SNP (Single Nucleotide Polymorphism) marker combination and probe group for assistant selection of disease resistance of apple branch ring spot and application of SNP marker combination and probe group

Abstract

The invention belongs to the technical field of plant molecular breeding, and particularly relates to an SNP (Single Nucleotide Polymorphism) marker combination for assistant selection of apple branch and trunk ring spot disease resistance, a probe group and application thereof. The SNP marker combination is composed of SNP1 to SNP4; the nucleotide sequences of the SNP1 to the SNP4 are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 4. The invention provides an SNP (Single Nucleotide Polymorphism) marker combination for apple branch and trunk ring spot disease resistance, which is suitable for molecular auxiliary evaluation of apple tree germplasm resources on the apple branch and trunk ring spot disease resistance, excellent disease-resistant germplasm screening and breeding scheme design.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular breeding, and particularly relates to an SNP marker combination, a probe set and their applications for the assistant selection of apple Botryosphaeria canker resistance. Background Art

[0002] Apple Botryosphaeria canker, Botryosphearia canke r , Bot canker is one of the three major diseases currently harming the main apple production areas in China. The main pathogen of apple ring rot is Botryosphaeria dothidea ( Botryosphaeria dothidea ). The disease harms the branches and sometimes is called dry rot or rough bark disease. In severe cases, it even causes branch death, seriously restricting the development of China's apple industry. At present, the combination of chemical control and agricultural measures can control the incidence of apple ring rot to a certain extent. For chemical control, lime sulfur can be selected as the orchard early spring management agent, and carbendazim and dithianon are also commonly used agents by fruit farmers. For agricultural measure management and control, for example, cleaning the orchard in winter, thoroughly removing the diseased leaves in the orchard, increasing the application of organic fertilizers, appropriately reducing the use of nitrogen fertilizers, paying attention to pruning, and improving the ventilation and light transmission conditions of the orchard. However, the existing control methods have disadvantages such as high cost and unstable effects. Therefore, improving the disease resistance of cultivated varieties and breeding resistant varieties are the most effective control methods.

[0003] Traditional apple breeding methods usually require a long time and a large amount of resources, and it is difficult to accurately predict the trait performance of new varieties. Using molecular marker-assisted breeding can improve breeding efficiency, shorten the breeding cycle, reduce costs, and improve the accuracy and stability of varieties. Therefore, developing relevant molecular markers is very important for apple disease resistance breeding.

[0004] The resistance of apple Botryosphaeria canker shows a quantitative trait controlled by minor polygenes, which contains major genes with significant effects. In the research on the genetic analysis and molecular markers of apple Botryosphaeria canker resistance, by investigating the field inoculation data of 5 Botryosphaeria canker pathogens on the branches of the hybrid offspring population of 'Jonathan' × 'Golden Delicious', and using SSR primers for segregation analysis, 14 pairs of SSR markers related to apple Botryosphaeria canker resistance genes were screened out. The fruits of 1733 F1 generation offspring lines in the 'Jonathan' × 'Golden Delicious' hybrid population were inoculated with 4 Botryosphaeria canker pathogen strains Zz26, Ls1, Lw023 and Lw048. Using BSA-seq, a total of 46 disease resistance QTLs were detected. Combining the resequencing and transcriptome analysis of the two parents, 57 candidate genes for fruit ring rot resistance / susceptibility were predicted. However, these current SSR markers are not accurate enough for the development and application of molecular markers for apple resistance to Botryosphaeria canker, and cannot accurately determine the resistance or susceptibility of the plant to Botryosphaeria canker. Summary of the Invention

[0005] To solve the problem that the current SSR markers in the existing technology are not accurate enough for the development and application of molecular markers for apple resistance to Botryosphaeria dothidea, and cannot accurately determine the resistance and susceptibility of the plant to Botryosphaeria dothidea, the purpose of the present invention is to develop a set of molecular markers suitable for marker-assisted selection of apple resistance to Botryosphaeria dothidea, so as to conduct marker-assisted selection of apple resistance to Botryosphaeria dothidea at the seedling stage of hybrid offspring seedlings, and achieve the purpose of improving breeding efficiency. To achieve the above purpose, the present invention adopts the following technical solutions.

[0006] One of the purposes of the present invention is to provide an SNP marker for assisting in the selection of apple resistance to Botryosphaeria dothidea, and the SNP marker includes SNP1 to SNP4.

