Molecular marker probe combination for auxiliary selection of characters in mature period of apple fruits and application of molecular marker probe combination

By developing a molecular marker probe combo for apple fruit ripening trait-assisted selection and integrating eight key SNP markers, we have achieved refined grading and prediction of apple fruit ripening trait, solving the problem of inaccurate ripening prediction in existing technologies and improving the accuracy and efficiency of breeding selection.

CN121780753APending Publication Date: 2026-04-03CHINA AGRI UNIV
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Patent Information

Application Number
CN202610005611.8
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

Existing commercially available or literature-reported detection products are mostly simple typing systems targeting single or a few markers, which cannot accurately predict the ripening period of apple fruits and cannot meet the refined needs of modern apple breeding for varieties with different ripening periods.

Method used

To develop a molecular marker probe combo for apple fruit ripening trait-assisted selection, a systematic probe combo is formed by integrating eight key SNP markers. The genetic variation during ripening is precisely analyzed by utilizing the synergistic effect of multiple markers, providing a refined grading prediction of fruit ripening traits.

Benefits of technology

It significantly improves the accuracy of apple fruit ripening period selection, shortens the selection cycle, saves resource consumption, and is suitable for refined and targeted selection of apple breeding, evaluation and screening of apple germplasm resources, and guidance for breeding program design.

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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 characters in an apple fruit maturation period and application of the molecular marker probe combination. The probe combination comprises eight probes for auxiliary selection of characters in the mature period of the apple fruits; the nucleotide sequences of the eight probes for auxiliary selection of the characters of the apple fruits in the mature period are sequentially shown as SEQ ID NO.9 to SEQ ID NO.16. The DNA probe combination corresponding to the SNP marker combination provided by the invention can be applied to molecular marker-assisted selection of mature characters of apples 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 auxiliary selection of apple fruit ripening traits and their applications. Background Technology

[0002] apple( Malus domestica Borkh. Apples are one of China's most important economic crops, with the highest yield in the world. The maturity period of apples is a crucial trait for fruit storage quality, and breeding a diverse range of superior varieties, combining early, mid, and late-maturing types, remains a pressing need for my country's apple industry. Early-maturing varieties with excellent fruit quality can help save on orchard management costs, and breeding early-maturing varieties is less costly than year-round storage. However, some early-maturing varieties have poor fruit quality and storage tolerance, while high-quality, storage-tolerant varieties often mature later. Therefore, breeding new early-maturing varieties is an effective means to achieve year-round apple supply and reduce cold storage costs.

[0003] Currently, most commercially available or literature-reported testing products are simple typing systems targeting single or a few markers, which can only roughly distinguish between early-maturing and late-maturing types. This makes it impossible to accurately predict the fruit ripening time window, and it is difficult to meet the refined needs of modern apple breeding for targeted selection of varieties with different ripening periods. Summary of the Invention

[0004] To address the problem that existing commercially available or literature-reported detection products are mostly simple genotyping systems targeting single or a few markers, only roughly distinguishing between early-maturing and late-maturing types, thus failing to accurately predict fruit ripening time windows and failing to meet the refined requirements of modern apple breeding for targeted selection of varieties with different ripening periods, this invention aims to develop a new strategy for efficient screening of ripening period traits, providing a probe combination of molecular markers for auxiliary selection of apple fruit ripening period traits and its application. To achieve the above objectives, this invention adopts the following technical solution.

[0005] The ripening period of apple fruits is evaluated using the number of days after full bloom, i.e., the time it takes for the fruit to ripen after full bloom, measured in days. This invention conducted a phenotypic investigation on the fruit ripening period of 2664 offspring plants from crosses of apple varieties 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Jinguan'. Forty-eight SNP markers were developed using methods such as GWAS, BSA-seq, and RNA-seq. However, using 48 molecular markers for marker-assisted selection of apple fruit ripening traits is extremely difficult; therefore, this invention conducts the following experimental research.

[0006] This invention screens for markers with an effect value greater than 15 days on fruit ripening period from 48 SNP markers. A total of 8 SNP markers were selected, including SNP1 to SNP8. Based on this, this invention provides a probe array of molecular markers for assisting selection of apple fruit ripening period traits, the probe array comprising 8 probes for assisting selection of apple fruit ripening period traits.

[0007] The nucleotide sequences of the eight probes for the selection of apple fruit ripening traits are shown in SEQ ID NO.9~SEQ ID NO.16, respectively.

[0008] The eight probes used for auxiliary selection of apple fruit ripening traits were probes for detecting SNP1 to SNP8. The nucleotide sequence of the probe (GenoBaits DNA) used to detect SNP1 is shown in SEQ ID NO. 9:

[0009] ACCAATGATACCATGGGGTACAAAAAACCAATTTAAATATCATTAATCAATTAATCAGTTAATTATACACGTGCTTAGTGTTGGATTACCCTCATGGAGTTCTTCTTGTT.

[0010] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP2 is shown in SEQ ID NO.10: TGGAAGCGGAACCCGGGCGGCAGGTTTGGCTGCGGAGGTGGCGGCTGCTGGTGCTGTTGCTGTTGTGAGCCCGCGGACGAGTCGGTGCACTCCATCCTTGGTTATTGGGT.

[0011] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP3 is shown in SEQ ID NO.11: AGAATTGTTAAAGTTATACAATTTTGGTATTAGGAGTGAATGTTACTCATATGGACCTGAATGTTGTAAACTGAAAACAATTCTTGTTCATCCGTGATGAGGGGTCTCAC.

[0012] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP4 is shown in SEQ ID NO.12: CGGTGGAGTCATCATGGTCGAAGGAGCAGAGGTCGACGCTGGCGGAGGCGTGGGAGAAGAGGCTGAAGTTCGACTCTAAGGCGAAGATTACGGAGTCAGGGTTTGGGTCG.

[0013] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP5 is shown in SEQ ID NO.13: CGTCAACCATGCTCACACTGTTATCAAATGGCTCTCCTTGGGTTAATTGGATACCAGGTATGTGGTTTGTGCTGTTATCGTCAGGTTCCATGAGTGCAGATGATGTCTCC.

