Frl gene is derived from tomato wild species Peru tomato and is the only known resistance site of tomato neck rot and root rot. Tomato plants containing Frl genes show high resistance and even immunity to fusarium oxysporum tomato specialization, which shows that the genes have important prospects in breeding of tomato with neck rot resistance and root rot resistance. Genetic analysis showed that the Frl gene was located in the interval 4.2-5.1Mb of chromosome 9 (Devran, z., kahveci, e., hong, y., david, j.s., and Mahmut, t.2018.identifying molecular markers suitable for Frl selection in tomato marking.Theor.appl. Genet.131, 2099-2105). Although the Frl gene has not been cloned, some molecular markers linked to Frl have been developed and can be applied to selection of anti-influenza materials. The most widely used molecular marker in the current production is co-dominant CAPS molecular marker C2-25 (Staniaszek, M., szczechura, W., and Marczewski, W.2014.Identification of a new molecular marker C-25 linked to the Fusarium oxysporum f.sp.Radicis-lycopersici resistance Frl gene in chemical J.Genet.plant seed.50, 285-287), but the detection process needs restriction endonuclease for enzyme digestion detection, the process is complicated, and researches show that the identification result of C2-25 is only 95.83% and is insufficient for accurately identifying the resistance of the material (Liu Lei, wang Hui, li Wenli, wang Fu 2018. Markers linked with tomato neck rot disease resistance gene Frl and application of Jiangsu agricultural science 46,91-93). Therefore, it is necessary to develop a molecular marker that is more closely linked and co-isolated with the resistance gene Frl, so as to improve the accuracy of molecular marker-assisted selection breeding.
Disclosure of Invention
An object of the present invention is to provide the use of a substance for detecting whether a 165bp fragment is contained in the localization interval of the Frl gene in the genome of a tomato variety.
The invention provides an application of a substance for detecting whether 165bp fragments are contained in a positioning interval of Frl genes in a genome of a tomato variety in identification or auxiliary identification of tomato neck rot resistance of tomatoes;
or, the invention provides the application of a substance for detecting whether 165bp fragments are contained in the positioning interval of the Frl gene in the genome of the tomato variety in cultivation or assisted cultivation of tomato with tomato neck rot resistance and root rot resistance;
the positioning interval of the Frl gene is an interval of 4.2-5.1Mb of chromosome 9 of tomato;
the 165bp fragment is 1) or 2) as follows:
1) The nucleotide sequence is sequence 3;
2) And (3) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotide residues for the nucleotide sequence shown in the sequence 3 in the sequence table and has the same function.
In the above application, the substance for detecting whether the 165bp fragment is contained in the positioning region of the Frl gene in the genome of the tomato variety is as follows 1) or 2):
1) The primer set consists of a single-stranded DNA molecule or a derivative thereof shown as a sequence 1 in a sequence table and a single-stranded DNA molecule shown as a sequence 2 in the sequence table;
2) PCR reagents or kits containing the set of primers.
In the above, the derivative of the single-stranded DNA molecule shown in the sequence 1 in the sequence table is a DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotide residues in the nucleotide sequence shown in the sequence 1 in the sequence table and having the same function;
the derivative of the single-stranded DNA molecule shown in the sequence 2 in the sequence table is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotide residues for the nucleotide sequence shown in the sequence 2 in the sequence table and has the same function.
It is another object of the invention to provide a method of:
the invention provides a method for identifying or assisting in identifying tomato neck rot and root rot resistance of tomatoes, which is used for detecting whether 165bp fragments are contained in a positioning interval of Frl genes in a genome of tomatoes to be detected,
if the 165bp fragment is contained, the tomato resistance to be detected is or is candidate to resist the tomato neck rot and root rot;
if the 165bp fragment is not contained, the tomato to be detected is not resistant to the neck rot or the root rot of the candidate tomato;
the positioning interval of the Frl gene is an interval of 4.2-5.1Mb of chromosome 9 of tomato;
the 165bp fragment is 1) or 2) as follows:
1) The nucleotide sequence is sequence 3;
2) And (3) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotide residues for the nucleotide sequence shown in the sequence 3 in the sequence table and has the same function.