[0007] The SNP1 is Chr00_23716755 C / T, and the nucleotide sequence of Chr00_23716755 C / T is shown in SEQ ID NO.1, where there is a mutation from C to T at the 201bp position. The T allele variation of Chr00_23716755 C / T is the genotype resistant to Botryosphaeria dothidea.

[0008] The SNP1 is a mutation from C to T at the 23716755th position of apple chromosome No. 0.

[0009] The SNP2 is Chr02_20663119 A / C, and the nucleotide sequence of Chr02_20663119 A / C is shown in SEQ ID NO.2, where there is a mutation from A to C at the 201bp position. The C allele variation of Chr02_20663119 A / C is the genotype resistant to Botryosphaeria dothidea.

[0010] The SNP2 is a mutation from A to C at the 20663119th position of apple chromosome No. 2.

[0011] The SNP3 is Chr14_24248741 A / C, and the nucleotide sequence of Chr14_24248741 A / C is shown in SEQ ID NO.3, where there is a mutation from A to C at the 198bp position. The C allele variation of Chr14_24248741 A / C is the genotype resistant to Botryosphaeria dothidea.

[0012] The SNP3 is a mutation from A to C at the 24248741st position of apple chromosome No. 14.

[0013] The SNP4 is Chr14_29982606 A / G, and the nucleotide sequence of Chr14_29982606 A / G is shown in SEQ ID NO.4, where a mutation from A to G exists at the 201bp position. The G allele variation of Chr14_29982606 A / G is the genotype resistant to apple ring rot of branches and trunks.

[0014] The SNP4 is a mutation from A to G at the 29982606th position of the fourteenth chromosome of apple.

[0015] The SNP marker combination provided by the present invention has the advantages of being able to simply and quickly determine the resistance and susceptibility of a certain apple fruit tree plant to apple ring rot of branches and trunks, and the prediction result has extremely high accuracy, which can solve the problem that the current SSR markers are not accurate enough for the development and application of molecular markers for apple resistance to apple ring rot of branches and trunks and cannot accurately determine the resistance and susceptibility of the plant to apple ring rot of branches and trunks.

[0016] The second object of the present invention is to provide a probe set for an SNP marker combination for assisting in the selection of apple ring rot disease resistance, including GenoBaits DNA probes for detecting the SNP1 to the SNP4.

[0017] The nucleotide sequence of the GenoBaits DNA probe for detecting the SNP1 is shown in SEQ ID NO.5 in sequence.

[0018] The nucleotide sequence of the GenoBaits DNA probe for detecting the SNP2 is shown in SEQ ID NO.6 in sequence.

[0019] The nucleotide sequence of the GenoBaits DNA probe for detecting the SNP3 is shown in SEQ ID NO.7 in sequence.

[0020] The nucleotide sequence of the GenoBaits DNA probe for detecting the SNP4 is shown in SEQ ID NO.8 in sequence.

[0021] The third object of the present invention is to use the SNP marker combination for replicating and selecting apple ring rot disease resistance to identify the disease resistance of germplasm resources of the genus Malus, excavate excellent disease-resistant germplasm, create new disease-resistant materials, and at the same time improve the working efficiency of parental selection and combination matching, and further improve the breeding efficiency.

[0022] The fourth object of the present invention is to provide the application of the SNP marker combination or the probe set in the preparation of a kit for determining apple ring rot disease resistance.

[0023] Preferably, the kit includes the SNP marker combination or the probe set.

[0024] A fifth object of the present invention is to provide the application of the SNP marker combination, the probe set or the kit in determining the resistance to apple ring rot of apple branches.

[0025] Preferably, it includes the following steps: Extract the genomic DNA of the apple plant to be tested.

[0026] Using the genomic DNA of the apple plant to be tested as a template, perform PCR amplification with the probe set to obtain a PCR amplification product.

[0027] Perform genotyping detection on the PCR amplification product.

[0028] Determine the resistance to apple ring rot of the branches of the apple plant to be tested.

[0029] Preferably, resistance allele variations can occur at each of the SNP1 to SNP4: among them, the resistance in the resistance allele variation refers to the resistance to apple ring rot of branches.

[0030] The resistance allele variation at SNP1 is the T allele variation; when the T allele variation occurs at SNP1, it is the genotype resistant to apple ring rot of branches.

[0031] The resistance allele variation at SNP2 is the C allele variation; when the C allele variation occurs at SNP2, it is the genotype resistant to apple ring rot of branches.