[0014] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP6 is shown in SEQ ID NO.14: TTTTATATGATGTGTAATTACTTCGTTAACCATGATTAAGGCGGGGGCCTAATTTTGTGCATGGGATTGAGAGTGACTGTGTGGTGGTTAGCATAGGGACAGGTAGGGAA.

[0015] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP7 is shown in SEQ ID NO.15: TACGTGGATTTTCTACCCAAATGCACTCCCTCGGGCCTCGGTCCTTTGCTGCTTCCATTTATTTTCGACTTCTGGCTTTTGCCTTTACTGCCAATGCCCGAACCCGCTGG.

[0016] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP8 is shown in SEQ ID NO.16: CCTGGATGGGTAGTTCCAGTCGCTCCACCCTGGAGGAGTTATTATATCATCGAAATGACAGTAGGAATACACCGCTCTTGAATAATTTCCCCAAGCTCTTCCCAAGTAGA.

[0017] Existing detection products rely on only one or a few markers, allowing for a rough binary classification of maturity traits (such as early-maturing versus late-maturing). They cannot analyze the continuous variation characteristics of complex traits controlled by multiple genes, resulting in low resolution predictions and difficulty in accurately classifying maturity levels and predicting specific maturity time windows. This invention integrates eight key SNP markers to form a systematic probe combination. Utilizing the synergistic effect of multiple markers, it can finely analyze genetic variations in maturity, achieving precise grading predictions from very early to very late maturity. This significantly improves selection accuracy, thus meeting the requirements of modern apple breeding for precise targeted selection of maturity.

[0018] Preferably, the above 8 SNPs are labeled as SNP1 to SNP8; SNP1 to SNP8 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.8, respectively.

[0019] SNP1 is an A-to-T mutation located at position 30697394 on apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.1: GAAGTACCCGAACGGAGCGAGACCCAATAGGGCAGCGACTTCCGG(G / A)TATTGGAAAGCGACGGGGACTGATAAGCCGGTGCTGACTTCCGGCGATACTCAGAAAGTTGGTGTGAAAAAAGCACT TGGTGTTCTATGGAGGAAAACCCCCAAAAGGAATTAAAACCAATTGGATTATGCACGAGTACAGGCTTGCTGATAACAAGCCCAACAACAAGCCACCTGGGTGTGACTTGGGCAACAAGAAGAACTCC[ A / T]TGAGGGTAATCCAACACTAAGCACGTGTATAATTAACTGATTAATTGATTAATGATATTTAAATTGGTTTTTTGTACCCCATGGTATCATTGGTACTTACCTTTTATTTGTTAATTTAATTTGGAGATTAGTAAATTGATTAATATGTTTGTGTTCATGTAGCTTGATGATTGGGTGCTGTGTAGAATTTACAAGAAGAACAACACGCATAGGCCGATGGATCATGAGAGGGAGGGAGATTCCATGGAGGACA. The position of SNP1 is marked with square brackets "[ ]", where the non-variant base "A" comes first and the variant base "T" comes second, separated by " / ", the same below).

[0020] SNP2 is a G-to-T mutation located at position 30698039 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.2: ATCCCATTACG(T / G)GGCCTT(G / T)ACCATTGGTTCCCTAGTTTAAAAACCA(T / C)GCAGGCAACTGCAGCGGAATATT(GC / TT)CCCCTCACAT(A / G)CTTAAAACAGGGG CCCTGCCAGTCTGCCACCTCCAAACGACATTGAAGGTCTATATGTAATATGTGCTTGGCTGCACAGAAACCCTCCGAACCCAATAACCAA(G / A)GATGGAG(T / A)GCACC[G / T]A CTCGTCCGCGGGCTCACAACAGCAAC(A / G)GCACCAGCAGCCGC(- / AGCCGCAGCCGC)CACCTCCGCAGCCAAACCTGCCGCCCGGGTTCCGCTTCCACCC(G / T)ACGGACGAA GAGCTAGTCGTCCACTATCTCAAGAAAAAGGCCACC(T / G)CCGCTCCCCTCCCGGTTGCTAT(T / C)AT(C / A)GCTGAAGTCGACCTTTACAAGTTCGACCCCTGGGAGCTCCCA.

[0021] SNP3 is a C-to-T mutation located at position 21452649 on chromosome 10 of the apple chromosome, and its nucleotide sequence is shown in SEQ ID NO.3: TATTTTCTTGACTTTCTTTTACATATATATTTTGGATTTCGTATATGGAGAATTTTTGTTTTATTAATTCGTTCGCATGAGATAACCCGGAAGCAAATTGGAATGAAAATGAATTGTCAGAATTGTTAAAGTTATACAATTTTGGTATTAGGAGTGAATGTTACTCATATGGACCTGAATGTTGTAAACTGAAAACAATT[C / T]TTGTTCATCCGTGATGAGGGGTCTCACTTTTATCTGGTTCTCTAGCAAGCCTTCTTGCAGCTTCACATCATAATTGTGCCAGATTTCGCTAATTTTTTTTTATTTTTTATTGCATGATGTTAACTTGGCTAGTTCAGTGCTCTATATTGACTTCAAGTTCATGCACTAGTTTCTGAACATCCACGATATTGGGGATTTTT.

[0022] SNP4 is a G-to-A mutation at position 29106042 of apple chromosome 16, and its nucleotide sequence is shown in SEQ ID NO.4: GGTGGCAACGAGAACCGCCAAGAACTAGGATTCGAATTCGGATTGCGATCTGGACCACTCGAGCTTTCCTCAGGGCGGCGATCGCGTGGGGCTGAGTTATGCGAAGAGATCTAGGAGGCGGTGGAGTCATCATGGTCGAAGGAGCAGAGGTCGACGCTGGCGGAGGCGTGGGAGAAGAGGCTGAAGTTCGACTCTAAGGC[G / A]AAGATTACGGAGTCAGGGTTTGGGTCGTAGACTCGGAAACCCGGTCTCTGCTCCTGCATTTTTCAGAATACCAAATTTATAGAGTTCTTTGAAGATCTGGAGATCTGGAGCACCTTGGAGATCATGGACGAGTCAACTCGATGACTCGACGACTTCACGATCAGCTTCTGCTTGAGCAAAATTAGGGCTTGGGTCGGGTC.

