Molecular marker probe combination for auxiliary selection of apple sugar content character and application of molecular marker probe combination

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By developing a molecular marker probe combination for the selection of apple sugar content traits, integrating nine key SNP markers for simultaneous multi-site detection, the problem of insufficient accuracy in traditional apple breeding was solved, and efficient identification of fruit sugar content traits and breeding guidance were achieved.

CN121780752APending Publication Date: 2026-04-03CHINA AGRI UNIV

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Patent Information

Authority / Receiving Office: CN · China
Patent Type: Applications(China)
Current Assignee / Owner
Filing Date: 2026-01-05
Publication Date: 2026-04-03

AI Technical Summary

Technical Problem

In current apple breeding, traditional testing products have low accuracy in detecting the sugar content trait of apple fruits, making it difficult to fully capture genetic variations through a single or a few SNP markers. This results in long breeding cycles and difficulty in predicting the traits of new varieties.

Method used

To develop a molecular marker probe combo for apple sugar content trait-assisted selection, a systematic probe combo is formed by integrating nine key SNP markers to achieve simultaneous detection at multiple sites, thereby improving genetic resolution and predictive robustness.

Benefits of technology

It significantly improved the accuracy of detecting sugar content in apple fruits, shortened the breeding cycle, saved resources, and improved breeding efficiency and selection accuracy.

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Abstract

The invention belongs to the technical field of plant molecular breeding, and particularly relates to a molecular marker probe combination for auxiliary selection of apple sugar content characters and application of the molecular marker probe combination. The probe combination comprises nine probes related to auxiliary selection of the apple sugar content character; nucleotide sequences of the nine probes related to auxiliary selection of the apple sugar content character are sequentially shown as SEQ ID NO. 10 to SEQ ID NO. 18. The DNA probe combination corresponding to the SNP marker combination provided by the invention can be applied to marker-assisted selection of apple sugar content traits in the seedling stage of apple filial generations.

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Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to probe combinations of molecular markers for the auxiliary selection of apple sugar content traits and their applications. Background Technology

[0002] apple( Malus domestica Borkh. Apples are an important economic crop, with the world's highest yield. Their post-harvest quality plays a crucial role in maintaining market competitiveness and economic value. Sugar content in apples is another decisive indicator of their flavor; the sugar content of the fruit is mainly determined by the content of fructose, sucrose, and glucose. Soluble sugar content in apples is a typical quantitative trait influenced by multiple loci, with each locus having a relatively small impact. Conventional hybridization breeding is the main approach in apple breeding, relying on phenotypic selection to breed offspring with high sugar content.

[0003] my country possesses abundant apple germplasm resources, many of which carry a wealth of sugar content-related genes. Traditional breeding methods suffer from long cycles and difficulty in predicting new varietal traits. Molecular marker-assisted breeding can reduce breeding costs and improve efficiency. Therefore, detection products developed based on sugar content-related genes can overcome the shortcomings of traditional breeding techniques. However, most commercially available or literature-reported detection products are independent detection systems targeting single or a few SNP markers. This fragmented detection approach leads to low accuracy in these products. Summary of the Invention

[0004] To address the issue of low accuracy in existing commercially available or literature-reported detection products, this invention aims to develop an efficient screening strategy for sugar content traits, providing a probe combination of molecular markers for auxiliary selection of apple sugar content traits and its application. To achieve the above objective, this invention adopts the following technical solution.

[0005] Sugar content in apples is evaluated using the soluble solids content of the pulp as an indicator. This invention investigated the phenotypic retention trait of pulp crispness in 2664 hybrid offspring of apples 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Jinguan'. 134 SNPs and InDel markers were developed using GWAS, BSA-seq, and RNA-seq methods. However, using 134 molecular markers for marker-assisted selection of apple fruit sugar content is extremely difficult; therefore, this invention conducts the following experimental research.

[0006] This invention screened for markers with an effect value greater than 1°Brix on soluble solids in apple pulp from 134 SNPs and InDel markers. Nine SNP markers were identified, including SNP1 to SNP9. Based on this, this invention provides a probe combination of molecular markers for the auxiliary selection of apple sugar content traits. The probe combination includes nine probes related to the auxiliary selection of apple sugar content traits.

[0007] The nucleotide sequences of the nine apple sugar content trait-assisted selection probes are shown in SEQ ID NO.10~SEQ ID NO.18. These nine apple sugar content trait-assisted selection probes are used to detect SNP1~SNP9.

[0008] The nucleotide sequence of the probe used to detect SNP1 is shown in SEQ ID NO.10: ACCTCCATCTTCCGTCGAACCGATCCCCAATTCCTCCTTCGTGGAGCCATCCCCAATCCCTCATCCGTCCTCTCCTCCATTTGATTCACAGTCGACCTCACTCATCCAC.

[0009] The nucleotide sequence of the probe used to detect SNP2 is shown in SEQ ID NO.11: AGCCATCTTGGCTGATTCTAGCTCAACGTTGAGCTGTTCAATGGAAGCCTCTTTCTATCAATCTCTCCTCGTAACCTTTTGCTTTCTCAAGTTCTTGTTTGAGAAAGT.

[0010] The nucleotide sequence of the probe used to detect SNP3 is shown in SEQ ID NO.12: CAGACCGAAATGCGAACTGCACGACCTTCTCGTCATTCAGATTCATCCCGGTCATTGCCTCGCCCAATCCGCAGCTGATCTCCGCCAGCACATCCGGGTCACTATAATAG.

[0011] The nucleotide sequence of the probe used to detect SNP4 is shown in SEQ ID NO.13: ACAATACCATCTTCGTTCATCACCAAGTTTCCCTCTCCTCATAGCTCCTGAATCTTTGGCTAGATTTTCTTATCAAAATCTCCCATTTTCTCGGAAACCCCATCAACAG.

[0012] The nucleotide sequence of the probe used to detect SNP5 is shown in SEQ ID NO.14: GTGCAGCACAAACTGTTGCACTTGTAACTCAGAACCAATCCAAACTAAAACGTTCATTTCTCCGGCAAGCAATCGGCAGCAAGCATGAAGTACCACTCATGCGACTCACG.

[0013] The nucleotide sequence of the probe used to detect SNP6 is shown in SEQ ID NO.15: AAAAAATTAAATGCAAAGTGAAAGTTATCTATCTAAGTGAGTCGCAATGTAGTCACCCTTCTTAATTGCCATATGAAAACCCTTTTCTTTTTTATTGACATGAGATGTAG.

