Structural variation markers in apple (malus domestica) resistance breeding and their applications

Abstract

The application discloses a structural variation marker in apple scab resistance breeding and application thereof. The application claims the application of a specific fragment as a molecular marker in detection or assisted detection of apple plant resistance to scab; the nucleotide sequence of the specific fragment is as shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No. 1. Experiments prove that the absence and presence of the SCAB-R fragment (SEQ ID No. 1) can be used to quickly screen the scab resistance potential of natural plants or hybrid offspring, which is of great significance to the scab resistance breeding of apple plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a structural variation marker and its application in apple black spot disease resistance breeding. Background Technology

[0002] Apple scab, caused by the fungus *Venturia inaequalis*, is a serious disease in apple production, primarily affecting leaves, inflorescences, and fruits. Infected plants exhibit black lesions, and in severe cases, this leads to fruit deformities and drop, impacting the marketability and economic value of apples. The prevalence of apple scab is closely related to climatic conditions; in humid and rainy environments, the spread of pathogen spores is significantly enhanced, posing a considerable challenge to control in apple-producing areas.

[0003] Traditional control methods mainly rely on the use of pesticides. However, the long-term and large-scale use of chemical pesticides not only increases production costs but also poses a threat to the ecological environment and food safety. Meanwhile, due to the strong variability of pathogens, the pathogenic population of apple scab exhibits diversity and complexity, with some pathogens developing resistance to conventional pesticides, further increasing the difficulty of control. Therefore, breeding disease-resistant apple varieties has become the fundamental way to solve this problem.

[0004] Disease resistance breeding involves screening for and introducing disease-resistant genes to cultivate new varieties that are effectively resistant to diseases. Starting in the 1950s, the United States introduced disease-resistant genes from apples, particularly the Rvi6 (formerly known as Vf) gene from the Malus floribunda (Chinese crabapple), to cultivate apple varieties with natural resistance to apple scab. This resistance gene allows apple varieties to effectively resist the infection of apple scab pathogens without relying on chemical pesticides. After the introduction of Rvi6, breeders successfully developed many apple varieties resistant to apple scab through continuous hybridization and backcrossing.

[0005] Molecular marker-assisted selection (MAS) technology has become an important technique in plant breeding in recent years, especially in the breeding of perennial fruit trees. Due to the long generational alternation cycle of fruit trees such as apples, traditional phenotypic screening methods are time-consuming, labor-intensive, and easily affected by environmental factors, making it difficult to guarantee breeding efficiency. The introduction of molecular marker technology has significantly improved the efficiency of disease resistance breeding. By screening breeding materials carrying disease resistance genes at an early stage, the breeding process can be accelerated and the selection cycle shortened. Currently, in apple scab resistance breeding, various molecular markers have been developed, mainly based on simple sequence repeats (SSRs). Rvi6 gene-related SSR markers have been widely used in apple disease resistance screening.

[0006] While existing SSR markers for resistance to Alternaria solani have played an important role in marker-assisted selection, their limitations have become increasingly apparent with technological advancements. Insufficient marker polymorphism, low resolution, complex development, and high detection costs limit their application in modern large-scale breeding.

[0007] First, the resolution is low, making it impossible to accurately locate gene loci. SSR markers typically only detect repetitive sequences within a few dozen base pairs, resulting in relatively low resolution when accurately locating gene loci, especially regulatory regions. This makes existing SSR markers for resistance to black spot disease less accurate in gene localization, and makes it difficult to precisely distinguish subtle genomic variations associated with resistance, especially small fragment variations in complex regulatory regions.

[0008] Second, the development and detection of SSR markers is cumbersome and costly. Developing SSR markers requires amplification of repetitive sequences, and the amplification efficiency can vary significantly between different germplasms, increasing the difficulty of development and validation. While SSR markers are relatively economical for small sample sizes, large-scale screening requires extensive PCR amplification, making them unsuitable for the demands of high-throughput, automated breeding.