[0023] SNP5 is a mutation from A to G at position 30,498,789 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO. 5: GGAACAAGATTCAGATAGGCAAAGGATCTGTCACCTCATCTTCCGCGAGGATTAACAAGCTGAGAAAACACAGCACTTGTGGCAGCATTTTCTGTGTCATCTGTGCCGGAGTGTCCCCCGTCAACCATGCTCACACTGTTATCAAATGGCTCTCCTTGGGTTAATTGGATACCAGGTATGTGGTTTGTGCTGTTATCGTC[A / G]GGTTCCATGAGTGCAGATGATGTCTCCTCGAGCTGAAGAGCATATTCAAGATTCCACAAGACATCTCCCATTGATGGCCTGTCAACACCATACTCAGCAAGGCACTTCTCAGCTGTCTCCCCGAACTTCTTAAGAGAAGCTGGATTTACCTTCCCCGCCAGATTAGGGTCCATGATTTGGTCTAGCATGCCCTTCTTCTG.

[0024] SNP6 is a T-to-A mutation located at position 30698350 on apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO. 6: GCTTGTTCCCACCCCAGGCTTATCCGTCTGTATAATTTTGTTGCCTCG(C / T)GTGGTCCGTGGGCCCCACGGAA(- / C)GTCCACCACACGTGTCGACCTTTCTCTCGTTTTCGAATTACACGTGTCGGCCTATCAACGGCCCACTTTTCAGTTCGCTTCGTCCTAGGGTTTTCCATCACCTCCATATCCCCTTTCCCTACCTGTCCC[T / A]ATGCTAACCACCACACAGTCACTCTCAATCCCATGCACAAAATTAGGCCCCCGCCTTAATCATGGTTAACGAAGTAATTACACATCATATAAAATGGCTAATCTACCCTGATCCCATTACGTGGCCTTGACCATTGGTTCCCTAGTTTAAAAACCA(T / C)GCAGGCAACTGCAGCGGAATATT(GC / TT)CCCCTCACATACTTAAAA.

[0025] SNP7 is an A-to-T mutation located at position 30739052 on the third chromosome of apple, and its nucleotide sequence is shown in SEQ ID NO.7: GCGCCAGAACTCAATCT(C / G)AATAATCAGACTCAGCAAGGCCAAGGACAAATGAATTTCAATTTCAATAACAATG(A / G)CGTGCCTCCCTTCAACCCACCTCTCTGCCACGTGGAATGGCCGCCGG AGAGTTTGGGAAGACGGCGGAGGAGGAGGTGCAGAGCGGACTCAGAACTCAGCGGGTGGATAACTCCAACTTCTTCCAGCAACACTCTCAGAACGTTTGTTACCCGGTCCGGCCCAGCGGGTTCGGGC[A / T]TTGGCAGTAAA(GG / AT)CAAAAGCCAGAAGTCGAAAATAAATGGAAGCAG(C / G)AAAGGACCGAGGCCCGAGGGAGTGCATTTGGGTAGAAAATCCACGTAGGAGTGCATTTCTGTAAAATAATTTGG ATTTTTTGGGCTAACATTTTGGGGTTAAATTATAAAAAAAAAGTCTGGGAGGGGGTGGCATGAAAAAAGTGGATGGAGATTTGTCCGTTGTTTTTGCTAGTTTGAGGCCGCAGATTCAACCAACTCGAAA.

[0026] SNP8 is a mutation from G to C at position 25532731 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.8.

[0027] AATCCACTGTCCTGTGTCAACGAAAACTTTCAGTTTTAGAAACAATAGAGATAATCTGATAAGATGAAATATTGTTCGAGTGAAAAGAGGTTATATTTACTTCTGCCTGGATGGGTAGTTCCAGTCGCTCCACCCTGGAGGAGTTATTATATCATCGAAATGACAGTAGGAATACACCGCTCTTGAATAATTTCCCCAAG[G / C]CTCTTCCCAAGTAGATGTAGCCGGTTCCGATGATTTTGCAGTTTACAAAAGAGAATCCCGTGTCTTCATACGGTGAGTCCCTGTGATGAGCTGCTATTGCTCCCGAGTTCTCAGCCGTTGATTGAATAACACACTCCTGATTGCAAGGGCCAGATTCAAGATTTAGTATCATGAAGAAACTTGGGGACCAAGTTACAGAA.

[0028] Among them, the effect value of SNP1 on the fruit ripening period was 27.3 days; the effect value of SNP2 on the fruit ripening period was 50.7 days; the effect value of SNP3 on the fruit ripening period was 51.8 days; the effect value of SNP4 on the fruit ripening period was 49.6 days; the effect value of SNP5 on the fruit ripening period was 37.1 days; the effect value of SNP6 on the fruit ripening period was 19.8 days; the effect value of SNP7 on the fruit ripening period was 30.3 days; and the effect value of SNP8 on the fruit ripening period was 16.6 days.

[0029] The present invention also provides a kit for identifying the ripening characteristics of apple fruits, the kit comprising the probe combination.

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

[0031] Preferably, the steps for identifying apple fruit ripening characteristics 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 fruit ripening period traits of the tested Malus species were identified.

[0038] Preferably, based on the obtained genotyping data, the sum of the number of alleles in the genotypes of SNP1 to SNP8 that have a positive effect on fruit ripening period is calculated and denoted as NPA.

[0039] The NPA was used as an indicator of the fruit ripening period of the tested Malus species; the higher the NPA value, the stronger the fruit ripening period of the tested Malus species.

[0040] Preferably, if the NPA is -6 to -2, the fruit of the tested *Malus* species is very early-maturing, with a maturity period of less than 150 days; if the NPA is -1 to 2, the fruit of the tested *Malus* species is relatively early-maturing, with a maturity period between 150 and 170 days; if the NPA is 3 to 6, the fruit of the tested *Malus* species is late-maturing, with a maturity period between 170 and 190 days; if the NPA is 7, the fruit of the tested *Malus* species is very late-maturing, with a maturity period of more than 190 days.

