SSR Molecular Markers of Malus Plants and Their Applications

Abstract

The present invention relates to the technical field of plant breeding, and particularly to SSR molecular markers of Malus plants and their applications. The molecular markers include 17 SSR marker loci on chromosomes 1-17 of Malus. The present invention screened 17 SSR markers with high polymorphism, high reliability, good repeatability and good universality from the seven published whole-genome sequences of Malus plants. These molecular markers can be used for genetic research and applications such as DNA fingerprinting construction, molecular identification, diversity assessment, and resource protection of Malus plants, and have important significance in the field of Malus plant breeding.

Description

Technical Field

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

[0002] The genus Malus Malus Miller belongs to the subfamily Maloideae of the family Rosaceae, is a genus in the north temperate zone, and is widely distributed in North America, Asia and Europe. According to the fruit diameter and function, Malus plants can be divided into two categories: apples (>5 cm, for consumption) and Chinese flowering crabapples (≤5 cm, for ornamental purposes). Apples are one of the important fruits in the world, while Chinese flowering crabapples can be admired for their flowers in spring and fruits in autumn and winter, and are important landscape plants. Malus plants are easy to hybridize among individuals. Through the long-term efforts of breeders and horticulturists, a large number of cultivated varieties have been bred. Due to factors such as regional differences, misunderstandings and confusions, different naming standards, variations and hybridizations, the phenomena of different plants with the same name and the same plant with different names are widespread within the genus Malus, which hinders the identification, protection and industrial development of germplasm resources.

[0003] In the prior art, the comparative morphological method is usually used to classify and identify the cultivated varieties of Malus plants. With the gradual increase in the number of varieties, the morphological differences between new varieties and existing varieties may not be obvious enough, and the vast majority of differential traits are quantitative traits controlled by minor polygenes, and are also affected by epistatic effects and environmental factors. In this context, it is necessary to add additional or seek alternative classification and identification means, 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 the existing comparative morphological method. The molecular marker mainly used for current variety identification is the SSR marker. The SSR marker is also called microsatellite or short tandem repeat sequence (STRs), which is composed of 1 to 6 nucleotide tandem repeats. The number of these repeats is different among individuals, so length polymorphism will be generated through PCR amplification. The SSR marker has the advantages of codominance, high polymorphism, low cost, good transferability among related species, etc., and is a reliable method for variety identification or pedigree tracing.

[0005] In Malus plants, previous studies usually relied on microsatellite enrichment library sequences, EST sequences or transcriptome sequences, but these sequences only cover a small part of the genome. Moreover, due to limited sequence information, the vast majority of the developed SSR markers are of the more abundant 2-nucleotide repeat type, and the number of long repeat types (more than 2 nucleotides) is small. This easily leads to serious false peak phenomena, thereby increasing 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 their applications.

[0007] Most of the currently widely used SSR markers of Malus plants are of the 2-nucleotide repeat type, which is prone to serious false peak phenomena, resulting in a high typing error rate. In order to solve this technical problem, the present invention provides a group of SSR markers with good polymorphism and high accuracy of long repeat motifs (4-6 nucleotide repeats), and develops and designs corresponding SSR marker primers. The applications of these SSR markers and primers can provide more reliable results for molecular identification and genetic research of germplasms such as cultivated apples and ornamental crabapples.

[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;

[0009] Among them, D1L80M4 is located at positions 25873955 - 25874479 on chromosome 1 of apple; D2L23M4 is located at positions 8189707 - 8190228 on chromosome 2 of apple; D3L66M4 is located at positions 24094463 - 24095000 on chromosome 3 of apple; D4L90M5 is located at positions 28327304 - 28327827 on chromosome 4 of apple; D5L99M4 is located at positions 45659776 - 45660306 on chromosome 5 of apple; D6L12M4 is located at positions 4401044 - 4401565 on chromosome 6 of apple; D7L74M4 is located at positions 27323858 - 27324416 on chromosome 7 of apple; D8L26M5 is located at positions 8431509 - 8432031 on chromosome 8 of apple; D9L82M4 is located at positions 29336240 - 29336778 on chromosome 9 of apple; D10L24M4 is located at positions 10692000 - 10692383 on chromosome 10 of apple; D11L51M5 is located at positions 19864815 - 19865331 on chromosome 11 of apple; D12L43M4 is located at positions 13575540 - 13576097 on chromosome 12 of apple; D13L30M5 is located at positions 13747175 - 13747711 on chromosome 13 of apple; D14L8M4 is located at positions 3618789 - 3619308 on chromosome 14 of apple; D15L2M4 is located at positions 1024164 - 1024705 on chromosome 15 of apple; D16L85M4 is located at positions 35310591 - 35311113 on chromosome 16 of apple; D17L41M4 is located at positions 13895588 - 13896141 on chromosome 17 of apple.

