SSR molecular marker of malus plant and application of SSR molecular marker

Abstract

The invention relates to the technical field of plant breeding, in particular to SSR molecular markers of malus plants and application of the SSR molecular markers. The molecular marker comprises 17 SSR (Simple Sequence Repeat) marker sites on chromosomes 1-17 of an apple. 17 long repeat motif (4-6 nucleotide repeat) SSR markers with high polymorphism, high reliability, good repeatability and good universality are screened from seven published whole genome sequences of Malus plants, and the molecular markers can be used for genetic research and application of DNA fingerprint spectrum construction, molecular identification, diversity evaluation, resource protection and the like of the Malus plants. The method has important significance in the field of apple plant breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to SSR molecular markers of Malus plants and applications thereof. Background Art

[0002] Malus Malus Miller belongs to the Maloidae subfamily of the Rosaceae family. It is a north temperate genus widely distributed in North America, Asia and Europe. According to the diameter and function of the fruit, plants of the genus Malus can be divided into two categories: apple (>5cm, edible) and crabapple (≤5cm, ornamental). Apple is one of the important fruits in the world, and crabapple is an important landscape plant for its flowers in spring and fruits in autumn and winter. Individuals of the genus Malus are easy to hybridize, and a large number of cultivated varieties have been cultivated through the long-term efforts of breeders and horticulturists. Due to regional differences, misunderstandings and confusion, different naming standards, variation and hybridization, the phenomenon of homonyms and homonyms is common in the genus Malus, which hinders the identification, protection and industrial development of germplasm resources.

[0003] In the prior art, comparative morphology is usually used to classify and identify cultivars of the genus Malus. As the number of varieties increases, the morphological differences between new varieties and existing varieties may not be obvious enough, and most of the differential traits are quantitative traits controlled by micro-effect polygenes, and are also affected by epistatic effects and environmental factors. In this context, it is necessary to add additional or alternative classification and identification methods, such as molecular marker technology.

[0004] Molecular marker technology uses specific DNA sequences or molecular markers to quickly identify and distinguish species, varieties or individuals. Variety identification based on DNA molecular markers has been recognized by the International Union for the Protection of New Varieties of Plants and can be used to supplement or even completely replace existing comparative morphological methods. The main molecular marker currently used for variety identification is SSR markers. SSR markers, also known as microsatellites or short tandem repeats (STRs), are composed of 1 to 6 nucleotides repeated in tandem. The number of these repeats varies between individuals, so length polymorphism will occur through PCR amplification. SSR markers have the advantages of co-dominance, high polymorphism, low cost, and good transferability between closely related species. They are a reliable method for variety identification or pedigree tracing.

[0005] In apple plants, previous studies usually rely on microsatellite enrichment library sequences, EST sequences or transcriptome sequences, but these sequences only cover a small part of the genome. In addition, due to limited sequence information, the majority of SSR markers developed are more abundant 2-nucleotide repeat types, and the number of long repeat types (greater than 2 nucleotides) is relatively small. This can easily lead to serious pseudo-peaks, which in turn increases the typing error rate. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides SSR molecular markers of Malus plants and applications thereof.

[0007] Most of the SSR markers currently widely used in apple plants are of the 2-nucleotide repeat type, which are prone to serious pseudo-peak phenomenon, resulting in a high typing error rate. In order to solve this technical problem, the present invention provides a group of long repeat motif (4-6 nucleotide repeat) SSR markers with good polymorphism and high accuracy, and develops and designs corresponding SSR marker primers. The application of these SSR markers and primers can provide more reliable results for the molecular identification and genetic research of cultivated apple and ornamental crabapple germplasm.