[0041] Preferably, allelic variations can occur at SNP1 through SNP8; Among them, the T allele variation at SNP1 has a positive effect on fruit ripening period; if the genotype at SNP1 is TT, then the number of alleles in the genotype of SNP1 that have a positive effect on fruit ripening period is 2; if the genotype at SNP1 is TA, then the number of alleles in the genotype of SNP1 that have a positive effect on fruit ripening period is 1; if the genotype at SNP1 is AA, then the number of alleles in the genotype of SNP1 that have a positive effect on fruit ripening period is 0.

[0042] The T allele variation at SNP2 has a negative effect on fruit ripening time; if the genotype at SNP2 is GG, then the number of alleles in the genotype of SNP2 that have a positive effect on fruit ripening time is 0; if the genotype at SNP2 is GT, then the number of alleles in the genotype of SNP2 that have a positive effect on fruit ripening time is -1; if the genotype at SNP2 is TT, then the number of alleles in the genotype of SNP2 that have a positive effect on fruit ripening time is -2.

[0043] The T allele variation at SNP3 has a negative effect on fruit ripening time; if the genotype at SNP3 is CC, then the number of alleles in the SNP3 genotype that have a positive effect on fruit ripening time is 0; if the genotype at SNP3 is CT, then the number of alleles in the SNP3 genotype that have a positive effect on fruit ripening time is -1; if the genotype at SNP3 is TT, then the number of alleles in the SNP3 genotype that have a positive effect on fruit ripening time is -2.

[0044] The A allele at SNP4 has a negative effect on fruit ripening time; if the genotype at SNP4 is GG, then the number of alleles in the SNP4 genotype that have a positive effect on fruit ripening time is 0; if the genotype at SNP4 is GA, then the number of alleles in the SNP4 genotype that have a positive effect on fruit ripening time is -1; if the genotype at SNP4 is AA, then the number of alleles in the SNP4 genotype that have a positive effect on fruit ripening time is -2.

[0045] The G allele at SNP5 has a positive effect on fruit ripening time; if the genotype at SNP5 is AA, then the number of alleles in the SNP5 genotype that have a positive effect on fruit ripening time is 0; if the genotype at SNP5 is AG, then the number of alleles in the SNP5 genotype that have a positive effect on fruit ripening time is 1; if the genotype at SNP5 is GG, then the number of alleles in the SNP5 genotype that have a positive effect on fruit ripening time is 2.

[0046] The T allele at SNP6 has a positive effect on fruit ripening time; if the genotype at SNP6 is AA, then the number of alleles in the genotype at SNP6 that have a positive effect on fruit ripening time is 0; if the genotype at SNP6 is AT, then the number of alleles in the genotype at SNP6 that have a positive effect on fruit ripening time is 1; if the genotype at SNP6 is TT, then the number of alleles in the genotype at SNP6 that have a positive effect on fruit ripening time is 2.

[0047] The T allele at SNP7 has a positive effect on fruit ripening time; if the genotype at SNP7 is TT, then the number of alleles in the genotype of SNP7 that have a positive effect on fruit ripening time is 2; if the genotype at SNP7 is TA, then the number of alleles in the genotype of SNP7 that have a positive effect on fruit ripening time is 1; if the genotype at SNP7 is AA, then the number of alleles in the genotype of SNP7 that have a positive effect on fruit ripening time is 0.

[0048] The C allele at SNP8 has a positive effect on fruit ripening time; if the genotype at SNP8 is GG, then the number of alleles in the SNP8 genotype that have a positive effect on fruit ripening time is 0; if the genotype at SNP8 is GC, then the number of alleles in the SNP8 genotype that have a positive effect on fruit ripening time is 1; if the genotype at SNP8 is CC, then the number of alleles in the SNP8 genotype that have a positive effect on fruit ripening time is 2.

[0049] Preferably, the connector sequence includes a sample identification barcode, and the sample marking process is completed using a barcode connector kit.

[0050] Preferably, the target sequence is captured using a liquid-phase hybridization-based targeted capture technique.

[0051] Preferably, the precise quantification is performed using double-stranded DNA fluorescence quantification.

[0052] Preferably, the DNA library construction is performed using an enzyme digestion library construction kit suitable for the Illumina platform.

[0053] Preferably, the second-generation sequencing is performed using the Illumina sequencing platform for resequencing.

[0054] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a probe combination of molecular markers for auxiliary selection of apple fruit ripening period traits. This probe combination is suitable for auxiliary selection of fruit ripening period traits in apple hybrid offspring. Auxiliary selection of fruit ripening period traits in apple hybrid offspring can be carried out at the 6-week seedling stage. GenoBaits marker detection requires 4 weeks, meaning auxiliary selection of fruit ripening period traits in hybrid offspring is completed at the 11th week after sowing. Traditional phenotypic selection requires 8-10 years. Therefore, the cycle of auxiliary selection of hybrid offspring using molecular markers and probe combinations for apple fruit ripening period traits is shortened by 8-10 years compared to traditional phenotypic selection methods. Furthermore, since auxiliary selection of hybrid offspring using molecular markers and probe combinations for apple fruit ripening period traits is completed at the 11-week seedling stage, selected superior plants directly enter the re-selection process, while hybrid offspring that do not pass the selection are directly eliminated. This significantly saves resources and breeding workload in seedling propagation, hybrid primary selection nursery management, and phenotypic selection. Using the above probe combination, the ripening period of apple fruits can be predicted comprehensively and accurately.

[0055] Existing detection products rely on only one or a few markers, allowing for a rough binary classification of maturity traits (such as early-maturing versus late-maturing). They cannot analyze the continuous variation characteristics of complex traits controlled by multiple genes, resulting in low resolution predictions and difficulty in accurately classifying maturity levels and predicting specific maturity time windows. This invention integrates eight key SNP markers to form a systematic probe combination. Utilizing the synergistic effect of multiple markers, it can finely analyze genetic variations in maturity, achieving precise grading predictions from very early to very late maturity. This significantly improves selection accuracy, thus meeting the requirements of modern apple breeding for precise targeted selection of maturity.

[0056] 2. The molecular marker and probe combination developed in this invention is applicable to the evaluation and screening of fruit ripening traits in apple germplasm resources. The scope of application includes 25 species of apple plants. The selected superior fruit ripening 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.

