Structural variation marker in apple scab resistance breeding and application thereof

Abstract

The invention discloses a structure variation marker in apple scab resistance breeding and application thereof. The invention claims to protect application of a specific fragment as a molecular marker in detection or auxiliary detection of resistance of apple plants to scab; the nucleotide sequence of the specific fragment is as shown in SEQ ID No. 1 or the 393 to 1166 sites of SEQ ID No. 1. Experiments prove that the scab resistance potential in natural plants or filial generations can be quickly screened by utilizing the deletion and existence of the SCAB-R fragment (SEQ ID No.1), which has important significance on scab resistance breeding of malus plants.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a structural variation marker in apple scab resistance breeding and application thereof. Background Art

[0002] Apple scab is caused by the fungus Venturia inaequalis and is a serious disease in apple production, mainly harming leaves, inflorescences and fruits. Infected plants will show black spots, which in severe cases will cause fruit deformity and fall off, affecting the commercial quality and economic value of apples. The prevalence of apple scab is closely related to climatic conditions. In particular, in humid and rainy environments, the ability of pathogen spores to spread is significantly enhanced, thus posing a great challenge to prevention and control in apple-producing areas.

[0003] Traditional prevention and 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. At the same time, due to the strong mutation ability of pathogens, the pathogenic bacteria population of apple scab presents diversity and complexity, and some pathogens have developed resistance to conventional pesticides, further increasing the difficulty of prevention and control. Therefore, breeding disease-resistant apple varieties has become a fundamental way to solve this problem.

[0004] Disease-resistant breeding is to cultivate new varieties that can effectively resist diseases by screening and introducing disease-resistant genes. Since the 1950s, the United States has introduced disease-resistant genes from apples, especially the black scab resistance gene Rvi6 (formerly known as Vf) from Malus floribunda, to cultivate apple varieties with natural resistance to black scab. This disease-resistant gene can enable apple varieties to effectively resist the invasion of apple black scab pathogens without relying on chemical pesticides. After the introduction of Rvi6, breeders have successfully cultivated many black scab-resistant apple varieties through continuous hybridization and backcrossing.

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

[0006] Although the existing SSR markers for resistance to black spot have played an important role in molecular marker-assisted selection, the limitations of SSR markers have gradually been exposed with the advancement of technology. Their lack of marker polymorphism, low resolution, complex development and high detection costs have limited their application in modern large-scale breeding.

[0007] First, the resolution is low and the gene locus cannot be accurately located. SSR markers can usually only detect repetitive sequences within a few dozen base pairs, so their resolution is relatively low when accurately locating gene loci, especially regulatory regions. This makes the existing black spot resistance SSR markers less accurate in gene positioning and difficult to accurately distinguish minor genomic variations associated with disease resistance, especially small fragment variations in complex regulatory regions.

[0008] Second, the marker development and detection process is cumbersome and costly. The development of SSR markers requires the amplification of repetitive sequences, and the amplification efficiency between different germplasms may vary greatly, which increases the difficulty of development and verification. Although SSR markers are relatively economical in a small number of samples, they require a large amount of PCR amplification for large-scale screening, which is difficult to adapt to the breeding needs of high-throughput automation.

[0009] Third, polymorphism is unstable. The polymorphism of SSR markers is unstable under different genetic backgrounds and environmental conditions, which affects the application of markers in diversified germplasm resources. Especially in a highly heterogeneous genome such as apple, 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 replaced by more efficient molecular markers. Modern black spot disease resistance breeding urgently needs 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 refers to large-scale variations such as large insertions, deletions, inversions, duplications and translocations in the genome. These variations play an important role in regulating gene expression, generating new gene functions and regulating complex traits. In disease-resistant breeding, SV markers can more accurately reveal gene loci and regulatory regions related to disease-resistant traits, so SV markers have gradually become a hot spot in molecular breeding. Compared with SSR markers, SV markers can reveal a wider range of genomic variations, especially when identifying complex gene regulatory regions that are difficult to locate with traditional molecular markers. SV markers show a high degree of accuracy. Since pathogen infection and disease resistance responses usually involve complex gene regulatory networks, SV (such as large insertions and deletions) may directly affect the expression and function of disease-resistant genes. Therefore, the detection and use of SV markers can more accurately select breeding materials with disease resistance. Summary of the invention

[0012] The purpose of the present invention is to provide a structural variation marker in apple scab resistance breeding and its application.