[0008] In a first aspect, the present invention provides a molecular marker, characterized in that the molecular marker comprises one or more of D1L80M4, D2L23M4, D3L66M4, D4L90M5, D5L99M4, D6L12M4, D7L74M4, D8L26M5, D9L82M4, D10L24M4, D11L51M5, D12L43M4, D13L30M5, D14L8M4, D15L2M4, D16L85M4 or D17L41M4; Among them, D1L80M4 is located at positions 25873955-25874479 of Apple chromosome 1; D2L23M4 is located at positions 8189707-8190228 of Apple chromosome 2; D3L66M4 is located at positions 24094463-24095000 of Apple chromosome 3; D4L90M5 is located at positions 28327304-28327827 of Apple chromosome 4; and D5L99M4 is located at positions 28327304-28327827 of Apple chromosome 5. D6L12M4 is located at position 4401044-4401565 on chromosome 6 of apple; D7L74M4 is located at position 27323858-27324416 on chromosome 7 of apple; D8L26M5 is located at position 8431509-8432031 on chromosome 8 of apple; D9L82M4 is located at position 2933624 on chromosome 9 of apple 0-29336778; D10L24M4 is located on the apple chromosome 10, 10692000-10692383; D11L51M5 is located on the apple chromosome 11, 19864815-19865331; D12L43M4 is located on the apple chromosome 12, 13575540-13576097; D13L30M5 is located on the apple chromosome 13, 13747175-1 3747711; D14L8M4 is located at positions 3618789-3619308 on apple chromosome 14; D15L2M4 is located at positions 1024164-1024705 on apple chromosome 15; D16L85M4 is located at positions 35310591-35311113 on apple chromosome 16; D17L41M4 is located at positions 13895588-13896141 on apple chromosome 17.

[0009] Further, the molecular marker includes any one of the nucleic acids shown in SEQ ID NO.1-17; The nucleic acid shown in SEQ ID NO.1 includes a repeating sequence TCCA, which is repeated 6 times; The nucleic acid shown in SEQ ID NO.2 includes a repeating sequence AACA, which is repeated 5 times; The nucleic acid shown in SEQ ID NO. 3 includes a repeating sequence TGTA, which is repeated 9 times; The nucleic acid shown in SEQ ID NO. 4 includes a repeating sequence CCAAC, which is repeated 4 times; The nucleic acid shown in SEQ ID NO.5 includes a repeating sequence TCTT, which is repeated 7 times; The nucleic acid shown in SEQ ID NO.6 includes a repeating sequence TCAA, which is repeated 5 times; The nucleic acid shown in SEQ ID NO.7 includes a repeating sequence ATAG, which is repeated 14 times; The nucleic acid shown in SEQ ID NO.8 includes a repeating sequence CCAAT, which is repeated 4 times; The nucleic acid shown in SEQ ID NO.9 includes a repeating sequence ATAC, which is repeated 9 times; The nucleic acid shown in SEQ ID NO. 10 includes a repeating sequence TACA, which is repeated 10 times; The nucleic acid shown in SEQ ID NO. 11 includes a repeating sequence TGGAT, which is repeated three times; The nucleic acid shown in SEQ ID NO. 12 includes a repeating sequence ATGA, which is repeated 4 times; The nucleic acid shown in SEQ ID NO. 13 includes a repeating sequence GGGCT, which is repeated 7 times; The nucleic acid shown in SEQ ID NO. 14 includes a repeating sequence TGCT, which is repeated 5 times; The nucleic acid shown in SEQ ID NO. 15 includes a repeating sequence TCCA, which is repeated 10 times; The nucleic acid shown in SEQ ID NO. 16 includes a repeating sequence ATAC, which is repeated 5 times; The nucleic acid shown in SEQ ID NO.17 includes the repeating sequence TCCT, which is repeated 13 times.

[0010] In a second aspect, the present invention provides a primer pair, wherein the primer pair comprises any one or more of the following: (1) the nucleotide sequences shown in SEQ ID NO.18 and SEQ ID NO.19; (2) the nucleotide sequences shown in SEQ ID NO.20 and SEQ ID NO.21; (3) the nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23; (4) the nucleotide sequences shown in SEQ ID NO.24 and SEQ ID NO.25; (5) the nucleotide sequences shown in SEQ ID NO.26 and SEQ ID NO.27; (6) the nucleotide sequences shown in SEQ ID NO.28 and SEQ ID NO.29; (7) the nucleotide sequences shown in SEQ ID NO.30 and SEQ ID NO.31; (8) the nucleotide sequences shown in SEQ ID NO.32 and SEQ ID NO.33; (9) the nucleotide sequences shown in SEQ ID NO.34 and SEQ ID NO.35; (10) the nucleotide sequences shown in SEQ ID NO.36 and SEQ ID NO.37; (11) the nucleotide sequences shown in SEQ ID NO.38 and SEQ ID NO.39; (12) the nucleotide sequences shown in SEQ ID NO.40 and SEQ ID NO.41; (13) the nucleotide sequences shown in SEQ ID NO.42 and SEQ ID NO.43; (14) the nucleotide sequences shown in SEQ ID NO.44 and SEQ ID NO.45; (15) the nucleotide sequences shown in SEQ ID NO.46 and SEQ ID NO.47; (16) the nucleotide sequences shown in SEQ ID NO.48 and SEQ ID NO.49; (17) The nucleotide sequences shown in SEQ ID NO.50 and SEQ ID NO.51.

