Molecular marker probe combination for auxiliary selection of storage endurance character of apple fruit and application of molecular marker probe combination

Abstract

The invention belongs to the technical field of molecular breeding of plants, and particularly relates to a probe combination of molecular markers for auxiliary selection of storage resistance characters of apple fruits and application of the probe combination. The probe combination comprises eight probes for auxiliary selection of the storage-resistant character of the apple fruits, and the nucleotide sequences of the eight probes for auxiliary selection of the storage-resistant character of the apple fruits are shown as SEQ ID NO.9-SEQ ID NO.16 in sequence. The DNA probe combination corresponding to the molecular marker provided by the invention can be applied to the apple filial generation seedling stage to carry out molecular marker-assisted selection on the storage endurance character of the apple.

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 storage-resistant traits in apple fruits and their applications. Background Technology

[0002] apple( Malus domestica Borkh. Apples are an important economic crop, and one of the most important economic crops in my country, with the highest yield in the world. Their post-harvest quality plays a crucial role in maintaining market competitiveness and economic value. Apples are among the most storable fruits, but their storability varies greatly among different varieties. Changes in fruit texture during storage are a key characteristic of post-harvest quality changes. Fruit texture is an important indicator for evaluating the storability of apples and also an important goal for apple quality breeding.

[0003] my country possesses abundant apple germplasm resources, many of which carry a wealth of genes related to storage tolerance. Traditional breeding methods suffer from long cycles and difficulty in predicting the traits of new varieties. Molecular marker-assisted breeding can reduce breeding costs and improve efficiency. Therefore, detection products developed based on storage tolerance-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 a novel strategy for efficient screening of storage-resistance traits. It provides a probe combination of molecular markers for the auxiliary selection of apple fruit storage-resistance traits and its application. To achieve the above objective, this invention employs the following technical solution.

[0005] The storability of apple fruit is evaluated by the retention of flesh crispness, specifically, the apple flesh crispness is maintained at 0.7 kg·cm² under cold storage conditions. -2 The above longest timeframes are in months. This invention conducted a phenotypic investigation on the fruit crispness retention trait of 2664 offspring plants from crosses of apple varieties 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Jinguan'. 454 SNPs and InDel markers were developed using methods such as GWAS, BSA-seq, and RNA-seq. However, using 454 molecular markers for marker-assisted selection of apple fruit storage tolerance traits is extremely difficult; therefore, this invention conducts the following experimental research.

[0006] This invention screens 454 SNP and InDel markers for markers with an effect value greater than 1.5 months on the retention of fruit crispness, including 6 SNP markers and 2 InDel markers; wherein the 6 SNP markers include SNP1~SNP6, and the 2 InDel markers include InDel1~InDel2. Based on this, this invention provides a probe combination of molecular markers for the auxiliary selection of apple fruit storage-resistant traits, the probe combination including probes for detecting SNP1~SNP8 and InDel1~InDel2.

[0007] The probe set includes eight probes for assisting in the selection of apple fruit storage traits.

[0008] The nucleotide sequences of the probes used for the selection of eight apple fruit storage-resistant traits are shown in SEQ ID NO.9~SEQ ID NO.16.

[0009] The probes used for the auxiliary selection of eight apple fruit storage resistance traits were probes for detecting six SNP markers and two InDel markers; the six SNP markers were designated as SNP1 to SNP6, and the two InDel markers were designated as InDel1 to InDel2.

[0010] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP1 is shown in SEQ ID NO.9: TTCACCCTCACACTGCACACACTATAACGTGCGCCCAGTACAGTTGCTTGATCAAATACCAATTCAGCTTTGAACTTTGCTTTAATGCTCTTTCAATTGAATTATGTCAG.

[0011] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP2 is shown in SEQ ID NO.10: TCATTACTGGGCATATTCAGCTGGTCAGGTCAAGAAAGCTGAAGCTGATTCCTCTGAGTTTGAAGTTCTTGATCCCATTTCAAGTATCTTCGTTTCAGCTTTCACTCCTC.

[0012] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP3 is shown in SEQ ID NO.11: ATTTCCTGTGGGAAAGTGTAGTACCTTCAAAGTTTGCCAAGCAAGCTGCAAATAATGAACCTTGAGGCCTTATCTGACTGTTGATTTTTGTTTTTGTTTTCGTTGACATT.

[0013] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP4 is shown in SEQ ID NO.12: CATTCAAGTAAGGTCAAATTAAACAAAGTACAGGTACTGGATACCAGGTGTCAAGAATGTTCTTTCTCAGCATGAATATCATCGCTTCTATTTATGATGCACAGAGAAAT.

[0014] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP5 is shown in SEQ ID NO.13: TTGATTGTCAAATTGATCAGATAACCATTATAGACCCACCATATCACCCATTTGTTAGTTTAGCACGGACTCAGATCCTCTCCGGATCATTTCCGTGGGATCCTCCTGACC.

[0015] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP6 is shown in SEQ ID NO.14: CCTATTAGATCAGCTTCTTCACATGAGTTCTGGATGGAGATAAAAGTTACCTTTCGATGATTTGCAGGAAGATATTCATTCCATAGGAAAAGCCGTCATGCAATTGGTAG.

[0016] The nucleotide sequence of the (GenoBaits DNA) probe used to detect InDel1 is shown in SEQ ID NO.15: TTATGATGGAACCCAATTTCATCATGGTATTAGAGCAAGGTTATCCACGTGTGAAGCCCAACGGCCACATTGAAATCTGCCACACGTGCGTGGGCGCGTTGAGAGTTGT.

[0017] The nucleotide sequence of the (GenoBaits DNA) probe used to detect InDel2 is shown in SEQ ID NO.16: CCATAACTAAATGTTGATGACAGAGGCTACAACCGTAATTTCACCCAATGACTTTTAAGGCTTTTGGTCCAGTACAGCTGTCAGCATGACATAACGTGACTTCATCCTTA.

[0018] Existing detection products suffer from low accuracy primarily because they rely on single or a few markers to predict storage-resistant traits controlled by multiple genes, making it difficult to comprehensively capture genetic information and susceptible to interference from individual variations. This invention addresses this issue by integrating a systematic probe combination of six SNPs and two InDel markers to achieve simultaneous detection of key loci. Utilizing the synergistic effect of multiple markers significantly improves genetic resolution and predictive robustness, thereby effectively solving the problem of insufficient accuracy.