[0057] 3. The molecular marker and probe combination developed in this invention for assisted selection of apple fruit ripening traits has been applied in molecular breeding practices for apple fruit ripening traits. Compared with high-density chips and whole-genome selection, the marker detection cost is low and the selection accuracy is high. Attached Figure Description

[0058] Figure 1 The relationship between the number of positive-effect alleles of molecular markers and fruit ripening period in the auxiliary selection of apple fruit ripening period traits in this invention.

[0059] Figure 2 The results of the training population validation for molecular marker combinations used in the auxiliary selection of apple fruit ripening traits in this invention. Detailed Implementation

[0060] 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.

[0061] 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).

[0062] Using probe combinations of molecular markers selected with the aid of the above-mentioned apple fruit ripening traits, molecular hybridization and target site capture were performed using the GenoBaits® DNA Hybridization kit for ILM.

[0063] Among them, the materials to be tested (Malus species to be tested) refer to 335 germplasm resources of 25 species of Malus species, 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.

[0064] The method used to extract genomic DNA was the CTAB plant genome extraction method.

[0065] The GenoBaits probe suite for apple fruit ripening trait-assisted selection includes probes for detecting SNP1 to SNP8.

[0066] The nucleotide sequences of the probes used to detect SNP1 to SNP8 are shown in SEQ ID NO.9 to SEQ ID NO.16, respectively.

[0067] The nucleotide sequence of the GenoBaits DNA probe used to detect SNP1 is shown in SEQ ID NO.9: ACCAATGATACCATGGGGTACAAAAAACCAATTTAAATATCATTAATCAATTAATCAGTTAATTATACACGTGCTTAGTGTTGGATTACCCTCATGGAGTTCTTCTTGTT.

[0068] The nucleotide sequence of the GenoBaits DNA probe used to detect SNP2 is shown in SEQ ID NO.10: TGGAAGCGGAACCCGGGCGGCAGGTTTGGCTGCGGAGGTGGCGGCTGCTGGTGCTGTTGCTGTTGTGAGCCCGCGGACGAGTCGGTGCACTCCATCCTTGGTTATTGGGT.

[0069] The nucleotide sequence of the GenoBaits DNA probe used to detect SNP3 is shown in SEQ ID NO.11: AGAATTGTTAAAGTTATACAATTTTGGTATTAGGAGTGAATGTTACTCATATGGACCTGAATGTTGTAAACTGAAAACAATTCTTGTTCATCCGTGATGAGGGGTCTCAC.

[0070] The nucleotide sequence of the GenoBaits DNA probe used to detect SNP4 is shown in SEQ ID NO.12: CGGTGGAGTCATCATGGTCGAAGGAGCAGAGGTCGACGCTGGCGGAGGCGTGGGAGAAGAGGCTGAAGTTCGACTCTAAGGCGAAGATTACGGAGTCAGGGTTTGGGTCG.

[0071] The nucleotide sequence of the GenoBaits DNA probe used to detect SNP5 is shown in SEQ ID NO.13: CGTCAACCATGCTCACACTGTTATCAAATGGCTCTCCTTGGGTTAATTGGATACCAGGTATGTGGTTTGTGCTGTTATCGTCAGGTTCCATGAGTGCAGATGATGTCTCC.

[0072] The nucleotide sequence of the GenoBaits DNA probe used to detect SNP6 is shown in SEQ ID NO.14: TTTTATATGATGTGTAATTACTTCGTTAACCATGATTAAGGCGGGGGCCTAATTTTGTGCATGGGATTGAGAGTGACTGTGTGGTGGTTAGCATAGGGACAGGTAGGGAA.

[0073] The nucleotide sequence of the GenoBaits DNA probe used to detect SNP7 is shown in SEQ ID NO.15: TACGTGGATTTTCTACCCAAATGCACTCCCTCGGGCCTCGGTCCTTTGCTGCTTCCATTTATTTTCGACTTCTGGCTTTTGCCTTTACTGCCAATGCCCGAACCCGCTGG.

[0074] The nucleotide sequence of the GenoBaits DNA probe used to detect SNP8 is shown in SEQ ID NO.16: CCTGGATGGGTAGTTCCAGTCGCTCCACCCTGGAGGAGTTATTATATCATCGAAATGACAGTAGGAATACACCGCTCTTGAATAATTTCCCCAAGCTCTTCCCAAGTAGA.

[0075] The above 8 SNPs are labeled as SNP1 to SNP8; SNP1 to SNP8 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.8, respectively.

[0076] SNP1 is an A-to-T mutation located at position 30697394 on apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.1: GAAGTACCCGAACGGAGCGAGACCCAATAGGGCAGCGACTTCCGG(G / A)TATTGGAAAGCGACGGGGACTGATAAGCCGGTGCTGACTTCCGGCGATACTCAGAAAGTTGGTGTGAAAAAAGCACT TGGTGTTCTATGGAGGAAAACCCCCAAAAGGAATTAAAACCAATTGGATTATGCACGAGTACAGGCTTGCTGATAACAAGCCCAACAACAAGCCACCTGGGTGTGACTTGGGCAACAAGAAGAACTCC[ The position of SNP1 is marked with square brackets "[ ]", where the non-variant base "A" comes first, followed by the variant base "T", separated by " / ", and the same applies below. The positions marked with "()" are interference sites, where the non-variant base G comes first, followed by the variant base A, separated by " / ", indicating a base variation (G / A) at that position. The same applies below.