Or the invention provides a method for identifying or assisting in identifying whether tomatoes are resistant to tomato neck rot and root rot, in order to detect whether 165bp fragments are contained in a positioning interval of Frl genes in a genome of tomatoes to be detected,
if the 165bp fragment is contained, the tomato to be detected is or is candidate to be tomato with tomato neck rot resistance and root rot resistance;
if the tomato does not contain the 165bp fragment, the tomato to be detected is not or is not a candidate tomato for resisting the tomato neck rot and root rot;
the positioning interval of the Frl gene is an interval of 4.2-5.1Mb of chromosome 9 of tomato;
the 165bp fragment is 1) or 2) as follows:
1) The nucleotide sequence is sequence 3;
2) And (3) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotide residues for the nucleotide sequence shown in the sequence 3 in the sequence table and has the same function.
Or the invention provides a method for cultivating or assisting in cultivating tomato with tomato neck rot and root rot resistance, which is used for detecting whether 165bp fragments are contained in a positioning interval of Frl genes in a genome of a tomato to be detected, and selecting the tomato to be detected containing 165bp fragments for cultivation to obtain the tomato with tomato neck rot and root rot resistance;
the positioning interval of the Frl gene is an interval of 4.2-5.1Mb of chromosome 9 of tomato;
the 165bp fragment is 1) or 2) as follows:
1) The nucleotide sequence is sequence 3;
2) And (3) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotide residues for the nucleotide sequence shown in the sequence 3 in the sequence table and has the same function.
In the method, whether 165bp fragments are contained in the positioning interval of the Frl gene in the tomato genome to be detected or not is detected by using the set of primers in claim 2 to carry out PCR amplification on the tomato genome DNA to be detected,
detecting the size of a PCR product; judged according to the following standard a or standard B:
the standard a is as follows:
if the product only has a single fragment of 450-440bp, the tomato to be detected is not resistant to or candidate for not being resistant to the tomato neck rot and root rot;
if the product only has a single fragment of 605-615bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 605-615bp and 450-440bp, the tomato resistance to be detected is or is candidate to resist the tomato neck rot and root rot;
the standard B is as follows:
if the product only has a single fragment of 444bp, the tomato to be detected is not resistant to or candidate for not resisting the neck rot and root rot of the tomato;
if the product only has a single fragment of 609bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 609bp and 444bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot.
Or the invention provides a method for identifying or assisting in identifying the resistance of tomatoes to neck rot and root rot of tomatoes, which comprises the following steps:
PCR amplification is carried out on the tomato genome DNA to be detected by using the complete set of primers,
detecting the size of a PCR product; judged according to the following standard a or standard B:
the standard a is as follows:
if the product only has a single fragment of 450-440bp, the tomato to be detected is not resistant to or candidate for not being resistant to the tomato neck rot and root rot;
if the product only has a single fragment of 605-615bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 605-615bp and 450-440bp, the tomato resistance to be detected is or is candidate to resist the tomato neck rot and root rot;
the standard B is as follows:
if the product only has a single fragment of 444bp, the tomato to be detected is not resistant to or candidate for not resisting the neck rot and root rot of the tomato;
if the product only has a single fragment of 609bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 609bp and 444bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot.
Or the invention provides a method for identifying or assisting in identifying whether tomatoes are tomatoes resistant to neck rot and root rot, which comprises the following steps:
PCR amplification is carried out on the tomato genome DNA to be detected by using the complete set of primers,
detecting the size of a PCR product; judged according to the following standard a or standard B:
the standard a is as follows:
if the product only has a single fragment of 450-440bp, the tomato to be detected is not resistant to or candidate for not being resistant to the tomato neck rot and root rot;
if the product only has a single fragment of 605-615bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 605-615bp and 450-440bp, the tomato resistance to be detected is or is candidate to resist the tomato neck rot and root rot;
the standard B is as follows:
if the product only has a single fragment of 444bp, the tomato to be detected is not resistant to or candidate for not resisting the neck rot and root rot of the tomato;
if the product only has a single fragment of 609bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 609bp and 444bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot.
Or the invention provides a method for cultivating or assisting in cultivating tomatoes resistant to the neck rot and root rot of the tomatoes, which comprises the following steps of:
PCR amplification is carried out on the tomato genome DNA to be detected by using the complete set of primers,
detecting the size of a PCR product; judged according to the following standard a or standard B:
the standard a is as follows:
if the product only has a single fragment of 450-440bp, the tomato to be detected is not resistant to or candidate for not being resistant to the tomato neck rot and root rot;
if the product only has a single fragment of 605-615bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 605-615bp and 450-440bp, the tomato resistance to be detected is or is candidate to resist the tomato neck rot and root rot;
the standard B is as follows:
if the product only has a single fragment of 444bp, the tomato to be detected is not resistant to or candidate for not resisting the neck rot and root rot of the tomato;
if the product only has a single fragment of 609bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 609bp and 444bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot.