[0019] Preferably, the molecular markers used for the auxiliary selection of the above-mentioned apple fruit storage resistance traits are SNP1~SNP8 and InDel1~InDel2; wherein SNP1~SNP6 are in apple 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.6, respectively.

[0020] Among them, SNP1 is a T-to-C mutation at position 22771272 on chromosome 4 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.1: GTTTTTTTGACATGTTTGTCGATTAACTCTCTCTGACTCTCTCTCTCACGGCTAAGATGATGGAAGAGCTTCTCACACTCGTGTTGTTTTGTGCTAGGCTCAAGCTTCTAGCTTTTCACCCTCACACTGCACACACTATAACGTGCGCCCAGTACAGTTGCTTGATCAAATACCAATTCAGCTTTGAACTTTGCTT[T / C]AATGCTCTTTCAATTGAATTATGTCAGATGCTGGAAATCTACACTTTGCAGACTTGATTTTTGCAGAGAGCAAATCTTTGCTGCAGTTTTTTCTCTACTCTTTGAAGGGTGTGGCTCTTATTTTCTGAACTCTCGTTCACTCACAAATGAAGGGTTCAACAAGCTCTTTTCCAAAGCACTTTGCACTCTTGGTGCTCATG. The position of SNP1 is marked with square brackets "[ ]", with the non-variant base "T" first and the variant base "C" second, separated by " / " (the same applies below).

[0021] SNP2 is a mutation from A to G located at position 201180 on chromosome 7 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.2: TATGCCTTATATTTACGTAAGAGTAAAGAAAGAAAGAAAAGGAAAAAGAAACGAAAATTGGCTCCAGCCTTTCATCCTGGCTACAGAGTGCAGGCAAGAGTATATTTGACGATCAGTCATTACTGGGCATATTCAGCTGGTCAGGTCAAGAAAGCTGAAGCTGATTCCTCTGAGTTTGAAGTTCTTGATCCCATTTCA[A / G]GTATCTTCGTTTCAGCTTTCACTCCTCTGCACGATTTCATGTGTGAGTGCAATCTTTCTGGCTTAAACAGCTTCCTGCACTGCCTGCATTAACATCAAGTCTTTCGAATTAATCGCTTGTTTAGATTTTCCACATAAATGTTCTCTTGGTCACCAAGTTTTATGAATGCCAACGTTGACCACATTCACTGTTTTGGGAGA.

[0022] SNP3 is a T-to-G mutation located at position 30980600 on chromosome 10 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.3: TAGAGTTGGCAGAAGAATTTGGGCTGCATTTGGTTGAGGAATCAGAGTTATCACTATATGATTATCCAGGTTATTTAAACAGGAGAGGAAGTGGGATTGCTCTATGGAATTTCCTGTGGGAAAGTGTAGTACCTTCAAAGTTTGCCAAGCAAGCTGCAAATAATGAACCTTGAGGCCTTATCTGACTGTTGATTTTTGTT[T / G]TTGTTTTCGTTGACATTGGAATTTACCAGAACAAAATTGTCCCAATTAAGTTTACTTCAATATTTTGAAGCAATGAGAAGTGAAGATGTTGGATTGTATTTATATCAATTTTATCATGGTTTCACGTAGACACTGTCCACTTTTGGAGAGATTTTTCAGTATGATCGATACACGAGATGATACTTCACGTGTCACTATAC.

[0023] SNP4 is a mutation from C to A at position 454583 on chromosome 13 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.4: AAGCATCACAACTCATCCCCACGTATTCTAGCTAATGGTCTGTTTTATGAGAAAAGTTATTTAGCTAATTGTCAAGATCATGTTCCAACAAACTCGAGCAGCATCTGTGATCAACTTACATTCAAGTAAGGTCAAATTAAACAAAGTACAGGTACTGGATACCAGGTGTCAAGAATGTTCTTTCTCAGCATGAATATCAT[C / A]GCTTCTATTTATGATGCACAGAGAAATATCTGTGTAGGACTTGGTTTCTGTTGTTAAATTAGTTTTCTACCAACTAGATATTATAAAAGACTGCATTTTCAGTAACTTTAGAAGAAGCATTGCAGTGGTGTTAAATGAAAGGTTGGAACAATGAAAAGTTTCTCCCTTTCTTTTGTGAGAATAATACTCACAGCAATGAC.

[0024] SNP5 is a mutation from G to A at position 8210788 on chromosome 15 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.5: TAGTTCTTGATAGAATAATAAAAACGAAAAAAGAGGTTAGGCTTAGGATCAAAGATTTGACTCCTCCGTTGAGACAAAAAAAAAAAAAATCATTAAAATGATCAGAAACTAAATCTTGATTGTCAAATTGATCAGATACATTATAGACCCACCATATCACCCATTTGTTAGTTTAGCACGGACTCAGATCCTCTCC[C / A]GATCATTTCCGTGGGATCCTCCTGACCGATTAATCTCATCCGTTGATACTAATCCTTTGCGCTCTTCCCCTCAACTCCTCTTTCTCCCTCAATTCGAAACACCCGACGCGCCCGCCACCAAGTTAAGAACTGGATTTGAACCAACATCCCAAACTTCGCCGTAAAACTTCCATTTTTCTTTTGATCCTGACTATGGTCTTCC.

[0025] SNP6 is a G-to-T mutation located at position 38704813 on chromosome 16 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.6: TAACCTGAAGCATTTATATATTCCCAGGTTATTTCGGCATTGAGAGAAGCTGGCCTTGAATCATCAAATTTAATACTTGGTGTCGACTTCACAAAGAGCAATGAATGGACAGGTAATTTTCC TATTAGATCAGCTTCTTCACATGAGTTCTGGATGGAGATAAAAGTTACCTTTCGATGATTTGCAGGAAGATATTCATT(C / G)CATAGGAAAAGCC[G / T]TCATGCAATTGGTAGCACATC TAATCCTTACGAGCATGCGATCTCAATAATTGGCCGTA(T / C)ATTATCTCCATTTGATGAAGATAATCTAATACCTTGTTTTGGATTCGGCGAAGGTTAGTTTTGAACCACAACTCTACTT TGGTTGAAAGTACCTTGTGAAAATTGTCTGATATTACCTTCTTGTGTAGCATCAACGCATGATCAATGCGTGTTCAGTTTCTATCCTGAGGGCCGATATTGTCATGGTTTCGAGGAAGCTC.