[0032] The resistance allele variation at SNP3 is the C allele variation; when the C allele variation occurs at SNP3, it is the genotype resistant to apple ring rot of branches.

[0033] The resistance allele variation at SNP4 is the G allele variation. When the G allele variation occurs at SNP4, it is the genotype resistant to apple ring rot of branches.

[0034] When the number of resistance allele genes at SNP1 to SNP4 is 7 or 8, the apple plant to be tested is highly resistant to apple ring rot of branches.

[0035] After excluding the above-mentioned single plants highly resistant to apple ring rot of branches, if the number of resistance allele genes at SNP1 to SNP4 is 4 to 6, the apple plant to be tested is resistant to apple ring rot of branches.

[0036] After excluding the above-mentioned single plants highly resistant to apple ring rot of branches and resistant to apple ring rot of branches, if the number of resistance allele genes at SNP1 to SNP4 is 1 to 3, the apple plant to be tested is susceptible to apple ring rot of branches.

[0037] When the number of resistance alleles at the SNP1 to SNP4 is 0, the apple plant to be tested is highly susceptible to Botryosphaeria dothidea.

[0038] Preferably, the reaction conditions for the PCR amplification are 98°C for 2 min; 98°C for 30 s, 65°C for 30 s, 72°C for 40 s for 7 cycles; 72°C for 4 min.

[0039] Preferably, the reaction system for the PCR amplification is 10 μL of GenoBaits PCR Master Mix, 1 μL of GenoBaitsUni_oligo, 5 μL of GenoBaits DNA index, and 4 μL of nuclease-free water.

[0040] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a SNP marker combination for the assistant selection of the resistance to Botryosphaeria dothidea in apples. The SNP marker combination includes 4 major SNP markers related to the resistance to Botryosphaeria dothidea in apples, namely 4 SNP markers; the 4 SNP markers include SNP1 to SNP4. The nucleotide sequences of the SNP1 to SNP4 are shown in SEQ ID NO.1 to SEQ ID NO.4 in sequence. The SNP marker combination is applicable to the molecular assistant evaluation, screening, and breeding program design of apple plant germplasm resources. The SNP marker combination provided by the present invention has the advantages of simply and quickly determining the resistance and susceptibility of a certain apple tree plant to Botryosphaeria dothidea, and the prediction result has extremely high accuracy, and can solve the problem that the current SSR markers are not accurate enough for the molecular marker development and application of apple resistance to Botryosphaeria dothidea and cannot accurately determine the resistance and susceptibility of the plant to Botryosphaeria dothidea.

[0041] 2. The present invention has developed 4 major SNP markers related to the resistance to Botryosphaeria dothidea in apples, which are applicable to marker-assisted selection of the resistance to Botryosphaeria dothidea in apple breeding materials at the seedling stage.

[0042] 3. The present invention has developed 4 major SNP markers related to the resistance to Botryosphaeria dothidea in apples, which are applicable to the molecular assistant evaluation of the resistance to Botryosphaeria dothidea in apple germplasm resources, as well as the screening of excellent disease-resistant germplasms and the design of breeding programs.

[0043] 4. The present invention has developed 4 major-effect SNP markers related to the resistance to apple ring rot on branches, which have low cost and high accuracy. Using the above 4 major-effect SNP markers related to the resistance to apple ring rot on branches, the severity of apple ring rot on branches of 1,506 hybrid offspring and germplasm resource materials of the genus Malus was predicted. The Pearson correlation coefficient r between the number of resistance alleles and the severity of apple ring rot on branches was 0.925 (n = 1,055). BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the linear relationship between the number of resistance alleles and the severity of apple ring rot on branches in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] The present invention will be described in detail below with reference to the drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0046] Example 1 1. Phenotypic identification of the resistance to apple ring rot on branches The resistance to apple ring rot on branches of 361 germplasm resources of the genus Malus and 280 offspring lines of the hybrid population of 'Jonathan' × 'Golden Delicious' was inoculated and identified continuously for 2 years. The strains Zz26, Ls1, LW048, and LW023 were used for inoculation respectively. The incidence rate of apple ring rot on branches and the lesion diameter were used as the resistance indexes for the resistance inoculation identification.

[0047] 2. Mining of the variation points of the resistance to apple ring rot on branches For the hybrid offspring population, BSA-seq was used to mine the QTL related to the resistance to apple ring rot on branches in the hybrid population of 'Jonathan' × 'Golden Delicious'. Genome-wide association analysis was performed on the resistance to apple ring rot on branches of 253 germplasm resources of the genus Malus, and a total of 40 significant association intervals related to the resistance to apple ring rot on branches were located.