[0010] Furthermore, the molecular marker includes any one of the nucleic acids shown in SEQ ID NO.1 - 17;

[0011] The nucleic acid shown in SEQ ID NO.1 includes the repeat sequence TCCA, repeated 6 times;

[0012] The nucleic acid shown in SEQ ID NO.2 includes the repeat sequence AACA, repeated 5 times;

[0013] The nucleic acid shown in SEQ ID NO.3 includes the repeat sequence TGTA, repeated 9 times;

[0014] The nucleic acid shown in SEQ ID NO.4 includes the repeat sequence CCAAC, repeated 4 times;

[0015] The nucleic acid shown in SEQ ID NO.5 includes the repeat sequence TCTT, repeated 7 times;

[0016] The nucleic acid shown in SEQ ID NO.6 includes the repeated sequence TCAA, repeated 5 times;

[0017] The nucleic acid shown in SEQ ID NO.7 includes the repeated sequence ATAG, repeated 14 times;

[0018] The nucleic acid shown in SEQ ID NO.8 includes the repeated sequence CCAAT, repeated 4 times;

[0019] The nucleic acid shown in SEQ ID NO.9 includes the repeated sequence ATAC, repeated 9 times;

[0020] The nucleic acid shown in SEQ ID NO.10 includes the repeated sequence TACA, repeated 10 times;

[0021] The nucleic acid shown in SEQ ID NO.11 includes the repeated sequence TGGAT, repeated 3 times;

[0022] The nucleic acid shown in SEQ ID NO.12 includes the repeated sequence ATGA, repeated 4 times;

[0023] The nucleic acid shown in SEQ ID NO.13 includes the repeated sequence GGGCT, repeated 7 times;

[0024] The nucleic acid shown in SEQ ID NO.14 includes the repeated sequence TGCT, repeated 5 times;

[0025] The nucleic acid shown in SEQ ID NO.15 includes the repeated sequence TCCA, repeated 10 times;

[0026] The nucleic acid shown in SEQ ID NO.16 includes the repeated sequence ATAC, repeated 5 times;

[0027] The nucleic acid shown in SEQ ID NO.17 includes the repeated sequence TCCT, repeated 13 times.

[0028] In a second aspect, the present invention provides a primer pair, which includes any one or more of the following:

[0029] (1) The nucleotide sequences shown in SEQ ID NO.18 and SEQ ID NO.19;

[0030] (2) The nucleotide sequences shown in SEQ ID NO.20 and SEQ ID NO.21;

[0031] (3) The nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23;

[0032] (4) The nucleotide sequences shown in SEQ ID NO.24 and SEQ ID NO.25;

[0033] (5) The nucleotide sequences shown in SEQ ID NO.26 and SEQ ID NO.27;

[0034] (6) The nucleotide sequences shown in SEQ ID NO.28 and SEQ ID NO.29;

[0035] (7) The nucleotide sequences shown in SEQ ID NO.30 and SEQ ID NO.31;

[0036] (8) The nucleotide sequences shown in SEQ ID NO.32 and SEQ ID NO.33;

[0037] (9) The nucleotide sequences shown in SEQ ID NO.34 and SEQ ID NO.35;

[0038] (10) The nucleotide sequences shown in SEQ ID NO.36 and SEQ ID NO.37;

[0039] (11) The nucleotide sequences shown in SEQ ID NO.38 and SEQ ID NO.39;

[0040] (12) The nucleotide sequences shown in SEQ ID NO.40 and SEQ ID NO.41;

[0041] (13) The nucleotide sequences shown in SEQ ID NO.42 and SEQ ID NO.43;

[0042] (14) The nucleotide sequences shown in SEQ ID NO.44 and SEQ ID NO.45;

[0043] (15) The nucleotide sequences shown in SEQ ID NO.46 and SEQ ID NO.47;

[0044] (16) The nucleotide sequences shown in SEQ ID NO.48 and SEQ ID NO.49;

[0045] (17) The nucleotide sequences shown in SEQ ID NO.50 and SEQ ID NO.51.