[0009] Third, the polymorphism of SSR markers is unstable. The polymorphism of SSR markers is unstable under different genetic backgrounds and environmental conditions, affecting their application in diverse germplasm resources. Especially in highly heterogeneous genomes like apples, the polymorphism of SSR markers may not be fully expressed in some varieties, affecting the reliability of the markers.

[0010] With the development of structural variation (SV) marker technology, SSR markers are gradually being replaced by more efficient molecular markers. Modern breeding for resistance to black spot disease urgently requires markers with high specificity, high resolution, comprehensive coverage, and low detection cost to meet the needs of complex trait screening and rapid breeding.

[0011] SV (Segmented Variation) refers to large-scale variations in the genome, such as insertions, deletions, inversions, duplications, and translocations. These variations play important roles in regulating gene expression, generating new gene functions, and regulating complex traits. In disease resistance breeding, SV markers can more accurately reveal gene loci and regulatory regions associated with disease resistance traits, thus SV markers have gradually become a hot topic in molecular breeding. Compared to SSR (Segmented Spectrum Reduction) markers, SV markers can reveal a wider range of genomic variations, especially in identifying complex gene regulatory regions that are difficult to locate with traditional molecular markers, where SV markers demonstrate high precision. Since pathogen infection and disease resistance responses usually involve complex gene regulatory networks, SVs (such as large-segment insertions and deletions) may directly affect the expression and function of disease resistance genes. Therefore, detecting and utilizing SV markers can more accurately select breeding materials with disease resistance. Summary of the Invention

[0012] The purpose of this invention is to provide a structural variation marker and its application in apple black spot disease resistance breeding.

[0013] Firstly, the present invention claims protection for the use of a specific fragment as a molecular marker in any of the following:

[0014] (A1) To detect or assist in the detection of resistance of apple plants to black spot disease;

[0015] (A2) Prepare products for detecting or assisting in the detection of resistance levels of apple plants to black spot disease;

[0016] (A3) To detect or assist in the detection of whether apple plants are resistant to black spot disease;

[0017] (A4) Prepare products for detecting or assisting in the detection of whether apple plants are resistant to black spot disease;

[0018] (A5) Develop apple varieties resistant to black spot disease;

[0019] (A6) Prepare products for breeding apple scab resistant varieties;

[0020] (A7) Screen out susceptible individual plants of the genus *Malus* to black spot disease;

[0021] (A8) Prepare a product for screening and eliminating susceptible individual plants of Maloides scab disease;

[0022] The nucleotide sequence of the specific fragment is shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No. 1.

[0023] Secondly, the present invention claims protection for the use of a substance for detecting whether a specific fragment is present in the genome of a Malus species in any of the following:

[0024] (A1) To detect or assist in the detection of resistance of apple plants to black spot disease;

[0025] (A2) Prepare products for detecting or assisting in the detection of resistance levels of apple plants to black spot disease;

[0026] (A3) To detect or assist in the detection of whether apple plants are resistant to black spot disease;

[0027] (A4) Prepare products for detecting or assisting in the detection of whether apple plants are resistant to black spot disease;

[0028] (A5) Develop apple varieties resistant to black spot disease;

[0029] (A6) Prepare products for breeding apple scab resistant varieties;

[0030] (A7) Screen out susceptible individual plants of the genus *Malus* to black spot disease;

[0031] (A8) Prepare a product for screening and eliminating susceptible individual plants of Maloides scab disease;

[0032] The nucleotide sequence of the specific fragment is shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No. 1.

[0033] Furthermore, the substance used to detect whether a specific fragment is present in the genome of a Malus species is a primer pair used to specifically amplify the full length or a portion of the specific fragment.

[0034] In one embodiment of the present invention, the primer pair consists of a single-stranded DNA molecule as shown in SEQ ID No. 2 and a single-stranded DNA molecule as shown in SEQ ID No. 3. This primer pair is used to specifically amplify positions 393-1166 of SEQ ID No. 1.

[0035] Thirdly, the present invention claims a method for detecting or assisting in the detection of resistance levels of apple plants to black spot disease.