[0014] The nucleotide sequence of the probe used to detect SNP7 is shown in SEQ ID NO.16: GAACCAAAGAGAGGCGCGATGTCGCTGCCCTAAAGTCGTTGACGCCTCCCTACGTGCAGGTTATGGCATAATCAAAGAGTAATATAGGCTCTTTCCTTCTTTTCTTCTGT.

[0015] The nucleotide sequence of the probe used to detect SNP8 is shown in SEQ ID NO.17: TGATCAAGGATAGTTTTCCTTGGTTAACCTCACCGTTGATGATAACCGACTCATCACGCTCCTGCATTTTGCCTCCAAACATGCAACAAGCAGCTAGCAATACCGATCGA.

[0016] The nucleotide sequence of the probe used to detect SNP9 is shown in SEQ ID NO.18: ATGCTCATTGCCCGTGTTGCAGAATGGAGATTTCTCCTAGTAATTGTATTGGTAATTTTGTAAATTAAAATTGTAAAATATAGATTGAGCTTGCAGTTGCAGTTGTAGAG.

[0017] Existing detection products suffer from low accuracy primarily because they rely on single or a few SNP markers to predict sugar content traits controlled by multiple genes, making it difficult to comprehensively capture genetic variations. This invention integrates nine key SNP markers to form a systematic probe combination, enabling simultaneous detection at multiple sites. By leveraging the synergistic effect of multiple markers, it significantly improves genetic resolution and predictive robustness, thereby effectively solving the problem of insufficient accuracy.

[0018] Preferably, the above 9 SNPs are labeled as SNP1 to SNP9. SNP1 to SNP9 are found in apples... GDDH13.1 The locations on the genome, the variant bases or variant sequences and their flanking sequences are shown in SEQ ID NO.1 to SEQ ID NO.9, respectively.

[0019] SNP1 is a G-to-T mutation located at position 25310876 on chromosome 0 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.1: GCAAATCTCACCCTCTGGTACCACTCTCCTCCGTCGAACACTAACGTACCCCTTCCTCCAACCTTTCCGTTGATTCTAGCCCTAGTAGTTCTACCAACCTCTCTTCTCTGTCGAACCCTCTCCTCCGCTGAAACCTCCATCTTCCGTCGAACCGATCCCCAATTCCTCCTTCGTGGAGCCATCCCCAATCCCTCATCC[G / T]TCCTCTCCTCCATTTGATTCACAGTCGACCTCACTCATCCACCGAATCAGATCACCTAATAAACTAAATCAGTCATTTCTAATGAACTAGAAGCAACAATATACATGGTTTCAATGATTTTGAGGTTAAGGTCATTTTCTAAAAAATCATGCATCAAATTGTATCTGGGATGCGCAGAACTATGGTTGCTCGCGAGAAAG. The position of SNP1 is marked with square brackets "[ ]", with the non-variant base "G" first and the variant base "T" second, separated by " / " (the same applies below).

[0020] SNP2 is a G-to-C mutation located at position 32459528 on chromosome 1 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.2: GACTGAATCTAAAGACTCTGATGCAGACCTCTCCAACTTATTTGCTTCTTCGACCTGCATCTCTAATTCCTCAACCCTAATTTTCCACTCCTCAACTACACTACGTGCATAAGATTCAGCCATCTTGGCTGATTCTAGCTCAACGTTGAGCTGTTCAATGGAAGCCTCTTTCTCTATCAATCTCTCCTCGTAACCTTTT[G / C]CTTTCTCAAGTTCTTGTTTGAGAAAGTCTACCTCCGATTTAAGGCTAACCACCATCTTGTTGCTCTCGCTAGCCTCTGTTTCAAGCTTTGCATCTAGCAAGGCCCTTAACTGAGTCAACTCAGCTAAAAGAATCTCCACCTTATTGGCATGGATCTCGGCAATCTTGGTTGCATCATCAGCATGAGTCAGTGCCTGGTTT。

[0021] SNP3 is a C to T mutation at position 5,665,814 on chromosome 3 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.3: AAATATGTAAAATCGTATATATGGGCAATGTTCGTTTTCTTCTAAAAGTTGGAACCCTTAGCTTTTGGTCATCTGGAGATCACCAGAAAGCTCTAAACTCGAATCTTACTTTTCAATCAGACCGAAATGCGAACTGCACGACCTTCTCGTCATTCAGATTCATCCCGGTCATTGCCTCGCCCAATCCGCAGCTGATCTC[C / T]GCCAGCACATCCGGGTCACTATAATAGGTTGAAGCCTGCACAATAGCCTAGGCTCTCCGGGCAGGGTCACTGCTATTGAACATGCCCGACCTGACGAAAACCCCATTGCACCTCTACTGCATCATCAGCTTCGCGTCTGCGGGAGTTGGCACGCCACCAATGGCAAAATGCACCACCGGCAGCCTCTCGAGCTGCTTCGT。

[0022] SNP4 is a G-to-T mutation at position 23915298 on chromosome 3 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.4: GTCTTGGAACGTTTGAGTTGGAGTAATTTGGACCTCAAAATTTGTCTGGTTGATGTTCACACACCTTCAAAACCTCCCAGATTCTCCCACACCTTTACTTGCACCACAATACCATCTTCGTTCATCACCAAGTTTTCCCTCTCCTCATAGCTCCTGAATCTTTGGCTAGATTTTCTTATCAAAATCTCCCATTTTCTCG[G / T]AAACCCCATCAACAGATTCTTGGCATTTCCCTGAATCCAGCTGCCAAGATTGTTTTTTGGGAAAAATTCTGATCTGGGATTTTCACAAAAAGGTGGTTTGAGCTTTTGAGAGAGAGGATGGTGGGAGCTGCTCTGTCTCTGGTAGGTTCGTCGGTGGTGGACTCGCACACTAGTCCTTGCCTGTGTTTGGATGCGCTG.

[0023] SNP5 is a mutation from G to A at position 27405697 on chromosome 3 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.5: TTATTTCTCTTCATTTATTAATGTGAAAAAGAATGTACGGAGCCAAACATGATAAAAACAAGACAACAAAGATAAATACAAGATAAAAAAGAGATGCTGACTTGTTATGCGCAATTTGTGCAGCACAAACTGTTGCACTTGTAACTCAGAACCAATCCAAACTAAAACGTTCATTTCTCCGGCAAGCAATCGGCAGCAA[G / A]CATGAAGTACCACTCATGCGACTCACGGTCCTCCCTCGGTTTACACATGTATATTCTACGAGGGGAACTTATGGCTTGCAACAGTCGGAGAATGGCATCAGAAAGAAACTTTAGAATAGCTTGGCCTGCGGTTGCATTCCACTTTTCATCATCGTGGAGAATTCTTCATAGTTGATCCGACCATCCTGTAATCCCAGTGG.