The detection of whether the 165bp fragment is contained in the positioning region of the Frl gene in the genome of the tomato variety is also the protection scope of the invention.
The invention discovers that a 165bp large fragment inserted in a gene interval region in a Frl gene positioning region (in a region of 4.2-5.1Mb of a No. 9 chromosome of a tomato) in a genome of a tomato variety is related to resistance to tomato neck rot and root rot, and according to the fragment, a PCR-based molecular marker is designed, so that whether a tomato material to be detected contains the Frl gene and whether the Frl gene is homozygous or not can be detected, the breeding period can be shortened, and the accuracy of breeding selection can be improved.
Drawings
FIG. 1 is an electrophoretogram of molecular markers closely linked to the Frl gene for detection of tomato resistance, inbred lines and commercial varieties.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The invention is further illustrated by the following specific embodiments, which are not intended to limit the scope of the invention. The experimental methods used in the examples below were according to conventional conditions, or according to the conditions recommended by the manufacturer, unless otherwise specified.
Tomato varieties Ailsa Craig (LA 2838A), LA3769, LA3292, LA3247, LA2706, LA2827, LA2828, LA2829, LA2830 used in the examples below were all obtained from the American tomato genetic resources center (TGRC, http:// TGRC. Ucdavis. Edu /), the commercial variety experimental materials Kjeldahl, pizza, and Zhuli 2 were purchased from the seed company of Tageta, and European were purchased from the seed company of the agricultural technology, inc. of Beijing and Zener, the manufacturing process thereof was carried out in the examples below:
wherein tomato varieties LA3292, LA3273, LA2827, LA2828, LA2829 and LA2830 and commercial varieties Kwando, pizza, zhuli No. 2 and European scallop contain Frl sites and have high resistance to tomato neck rot and root rot; commercial varieties of Kavin, pizza, zhuli No. 2 and European scallopFor hybridization F 1 And (3) replacing. Tomato varieties AC, LA3769, LA2706 and commercial varieties euclidean do not contain the Frl site, and are highly susceptible to neck rot and root rot.
Example 1 acquisition of molecular markers and establishment of method for identifying tomato neck rot and root rot resistance
1. Obtaining of molecular marker related fragments
By comparing tomato varieties containing the Frl locus of tomato neck rot with tomato varieties not containing the Frl locus of tomato neck rot, a 165bp large fragment is inserted into the gene interval region (in the region of 4.2-5.1Mb of chromosome 9 of tomato) of the Frl gene located in the genome of the tomato varieties containing the Frl locus of tomato neck rot than the tomato varieties not containing the Frl locus of tomato neck rot.
Through sequencing, the nucleotide sequence of the 165bp large fragment is sequence 3 in a sequence table.
Therefore, whether the tomato to be detected is resistant to the tomato neck rot and root rot can be judged by judging whether the positioning interval of the Frl gene of the tomato variety to be detected contains a 165bp large fragment or not, and the method specifically comprises the following steps:
detecting whether a large fragment of 165bp is contained in a positioning interval of Frl genes of the tomato variety to be detected, if so, resisting or candidate tomato neck rot and root rot of the tomato to be detected, and if not, not resisting or candidate tomato neck rot and root rot of the tomato to be detected.
2. Design of molecular marker related primer and establishment of method for identifying whether tomato to be detected is resistant to tomato neck rot and root rot
1. Design of molecular marker related primer
Based on the flanking sequences at the two ends of the 165bp large fragment, a PCR-based molecular marker was developed, and PCR amplification primers were located on the two sides of the 165bp insert.
The primer sequences for PCR-based molecular markers are as follows:
primer 1:5 '-ACAATTTTGCCTTTAGTGTTGG-3' (sequence 1 in the sequence Listing)
Primer 2:5 '-CCTAAAGTAGTGAAACTTATGGTG-3' (sequence 2 in the sequence Listing)
2. Establishment of method for identifying whether tomatoes to be detected resist tomato neck rot and root rot
1) Extracting genome DNA of tomato to be detected
2) PCR amplification
And (3) taking the genome DNA as a template, and carrying out PCR amplification by using the primer 1 and the primer 2 of the designed PCR-based molecular marker to obtain a PCR amplification product.
