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

Abstract

The invention belongs to the technical field of plant molecular breeding, and particularly relates to a molecular marker probe combination for apple fruit acidity character auxiliary selection and application of the molecular marker probe combination. The probe combination comprises five probes for auxiliary selection of the acidity character of the apple fruit; the nucleotide sequences of the five probes for auxiliary selection of the acidity character of the apple fruit are sequentially shown as SEQ ID NO. 7 to SEQ ID NO. 11. The DNA probe combination corresponding to the SNP marker combination provided by the invention can be applied to auxiliary selection of apple fruit acidity characters in the seedling stage of apple filial generations.

Description

Technical Field

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

[0002] apple( Malus domestica Borkh. Apples are an important economic crop, with the world's largest production. Their post-harvest quality plays a crucial role in maintaining market competitiveness and economic value. The acidity of apple fruit is another decisive indicator of its flavor.

[0003] Currently, there is a lack of probe combinations that can systematically cover key SNPs and InDel sites in apple fruit acidity trait detection products, resulting in low throughput of final detection information and failing to meet the needs of precise auxiliary selection of acidity traits in apple molecular breeding. Summary of the Invention

[0004] To address the problem that existing detection products for apple fruit acidity traits lack probe combinations that can systematically cover key SNPs and InDel sites, resulting in low throughput and failing to meet the needs of precise assisted selection of acidity traits in apple molecular breeding, this invention aims to develop a novel strategy for efficient screening of acidity traits, providing a probe combination of molecular markers for assisted selection of apple fruit acidity traits and its application. To achieve the above objective, this invention adopts the following technical solution.

[0005] This invention investigated the phenotypic characteristics of fruit acidity in 2664 offspring plants of crosses between apple varieties 'Zise Mingzhu' × 'Hongfushi', 'Zise Mingzhu' × 'Jinguan', and 'Hongyu' × 'Jinguan'. Ninety SNP markers were developed using methods such as GWAS, BSA-seq, and RNA-seq. However, using 90 molecular markers for marker-assisted selection of apple fruit acidity traits is extremely difficult; therefore, this invention conducts the following experimental research.

[0006] This invention screens 90 SNP markers for markers with an effect value greater than 2 mg / g FW on malic acid content in apple pulp. A total of 5 SNP markers and 1 InDel marker were screened, including SNP1 to SNP5 and InDel1. Based on this, this invention provides a probe array of molecular markers for the auxiliary selection of apple fruit acidity traits, the probe array comprising probes for detecting the molecular marker array for the auxiliary selection of apple fruit acidity traits.

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

[0008] The nucleotide sequences of the probes used for selection of five apple fruit acidity traits are shown in SEQ ID NO.7~SEQ ID NO.11.

[0009] The probes used for the auxiliary selection of five apple fruit acidity traits were probes for detecting five SNP markers and one InDel marker; the five SNP markers included SNP1 to SNP5, and the one InDel marker was InDel1; the nucleotide sequence of the probe (GenoBaits DNA) used to detect SNP1 is shown in SEQ ID NO.7: TTCCAAAATCTTAAAATAATAGTACGAACATCACGTAACAGTACGACAACCGCACTCGTTAAACTTTGTTGGATAACTCTTATCGTCTTTATCGCAAAAAAAAAAAATAA.

[0010] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP2 is shown in SEQ ID NO.8: AAGAGAATTAAGAGAACAAAATCGCTTGAATTCTAATGAGGTAAAAATTTCTAGCTTAATGGTTGCATATGAAGAAGCAACTCTAAAACTAGCTGACAGTTCATTAAATC.

[0011] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP3 is shown in SEQ ID NO.9: AATATAAAATATTGACGTGACTTAACCGTGACCACACAGGAGGGCACGGGAGGGCAGACAATCTTTGTCCTCTTTGTATATATGAAACTGTCAACCTTTTGATAGGGTTG.

[0012] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP4 is shown in SEQ ID NO.10: CTACAAATTATAGGCATGATTACCTTTATAATGTGCCTGCCTCTCATCTCTTATTGCCATTGTCTCTTACACAAACATGTTACATTTATTGTATGAATTACTCATAACTA.