[0013] In a first aspect, the present invention claims the use of a specific fragment as a molecular marker in any of the following:

[0014] (A1) Detecting or assisting in detecting the resistance of apple plants to black scab;

[0015] (A2) preparing a product for detecting or assisting in detecting the resistance of apple plants to black scab;

[0016] (A3) detecting or assisting in detecting whether apple plants are scab-resistant varieties;

[0017] (A4) preparing a product for detecting or assisting in detecting whether a plant of the genus Apple is a scab-resistant variety;

[0018] (A5) breeding apple varieties resistant to black scab;

[0019] (A6) preparing a product for breeding apple plant varieties resistant to black spot disease;

[0020] (A7) Screening and eliminating individual plants of the genus Malus susceptible to black scab;

[0021] (A8) preparing a product for screening and eliminating individual plants of the genus Malus susceptible to black scab;

[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] In a second aspect, the present invention claims the use of a substance for detecting whether a specific fragment is contained in the genome of a plant of the genus Malus in any of the following:

[0024] (A1) Detecting or assisting in detecting the resistance of apple plants to black scab;

[0025] (A2) preparing a product for detecting or assisting in detecting the resistance of apple plants to black scab;

[0026] (A3) detecting or assisting in detecting whether apple plants are scab-resistant varieties;

[0027] (A4) preparing a product for detecting or assisting in detecting whether a plant of the genus Apple is a scab-resistant variety;

[0028] (A5) breeding apple varieties resistant to scab;

[0029] (A6) preparing a product for breeding apple plant varieties resistant to black spot disease;

[0030] (A7) Screening and eliminating individual plants of the genus Malus susceptible to black scab;

[0031] (A8) preparing a product for screening and eliminating individual plants of the genus Malus susceptible to black scab;

[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 the genome of the apple plant contains a specific fragment is a primer pair used to specifically amplify the full-length or partial fragment of the specific fragment.

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

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

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

[0037] (B1) detecting whether the genome of the tested Malus plant contains a specific fragment;

[0038] (B2) according to the result of (B1), determining the resistance of the tested apple plant to black scab as follows: the tested apple plant containing the specific fragment in its genome has a higher or candidate higher resistance to black scab than the tested apple plant not containing the specific fragment in its genome;

[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] In a fourth aspect, the present invention claims a method for detecting or assisting in detecting whether a plant of the genus Malus is a scab-resistant variety.

[0041] The method for detecting or assisting in detecting whether a plant of the genus Malus is a scab-resistant variety claimed in the present invention may include the following steps:

[0042] (C1) detecting whether the genome of the tested Malus plant contains a specific fragment;

[0043] (C2) according to the result of (C1), determining whether the tested apple plant is a black scab resistant variety as follows: if the genome of the tested apple plant contains the specific fragment, the tested apple plant is or is a candidate for being a black scab resistant variety; if the genome of the tested apple plant does not contain the specific fragment, the tested apple plant is not or is not a candidate for being a black scab resistant variety;

[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] In a fifth aspect, the present invention claims a method for breeding a scab-resistant variety of apple plants.

[0046] The method for cultivating a black spot disease resistant variety of apple plants claimed in the present invention may include the following steps:

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

[0048] (D2) using apple plants 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, and ultimately obtaining apple plant varieties resistant to black spot disease;

[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] In a sixth aspect, the present invention claims a method for screening and eliminating individual plants susceptible to black spot disease in the genus Malus.