[0046] In a third aspect, the present invention provides a kit comprising the molecular marker or the primer pair described above.

[0047] Fourthly, the present invention provides the use of the molecular marker, or the primer pair, or the kit in the variety identification of Malus plants.

[0048] The present invention further provides the use of the molecular marker, or the primer pair, or the kit in molecular marker-assisted breeding, genome-wide association analysis or construction of DNA fingerprint maps of Malus plants.

[0049] Fifthly, the present invention provides a method for identifying the variety of Malus plants, comprising:

[0050] Detecting the polymorphism of the molecular marker in the sample to be tested, and judging the variety of the sample to be tested according to the detection result.

[0051] Further, the method comprises: extracting the genomic DNA of the sample to be tested, performing PCR amplification using the primer pair or the kit, and judging the variety of the sample to be tested according to the amplification result.

[0052] Further, taking a 10 μL system as an example, the PCR amplification system comprises:

[0053] 4 - 6 μL of 2×Master Mix, 0.2 - 0.5 μL of 10 mM upstream and downstream primers respectively, 0.5 - 1.5 μL of 20 ng / μL DNA template, and the balance is water;

[0054] The PCR amplification program comprises:

[0055] Pre-denaturation at 94 - 96 °C for 5 - 10 min;

[0056] Denaturation at 94 - 96 °C for 30 - 60 s, annealing at 57 - 62 °C for 30 - 60 s, extension at 70 - 75 °C for 45 - 90 s, for a total of 28 - 35 reaction cycles; extension at 70 - 75 °C for 10 - 15 min, and preservation at 0 - 4 °C.

[0057] Further, judging the variety of the sample to be tested according to the detection result includes: comparing the detection result with the DNA fingerprint map, and judging the variety of the sample to be tested according to the comparison result;

[0058] The DNA fingerprint map is constructed based on the polymorphism of the aforementioned molecular marker in each Malus plant.

[0059] The present invention has the following beneficial effects:

[0060] The present invention takes the seven publicly available whole-genome sequences of Malus plants as objects, retrieves and screens to obtain 17 SSR markers of long repeat motifs (4-6 nucleotide repeats) in Malus plants, and simultaneously 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 universality, and can be applied to the construction of fingerprint maps and the identification of germplasm resources of Malus plants.

[0061] The present invention proposes 17 SSR markers and primers of long repeat motifs (4-6 nucleotide repeats) in Malus plants, establishes a technical system for the development of SSR markers in Malus plants, and provides new marker selections for the molecular genetic analysis of Malus plants. Brief Description of the Drawings

[0062] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0063] Figure 1 It is the fluorescence capillary electrophoresis map of the primer amplification product of the SSR marker D1L80M4 provided in Example 1 of the present invention.

[0064] Figure 2 It is the fluorescence capillary electrophoresis map of the primer amplification product of the SSR marker D2L23M4 provided in Example 1 of the present invention.

[0065] Figure 3 It is the fluorescence capillary electrophoresis map of the primer amplification product of the SSR marker D3L66M4 provided in Example 1 of the present invention.

[0066] Figure 4 It is the fluorescence capillary electrophoresis map of the primer amplification product of the SSR marker D4L90M5 provided in Example 1 of the present invention.

[0067] Figure 5 It is the fluorescence capillary electrophoresis map of the primer amplification product of the SSR marker D5L99M4 provided in Example 1 of the present invention.

[0068] Figure 6 It is the fluorescence capillary electrophoresis map of the primer amplification product of the SSR marker D6L12M4 provided in Example 1 of the present invention.

[0069] Figure 7 It is the fluorescence capillary electrophoresis map of the primer amplification product of the SSR marker D7L74M4 provided in Example 1 of the present invention.

[0070] Figure 8 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D8L26M5 provided in Example 1 of the present invention.

[0071] Figure 9 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D9L82M4 provided in Example 1 of the present invention.

[0072] Figure 10 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D10L24M4 provided in Example 1 of the present invention.

[0073] Figure 11 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D11L51M5 provided in Example 1 of the present invention.

[0074] Figure 12 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D12L43M4 provided in Example 1 of the present invention.

[0075] Figure 13 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D13L30M5 provided in Example 1 of the present invention.

[0076] Figure 14 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D14L8M4 provided in Example 1 of the present invention.

[0077] Figure 15 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D15L2M4 provided in Example 1 of the present invention.