[0013] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP5 is shown in SEQ ID NO.11: AATATTGGGATTCTCAGAATCCAAGCACGACTGCTGATCCTTCCAATCAACAATGGATATCTACAGAAAGTCTTTTAAAACGACCAATATCATGGCCACGCCTCTCATTC.

[0014] The nucleotide sequence of the (GenoBaits DNA) probe used to detect InDel1 is shown in SEQ ID NO.7.

[0015] Existing technologies suffer from low detection throughput and insufficient predictive ability due to the lack of systematic multi-marker probe combinations. This invention integrates five key SNPs with one InDel marker to form a specific probe combination, enabling simultaneous detection of multiple targets. This significantly improves the information throughput and genetic resolution of a single detection and enhances the accuracy of acidity trait prediction by utilizing the synergistic effect of multiple markers, thereby meeting the needs of apple molecular breeding for efficient and precise assisted selection.

[0016] Preferably, the above six markers are SNP1 to SNP5 and InDel1, wherein SNP1 to SNP5 are 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.5, respectively.

[0017] Among them, SNP1 is a mutation from A to T at position 7674347 on chromosome 8 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.1: GTAGAATTTTAGATAACGGATATATGTGAGTGTCGCAATCAAAGCAATCACATTTTATCAATAACGCACTAATATAATATGATGAGACAAAATAATCTCAAAATCTTAAAATAATAGTACGAACATCACGTAACAGTACGACAACCGCACTCGTTAAACTTTGTTGGATAACTCTTATCGTCTTTATCGCAAAAAAAAA[A / T]AATAATAATAAATAAATTGAAATTTTGGAAAATGAAATTGGATTAAATTAACAGGAAGGTGTACGGTATGGTTTAGTTGGGAGGTGGGGTCGGAAATGGATGGGGAGCGTAAGCAAACGGATGACTTTGACCAAAAGCCAGGCTGGCGAGCATTTTGGGGCGGGGGAGAAAATGATTTTTTTAATTGAAATAGAAACGG. The position of SNP1 is marked with square brackets "[ ]", with the non-variant base "A" first and the variant base "T" second, separated by " / " (the same applies below).

[0018] SNP2 is a G-to-T mutation located at position 8728049 on chromosome 8 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.2: ATGTAATTTTCTCGGAAAAATAAAGCTCATCGCTTAACGGATGGTCTGTCCAAAATCCTTTTTACCTGCAATAGTAAGTAAAAGAGCAATGTGAGAGAGAAGACAAGAGAATTAAGAGAACAAAATCGCTTGAATTCTAATGAGGTAAAAATTTCTAGCTTAATGGTTGCATATGAAGAAGCAACTCTAAAACTAGCT[G / T]ACAGTTCATTAAATCACAGCTGTGTAGGCTTTCTATGTATATGGTCAACTAACCTATCTCAATCTCGCAGCAGAACAAACGTTGGGCTTGATATGTTGAAGAACTTGGGAACCCATAAGCAAAAGAGTAAATGTATGACTTGGTCAAGAGGCTACGTATGAAATGTGCATTAACAAACCGTAGAAAATATCACTAGCGGT.

[0019] SNP3 is a G-to-A mutation located at position 11654518 on chromosome 8 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.3: ATTGCACGTTACGAGAATAGCCCATCTCAATCAGTCCCGAGACCTGAGCTTCTTTGTATGGACAAGGATTGTCTGCCCTTTTTGTTCTTGTGTCCTTTCATGTCTTTCTATTTATGTGGTTACGATTAAATCATGTCAATATTTTATATTACTATTCATTTTTGTCTTATTATCTCAATAAAAAACAATATAAAATATT[G / A]ACGTGACTTAACCGTGACCACACAGGAGGGCACGGGAGGGCAGACAATCTTTGTCCTCTTTGTATATATGAAACTGTCAACCTTTTGATAGGGTTGTTACATAACTTTTTATATATATATATATATATATCGAATTAGTTTATATAGAAAAATGTATATTAAATATTAATCTTAAGGTCCTCAACCACTATAAATTGA.