[0026] InDel1 is a C deletion mutation at position 10441619 on chromosome 2 of the apple reference genome GDDH13.1. The non-variant base is C, and the allele is denoted as Ins. The variant base is the deletion of C, and the allele is denoted as Del. Its nucleotide sequence is shown in SEQ ID NO.7. GGCGACATAGAGGGAGAGAGGATGGGGCTGATGAAATTGGGGTTCCACCATAAAACCAATTGGCAATATGAGGAATAGCCCAAGATCACATAAGCACATAGCAAACTTTGTCCCTCACCAATGTGGGACAACTCTCAACGCGCCCACGCACGTGTGGCAGATTTTCAATGTGGCCGTTGGGCTTCACACGTGGATAACCTTGCTCTAATACCATGATGAAATT[G / -]GGTTCCATCATAAAACCAATTGACAATATGGAGAATAGCCCAAGATCATATAAGCACATAGTAAACCTTATCTCTCACCAATGTGGGACAACTCTCAACAGGGGCATGTATAAGCTTGCAGGAGGGGTGGGTGGGGAGCGTTTTGCTGCTGCAATTATGGTGGATGCTGAGGCGATTAGGCAAGGGATGGAGATGATTATTGCTAGTGATGTTATGGAGCCTGGGATTAG。

[0027] InDel2 is a 215 bp deletion mutation on chromosome 3 of the apple reference genome GDDH13.1, located at positions 9126219J~9126434. The non-variant base sequence is TGACCAGATTCTCTCATGAGCAATTCCCTAGGGATTCTAGGGATCTCGCAATCTTGTCCGTTCATTTTATATCGTGTGGTCAGTTTTCGTTAAATACTATTCATATTTAAATTTTAAATAATTTCTGACCGCACGATATACGATGAACGGACATGATTGCGGGATTCCTAGGATCCCTAGGGAATTGCTCAGGAGAGGATCCTTGTCCATTCCCTC. The allele is denoted as Ins. The variant base is TGACCAGATTCTCTCATGAGCAATTCCCTAGGGATTCTAGGGATCTCGCAATCTTGTCCGTTCATTTTATATCGTGTGGTCAGTTTTCGTTAAATACTATTCATATTTAAATTTTAAATAATTTCTGACCGCACGATATACGATGAACGGACATGATTGCGGGATTCCTAGGATCCCTAGGGAATTGCTCAGGAGAGGATCCTTGTCCATTCCCTC. The allele is denoted as Del, and its nucleotide sequence is shown in SEQ ID NO.8.

[0028] GTTTACCCGGCAAATGGCACCATAACTAAATGTTGATGACAGAGGCTACAACCGTAATTTCACCCAATGACTTTTAAGGCTTTTGGTCCAGTACAGCTGTCAGCATGACATAACGTGACTTCATCCTTACAGGGCCGTAACCTTTTGCTGCCTGCAACTATTACCCTCGGAGTATGATTTTTTTTCTTCCTATTCTCATCTCATTCCCTC[TGACCAGATTCTCTCATGAGCAATTCCCTAGGGATTCTAGGGATCTCGCAATCTTGTCCGTTCATTTTATATCGTGTGGTCAGTTTTCGTTAAATACTATTCATATTTAAATTTTAAATAATTTCTGACCGCACGATATACGATGAACGGACATGATTGCGGGATTCCTAGGATCCCTAGGGAATTGCTCAGGAGAGGATCCTTGTCCATTCCCTC / -]TCATTCTTT(T / C)ACATATTATTTTTTGTCTTA(A / -)TTATCTTTATAAAAAAATCAATATAAGATATTGACGTG(A / G)CT(C / A)AA(T / C)CATAACCATTCAAATAAAAGAAAAAGAAA(A / -)G(G / A)G(A / G)AAAAAATTATGAGGAGAGAGAATCCTCCTCCAAT(G / A)CC(G / A)TCTGCCTTTCGTACCAG(A / T)ACTTGGCCTTTGCTCATCAGACTTCGGTACAAAGACCATAATACCCTT(TG / CA)AGCATTTCCTCT(G / C)TCAATTGTACGGAAAATC(T / G)ACCTACAA(C / T)GAGGGTGTTATTCTAGCAGTATTCTAAATTAATCAT。

[0029] Among them, the effect value of SNP1 on the retention of fruit crispness was 1.63 months; the effect value of SNP2 on the retention of fruit crispness was 1.82 months; the effect value of SNP3 on the retention of fruit crispness was 1.79 months; the effect value of SNP4 on the retention of fruit crispness was 1.90 months; the effect value of SNP5 on the retention of fruit crispness was 1.89 months; the effect value of SNP6 on the retention of fruit crispness was 1.69 months; the effect value of InDel1 on the retention of fruit crispness was 1.58 months; and the effect value of InDel2 on the retention of fruit crispness was 1.65 months.

[0030] The present invention also provides a kit for identifying the storage resistance traits of apple fruits, including the probe combination.

[0031] The present invention also provides the application of the probe combination or the kit in identifying the storage resistance traits of apple fruits.

[0032] Preferably, the steps for identifying the storage resistance traits of apple fruits are as follows: Genomic DNA was extracted from the apple species to be tested.

[0033] The extracted genomic DNA was precisely quantified.

[0034] Enzymatic digestion was used to fragment the quantified genomic DNA, and a DNA library was constructed.

[0035] Adapter sequences were added to both ends of the DNA library fragments for sample labeling.

[0036] The target sequence is captured using the probe combination described above.

[0037] 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.

[0038] Based on the genotyping data, the storage tolerance traits of the fruits of the tested Malus species were identified.

[0039] Preferably, based on the obtained genotyping data, the sum of the number of alleles that have a positive effect on maintaining the crispness of the fruit flesh in the genotypes of SNP1~SNP6 and InDel1~InDel2 is calculated and denoted as NPA.

[0040] The NPA was used as an indicator of the preservation of flesh crispness in the tested Malo species; the higher the NPA value, the stronger the preservation of flesh crispness in the tested Malo species.

