SNP (Single Nucleotide Polymorphism) marker combination and probe group for single apple remarking assisted selection and application of SNP marker combination and probe group

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to an SNP (Single Nucleotide Polymorphism) marker combination for single apple remarking assisted selection, a probe group and application of the SNP marker combination and the probe group. The SNP marker combination is composed of 16 SNP markers; the 16 SNP markers comprise SNP1-SNP16, and the number of the SNP markers is 16; the nucleotide sequences of the SNP1-SNP16 are sequentially as shown in SEQ ID NO. 1 to SEQ ID NO. 16. The invention focuses on controlling major variation sites of apple plant single fruit weight, develops 16 major SNP markers linked with apple single fruit weight traits, and is suitable for marker-assisted selection of apple single fruit weight traits in the seedling stage of breeding materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a group of SNP marker combinations, probe sets and applications thereof for apple single fruit weight marker-assisted selection. Background Art

[0002] apple, Malus domestica Apple is an important economic crop. my country's apple cultivation area covers 29.01 million mu (apple acres), with a production of 49.6017 million tons, representing 44.12% of the global cultivated area and 49.64% of the global production. The genus Malus is extremely diverse, with 35 species worldwide, including 27 wild species. Approximately 10,000 cultivated varieties have been developed through artificial selection, hybridization, and clonal propagation. Apple fruit weight is a key quality trait, and breeding new apple varieties with appropriate fruit weights is a key breeding goal and industry need. For a long time, breeders have focused on breeding new varieties based on a small number of high-quality parents. This limited genetic background has limited available variants, resulting in reduced genetic diversity. Furthermore, cultivated varieties lack resistance genes, making hybridization between mainstream varieties difficult to meet the breeding needs of achieving both resistance and excellent fruit quality. Currently, the lack of methods to identify undesirable variants in wild apple germplasm resources limits the full utilization of these genes. Apple fruit weight is a complex quantitative trait controlled by multiple genes.

[0003] To date, multiple QTLs for fruit size have been reported in apple, covering almost all chromosomes. Previous researchers developed molecular markers near the peak of the significant QTL interval and established a genome-wide additive model for predicting single fruit weight in apple, with a prediction accuracy of 0.7568. MdARF106 It is a major QTL for single fruit weight located on chromosome 15. miRNA172p Colocalizing with a significant fruit size QTL on chromosome 11, the gene explains 13.9% of the phenotypic variation. However, the genetic mechanism governing the small fruit trait remains unclear, severely hindering the creation of new, resistant varieties through hybridization between cultivars and closely related species. Therefore, there is a pressing need for marker-assisted selection to predict apple fruit weight and fully utilize closely related apple species. However, molecular markers that accurately predict apple fruit weight are currently lacking. Therefore, the discovery of new molecular markers that can accurately predict apple fruit weight is urgently needed. Summary of the Invention

[0004] In order to discover new molecular markers that can accurately predict apple fruit weight and implement marker-assisted selection for the apple fruit weight trait, the present invention aims to provide a major SNP marker combination suitable for marker-assisted selection of apple fruit weight. This combination can be applied to marker-assisted selection of apple fruit weight in hybrid offspring seedlings during the seedling stage, thereby improving breeding efficiency and reducing breeding costs. The present invention provides a set of SNP marker combinations, primer probe sets, and applications thereof for marker-assisted selection of apple fruit weight. To achieve the above objectives, the present invention adopts the following technical solutions.

[0005] One of the objectives of the present invention is to provide a set of SNP marker combinations for marker-assisted selection of single fruit weight in apple, wherein the SNP marker combination consists of 16 SNP markers; the 16 SNP markers include SNP1 to SNP16.

[0006] The SNP1 is Chr02_23876017 C / A, and its nucleotide sequence is shown in SEQ ID NO.1; the SNP1 is a mutation from C to A at 23876017bp of apple chromosome 2.

[0007] The SNP2 is Chr11_281738 C / T, and its nucleotide sequence is shown in SEQ ID NO.5. The SNP2 is a mutation from C to T at the 281738bp position of the 11th chromosome of apple.

[0008] The SNP3 is Chr12_17104265 T / G, and its nucleotide sequence is shown in SEQ ID NO.6. The SNP3 is a mutation from T to G at 17104265bp of apple chromosome 12.

[0009] The SNP4 is Chr12_23710003 T / G, and its nucleotide sequence is shown in SEQ ID NO.7. The SNP4 is a mutation from T to G at 23710003bp on apple chromosome 12.

[0010] The SNP5 is Chr14_20448307 A / G, and its nucleotide sequence is shown in SEQ ID NO.9. The SNP5 is a mutation from A to G at 20448307bp on the fourteenth chromosome of apple.

[0011] The SNP6 is Chr15_10278900 T / G, and its nucleotide sequence is shown in SEQ ID NO.10. The SNP6 is a mutation from T to G at 10278900bp on apple chromosome 15.

[0012] The SNP7 is Chr15_22313813 A / T, and its nucleotide sequence is shown in SEQ ID NO.12. The SNP7 is a mutation from A to T at 22313813bp on apple chromosome 15.

[0013] The SNP8 is Chr15_26425887 G / A, and its nucleotide sequence is shown in SEQ ID NO.13. The SNP8 is a mutation from G to A at 26425887bp on apple chromosome 15.

[0014] The SNP9 is Chr15_38111629 G / T, and its nucleotide sequence is shown in SEQ ID NO.14. The SNP9 is a mutation from G to T at 38111629bp of apple chromosome 15.

[0015] The SNP10 is Chr03_23985991 A / G, and its nucleotide sequence is shown in SEQ ID NO.2. The SNP10 is a mutation from A to G at 23985991bp of apple chromosome 3.

[0016] The SNP11 is Chr09_7495024 A / G, and its nucleotide sequence is shown in SEQ ID NO.3. The SNP11 is a mutation from A to G at 7495024bp of apple chromosome 9.

[0017] The SNP12 is Chr10_39740659 G / T / A:, and its nucleotide sequence is shown in SEQ ID NO.4. The SNP12 is a mutation from G to A at 39740659bp of the tenth chromosome of apple.

[0018] The SNP13 is Chr13_13933420 C / G, and its nucleotide sequence is shown in SEQ ID NO.8. The SNP13 is a mutation from C to G at 13933420 bp of apple chromosome 13.

[0019] The SNP14 is Chr15_14065871 G / A, and its nucleotide sequence is shown in SEQ ID NO.11. The SNP14 is a mutation from G to A at 14065871 bp of apple chromosome 15.

[0020] The SNP15 is Chr16_6334724 C / T, and its nucleotide sequence is shown in SEQ ID NO.15. The SNP15 is a mutation from C to T at 6334724bp of apple chromosome 16.

[0021] The SNP16 is Chr17_1867893 T / G, and its nucleotide sequence is shown in SEQ ID NO.16. The SNP16 is a mutation from T to G at 1867893bp of apple chromosome 17.

[0022] The present invention provides a set of SNP marker combinations for marker-assisted selection of apple fruit weight. The SNP marker combination includes 16 major SNP markers related to apple fruit weight and is suitable for molecular-assisted evaluation, screening, and breeding program design of Malus germplasm resources. The SNP marker combination provided by the present invention can accurately predict apple fruit weight, thereby enabling molecular marker-assisted selection for the apple fruit weight trait. This can address the technical deficiency of the existing art that still lacks molecular markers for relatively accurate prediction of apple fruit weight.