[0048] Among them, the full English name of BSA-seq is bulked segregant analysis sequencing, that is, the bulked segregant analysis method.

[0049] The English abbreviation of genome-wide association analysis is GWAS, and its full English name is Genome Wide Association Study.

[0050] 3. Development of molecular markers for the resistance to apple ring rot on branches According to the mutation type, gene expression level, and functional annotation, mutation sites of candidate genes were selected in each QTL and GWAS interval related to the resistance to apple ring rot on branches and trunks, and a total of 40 SNP and InDel molecular markers were designed and distributed on 8 chromosomes, namely Chr00_11231274, Chr00_11240324, Chr00_23716755, Chr00_45990013, Chr02_13403916, Chr02_13473882, Chr02_16535802, Chr02_17279645Chr02_17693842, Chr02_18601387, Chr02_18657362, Chr02_20663119, Chr02_21547202, Chr02_21856203, Chr02_22956039, Chr02_23603449, Chr02_25350300, Chr02_28342525, Chr04_28954120, Chr05_5094005, Chr05_47489677, Chr10_24655771, Chr10_27164253, Chr10_27354881, Chr10_27706538, Chr10_28057784, Chr10_29638773, Chr10_30268315, Chr10_31603916, Chr10_34064311, Chr13_2622717, Chr14_24248741, Chr14_27043108, Chr14_29624750, Chr14_29833236, Chr14_29982606, Chr14_29996846, Chr14_30317685, Chr14_31432200, Chr17_22317354. The markers adopted the GenoBaits strategy, captured the target sequences through DNA probes, and then performed marker genotyping using Illumina second-generation resequencing.

[0051] 4. Estimation of the genotype effect value of the apple ring rot resistance marker on branches and trunks and screening of the major-effect markers A training population was constructed using 128 germplasm resources of Malus and 208 hybrid progeny lines of 'Jonathan' × 'Golden Delicious'. The GenoBaits strategy was used to genotype the above 40 molecular markers. By calculating the difference between the phenotypic mean of the same genotype at a certain locus in the training population and the phenotypic mean of the total training population, the genotypic effect value and marker effect value of each marker on the resistance to Botryosphaeria dothidea in the training population were estimated. Markers with larger marker effect values were screened out, and a total of 4 SNP markers were selected as the major-effect markers for the resistance to Botryosphaeria dothidea in apples.

[0052] Among them, the major-effect markers for the resistance to Botryosphaeria dothidea in apples are Chr00_23716755 C / T, Chr02_20663119 A / C, Chr14_24248741 A / C, and Chr14_29982606 A / G, respectively.

[0053] Chr00_23716755 C / T is SNP1, and its nucleotide sequence is shown in SEQ ID NO.1: TTTTTTAGTTGAATATAATATATAAATTGGATTCTCTACAAATCCTAAAAATAAGAGCTAATTCCATTTTACATGATTTAAGTTTGAGGTAATTCTTAAAATGGGTATGAACTTAAATTTTCCCACATTTGATGTTTTGATTTTTTTTATTATTTTATATATATTTGTCTCAGGCCCTGTTTGATATTATTTTTTCTCAT[C / T]AAAAACTACTTCACTTTACTGCCGCAACCATCTCTACCACTGCTGTCATTGTCACCACCCCCACTACAATGTCCACTTTTGTCACTATATACAATTATATCACTTTCACATTAAAATTTACCGAACACTGCTTTTCATACTTGCAACACTATTAAAAATACATTTTATCAAACGCTCAATTGCTTTATTTTACAAATGATT; among them, there is a mutation from C to T at the 201bp position. The T allele variation of Chr00_23716755 C / T is the genotype resistant to Botryosphaeria dothidea.

[0054] SNP1 is a mutation from C to T at the 23716755th position on chromosome 0 of apples.