[0020] SNP4 is a mutation from C to A at position 1855043 on chromosome 15 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.4: CTGGTGGGTTGGCCTTTAGCCAACATAGGTTGTCGAAAACACATAGAGGGACTTTCTCATTAACCTAATCTGTTGGCCCATCTGTGGTTTCGCTTATTCAGTCATACAGTGTCATCAGCCTACAAATTATAGGCATGATTACCTTTATAATGTGCCTGCCTCTCATCTCTTATTGCCATTGTCTCTTACACAAACATGTTA[C / A]ATTTATTGTATGAATTACTCATAACTACACTGACATTTCATCTTTAAAAGAATTGATTGTTATTTCTATGCTTTAGGAAAATGGATATCAATTGCTTTTTTTGAGTGCACGTGCCATTTCTCAAGCCCTGTCACCAGACAATTTTTATTCAACCTTAAGCAGGTTATGCTTAACCCATGCTAAATCCTTGATTTTCATT.

[0021] SNP5 is a mutation from G to A at position 3179027 on chromosome 16 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.5: ATTCAGAGAGCTGGGGACCTGCAGTACGTCCCAAGGAATACGAAGATCATGGGCATTTTGTCGATAAAGACAACGAAAATAAACAGGTGGTGATCGATTCTCTCAGTGAATATTGGGATTCTCAGAATCCAAGCACGACTGCTGATCCTTCCAATCAACAATGGATATCTACAGAAAGTC TTTTAAACGACCAATATCATG[G / A]CCACGCCTCTCATTCAACGCGCACGCTGTGCAGCAGGAACCTGAAGAATCAAAAGTTTACGAAAGCGCCAGCTCCTTATCTTTTGGCAACATTTGCATCGCTTCTGATTGAGTTTGTGGCTCGGCTTCAAAATCTGGTGGATGAATTCGAAGAG.

[0022] InDel1 contains a T insertion mutation at position 7674347 on chromosome 8 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.6: GTAGAATTTTAGATAACGGATATATGTGAGTGTCGCAATCAAAGCAATCACATTTTATCAATAACGCACTAATATAATATGATGAGACAAAATAATCTCAAAATCTTAAAATAATAGTACGAACATCACGTAACAGTACGACAACCGCACTCGTTAAACTTTGTTGGATAACTCTTATCGTCTTTATCGCAAAAAAAAAA[- / T]AATAATAATAAATAAATTGAAATTTTGGAAAATGAAATTGGATTAAATTAACAGGAAGGTGTACGGTATGGTTTAGTTGGGAGGTGGGGTCGGAAATGGATGGGGAGCGTAAGCAAACGGATGACTTTGACCAAAAGCCAGGCTGGCGAGCATTTTGGGGCGGGGGAGAAAATGATTTTTTTAATTGAAATAGAAAACG. The position of InDel1 is marked with square brackets "[ ]", where the non-mutant is indicated by "-" first, and the mutant base "T" is indicated by " / " second.

[0023] Among them, SNP1 had an effect of 5.07 mg / g FW on malic acid content in the fruit; SNP2 had an effect of 2.36 mg / g FW on the malic acid content in the pulp; SNP3 had an effect of 4.85 mg / g FW on the malic acid content; SNP4 had an effect of 6.41 mg / g FW on the malic acid content; and SNP5 had an effect of 4.76 mg / g FW on the malic acid content. InDel1 had an effect of 5.07 mg / g FW on the malic acid content in the fruit.

[0024] The present invention also provides a kit for identifying the acidity trait of apple fruit, including the probe combination.

[0025] The present invention also provides the application of the probe combination or the kit in identifying the acidity trait of apple fruit.

[0026] Preferably, the steps for identifying the acidity trait of apple fruit are as follows: Genomic DNA was extracted from the apple species to be tested.

[0027] The extracted genomic DNA was precisely quantified.

[0028] Enzymatic digestion was used to fragment the quantified genomic DNA, and a DNA library was constructed.

[0029] Adapter sequences were added to both ends of the DNA library fragments for sample labeling.

[0030] The target sequence is captured using the probe combination described above.

[0031] 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.

[0032] Based on the genotyping data, the acidity trait of the fruit of the tested Malus species was identified.