[0051] The method for screening and eliminating individual plants susceptible to black scab in apple plants claimed in the present invention may include the following steps:

[0052] (E1) Detecting whether a specific fragment is contained in the genome of Malus plants;

[0053] (E2) eliminating individual plants of the genus Malus whose genomes do not contain the specific fragment;

[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 above-mentioned related aspects, the method for detecting whether the genome of the tested Malus plant contains the specific fragment may be any of the following:

[0056] (F1) direct sequencing (i.e. directly sequencing the genome of the tested Malus plant);

[0057] (F2) Using the genome of the test Malus plant as a template, PCR amplification is performed using the primer pair described in the second aspect above (such as the primer pair shown in SEQ ID No. 2 and SEQ ID No. 3), and determining whether the genome of the test Malus plant contains the specific fragment based on the amplification result.

[0058] In a seventh aspect, the present invention claims protection for any of the following biological materials:

[0059] (H1) a DNA fragment, the nucleotide sequence of which is shown in SEQ ID No. 1 or positions 393 to 1166 of SEQ ID No. 1;

[0060] (H2) A primer pair consisting of a single-stranded DNA molecule shown in SEQ ID No.2 and a single-stranded DNA molecule shown in SEQ ID No.3.

[0061] In the implementation case of the present invention, the Malus plant is selected from 104 materials in Table 1, such as Malus multiflora, 'Sir Prize', 'Britegold', 'Liberty', 'Florina', 'Pionier', 'Prima', 'Moira', 'Primiera', 'Juliet', 'Sundance', 'Priscilla', 'Pristine', 'Enterprise', 'Goldrush', 'Dayton', 'Trent', 'Fuji', 'Gala', 'Golden Crown', Xinjiang wild apple, Catalpa ovata, Henan crabapple, Dianchi crabapple, grassland crabapple, etc.

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

[0063] The SV markers (absence and presence of SCAB-R fragments) developed in the present invention have significant advantages over SSR markers in terms of resolution, correlation with functional variation, detection stability and application in the context of big data, and are 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, with significantly better resolution than SSR markers. SSR markers usually detect shorter repetitive sequences and are limited in characterizing complex genomic variations, while SV markers can more comprehensively reflect the structural differences of the genome.

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

[0066] (3) Detection sensitivity and stability: SV markers can identify structural variations in longer fragments, 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 better combine high-throughput sequencing data for whole genome variation analysis. In contrast, the detection technology of SSR markers is relatively traditional and difficult to adapt to the needs of large-scale data analysis.

[0068] (5) Cross-species applicability: Since SV markers can capture a wide range of genomic variations, they are applicable across species and are suitable for multi-species genomic studies. The polymorphism of SSR markers is often limited to specific species and is less applicable in cross-species studies. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0070] Figure 2 The Rvi6 region in the pan-genome map. Red represents the 'Fuji' reference genome; gray represents alternative pathways; fragments unique to M. floribunda are shown in blue; the location and orientation of the HcrVf2 (Rvi6) gene are marked in purple.

[0071] Figure 3 PCR detection of SCAB-R insertion in some black scab-resistant and non-resistant varieties. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0073] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0074] The black scab-resistant apple varieties involved in the following examples are all recorded in “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 can only be used to repeat the experiments of the present invention and cannot be used for other purposes.

[0075] Example 1. Development of SV markers associated with apple scab resistance (absence and presence of SCAB-R fragment)

[0076] 1. Experimental Methods

[0077] 1. Genome map construction and basic description

[0078] First, the apple genome was constructed as a genome map file using the Minigraph software, with the 'Fuji' genome as a reference, and the parameters were: "minigraph-xggs refrence-genome.fa genome2.fa...genomeN.fa>graph.gfa". The total length of the genome map, the total length of each genome path, the common length of each genome, the unique length, and other basic information were statistically generated. The path information file of each genome at the map variation site was generated using the Minigraph software, with the parameters: "minigraph-xasm-l10k--call graph.gfa target-genome.fa>target-genome.bed". Based on the information in this file, the common path sequence length, the total sequence length of each genome in the map, and the unique sequence length were statistically generated.