[0077] SNP2 is a G-to-T mutation located at position 30698039 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.2: ATCCCATTACG(T / G)GGCCTT(G / T)ACCATTGGTTCCCTAGTTTAAAAACCA(T / C)GCAGGCAACTGCAGCGGAATATT(GC / TT)CCCCTCACAT(A / G)CTTAAAACAGGGGCCCTGCCAGTCTGCCACCTCCAAACGACATTGAAGGTCTATATGTAATATGTGCTTGGCTGCACAGAAACCCTCCGAACCCAATAACCAA(G / A)GATGGAG(T / A)GCACC[G / T]ACTCGTCCGCGGGCTCACAACAGCAAC(A / G)GCACCAGCAGCCGC(- / AGCCGCAGCCGC)CACCTCCGCAGCCAAACCTGCCGCCCGGGTTCCGCTTCCACCC(G / T)ACGGACGAAGAGCTAGTCGTCCACTATCTCAAGAAAAAGGCCACC(T / G)CCGCTCCCCTCCCGGTTGCTAT(T / C)AT(C / A)GCTGAAGTCGACCTTTACAAGTTCGACCCCTGGGAGCTCCCA。

[0078] SNP3 is a C to T mutation at position 21452649 on chromosome 10 of apple, and its nucleotide sequence is shown in SEQ ID NO.3: TATTTTCTTGACTTTCTTTTACATATATATTTTGGATTTCGTATATGGAGAATTTTTGTTTTATTAATTCGTTCGCATGAGATAACCCGGAAGCAAATTGGAATGAAAATGAATTGTCAGAATTGTTAAAGTTATACAATTTTGGTATTAGGAGTGAATGTTACTCATATGGACCTGAATGTTGTAAACTGAAAACAATT[C / T]TTGTTCATCCGTGATGAGGGGTCTCACTTTTATCTGGTTCTCTAGCAAGCCTTCTTGCAGCTTCACATCATAATTGTGCCAGATTTCGCTAATTTTTTTTTATTTTTTATTGCATGATGTTAACTTGGCTAGTTCAGTGCTCTATATTGACTTCAAGTTCATGCACTAGTTTCTGAACATCCACGATATTGGGGATTTTT。

[0079] SNP4 is a G-to-A mutation at position 29106042 of apple chromosome 16, and its nucleotide sequence is shown in SEQ ID NO.4: GGTGGCAACGAGAACCGCCAAGAACTAGGATTCGAATTCGGATTGCGATCTGGACCACTCGAGCTTTCCTCAGGGCGGCGATCGCGTGGGGCTGAGTTATGCGAAGAGATCTAGGAGGCGGTGGAGTCATCATGGTCGAAGGAGCAGAGGTCGACGCTGGCGGAGGCGTGGGAGAAGAGGCTGAAGTTCGACTCTAAGGC[G / A]AAGATTACGGAGTCAGGGTTTGGGTCGTAGACTCGGAAACCCGGTCTCTGCTCCTGCATTTTTCAGAATACCAAATTTATAGAGTTCTTTGAAGATCTGGAGATCTGGAGCACCTTGGAGATCATGGACGAGTCAACTCGATGACTCGACGACTTCACGATCAGCTTCTGCTTGAGCAAAATTAGGGCTTGGGTCGGGTC.

[0080] SNP5 is a mutation from A to G at position 30,498,789 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO. 5: GGAACAAGATTCAGATAGGCAAAGGATCTGTCACCTCATCTTCCGCGAGGATTAACAAGCTGAGAAAACACAGCACTTGTGGCAGCATTTTCTGTGTCATCTGTGCCGGAGTGTCCCCCGTCAACCATGCTCACACTGTTATCAAATGGCTCTCCTTGGGTTAATTGGATACCAGGTATGTGGTTTGTGCTGTTATCGTC[A / G]GGTTCCATGAGTGCAGATGATGTCTCCTCGAGCTGAAGAGCATATTCAAGATTCCACAAGACATCTCCCATTGATGGCCTGTCAACACCATACTCAGCAAGGCACTTCTCAGCTGTCTCCCCGAACTTCTTAAGAGAAGCTGGATTTACCTTCCCCGCCAGATTAGGGTCCATGATTTGGTCTAGCATGCCCTTCTTCTG.

[0081] SNP6 is a T-to-A mutation located at position 30698350 on apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO. 6: GCTTGTTCCCACCCCAGGCTTATCCGTCTGTATAATTTTGTTGCCTCG(C / T)GTGGTCCGTGGGCCCCACGGAA(- / C)GTCCACCACACGTGTCGACCTTTCTCTCGTTTTCGAATTACACGTGTCGGCCTATCAACGGCCCACTTTTCAGTTCGCTTCGTCCTAGGGTTTTCCATCACCTCCATATCCCCTTTCCCTACCTGTCCC[T / A]ATGCTAACCACCACACAGTCACTCTCAATCCCATGCACAAAATTAGGCCCCCGCCTTAATCATGGTTAACGAAGTAATTACACATCATATAAAATGGCTAATCTACCCTGATCCCATTACGTGGCCTTGACCATTGGTTCCCTAGTTTAAAAACCA(T / C)GCAGGCAACTGCAGCGGAATATT(GC / TT)CCCCTCACATACTTAAAA.

[0082] SNP7 is an A-to-T mutation located at position 30739052 on the third chromosome of apple, and its nucleotide sequence is shown in SEQ ID NO.7: GCGCCAGAACTCAATCT(C / G)AATAATCAGACTCAGCAAGGCCAAGGACAAATGAATTTCAATTTCAATAACAATG(A / G)CGTGCCTCCCTTCAACCCACCTCTCTGCCACGTGGAATGGCCGCCGG AGAGTTTGGGAAGACGGCGGAGGAGGAGGTGCAGAGCGGACTCAGAACTCAGCGGGTGGATAACTCCAACTTCTTCCAGCAACACTCTCAGAACGTTTGTTACCCGGTCCGGCCCAGCGGGTTCGGGC[A / T]TTGGCAGTAAA(GG / AT)CAAAAGCCAGAAGTCGAAAATAAATGGAAGCAG(C / G)AAAGGACCGAGGCCCGAGGGAGTGCATTTGGGTAGAAAATCCACGTAGGAGTGCATTTCTGTAAAATAATTTGG ATTTTTTGGGCTAACATTTTGGGGTTAAATTATAAAAAAAAAGTCTGGGAGGGGGTGGCATGAAAAAAGTGGATGGAGATTTGTCCGTTGTTTTTGCTAGTTTGAGGCCGCAGATTCAACCAACTCGAAA.

[0083] SNP8 is a mutation from G to C at position 25532731 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.8.