[0024] SNP6 is a G-to-T mutation located at position 28603676 on chromosome 10 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.6: AATTCATTATATTTTGTGTAAATAAGTGTCTGTTTATACTTAAAATATATATAATTTCATCATAAACTATAAAATAGAATGATATATGTATTATGAAGTACTGGAACATAATGAAAACATGGGGAACAAGCATATCGTGTGTGCTCATTTAAATATTCAACAATTCTCTTACAATTTATTGAAAAAAATTAAATGCAAA[G / T]TGAAAGTTATCTATCTAAGTGAGTCGCAATGTAGTCACCCTTCTTAATTGCCATATGAAAACCCTTTTCTTTTTTGACATGAGATGTAGATTATCAATAATGGGAGGCTGAAAAGTGCTTAACATCGGGCAGTGACGAGTTCAAGCCATTCCAGGACATCTGCTGCATTTTTCATCGCGCCTTCAGACGATTGCATT.

[0025] SNP7 is a T-to-C mutation located at position 36,811,784 on chromosome 11 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.7: ATATTGCATAAATATAACCAATTATTTATATGATCAAATATATCAATTCAAATGTATAATCACTATCAGACTTATAAAAATAAAATTAGCAAAAAGAGAATAAGAATATCTAGCCTGTGAACCAAAGAGAGGCGCGATGTCGCTGCCCTAAAGTCGTTGACGCCTCCCTACGTGCAGGTTATGGCATAATCAAAGAGTAA[T / C]ATAGGCTCTTTCCTTCTTCTTCTGTATACAATTCTCAGAGAGTAAACACACAAAGCAATTCGCTCGAATTCTATAATTCAGTGCGGGTTGTAAAATTAGGTAGTTGTATCCTGATTGAGCAGACGTTGTAAAACCACAAGCACAAGAGTGTGACGGAAATACTGTTCAAATGACATAGCTTTTGCGCTATCCTCTACA.

[0026] SNP8 is a G-to-T mutation located at position 6378877 on chromosome 14 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.8: ATGAAATAAGAACTTTTGGTAAGCGCATACCACGCTTTTGAGACACATGAAAATCTAATCAAAGACTTTGCAGGTAGTTTTGAGAGAATTTCTACTAGGACATCTTGGTGTAAACACTTGATCAAGGATAGTTTTCCTTGGTTAACCTCACCGTTGATGATAACCGACTCATCACGCTCCTGCATTTTGCCTCCAAACAT[G / T]CAACAAGCAGCTAGCAATACCGATCGAGAAGAGCCTTTTGGTCCAAAACCCTAGCAGTACCAGTATTATTTATATAGGATTTCGTCTACTAGAAACTAAATAGAACCCGTACTGGATTCAGTCAACAAATACATTGCGGAGTAGGAGTTTGAATAGTAAAAGGGAAAGGAAATTGACAAATACTTATGCTAATGGGAAAA.

[0027] SNP9 is a mutation from A to G located at position 11982626 on chromosome 15 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.9: AGTGTGCAGTCTGCCTGGAAAGGTTCAGTCCAGGTAAGACCCTGGTGCACCTGCCCTGTGCCCATAGGTTCCACTCTGGTTGCATGGTTCCATGGCTTCACAGCAATGCTCATTGCCCGTGTTGCAGAATGGAGATTTCTCCTAGTAATTGTATTGGTAATTTTGTAAATTAAAATTGTAAAATATAGATTGAGCTTGC[A / G]GTTGCAGTTGTAGAGTTGGTTTGAAGGATTTGACCTTGTGTGCTAATTGTTTAGCTACCATATGTTTTTGTTGAAGTGAATAGTTGCCAAGTATGAGTAAATAGTTTATGCATATTGATGGGAGATCATAATCCCACACATTGATCACTTCCACAAGTATTTTGGAAAAATACTTTGACTCGTTACATGTAAGTAGCATT.

[0028] Among them, SNP1 had an effect value of 1.46°Brix on soluble solids in pulp; SNP2 had an effect value of 2.17°Brix; SNP3 had an effect value of 2.12°Brix; SNP4 had an effect value of 1.36°Brix; SNP5 had an effect value of 1.81°Brix; SNP6 had an effect value of 1.07°Brix; SNP7 had an effect value of 1.91°Brix; SNP8 had an effect value of 1.98°Brix; and SNP9 had an effect value of 1.69°Brix.

[0029] The present invention also provides a kit for identifying the sugar content trait of apple fruit, including the probe combination.

[0030] The present invention also provides the application of the probe combination or the kit in identifying the sugar content trait of apple fruit.

[0031] Preferably, the steps for identifying the sugar content trait of apple fruit are as follows: Genomic DNA was extracted from the apple species to be tested.

[0032] The extracted genomic DNA was precisely quantified.

[0033] Enzymatic digestion was used to fragment the quantified genomic DNA, and a DNA library was constructed.

[0034] Adapter sequences were added to both ends of the DNA library fragments for sample labeling.

[0035] The target sequence is captured using the probe combination described above.

[0036] The captured target sequence was subjected to next-generation sequencing. The sequencing data was compared and analyzed with the apple reference genome GDDH13.1 to obtain genotyping data.

[0037] Based on the genotyping data, the sugar content trait of the fruit of the tested Malus species was identified.

[0038] Preferably, based on the obtained genotyping data, the sum of the number of alleles in the SNP1~SNP9 genotypes that have a positive effect on the retention of fruit sugar content is calculated and denoted as NPA.

[0039] The NPA was used as an indicator of the sugar content retention of the fruit of the tested Malus species. The higher the NPA value, the stronger the sugar content retention of the fruit of the tested Malus species.

[0040] Preferably, if the NPA is -5 to -2, the fruit of the tested *Malus* species has a low sugar content and the soluble solids content is between 11°Brix and 14°Brix; if the NPA is -1 to 5, the soluble solids content of the fruit of the tested *Malus* species is between 11°Brix and 18.5°Brix; if the NPA is 5 to 8, the fruit of the tested *Malus* species has a high sugar content and the soluble solids content is between 13.8°Brix and 22°Brix.

[0041] Preferably, allelic variations can occur at SNP1 to SNP9.

[0042] Specifically, the T allele variation at SNP1 has a positive effect on the retention of fruit sugar content; if the genotype at SNP1 is GG, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP1 is GT, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP1 is TT, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0043] The C allele variation at SNP2 has a negative effect on the retention of fruit sugar content; if the genotype at SNP2 is GG, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP2 is GC, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP2 is CC, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit sugar content is -2.

[0044] The T allele at SNP3 has a negative effect on the retention of fruit sugar content; if the genotype at SNP3 is CC, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP3 is CT, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP3 is TT, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit sugar content is -2.