[0023] A second object of the present invention is to provide a probe set for detecting the SNP marker combination for marker-assisted selection of single fruit weight in apple, comprising GenoBaits DNA probes for detecting SNP1 to SNP16.

[0024] The nucleotide sequences of the GenoBaits DNA probes used to detect SNP1 to SNP16 are shown in SEQ ID NO.17 to SEQ ID NO.32, respectively.

[0025] The third purpose of the present invention is to use this group of major effect SNP marker combinations related to apple single fruit weight traits to identify apple germplasm resources, explore high-quality disease-resistant sites while eliminating undesirable allelic variation sites, and create new disease-resistant and excellent varieties.

[0026] The fourth object of the present invention is to use this group of apple single fruit weight molecular marker combinations in the molecular design of breeding programs, such as parent selection and combination matching, to further improve breeding efficiency.

[0027] A fifth object of the present invention is to provide a use of the SNP marker combination or the probe group in preparing a kit for determining the single fruit weight trait of apple.

[0028] Preferably, the kit comprises the SNP marker combination or the probe set.

[0029] A sixth object of the present invention is to provide the use of the SNP marker combination, the probe set or the kit in determining the single fruit weight trait of apple.

[0030] Preferably, the method comprises the following steps: Extract genomic DNA from the tested Malus plants.

[0031] The probe group is used to perform typing detection on the tested Malus plant.

[0032] According to the result of the typing test, the single fruit weight trait of the tested Malus plant is determined.

[0033] Preferably, the single fruit weight trait includes small fruit type, medium-small fruit type, large fruit type, medium fruit type and large-medium fruit type.

[0034] The small fruit type is recorded as S, and the average single fruit weight is 9.4 grams to 9.6 grams; the medium and small fruit type is recorded as MS, and the average single fruit weight is 68.4 grams to 68.6 grams; the large fruit type is recorded as L, and the average single fruit weight is 163.9 grams to 164.1 grams; the medium fruit type is recorded as M, and the average single fruit weight is 80.64 grams to 82.64 grams; the medium and large fruit type is recorded as LM, and the average single fruit weight is 131 grams to 133 grams.

[0035] Preferably, when the genotype at the SNP1 is AA, the genotype at the SNP10 is TT, the genotype at the SNP3 is GG, the genotype at the SNP4 is GG, the genotype at the SNP5 is GG, the genotype at the SNP6 is GG, the genotype at the SNP7 is TT, the genotype at the SNP8 is AA or the genotype at the SNP9 is TT, it is determined that the single fruit weight trait of the tested apple plant is a small fruit type.

[0036] When the genotype at SNP1 is CA, the genotype at SNP10 is CT, the genotype at SNP3 is TG, the genotype at SNP4 is TG, the genotype at SNP5 is AG, the genotype at SNP6 is TG, the genotype at SNP7 is AT, the genotype at SNP8 is GA or the genotype at SNP9 is GT, it is determined that the single fruit weight trait of the tested apple plant is a medium or small fruit type.

[0037] When the prerequisite that the genotype at SNP1 is CC, the genotype at SNP10 is CC, the genotype at SNP3 is TT, the genotype at SNP4 is TT, the genotype at SNP5 is AA, the genotype at SNP6 is TT, the genotype at SNP7 is AA, the genotype at SNP8 is GG, and the genotype at SNP9 is GG is satisfied at the same time, and the genotype at SNP10 is GG, the genotype at SNP11 is AG, the genotype at SNP14 is AA, or the genotype at SNP16 is TG, then it is determined that the single fruit weight trait of the tested apple plant is a large fruit type.

[0038] When the genotype at SNP1 is CC, the genotype at SNP10 is CC, the genotype at SNP3 is TT, the genotype at SNP4 is TT, the genotype at SNP5 is AA, the genotype at SNP6 is TT, the genotype at SNP7 is AA, the genotype at SNP8 is GG, and the genotype at SNP9 is GG is satisfied at the same time, and any one of the T allele variation at SNP12, the G allele variation at SNP13, and the T allele variation at SNP15 is satisfied, it is determined that the single fruit weight trait of the tested apple plant is medium fruit type.

[0039] When the tested Malus plant does not meet any of the above conditions, it is determined that its single fruit weight trait is a large-medium fruit type.

[0040] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a set of SNP marker combinations for marker-assisted selection of apple fruit weight, comprising 16 major SNP markers linked to the apple fruit weight trait, and suitable for molecular-assisted evaluation, screening, and breeding program design of Malus germplasm resources. The SNP marker combination provided by the present invention is composed of 16 SNP markers; the 16 SNP markers include SNP1 to SNP16; the nucleotide sequences of SNP1 to SNP16 are shown in SEQ ID NO.1 to SEQ ID NO.16, respectively; the SNP marker combination can accurately predict apple fruit weight, thereby achieving molecular marker-assisted selection for the apple fruit weight trait, and can address the technical defect in the prior art that there is still a lack of molecular markers that can more accurately predict apple fruit weight.

[0041] 2. The SNP marker combination provided by the present invention has the function of predicting single fruit weight, which is specifically manifested as follows: When the genotype at SNP1 is AA, or the genotype at SNP2 is TT, or the genotype at SNP3 is GG, or the genotype at SNP4 is , or the genotype at SNP5 is GG, or the genotype at SNP6 is GG, or the genotype at SNP7 is TT, or the genotype at SNP8 is AA, or the genotype at SNP9 is TT, then the single fruit weight trait of the tested apple plant is small fruit, that is, S. When the genotype at SNP1 is CA, or the genotype at SNP2 is CT, or the genotype at SNP3 is TG, or the genotype at SNP4 is TG, or the genotype at SNP5 is AG, or the genotype at SNP6 is TG, or the genotype at SNP7 is AT, or the genotype at SNP8 is GA, or the genotype at SNP9 is GT, then the single fruit weight trait of the tested apple plant is small to medium fruit, that is, MS. When the prerequisite that the genotype at SNP1 is CC, the genotype at SNP2 is CC, the genotype at SNP3 is TT, the genotype at SNP4 is TT, the genotype at SNP5 is AA, the genotype at SNP6 is TT, the genotype at SNP7 is AA, the genotype at SNP8 is GG, and the genotype at SNP9 is GG is met, and the genotype at SNP10 is GG or the genotype at SNP11 is AG or the genotype at SNP14 is AA or the genotype at SNP16 is TG, then the single fruit weight trait of the tested apple plant is large fruit, i.e. L. When the conditions that the genotype at SNP1 is CC, the genotype at SNP2 is CC, the genotype at SNP3 is TT, the genotype at SNP4 is TT, the genotype at SNP5 is AA, the genotype at SNP6 is TT, the genotype at SNP7 is AA, the genotype at SNP8 is GG, and the genotype at SNP9 is GG are met simultaneously, and SNP12 contains a T allele variation, or SNP13 contains a G allele variation, or SNP15 contains a T allele variation, then the single fruit weight trait of the tested apple plant is medium fruit, i.e., M. When the tested apple plant does not meet any of the above conditions, then the single fruit weight trait of the tested apple plant is large medium fruit, i.e., LM.

[0042] From the above, it can be seen that the SNP marker combination provided by the present invention can more accurately predict the weight of single apple fruit.