[0011] In a third aspect, the present invention provides a kit comprising the molecular marker or the primer pair.

[0012] In a fourth aspect, the present invention provides the use of the molecular marker, or the primer pair, or the kit in identifying varieties of plants of the genus Malus.

[0013] The present invention further provides the use of the molecular marker, the primer pair, or the kit in molecular marker-assisted breeding, whole genome association analysis, or construction of DNA fingerprints of plants of the genus Malus.

[0014] In a fifth aspect, the present invention provides a method for identifying a variety of a plant of the genus Malus, comprising: The sample to be tested is tested for polymorphism of the molecular marker, and the variety of the sample to be tested is determined according to the test result.

[0015] Furthermore, the method includes: extracting genomic DNA of the sample to be tested, performing PCR amplification using the primer pair or the kit, and determining the variety of the sample to be tested according to the amplification result.

[0016] Furthermore, the PCR amplification system is based on a 10 μL system and includes: 2× Master Mix 4~6μL, 10mM upstream and downstream primers 0.2~0.5μL each, 20ng / μL DNA template 0.5~1.5μL, the rest is water; The procedure of the PCR amplification includes: Pre-denaturation at 94-96℃ for 5-10 min; Denaturation at 94~96℃ for 30~60S, annealing at 57~62℃ for 30~60S, extension at 70~75℃ for 45~90S, for a total of 28~35 reaction cycles; extension at 70~75℃ for 10~15min, and storage at 0~4℃.

[0017] Further, judging the variety of the sample to be tested according to the test result includes: comparing the test result with the DNA fingerprint, and judging the variety of the sample to be tested according to the comparison result; The DNA fingerprint is constructed based on the polymorphism of the aforementioned molecular markers in various Malus plants.

[0018] The present invention has the following beneficial effects: The present invention uses the published whole genome sequences of seven apple plants as the object, obtains 17 apple plant long repeat motif (4-6 nucleotide repeat) SSR markers through retrieval and screening, and designs corresponding SSR primer pairs based on these SSR markers. The SSR primers provided by the present invention have high polymorphism, high reliability, good repeatability and good versatility, and can be applied to the fingerprint map construction and germplasm resource identification of apple plants.

[0019] The present invention proposes 17 SSR markers and primers with long repeat motifs (4-6 nucleotide repeats) in apple plants, establishes a technical system for the development of SSR markers in apple plants, and provides new marker selection for the molecular genetic analysis of apple plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D1L80M4 provided in Example 1 of the present invention.

[0022] Figure 2 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D2L23M4 provided in Example 1 of the present invention.

[0023] Figure 3 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D3L66M4 provided in Example 1 of the present invention.

[0024] Figure 4 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D4L90M5 provided in Example 1 of the present invention.

[0025] Figure 5 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D5L99M4 provided in Example 1 of the present invention.

[0026] Figure 6 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D6L12M4 provided in Example 1 of the present invention.

[0027] Figure 7 It is a fluorescent capillary electrophoresis diagram of the primer amplification product of the SSR marker D7L74M4 provided in Example 1 of the present invention.

[0028] Figure 8 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D8L26M5 provided in Example 1 of the present invention.

[0029] Fig. 9 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D9L82M4 provided in Example 1 of the present invention.

[0030] Fig.10 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D10L24M4 provided in Example 1 of the present invention.

[0031] Fig.11 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D11L51M5 provided in Example 1 of the present invention.

[0032] Fig.12 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D12L43M4 provided in Example 1 of the present invention.

[0033] Fig.13 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D13L30M5 provided in Example 1 of the present invention.

[0034] Fig.14 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D14L8M4 provided in Example 1 of the present invention.

[0035] Fig.15It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D15L2M4 provided in Example 1 of the present invention.

[0036] Fig.16 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D16L85M4 provided in Example 1 of the present invention.