[0079] 2. Classification of structural variation sites

[0080] The bubble region file was generated using the genomic map generated in step 1 using the Gfatools software (https: / / github.com / lh3 / gfatools) with the following parameters: "gfatools bubble graph.gfa>graph.bed". The bubble regions were classified according to the information in this file to determine the SV sites: 1) Insertion: There are only two paths in the region, the shorter path is less than 50 bp and comes from the reference genome, and the longer path is greater than or equal to 50 bp and comes from the non-reference genome; 2) Deletion: There are only two paths in the region, the shorter path is less than 50 bp and comes from the non-reference genome, and the longer path is greater than or equal to 50 bp and comes from the reference genome; 3) Divergent: There are only two paths in the region, the shorter path is greater than or equal to 50 bp; 4) Multi-allelic: There are three or more paths in the region, and the shortest path is greater than or equal to 50 bp. The number of SV sites of each type was counted.

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

[0082] The genome path information files generated in step 1 were merged to obtain the distribution information of each fragment in each genome in the genome map; the target fragments unique to the Malus multiflora genome were screened in the SV sites counted in step 2. The BLASTP software was used to annotate the gene function of the 'Fuji' genome using the gene function information of the NCBI-nr database. The position information of the target fragment was intersected with the position information of 5,000 bp upstream and downstream of the 'Fuji' gene to determine the gene function that each fragment may affect.

[0083] 2. Results and Analysis

[0084] Species of the genus Malus exhibit a high degree of allelic diversity, which is closely related to their morphological characteristics and environmental adaptability. These rich genetic resources provide a valuable gene pool for apple scion and rootstock breeding. Pan-genome maps can reveal SVs that are difficult to capture with traditional linear comparative genomics methods, and are particularly suitable for revealing genomic regions containing a large number of small variations, which usually show complex diversity and multi-allelic pathways. In order to further explore the genetic diversity of the genus Malus, the present invention constructed a pan-genome map containing 3.15 million fragments with a total length of 3.18Gb, covering 32 species of the genus Malus, including 1.83 million fragments that do not exist in the 'Fuji' reference genome, with a total length of 2.45Gb. These non-reference fragments were divided into four categories: core (present in all 36 species, a total of 253,252 fragments), soft-core (present in 33-35 species, a total of 406,680 fragments), shell (present in 2-32 species, a total of 1,805,311 fragments), specific (present in only one species, a total of 515,529 fragments). In-depth research on these species-specific fragments or SVs will provide important clues for the genetic analysis of important agronomic traits. Through this map-based approach, we identified a total of 117,246 structural variants (SVs) with a length of no less than 50 bp. These SVs were further divided into four categories: Insertion (31,989), Deletion (21,969), Divergent (9,577), and Multi-allelic (53,711).

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

[0086] Take apple scab as an example. This disease is one of the most serious apple diseases worldwide. As the source of the main locus Rvi6 for resistance to scab, Malus multiflora has been widely used in breeding. Figure 2 ), the present invention discovered two Malus multiflora-specific fragments (s2410094 and s2410096). It is worth noting that these two fragments only exist in the B haploid of the diploid genome of Malus multiflora, which highlights the great potential of haploid genome assembly technology in analyzing the functional and structural diversity of apple genomes. Since 1970, almost all scab-resistant varieties have been derived from Malus multiflora. Among them, the nucleotide sequence of the s2410094 fragment (named as SCAB-R fragment) is shown in SEQ ID No.1.