[0043] 3. The present invention focuses on the major effect variation sites that control the single fruit weight of apple plants, and develops 16 major effect SNP markers linked to the apple single fruit weight trait. These 16 major effect SNP markers include SNP1~SNP16, which constitute the SNP marker combination, which is suitable for marker-assisted selection of the apple single fruit weight trait during the seedling stage of breeding materials.

[0044] 4. This study developed 16 major SNP markers linked to the apple fruit weight trait, demonstrating low cost, simple operation, and high accuracy. Furthermore, 1,808 apple accessions were graded into five categories: small, small-medium, medium, large-medium, and large. The Pearson correlation coefficient between the predicted and measured phenotypic values ​​for apple fruit weight was r=0.8423 (n=1,808). BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 These are the results of marker-assisted selection of 16 major-effect SNPs linked to the apple fruit weight trait in the present invention. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0047] The test materials used in the examples of the present invention are as follows: The apple materials used in the embodiments of the present invention include 'Purple Pearl', 'Red Fuji', 'Golden Crown', 'Red Jade' and 'Tsugaru', as well as hybrid populations constructed using the above parent materials, which are sourced from the apple breeding base in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0048] The 361 germplasm resources used in the examples were sourced from the apple breeding base in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0049] Example 1 1. Phenotypic identification of single fruit weight of apple fruit A phenotypic investigation of single fruit weight of apple was conducted for 10 consecutive years using 4792 hybrid offspring lines from four hybrid populations, namely 'Purple Pearl' × 'Red Fuji', 'Purple Pearl' × 'Golden Crown', 'Red Jade' × 'Golden Crown' and 'Red Jade' × 'Tsugaru', and 361 germplasm resources.

[0050] The specific method is to collect mature fruits, with the sample size of a single plant being greater than 5 fruits, and use a precision electronic balance (0.01g) to complete the mass measurement.

[0051] 2. Mining of variation sites for single fruit weight in apple Using BSA-seq and QTL mapping techniques on hybrid offspring populations, the QTL peaks mapped by BSA were integrated and aligned for trait association. Regions in the peaks exceeding the threshold indicate regions on the chromosome with significant genotype differences. Different parent combinations and traits have different thresholds.

[0052] Using a modified G' value method, we prioritized candidate genes with the highest G' values, expressed genes, and genes associated with the target trait. Molecular markers were developed based on the variant loci of these candidate genes. The marker effect sizes were calculated across the entire training population. Based on the contribution of the markers, QTLs associated with single fruit weight in apples were discovered.

[0053] A total of 90 QTL loci related to single fruit weight were obtained from the above four populations, distributed on chromosomes except apple chromosome 3, apple chromosome 4 and apple chromosome 8.

[0054] A genome-wide association study was conducted using 253 apple germplasm accessions. GWAS analysis was performed using EMMAX software with a linear mixed model. The 99.0% percentile of all sliding window association values ​​was used as the unified significance screening threshold, i.e., -log10(P_value) = 0.788. Sites above this threshold were identified as significant SNPs highly associated with the target trait, and 37 significant association intervals related to apple fruit weight were identified.

[0055] Among them, the full English name of BSA-seq is bulked segregant analysis sequencing, which means bulked segregant group mixed analysis method.

[0056] The full name of QTL in English is Quantitative trait loci mapping, which means quantitative trait locus.

[0057] The English abbreviation for genome-wide association study is GWAS, and its full English name is Genome Wide Association Study.

[0058] 3. Development of molecular markers for apple fruit weight Based on variant type, gene expression, and functional annotation, candidate genes were selected within each QTL and GWAS interval associated with apple fruit weight. GenoBaits primers were designed based on the variants identified, and molecular markers were developed. A total of 147 single-nucleotide polymorphisms (SNPs) and indels (InDel) were designed, distributed across 17 apple chromosomes. The GenoBaits strategy was employed for marker design. The GenoBaits primer design method is characterized by truncating 200 base pairs of sequence before and after the variant site. The variant site is retained and highlighted with square brackets ([ ]). The non-variant bases (before and after the variant bases) are indicated within the brackets, separated by a slash ( / ).

[0059] Among them, GenoBaits primers were synthesized by Shijiazhuang Boridi Biotechnology Co., Ltd. in Hebei Province. The target sequence was captured by DNA probes, and then the marker genotyping was performed using Illumina second-generation resequencing.

[0060] 4. Estimation of genotype effect values ​​of molecular markers for apple fruit weight and screening of major effect markers A total of 2,264 individual plants from the four hybrid combinations and apple germplasm resources were selected as a training population. The 147 SNP and InDel markers were genotyped using the GenoBaits strategy. The genotypic and marker effects of each marker on the apple fruit weight trait in the training population were estimated. The genotypic effect of a specific molecular marker was calculated as the difference between the mean fruit weight phenotype of individuals with the same genotype and the mean fruit weight phenotype of the total population. Sixteen major SNP markers linked to the apple fruit weight trait were identified, including nine SNP markers significantly associated with the small fruit phenotype and seven SNP markers significantly associated with the large fruit phenotype.

[0061] The GenoBaits strategy is the same as step 3.

[0062] The nine SNP markers significantly associated with the small fruit phenotype of apple were Chr02_23876017 C / A, Chr11_281738 C / T, Chr12_17104265 T / G, Chr12_23710003 T / G, Chr14_20448307 A / G, Chr15_10278900 T / G, Chr15_22313813 A / T, Chr15_26425887 G / A and Chr15_38111629 G / T / A.

[0063] Chr02_23876017 C / A is SNP1; Chr11_281738 C / T is SNP2; Chr12_17104265 T / G is SNP3; Chr12_23710003 T / G is SNP4; Chr14_20448307 A / G is SNP5; Chr15_10278900 T / G is SNP6; Chr15_22313813 A / T is SNP7; Chr15_26425887 G / A is SNP8; Chr15_38111629 G / T is SNP9.

[0064] Among them, the seven SNP markers significantly associated with the large fruit phenotype of apple were Chr03_23985991 A / G, Chr09_7495024 A / G, Chr10_39740659 G / T / A, Chr13_13933420 C / G, Chr15_14065871 G / A, Chr16_6334724 C / T and Chr17_1867893 T / G.

[0065] Chr03_23985991 A / G is SNP10; Chr09_7495024 A / G is SNP11; Chr10_39740659 G / A is SNP12; Chr13_13933420 C / G is SNP13; Chr15_14065871 G / A is SNP14; Chr16_6334724 C / T is SNP15; Chr17_1867893 T / G is SNP16.

[0066] SNP1 to SNP16 are the 16 major SNP markers linked to apple fruit weight identified in the screening, referred to as the 16 major SNP markers. Their flanking nucleotide sequence information is as follows. The brackets "[ ]" indicate the non-variable base (before) and the variant base (after), separated by a " / ".

[0067] The nucleotide sequence of Chr02_23876017 C / A is shown in SEQ ID NO.1: TGTTGATTTACCATCAAATAAATATTACTCGAACTCAAA[C / A]TTCTACACCTTATAATGCAAAACATGTACAAGGAAAATGATTTTTGCATG; among them, there is a C to A mutation at the 40bp position, that is, there is a C to A mutation at the 23876017bp position of apple chromosome 2.