[0037] Fig.17 It is a fluorescence capillary electrophoresis diagram of the primer amplification product of the SSR marker D17L41M4 provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0040] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0041] Example 1 The present invention proposes a method for developing SSR markers based on multi-sample gene sequences, comprising: 1. SSR marker detection: Use SciRoKo and other SSR marker analysis software to detect SSR sites in multi-sample gene sequences.

[0042] In this step, SciRoKo software was used to detect SSR loci of the long repeat motif (4-6 nucleotide repeats) type in the published whole genome sequences of seven apple plants. The minimum number of repeats was 3 times, and the length of the SSR locus flanking sequence was ≥50bp.

[0043] 2. Primer design: Extract the flanking sequences of all SSR sites, including 250 bp upstream and downstream, and design primers using Primer software. In this step, the principles of primer design using Primer3.0 software are: primer length is 18-25 bp, optimal 20 bp; PCR amplification product length is 80-500 bp; GC content is 40%-60%, optimal 50%; primer Tm value is 58.0-62.0℃, optimal 60.0℃.

[0044] 3. Computer screening of primers: Electronic PCR technology (e-PCR) was used to evaluate the amplification effect of the designed SSR primers, and primers with specific amplification, high polymorphism, high amplification rate, good versatility, and the difference between allele lengths being a multiple of the repeat unit length were retained.

[0045] In this step, the e-PCR program was used to obtain the amplification results of each pair of primers on each sample gene sequence, with multiple sample gene sequences as the object. The following criteria were used for primer screening: the number of amplified products ≤ 1, the product length was 80-500bp, the amplified fragment sequence only contained SSR markers of the long repeat motif (4-6 nucleotide repeats); the amplification success rate in multiple samples was ≥ 80%; the proportion of allele differences caused by mutations and other reasons that were integer times the length of the repeat motif was ≥ 75%. A total of 100 long repeat motif (4-6 nucleotide repeats) SSR markers with the best polymorphism were screened, and the designed primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0046] 4. PCR primer amplification: Set up the PCR reaction system and reaction program, and use genomic DNA of 4 individual apple plants to perform PCR amplification of SSR primers.

[0047] In this step, the PCR amplification product adopts a 10 μL reaction system. Specifically, 5 μL of 2×Master Mix, 0.2 μL of 10 mM upstream and downstream primers, 1 μL of 20 ng / μL DNA template, and 3.6 μL of ultrapure water are added to the 10 μL reaction system.

[0048] It should be pointed out here that the above-mentioned DNA template is: the genomic DNA of apple plants was extracted by the improved CTAB method, and the genomic DNA of four apple plant individuals (Xinjiang wild apple, Vitex schrenkiana, Prairie Fire Begonia and Weeping Begonia) was used as template.

[0049] Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 45 s, for a total of 30 reaction cycles; extension at 72°C for 12 min, and storage at 4°C.

[0050] 5. Agarose gel electrophoresis screening of primers: Use agarose gel to separate the PCR amplification products and obtain the gel electrophoresis pattern for SSR marker primer screening.

[0051] In this step, 2% agarose gel was used, the electrophoresis buffer was 1×TAE, and the dye was SYBR Green. 2 μL of PCR product was loaded into the sample well, and after 15 minutes of electrophoresis at 100W constant voltage, the gel was placed in an electrophoresis imaging system to observe the electrophoresis results and take photos for preservation.

[0052] 6. Screening of primers by fluorescent capillary electrophoresis: The 5' end of one of the SSR primers in each pair obtained in the previous step was fluorescently labeled, PCR amplification was performed using genomic DNA from four individual Malus plants, and then the PCR amplification products were subjected to fluorescent capillary electrophoresis.

[0053] In this step, after deionized formamide and the molecular mass internal standard are mixed at a volume ratio of 100:1, 15 μL is taken and added to the sample plate, and then 1 μL of the PCR product diluted 10 times is added, and then the fluorescence signal is detected using the capillary automatic fluorescence electrophoresis system ABI3730XL. The fluorescence signal is processed using Genemarker software, and the allele numerical results are output in an Excel table. According to the method for developing SSR markers based on multi-sample gene sequences proposed in this embodiment, 17 pairs of SSR primers with high polymorphism, high reliability, good repeatability and good versatility were screened from 100 pairs of SSR primers, and their fluorescence capillary electrophoresis diagrams are as follows Figures 1 to 17 shown.