[0078] Figure 16 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D16L85M4 provided in Example 1 of the present invention.

[0079] Figure 17 It is the fluorescence capillary electrophoresis map of the primer amplification product of SSR marker D17L41M4 provided in Example 1 of the present invention. Detailed implementation manners

[0080] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts fall within the scope of protection of the present invention.

[0081] For the experimental methods involved in the following examples, unless otherwise specified, they are all conventional methods in the art. For example, reference can be made to the experimental manuals in the art or the conditions recommended in the manufacturer's instructions.

[0082] For the experimental materials and reagents involved in the following examples, unless otherwise specified, they can all be obtained commercially.

[0083] Example 1

[0084] The present invention provides a method for developing SSR markers based on multi-sample gene sequences, including:

[0085] 1. SSR marker detection: Use SSR marker analysis software such as SciRoKo to detect SSR loci in multi-sample gene sequences.

[0086] In this step, SciRoKo software is used to detect SSR loci of long repeat motif (4-6 nucleotide repeats) type in the publicly available whole genome sequences of seven Malus plants. The minimum number of repeats is 3 times, and the length of the flanking sequences of SSR loci is ≥50 bp.

[0087] 2. Primer design: Extract the flanking sequences of all SSR loci, including 250 bp each for upstream and downstream, and use Primer software to design primers. In this step, the principles for primer design using Primer3.0 software are: primer length is 18-25 bp, optimally 20 bp; PCR amplification product length is 80-500 bp; GC content is 40%-60%, optimally 50%; primer Tm value is 58.0-62.0 °C, optimally 60.0 °C.

[0088] 3. Computer screening of primers: Use in silico PCR (e-PCR) technology to evaluate the amplification effect of the designed SSR primers, and retain primers with specific amplification, high polymorphism, high amplification rate, good universality, and the difference between allele lengths being a multiple of the repeat unit length.

[0089] In this step, taking the multi-sample gene sequences as the object, the e-PCR program is used to obtain the amplification results of each pair of primers on each sample gene sequence. The following criteria are used for primer screening: the number of amplification products ≤1, the product length is 80-500 bp, and the amplified fragment sequence only contains SSR markers of long repeat motif (4-6 nucleotide repeats) type; the amplification success rate in multiple samples ≥80%; the proportion of the difference between alleles caused by mutations and other reasons being an integer multiple of the repeat motif length ≥75%. A total of 100 SSR markers with the best polymorphism of long repeat motif (4-6 nucleotide repeats) are screened out, and the designed primers are synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0090] 4. PCR Primer Amplification: Set up the PCR reaction system and reaction program, and perform PCR amplification of SSR primers using the genomic DNA of 4 individuals of Malus plants.

[0091] In this step, the PCR amplification product uses a 10 μL reaction system. Specifically, in a 10 μL reaction system, add 5 μL of 2×Master Mix, 0.2 μL each of 10 mM upstream and downstream primers, 1 μL of 20 ng / μL DNA template, and 3.6 μL of ultrapure water.

[0092] It should be noted here that the above DNA template is: Genomic DNA of Malus plants was extracted using the modified CTAB method, and the genomic DNA of 4 individuals of Malus plants (Malus sieversii, Malus baccata, Malus 'Prairifire', and Malus halliana) was used as the template.

[0093] 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 store at 4°C.

[0094] 5. Screening of Primers by Agarose Gel Electrophoresis: Use agarose gel to screen SSR marker primers from the gel electrophoresis pattern obtained after electrophoretic separation of the PCR amplification product.

[0095] In this step, a 2% agarose gel is used, the electrophoresis buffer is 1×TAE, and the dye is SYBR Green. Take 2 μL of the PCR product and load it into the sample well. After electrophoresis at a constant voltage of 100 W for 15 min, place the gel in an electrophoresis imaging system, observe the electrophoresis results and take pictures for preservation.

[0096] 6. Screening of Primers by Fluorescent Capillary Electrophoresis: Fluorescently label the 5' end of one of each pair of SSR primers obtained in the previous step, perform PCR amplification using the genomic DNA of 4 individuals of Malus plants, and then perform fluorescent capillary electrophoresis on the PCR amplification product.