[0068] The nucleotide sequence of Chr03_23985991 A / G is shown in SEQ ID NO.2: ACCTCCACAGGTTCCAAGCAGTCACTCATGGAAGCCGCGACGGCTATTTCTGATGGGAAATTCGAGGCCGCGGTGGAGATCCTCACCCGGATGACTCCGACCCGGGTCACGAATCCTAGACCCAGTTCAGAAGAAAGGCTTTTGGAGTTCATGGGTTTAGCTTTGAAGTCTCGGGTCAACCCGATTGATAACCCGCCACC[A / G]ATAGCGAGAGACTTCTGCAATTCATAAAGTTTCAATTTTTATCGAAGAAGAGAGAGAGGGCGTCTGAGTCATAGACAGTTCTCCTTCTAAGATCTCGAAGCGAATCCGCCTATCAAGCTCTCAGTCGCCATCGCTGGAAAATCCATTGAAATCTGCAACTTTTAAGTGAGTCTCAAATTTCCCAATTCGGAAATTGCTAC; among them, there is a mutation from A to G at the 201bp position, that is, there is a mutation from A to G at the 23985991bp position on apple chromosome 3.

[0069] The nucleotide sequence of Chr09_7495024 A / G is shown in SEQ ID NO.3: AACTGAAAAGAAAACATTTTTTGGTATCGATTGGTATTTTTTTTTTTTGTGTTGGGAGATTTTAATATACCGGAAAAGTTGTCGAGTTTCATGTGAATTTGCTCTCCCTCGTAGGTGAAATGATCGAAAGCCCAGGTTGGCTGGAAAAGCTCATCAAATTTTGCTGAGCTGACCAAACCCAGCAATGCAAGACTCAC[A / G]AAAGCACTTAGAGAAAAAGCCATTATTCCAAAAGCTGAACCAGCCATGGAAGCAGAAGAAGAGAGTTGAGTTGAGCAGAAGAAGGGAGTGGAGCAGAAGAAGAAGAGTGCAGAGAAAGTGTGGATCTTCTTCACTGAACTGCTCTGAGTCTGTGGAAGCACAAACCGAAAACACTGGCTTTTATAGCCCAATTTTCATTAGGC; among them, there is a mutation from A to G at the 198bp position, that is, there is a mutation from A to G at the 7495024bp position of apple chromosome 9.

[0070] The nucleotide sequence of Chr10_39740659 G / A is shown in SEQ ID NO.4: TCTTCCTTCCAAAGTGAGCTCTGGTCATCTTGATTTTATTCCGGAAGATGGTGCTATCAAGGTTGGCCCGCATCAAGGCGATTTATTTCAGGCTTATACTCTGATGAAAGTGAACAATGAGGCTGATGGAGACACAGTTTAAAGTTGATACTAATTTTTTTCCGCTCTTACACAAAATGATCCGAGAGAAGACAG[G / A]TTGGGGTTTTGAGTGTCAAGGTCACAG GTTGTGCTACTTTTCAGGTTGGTGTGCTGGAGTGGACAGAGACTACTTTACGAACTTATGAGAATGTGCTGCGTCAAGCAGATATTTATGGTGCTGTGGCAATGTCCCGTTATCCTTACAAATACTCATCTAATGTATGGAAAGCTTTTTGTGAGCTATGGGGGCCTTTAACCAACA; among them, there is a mutation from G to A at the 198bp position, that is, there is a mutation from G to A at the 39740659bp position of apple chromosome 10.

[0071] The nucleotide sequence of Chr11_281738 C / T is shown in SEQ ID NO.5: AAAGGCACAGAGGTAGCTGTTGTTTTATGGCCACTCTTCTAACTTTTTAATTTTTTTAATTTTTATTTTCAATATATTGTTTTTATTTTGATCTTTTGCCTGTTTTCTCTCTCCAAAGGAGCTTCCTTTTTCACCTCACACAGTTCACACAAAGATATCTGTGAGTCAGTAGGGTTTTTTTAATCTTTACTCTTTCTCCC[C / T]CATCCCTCTCTGTGAACCTACAATCTTGACTCGTGAGAGACAAACCCAACACAACACAAAACAAACAAACATAAAGCCAAAATCTTTTTCAACCTAAAACAAACCCTAATTCCCAAATCATAGTCGTCTTCTTCTTCCTTTCTTCCTGCTATTGTTGACTCAAAAACCTTGGCCTTTCACTCTTGTTCAATTTCTGCCAC; among them, there is a C to T mutation at the 201bp position, that is, there is a C to T mutation at the 281738bp position of apple chromosome 11.

[0072] The nucleotide sequence of Chr12_17104265 T / G is shown in SEQ ID NO.6: TGGAAGGGGGAAAACACTAATGAAAATGAATACTATTTCAACTAAACCTA[T / G]ACCCTACCAAGCAAACCAAGTGCATACTAACAAGAGGAATGCTAGCTACT; among them, there is a T to G mutation at the 51bp position, that is, there is a T to G mutation at the 17104265bp position of apple chromosome 12.

[0073] The nucleotide sequence of Chr12_23710003 T / G is shown in SEQ ID NO.7: GGTTGGTTGCATTGTGAACTTTGAGAGAAGATGTATACTTTTGAGCATTG[T / G]AGGAAGTTCCTATAAGTTTATTAGTGTGATTAGTGTTAAACTTGAAGTTC; among them, there is a mutation from T to G at the 51bp position, that is, there is a mutation from T to G at the 23710003bp position of apple chromosome 12.

[0074] The nucleotide sequence of Chr13_13933420 C / G is shown in SEQ ID NO.8: CACGTTTGCAATCTAGTTCATCAAAGGGTACAAGTTTTGCCTTAAAACTT[C / G]TTCCATTCTAAATGTGGACTCAGATTGAAACTGATG; among them, there is a C to G mutation at the 51bp position, that is, there is a C to G mutation at the 13933420bp position of apple chromosome 13.

[0075] The nucleotide sequence of Chr14_20448307 A / G is shown in SEQ ID NO.9: GGTGGAATTGGTGGAGCTCTGCATACAAGCCGCCTCTGAAAGCAGAGACA[A / G]CGTCGAGAAATGGCGGAGGCAGCGACGGAGTCTCGAGCGCTTGCCTCCTC; among them, there is a mutation from A to G at the 51bp position, that is, there is a mutation from A to G at the 20448307bp position of apple chromosome 14.

[0076] The nucleotide sequence of Chr15_10278900 T / G is shown in SEQ ID NO.10: ACCAGTTTTATTTATTCTTTTTATTTGTCGAATTGTACTAGAATCTGCGGTATCCACATTTATAAACCGCCTTTTCCCTGTGCTGTATTGTTATCTTGTATAATCGAAACCCCTTCAGTCTCCACACACAAAAATACTGTAGTCTCGATTTATCCAGTTGGTGCATGATGGCTAGTCCTCCTCCAAACCCAGTGC[T / G]GATTGCATACCAGGGAGGGGCGGGGGC AGTGCCAGACTGGTTGAACAAGGGGGACAACGCATGGCAAATGATATCTGCCACCCTCGTTGGTCTGCAGAGTGTGCCGGGTCTCGTCATCCTCTACGGCAGTATCGTTAAAAAGAAATGGGCGGTTAACTCAGCCTTCATGGCTCTCTACGCTTTTGCCGCTGTGGTCCTCTGCT; among them, there is a T to G mutation at the 198bp position, that is, there is a T to G mutation at the 10278900bp position of apple chromosome 15.