[0033] Preferably, based on the obtained genotyping data, the sum of the number of alleles with a positive effect on fruit acidity in the genotypes of SNP1 to SNP5 is calculated and denoted as NPA.

[0034] The NPA (Neural Protein Acidity) was used as an indicator of the acidity of the fruit of the tested Malus species. The higher the NPA value, the higher the acidity of the fruit of the tested Malus species.

[0035] Preferably, if the NPA is less than 3.5, the fruit acidity of the tested *Malus* species is very low, and the malic acid content is less than 4.0 mg / g fresh weight; if the NPA is 4.0 to 6.5, the fruit acidity of the tested *Malus* species is low, and the malic acid content is between 4.0 mg / g and 7.5 mg / g fresh weight; if the NPA is 8.0 to 9.0, the fruit acidity of the tested *Malus* species is high, and the malic acid content is between 8.0 mg / g and 10.0 mg / g fresh weight; if the NPA is greater than 10.0, the fruit acidity of the tested *Malus* species is very high, and the malic acid content is 10.0 mg / g fresh weight.

[0036] Preferably, the T allele variation at SNP1 has a positive effect on fruit acidity; if the genotype at SNP1 is AA, then the number of alleles in the genotype of SNP1 that have a positive effect on fruit acidity is 0; if the genotype at SNP1 is AT, then the number of alleles in the genotype of SNP1 that have a positive effect on fruit acidity is 1; if the genotype at SNP1 is TT, then the number of alleles in the genotype of SNP1 that have a positive effect on fruit acidity is 2.

[0037] The insT allele variation at InDel1 has a positive effect on fruit acidity; if the genotype at InDel1 is AA (without insT), then the number of alleles in the InDel1 genotype that have a positive effect on fruit acidity is 0; if the genotype at InDel1 is A / insT, then the number of alleles in the InDel1 genotype that have a positive effect on fruit acidity is 1; if the genotype at InDel1 is insT / insT, then the number of alleles in the InDel1 genotype that have a positive effect on fruit acidity is 2.

[0038] The T allele at SNP2 has a positive effect on fruit acidity; when the genotype at SNP5 is not AA and the genotype at SNP2 is GG, the number of alleles in the SNP2 genotype that have a positive effect on fruit acidity is 0; when the genotype at SNP5 is not AA and the genotype at SNP2 is GT, the number of alleles in the SNP2 genotype that have a positive effect on fruit acidity is 1; when the genotype at SNP5 is not AA and the genotype at SNP2 is TT, the number of alleles in the SNP2 genotype that have a positive effect on fruit acidity is... The number of alleles with a positive effect on fruit acidity is 2; when the genotype at SNP5 is AA and the genotype at SNP2 is GG, the number of alleles with a positive effect on fruit acidity in the genotype of SNP2 is 0; when the genotype at SNP5 is AA and the genotype at SNP2 is GT, the number of alleles with a positive effect on fruit acidity in the genotype of SNP2 is 0.5; when the genotype at SNP5 is AA and the genotype at SNP2 is TT, the number of alleles with a positive effect on fruit acidity in the genotype of SNP2 is 1.

[0039] The A allele at SNP3 has a positive effect on fruit acidity. When the genotype at SNP5 is not AA and the genotype at SNP3 is GG, the number of alleles in the SNP3 genotype that have a positive effect on fruit acidity is 0. When the genotype at SNP5 is not AA and the genotype at SNP3 is GA, the number of alleles in the SNP3 genotype that have a positive effect on fruit acidity is 1. When the genotype at SNP5 is not AA and the genotype at SNP3 is AA, the number of alleles in the SNP3 genotype that have a positive effect on fruit acidity is... The number of alleles with a positive effect on fruit acidity is 2; when the genotype at SNP5 is AA and the genotype at SNP3 is GG, the number of alleles with a positive effect on fruit acidity in the SNP3 genotype is 0; when the genotype at SNP5 is AA and the genotype at SNP3 is GA, the number of alleles with a positive effect on fruit acidity in the SNP3 genotype is 0.5; when the genotype at SNP5 is AA and the genotype at SNP3 is AA, the number of alleles with a positive effect on fruit acidity in the SNP3 genotype is 1.