[0045] The T allele variation at SNP4 has a negative effect on the retention of fruit sugar content; if the genotype at SNP4 is GG, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP4 is GT, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP4 is TT, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit sugar content is -2.

[0046] The A allele at SNP5 has a positive effect on the retention of fruit sugar content; if the genotype at SNP5 is GG, then the number of alleles in the SNP5 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP5 is GA, then the number of alleles in the SNP5 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP5 is AA, then the number of alleles in the SNP5 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0047] The C allele variation at SNP6 has a positive effect on the retention of fruit sugar content; if the genotype at SNP6 is GG, then the number of alleles in the SNP6 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP6 is GT, then the number of alleles in the SNP6 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP6 is TT, then the number of alleles in the SNP6 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0048] The C allele at SNP7 has a negative effect on the retention of fruit sugar content; if the genotype at SNP7 is TT, then the number of alleles in the SNP7 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP7 is TC, then the number of alleles in the SNP7 genotype that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP7 is CC, then the number of alleles in the SNP7 genotype that have a positive effect on the retention of fruit sugar content is -2.

[0049] The T allele variation at SNP8 has a positive effect on the retention of fruit sugar content; if the genotype at SNP8 is GG, then the number of alleles in the SNP8 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP8 is GT, then the number of alleles in the SNP8 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP8 is TT, then the number of alleles in the SNP8 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0050] The A allele at SNP9 has a positive effect on the retention of fruit sugar content; if the genotype at SNP9 is GG, then the number of alleles in the SNP9 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP9 is GA, then the number of alleles in the SNP9 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP9 is AA, then the number of alleles in the SNP9 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0051] Preferably, the connector sequence includes a sample identification barcode, and the sample marking process is completed using a barcode connector kit.

[0052] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a probe combination of molecular markers for the auxiliary selection of sugar content traits in apples. The probe combination provided by this invention is suitable for the auxiliary selection of sugar content traits in apple hybrid offspring. The auxiliary selection of sugar content traits in apple hybrid offspring can be carried out at the 6-week-old seedling stage. GenoBaits marker detection requires 4 weeks, meaning the auxiliary selection of sugar content traits in hybrid offspring can be completed in the 11th week after sowing. Traditional phenotypic selection requires 8 to 10 years to complete. Therefore, the cycle of auxiliary selection of hybrid offspring using the molecular marker and probe combination for the auxiliary selection of apple fruit sugar content traits is shortened by 8 to 10 years compared to traditional phenotypic selection methods. Secondly, since the selection of hybrid offspring using molecular markers and probes for sugar content trait-assisted selection of apple fruits was completed during the 11-week seedling stage, the selected superior individual plants directly entered the re-selection process, while the hybrid offspring that did not pass the selection were directly eliminated. This significantly reduced the resource consumption and breeding workload of seedling propagation, hybrid primary selection nursery management, and phenotypic selection. The above-mentioned molecular marker and probe combination can comprehensively and accurately predict the sugar content of apple fruits.

[0053] Existing detection products suffer from low accuracy primarily because they rely on single or a few SNP markers to predict sugar content traits controlled by multiple genes, making it difficult to comprehensively capture genetic variations. This invention integrates nine key SNP markers to form a systematic probe combination, enabling simultaneous detection at multiple sites. By leveraging the synergistic effect of multiple markers, it significantly improves genetic resolution and predictive robustness, thereby effectively solving the problem of insufficient accuracy.

[0054] 2. The molecular marker and probe combination developed in this invention is applicable to the evaluation and screening of fruit sugar content traits in apple germplasm resources. The scope of application includes 25 species of apple plants. The selected superior fruit sugar content germplasm resources are used for the design of breeding programs, the selection of hybrid parents and the matching of hybrid combinations, and to guide apple molecular breeding.

[0055] 3. The molecular marker and probe combination developed in this invention for the auxiliary selection of apple fruit sugar content trait is applied to the molecular breeding practice of apple fruit sugar content trait. Compared with high-density chips and whole genome selection, the marker detection cost is low and the selection accuracy is high. Attached Figure Description

[0056] Figure 1 The relationship between the number of positive-effect alleles of molecular markers for the auxiliary selection of soluble solids traits in apple fruit and the sugar content of the fruit in this invention.

[0057] Figure 2 This is the validation result of the training population for the molecular marker combination used in the selection of apple fruit sugar content in this invention. Detailed Implementation

[0058] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0059] Example 1 (1) DNA extraction, library construction and target site capture sequencing Leaves of the material to be tested were taken, genomic DNA was extracted, and the genome was accurately quantified using the Qubit® dsDNAHSAssayKit. 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 to obtain the target library (DNA library).

[0060] Using probe combinations of molecular markers selected with the help of the above-mentioned apple sugar content trait, molecular hybridization and target site capture were performed using the GenoBaits® DNAHybridization kit for ILM.

[0061] Among them, the materials to be tested (the apple species to be tested) refer to 335 germplasm resources of 25 species of apple, as well as 1073 hybrid offspring from 4 half-sib families, namely 'Hongyu' × 'Jinguan', 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Hongjinqing', to construct a training population. The total number of individuals in the training population is 1408, and the source is the apple breeding base in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0062] The method used to extract genomic DNA was the CTAB plant genome extraction method.

[0063] The GenoBaits probe suite for apple sugar content trait-assisted selection includes probes for detecting SNP1 to SNP9.

[0064] The nucleotide sequences of the probes used to detect SNP1 to SNP9 are shown in SEQ ID NO.10 to SEQ ID NO.18, respectively.

[0065] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP1 is shown in SEQ ID NO.10: ACCTCCATCTTCCGTCGAACCGATCCCCAATTCCTCCTTCGTGGAGCCATCCCCAATCCCTCATCCGTCCTCTCCTCCATTTGATTCACAGTCGACCTCACTCATCCAC.

[0066] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP2 is shown in SEQ ID NO.11: AGCCATCTTGGCTGATTCTAGCTCAACGTTGAGCTGTTCAATGGAAGCCTCTTTCTATCAATCTCTCCTCGTAACCTTTTGCTTTCTCAAGTTCTTGTTTGAGAAAGT.

[0067] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP3 is shown in SEQ ID NO.12: CAGACCGAAATGCGAACTGCACGACCTTCTCGTCATTCAGATTCATCCCGGTCATTGCCTCGCCCAATCCGCAGCTGATCTCCGCCAGCACATCCGGGTCACTATAATAG.