[0084] AATCCACTGTCCTGTGTCAACGAAAACTTTCAGTTTTAGAAACAATAGAGATAATCTGATAAGATGAAATATTGTTCGAGTGAAAAGAGGTTATATTTACTTCTGCCTGGATGGGTAGTTCCAGTCGCTCCACCCTGGAGGAGTTATTATATCATCGAAATGACAGTAGGAATACACCGCTCTTGAATAATTTCCCCAAG[G / C]CTCTTCCCAAGTAGATGTAGCCGGTTCCGATGATTTTGCAGTTTACAAAAGAGAATCCCGTGTCTTCATACGGTGAGTCCCTGTGATGAGCTGCTATTGCTCCCGAGTTCTCAGCCGTTGATTGAATAACACACTCCTGATTGCAAGGGCCAGATTCAAGATTTAGTATCATGAAGAAACTTGGGGACCAAGTTACAGAA.

[0085] Among them, the effect value of SNP1 on the fruit ripening period was 27.3 days; the effect value of SNP2 on the fruit ripening period was 50.7 days; the effect value of SNP3 on the fruit ripening period was 51.8 days; the effect value of SNP4 on the fruit ripening period was 49.6 days; the effect value of SNP5 on the fruit ripening period was 37.1 days; the effect value of SNP6 on the fruit ripening period was 19.8 days; the effect value of SNP7 on the fruit ripening period was 30.3 days; and the effect value of SNP8 on the fruit ripening period was 16.6 days.

[0086] (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.

[0087] (3) Calculation of the number of positive effect sites of apple fruit ripening period by the marker combination SNP1~SNP8 (probe combination of molecular markers for auxiliary selection of apple fruit ripening period traits).

[0088] The T allele variation of SNP1 A / T has a positive effect on fruit ripening time. The number of alleles with positive effects in the three test sample genotypes TT, TA and AA are 2, 1 and 0, respectively.

[0089] The T allele variation of SNP2 G / T has a negative effect on fruit ripening time. The number of alleles with positive effects in the three test sample genotypes, GG, GT and TT, are 0, -1 and -2, respectively.

[0090] The T allele variation of SNP3 C / T has a negative effect on fruit ripening time, and the number of positive effect alleles in the test samples of the three genotypes CC, CT and TT are 0, -1 and -2, respectively.

[0091] The A allele variation of SNP4 G / A has a negative effect on fruit ripening time, and the number of positive effect alleles in the test samples of the three genotypes GG, GA and AA are 0, -1 and -2, respectively.

[0092] The G allele variation of SNP5 A / G has a positive effect on fruit ripening time. The number of positive effect alleles in the test samples of the three genotypes AA, AG and GG are 0, 1 and 2, respectively.

[0093] The T allele variation of SNP6 T / A has a positive effect on fruit ripening time. The number of positive effect alleles in the test samples of the three genotypes AA, AT and TT are 0, 1 and 2, respectively.

[0094] The T allele variation of SNP7 A / T has a positive effect on fruit ripening time. The number of positive effect alleles in the test samples of the three genotypes TT, TA and AA are 2, 1 and 0, respectively.

[0095] The C allele variation of SNP8 G / C has a positive effect on fruit ripening time. The number of positive effect alleles in the test samples of the three genotypes, GG, GC and CC, are 0, 1 and 2, respectively.

[0096] (4) Auxiliary selection of apple fruit ripening traits The steps for identifying apple fruit ripening characteristics 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 fruit ripening period traits of the tested Malus species were identified.

[0103] The identification process is based on genotyping data from SNP1 to SNP8. Based on the obtained genotyping data, the sum of the number of alleles in the SNP1 to SNP8 genotypes that have a positive effect on fruit ripening time is calculated and denoted as NPA.

[0104] NPA was used as an indicator of the fruit ripening period of the tested *Malus* species. A higher NPA value indicates a stronger fruit ripening period in the tested *Malus* species. Specific identification criteria are as follows:

[0105] ①If the NPA is -6 to -2, then the fruit of the tested Malus species is extremely early-maturing, with a maturity period of less than 150 days.

[0106] ②If the NPA is -1 to 2, then the fruit of the tested Malus species matures relatively early, with a maturity period of 150 to 170 days.

[0107] ③ If the NPA is 3~6, ​​then the fruit of the tested Malus species is late-maturing, with a ripening period of 170 to 190 days.

[0108] ④ If the NPA is 7, then the fruit of the tested Malus species is extremely late-maturing, with a ripening period of more than 190 days.

[0109] Allelic variations can occur at all SNP1 to SNP8: Among them, the T allele variation at SNP1 has a positive effect on fruit ripening period; if the genotype at SNP1 is TT, then the number of alleles in the genotype of SNP1 that have a positive effect on fruit ripening period is 2; if the genotype at SNP1 is TA, then the number of alleles in the genotype of SNP1 that have a positive effect on fruit ripening period is 1; if the genotype at SNP1 is AA, then the number of alleles in the genotype of SNP1 that have a positive effect on fruit ripening period is 0.

[0110] The T allele variation at SNP2 has a negative effect on fruit ripening time; if the genotype at SNP2 is GG, then the number of alleles in the genotype of SNP2 that have a positive effect on fruit ripening time is 0; if the genotype at SNP2 is GT, then the number of alleles in the genotype of SNP2 that have a positive effect on fruit ripening time is -1; if the genotype at SNP2 is TT, then the number of alleles in the genotype of SNP2 that have a positive effect on fruit ripening time is -2.

[0111] The T allele variation at SNP3 has a negative effect on fruit ripening time; if the genotype at SNP3 is CC, then the number of alleles in the SNP3 genotype that have a positive effect on fruit ripening time is 0; if the genotype at SNP3 is CT, then the number of alleles in the SNP3 genotype that have a positive effect on fruit ripening time is -1; if the genotype at SNP3 is TT, then the number of alleles in the SNP3 genotype that have a positive effect on fruit ripening time is -2.

[0112] The A allele at SNP4 has a negative effect on fruit ripening time; if the genotype at SNP4 is GG, then the number of alleles in the SNP4 genotype that have a positive effect on fruit ripening time is 0; if the genotype at SNP4 is GA, then the number of alleles in the SNP4 genotype that have a positive effect on fruit ripening time is -1; if the genotype at SNP4 is AA, then the number of alleles in the SNP4 genotype that have a positive effect on fruit ripening time is -2.