[0041] Preferably, if the NPA is -6 to -2, the fruit of the tested *Malus* species is extremely intolerant to storage, with a cold storage period of less than 2 months; if the NPA is -1 to 3, the fruit of the tested *Malus* species is relatively intolerant to storage, with a cold storage period of 2.5 to 3.5 months; if the NPA is 4 to 6, the fruit of the tested *Malus* species is tolerant to storage, with a cold storage period of 4 to 4.5 months; if the NPA is 7 or 8, the fruit of the tested *Malus* species is extremely tolerant to storage, with a cold storage period of more than 5 months.

[0042] Preferably, the C allelic variation at SNP1 has a positive effect on the retention of fruit crispness; if the genotype at SNP1 is TT, then the number of alleles in the genotype of SNP1 that have a positive effect on the retention of fruit crispness is 0; if the genotype at SNP1 is TC, then the number of alleles in the genotype of SNP1 that have a positive effect on the retention of fruit crispness is 1; if the genotype at SNP1 is CC, then the number of alleles in the genotype of SNP1 that have a positive effect on the retention of fruit crispness is 2.

[0043] The A allele variation at SNP2 has a negative effect on the retention of fruit crispness; if the genotype at SNP2 is AA, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit crispness is -2; if the genotype at SNP2 is AG, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit crispness is -1; 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 crispness is 0.

[0044] The G allele at SNP3 has a negative effect on the retention of fruit crispness; 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 crispness is 0; if the genotype at SNP3 is TG, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit crispness is -1; if the genotype at SNP3 is GG, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit crispness is -2. The C allele at SNP4 has a negative effect on the retention of fruit crispness; if the genotype at SNP4 is CC, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit crispness is -2; if the genotype at SNP4 is CA, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit crispness is -1; if the genotype at SNP4 is AA, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit crispness is 0.

[0045] The A allele variation at SNP5 has a negative effect on the retention of fruit crispness; 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 crispness 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 crispness 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 crispness is -2.

[0046] The T allele at SNP6 has a positive effect on the retention of fruit crispness; 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 crispness 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 crispness 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 crispness is 2.

[0047] The ins allelic variation at InDel1 has a positive effect on the retention of fruit crispness. If the ins allelic variation occurs at InDel1, the number of alleles in the InDel1 genotype that have a positive effect on the retention of fruit crispness is 2. If the ins / del allelic variation occurs at InDel1, the number of alleles in the InDel1 genotype that have a positive effect on the retention of fruit crispness is 1. If the del allelic variation occurs at InDel1, the number of alleles in the InDel1 genotype that have a positive effect on the retention of fruit crispness is 0.

[0048] The idel allelic variation at InDel2 has a positive effect on the retention of fruit crispness; if the ins allelic variation occurs at InDel2, the number of alleles with a positive effect on the retention of fruit crispness in the InDel2 genotype is 0; if the ins / del allelic variation occurs at InDel2, the number of alleles with a positive effect on the retention of fruit crispness in the InDel2 genotype is 1; if the del allelic variation occurs at InDel2, the number of alleles with a positive effect on the retention of fruit crispness in the InDel2 genotype 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 the auxiliary selection of storage-resistant traits in apple fruits. This probe combination is suitable for the auxiliary selection of storage-resistant traits in apple hybrid offspring. The auxiliary selection of storage-resistant traits in apple hybrid offspring can be carried out during the 6-week seedling stage of the hybrids. GenoBaits marker detection requires 4 weeks, meaning the auxiliary selection of storage-resistant traits in hybrid offspring is completed in the 11th week after sowing. Traditional phenotypic selection takes 8-10 years. Therefore, the cycle of auxiliary selection of hybrid offspring using the molecular marker and probe combination for apple fruit storage-resistant traits is shortened by 8-10 years compared to traditional phenotypic selection. Furthermore, since the auxiliary selection of hybrid offspring using the molecular marker and probe combination for apple fruit storage-resistant traits is completed during 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 storage period of apple fruits can be predicted comprehensively and accurately.

[0055] Existing detection products suffer from low accuracy primarily because they rely on single or a few markers to predict storage-resistant traits controlled by multiple genes, making it difficult to comprehensively capture genetic information and susceptible to interference from individual variations. This invention addresses this issue by integrating a systematic probe combination of six SNPs and two InDel markers to achieve simultaneous detection of key loci. Utilizing the synergistic effect of multiple markers significantly improves genetic resolution and predictive robustness, thereby effectively solving the problem of insufficient accuracy.

[0056] 2. The molecular marker and probe combination developed in this invention is applicable to the evaluation and screening of storage-resistant traits of apple fruit germplasm resources. The scope of application includes 25 species of apple plants. The selected superior storage-resistant 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 the auxiliary selection of apple fruit storage tolerance traits has been applied to the molecular breeding practice of apple fruit storage tolerance 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 for the auxiliary selection of apple fruit storage resistance traits in this invention and the retention of fruit crispness.

[0059] Figure 2 The results of the training population validation of molecular marker combinations for the auxiliary selection of apple fruit storage resistance 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] Probes for the molecular markers (SNP1~SNP6 and InDel1~InDel2) selected with the help of the above-mentioned apple fruit storage tolerance traits were used to perform molecular hybridization and capture of target sites 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 ensemble for auxiliary selection of apple fruit storage tolerance traits includes probes for detecting SNP combinations (SNP1~SNP8 and InDel1~InDel2). It comprises 6 SNP markers and 2 InDel markers; the 6 SNP markers include SNP1~SNP6, and the 2 InDel markers include InDel1~InDel2.

[0066] The nucleotide sequences of the probes used to detect the molecular markers (SNP1~SNP6 and InDel1~InDel2) for the auxiliary selection of the storage resistance trait of the apple fruit are shown in SEQ ID NO.9~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: TTCACCCTCACACTGCACACACTATAACGTGCGCCCAGTACAGTTGCTTGATCAAATACCAATTCAGCTTTGAACTTTGCTTTAATGCTCTTTCAATTGAATTATGTCAG.

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

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

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

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

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

[0073] The nucleotide sequence of the GenoBaits DNA probe used to detect InDel1 is shown in SEQ ID NO.15: TTATGATGGAACCCAATTTCATCATGGTATTAGAGCAAGGTTATCCACGTGTGAAGCCCAACGGCCACATTGAAATCTGCCACACGTGCGTGGGCGCGTTGAGAGTTGT.