[0068] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP4 is shown in SEQ ID NO.13: ACAATACCATCTTCGTTCATCACCAAGTTTCCCTCTCCTCATAGCTCCTGAATCTTTGGCTAGATTTTCTTATCAAAATCTCCCATTTTCTCGGAAACCCCATCAACAG.

[0069] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP5 is shown in SEQ ID NO.14: GTGCAGCACAAACTGTTGCACTTGTAACTCAGAACCAATCCAAACTAAAACGTTCATTTCTCCGGCAAGCAATCGGCAGCAAGCATGAAGTACCACTCATGCGACTCACG.

[0070] The nucleotide sequence of the probe used to detect SNP6 (GenoBaits DNA) is shown in SEQ ID NO.15: AAAAAATTAAATGCAAAGTGAAAGTTATCTATCTAAGTGAGTCGCAATGTAGTCACCCTTCTTAATTGCCATATGAAAACCCTTTTCTTTTTTATTGACATGAGATGTAG.

[0071] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP7 is shown in SEQ ID NO.16: GAACCAAAGAGAGGCGCGATGTCGCTGCCCTAAAGTCGTTGACGCCTCCCTACGTGCAGGTTATGGCATAATCAAAGAGTAATATAGGCTCTTTCCTTCTTTTCTTCTGT.

[0072] The nucleotide sequence of the probe used to detect SNP8 (GenoBaits DNA) is shown in SEQ ID NO.17: TGATCAAGGATAGTTTTCCTTGGTTAACCTCACCGTTGATGATAACCGACTCATCACGCTCCTGCATTTTGCCTCCAAACATGCAACAAGCAGCTAGCAATACCGATCGA.

[0073] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP9 is shown in SEQ ID NO.18: ATGCTCATTGCCCGTGTTGCAGAATGGAGATTTCTCCTAGTAATTGTATTGGTAATTTTGTAAATTAAAATTGTAAAATATAGATTGAGCTTGCAGTTGCAGTTGTAGAG.

[0074] The above nine SNPs are labeled SNP1 to SNP9. SNP1 to SNP9 are found in apples... GDDH13.1 The locations on the genome, the variant bases or variant sequences and their flanking sequences are shown in SEQ ID NO.1 to SEQ ID NO.9, respectively.

[0075] SNP1 is a G-to-T mutation located at position 25310876 on chromosome 0 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.1: GCAAATCTCACCCTCTGGTACCACTCTCCTCCGTCGAACACTAACGTACCCCTTCCTCCAACCTTTCCGTTGATTCTAGCCCTAGTAGTTCTACCAACCTCTCTTCTCTGTCGAACCCTCTCCTCCGCTGAAACCTCCATCTTCCGTCGAACCGATCCCCAATTCCTCCTTCGTGGAGCCATCCCCAATCCCTCATCC[G / T]TCCTCTCCTCCATTTGATTCACAGTCGACCTCACTCATCCACCGAATCAGATCACCTAATAAACTAAATCAGTCATTTCTAATGAACTAGAAGCAACAATATACATGGTTTCAATGATTTTGAGGTTAAGGTCATTTTCTAAAAAATCATGCATCAAATTGTATCTGGGATGCGCAGAACTATGGTTGCTCGCGAGAAAG. The position of SNP1 is marked with square brackets "[ ]", with the non-variant base "G" first and the variant base "T" second, separated by " / " (the same applies below).

[0076] SNP2 is a G-to-C mutation located at position 32459528 on chromosome 1 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.2: GACTGAATCTAAAGACTCTGATGCAGACCTCTCCAACTTATTTGCTTCTTCGACCTGCATCTCTAATTCCTCAACCCTAATTTTCCACTCCTCAACTACACTACGTGCATAAGATTCAGCCATCTTGGCTGATTCTAGCTCAACGTTGAGCTGTTCAATGGAAGCCTCTTTCTCTATCAATCTCTCCTCGTAACCTTTT[G / C]CTTTCTCAAGTTCTTGTTTGAGAAAGTCTACCTCCGATTTAAGGCTAACCACCATCTTGTTGCTCTCGCTAGCCTCTGTTTCAAGCTTTGCATCTAGCAAGGCCCTTAACTGAGTCAACTCAGCTAAAAGAATCTCCACCTTATTGGCATGGATCTCGGCAATCTTGGTTGCATCATCAGCATGAGTCAGTGCCTGGTTT.

[0077] SNP3 is a C-to-T mutation located at position 5665814 on chromosome 3 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.3: AAATATGTAAAATCGTATATATGGGCAATGTTCGTTTTCTTCTAAAAGTTGGAACCCTTAGCTTTTGGTCATCTGGAGATCACCAGAAAGCTCTAAACTCGAATCTTACTTTTCAATCAGACCGAAATGCGAACTGCACGACCTTCTCGTCATTCAGATTCATCCCGGTCATTGCCTCGCCCAATCCGCAGCTGATCTC[C / T]GCCAGCACATCCGGGTCACTATAATAGGTTGAAGCCTGCACAATAGCCTAGGCTCTCCGGGCAGGGTCACTGCTATTGAACATGCCCGACCTGACGAAAACCCCATTGCACCTCTACTGCATCATCAGCTTCGCGTCTGCGGGAGTTGGCACGCCACCAATGGCAAAATGCACCACCGGCAGCCTCTCGAGCTGCTTCGT.

[0078] SNP4 is a G-to-T mutation at position 23915298 on chromosome 3 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.4: GTCTTGGAACGTTTGAGTTGGAGTAATTTGGACCTCAAAATTTGTCTGGTTGATGTTCACACACCTTCAAAACCTCCCAGATTCTCCCACACCTTTACTTGCACCACAATACCATCTTCGTTCATCACCAAGTTTTCCCTCTCCTCATAGCTCCTGAATCTTTGGCTAGATTTTCTTATCAAAATCTCCCATTTTCTCG[G / T]AAACCCCATCAACAGATTCTTGGCATTTCCCTGAATCCAGCTGCCAAGATTGTTTTTTGGGAAAAATTCTGATCTGGGATTTTCACAAAAAGGTGGTTTGAGCTTTTGAGAGAGAGGATGGTGGGAGCTGCTCTGTCTCTGGTAGGTTCGTCGGTGGTGGACTCGCACACTAGTCCTTGCCTGTGTTTGGATGCGCTG.