The PCR amplification system comprises: premix Taq DNA polymerase Mix (Nanjinouzan Biotechnology Co., ltd. P222-01) 10. Mu.L, primer 1 and primer 2 (final concentration in the system: 10. Mu.M) each 0.8. Mu.L, genomic DNA 1.5. Mu.L, and double distilled water to 20. Mu.L.
The reaction conditions for the PCR amplification are as follows: denaturation at 94℃for 20 sec, annealing at 56℃for 20 sec, extension at 72℃for 20 sec for a total of 35 cycles.
Electrophoresis is carried out on the PCR product by using 1% agarose gel, and the size of the PCR product is detected;
if the product only has a single fragment of 440-450bp, the genome of the tomato to be detected does not contain a large fragment of 165bp, the genotype is frl/frl, and the tomato to be detected is not resistant to or candidate for not being resistant to the tomato neck rot and root rot;
if the product only has a single fragment of 605-615bp, the 2 homologous chromosomes of the genome of the tomato to be detected all contain 165bp large fragments, the genotype of the tomato to be detected is Frl/Frl, and the resistance of the tomato to be detected is or is candidate for resisting the tomato neck rot root rot;
if the product contains 2 fragments of 605-615bp and 440-450bp, 1 homologous chromosome of the tomato genome to be detected contains 165bp large fragment, the other homologous chromosome does not contain 165bp large fragment, the genotype of the tomato to be detected is Frl/Frl, and the tomato to be detected is resistant to or candidate for resisting tomato neck rot and root rot.
Alternatively, the PCR product is sequenced,
if the product only has a single fragment of 444bp (sequence 4), the genome of the tomato to be detected does not contain a large fragment of 165bp, the genotype is frl/frl, and the tomato to be detected is not resistant to or candidate for not being resistant to the tomato neck rot and root rot;
if the product only has a single fragment of 609bp (sequence 5), the 2 homologous chromosomes of the genome of the tomato to be detected all contain large fragments of 165bp, the genotype of the tomato to be detected is Frl/Frl, and the resistance of the tomato to be detected is or is candidate for resisting the neck rot root rot of the tomato;
if the product contains 2 fragments of 609bp and 444bp, 1 homologous chromosome of the genome of the tomato to be detected contains a 165bp large fragment, the other homologous chromosome does not contain a 165bp large fragment, the genotype of the tomato to be detected is Frl/Frl, and the tomato to be detected is resistant or candidate to be resistant to the tomato neck rot root rot.
Example 2 application of molecular marker related fragment or primer in identifying whether or not tomato to be tested is neck rot and root rot resistant tomato
1. Detecting whether the tomato to be detected is resistant to the tomato neck rot and root rot
1. Extracting genome DNA of tomato to be detected
Genomic DNA of tomato anti-cold homozygous inbred line and commercial variety leaves shown in Table 1 was extracted using a plant DNA rapid extraction kit to obtain genomic DNA of each tomato variety.
The resistance of tomato resistant homozygous inbred lines and commercial varieties shown in table 1 is shown in column 4 of table 1, and is known.
2. PCR amplification
And respectively taking genome DNA of each tomato variety as a template, and carrying out PCR amplification by using the primer 1 and the primer 2 of the PCR-based molecular marker designed by the 1 to obtain PCR amplification products of each tomato variety.
The PCR amplification system comprises: premix Taq DNA polymerase Mix (Nanjinouzan Biotechnology Co., ltd. P222-01) 10. Mu.L, primer 1 and primer 2 (final concentration in the system: 10. Mu.L) each 0.8. Mu.L, genomic DNA 1.5. Mu.L, and double distilled water was added to 20. Mu.L.
The reaction conditions for the PCR amplification are as follows: denaturation at 94℃for 20 sec, annealing at 56℃for 20 sec, extension at 72℃for 20 sec for a total of 35 cycles.