[0077] The nucleotide sequence of Chr15_14065871 G / A is shown in SEQ ID NO.11: TGGTGATTGGGTCCCTGAACTTGACACGCTCATCGTACGCCGTCCTATCAATTCCCCGATCATCAAAAAGATGAACCAGATCCTCATACAAAAACCCTACCAGCTGTTCCACGTCAAACGTTGATTTTGGCGGGCTTTGGTCCACTAAGCTTAGCCTAACAGCCCACCTGGATTTTTGGGTTAAGTGTTTGTGTTTC[G / A]AAGATTTAGGGCGTGGGCAAGGCGGTA GTTTGGGCTTAGTGGTGCTCAGCTGCCGGAAACTGACACACGCTGCGGGGATTAATGGCCGGAAGATTTGGTCTGAAAGTTGTGTGCTGCTCATTTTGTGGCTGAGCTGCTAAGGTTGTAGTGGTTGTGTTAGTTTTGGTTTTGGCATATGAAATCTTAACTAAAAATTGATTGGT; among them, there is a mutation from G to A at the 198bp position, that is, there is a mutation from G to A at the 14065871bp position of apple chromosome 15.

[0078] The nucleotide sequence of Chr15_22313813 A / T is shown in SEQ ID NO.12: GATGTTGTTACCATCAATGTTGGTAAGGGATCGATTTTGATGGATTCCAT[A / T]CTCTGTAGCAACCAACTTAAGTAGTCATTGTATTGCTGAATAAATACTAC; among them, there is a mutation from A to T at the 51bp position, that is, there is a mutation from A to T at the 22313813bp position of apple chromosome 15.

[0079] The nucleotide sequence of Chr15_26425887 G / A is shown in SEQ ID NO.13: TTGCGTGCATTAAAACCAAAGCCTTCCCTGTCCGAAATCGCACAAAGCCC[G / A]TCTGCGCCTTTCTCTCTCCTCTGTTTGAAAGTGGACAAAGAAATGCTGAG; among them, there is a mutation from G to A at the 51bp position, that is, there is a mutation from G to A at the 26425887bp position of apple chromosome 15.

[0080] The nucleotide sequence of Chr15_38111629 G / T is shown in SEQ ID NO.14: CAGCCTCTACCGTAAAACTACATAAAAATAAATGAATGAATTCAACACAA[G / T]CCTAACTCAATATGCAGGTACCATATAAATCAGGGGTTAGTAACAGTAAA; among them, there is a G to T mutation at the 51bp position, that is, there is a G to T mutation at the 38111629bp position of apple chromosome 15.

[0081] The nucleotide sequence of Chr16_6334724 C / T is shown in SEQ ID NO.15: CATTGCGCCACTTTGTTGGCTAGACAACTTAACAAACTCCGGCCCCTCGA[C / T]GGSGTCCACGCTCGAGGGATTGCTCCGAGCACACGACGTCACTGTTCCGG; among them, there is a C to T mutation at the 51bp position, that is, there is a C to T mutation at the 6334724bp position of apple chromosome 16.

[0082] The nucleotide sequence of Chr17_1867893 T / G is shown in SEQ ID NO.16: GGGAGTTGGAGATAAGAGCGTGCACAAATTTGAAGGTCCCAAGTGGGTTGAGGCACTTGAAGAATCTCACTCAGCTGAAATTAACTAATATGCCGGAAGAATTTGTTGCAACCATTGCGACAACTAAGGTGCAAATTTGGGATGACATAACTCACTCGCCAACAGTCATTACCGATAGCTGGTAGTCTATGCGGAAT[T / G]AGTCACAGGCTCTCTCTCCAGTAATC GGGTATGTTTTGGTTTTTTTGTACTGTGTTTATGTGTACAAGTACTGCGTAATGATGCTTGTTTTCTACATGGAAATCATGTTCTAGATCGAGTGACGCTCTTACTGGTTTATTGCTTTCAGGTAACATCGCCCAATGCATTTGACGCATAGGGAACCTGACAGCCTAGTCCATGTT; among them, there is a T to G mutation at the 198bp position, that is, there is a T to G mutation at the 1867893bp position of apple chromosome 17.

[0083] The GenoBaits DNA probe sequence information corresponding to the 16 major SNP markers linked to the apple fruit weight trait is as follows: The GenoBaits DNA probe used to detect SNP1 is Chr02_23876017:, and its nucleotide sequence is shown in SEQ ID NO.17: TGTTGATTTACCATCAAATAAATATTACTCGAACTCAAACTTCTACACCTTATAATGCAAAACATGTACAAGGAAAATGATTTTTGCATG.

[0084] The GenoBaits DNA probe used to detect SNP2 is Chr11_281738, and its nucleotide sequence is shown in SEQ ID NO.18: AAAGGCACAGAGGTAGCTGTTGTTTTATGGCCACTCTTCTAACTTTTTAATTTTTTTAATTTTTATTTTCAATATATTGTTTTTATTTTGATCTTTTGCCTGTTTTCTCTCCAAAGGAGCTTCCTTTTTCACCTCACACAGTTCACACAAAGATATCTGTGAGTCAGTAGGGTTTTTTTAATCTTTACTCTTTCTCCCC CATCCCTCTCTGTGAACCTACAATCTTGACTCGTGAGAGACAAACCCAACACAACACAAAACAAACAAACATAAAGCCAAAATCTTTTTCAACCTAAAACAAACCCTAATTCCCAAATCATAGTCGTCTTCTTCTTCCTTTCTTCCTGCTATTGTTGACTCAAAAACCTTGGCCTTTCACTCTTGTTCAATTTCTGCCAC.

[0085] The GenoBaits DNA probe used to detect SNP3 is Chr12_17104265, and its nucleotide sequence is shown in SEQ ID NO. 19: TGGAAGGGGGAAAACACTAATGAAAATGAATACTATTTCAACTAAACCTATACCCTACCAAGCAAACCAAGTGCATACTAACAAGAGGAATGCTAGCTACT.

[0086] The GenoBaits DNA probe used to detect SNP4 is Chr12_23710003, and its nucleotide sequence is shown in SEQ ID NO. 20: GGTTGGTTGCATTGTGAACTTTGAGAGAAGATGTATACTTTTGAGCATTGTAGGAAGTTCCTATAAGTTTATTAGTGTGATTAGTGTTAAACTTGAAGTTC.

[0087] The GenoBaits DNA probe used to detect SNP5 is Chr14_20448307, and its nucleotide sequence is shown in SEQ ID NO.21: GGTGGAATTGGTGGAGCTCTGCATACAAGCCGCCTCTGAAAGCAGAGACAACGTCGAGAAATGGCGGAGGCAGCGACGGAGTCTCGAGCGCTTGCCTCCTC.