[0079] SNP5 is a mutation from G to A at position 27405697 on chromosome 3 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.5: TTATTTCTCTTCATTTATTAATGTGAAAAAGAATGTACGGAGCCAAACATGATAAAAACAAGACAACAAAGATAAATACAAGATAAAAAAGAGATGCTGACTTGTTATGCGCAATTTGTGCAGCACAAACTGTTGCACTTGTAACTCAGAACCAATCCAAACTAAAACGTTCATTTCTCCGGCAAGCAATCGGCAGCAA[G / A]CATGAAGTACCACTCATGCGACTCACGGTCCTCCCTCGGTTTACACATGTATATTCTACGAGGGGAACTTATGGCTTGCAACAGTCGGAGAATGGCATCAGAAAGAAACTTTAGAATAGCTTGGCCTGCGGTTGCATTCCACTTTTCATCATCGTGGAGAATTCTTCATAGTTGATCCGACCATCCTGTAATCCCAGTGG.

[0080] SNP6 is a G-to-T mutation located at position 28603676 on chromosome 10 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.6: AATTCATTATATTTTGTGTAAATAAGTGTCTGTTTATACTTAAAATATATATAATTTCATCATAAACTATAAAATAGAATGATATATGTATTATGAAGTACTGGAACATAATGAAAACATGGGGAACAAGCATATCGTGTGTGCTCATTTAAATATTCAACAATTCTCTTACAATTTATTGAAAAAAATTAAATGCAAA[G / T]TGAAAGTTATCTATCTAAGTGAGTCGCAATGTAGTCACCCTTCTTAATTGCCATATGAAAACCCTTTTCTTTTTTGACATGAGATGTAGATTATCAATAATGGGAGGCTGAAAAGTGCTTAACATCGGGCAGTGACGAGTTCAAGCCATTCCAGGACATCTGCTGCATTTTTCATCGCGCCTTCAGACGATTGCATT.

[0081] SNP7 is a T-to-C mutation located at position 36,811,784 on chromosome 11 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.7: ATATTGCATAAATATAACCAATTATTTATATGATCAAATATATCAATTCAAATGTATAATCACTATCAGACTTATAAAAATAAAATTAGCAAAAAGAGAATAAGAATATCTAGCCTGTGAACCAAAGAGAGGCGCGATGTCGCTGCCCTAAAGTCGTTGACGCCTCCCTACGTGCAGGTTATGGCATAATCAAAGAGTAA[T / C]ATAGGCTCTTTCCTTCTTCTTCTGTATACAATTCTCAGAGAGTAAACACACAAAGCAATTCGCTCGAATTCTATAATTCAGTGCGGGTTGTAAAATTAGGTAGTTGTATCCTGATTGAGCAGACGTTGTAAAACCACAAGCACAAGAGTGTGACGGAAATACTGTTCAAATGACATAGCTTTTGCGCTATCCTCTACA.

[0082] SNP8 is a G-to-T mutation located at position 6378877 on chromosome 14 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.8: ATGAAATAAGAACTTTTGGTAAGCGCATACCACGCTTTTGAGACACATGAAAATCTAATCAAAGACTTTGCAGGTAGTTTTGAGAGAATTTCTACTAGGACATCTTGGTGTAAACACTTGATCAAGGATAGTTTTCCTTGGTTAACCTCACCGTTGATGATAACCGACTCATCACGCTCCTGCATTTTGCCTCCAAACAT[G / T]CAACAAGCAGCTAGCAATACCGATCGAGAAGAGCCTTTTGGTCCAAAACCCTAGCAGTACCAGTATTATTTATATAGGATTTCGTCTACTAGAAACTAAATAGAACCCGTACTGGATTCAGTCAACAAATACATTGCGGAGTAGGAGTTTGAATAGTAAAAGGGAAAGGAAATTGACAAATACTTATGCTAATGGGAAAA.

[0083] SNP9 is a mutation from A to G located at position 11982626 on chromosome 15 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.9: AGTGTGCAGTCTGCCTGGAAAGGTTCAGTCCAGGTAAGACCCTGGTGCACCTGCCCTGTGCCCATAGGTTCCACTCTGGTTGCATGGTTCCATGGCTTCACAGCAATGCTCATTGCCCGTGTTGCAGAATGGAGATTTCTCCTAGTAATTGTATTGGTAATTTTGTAAATTAAAATTGTAAAATATAGATTGAGCTTGC[A / G]GTTGCAGTTGTAGAGTTGGTTTGAAGGATTTGACCTTGTGTGCTAATTGTTTAGCTACCATATGTTTTTGTTGAAGTGAATAGTTGCCAAGTATGAGTAAATAGTTTATGCATATTGATGGGAGATCATAATCCCACACATTGATCACTTCCACAAGTATTTTGGAAAAATACTTTGACTCGTTACATGTAAGTAGCATT.

[0084] Among them, SNP1 had an effect value of 1.46°Brix on soluble solids in pulp; SNP2 had an effect value of 2.17°Brix; SNP3 had an effect value of 2.12°Brix; SNP4 had an effect value of 1.36°Brix; SNP5 had an effect value of 1.81°Brix; SNP6 had an effect value of 1.07°Brix; SNP7 had an effect value of 1.91°Brix; SNP8 had an effect value of 1.98°Brix; and SNP9 had an effect value of 1.69°Brix.

[0085] (2) Sequencing of target sites and marker genotyping The captured target library was sequenced using an Illumina sequencer with a PE150 strategy at a sequencing depth of 1000×~1200×. The obtained reads were then used... GDDH13.1 Data analysis and genotyping were performed using a reference genome.

[0086] (3) Calculation of the number of positive effect sites for maintaining apple fruit sugar content by the marker combination SNP1~SNP9 (probe combination of molecular markers for auxiliary selection of apple sugar content trait).

[0087] The T allele variation of SNP1 G / T has a positive effect on the retention of fruit sugar content. The number of alleles with positive effects in the three test sample genotypes, namely GG, GT and TT, are 0, 1 and 2, respectively.

[0088] The C allelic variation of SNP2 G / C has a negative effect on the retention of fruit sugar content. The number of alleles with positive effects in the three test sample genotypes, GG, GC and CC, are 0, -1 and -2, respectively.

[0089] The T allelic variation of SNP3 C / T has a negative effect on the retention of fruit sugar content. The number of alleles with positive effects in the three test sample genotypes, CC, CT and TT, are 0, -1 and -2, respectively.

[0090] The T allelic variation of SNP4 G / T has a negative effect on the retention of fruit sugar content. The number of alleles with positive effects in the three test sample genotypes, GG, GT and TT, are 0, -1 and -2, respectively.

[0091] The A allele variation of SNP5 G / A has a positive effect on the retention of fruit sugar content. The number of alleles with positive effects in the three test sample genotypes, namely GG, GA and AA, are 0, 1 and 2, respectively.

[0092] The T allele variation of SNP6 G / T has a positive effect on the retention of fruit sugar content. The number of alleles with positive effects in the three test sample genotypes, namely GG, GT and TT, are 0, 1 and 2, respectively.