[0074] The nucleotide sequence of the GenoBaits DNA probe used to detect InDel2 is shown in SEQ ID NO.16: CCATAACTAAATGTTGATGACAGAGGCTACAACCGTAATTTCACCCAATGACTTTTAAGGCTTTTGGTCCAGTACAGCTGTCAGCATGACATAACGTGACTTCATCCTTA.

[0075] The above 8 SNPs are labeled as SNP1~SNP8 and InDel1~InDel2; SNP1~SNP6 are found in apples GDDH13.1The 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.6, respectively.

[0076] Among them, SNP1 is a T-to-C mutation at position 22771272 on chromosome 4 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.1: GTTTTTTTGACATGTTTGTCGATTAACTCTCTCTGACTCTCTCTCTCACGGCTAAGATGATGGAAGAGCTTCTCACACTCGTGTTGTTTTGTGCTAGGCTCAAGCTTCTAGCTTTTCACCCTCACACTGCACACACTATAACGTGCGCCCAGTACAGTTGCTTGATCAAATACCAATTCAGCTTTGAACTTTGCTT[T / C]AATGCTCTTTCAATTGAATTATGTCAGATGCTGGAAATCTACACTTTGCAGACTTGATTTTTGCAGAGAGCAAATCTTTGCTGCAGTTTTTTCTCTACTCTTTGAAGGGTGTGGCTCTTATTTTCTGAACTCTCGTTCACTCACAAATGAAGGGTTCAACAAGCTCTTTTCCAAAGCACTTTGCACTCTTGGTGCTCATG. The position of SNP1 is marked with square brackets "[ ]", with the non-variant base "T" first and the variant base "C" second, separated by " / " (the same applies below).

[0077] SNP2 is a mutation from A to G located at position 201180 on chromosome 7 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.2: TATGCCTTATATTTACGTAAGAGTAAAGAAAGAAAGAAAAGGAAGAAAAGAAACGAAAATTGGCTCCAGCCTTTCATCCTGGCTACAGAGTGCAGGCAAGAGTATATTTGACGATCAGTCATTACTGGGCATATTCAGCTGGTCAGGTCAAGAAAGCTGAAGCTGATTCCTCTGAGTTTGAAGTTCTTGATCCCATTTCA[A / G]GTATCTTCGTTTCAGCTTTCACTCCTCTGCACGATTTCATGTGTGAGTGCAATCTTTCTGGCTTAAACAGCTTCCTGCACTGCCTGCATTAACATCAAGTCTTTCGAATTAATCGCTTGTTTAGATTTTCCACATAAATGTTCTCTTGGTCACCAAGTTTTATGAATGCCAACGTTGACCACATTCACTGTTTTGGGAGA。

[0078] SNP3 is a mutation from T to G at position 30980600 on chromosome 10 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.3: TAGAGTTGGCAGAAGAATTTGGGCTGCATTTGGTTGAGGAATCAGAGTTATCACTATATGATTATCCAGGTTATTTAAACAGGAGAGGAAGTGGGATTGCTCTATGGAATTTCCTGTGGGAAAGTGTAGTACCTTCAAAGTTTGCCAAGCAAGCTGCAAATAATGAACCTTGAGGCCTTATCTGACTGTTGATTTTTGTT[T / G]TTGTTTTCGTTGACATTGGAATTTACCAGAACAAAATTGTCCCAATTAAGTTTACTTCAATATTTTGAAGCAATGAGAAGTGAAGATGTTGGATTGTATTTATATCAATTTTATCATGGTTTCACGTAGACACTGTCCACTTTTGGAGAGATTTTTCAGTATGATCGATACACGAGATGATACTTCACGTGTCACTATAC。

[0079] SNP4 is a mutation from C to A at position 454583 on chromosome 13 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.4: AAGCATCACAACTCATCCCCACGTATTCTAGCTAATGGTCTGTTTTATGAGAAAAGTTATTTAGCTAATTGTCAAGATCATGTTCCAACAAACTCGAGCAGCATCTGTGATCAACTTACATTCAAGTAAGGTCAAATTAAACAAAGTACAGGTACTGGATACCAGGTGTCAAGAATGTTCTTTCTCAGCATGAATATCAT[C / A]GCTTCTATTTATGATGCACAGAGAAATATCTGTGTAGGACTTGGTTTCTGTTGTTAAATTAGTTTTCTACCAACTAGATATTATAAAAGACTGCATTTTCAGTAACTTTAGAAGAAGCATTGCAGTGGTGTTAAATGAAAGGTTGGAACAATGAAAAGTTTCTCCCTTTCTTTTGTGAGAATAATACTCACAGCAATGAC.

[0080] SNP5 is a mutation from G to A at position 8210788 on chromosome 15 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.5: TAGTTCTTGATAGAATAATAAAAACGAAAAAAGAGGTTAGGCTTAGGATCAAAGATTTGACTCCTCCGTTGAGACAAAAAAAAAAAAAATCATTAAAATGATCAGAAACTAAATCTTGATTGTCAAATTGATCAGATACATTATAGACCCACCATATCACCCATTTGTTAGTTTAGCACGGACTCAGATCCTCTCC[G / A]GATCATTTCCGTGGGATCCTCCTGACCGATTAATCTCATCCGTTGATACTAATCCTTTGCGCTCTTCCCCTCAACTCCTCTTTCTCCCTCAATTCGAAACACCCGACGCGCCCGCCACCAAGTTAAGAACTGGATTTGAACCAACATCCCAAACTTCGCCGTAAAACTTCCATTTTTCTTTTGATCCTGACTATGGTCTTCC.

[0081] SNP6 is a G-to-T mutation located at position 38704813 on chromosome 16 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.6: TAACCTGAAGCATTTATATATTCCCAGGTTATTTCGGCATTGAGAGAAGCTGGCCTTGAATCATCAAATTTAATACTTGGTGTCGACTTCACAAAGAGCAATGAATGGACAGGTAATTTTCC TATTAGATCAGCTTCTTCACATGAGTTCTGGATGGAGATAAAAGTTACCTTTCGATGATTTGCAGGAAGATATTCATT(C / G)CATAGGAAAAGCC[G / T]TCATGCAATTGGTAGCACATC TAATCCTTACGAGCATGCGATCTCAATAATTGGCCGTA(T / C)ATTATCTCCATTTGATGAAGATAATCTAATACCTTGTTTTGGATTCGGCGAAGGTTAGTTTTGAACCACAACTCTACTT TGGTTGAAAGTACCTTGTGAAAATTGTCTGATATTACCTTCTTGTGTAGCATCAACGCATGATCAATGCGTGTTCAGTTTCTATCCTGAGGGCCGATATTGTCATGGTTTCGAGGAAGCTC.