[0088] The GenoBaits DNA probe used to detect SNP6 is Chr15_10278900, and its nucleotide sequence is shown in SEQ ID NO. 22: ACCAGTTTTATTTATTCTTTTTATTTGTCGAATTGTACTAGAATCTGCGGTATCCACATTTATAAACCGCCTTTTCCCTGTGCTGTATTGTTATCTTGTATAATCGAAACCCCTTCAGTCTCCACACACAAAAATACTGTAGTCTCGATTTATCCAGTTGGTGCATGATGGCTAGTCCTCCTCCAAACCCAGTGCTGAT TGCATACCAGGGAGGGGCGGGGGCAGTGCCAGACTGGTTGAACAAGGGGGACAACGCATGGCAAATGATATCTGCCACCCTCGTTGGTCTGCAGAGTGTGCCGGGTCTCGTCATCCTCTACGGCAGTATCGTTAAAAAGAAATGGGCGGTTAACTCAGCCTTCATGGCTCTCTACGCTTTTGCCGCTGTGGTCCTCTGCT.

[0089] The GenoBaits DNA probe used to detect SNP7 is Chr15_22313813, and its nucleotide sequence is shown in SEQ ID NO. 23: GATGTTGTTACCATCAATGTTGGTAAGGGATCGATTTTGATGGATTCCATACTCTGTAGCAACCAACTTAAGTAGTCATTGTATTGCTGAATAAATACTAC.

[0090] The GenoBaits DNA probe used to detect SNP8 is Chr15_26425887, and its nucleotide sequence is shown in SEQ ID NO. 24: TTGCGTGCATTAAAACCAAAGCCTTCCCTGTCCGAAATCGCACAAAGCCCGTCTGCGCCTTTCTCTCTCCTCTGTTTGAAAGTGGACAAAGAAATGCTGAG.

[0091] The GenoBaits DNA probe used to detect SNP9 is: Chr15_38111629, and its nucleotide sequence is shown in SEQ ID NO.25: CAGCCTCTACCGTAAAACTACATAAAAATAAATGAATGAATTCAACACAAGCCTAACTCAATATGCAGGTACCATATAAATCAGGGGTTAGTAACAGTAAA.

[0092] The GenoBaits DNA probe used to detect SNP10 is Chr03_23985991, and its nucleotide sequence is shown in SEQ ID NO. 26: ACCTCCACAGGTTCCAAGCAGTCACTCATGGAAGCCGCGACGGCTATTTCTGATGGGAAATTCGAGGCCGCGGTGGAGATCCTCACCCGGATGACTCCGACCCGGGTCACGAATCCTAGACCCAGTTCAGAAGAAAGGCTTTTGGAGTTCATGGGTTTAGCTTTGAAGTCTCGGGTCAACCCGATTGATAACCCGCCACCAATAGCGAGAGACTTCTGCAATTCATAAAGTTTCAATTTTTATCGAAGAAGAGAGAGAGGGCGTCTGAGTCATAGACAGTTCTCCTTCTAAGATCTCGAAGCGAATCCGCCTATCAAGCTCTCAGTCGCCATCGCTGGAAAATCCATTGAAATCTGCAACTTTTAAGTGAGTCTCAAATTTCCCAATTCGGAAATTGCTAC。

[0093] The GenoBaits DNA probe for detecting SNP11 is Chr09_7495024, and its nucleotide sequence is shown in SEQ ID NO.27: AACTGAAAAGAAAACATTTTTTGGTATCGATTGGTATTTTTTTTTTTTGTGTTGGGAGATTTTAATATACCGGAAAAGTTGTCGAGTTTCATGTGAATTTGCTCTCCCTCGTAGGTGAAATGATCGAAAGCCCAGGTTGGCTGGAAAAGCTCATCAAATTTTGCTGAGCTGACCAAACCCAGCAATGCAAGACTCACAAAAGCACTTAGAGAAAAAGCCATTATTCCAAAAGCTGAACCAGCCATGGAAGCAGAAGAAGAGAGTTGAGTTGAGCAGAAGAAGGGAGTGGAGCAGAAGAAGAAGAGTGCAGAGAAAGTGTGGATCTTCTTCACTGAACTGCTCTGAGTCTGTGGAAGCACAAACCGAAAACACTGGCTTTTATAGCCCAATTTTCATTAGGC。

[0094] The GenoBaits DNA probe for detecting SNP12 is Chr10_39740659, and its nucleotide sequence is shown in SEQ ID NO.28: TCTTCCTTCCAAAGTGAGCTCTGGTCATCTTGATTTTATTCCGGAAGATGGTGCTATCAAGGTTGGCCCGCATCAAGGCGATTTATTTCAGGCTTATACTCTGATGAAAGTGAACAATGAGGCTGATGGAGACACCAAGTTTAAAGTTGATACTAATTTTTTTCCGCTCTTACAAAATGATCCGAGAGAAGACAGGTTG GGGTTTGAGTGTCAAGGTCACAGGTTGTGCTACTTTTCAGGTTGGTGTGCTGGAGTGGACAGAGACTACTTTACGAACTTATGAGAATGTGCTGCGTCAAGCAGATATTTATGGTGCTGTGGCAATGTCCCGTTATCCTTACAAATACTCATCTAATGTATGGAAAGCTTTTTGTGAGCTATGGGGGCCTTTAACCAACA.

[0095] The GenoBaits DNA probe used to detect SNP13 is Chr13_13933420, and its nucleotide sequence is shown in SEQ ID NO.29: CACGTTTGCAATCTAGTTCATCAAAGGGTACAAGTTTTGCCTTAAAACTTCTTCCATTCTAAATGTGGACTCAGATTGAAACTGATG.

[0096] The GenoBaits DNA probe used to detect SNP14 is Chr15_14065871:, and its nucleotide sequence is shown in SEQ ID NO.30: TGGTGATTGGGTCCCTGAACTTGACACGCTCATCGTACGCCGTCCTATCAATTCCCCGATCATCAAAAAGATGAACCAGATCCTCATACAAAAACCCTACCAGCTGTTCCACGTCAAACGTTGATTTTGGCGGGCTTTGGTCCACTAAGCTTAGCCTAACAGCCCACCTGGATTTTTGGGTTAAGTGTTTGTGTTTCGAAG ATTTAGGGCGTGGGCAAGGCGGTAGTTTGGGCTTAGTGGTGCTCAGCTGCCGGAAACTGACACACGCTGCGGGGATTAATGGCCGGAAGATTTGGTCTGAAAGTTGTGTGCTGCTCATTTTGTGGCTGAGCTGCTAAGGTTGTAGTGGTTGTGTTAGTTTTGGTTTTGGCATATGAAATCTTAACTAAAAATTGATTGGT.

[0097] The GenoBaits DNA probe used to detect SNP15 is Chr16_6334724, and its nucleotide sequence is shown in SEQ ID NO.31: CATTGCGCCACTTTGTTGGCTAGACAACTTAACAAACTCCGGCCCCTCGACGGSGTCCACGCTCGAGGGATTGCTCCGAGCACACGACGTCACTGTTCCGG.

[0098] The GenoBaits DNA probe used to detect SNP16 is Chr17_1867893, and its nucleotide sequence is shown in SEQ ID NO.32: GGGAGTTGGAGATAAGAGCGTGCACAAATTTGAAGGTCCCAAGTGGGTTGAGGCACTTGAAGAATCTCACTCAGCTGAAATTAACTAATATGCCGGAAGAATTTGTTGCAACCATTGCGACAACTAAGGTGCAAATTTGGGATGACATAACTCACTCGCCAACAGTCATTACCGATAGCTGGTAGTCTATGCGGAATTAGT CACAGGCTTCTCTCCAATCGGGTATGTTTTGGTTTTTTGTACTGTGTTTATGTGTACAAGTACTGCGTAATGATGCTTGTTTTCTACATGGAAATCATGTTCTAGATCGAGTGACGCTCTTACTGGTTTTATTGCTTTCAGGTAACATCGCCCAATGCATTTGACGCATAGGGAACCTGACAGCCTAGTCCATGTT.