[0093] The C allelic variation of SNP7 T / C has a negative effect on the retention of fruit sugar content. The number of alleles with positive effects in the three test sample genotypes, TT, TC and CC, are 0, -1 and -2, respectively.

[0094] The T allele variation of SNP8 G / T has a positive effect on the retention of fruit sugar content. The number of alleles with positive effects in the three test sample genotypes, namely GG, GT and TT, are 0, 1 and 2, respectively.

[0095] The A allele variation of SNP9 G / A has a positive effect on the retention of fruit sugar content. The number of alleles with positive effects in the three test sample genotypes, namely GG, GA and AA, are 0, 1 and 2, respectively.

[0096] (4) Auxiliary selection of sugar content trait in apple fruit The steps for identifying the sugar content trait in apples are as follows: Genomic DNA was extracted from the apple species to be tested.

[0097] The extracted genomic DNA was precisely quantified.

[0098] Enzymatic digestion was used to fragment the quantified genomic DNA, and a DNA library was constructed.

[0099] Adapter sequences were added to both ends of the DNA library fragments for sample labeling.

[0100] The target sequence is captured using the probe combination described above.

[0101] The captured target sequence was subjected to next-generation sequencing. The sequencing data was compared and analyzed with the apple reference genome GDDH13.1 to obtain genotyping data.

[0102] Based on the genotyping data, the sugar content trait of the fruit of the tested Malus species was identified.

[0103] The identification process is based on the genotyping data of SNP1 to SNP9. Based on the obtained genotyping data, the sum of the number of alleles in the SNP1 to SNP9 genotypes that have a positive effect on the preservation of fruit sugar content is calculated and denoted as NPA.

[0104] NPA was used as an indicator of the sugar content retention of the fruit of the tested *Malus* species. A higher NPA value indicates a higher sugar content in the fruit of the tested *Malus* species. The specific identification criteria are as follows:

[0105] ①If the NPA is -5 to -2, then the sugar content of the fruit of the tested apple species is low, and the soluble solids content is between 11°Brix and 14°Brix.

[0106] ②If the NPA is -1 to 5, then the soluble solids content of the fruit of the tested Malus species is between 11°Brix and 18.5°Brix.

[0107] ③ If the NPA is 5~8, then the fruit of the tested apple species has a high sugar content and the soluble solids content is between 13.8°Brix and 22°Brix.

[0108] Allelic variations can occur at all SNP1 to SNP9; Specifically, the T allele variation at SNP1 has a positive effect on the retention of fruit sugar content; if the genotype at SNP1 is GG, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP1 is GT, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP1 is TT, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0109] The C allele variation at SNP2 has a negative effect on the retention of fruit sugar content; if the genotype at SNP2 is GG, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP2 is GC, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP2 is CC, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit sugar content is -2.

[0110] The T allele at SNP3 has a negative effect on the retention of fruit sugar content; if the genotype at SNP3 is CC, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP3 is CT, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP3 is TT, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit sugar content is -2.

[0111] The T allele variation at SNP4 has a negative effect on the retention of fruit sugar content; if the genotype at SNP4 is GG, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP4 is GT, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP4 is TT, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit sugar content is -2.

[0112] The A allele at SNP5 has a positive effect on the retention of fruit sugar content; if the genotype at SNP5 is GG, then the number of alleles in the SNP5 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP5 is GA, then the number of alleles in the SNP5 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP5 is AA, then the number of alleles in the SNP5 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0113] The C allele variation at SNP6 has a positive effect on the retention of fruit sugar content; if the genotype at SNP6 is GG, then the number of alleles in the SNP6 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP6 is GT, then the number of alleles in the SNP6 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP6 is TT, then the number of alleles in the SNP6 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0114] The C allele at SNP7 has a negative effect on the retention of fruit sugar content; if the genotype at SNP7 is TT, then the number of alleles in the SNP7 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP7 is TC, then the number of alleles in the SNP7 genotype that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP7 is CC, then the number of alleles in the SNP7 genotype that have a positive effect on the retention of fruit sugar content is -2.

[0115] The T allele variation at SNP8 has a positive effect on the retention of fruit sugar content; if the genotype at SNP8 is GG, then the number of alleles in the SNP8 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP8 is GT, then the number of alleles in the SNP8 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP8 is TT, then the number of alleles in the SNP8 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0116] The A allele at SNP9 has a positive effect on the retention of fruit sugar content; if the genotype at SNP9 is GG, then the number of alleles in the SNP9 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP9 is GA, then the number of alleles in the SNP9 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP9 is AA, then the number of alleles in the SNP9 genotype that have a positive effect on the retention of fruit sugar content is 2.

[0117] The target sequences were obtained by testing the samples according to the above method. The samples to be tested refer to 335 germplasm resources of 25 species of apple plants, as well as 1073 hybrid offspring from four half-sib families, namely 'Hongyu' × 'Jinguan', 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Hongjinqing', to construct a training population. The total number of individuals in the training population is 1408, and the source is the apple breeding base in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0118] The generational sum of the number of alleles with a positive effect on fruit sugar content retention in each marker genotype of the SNP1~SNP9 marker combinations in the test sample is used as an indicator of fruit sugar content retention. A higher generational sum indicates a higher fruit sugar content in the test sample. Figure 1 .

[0119] Depend on Figure 1 It can be seen that the sugar content of apples can be predicted comprehensively and accurately by using the above SNP1~SNP9 marker combination (9 SNP markers).

[0120] Example 2 Using 335 germplasm resources from 25 species of *Malus* with phenotypic data on fruit sugar content obtained for 2-5 years, and 1073 hybrid progeny from four half-sib families ('Hongyu' × 'Jinguan', 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Hongjinqing'), a training population was constructed, with a total of 1408 individuals. GenoBaits DNA probe combinations corresponding to the molecular markers for apple fruit sugar content trait-assisted selection were used to genotype molecular marker combinations (SNP1-SNP9). Then, the generational sum (NPA) of the number of alleles with positive effects on fruit sugar content in the five high-sugar molecular marker genotypes in each individual of the training population was calculated. Finally, linear regression analysis was performed using the NPA values and the average measured individual fruit sugar content. The results are shown below. Figure 2 ,

[0121] Among them, 335 germplasm resources of 25 species originated from the apple breeding base in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0122] The training population of 1,073 hybrid offspring from four half-sib families, namely 'Hongyu' × 'Jinguan', 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Hongjinqing', originated from the apple breeding base in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0123] The coefficients of determination for the linear regression equation between NPA value and fruit sugar content are R0 and R1, respectively. 2 =0.42. This indicates that the combination of molecular markers and probes for assisted selection of sugar content traits in apple fruits is feasible.