[0055] Chr02_20663119 A / C is SNP2, and its nucleotide sequence is shown in SEQ ID NO.2: TTGTTTTTCTCAACCGGGTTCTGACCCAACCGCTTGTAACCCGACCCTGGCTTGCTGCAGCACAAAGATTTAGCCCCAGCGGACAGCTTCCGACCCCAGGTGAGCAGCCTTGACATGGGTTTGTAGCTGCAAGCCGAGGGAGAACCCGGGTTGAGCCGGGCATATTCGGGTCGGGTTCGGGTCCCACGGACGAAGAACCA[A / C]CGGCTGACCCGGCAAAGGCGCTTTCCGAGCTTGAATCCTCTGAACCTTGCCATTTTTAAGGGAGAAAAAGGAGGAAAATCTGTCTTGTGCGTTCTGAAATTTGGGTTGAGAAGTGAATGGATTTTCGTCAGAGATTGACTTGGTTCAAGAAAATGGCCACGGGTCGGCCTCGGACATGGTGAGACGAGAGAGAGTTAAGC; Among them, there is a mutation from A to C at the 201bp position. The C allele variation of Chr02_20663119A / C is the genotype resistant to apple ring rot of branches.

[0056] SNP2 is a mutation from A to C at the 20663119th position of the second chromosome of apple.

[0057] Chr14_24248741 A / C is SNP3, and its nucleotide sequence is shown in SEQ ID NO.3: TTTTCTTGTTCGATCAGTTCCTCTCACTGAAACTTGCTTCTTTGAATCCAATGTTCTTTGACTTTGGAATGGACATGGGTGCTTTAGTGGTTAAACCAGAGGTACATAGTTAAAAAGTATCAGTTATGTTCAATCCCACTTCACAGCGTTTTCAAACACAGGAATGAAAATCTTCCGTCCGTTTCTAGTCGGAATAG[A / C]CGAGAGTTTCATTCTCAAAAAACCTAATTCATGAACCAAAAATTACTAATGTAATTACAATTTGGTTTTTATTTTTTGCCATGCAGAGATTGTGTAGCATGGAATCACCATATCCATCTGTGCAACAATGCAACCCAATACAGTCCACACCTTTTGCTGATACATCCACCAATATCACTGCCACCACTGCCGCTGCCACT; Among them, there is a mutation from A to C at the 198bp position. The C allele variation of Chr14_24248741 A / C is the genotype resistant to apple ring rot of branches.

[0058] SNP3 is a mutation from A to C at the 24,248,741st position of the fourteenth chromosome of apple.

[0059] Chr14_29982606 A / G, namely SNP4, and its nucleotide sequence is shown in SEQ ID NO.4: AGATAGCAAATGTACTGTTAATGTGCATTATAGTTTACCTGAAAGAAAGCCTTGCCCTCCTCGATCTCTTCAAGGATTTCTTTCTTTTGCTGGTCTATAACTTTGTATTTCTCAGAATCTATTGATCGGATGCTTACTGAACGTGAAGGCATTTTCTAGAAAAATATGCACAAATAATGTCATGTTAAAAACCAAAGCCA[A / G]TCTTCCTTAAAATATATACACAGAGCTCACAGGAGCATATGCTACAAGAGGGAAAGAAACTATAACCAAAAGAACGGAAAAATCAACTCAGATAATGCAAAGAAATTGTGTACCTCTTGCCCAGTATAGTTCCAAATCTCGGCAGACGAATTACACGATGGATCAAGACTCAAATACCCTTTATTTTTAAGAGGTACTATG; Among them, there is a mutation from A to G at the 201bp position. The G allele variation of Chr14_29982606 A / G is the genotype resistant to apple ring rot disease of branches and trunks.

[0060] SNP4 is a mutation from A to G at the 29982606th position of the fourteenth chromosome of apple.

[0061] The flanking sequence information of the SNP variant sites corresponding to the above 4 major SNP markers related to the resistance of apple ring rot disease of branches and trunks is as follows. The non-variant base (before) and the variant base (after) are marked in the square brackets "[ ]", and are separated by " / ".

[0062] The GenoBaits DNA probe sequence information corresponding to the above 4 major SNP markers related to the resistance of apple ring rot disease of branches and trunks is as follows: The nucleotide sequence of the DNA probe for detecting SNP1 is shown in SEQ ID NO.5: GCCCTGTTTGATATTATTTTTTCTCATCAAAAACTACTTCACTTTACTGCCGCAACCATCTCTACCACTGCTGTCATTGTCACCACCCCCACTACAATGTCCACTTTTGT.