[0099] 5. Marker-assisted selection method for single fruit weight trait of apple fruit (1) DNA extraction, library construction, and target site capture sequencing Genomic DNA was extracted from leaves of the material to be assayed. The genome was accurately quantified using the Qubit® dsDNA HS Assay Kit. Enzyme digestion and DNA library construction were performed using the GenoBaits® DNA Library Prep Kit for ILM. Adapters were added using the GenoBaits® Barcode for ILM Kits. Molecular hybridization and target site capture were then performed using the GenoBaits® DNA Hybridization Kit for ILM.

[0100] (2) Target site sequencing and marker genotyping The captured target library was sequenced using an Illumina sequencer using the PE150 strategy at a sequencing depth of 1000× to 1200×. Sequencing reads were analyzed and genotyped using the GDDH13.1 reference genome.

[0101] (3) Marker-assisted prediction of single apple weight In the training population of this embodiment, the genotype distribution and phenotypic allelic variation of the 16 major SNP markers linked to the apple fruit weight trait are as follows: SNP1, Chr02_23876017, contains three genotypes: CC, CA, and AA. Individuals with the CA heterozygous genotype exhibit small to medium fruit, while individuals with the AA homozygous mutation exhibit small fruit.

[0102] SNP2, Chr14_20448307, contains three genotypes: AA, AG, and GG. Individuals with the AG heterozygous genotype exhibit small to medium-sized fruit, while individuals with the GG homozygous genotype exhibit small fruit.

[0103] SNP3, Chr15_10278900, contains three genotypes: TT, TG, and GG. Individuals with the TG heterozygous genotype exhibit small to medium-sized fruit, while individuals with the GG homozygous mutation exhibit small fruit.

[0104] SNP4, Chr15_22313813, contains three genotypes: AA, AT, and TT. Among them, individuals with the AT heterozygous genotype exhibit small to medium fruit, while individuals with the TT homozygous mutation genotype exhibit small fruit.

[0105] SNP5, Chr15_38111629, contains three genotypes: GG, GT, and TT. Individuals with the GT heterozygous genotype exhibit small to medium-sized fruit, while individuals with the TT homozygous mutation exhibit small fruit.

[0106] SNP6, Chr03_23985991, contains three genotypes: AA, AG, and GG. Among them, individuals with the GG genotype exhibit large fruit.

[0107] SNP7, Chr10_39740659, contains six genotypes: GG, GA, AT, GT, TT, and AA. Among them, individuals carrying the T allele exhibit medium fruit type.

[0108] SNP8, Chr13_13933420, has three genotypes: CC, CG, and GG. Individuals carrying the G allele exhibit a medium fruit type.

[0109] SNP9, Chr15_14065871, has three genotypes: GG, GA, and AA. Among them, individuals with the AA genotype exhibit large fruit.

[0110] SNP10, Chr11_281738, contains three genotypes: CC, CT, and TT. Individuals with the CT heterozygous genotype exhibit small to medium-sized fruit, while individuals with the TT homozygous mutation exhibit small fruit.

[0111] SNP11, Chr12_17104265, contains three genotypes: TT, TG, and GG. Individuals with the TG heterozygous genotype exhibit small to medium-sized fruit, while individuals with the GG homozygous genotype exhibit small fruit.

[0112] SNP12, Chr12_23710003, contains three genotypes: TT, TG, and GG. Individuals with the TG heterozygous genotype exhibit small to medium-sized fruit, while individuals with the GG homozygous genotype exhibit small fruit.

[0113] SNP13, Chr15_26425887, contains three genotypes: GG, GA, and AA. Individuals with the GA heterozygous genotype exhibit small to medium-sized fruit, while individuals with the AA homozygous genotype exhibit small fruit.

[0114] SNP14, Chr09_7495024, has two genotypes: AA and AG. Individuals with the AG genotype exhibit large fruit.

[0115] SNP15, Chr16_6334724, includes CC and CT genotypes. Among them, individuals with the CT genotype exhibit medium fruit type.

[0116] SNP16, Chr17_1867893, has three genotypes: TT, TG, and GG. Individuals with the TG genotype exhibit large fruit.

[0117] Among them, the small fruit type is recorded as S, with an average single fruit weight of 9.5 grams; the medium and small fruit type is recorded as MS, with an average single fruit weight of 68.5 grams; the large fruit type is recorded as L, with an average single fruit weight of 164.0 grams; the medium fruit type is recorded as M, with an average single fruit weight of 81.64 grams; and the medium and large fruit type is recorded as LM, with an average single fruit weight of 132 grams.

[0118] The 16 major SNP markers linked to the apple fruit weight trait have complementary epistatic effects. Based on their non-allelic interaction effects, the following criteria were used to judge and select apple fruit weight for apple germplasm resources or hybrid progeny: ① When the genotype at Chr02_23876017 is AA, or the genotype at Chr11_281738 is TT, or the genotype at Chr12_17104265 is GG, or the genotype at Chr12_23710003 is GG, or the genotype at Chr14_20448307 is GG, or the genotype at Chr15_10278900 is GG, or the genotype at Chr15_22313813 is TT, or the genotype at Chr15_26425887 is AA, or the genotype at Chr15_3811162 is TT, the germplasm resources or hybrid offspring all show small fruit type, i.e. S.

[0119] ② When the genotype at Chr02_23876017 is CA, or the genotype at Chr11_281738 is CT, or the genotype at Chr12_17104265 is TG, or the genotype at Chr12_23710003 is TG, or the genotype at Chr14_20448307 is AG, or the genotype at Chr15_10278900 is TG, or the genotype at Chr15_22313813 is AT, or the genotype at Chr15_26425887 is GA, or the genotype at Chr15_3811162 is GT, the germplasm resources or hybrid offspring all show small to medium fruit type, i.e. MS.

[0120] ③ When the genotype at Chr02_23876017 is CC, the genotype at Chr11_281738 is CC, the genotype at Chr12_17104265 is TT, the genotype at Chr12_23710003 is TT, the genotype at Chr14_20448307 is AA, the genotype at Chr15_10278900 is TT, and the genotype at Chr15_22313813 is AA Under the conditions that the genotype at Chr15_26425887 is GG, the genotype at Chr15_38111629 is GG, the genotype at Chr03_23985991 is GG, or the genotype at Chr09_7495024 is AG, or the genotype at Chr15_14065871 is AA, or the genotype at Chr17_1867893 is TG, the germplasm resources or hybrid offspring show a large fruit phenotype, i.e., L.

[0121] ④ When the genotype at Chr02_23876017 is CC, the genotype at Chr11_281738 is CC, the genotype at Chr12_17104265 is TT, the genotype at Chr12_23710003 is TT, the genotype at Chr14_20448307 is AA, the genotype at Chr15_10278900 is TT, and the genotype at Chr15_2231 is TT. Under the conditions that the genotype at Chr15_3813 is AA, the genotype at Chr15_26425887 is GG, and the genotype at Chr15_38111629 is GG, the germplasm resources or hybrid offspring containing the T allele variation at Chr10_39740659, or the G allele variation at Chr13_13933420, or the T allele variation at Chr16_6334724 exhibit medium fruit type, i.e., M.