[0097] In this step, after mixing deionized formamide and molecular weight internal standard in a volume ratio of 100:1, take 15 μL and add it to the upper sample plate, then add 1 μL of the PCR product diluted 10 times, and then use the capillary automatic fluorescence electrophoresis system ABI3730XL to detect the fluorescence signal. Use Genemarker software to process the fluorescence signal and output the allele numerical results in an Excel table. According to the method for developing SSR markers based on multi-sample gene sequences proposed in this example, 17 pairs of SSR primers with high polymorphism, high reliability, good repeatability, and good universality were screened from 100 pairs of SSR primers, and their fluorescent capillary electrophoresis patterns are respectively as Figures 1 to 17 shown.

[0098] Example 2

[0099] As described in Example 1, 17 SSR markers with high polymorphism, high reliability, good repeatability, and good universality (4 - 6 nucleotide repeat long repeat motifs) were screened from the seven published whole - genome sequences of Malus plants, and the corresponding primer sequences were developed. These markers can be used for genetic research and applications such as molecular identification, diversity assessment, and resource protection of Malus plants.

[0100] These molecular markers include: D1L80M4, D2L23M4, D3L66M4, D4L90M5, D5L99M4, D6L12M4, D7L74M4, D8L26M5, D9L82M4, D10L24M4, D11L51M5, D12L43M4, D13L30M5, D14L8M4, D15L2M4, D16L85M4, and D17L41M4.

[0101] D1L80M4 is located at positions 25873955 - 25874479 on chromosome 1 of apple, and includes the nucleic acid shown in SEQ ID NO.1, including the repeat sequence TCCA, repeated 6 times;

[0102] D2L23M4 is located at positions 8189707 - 8190228 on chromosome 2 of apple, and includes the nucleic acid shown in SEQ ID NO.2, including the repeat sequence AACA, repeated 5 times;

[0103] D3L66M4 is located at positions 24094463 - 24095000 on chromosome 3 of apple, and includes the nucleic acid shown in SEQ ID NO.3, including the repeat sequence TGTA, repeated 9 times;

[0104] D4L90M5 is located at positions 28327304 - 28327827 on chromosome 4 of apple, and includes the nucleic acid shown in SEQ ID NO.4, including the repeat sequence CCAAC, repeated 4 times;

[0105] D5L99M4 is located at positions 45659776 - 45660306 on chromosome 5 of apple, and includes the nucleic acid shown in SEQ ID NO.5, including the repeat sequence TCTT, repeated 7 times;

[0106] D6L12M4 is located at positions 4401044 - 4401565 on chromosome 6 of apple, and includes the nucleic acid shown in SEQ ID NO.6, including the repeat sequence TCAA, repeated 5 times;

[0107] D7L74M4 is located at positions 27323858 - 27324416 on chromosome 7 of apple, and includes the nucleic acid shown in SEQ ID NO.7, including the repeat sequence ATAG, repeated 14 times;

[0108] D8L26M5 is located at positions 8431509 - 8432031 on chromosome 8 of apple, and includes the nucleic acid shown in SEQ ID NO.8, including the repeat sequence CCAAT, repeated 4 times;

[0109] D9L82M4 is located at positions 29336240 - 29336778 on chromosome 9 of apple, and includes the nucleic acid shown in SEQ ID NO.9, including the repeat sequence ATAC, repeated 9 times;

[0110] D10L24M4 is located at positions 10692000 - 10692383 on chromosome 10 of apple, and includes the nucleic acid shown in SEQ ID NO.10, including the repeat sequence TACA, repeated 10 times;

[0111] D11L51M5 is located at positions 19864815 - 19865331 on chromosome 11 of apple, and includes the nucleic acid shown in SEQ ID NO.11, including the repeat sequence TGGAT, repeated 3 times;

[0112] D12L43M4 is located at positions 13575540 - 13576097 on chromosome 12 of apple, and includes the nucleic acid shown in SEQ ID NO.12, including the repeat sequence ATGA, repeated 4 times;

[0113] D13L30M5 is located at positions 13747175 - 13747711 on chromosome 13 of apple, and includes the nucleic acid shown in SEQ ID NO.13, including the repeat sequence GGGCT, repeated 7 times;

[0114] D14L8M4 is located at positions 3618789 - 3619308 on chromosome 14 of apple, and includes the nucleic acid shown in SEQ ID NO.14, including the repeat sequence TGCT, repeated 5 times;

[0115] D15L2M4 is located at positions 1024164 - 1024705 on chromosome 15 of apple, and includes the nucleic acid shown in SEQ ID NO.15, including the repeat sequence TCCA, repeated 10 times;

[0116] D16L85M4 is located at positions 35310591 - 35311113 on chromosome 16 of apple, and includes the nucleic acid shown in SEQ ID NO.16, including the repeat sequence ATAC, repeated 5 times;

[0117] D17L41M4 is located at positions 13895588 - 13896141 on chromosome 17 of apple, and includes the nucleic acid shown in SEQ ID NO.17, including the repeat sequence TCCT, which repeats 13 times.