[0087] Example 2: Application of SV markers associated with apple scab resistance (absence and presence of SCAB-R fragment)

[0088] The present invention detected the species-specific fragment s2410094 (i.e., SCAB-R) in 16 scab-resistant varieties bred with Malus multiflorus as one of the parents, and found that all 16 varieties carried the SCAB-R fragment, indicating that SCAB-R and the scab-resistant trait were highly co-segregated. This result demonstrates the application potential of the SCAB-R fragment in molecular marker-assisted selection, especially in the offspring with Malus multiflorus as the parent. The details are as follows:

[0089] 1. Detection of resistance to black spot disease

[0090] For the criteria for identifying scab resistance, please refer to the article "Papp D., Gao L., Thapa R., Olmstead D. & Khan A, 2020. Field apple scab susceptibility of a diverse Malus germ plasm collection identifies potential sources of resistance for apple breeding. CABI Agriculture and Bioscience 1, 16", which are as follows:

[0091] The black spot disease traits on leaves of various qualities were evaluated separately, and the black spot disease symptom ranking and scale used in the evaluation were as follows:

[0092] 0: no symptoms on leaves; 1: needle-like pits appear on leaves; 2: chlorotic lesions appear on leaves; 3a: necrotic and chlorotic lesions appear on leaves, with weak spore formation ability; 3b: chlorotic and necrotic lesions with obvious spore formation on leaves; 4: a large number of spore lesions on leaves cover most of the leaf area. According to the symptom category, the germplasms with grades 0, 1 and 2 were identified as resistant to black scab (i.e., resistant to black scab), and the germplasms with grades 3a, 3b and 4 were identified as not resistant to black scab (i.e., not resistant to black scab).

[0093] 2. 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] The primer pair is used to specifically amplify positions 393-1166 of the SCAB-R fragment, namely positions 393-1166 of SEQ ID No. 1, and the theoretical amplification product size is 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] 3. PCR Testing

[0102] The Taq enzyme used for PCR amplification was 2×DreamTaq Green PCR Master Mix (Thermo scientific, K1081), and the amplification steps and conditions were: (1) 95°C, 3 min; (2) 95°C, 15 s; (3) 62°C, 15 s; (4) 72°C, 15 s; (5) 72°C for 5 min; steps 2-4 were cycled 36 times. PCR products were visualized using 1% agarose gel.

[0103] Using the primers designed in step 2, PCR amplification was performed on a 774 bp fragment of a portion 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. The PCR amplification and electrophoresis experiments were repeated three times independently.

[0104] IV. Practical Application

[0105] The genomic DNA of 104 materials, including Malus multiflora confirmed to have scab resistance according to step 1, scab-resistant varieties bred with Malus multiflora as one of the parents ('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 Crown', Xinjiang Wild Apple, Catalpa ovata, Malus Henan, Malus Dianchi, Malus prairie, etc.), were used as templates, and PCR amplification was performed according to step 3 using the primers designed in step 2 to detect whether there was an inserted fragment (SCAB-R fragment, i.e., SEQ ID No. 1).

[0106] The PCR test results of some materials are as follows Figure 3 As shown, except for the third lane which was loaded with water, all lanes had a band at 174 bp, indicating that the sample DNA used could amplify the MdActin sequence and the DNA quality was qualified; among all the materials tested, the materials identified as having black scab resistance using the step one method, such as Malus multiflora, 'Sir Prize', and 'Britegold', had a band at 774 bp, while the materials identified as not having black scab resistance using the step one method, such as 'Fuji', 'Gala', and 'Golden Crown', had no band at 774 bp, indicating the accuracy of the SCAB-R fragment in detecting whether the apple material is a black scab resistant variety, and the stability of the PCR amplification system in verifying whether the apple material contains the SCAB-R fragment.

[0107] The results of whether the 104 apple materials contain SCAB-R fragments and whether they are resistant to black scab are summarized in Table 1. As can be seen from the table, any material containing SCAB-R fragments is resistant to black scab, and any material without SCAB-R fragments is not resistant to black scab. This further proves the accuracy of SCAB-R fragments in detecting whether apple materials are black scab-resistant varieties.