[0036] The method for detecting or assisting in the detection of resistance to black spot disease in apple plants, as claimed in this invention, may include the following steps:

[0037] (B1) Detect whether the genome of the apple plant to be tested contains a specific segment;

[0038] (B2) Based on the results of (B1), the resistance of the tested apple species to black spot disease is determined as follows: the resistance of the tested apple species containing the specific fragment in the genome to black spot disease is higher or higher than that of the tested apple species not containing the specific fragment in the genome to black spot disease.

[0039] The nucleotide sequence of the specific fragment is shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No. 1.

[0040] Fourthly, the present invention claims a method for detecting or assisting in the detection of whether an apple plant is a resistant variety to black spot disease.

[0041] The method for detecting or assisting in the detection of whether an apple plant is a resistant variety to black spot disease, as claimed in this invention, may include the following steps:

[0042] (C1) Detect whether the genome of the apple plant to be tested contains a specific segment;

[0043] (C2) Based on the results of (C1), determine whether the tested apple species is a resistant variety of black spot disease as follows: if the genome of the tested apple species contains the specific fragment, then the tested apple species is or is a candidate for a resistant variety of black spot disease; if the genome of the tested apple species does not contain the specific fragment, then the tested apple species is not or is not a candidate for a resistant variety of black spot disease.

[0044] The nucleotide sequence of the specific fragment is shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No. 1.

[0045] Fifthly, the present invention claims a method for breeding apple scab-resistant varieties.

[0046] The method for cultivating apple scab-resistant varieties, as claimed in this invention, may include the following steps:

[0047] (D1) Detect whether the genome of apple plants contains a specific segment;

[0048] (D2) Using apple plants whose genomes contain the specific fragment as parents for breeding, and selecting plants whose genomes contain the specific fragment in each generation of breeding, finally obtaining apple scab resistant varieties.

[0049] The nucleotide sequence of the specific fragment is shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No. 1.

[0050] Sixthly, the present invention claims a method for screening and removing susceptible individual plants of the genus *Malus* to be infected with black spot disease.

[0051] The method for screening and removing susceptible individual plants of the genus *Malus* scab, as claimed in this invention, may include the following steps:

[0052] (E1) Detect whether the genome of apple plants contains a specific segment;

[0053] (E2) Eliminate individual Malus species whose genomes do not contain the specific fragments described above;

[0054] The nucleotide sequence of the specific fragment is shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No. 1.

[0055] In the aforementioned aspects, any of the methods for detecting whether the genome of the target Malus species contains the specific fragment may be as follows:

[0056] (F1) Direct sequencing (i.e., directly sequencing the genome of the apple plant to be tested);

[0057] (F2) Using the genome of the apple plant to be tested as a template, PCR amplification is performed using the primer pairs described in the second aspect above (such as the primer pairs shown in SEQ ID No. 2 and SEQ ID No. 3), and the specific fragment is determined based on the amplification results.

[0058] Seventhly, the present invention claims protection for any of the following biological materials:

[0059] (H1) DNA fragment, nucleotide sequence as shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No. 1;

[0060] (H2) Primer pair, consisting of the single-stranded DNA molecule shown in SEQ ID No. 2 and the single-stranded DNA molecule described in SEQ ID No. 3.

[0061] In the embodiments of the present invention, the apple plants are selected from 104 materials in Table 1, such as Malus spectabilis, 'Sir Prize', 'Britegold', 'Liberty', 'Florina', 'Pionier', 'Prima', 'Moira', 'Primiera', 'Juliet', 'Sundance', 'Priscilla', 'Pristine', 'Enterprise', 'Goldrush', 'Dayton', 'Trent', 'Fuji', 'Gala', 'Golden Delicious', Xinjiang wild apple, Catalpa, Henan crabapple, Dianchi crabapple, Prairie crabapple, etc.

[0062] Experiments have shown that the deletion and presence of the SCAB-R fragment (SEQ ID No. 1) of this invention can be used to quickly screen for black spot disease resistance in natural plants or hybrid offspring, which is of great significance for black spot disease resistance breeding in apple plants.