[0082] InDel1 is a C deletion mutation at position 10441619 on chromosome 2 of the apple reference genome GDDH13.1. The non-variant base is C, and the allele is denoted as Ins. The variant base is the deletion of C, and the allele is denoted as Del. Its nucleotide sequence is shown in SEQ ID NO.7. SEQ ID NO.7: GGCGACATAGAGGGAGAGAGGATGGGGCTGATGAAATTGGGGTTCCACCATAAAACCAATTGGCAATATGAGGAATAGCCCAAGATCACATAAGCACATAGCAAACTTTGTCCCTCACCAATGTGGGACAACTCTCAACGCGCCCACGCACGTGTGGCAGATTTTCAATGTGGCCGTTGGGCTTCACACGTGGATAACCTTGCTCTAATACCATGATGAAATT[G / -]GGTTCCATCATAAAACCAATTGACAATATGGAGAATAGCCCAAGATCATATAAGCACATAGTAAACCTTATCTCTCACCAATGTGGGACAACTCTCAACAGGGGCATGTATAAGCTTGCAGGAGGGGTGGGTGGGGAGCGTTTTGCTGCTGCAATTATGGTGGATGCTGAGGCGATTAGGCAAGGGATGGAGATGATTATTGCTAGTGATGTTATGGAGCCTGGGATTAG。

[0083] InDel2 is a 215 bp deletion mutation on chromosome 3 of the apple reference genome GDDH13.1, located at positions 9126219J~9126434. The non-variant base sequence is TGACCAGATTCTCTCATGAGCAATTCCCTAGGGATTCTAGGGATCTCGCAATCTTGTCCGTTCATTTTATATCGTGTGGTCAGTTTTCGTTAAATACTATTCATATTTAAATTTTAAATAATTTCTGACCGCACGATATACGATGAACGGACATGATTGCGGGATTCCTAGGATCCCTAGGGAATTGCTCAGGAGAGGATCCTTGTCCATTCCCTC. The allele is denoted as Ins. The variant base is TGACCAGATTCTCTCATGAGCAATTCCCTAGGGATTCTAGGGATCTCGCAATCTTGTCCGTTCATTTTATATCGTGTGGTCAGTTTTCGTTAAATACTATTCATATTTAAATTTTAAATAATTTCTGACCGCACGATATACGATGAACGGACATGATTGCGGGATTCCTAGGATCCCTAGGGAATTGCTCAGGAGAGGATCCTTGTCCATTCCCTC. The allele is denoted as Del, and its nucleotide sequence is shown in SEQ ID NO.8.

[0084] GTTTACCCGGCAAATGGCACCATAACTAAATGTTGATGACAGAGGCTACAACCGTAATTTCACCCAATGACTTTTAAGGCTTTTGGTCCAGTACAGCTGTCAGCATGACATAACGTGACTTCATCCTTACAGGGCCGTAACCTTTTGCTGCCTGCAACTATTACCCTCGGAGTATGATTTTTTTTCTTCCTATTCTCATCTCATTCCCTC[TGACCAGATTCTCTCATGAGCAATTCCCTAGGGATTCTAGGGATCTCGCAATCTTGTCCGTTCATTTTATATCGTGTGGTCAGTTTTCGTTAAATACTATTCATATTTAAATTTTAAATAATTTCTGACCGCACGATATACGATGAACGGACATGATTGCGGGATTCCTAGGATCCCTAGGGAATTGCTCAGGAGAGGATCCTTGTCCATTCCCTC / -]TCATTCTTT(T / C)ACATATTATTTTTTGTCTTA(A / -)TTATCTTTATAAAAAAATCAATATAAGATATTGACGTG(A / G)CT(C / A)AA(T / C)CATAACCATTCAAATAAAAGAAAAAGAAA(A / -)G(G / A)G(A / G)AAAAAATTATGAGGAGAGAGAATCCTCCTCCAAT(G / A)CC(G / A)TCTGCCTTTCGTACCAG(A / T)ACTTGGCCTTTGCTCATCAGACTTCGGTACAAAGACCATAATACCCTT(TG / CA)AGCATTTCCTCT(G / C)TCAATTGTACGGAAAATC(T / G)ACCTACAA(C / T)GAGGGTGTTATTCTAGCAGTATTCTAAATTAATCAT。

[0085] Among them, the effect value of SNP1 on the retention of fruit crispness was 1.63 months; the effect value of SNP2 on the retention of fruit crispness was 1.82 months; the effect value of SNP3 on the retention of fruit crispness was 1.79 months; the effect value of SNP4 on the retention of fruit crispness was 1.90 months; the effect value of SNP5 on the retention of fruit crispness was 1.89 months; the effect value of SNP6 on the retention of fruit crispness was 1.69 months; the effect value of InDel1 on the retention of fruit crispness was 1.58 months; and the effect value of InDel2 on the retention of fruit crispness was 1.65 months.

[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 on the preservation of apple flesh crispness by the marker combination SNP1~SNP6 and InDel1~InDel2 (probe combination of molecular markers for auxiliary selection of apple fruit storage resistance).

[0088] The C allelic variation of SNP1 T / C has a positive effect on the preservation of fruit crispness. The number of alleles with positive effects in the three test sample genotypes, TT, TC and CC, are 0, 1 and 2, respectively.

[0089] The A allele variation of SNP2 A / G has a negative effect on the preservation of fruit crispness. The number of alleles with positive effects in the three test sample genotypes, AA, AG and GG, are -2, -1 and 0, respectively.

[0090] The G allele variation of SNP3 T / G has a negative effect on the preservation of fruit crispness. The number of positive effect alleles in the test samples of the three genotypes TT, TG and GG are 0, -1 and -2, respectively.

[0091] The C allele variation of SNP4 C / A has a negative effect on the preservation of fruit crispness. The number of positive alleles in the test samples of the three genotypes CC, CA and AA are -2, -1 and 0, respectively.

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

[0093] The T allele variation of SNP6 G / T has a positive effect on the preservation of fruit crispness. The number of positive effect alleles in the test samples of the three genotypes, GG, GT and TT, are 0, 1 and 2, respectively.