[0040] The A allele at SNP4 has a positive effect on fruit acidity; when the genotype at SNP5 is not AA and the genotype at SNP4 is CC, the number of alleles in the SNP4 genotype that have a positive effect on fruit acidity is 0; when the genotype at SNP5 is not AA and the genotype at SNP4 is CA, the number of alleles in the SNP4 genotype that have a positive effect on fruit acidity is 1.5; when the genotype at SNP5 is not AA and the genotype at SNP4 is AA, the number of alleles in the SNP4 genotype that have a positive effect on fruit acidity is... The number of alleles with a positive effect is 3; when the genotype at SNP5 is AA and the genotype at SNP4 is CC, the number of alleles with a positive effect on fruit acidity in the SNP4 genotype is 0; when the genotype at SNP5 is AA and the genotype at SNP4 is CA, the number of alleles with a positive effect on fruit acidity in the SNP4 genotype is 0.5; when the genotype at SNP5 is AA and the genotype at SNP4 is AA, the number of alleles with a positive effect on fruit acidity in the SNP4 genotype is 1.

[0041] The G allele at SNP5 has a positive effect on fruit acidity; when the genotype at SNP5 is AA, the number of alleles with a positive effect on fruit acidity in the SNP5 genotype is 0; when the genotype at SNP5 is GA, the number of alleles with a positive effect on fruit acidity in the SNP5 genotype is 1; when the genotype at SNP5 is GG, the number of alleles with a positive effect on fruit acidity in the SNP5 genotype is 2.

[0042] 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 apple fruit acidity traits. This probe combination is suitable for the auxiliary selection of fruit acidity in apple hybrid progeny. The auxiliary selection of fruit acidity in apple hybrid progeny can be carried out during the 6-week seedling stage of the hybrids. GenoBaits marker detection requires 4 weeks, meaning the auxiliary selection of fruit acidity in hybrid progeny is completed in the 11th week after sowing. Traditional phenotypic selection takes 8-10 years to complete. Therefore, the cycle of auxiliary selection of hybrid progeny using the molecular marker and probe combination for apple fruit acidity is shortened by 8-10 years compared to traditional phenotypic selection methods. Furthermore, since the auxiliary selection of hybrid progeny using the molecular marker and probe combination for apple fruit acidity is completed during the 11-week seedling stage, selected superior plants directly enter the re-selection process, while hybrid progeny 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 acidity trait of apple fruit can be predicted comprehensively and accurately.

[0043] Existing technologies suffer from low detection throughput and insufficient predictive ability due to the lack of systematic multi-marker probe combinations. This invention integrates five key SNPs with one InDel marker to form a specific probe combination, enabling simultaneous detection of multiple targets. This significantly improves the information throughput and genetic resolution of a single detection and enhances the accuracy of acidity trait prediction by utilizing the synergistic effect of multiple markers, thereby meeting the needs of apple molecular breeding for efficient and precise assisted selection.

[0044] 2. The molecular marker and probe combination for apple fruit acidity-assisted selection developed in this invention is applicable to the evaluation and screening of fruit acidity in apple germplasm resources. The scope of application includes 25 species of apple plants. The selected superior fruit storage-resistant germplasm resources can be used for the design of breeding programs, the selection of hybrid parents and the matching of hybrid combinations, and to guide apple molecular breeding.

[0045] 3. The combination of molecular markers and probes for apple fruit acidity-assisted selection developed in this invention is applied to apple fruit acidity molecular breeding practices. Compared with high-density chips and whole-genome selection, the marker detection cost is low and the selection accuracy is high. Attached Figure Description

[0046] Figure 1 The relationship between the number of positive-effect alleles of molecular markers for the auxiliary selection of malic acid content trait in apple fruit in this invention and fruit acidity.

[0047] Figure 2 This is the validation result of the training population for the molecular marker combination used in the apple fruit acidity-assisted selection of this invention. Detailed Implementation

[0048] 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.

[0049] 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).

[0050] Adapter sequences were added to both ends of the DNA library fragments for sample labeling.

[0051] The target sequence is captured using the probe combination described above.

[0052] 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.