[0063] The SV markers (deletion and presence of SCAB-R fragments) developed in this invention have significant advantages over SSR markers in terms of resolution, correlation with functional variations, detection stability, and application in the context of big data, making them more suitable for current high-throughput genome research and refined selection in breeding.

[0064] (1) Higher resolution: SV markers can detect larger structural variations, especially in regions spanning thousands to millions of bases, where their resolution is significantly better than that of SSR markers. SSR markers typically detect shorter repetitive sequences and are limited in characterizing complex genomic variations, while SV markers can more comprehensively reflect structural differences in the genome.

[0065] (2) Stronger correlation with functional variations: SV markers are usually closely related to gene functional variations, especially when gene addition, deletion, duplication and rearrangement are involved. This close connection with functional variations makes SV markers more valuable in genetic research and functional genomics, while SSR markers are usually only used as neutral markers and have less functional information.

[0066] (3) Detection sensitivity and stability: SV markers can identify structural variations in longer segments, so they have higher stability and detection sensitivity. Especially when studying species with high genomic complexity and polymorphism, SV markers are superior to SSR markers in terms of stability and consistency of results.

[0067] (4) Applicability in the context of big data: With the rapid development of genomics technology, especially the popularization of whole-genome sequencing and pan-genome analysis, SV markers can be better integrated with high-throughput sequencing data for whole-genome variation analysis. In contrast, SSR marker detection technology is relatively traditional and difficult to adapt to the needs of large-scale data analysis.

[0068] (5) Cross-species applicability: Because SV markers can capture a wide range of genomic variations, they are applicable across species and suitable for multi-species genome research. The polymorphism of SSR markers is often limited to specific species, and their applicability in cross-species research is weaker. Attached Figure Description

[0069] Figure 1 The structure and number of four SVs in the apple genome map.

[0070] Figure 2 This is the Rvi6 region in the pangenome map. Red represents the 'Fuji' reference genome; gray indicates the alternative pathway; fragments specific to M. floribunda are shown in blue; the location and orientation of the HcrVf2 (Rvi6) gene are indicated in purple.

[0071] Figure 3 This study describes the PCR detection of SCAB-R insertion in some black spot disease-resistant and non-resistant varieties. Detailed Implementation

[0072] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0073] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0074] All the black spot resistant apple varieties mentioned in the following examples are described in the article "Bannier, HJ, 2011. Moderne Apfelzüchtung: Genetische Verarmung und Tendenzen zur Inzucht. Erwerbs-Obstbau 52, 85-110.", which is available to the public from the applicant and may only be used to repeat the experiments of this invention and may not be used for other purposes.

[0075] Example 1: Development of SV markers (deletion and presence of SCAB-R fragments) associated with resistance to apple scab.

[0076] I. Experimental Methods

[0077] 1. Genome Map Construction and Basic Description

[0078] First, using the 'Fuji' genome as a reference, the apple genome was constructed into a genome map file using the Minigraph software with the parameters: "minigraph-xggs reference-genome.fa genome2.fa...genomeN.fa>graph.gfa". Basic information such as the total length of the generated genome map, the total length of each genome pathway, the shared length of each genome, and the unique length were calculated. Next, the Minigraph software was used to generate a pathway information file for each genome at the map's variant sites with the parameters: "minigraph-xasm-l10k--call graph.gfa target-genome.fa>target-genome.bed". Based on the information in this file, the length of shared pathway sequences, as well as the total sequence length and unique sequence length of each genome in the map, were calculated.

[0079] 2. Classification of structural variation sites

[0080] Using the Gfatools software (https: / / github.com / lh3 / gfatools), bubble region files were generated from the genome map produced in step 1, with the parameter: "gfatools bubble graph.gfa>graph.bed". Based on the information in this file, the bubble regions were classified as follows to determine SV sites: 1) Insertion: The region contains only two paths, where the shorter path is less than 50 bp and originates from the reference genome, and the longer path is greater than or equal to 50 bp and originates from a non-reference genome; 2) Deletion: The region contains only two paths, where the shorter path is less than 50 bp and originates from a non-reference genome, and the longer path is greater than or equal to 50 bp and originates from the reference genome; 3) Divergent: The region contains only two paths, where the shorter path is greater than or equal to 50 bp; 4) Multi-allelic: The region contains three or more paths, where the shortest path is greater than or equal to 50 bp. The number of SV sites in each category was then counted.