[0054] Example 2 As described in Example 1, the present invention screened 17 long repeat motif (4-6 nucleotide repeats) SSR markers with high polymorphism, high reliability, good repeatability and good versatility from the published whole genome sequences of seven apple plants and developed corresponding primer sequences. These markers can be used for genetic research and applications such as molecular identification, diversity assessment, and resource protection of apple plants.

[0055] These molecular markers include: D1L80M4, D2L23M4, D3L66M4, D4L90M5, D5L99M4, D6L12M4, D7L74M4, D8L26M5, D9L82M4, D10L24M4, D11L51M5, D12L43M4, D13L30M5, D14L8M4, D15L2M4, D16L85M4 and D17L41M4.

[0056] D1L80M4 is located at positions 25873955-25874479 of chromosome 1 of apple, and includes the nucleic acid shown in SEQ ID NO.1, including the repeating sequence TCCA, which is repeated 6 times; D2L23M4 is located at positions 8189707-8190228 of chromosome 2 of apple, and includes the nucleic acid shown in SEQ ID NO. 2, including the repeating sequence AACA, which is repeated 5 times; D3L66M4 is located at position 24094463-24095000 of chromosome 3 of apple, and includes the nucleic acid shown in SEQ ID NO.3, including the repeating sequence TGTA, which is repeated 9 times; D4L90M5 is located at positions 28327304-28327827 of chromosome 4 of apple, and includes the nucleic acid shown in SEQ ID NO. 4, including the repeating sequence CCAAC, which is repeated 4 times; D5L99M4 is located at position 45659776-45660306 of chromosome 5 of apple, and includes the nucleic acid shown in SEQ ID NO.5, including the repeating sequence TCTT, which is repeated 7 times; D6L12M4 is located at positions 4401044-4401565 of chromosome 6 of apple, and includes the nucleic acid shown in SEQ ID NO.6, including the repeating sequence TCAA, which is repeated 5 times; D7L74M4 is located at positions 27323858-27324416 of chromosome 7 of apple, and includes the nucleic acid shown in SEQ ID NO.7, including the repeating sequence ATAG, which is repeated 14 times; D8L26M5 is located at positions 8431509-8432031 of chromosome 8 of apple, and includes the nucleic acid shown in SEQ ID NO.8, including the repeating sequence CCAAT, which is repeated 4 times; D9L82M4 is located at positions 29336240-29336778 of chromosome 9 of apple, and includes the nucleic acid shown in SEQ ID NO. 9, including the repeat sequence ATAC, which is repeated 9 times; D10L24M4 is located at positions 10692000-10692383 of chromosome 10 of apple, and includes the nucleic acid shown in SEQ ID NO. 10, including the repeating sequence TACA, which is repeated 10 times; D11L51M5 is located at positions 19864815-19865331 of chromosome 11 of apple, and includes the nucleic acid shown in SEQ ID NO. 11, including the repeating sequence TGGAT, which is repeated 3 times; D12L43M4 is located at positions 13575540-13576097 of chromosome 12 of apple, and includes the nucleic acid shown in SEQ ID NO. 12, including the repeating sequence ATGA, which is repeated 4 times; D13L30M5 is located at positions 13747175-13747711 of chromosome 13 of apple, and includes the nucleic acid shown in SEQ ID NO. 13, including the repeating sequence GGGCT, which is repeated 7 times; D14L8M4 is located at positions 3618789-3619308 of chromosome 14 of apple, and includes the nucleic acid shown in SEQ ID NO. 14, including the repeating sequence TGCT, which is repeated 5 times; D15L2M4 is located at positions 1024164-1024705 of chromosome 15 of apple, and includes the nucleic acid shown in SEQ ID NO. 15, including the repeating sequence TCCA, which is repeated 10 times; D16L85M4 is located at positions 35310591-35311113 of chromosome 16 of apple, and includes the nucleic acid shown in SEQ ID NO. 16, including the repeat sequence ATAC, which is repeated 5 times; D17L41M4 is located at positions 13895588-13896141 of chromosome 17 of apple, and includes the nucleic acid shown in SEQ ID NO.17, including the repetitive sequence TCCT, which is repeated 13 times.