[0118] Accordingly, the present invention has developed primer sequences for the above 17 SSR markers, as shown in the following table:

[0119] Table 1 Primer sequences for 17 SSR markers

[0120]

[0121] Example 3

[0122] The present invention uses a group of the developed markers, namely D6L12M4, D15L2M4, D16L85M4, D13L30M5, D9L82M4, D5L99M4, D11L51M5, a total of 7, to construct DNA fingerprint maps of 26 germplasms of Malus, providing a basis for the molecular identification of Malus germplasm resources in the future.

[0123] The DNA fingerprint maps of the 26 Malus germplasms constructed are shown in the following table:

[0124] Table 2 Fingerprint maps of 26 Malus germplasms

[0125]

[0126] The test samples and control varieties are subjected to marker detection with the primer pairs shown in Table 2 to obtain the allele variation data of the test samples and control varieties at these loci, and these data are used for comparison. The determination method is as follows:

[0127] (1) If the number of different bands detected between samples > 2, it is determined as different varieties;

[0128] (2) If the number of different bands detected between samples = 1, it is determined as closely related varieties;

[0129] (3) If the number of different bands detected between samples = 0, it is determined as suspected identical varieties.

[0130] The present invention applies the DNA fingerprint maps obtained above to the variety identification of 6 Malus samples (including the following Malus varieties: Gala apple, Royalty crabapple, Flame crabapple, Prairie Fire crabapple, Sargent crabapple, Spring Snow crabapple), and the result shows that the discrimination accuracy rate reaches 100%, indicating that the DNA fingerprint maps constructed in this example can be used for the variety identification of Malus plants.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A molecular marker combination, characterized in that: The molecular marker combination includes: D6L12M4, D15L2M4, D16L85M4, D13L30M5, D9L82M4, D5L99M4 and D11L51M5; Among them, D5L99M4 is located at positions 45659776-45660306 of apple chromosome 5; D6L12M4 is located at positions 4401044-4401565 of apple chromosome 6; D9L82M4 is located at positions 29336240-29336778 of apple chromosome 9; D11L51M5 is located at positions 19864815-19865331 of apple chromosome 11; D13L30M5 is located at positions 13747175-13747711 of apple chromosome 13; D15L2M4 is located at positions 1024164-1024705 of apple chromosome 15; D16L85M4 is located at positions 35310591-35311113 of apple chromosome 16.

2. The molecular marker combination according to claim 1, characterized in that: The molecular marker combination further includes: one or more of D1L80M4, D2L23M4, D3L66M4, D4L90M5, D7L74M4, D8L26M5, D10L24M4, D12L43M4, D14L8M4 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; D7L74M4 is located at positions 28327304-28327827 of Apple chromosome 7; 27323858-27324416; D8L26M5 is located on apple chromosome 8 at position 8431509-8432031; D10L24M4 is located on apple chromosome 10 at position 10692000-10692383; D12L43M4 is located on apple chromosome 12 at position 13575540-13576097; D14L8M4 is located on apple chromosome 14 at position 3618789-3619308; D17L41M4 is located on apple chromosome 17 at position 13895588-13896141.

3. A molecular marker combination, characterized in that: The molecular marker combination includes: nucleic acid molecules shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15 and SEQ ID NO.16; Wherein, the nucleic acid molecule as shown in SEQ ID NO.5 includes a repeating sequence TCTT, which is repeated 7 times; The nucleic acid molecule shown in SEQ ID NO.6 includes a repeating sequence TCAA, which is repeated 5 times; The nucleic acid molecule shown in SEQ ID NO.9 includes a repeating sequence ATAC, which is repeated 9 times; The nucleic acid molecule shown in SEQ ID NO. 11 includes a repeating sequence TGGAT, which is repeated three times; The nucleic acid molecule shown in SEQ ID NO. 13 includes a repeating sequence GGGCT, which is repeated 7 times; The nucleic acid molecule shown in SEQ ID NO. 15 includes a repeating sequence TCCA, which is repeated 10 times; The nucleic acid molecule shown in SEQ ID NO. 16 includes the repeating sequence ATAC, which is repeated 5 times.