[0124] The experimental results above show that the molecular marker combination provided by the present invention has comprehensive superior alleles related to sugar content performance in wild species and closely related species of the genus Malus, with accurate prediction and coverage of multiple allele loci. It can integrate superior alleles related to sugar content in wild species and closely related species of the genus Malus, which can solve the problem that the existing molecular marker combination has limited predictive comprehensiveness and application effect in molecular-assisted breeding, and is difficult to meet the needs of precision breeding.

[0125] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0126] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A probe array of molecular markers for the selection of apples by means of sugar content trait, characterized in that, Including nine probes related to the selection of apple sugar content traits; The nucleotide sequences of the probes for the selection of sugar content traits in apples are shown in SEQ ID NO.10~SEQ ID NO.

18.

2. The probe combination of molecular markers for auxiliary selection of apple sugar content trait according to claim 1, characterized in that, Nine apple sugar content trait-assisted selection probes were used to detect SNP1 to SNP9; the nucleotide sequences of SNP1 to SNP9 are shown in SEQ ID NO.1 to SEQ ID NO.9, respectively. SNP1 is a G-to-T mutation at position 25310876 on chromosome 0 of the apple reference genome GDDH13.1; SNP2 is a G-to-C mutation at position 32459528 on chromosome 1 of the apple reference genome GDDH13.1; SNP3 is a C-to-T mutation at position 5665814 on chromosome 3 of the apple reference genome GDDH13.1; SNP4 is a G-to-T mutation at position 23915298 on chromosome 3 of the apple reference genome GDDH13.1; SNP5 is a G-to-T mutation at position 3 on chromosome 3 of the apple reference genome GDDH13.

1. A mutation from G to A exists at position 27,405,697 of the chromosome; SNP6 is a mutation from G to T at position 28,603,676 of chromosome 10 in the apple reference genome GDDH13.1; SNP7 is a mutation from T to C at position 36,811,784 of chromosome 11 in the apple reference genome GDDH13.1; SNP8 is a mutation from G to T at position 6,378,877 of chromosome 14 in the apple reference genome GDDH13.1; and SNP9 is a mutation from A to G at position 11,982,626 of chromosome 15 in the apple reference genome GDDH13.

1.

3. A kit for identifying the sugar content trait of apple fruit, characterized in that, Includes the probe combination as described in any one of claims 1 to 2.

4. The use of the probe combination of claim 2 or the kit of claim 3 in identifying the sugar content trait of apple fruit.

5. The application according to claim 4, characterized in that, The steps for identifying the sugar content trait in apples are as follows: Genomic DNA was extracted from the Malus species to be tested; Genomic DNA was fragmented using enzyme digestion and a DNA library was constructed. Adapter sequences were added to both ends of the DNA library fragments for sample labeling; The target sequence is captured using the probe combination described above; The captured target sequence was subjected to next-generation sequencing. The sequencing data was compared and analyzed with the apple reference genome GDDH13.1 to obtain genotyping data. Based on the genotyping data, the sugar content trait of the fruit of the tested Malus species was identified.

6. The application according to claim 5, characterized in that, Based on the obtained genotyping data, the sum of the number of alleles in the genotypes of SNP1 to SNP9 that have a positive effect on the preservation of fruit sugar content is calculated and denoted as NPA; NPA was used as an indicator of the sugar content retention of the fruit of the tested Malus species; the higher the NPA value, the stronger the sugar content retention of the fruit of the tested Malus species.

7. The application according to claim 6, characterized in that, If the NPA is -5 to -2, the fruit of the tested *Malus* species has a low sugar content, with soluble solids content between 11°Brix and 14°Brix; if the NPA is -1 to 5, the fruit of the tested *Malus* species has a soluble solids content between 11°Brix and 18.5°Brix; if the NPA is 5 to 8, the fruit of the tested *Malus* species has a high sugar content, with soluble solids content between 13.8°Brix and 22°Brix.

8. The application according to claim 6, characterized in that, Allelic variations can occur at all SNP1 to SNP9; Specifically, the T allele variation at SNP1 has a positive effect on the retention of fruit sugar content; if the genotype at SNP1 is GG, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP1 is GT, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP1 is TT, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit sugar content is 2. The C allele variation at SNP2 has a negative effect on the retention of fruit sugar content; if the genotype at SNP2 is GG, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP2 is GC, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP2 is CC, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit sugar content is -2. The T allele at SNP3 has a negative effect on the retention of fruit sugar content; if the genotype at SNP3 is CC, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP3 is CT, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP3 is TT, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit sugar content is -2. The T allele at SNP4 has a negative effect on the retention of fruit sugar content; if the genotype at SNP4 is GG, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP4 is GT, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP4 is TT, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit sugar content is -2. The A allele at SNP5 has a positive effect on the retention of fruit sugar content; if the genotype at SNP5 is GG, then the number of alleles in the SNP5 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP5 is GA, then the number of alleles in the SNP5 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP5 is AA, then the number of alleles in the SNP5 genotype that have a positive effect on the retention of fruit sugar content is 2. The C allele variation at SNP6 has a positive effect on the retention of fruit sugar content; if the genotype at SNP6 is GG, then the number of alleles in the SNP6 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP6 is GT, then the number of alleles in the SNP6 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP6 is TT, then the number of alleles in the SNP6 genotype that have a positive effect on the retention of fruit sugar content is 2. The C allele at SNP7 has a negative effect on the retention of fruit sugar content; if the genotype at SNP7 is TT, then the number of alleles in the genotype of SNP7 that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP7 is TC, then the number of alleles in the genotype of SNP7 that have a positive effect on the retention of fruit sugar content is -1; if the genotype at SNP7 is CC, then the number of alleles in the genotype of SNP7 that have a positive effect on the retention of fruit sugar content is -2. The T allele at SNP8 has a positive effect on the retention of fruit sugar content; if the genotype at SNP8 is GG, then the number of alleles in the SNP8 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP8 is GT, then the number of alleles in the SNP8 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP8 is TT, then the number of alleles in the SNP8 genotype that have a positive effect on the retention of fruit sugar content is 2. The A allele at SNP9 has a positive effect on the retention of fruit sugar content; if the genotype at SNP9 is GG, then the number of alleles in the SNP9 genotype that have a positive effect on the retention of fruit sugar content is 0; if the genotype at SNP9 is GA, then the number of alleles in the SNP9 genotype that have a positive effect on the retention of fruit sugar content is 1; if the genotype at SNP9 is AA, then the number of alleles in the SNP9 genotype that have a positive effect on the retention of fruit sugar content is 2.

9. The application according to claim 5, characterized in that, The connector sequence contains a sample identification barcode, and the sample marking process is completed using a barcode connector kit.