[0122] ⑤ After excluding the large fruit type, medium fruit type, small to medium fruit type and small fruit type mentioned above ① to ④, the remaining individuals are large medium fruit, namely LM.

[0123] Among them, the small fruit type is recorded as S, with an average single fruit weight of 9.5 grams; the medium and small fruit type is recorded as MS, with an average single fruit weight of 68.5 grams; the large fruit type is recorded as L, with an average single fruit weight of 164.0 grams; the medium fruit type is recorded as M, with an average single fruit weight of 81.64 grams; and the medium and large fruit type is recorded as LM, with an average single fruit weight of 132 grams.

[0124] As can be seen from the above, the present invention focuses on the main effect variation sites that control the single fruit weight of apple plants, and develops 16 major effect SNP markers linked to the apple single fruit weight trait. These 16 major effect SNP markers include SNP1~SNP16, which constitute the SNP marker combination, which is suitable for marker-assisted selection of the apple single fruit weight trait during the seedling stage of breeding materials.

[0125] Example 2 In order to verify the feasibility of the 16 major SNP markers linked to the apple fruit weight trait and their primer probe sets, the present invention conducted the following studies: Based on the genotyping data of 16 major effect markers from 1808 apple materials, the complementary epistatic effects between the 16 major effect SNP markers linked to the apple fruit weight trait and the judgment and selection criteria for apple fruit weight were used to screen and judge the Malus germplasm resources or hybrid offspring: like Figure 1 As shown in the figure, 1808 apple materials were divided into 5 grades, including 55 plants with small fruit weight, 1003 plants with small to medium fruit weight, 45 plants with medium fruit weight, 639 plants with medium to large fruit weight, and 66 plants with large fruit weight.

[0126] From the above experimental results, it can be seen that the SNP marker combination provided by the present invention can accurately predict the weight of apple single fruit, so as to realize the molecular marker-assisted selection of the apple single fruit weight trait, which can solve the technical defect of the existing technology that there is still a lack of molecular markers that can more accurately predict the weight of apple single fruit.

[0127] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes a preferred embodiment.

[0128] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A set of SNP marker combinations for apple single fruit weight marker-assisted selection, characterized in that: The SNP marker combination consists of 16 SNP markers; the 16 SNP markers include SNP1 to SNP16; The nucleotide sequences of SNP1 to SNP16 are shown in SEQ ID NO.1 to SEQ ID NO.16, respectively; Among them, the SNP1 is a C to A mutation at 23876017bp of apple chromosome 2; the SNP2 is a C to T mutation at 281738bp of apple chromosome 11; the SNP3 is a T to G mutation at 17104265bp of apple chromosome 12; the SNP4 is a T to G mutation at 23710003bp of apple chromosome 12; the SNP5 is an A to G mutation at 20448307bp of apple chromosome 14; the SNP6 is a T to G mutation at 10278900bp of apple chromosome 15; the SNP7 is an A to T mutation at 22313813bp of apple chromosome 15; and the SNP8 is a G to A mutation at 26425887bp of apple chromosome 15. The SNP9 is a G to T mutation at the 38111629bp position of apple chromosome 15; the SNP10 is an A to G mutation at the 23985991bp position of apple chromosome 3; the SNP11 is an A to G mutation at the 7495024bp position of apple chromosome 9; the SNP12 is a G to A mutation at the 39740659bp position of apple chromosome 10; the SNP13 is a C to G mutation at the 13933420bp position of apple chromosome 13; the SNP14 is a G to A mutation at the 14065871bp position of apple chromosome 15; the SNP15 is a C to T mutation at the 6334724bp position of apple chromosome 16; and the SNP16 is a T to G mutation at the 1867893bp position of apple chromosome 17.

2. A probe set for detecting the SNP marker combination for apple single fruit weight marker-assisted selection according to claim 1, characterized in that: Comprising GenoBaits DNA probes for detecting the SNP1 to the SNP16; The nucleotide sequences of the GenoBaits DNA probes used to detect SNP1 to SNP16 are shown in SEQ ID NO.17 to SEQ ID NO.32, respectively.

3. Use of the SNP marker combination according to claim 1 or the probe set according to claim 2 in the preparation of a kit for determining the single fruit weight trait of apple.

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

5. Use of the SNP marker combination according to claim 1, the probe set according to claim 2, or the kit according to claim 6 in determining the single fruit weight trait of apple.

6. The use according to claim 5, characterized in that The steps include: extracting genomic DNA from the tested Malus plant; Performing typing detection on the tested Malus plant using the probe set; According to the result of the typing test, the single fruit weight trait of the tested Malus plant is determined.

7. The use according to claim 6, characterized in that The single fruit weight trait includes small fruit type, medium-small fruit type, large fruit type, medium fruit type and large-medium fruit type; The small fruit type is recorded as S, and the average single fruit weight is 9.4 grams to 9.6 grams; the medium and small fruit type is recorded as MS, and the average single fruit weight is 68.4 grams to 68.6 grams; the large fruit type is recorded as L, and the average single fruit weight is 163.9 grams to 164.1 grams; the medium fruit type is recorded as M, and the average single fruit weight is 80.64 grams to 82.64 grams; the medium and large fruit type is recorded as LM, and the average single fruit weight is 131 grams to 133 grams.

8. The use according to claim 6, characterized in that When the genotype at the SNP1 is AA, the genotype at the SNP2 is TT, the genotype at the SNP3 is GG, the genotype at the SNP4 is GG, the genotype at the SNP5 is GG, the genotype at the SNP6 is GG, the genotype at the SNP7 is TT, the genotype at the SNP8 is AA, or the genotype at the SNP9 is TT, it is determined that the single fruit weight trait of the tested apple plant is a small fruit type; When the genotype at the SNP1 is CA, the genotype at the SNP2 is CT, the genotype at the SNP3 is TG, the genotype at the SNP4 is TG, the genotype at the SNP5 is AG, the genotype at the SNP6 is TG, the genotype at the SNP7 is AT, the genotype at the SNP8 is GA, or the genotype at the SNP9 is GT, it is determined that the single fruit weight trait of the tested apple plant is a small to medium fruit type; When the genotype at SNP1 is CC, the genotype at SNP2 is CC, the genotype at SNP3 is TT, the genotype at SNP4 is TT, the genotype at SNP5 is AA, the genotype at SNP6 is TT, the genotype at SNP7 is AA, the genotype at SNP8 is GG, and the genotype at SNP9 is GG, and the genotype at SNP10 is GG, the genotype at SNP11 is AG, the genotype at SNP14 is AA, or the genotype at SNP16 is TG, then it is determined that the single fruit weight trait of the tested apple plant is a large fruit type; When the genotype at SNP1 is CC, the genotype at SNP2 is CC, the genotype at SNP3 is TT, the genotype at SNP4 is TT, the genotype at SNP5 is AA, the genotype at SNP6 is TT, the genotype at SNP7 is AA, the genotype at SNP8 is GG, and the genotype at SNP9 is GG, and any one of the T allele variation at SNP12, the G allele variation at SNP13, and the T allele variation at SNP15 is contained, it is determined that the single fruit weight trait of the tested apple plant is medium fruit type; When the tested Malus plant does not meet any of the above conditions, it is determined that its single fruit weight trait is a large-medium fruit type.

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