3. Detection of
Respectively carrying out electrophoresis on the PCR amplification products of the tomato varieties by using 1% agarose gel, and detecting the PCR amplification products;
the PCR products described above were sequenced and,
if the product only has a single fragment of 444bp (sequence 4), the genome of the tomato to be detected does not contain a large fragment of 165bp, the genotype is frl/frl, and the tomato to be detected is not resistant to or candidate for not being resistant to the tomato neck rot and root rot;
if the product only has a single fragment of 609bp (sequence 5), the 2 homologous chromosomes of the genome of the tomato to be detected all contain large fragments of 165bp, the genotype of the tomato to be detected is Frl/Frl, and the resistance of the tomato to be detected is or is candidate for resisting the neck rot root rot of the tomato;
if the product contains 2 fragments of 609bp and 444bp, 1 homologous chromosome of the genome of the tomato to be detected contains a 165bp large fragment, the other homologous chromosome does not contain a 165bp large fragment, the genotype of the tomato to be detected is Frl/Frl, and the tomato to be detected is resistant or candidate to be resistant to the tomato neck rot root rot.
The results are shown in FIG. 1 and Table 1, M in FIG. 1: DL5000 DNA marker,1: AC,2: LA3769,3: LA3292,4: LA3247,5: LA2706,6: LA2827,7: LA2828,8: LA2829,9: LA2830, 10: kai, 11: pizza, 12: zhuli No. 2, 13: euroco, 14: the scallops; it can be seen that the PCR amplified products of the infectious inbred lines AC, LA3769, LA2706 and the commercial variety European-Ke tomato are 444bp single fragments, and the genotypes of the single fragments are estimated to be frl/frl, so that the tomato is not resistant to the neck rot and root rot of the tomato; the PCR amplified products of the disease-resistant inbred lines LA3292, LA3273, LA2827, LA2828, LA2829 and LA2830 tomato are single fragments of 609bp, and the genotype of the single fragments is presumed to be Frl/Frl, so that the tomato neck rot and root rot are resisted; commercial variety (hybrid F) 1 Generation), kavin, pizza, zhuli 2 and European shellfish are fragments of 609bp and 444bp, and the genotype of the tomato is presumed to be Frl/Frl, so that the tomato neck rot and root rot are resisted.
Table 1 shows the detection results of PCR-based molecular markers of tomatoes to be detected
In Table 1, lane number is listed in the first column, variety source is listed in the second column, variety name is listed in the third column, resistance of variety known to tomato neck rot root rot is listed in the fourth column, band size of cleavage product is listed in the fifth column, and genotype is listed in the fourth column.
The result shows that the PCR-based molecular marker judges that the resistance of the tomato to the neck rot and root rot is highly consistent with the resistance of the material, and can be used for identifying the anti-sensory material.
2. Detecting whether the tomato to be detected is resistant to the tomato neck rot and root rot by using the control mark
1. Obtaining tomato populations to be tested
LA2706 (non-resistant) and LA2828 (resistant) are used as parents to obtain F2 population, and 1005 strains are obtained.
2. Tomato neck rot and root rot resistance phenotype detection
The detection method comprises the following steps: slightly pulling up a piece of true leaf of tomato seedling, cleaning root system with clear water, and placing in concentration of 10 7 Individual/mL of tomato neck rot root rot pathogen (Fusarium oxysporum tomato neck rot root rot specialization Fusarium oxysporum f. Sp. Radicis-lycopersici; geng Lihua, li Changbao, wining, wang Lijun, chai Min. Identification of tomato neck rot pathogen and influence of different conditions on its growth [ J)]Plant pathology report, 2012,42 (05): 449-455.) spore suspension (solvent PDB medium (available from BD company, usa, cat No. 254920)) was root soaked for 10 min and transplanted into seedling pots containing sterilized soil. Placing into an incubator at 18 ℃ and culturing with 75% humidity. After 7 days of inoculation, the growth conditions of plants, including the growth state of the overground parts and whether the roots of the underground parts decay, are observed.
The main root is seriously rotten, and the stem base presents dark brown and slightly sunken spots, so that the tomato neck rot and root rot cannot be resisted. The main root does not rot, and the stem base part is white and has no scars, so that the tomato neck rot and root rot resistance is realized.
As a result, as shown in Table 2, it was found that 775 strains were resistant to tomato neck rot and root rot and 230 strains were not resistant to tomato neck rot and root rot in 1005 strain F2 population.
3. PCR-based molecular marker detection
The method is the same as the first method, and the result is shown as the PCR-based molecular marker in Table 2, and the accuracy is 100% consistent with the phenotype identification result.