[0113] The G allele at SNP5 has a positive effect on fruit ripening time; if the genotype at SNP5 is AA, then the number of alleles in the SNP5 genotype that have a positive effect on fruit ripening time is 0; if the genotype at SNP5 is AG, then the number of alleles in the SNP5 genotype that have a positive effect on fruit ripening time is 1; if the genotype at SNP5 is GG, then the number of alleles in the SNP5 genotype that have a positive effect on fruit ripening time is 2.

[0114] The T allele at SNP6 has a positive effect on fruit ripening time; if the genotype at SNP6 is AA, then the number of alleles in the genotype at SNP6 that have a positive effect on fruit ripening time is 0; if the genotype at SNP6 is AT, then the number of alleles in the genotype at SNP6 that have a positive effect on fruit ripening time is 1; if the genotype at SNP6 is TT, then the number of alleles in the genotype at SNP6 that have a positive effect on fruit ripening time is 2.

[0115] The T allele at SNP7 has a positive effect on fruit ripening time; if the genotype at SNP7 is TT, then the number of alleles in the genotype of SNP7 that have a positive effect on fruit ripening time is 2; if the genotype at SNP7 is TA, then the number of alleles in the genotype of SNP7 that have a positive effect on fruit ripening time is 1; if the genotype at SNP7 is AA, then the number of alleles in the genotype of SNP7 that have a positive effect on fruit ripening time is 0.

[0116] The C allele at SNP8 has a positive effect on fruit ripening time; if the genotype at SNP8 is GG, then the number of alleles in the SNP8 genotype that have a positive effect on fruit ripening time is 0; if the genotype at SNP8 is GC, then the number of alleles in the SNP8 genotype that have a positive effect on fruit ripening time is 1; if the genotype at SNP8 is CC, then the number of alleles in the SNP8 genotype that have a positive effect on fruit ripening time is 2.

[0117] The target sequences were obtained by testing the samples according to the above method. The samples to be tested (Malus species) refer to 335 germplasm resources from 25 species of Malus, and 1073 hybrid offspring from four half-sib families ('Hongyu' × 'Jinguan', 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Hongjinqing') to construct a training population, with a total of 1408 individuals in the training population. The source of these samples is the Beidaihe New District Apple Breeding Base in Qinhuangdao City, Hebei Province.

[0118] The number of alleles with a positive effect on fruit ripening time in each marker genotype of the SNP1~SNP8 marker combinations in the test sample is used as an indicator to judge the fruit ripening time characteristic. The larger the value of the generational sum, the stronger the fruit ripening time characteristic of the test sample. Figure 1 .

[0119] Depend on Figure 1 It can be seen that the above-mentioned SNP1~SNP8 marker combinations or probe combinations can comprehensively and accurately predict the ripening period of apple fruits.

[0120] Example 2 Using 335 germplasm resources from 25 species of *Malus* with 2-5 years of fruit ripening phenotypic data, and 1073 hybrid offspring 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. Molecular marker combinations (SNP1-SNP8) were genotyped using probe combinations of molecular markers for fruit ripening trait-assisted selection. The generational sum of alleles with positive effects on fruit ripening time (NPA) in each individual's eight molecular marker genotypes was then calculated. Finally, linear regression analysis was performed using the NPA values ​​and the average measured fruit ripening time of each individual. 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 was constructed from 1,073 hybrid offspring from four half-sib families: 'Hongyu' × 'Jinguan', 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Hongjinqing'. The source of the training population was the apple breeding base in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0123] The coefficient of determination R of the linear regression equation between NPA value and fruit ripening time is... 2 =0.32. This indicates that the combination of molecular markers and probes for assisted selection of fruit ripening traits in apple plants is feasible.

[0124] The experimental results above show that the molecular marker combination provided by the present invention has comprehensive superior alleles related to storage 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 storage performance 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 assisted selection of apple fruit ripening traits, characterized in that, Includes eight probes for assisting selection of apple fruit ripening traits; The nucleotide sequences of the eight probes for the selection of apple fruit ripening traits are shown in SEQ ID NO.9~SEQ ID NO.

16.

2. The probe combination of molecular markers for auxiliary selection of apple fruit ripening traits according to claim 1, characterized in that, The eight probes used for auxiliary selection of apple fruit ripening traits were probes for detecting SNP1 to SNP8; among them, SNP1 is a mutation from A to T at position 30697394 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.1; SNP2 is a G-to-T mutation at position 30698039 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.2; SNP3 is a C-to-T mutation at position 21452649 of chromosome 10 of the apple, and its nucleotide sequence is shown in SEQ ID NO.3; SNP4 is a mutation from G to A at position 29106042 of chromosome 16 of the apple, and its nucleotide sequence is shown in SEQ ID NO.4; SNP5 is a mutation from A to G at position 30498789 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.5; SNP6 is a T-to-A mutation at position 30698350 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO. 6; SNP7 is an A-to-T mutation at position 30739052 on apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.7; SNP8 is a mutation from G to C at position 25532731 of apple chromosome 3, and its nucleotide sequence is shown in SEQ ID NO.

8.

3. A kit for identifying the ripening characteristics of apple fruits, 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 ripening characteristics of apple fruit.

5. The application according to claim 4, characterized in that, The steps for identifying apple fruit ripening characteristics 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 fruit ripening period traits of the tested Malus species were identified.

6. The application according to claim 5, characterized in that, Based on the obtained genotyping data, the sum of the number of alleles with a positive effect on fruit ripening period in the genotypes of SNP1 to SNP8 is calculated and denoted as NPA. The NPA was used as an indicator of the fruit ripening period of the tested Malus species; the higher the NPA value, the stronger the fruit ripening period of the tested Malus species.

7. The application according to claim 6, characterized in that, If the NPA is -6 to -2, the fruit of the tested *Malus* species is very early-maturing, with a maturity period of less than 150 days; if the NPA is -1 to 2, the fruit of the tested *Malus* species is relatively early-maturing, with a maturity period between 150 and 170 days; if the NPA is 3 to 6, the fruit of the tested *Malus* species is late-maturing, with a maturity period between 170 and 190 days; if the NPA is 7, the fruit of the tested *Malus* species is very late-maturing, with a maturity period of more than 190 days.

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

10. The application according to claim 5, characterized in that, The target sequence was captured using a liquid-phase hybridization-based targeted capture technique.