[0094] The ins allelic variation of InDel1 has a positive effect on the preservation of fruit crispness. The number of positive effect alleles in the test samples of the three genotypes ins, ins / del and del are 2, 1 and 0, respectively.

[0095] The del allelic variation of InDel2 has a positive effect on the preservation of fruit crispness. The number of positive alleles in the test samples of the three genotypes ins, ins / del and del are 0, 1 and 2, respectively.

[0096] (4) Auxiliary selection of apple fruit storage traits The steps for identifying the storage properties of apple fruits 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 sequences were subjected to next-generation sequencing, and the sequencing data were compared and analyzed with the apple reference genome GDDH13.1. Genotyping data were obtained.

[0102] Based on the genotyping data, the storage tolerance of the fruits of the tested Malus species was identified.

[0103] The identification was based on the genotyping data of SNP1~SNP6 and InDel1~InDel2. Based on the obtained genotyping data, the sum of the number of alleles in the SNP1~SNP6 and InDel1~InDel2 genotypes that have a positive effect on maintaining fruit crispness was calculated and denoted as NPA.

[0104] NPA was used as an indicator of the ability of the tested *Malus* species to retain fruit crispness; the higher the NPA value, the stronger the ability of the tested *Malus* species to retain fruit crispness. The specific identification criteria are as follows:

[0105] ①If the NPA is -6 to -2, then the fruit of the tested apple species is extremely intolerant to storage, and the cold storage period is less than 2 months.

[0106] ②If the NPA is -1 to 3, the fruit of the tested apple species is not very resistant to storage, and the cold storage period is between 2.5 and 3.5 months.

[0107] ③ If the NPA is 4~6, then the fruit of the tested apple species is resistant to storage, and the cold storage period is between 4 and 4.5 months.

[0108] ④ If the NPA is 7 or 8, the fruit of the tested apple species is extremely resistant to storage, and the cold storage period can reach more than 5 months.

[0109] Specifically, the C allele variation at SNP1 has a positive effect on the retention of fruit crispness; 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 crispness is 0; if the genotype at SNP1 is TC, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit crispness is 1; if the genotype at SNP1 is CC, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit crispness is 2.

[0110] The A allele variation at SNP2 has a negative effect on the retention of fruit crispness; if the genotype at SNP2 is AA, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit crispness is -2; if the genotype at SNP2 is AG, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit crispness is -1; 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 crispness is 0.

[0111] The G allele at SNP3 has a negative effect on the retention of fruit crispness; 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 crispness is 0; if the genotype at SNP3 is TG, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit crispness is -1; if the genotype at SNP3 is GG, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit crispness is -2. The C allele at SNP4 has a negative effect on the retention of fruit crispness; if the genotype at SNP4 is CC, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit crispness is -2; if the genotype at SNP4 is CA, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit crispness is -1; if the genotype at SNP4 is AA, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit crispness is 0.

[0112] The A allele variation at SNP5 has a negative effect on the retention of fruit crispness; 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 crispness 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 crispness 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 crispness is -2.

[0113] The T allele at SNP6 has a positive effect on the retention of fruit crispness; 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 crispness 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 crispness 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 crispness is 2.

[0114] The ins allelic variation at InDel1 has a positive effect on the retention of fruit crispness. If the ins allelic variation occurs at InDel1, the number of alleles in the InDel1 genotype that have a positive effect on the retention of fruit crispness is 2. If the ins / del allelic variation occurs at InDel1, the number of alleles in the InDel1 genotype that have a positive effect on the retention of fruit crispness is 1. If the del allelic variation occurs at InDel1, the number of alleles in the InDel1 genotype that have a positive effect on the retention of fruit crispness is 0.

[0115] The idel allelic variation at InDel2 has a positive effect on the retention of fruit crispness; if the ins allelic variation occurs at InDel2, the number of alleles with a positive effect on the retention of fruit crispness in the InDel2 genotype is 0; if the ins / del allelic variation occurs at InDel2, the number of alleles with a positive effect on the retention of fruit crispness in the InDel2 genotype is 1; if the del allelic variation occurs at InDel2, the number of alleles with a positive effect on the retention of fruit crispness in the InDel2 genotype is 2.

[0116] 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.

[0117] The algebraic sum of the number of alleles with a positive effect on maintaining fruit crispness in each marker genotype of the SNP1~SNP6 and InDel1~InDel2 marker combinations in the test sample is used as an indicator of fruit crispness retention. The larger the algebraic sum, the stronger the fruit crispness retention of the test sample. Figure 1 .

[0118] Depend on Figure 1 It can be seen that by using the above 6 SNPs and 2 InDel markers (molecular markers for auxiliary selection of apple fruit storage tolerance traits), the storage period of apple fruit can be predicted comprehensively and accurately.

[0119] Example 2 In 2019, the molecular marker combination described in this case was used for assisted selection of the storage tolerance trait in apple fruit. This was employed to select 616 hybrid progeny plants of 'Zhongnong 31' × 'Hongfushi' and 'Yulinglong 1' × 'Hongfushi' for the storage tolerance trait. The selection criteria were an NPA value of 7 or 8, resulting in the selection of 13 hybrid progeny plants that met the criteria. These 13 initially selected superior lines were grafted and cultivated in a secondary selection nursery, bearing fruit for two consecutive years from 2023 to 2024. Fruit from these 13 initially selected superior lines was subjected to a cold storage test to evaluate their storage tolerance. The results showed that the retention of flesh crispness in the 13 initially selected superior lines ranged from 5.5 months to 8.0 months. This indicates that the molecular marker combination for assisted selection of the storage tolerance trait in apple fruit has excellent application effects in breeding practice.

[0120] Among them, the 616 hybrid offspring of 'Zhongnong 31' × 'Red Fuji' and 'Yulinglong 1' × 'Red Fuji' originated from the apple breeding base in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0121] The molecular marker combinations were used to assist in the selection of fruit storage tolerance traits in 616 hybrid progeny plants, as shown in Table 1.

[0122] Table 1. Auxiliary selection of apple fruit storage resistance traits As shown in Table 1, the above 6 SNPs and 2 InDel markers can be used to comprehensively and accurately assist in the selection of fruit storage resistance traits in hybrid offspring.