[0057] Accordingly, the present invention has developed primer sequences for the above 17 SSR markers, as shown in the following table: Table 1 Primer sequences for 17 SSR markers

[0058] Example 3 The present invention adopts a group of developed markers, D6L12M4, D15L2M4, D16L85M4, D13L30M5, D9L82M4, D5L99M4, and D11L51M5, a total of 7, to construct DNA fingerprints of 26 apple germplasms, providing a basis for the molecular identification of apple germplasm resources in the future.

[0059] The DNA fingerprints of 26 apple germplasms constructed are shown in the following table: Table 2 Fingerprints of 26 Malus germplasms

[0060] The samples to be tested and the control varieties were labeled and detected with the primer pairs shown in Table 2 to obtain the allelic variation data of the samples to be tested and the control varieties at these sites. These data were used for comparison. The determination method is as follows: (1) If the number of different spectral bands between the samples is greater than 2, they are judged to be different varieties; (2) The number of different spectral bands between the detected samples = 1, which means they are similar varieties; (3) If the number of different spectral bands between the detected samples is 0, they are judged to be suspected to be the same variety.

[0061] The present invention applies the DNA fingerprint obtained as above to the variety identification of 6 apple samples (including the following apple varieties: Gala apple, Royal Begonia, Flame Begonia, Prairie Fire Begonia, Salmonella Begonia, and Spring Snow Begonia). The results show that the discrimination accuracy rate reaches 100%, which indicates that the DNA fingerprint constructed in this embodiment can be used for variety identification of apple plants.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A molecular marker, characterized in that The molecular markers include: one or more of D1L80M4, D2L23M4, D3L66M4, D4L90M5, D5L99M4, D6L12M4, D7L74M4, D8L26M5, D9L82M4, D10L24M4, D11L51M5, D12L43M4, D13L30M5, D14L8M4, D15L2M4, D16L85M4 or D17L41M4; Among them, D1L80M4 is located at positions 25873955-25874479 of Apple chromosome 1; D2L23M4 is located at positions 8189707-8190228 of Apple chromosome 2; D3L66M4 is located at positions 24094463-24095000 of Apple chromosome 3; D4L90M5 is located at positions 28327304-28327827 of Apple chromosome 4; and D5L99M4 is located at positions 28327304-28327827 of Apple chromosome 5. D6L12M4 is located at position 4401044-4401565 on chromosome 6 of apple; D7L74M4 is located at position 27323858-27324416 on chromosome 7 of apple; D8L26M5 is located at position 8431509-8432031 on chromosome 8 of apple; D9L82M4 is located at position 2933624 on chromosome 9 of apple 0-29336778; D10L24M4 is located on the apple chromosome 10, 10692000-10692383; D11L51M5 is located on the apple chromosome 11, 19864815-19865331; D12L43M4 is located on the apple chromosome 12, 13575540-13576097; D13L30M5 is located on the apple chromosome 13, 13747175-1 3747711; D14L8M4 is located at positions 3618789-3619308 on apple chromosome 14; D15L2M4 is located at positions 1024164-1024705 on apple chromosome 15; D16L85M4 is located at positions 35310591-35311113 on apple chromosome 16; D17L41M4 is located at positions 13895588-13896141 on apple chromosome 17.

2. A molecular marker, characterized in that The molecular marker includes any one of the nucleic acids shown in SEQ ID NO.1-17; The nucleic acid shown in SEQ ID NO.1 includes a repeating sequence TCCA, which is repeated 6 times; The nucleic acid shown in SEQ ID NO.2 includes a repeating sequence AACA, which is repeated 5 times; The nucleic acid shown in SEQ ID NO. 3 includes a repeating sequence TGTA, which is repeated 9 times; The nucleic acid shown in SEQ ID NO. 4 includes a repeating sequence CCAAC, which is repeated 4 times; The nucleic acid shown in SEQ ID NO.5 includes a repeating sequence TCTT, which is repeated 7 times; The nucleic acid shown in SEQ ID NO.6 includes a repeating sequence TCAA, which is repeated 5 times; The nucleic acid shown in SEQ ID NO.7 includes a repeating sequence ATAG, which is repeated 14 times; The nucleic acid shown in SEQ ID NO.8 includes a repeating sequence CCAAT, which is repeated 4 times; The nucleic acid shown in SEQ ID NO.9 includes a repeating sequence ATAC, which is repeated 9 times; The nucleic acid shown in SEQ ID NO. 10 includes a repeating sequence TACA, which is repeated 10 times; The nucleic acid shown in SEQ ID NO. 11 includes a repeating sequence TGGAT, which is repeated three times; The nucleic acid shown in SEQ ID NO. 12 includes a repeating sequence ATGA, which is repeated 4 times; The nucleic acid shown in SEQ ID NO. 13 includes a repeating sequence GGGCT, which is repeated 7 times; The nucleic acid shown in SEQ ID NO. 14 includes a repeating sequence TGCT, which is repeated 5 times; The nucleic acid shown in SEQ ID NO. 15 includes a repeating sequence TCCA, which is repeated 10 times; The nucleic acid shown in SEQ ID NO. 16 includes a repeating sequence ATAC, which is repeated 5 times; The nucleic acid shown in SEQ ID NO.17 includes the repeating sequence TCCT, which is repeated 13 times.