[0081] 3. Efficient mining of functional sites based on graphs

[0082] The genomic pathway information files generated in step 1 were merged to obtain the distribution information of each fragment in each genome within the genomic map; target fragments specific to the *Malus spectabilis* genome were screened from the SV sites counted in step 2. Gene function annotation of the *Malus spectabilis* genome was performed using BLASTP software and gene function information from the NCBI-nr database. The intersection of the location information of the target fragments with the location information of the upstream and downstream 5,000 bp of the *Malus spectabilis* gene was calculated to determine the gene functions that each fragment might affect.

[0083] II. Results and Analysis

[0084] Apple species exhibit high allelic diversity, closely related to their morphological characteristics and environmental adaptability. This rich genetic resource provides a valuable gene pool for apple scion and rootstock breeding. Pan-genome mapping can reveal SVs that are difficult to capture using traditional linear comparative genomics methods, and is particularly suitable for revealing genomic regions containing a large number of small variations, which often exhibit complex diversity and multi-allelic pathways. To further explore the genetic diversity of the genus *Malus*, this invention constructs a pan-genome map containing 3.15 million fragments with a total length of 3.18 Gb, covering 32 species of *Malus*, including 1.83 million fragments not present in the 'Fuji' reference genome, with a total length of 2.45 Gb. These non-reference fragments were categorized into four types: core (present in all 36 species, totaling 253,252 fragments), soft-core (present in 33–35 species, totaling 406,680 fragments), shell (present in 2–32 species, totaling 1,805,311 fragments), and specific (present in only one species, totaling 515,529 fragments). In-depth study of these species-specific fragments, or SVs, will provide important clues for the genetic analysis of key agronomic traits. Using this map-based method, we identified a total of 117,246 structural variations (SVs) with a length of at least 50 bp. These SVs were further categorized into four types: insertion (31,989), deletion (21,969), divergent (9,577), and multi-allelic (53,711).

[0085] Figure 1 The structure and number of four SVs in the apple genome map.

[0086] Apple scab is one of the most serious apple diseases worldwide. As the source of Rvi6, a key locus for resistance to scab, crabapple has been widely used in breeding. In the Rvi6 region of the pan-genome map (… Figure 2 This invention discovered two Malus spectabilis-specific fragments (s2410094 and s2410096). Notably, these two fragments are found only in the B haploid of the Malus spectabilis diploid genome, highlighting the significant potential of haplotype genome assembly technology in elucidating the functional and structural diversity of the apple genome. Since 1970, almost all resistance to scab disease in apple varieties has originated from Malus spectabilis. The nucleotide sequence of the s2410094 fragment (named the SCAB-R fragment) is shown in SEQ ID No. 1.

[0087] Example 2: Application of SV markers (deletion and presence of SCAB-R fragments) related to resistance to apple scab disease.

[0088] This invention detected the species-specific fragment s2410094 (i.e., SCAB-R) in 16 scab-resistant varieties bred with *Malus spectabilis* as one of their parents. It was found that all 16 varieties carried the SCAB-R fragment, indicating a high degree of co-segregation between SCAB-R and scab resistance. This result demonstrates the potential of the SCAB-R fragment in marker-assisted selection, especially in offspring with *Malus spectabilis* as a parent. Details are as follows:

[0089] I. Black Star Disease Resistance Test

[0090] The criteria for identifying resistance to black spot disease can be found in the article "Papp D., Gao L., Thapa R., Olmstead D. & Khan A, 2020. Field apple scab susceptibility of a diverse Malus germplasm collection identifies potential sources of resistance for apple breeding. CABI Agriculture and Bioscience 1, 16", as follows:

[0091] The characteristics of black spot disease on leaves of various leaf types were assessed separately. The ranking and scale of black spot disease symptom levels used in the assessment are as follows:

[0092] 0: No symptoms on leaves; 1: Pinpoint pits on leaves; 2: Chlorotic lesions on leaves; 3a: Necrotic and chlorotic lesions on leaves, with weak spore formation ability; 3b: Obvious chlorotic and necrotic lesions on leaves producing spores; 4: Numerous spore-bearing spots covering most of the leaf area. Based on symptom categories, germplasm classified as 0, 1, and 2 is considered resistant to black spot (i.e., resistant to black spot), while germplasm classified as 3a, 3b, and 4 is considered non-resistant to black spot (i.e., lacks resistance to black spot).

[0093] II. Design and Synthesis of Primers for Specific Amplification of SCAB-R Fragments

[0094] The primer sequences used to amplify the SCAB-R fragment are as follows:

[0095] SCAB-RF: 5'-AAAATACAAGCAAGCCATACAT-3' (SEQ ID No. 2);

[0096] SCAB-RR: 5'-CACAATCACAACCAAGGAGG-3' (SEQ ID No. 3).

[0097] This primer pair is used to specifically amplify positions 393-1166 of the SCAB-R fragment, i.e., positions 393-1166 of SEQ ID No. 1, with a theoretical amplification product size of 774 bp.

[0098] The primer sequences used to amplify the internal reference gene MdActin are as follows:

[0099] MdActin-F: 5'-AGTCCTCTTCCAACCATCCAT-3';

[0100] MdActin-R: 5'-ATTTCCTTGCTCATTCGGTC-3'.

[0101] III. PCR Detection

[0102] The Taq enzyme used for PCR amplification was 2×DreamTaq Green PCR Master Mix (Thermo Scientific, K1081). The amplification steps and conditions were as follows: (1) 95℃, 3 min; (2) 95℃, 15 s; (3) 62℃, 15 s; (4) 72℃, 15 s; (5) 72℃ for a 5-minute extension; steps 2-4 were repeated 36 times. The PCR products were visualized using a 1% agarose gel.

[0103] Using the primers designed in step two, PCR amplification was performed on a 774 bp fragment of the 4,034 bp insert (SCAB-R fragment, i.e., SEQ ID No. 1) to verify the presence of the insert. A 174 bp MdActin was used as a positive control. PCR amplification and electrophoresis experiments were independently repeated three times.

[0104] IV. Practical Applications

[0105] Using genomic DNA from 104 materials—including *Malus spectabilis* var. *multiflora* (confirmed as resistant to scab in step one) and scab-resistant varieties bred from *Malus spectabilis* var. *multiflora* ('Sir Prize', 'Britegold', 'Liberty', 'Florina', 'Pionier', 'Prima', 'Moira', 'Primiera', 'Juliet', 'Sundance', 'Priscilla', 'Pristine', 'Enterprise', 'Goldrush', 'Dayton', 'Trent', etc.) and materials without scab resistance ('Fuji', 'Gala', 'Golden Delicious', Xinjiang Wild Apple, *Malus spectabilis*, *Malus henryi*, *Malus yunnanensis*, *Malus yunnanensis*, *Malus yunnanensis*, *Malus yunnanensis*, etc.)—as templates, PCR amplification was performed using primers designed in step two according to step three to detect the presence of an insert fragment (SCAB-R fragment, i.e., SEQ ID No. 1).

[0106] PCR test results for some materials are as follows: Figure 3 As shown, except for lane 3 where water was added, all lanes showed a band at 174bp, indicating that the sample DNA could amplify the MdActin sequence and the DNA quality was qualified. Among all the materials tested, the 'Malus spectabilis', 'Sir Prize', and 'Britegold' varieties, which were identified as having resistance to black spot disease using the method in step one, showed a band at 774bp, while the 'Fuji', 'Gala', and 'Golden Delicious' varieties, which were identified as not having resistance to black spot disease using the method in step one, showed no band at 774bp. This demonstrates the accuracy of using the SCAB-R fragment to detect whether apple materials are black spot disease resistant varieties, and the stability of this PCR amplification system for verifying whether apple materials contain the SCAB-R fragment.