3. Control C2-25 molecular marker detection
C2-25 molecular marker detection of the same samples, methods are shown in Liu Lei, wang Hui, li Wenli, wang Fu and 2018. Markers linked to Frl of tomato neck rot and root rot disease resistance gene and application. Jiangsu agricultural science 46,91-93.
C2-25 molecular marker:
primer 1: ATGGGCGCTGCATGTTTCGTG-
Primer 2: -ACACCTTTGTTGAAAGCCATCCC-
The results are shown in the C2-25 molecular markers in Table 2, and it can be seen that the accuracy is 98.12% compared with the phenotypic assay results.
TABLE 2
The result shows that the accuracy rate of the resistance identification of the molecular marker is higher than that of C2-25.
While the invention has been described in detail in the foregoing general description and specific embodiments, it is evident that modifications and improvements will readily occur to those skilled in the art based on the invention. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> Shandong Shou vegetable seed group Co., ltd, research institute of China academy of sciences and developmental biology
<120> molecular marker closely linked with tomato neck rot resistance gene Frl and application thereof
<160> 5
<170> PatentIn version 3.5
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<400> 4
acaattttgc ctttagtgtt ggatttgaaa gtgattagac gttatgcgaa agattataat 60
tgcaaataat ataattgata aattagatga caaaaatcat actaaaaact aatgttatta 120
tatgaaaact gatataaaga aaaatattaa aataattgag agattaaatc tcccaataaa 180
caaaactact aaacgactac tttgtggatt ctattgtgtt atgctatgag aaaaaagatt 240
ttcaatttat agatctccaa atttttcctc taagaaaata gtaaaccaaa catgaaaaaa 300
tataagagag tgaaaaagtg taccatattg aatattatta atatataagg ctaattcatc 360
tattattttt ttctaatatg ctctatcaaa caacttcaag gtgtctttaa ttagggtaaa 420
caccataagt ttcactactt tagg 444
<210> 5
<211> 609
<212> DNA
<213> Artificial sequence
<400> 5
acaattttgc ctttagtgtt gggtttgaaa gtgattagac gtcatgcgga aatttacaat 60
tgcaaataat gtagttgata aattagatga caaaaattat aataaaaaaa aattaatgtt 120
attatatgaa aactgatata aagaaaacta ttaaaataat tgagagatta aatctcccaa 180
taaacaaaac taactaaacg attactttgt ggattctatt atgttatgct atgagaataa 240
agattttcaa tttatagatc tccaaatttc ccctctaaga aaataataaa ccaaacatga 300
aagaaaattg tattttttct ttccgcaaaa ataaaacatt tatgataata ctttgacttt 360
tctttaagaa aaaaaattaa actttaaata aaaaaagaaa atcagtacaa aaccctaaca 420
agcatgcatt aaacccttgt taataccata gttctccata tatatatgga gagagtgaaa 480
aagtgtacca cattgaatat tattaatata taaagctaaa tcatctatta tttttttcta 540
atatgctcta tcaaacaaat tcaaggtgtc tttaattagg ataaacacca taagtttcac 600
tactttagg 609
Claims (10)
1. Detecting whether a substance containing 165bp fragments in a positioning interval of Frl genes in a genome of a tomato variety is applied to identification or auxiliary identification of tomato neck rot resistance of tomatoes;
the positioning interval of the Frl gene is an interval of 4.2-5.1Mb of chromosome 9 of tomato;
the nucleotide sequence of the 165bp fragment is a sequence 3;
the substances for detecting whether 165bp fragments are contained in the locating interval of the Frl gene in the genome of the tomato variety are 1) or 2):
1) The primer set consists of a single-stranded DNA molecule shown as a sequence 1 in a sequence table and a single-stranded DNA molecule shown as a sequence 2 in the sequence table;
2) PCR reagents or kits containing the kit of primers described in 1).
2. Detecting whether a 165bp fragment-containing substance is contained in a positioning interval of Frl genes in a genome of a tomato variety, and applying the substance to cultivation or auxiliary cultivation of tomatoes resistant to tomato neck rot and root rot;
the positioning interval of the Frl gene is an interval of 4.2-5.1Mb of chromosome 9 of tomato;
the nucleotide sequence of the 165bp fragment is a sequence 3;
the substances for detecting whether 165bp fragments are contained in the locating interval of the Frl gene in the genome of the tomato variety are 1) or 2):
1) The primer set consists of a single-stranded DNA molecule shown as a sequence 1 in a sequence table and a single-stranded DNA molecule shown as a sequence 2 in the sequence table;
2) PCR reagents or kits containing the kit of primers described in 1).