4. The molecular marker combination according to claim 3, characterized in that: The molecular marker combination also includes: one or more of the nucleic acid molecules shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14 or SEQ ID NO.17; the nucleic acid molecule shown in SEQ ID NO.1 includes a repeating sequence TCCA, which is repeated 6 times; The nucleic acid molecule shown in SEQ ID NO.2 includes a repeating sequence AACA, which is repeated 5 times; The nucleic acid molecule shown in SEQ ID NO. 3 includes a repeating sequence TGTA, which is repeated 9 times; The nucleic acid molecule shown in SEQ ID NO.4 includes a repeating sequence CCAAC, which is repeated 4 times; The nucleic acid molecule shown in SEQ ID NO.7 includes a repeating sequence ATAG, which is repeated 14 times; The nucleic acid molecule shown in SEQ ID NO.8 includes a repeating sequence CCAAT, which is repeated 4 times; The nucleic acid molecule shown in SEQ ID NO. 10 includes a repeating sequence TACA, which is repeated 10 times; The nucleic acid molecule shown in SEQ ID NO. 12 includes a repeating sequence ATGA, which is repeated 4 times; The nucleic acid molecule shown in SEQ ID NO. 14 includes a repeating sequence TGCT, which is repeated 5 times; The nucleic acid molecule shown in SEQ ID NO.17 includes the repeating sequence TCCT, which is repeated 13 times.

5. A primer pair combination, characterized in that: The primer pair combination comprises: (1) the nucleotide sequences shown in SEQ ID NO.26 and SEQ ID NO.27; (2) the nucleotide sequences shown in SEQ ID NO.28 and SEQ ID NO.29; (3) the nucleotide sequences shown in SEQ ID NO.34 and SEQ ID NO.35; (4) the nucleotide sequences shown in SEQ ID NO.38 and SEQ ID NO.39; (5) the nucleotide sequences shown in SEQ ID NO.42 and SEQ ID NO.43; (6) the nucleotide sequences shown in SEQ ID NO.46 and SEQ ID NO.47; and (7) The nucleotide sequences shown in SEQ ID NO.48 and SEQ ID NO.

49.

6. The primer pair combination according to claim 5, characterized in that: The primer pair combination also includes one or more of the following primer pairs: (8) nucleotide sequences as shown in SEQ ID NO.18 and SEQ ID NO.19; (9) the nucleotide sequences shown in SEQ ID NO.20 and SEQ ID NO.21; (10) the nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23; (11) the nucleotide sequences shown in SEQ ID NO.24 and SEQ ID NO.25; (12) the nucleotide sequences shown in SEQ ID NO.30 and SEQ ID NO.31; (13) the nucleotide sequences shown in SEQ ID NO.32 and SEQ ID NO.33; (14) the nucleotide sequences shown in SEQ ID NO.36 and SEQ ID NO.37; (15) the nucleotide sequences shown in SEQ ID NO.40 and SEQ ID NO.41; (16) the nucleotide sequences shown in SEQ ID NO.44 and SEQ ID NO.45; (17) The nucleotide sequences shown in SEQ ID NO.50 and SEQ ID NO.

51.

7. A kit, characterized in that: It comprises the molecular marker combination according to any one of claims 1 to 4, or the primer pair combination according to claim 5 or 6.

8. Use of the molecular marker combination according to any one of claims 1 to 4, or the primer pair combination according to claim 5 or 6, or the kit according to claim 7 in constructing a DNA fingerprint.

9. Use of the molecular marker combination according to any one of claims 1 to 4, or the primer pair combination according to claim 5 or 6, or the kit according to claim 7 in molecular marker-assisted breeding of apple plants, whole genome association analysis, or identification of apple plant varieties.

10. A method for identifying varieties of plants of the genus Malus, characterized in that: include: The sample to be tested is tested for the polymorphism of the molecular marker combination as claimed in any one of claims 1 to 4, and the variety of the apple plant is identified according to the test result.

11. The method according to claim 10, characterized in that include: Extract the genomic DNA of the sample to be tested, perform PCR amplification using the primer pair combination described in claim 5 or 6, and determine the variety of the sample to be tested based on the amplification result.

12. The method according to claim 11, 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℃.

13. The method according to any one of claims 10 to 12, 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 marker combination described in any one of claims 1 to 4 in various Malus plants.

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