[0108] Table 1 shows whether the 104 apple materials tested contain SCAB-R fragments and whether they are resistant to black spot disease

[0109]

[0110]

[0111]

[0112] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of specific fragments as molecular markers in any of the following: (A1) Detecting or assisting in detecting the resistance of apple plants to black scab; (A2) preparing a product for detecting or assisting in detecting the resistance of apple plants to black scab; (A3) detecting or assisting in detecting whether apple plants are scab-resistant varieties; (A4) preparing a product for detecting or assisting in detecting whether a plant of the genus Apple is a scab-resistant variety; (A5) breeding apple varieties resistant to black scab; (A6) preparing a product for breeding apple plant varieties resistant to black spot disease; (A7) Screening and eliminating individual plants of the genus Malus susceptible to black scab; (A8) preparing a product for screening and eliminating individual plants of the genus Apple that are susceptible to black scab; The nucleotide sequence of the specific fragment is such as SEQ ID No.1 or positions 393-1166 of SEQ ID No.

1.

2. Use of a substance for detecting whether a specific fragment is contained in the genome of a plant of the genus Malus in any of the following: (A1) Detecting or assisting in detecting the resistance of apple plants to black scab; (A2) preparing a product for detecting or assisting in detecting the resistance of apple plants to black scab; (A3) detecting or assisting in detecting whether apple plants are scab-resistant varieties; (A4) preparing a product for detecting or assisting in detecting whether a plant of the genus Apple is a scab-resistant variety; (A5) breeding apple varieties resistant to scab; (A6) preparing a product for breeding apple plant varieties resistant to black spot disease; (A7) Screening and eliminating individual plants of the genus Malus susceptible to black scab; (A8) preparing a product for screening and eliminating individual plants of the genus Apple that are susceptible to black scab; The nucleotide sequence of the specific fragment is shown in SEQ ID No.1 or positions 393-1166 of SEQ ID No.

1.

3. The use according to claim 2, characterized in that: The substance used to detect whether the genome of the apple plant contains a specific fragment is a primer pair used to specifically amplify the full length or partial fragment of the specific fragment.

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

3.

5. A method for detecting or assisting in detecting the resistance of apple plants to black scab, comprising the following steps: (B1) detecting whether the genome of the tested Malus plant contains a specific fragment; (B2) according to the result of (B1), determining the resistance of the tested apple plant to black scab as follows: the tested apple plant containing the specific fragment in its genome has a higher or candidate higher resistance to black scab than the tested apple plant not containing the specific fragment in its genome; The nucleotide sequence of the specific fragment is shown in SEQ ID No.1 or positions 393-1166 of SEQ ID No.

1.

6. A method for detecting or assisting in detecting whether a plant of the genus Malus is a scab-resistant variety, comprising the following steps: (C1) detecting whether the genome of the tested Malus plant contains a specific fragment; (C2) according to the result of (C1), determining whether the tested apple plant is a black scab resistant variety as follows: if the genome of the tested apple plant contains the specific fragment, the tested apple plant is or is a candidate for being a black scab resistant variety; if the genome of the tested apple plant does not contain the specific fragment, the tested apple plant is not or is not a candidate for being a black scab resistant variety; 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 cultivating a scab-resistant variety of apple plants, comprising the following steps: (D1) Detect whether the genome of Malus plants contains a specific fragment; (D2) using apple plants 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, and ultimately obtaining apple plant varieties resistant to black spot disease; The nucleotide sequence of the specific fragment is shown in SEQ ID No.1 or positions 393-1166 of SEQ ID No.

1.

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

1.

9. The method according to any one of claims 5 to 8, characterized in that: The method for detecting whether the genome of the tested Malus plant contains the specific fragment is any one of the following: (F1) direct sequencing; (F2) Using the genome of the test Malus plant as a template, performing PCR amplification using the primer pair described in claim 3 or 4, and determining whether the genome of the test Malus plant contains the specific fragment based on the amplification result.

10. Any of the following biological materials: (H1) a DNA fragment, the nucleotide sequence of which is shown in SEQ ID No. 1 or positions 393 to 1166 of SEQ ID No. 1; (H2) A primer pair consisting of a single-stranded DNA molecule shown in SEQ ID No.2 and a single-stranded DNA molecule shown in SEQ ID No.3.

Images