[0063] The nucleotide sequence of the DNA probe for detecting SNP2 is shown in SEQ ID NO.6: GGTGAGCAGCCTTGACATGGGTTTGTAGCTGCAAGCCGAGGGAGAACCCGGGTTGAGCCGGGCATATTCGGGTCGGGTTCGGGTCCCACGGACGAAGAACCAACGGCTGA。

[0064] The nucleotide sequence of the DNA probe for detecting SNP3 is shown in SEQ ID NO.7: ATCAGTTATGTTCAATCCCACTTCACAGCGTTTTCAAACACAGGAATGAAAATCTTCCGTCCGTTTCTAGTCGGAATAGAGAAACGAGAGTTTCATTCTCAAAAAACCTA。

[0065] The nucleotide sequence of the DNA probe for detecting SNP4 is shown in SEQ ID NO.8: CTATTGATCGGATGCTTACTGAACGTGAAGGCATTTTCTAGAAAAATATGCACAAATAATGTCATGTTAAAAACCAAAGCCAATCTTCCTTAAAATATATACACAGAGCT。

[0066] 5. Marker-assisted selection method for resistance to Botryosphaeria dothidea of apple branches In the training population of this example, the resistance allele variations of the above 4 major SNP markers related to the resistance to Botryosphaeria dothidea of apple branches are as follows: The resistance allele variation at SNP1 is the T allele variation; when the T allele variation occurs at SNP1, it is the genotype resistant to Botryosphaeria dothidea.

[0067] The resistance allele variation at SNP2 is the C allele variation; when the C allele variation occurs at SNP2, it is the genotype resistant to Botryosphaeria dothidea.

[0068] The resistance allele variation at SNP3 is the C allele variation; when the C allele variation occurs at SNP3, it is the genotype resistant to Botryosphaeria dothidea.

[0069] The resistance allele variation at SNP4 is the G allele variation. When the G allele variation occurs at SNP4, it is the genotype resistant to Botryosphaeria dothidea.

[0070] There is an additive effect among the above 4 major SNP markers related to the resistance to Botryosphaeria dothidea of apple branches. The resistance to Botryosphaeria dothidea of apple germplasm resources or hybrid offspring is judged and selected according to the following criteria: ① If the sum of the number of resistance alleles of the 4 major SNP markers related to the resistance of apple branch canker in the sample to be tested is 7 or 8, then the sample is determined to be highly resistant to apple branch canker.

[0071] ② If the sum of the number of resistance alleles of the 4 major SNP markers related to the resistance of apple branch canker in the sample to be tested is 4 to 6, then the sample is determined to be resistant to apple branch canker.

[0072] ③ If the sum of the number of resistance alleles of the 4 major SNP markers related to the resistance of apple branch canker in the sample to be tested is 1 to 3, then the sample is determined to be susceptible to apple branch canker.

[0073] ④ If the sum of the number of resistance alleles of the 4 major SNP markers related to the resistance of apple branch canker in the sample to be tested is 0, then the sample is determined to be highly susceptible to apple branch canker.

[0074] As can be seen from the above, according to the 4 major SNP markers related to the resistance of apple branch canker, the disease resistance of apple seedlings to apple branch canker can be predicted, which greatly saves the breeding time.

[0075] Example 2 In order to verify the feasibility of the 4 major SNP markers related to the resistance of apple branch canker and their probe sets of the present invention, the following research was carried out: According to the genotyping data of the 4 major SNP markers related to the resistance of apple branch canker of 1506 apple materials, and according to the complementary epistatic effect between the above 4 major SNP markers related to the resistance of apple branch canker, and the judgment and selection criteria of the resistance of apple branch canker, the germplasm resources or hybrid offspring of the genus Malus were screened and judged: As Figure 1 shown, 1506 apple materials were divided into 4 grades. Specifically, if the sum of the number of resistance alleles of the 4 major SNP markers related to the resistance of apple branch canker in the sample to be tested is 7 or 8, then the sample is determined to be highly resistant to apple branch canker; if the sum of the number of resistance alleles of the 4 major SNP markers related to the resistance of apple branch canker in the sample to be tested is 4 to 6, then the sample is determined to be resistant to apple branch canker; if the sum of the number of resistance alleles of the 4 major SNP markers related to the resistance of apple branch canker in the sample to be tested is 1 to 3, then the sample is determined to be susceptible to apple branch canker; if the sum of the number of resistance alleles of the 4 major SNP markers related to the resistance of apple branch canker in the sample to be tested is 0, then the sample is determined to be highly susceptible to apple branch canker.