3. A method for identifying or assisting in identifying tomato neck rot and root rot resistance of tomato is to detect whether 165bp fragments are contained in a positioning interval of Frl gene in a genome of tomato to be detected,
if the 165bp fragment is contained, the tomato resistance to be detected is or is candidate to resist the tomato neck rot and root rot;
if the 165bp fragment is not contained, the tomato to be detected is not resistant to the neck rot or the root rot of the candidate tomato;
the positioning interval of the Frl gene is an interval of 4.2-5.1Mb of chromosome 9 of tomato;
the nucleotide sequence of the 165bp fragment is sequence 3.
4. A method for identifying or assisting in identifying whether tomato is tomato resistant to tomato neck rot and root rot is provided, which is to detect whether 165bp fragments are contained in the positioning interval of Frl gene in the genome of tomato to be detected,
if the 165bp fragment is contained, the tomato to be detected is or is candidate to be tomato with tomato neck rot resistance and root rot resistance;
if the tomato does not contain the 165bp fragment, the tomato to be detected is not or is not a candidate tomato for resisting the tomato neck rot and root rot;
the positioning interval of the Frl gene is an interval of 4.2-5.1Mb of chromosome 9 of tomato;
the nucleotide sequence of the 165bp fragment is sequence 3.
5. A method for cultivating or assisting in cultivating tomato with tomato neck rot and root rot resistance is to detect whether 165bp fragments are contained in a positioning region of Frl genes in a genome of a tomato to be detected, and select the tomato to be detected containing 165bp fragments for cultivation to obtain tomato with tomato neck rot and root rot resistance;
the positioning interval of the Frl gene is an interval of 4.2-5.1Mb of chromosome 9 of tomato;
the nucleotide sequence of the 165bp fragment is sequence 3.
6. The method according to any one of claims 3-5, wherein:
the method for detecting whether 165bp fragments are contained in the positioning interval of the Frl gene in the tomato genome to be detected is to carry out PCR amplification on the tomato genome DNA to be detected by using the set of primers in claim 2,
detecting the size of a PCR product; judging according to the following standard:
if the product only has a single fragment of 444bp, the tomato to be detected is not resistant to or candidate for not resisting the neck rot and root rot of the tomato;
if the product only has a single fragment of 609bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 609bp and 444bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot.
7. A method for identifying or aiding in the identification of resistance to neck rot and root rot of tomatoes comprising the steps of:
PCR amplification of tomato genomic DNA to be tested using the set of primers of claim 2,
detecting the size of a PCR product; judging according to the following standard:
if the product only has a single fragment of 444bp, the tomato to be detected is not resistant to or candidate for not resisting the neck rot and root rot of the tomato;
if the product only has a single fragment of 609bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 609bp and 444bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot.
8. A method for identifying or aiding in the identification of whether tomatoes are resistant to neck rot and root rot, comprising the steps of:
PCR amplification of tomato genomic DNA to be tested using the set of primers of claim 2,
detecting the size of a PCR product; judging according to the following standard:
if the product only has a single fragment of 444bp, the tomato to be detected is not resistant to or candidate for not resisting the neck rot and root rot of the tomato;
if the product only has a single fragment of 609bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 609bp and 444bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot.
9. A method for cultivating or assisting in cultivating tomatoes resistant to neck rot and root rot of tomatoes, comprising the following steps:
PCR amplification of tomato genomic DNA to be tested using the set of primers of claim 2,
detecting the size of a PCR product; judging according to the following standard:
if the product only has a single fragment of 444bp, the tomato to be detected is not resistant to or candidate for not resisting the neck rot and root rot of the tomato;
if the product only has a single fragment of 609bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot;
if the product contains 2 fragments of 609bp and 444bp, the tomato to be detected is resistant to or candidate for resisting the tomato neck rot and root rot.
10. The method according to claim 1 or 2, wherein the presence or absence of a 165bp fragment in the localization interval of the Frl gene in the genome of the tomato variety is detected.
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