[0123] Example 3 Using 335 germplasm resources from 25 species of *Malus* with phenotypic data on fruit crispness retention 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 molecular markers for apple fruit storage tolerance were used to genotype molecular marker combinations (SNP1-SNP6 and InDel1-InDel2). Then, the generational sum (NPA) of the number of alleles with positive effects on fruit crispness retention in each individual's eight molecular marker genotypes was calculated. Finally, linear regression analysis was performed using the NPA values ​​and the average measured individual fruit crispness retention. The results are shown below. Figure 2 .

[0124] Among them, 335 germplasm resources of 25 species originated from the apple breeding base in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0125] 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.

[0126] The coefficient of determination R in the linear regression equation between NPA value and the retention of fruit crispness is... 2 =0.261. This indicates that the combination of molecular markers and probes for assisted selection of storage-resistant traits in apple fruits is feasible.

[0127] 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.

[0128] 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.

[0129] 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 auxiliary selection of apple fruit storage tolerance traits, characterized in that, Includes eight probes for assisting in the selection of apple fruit storage tolerance traits; The nucleotide sequences of the probes used for the selection of eight apple fruit storage-resistant traits are shown in SEQ ID NO.9~SEQ ID NO.

16.

2. The probe combination of molecular markers for the auxiliary selection of apple fruit storage resistance traits according to claim 1, characterized in that, The probes used for auxiliary selection of eight apple fruit storage tolerance traits were probes for detecting six SNP markers and two InDel markers; the six SNP markers were designated SNP1~SNP6, and the two InDel markers were designated InDel1~InDel2; the nucleotide sequences of SNP1~SNP6 are shown in SEQ ID NO.1~SEQ ID NO. As shown in NO.6; SNP1 is a mutation from T to C at position 22771272 on chromosome 4 of the apple reference genome GDDH13.1; SNP2 is a mutation from A to G at position 201180 on chromosome 7 of the apple reference genome GDDH13.1; SNP3 is a mutation from T to G at position 30980600 on chromosome 10 of the apple reference genome GDDH13.1; SNP4 is a mutation from C to A at position 454583 on chromosome 13 of the apple reference genome GDDH13.1; SNP5 is a mutation from G to A at position 8210788 on chromosome 15 of the apple reference genome GDDH13.1; SNP6 is a mutation from G to T at position 38704813 on chromosome 16 of the apple reference genome GDDH13.

1. The nucleotide sequences of InDel1 to InDel2 are shown in SEQ ID NO.7 to SEQ ID NO.8, respectively. InDel1 has a C deletion mutation at position 10441619 on chromosome 2 of the apple reference genome GDDH13.

1. InDel2 has a 215bp deletion mutation at positions 9126219J to 9126434 on chromosome 3 of the apple reference genome GDDH13.

1.

3. A kit for identifying the storage resistance traits 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 storage resistance of apple fruit.

5. The application according to claim 4, characterized in that, The steps for identifying the storage properties of apple fruits 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; Next-generation sequencing was performed on the captured target sequences to obtain genotyping data; Based on the genotyping data, the storage tolerance traits of the fruits 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 that have a positive effect on the preservation of fruit crispness in the genotypes of SNP1~SNP6 and InDel1~InDel2 is calculated and denoted as NPA. The NPA was used as an indicator of the preservation of flesh crispness in the tested Malo species; the higher the NPA value, the stronger the preservation of flesh crispness in the tested Malo 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 extremely intolerant to storage, with a cold storage period of less than 2 months; if the NPA is -1 to 3, the fruit of the tested *Malus* species is relatively intolerant to storage, with a cold storage period of 2.5 to 3.5 months; if the NPA is 4 to 6, the fruit of the tested *Malus* species is tolerant to storage, with a cold storage period of 4 to 4.5 months; if the NPA is 7 or 8, the fruit of the tested *Malus* species is extremely tolerant to storage, with a cold storage period of more than 5 months.

8. The application according to claim 6, characterized in that, The C allele variation at SNP1 has a positive effect on the retention of fruit crispness; 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 crispness is 0; if the genotype at SNP1 is TC, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit crispness is 1; if the genotype at SNP1 is CC, then the number of alleles in the SNP1 genotype that have a positive effect on the retention of fruit crispness is 2. The A allele variation at SNP2 has a negative effect on the retention of fruit crispness; if the genotype at SNP2 is AA, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit crispness is -2; if the genotype at SNP2 is AG, then the number of alleles in the genotype of SNP2 that have a positive effect on the retention of fruit crispness is -1; 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 crispness is 0. The G allele at SNP3 has a negative effect on the retention of fruit crispness; 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 crispness is 0; if the genotype at SNP3 is TG, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit crispness is -1; if the genotype at SNP3 is GG, then the number of alleles in the SNP3 genotype that have a positive effect on the retention of fruit crispness is -2. The C allele at SNP4 has a negative effect on the retention of fruit crispness; if the genotype at SNP4 is CC, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit crispness is -2; if the genotype at SNP4 is CA, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit crispness is -1; if the genotype at SNP4 is AA, then the number of alleles in the SNP4 genotype that have a positive effect on the retention of fruit crispness is 0. The A allele at SNP5 has a negative effect on the retention of fruit crispness; 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 crispness 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 crispness 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 crispness is -2. The T allele variation at SNP6 has a positive effect on the retention of fruit crispness; 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 crispness 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 crispness 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 crispness is 2. The ins allelic variation at InDel1 has a positive effect on the retention of fruit crispness; if the ins allelic variation occurs at InDel1, the number of alleles in the InDel1 genotype that have a positive effect on the retention of fruit crispness is 2; if the ins / del allelic variation occurs at InDel1, the number of alleles in the InDel1 genotype that have a positive effect on the retention of fruit crispness is 1; if the del allelic variation occurs at InDel1, the number of alleles in the InDel1 genotype that have a positive effect on the retention of fruit crispness is 0. The idel allelic variation at InDel2 has a positive effect on the retention of fruit crispness; if the ins allelic variation occurs at InDel2, the number of alleles with a positive effect on the retention of fruit crispness in the InDel2 genotype is 0; if the ins / del allelic variation occurs at InDel2, the number of alleles with a positive effect on the retention of fruit crispness in the InDel2 genotype is 1; if the del allelic variation occurs at InDel2, the number of alleles with a positive effect on the retention of fruit crispness in the InDel2 genotype is 2.

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