[0076] As can be seen from the above experimental results, the SNP marker combination provided by the present invention has comprehensive excellent alleles related to apple ring rot disease in wild species and related species of the genus Malus, with accurate prediction, covering multiple allele loci, and can integrate excellent alleles related to apple ring rot disease in wild species and related species of the genus Malus, which can solve the problems that the existing SNP marker combination has limitations in the comprehensiveness of prediction and application effect of apple ring rot disease in molecular assisted breeding and is difficult to meet the requirements of precision breeding.

[0077] It should be noted that when the present invention involves a numerical range, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent unnecessary repetition, preferred embodiments of the present invention are described.

[0078] Although the preferred embodiments of the present invention have been described, once those skilled in the art learn the basic creative concept, additional changes and modifications can be made to these embodiments, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A SNP marker combination for assisting in the selection of resistance to apple ring rot on branches and trunks, characterized in that, The SNP marker combination described above includes SNP1 to SNP4; SNP1 is a mutation from C to T at position 23716755 on chromosome 0 of apple, and its nucleotide sequence is as shown in SEQ ID NO.1; SNP2 is a mutation from A to C at position 20663119 on chromosome 2 of apple, and its nucleotide sequence is as shown in SEQ ID NO.2; SNP3 is a mutation from A to C at position 24248741 on chromosome 14 of apple, and its nucleotide sequence is as shown in SEQ ID NO.3; SNP4 is a mutation from A to G at position 29982606 on chromosome 14 of apple, and its nucleotide sequence is as shown in SEQ ID NO.

4.

2. A probe set for detecting the SNP marker combination for the auxiliary selection of the resistance to apple ring rot on apple branches as claimed in claim 1, characterized in that, It includes GenoBaits DNA probes for detecting SNP1 to SNP4; The nucleotide sequence of the GenoBaits DNA probe for detecting SNP1 is successively as shown in SEQ ID NO.5; The nucleotide sequence of the GenoBaits DNA probe for detecting SNP2 is successively as shown in SEQ ID NO.6; The nucleotide sequence of the GenoBaits DNA probe for detecting SNP3 is successively as shown in SEQ ID NO.7; The nucleotide sequence of the GenoBaits DNA probe for detecting SNP4 is successively as shown in SEQ ID NO.

8.

3. Use of the SNP marker combination according to claim 1 or the probe set according to claim 2 in the preparation of a kit for determining the resistance to apple ring rot of branches and trunks.

4. The application according to claim 3, characterized in that, The kit includes the SNP marker combination according to claim 1 or the probe set according to claim 2.

5. Use of the SNP marker combination according to claim 1 or the probe set according to claim 2 in determining the resistance to apple ring rot of branches and trunks.

6. The application according to claim 5, characterized in that, It includes the following steps: Extract the genomic DNA of the apple plant to be tested; Using the genomic DNA of the apple plant to be tested as a template, perform PCR amplification with the probe set to obtain a PCR amplification product; Perform genotyping detection on the PCR amplification product; Determine the resistance to apple ring rot of the branches and trunks of the apple plant to be tested.

7. The application according to claim 6, wherein Resistance allele variations can occur at SNP1 to SNP4: among them, the resistance in the resistance allele variation refers to the resistance to apple ring rot of branches and trunks; The resistance allele variation at SNP1 is the T allele variation; the resistance allele variation at SNP2 is the C allele variation; the resistance allele variation at SNP3 is the C allele variation; the resistance allele variation at SNP4 is the G allele variation; When the number of resistance alleles at SNP1 to SNP4 is 7 or 8, the apple plant to be tested is highly resistant to apple ring rot of branches and trunks; After excluding the above-mentioned single plants highly resistant to apple ring rot of branches and trunks, if the number of resistance alleles at SNP1 to SNP4 is 4 to 6, the apple plant to be tested is resistant to apple ring rot of branches and trunks; After excluding the single plants resistant to Botryosphaeria dothidea and highly resistant to Botryosphaeria dothidea mentioned above, if the number of resistant alleles at the SNP1 to SNP4 is 1 to 3, the apple plant to be tested is susceptible to Botryosphaeria dothidea; When the number of resistant alleles at the SNP1 to SNP4 is 0, the apple plant to be tested is highly susceptible to Botryosphaeria dothidea.

8. The application according to claim 6, wherein, The reaction conditions for the PCR amplification are as follows: 98°C for 2 min; 7 cycles of 98°C for 30 s, 65°C for 30 s, and 72°C for 40 s; 72°C for 4 min.

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