[0107] Table 1 summarizes the information on whether 104 apple samples contained the SCAB-R fragment and their resistance to black spot disease. The table shows that materials containing the SCAB-R fragment are resistant to black spot disease, while materials not containing the SCAB-R fragment are not. This further demonstrates the accuracy of using the SCAB-R fragment to detect whether apple materials are resistant to black spot disease.

[0108] Table 1 shows the presence of the SCAB-R fragment and resistance to black spot disease in the 104 apple samples tested.

[0109]

[0110]

[0111]

[0112] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of a substance used to detect the presence of a specific fragment in the apple genome in any of the following: (A1) To detect or assist in the detection of the resistance level of apples to black spot disease; (A2) Prepare products for detecting or assisting in the detection of apple resistance to black spot disease; (A3) Testing or auxiliary testing to determine whether apples are resistant to black spot disease; (A4) Prepare products for detecting or assisting in the detection of whether apples are resistant to black spot disease; (A5) Develop apple scab resistant varieties; (A6) Prepare products for breeding apple scab resistant varieties; (A7) Screening and removing apple scab susceptible individual plants; (A8) Prepare products for screening and eliminating susceptible individual plants of apple scab; The nucleotide sequence of the specific fragment is shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No.

1.

2. The application according to claim 1, characterized in that: The substance used to detect whether a specific fragment is present in the apple genome is a primer pair used to specifically amplify the full length of the specific fragment.

3. The application according to claim 2, characterized in that: The primer pair consists of a single-stranded DNA molecule as shown in SEQ ID No. 2 and a single-stranded DNA molecule as shown in SEQ ID No.

3.

4. A method for detecting or assisting in the detection of resistance to apple scab, comprising the following steps: (B1) Detect whether the genome of the apple to be tested contains a specific fragment; (B2) Based on the results of (B1), the resistance of the tested apples to black spot disease is determined as follows: the resistance of the tested apples containing the specific fragment in the genome to black spot disease is higher or higher than that of the tested apples that do not contain the specific fragment in the genome to black spot disease. The nucleotide sequence of the specific fragment is as shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No.

1.

5. A method for detecting or assisting in the detection of whether an apple is a resistant variety to black spot disease, comprising the following steps: (C1) Detect whether the genome of the apple to be tested contains a specific fragment; (C2) Based on the results of (C1), determine whether the apple to be tested is a resistant variety of black spot disease as follows: if the genome of the apple to be tested contains the specific segment, then the apple to be tested is or is a candidate for a resistant variety of black spot disease; if the genome of the apple to be tested does not contain the specific segment, then the apple to be tested is not or is not a candidate for a resistant variety of black spot disease. The nucleotide sequence of the specific fragment is as shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No.

1.

6. A method for breeding apple scab resistant varieties, comprising the following steps: (D1) Detect whether a specific fragment is present in the apple genome; (D2) Using apples containing the specific fragment in their genome as parents for breeding, and selecting plants containing the specific fragment in their genome in each generation of breeding, ultimately obtaining apple scab resistant varieties; The nucleotide sequence of the specific fragment is shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No.

1.

7. A method for screening and removing apple scab susceptible individual plants, comprising the following steps: (E1) Detecting whether a specific fragment is present in the apple genome; (E2) Eliminate apple plants whose genomes do not contain the specific fragment described above; The nucleotide sequence of the specific fragment is shown in SEQ ID No. 1 or positions 393-1166 of SEQ ID No.

1.

8. The method according to any one of claims 4-7, characterized in that: The method for detecting whether the specific fragment is present in the genome of the apple to be tested is any of the following: (F1) Direct sequencing; (F2) Using the genome of the apple to be tested as a template, perform PCR amplification with the primer pair described in claim 2 or 3, and determine whether the specific fragment is contained in the genome of the apple to be tested based on the amplification results.

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