3. A primer pair, characterized in that: The primer pair includes any one or more of the following: (1) the nucleotide sequences shown in SEQ ID NO.18 and SEQ ID NO.19; (2) the nucleotide sequences shown in SEQ ID NO.20 and SEQ ID NO.21; (3) the nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23; (4) the nucleotide sequences shown in SEQ ID NO.24 and SEQ ID NO.25; (5) the nucleotide sequences shown in SEQ ID NO.26 and SEQ ID NO.27; (6) the nucleotide sequences shown in SEQ ID NO.28 and SEQ ID NO.29; (7) the nucleotide sequences shown in SEQ ID NO.30 and SEQ ID NO.31; (8) the nucleotide sequences shown in SEQ ID NO.32 and SEQ ID NO.33; (9) the nucleotide sequences shown in SEQ ID NO.34 and SEQ ID NO.35; (10) the nucleotide sequences shown in SEQ ID NO.36 and SEQ ID NO.37; (11) the nucleotide sequences shown in SEQ ID NO.38 and SEQ ID NO.39; (12) the nucleotide sequences shown in SEQ ID NO.40 and SEQ ID NO.41; (13) the nucleotide sequences shown in SEQ ID NO.42 and SEQ ID NO.43; (14) the nucleotide sequences shown in SEQ ID NO.44 and SEQ ID NO.45; (15) the nucleotide sequences shown in SEQ ID NO.46 and SEQ ID NO.47; (16) the nucleotide sequences shown in SEQ ID NO.48 and SEQ ID NO.49; (17) The nucleotide sequences shown in SEQ ID NO.50 and SEQ ID NO.

51.

4. A kit, characterized in that: It comprises the molecular marker according to claim 1 or 2, or the primer pair according to claim 3.

5. Use of the molecular marker according to claim 1 or 2, or the primer pair according to claim 3, or the kit according to claim 4 in constructing a DNA fingerprint.

6. Use of the molecular marker according to claim 1 or 2, or the primer pair according to claim 3, or the kit according to claim 4 in molecular marker-assisted breeding of apple plants, whole genome association analysis, or identification of apple plant varieties.

7. A method for identifying varieties of apple plants, characterized in that: include: The sample to be tested is tested for the polymorphism of the molecular marker as claimed in claim 1 or 2, and the variety of the apple plant is identified according to the test result.

8. The method according to claim 7, characterized in that include: Extract the genomic DNA of the sample to be tested, perform PCR amplification using the primer pair described in claim 3 or the kit described in claim 4, and determine the variety of the sample to be tested based on the amplification result.

9. The method according to claim 8, characterized in that The PCR amplification system is based on a 10 μL system and includes: 2× Master Mix 4~6μL, 10mM upstream and downstream primers 0.2~0.5μL each, 20ng / μL DNA template 0.5~1.5μL, the rest is water; The procedure of the PCR amplification includes: Pre-denaturation at 94-96℃ for 5-10 min; Denaturation at 94~96℃ for 30~60S, annealing at 57~62℃ for 30~60S, extension at 70~75℃ for 45~90S, for a total of 28~35 reaction cycles; extension at 70~75℃ for 10~15min, and storage at 0~4℃.

10. The method according to any one of claims 7 to 9, characterized in that: The identifying the variety of the apple plant according to the test result comprises: comparing the test result with the DNA fingerprint spectrum, and determining the variety of the sample to be tested according to the comparison result; The DNA fingerprint is constructed based on the polymorphism of the molecular markers of claim 1 or 2 in various apple plants.

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