[0053] Based on the genotyping data, the acidity trait of the fruit of the tested Malus species was identified.

[0054] The probe combination of molecular markers was selected with the aid of the above-mentioned apple fruit acidity trait.

[0055] 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 training populations. The total number of individuals in the training population is 1408, and the source is the Apple Breeding Base of China Agricultural University in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0056] The method used to extract genomic DNA was the CTAB plant genome extraction method.

[0057] The GenoBaits probe suite for apple fruit acidity trait-assisted selection includes probes for detecting SNP1 to SNP5 and InDel1.

[0058] The nucleotide sequences of the probes used to detect SNP1 to SNP5 are shown in SEQ ID NO.7 to SEQ ID NO.11, respectively.

[0059] The nucleotide sequences of the probes used to detect the labeled combination are shown in SEQ ID NO.7 to SEQ ID NO.11, respectively: The tag combination includes 5 SNP tags and 1 InDel tag; wherein the 5 SNP tags include SNP1 to SNP5, and the 1 InDel tag is InDel1.

[0060] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP1 is shown in SEQ ID NO.7: TTCCAAAATCTTAAAATAATAGTACGAACATCACGTAACAGTACGACAACCGCACTCGTTAAACTTTGTTGGATAACTCTTATCGTCTTTATCGCAAAAAAAAAAAATAA.

[0061] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP2 is shown in SEQ ID NO.8: AAGAGAATTAAGAGAACAAAATCGCTTGAATTCTAATGAGGTAAAAATTTCTAGCTTAATGGTTGCATATGAAGAAGCAACTCTAAAACTAGCTGACAGTTCATTAAATC.

[0062] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP3 is shown in SEQ ID NO.9: AATATAAAATATTGACGTGACTTAACCGTGACCACACAGGAGGGCACGGGAGGGCAGACAATCTTTGTCCTCTTTGTATATATGAAACTGTCAACCTTTTGATAGGGTTG.

[0063] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP4 is shown in SEQ ID NO.10: CTACAAATTATAGGCATGATTACCTTTATAATGTGCCTGCCTCTCATCTCTTATTGCCATTGTCTCTTACACAAACATGTTACATTTATTGTATGAATTACTCATAACTA.

[0064] The nucleotide sequence of the (GenoBaits DNA) probe used to detect SNP5 is shown in SEQ ID NO.11: AATATTGGGATTCTCAGAATCCAAGCACGACTGCTGATCCTTCCAATCAACAATGGATATCTACAGAAAGTCTTTTAAAACGACCAATATCATGGCCACGCCTCTCATTC.

[0065] The nucleotide sequence of the (GenoBaits DNA) probe used to detect InDel1 is shown in SEQ ID NO.7.

[0066] The above six markers are SNP1 to SNP5 and InDel1, where SNP1 to SNP5 are 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.5, respectively.

[0067] Among them, SNP1 is a mutation from A to T at position 7674347 on chromosome 8 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.1: GTAGAATTTTAGATAACGGATATATGTGAGTGTCGCAATCAAAGCAATCACATTTTATCAATAACGCACTAATATAATATGATGAGACAAAATAATCTCAAAATCTTAAAATAATAGTACGAACATCACGTAACAGTACGACAACCGCACTCGTTAAACTTTGTTGGATAACTCTTATCGTCTTTATCGCAAAAAAAAA[A / T]AATAATAATAAATAAATTGAAATTTTGGAAAATGAAATTGGATTAAATTAACAGGAAGGTGTACGGTATGGTTTAGTTGGGAGGTGGGGTCGGAAATGGATGGGGAGCGTAAGCAAACGGATGACTTTGACCAAAAGCCAGGCTGGCGAGCATTTTGGGGCGGGGGAGAAAATGATTTTTTTAATTGAAATAGAAACGG. The position of SNP1 is marked with square brackets "[ ]", with the non-variant base "A" first and the variant base "T" second, separated by " / " (the same applies below).

[0068] SNP2 is a G-to-T mutation located at position 8728049 on chromosome 8 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.2: ATGTAATTTTCTCGGAACAAATAAAGCTCATCGCTTAACGGATGGTCTGTCCAAAATCCTTTTTACCTGCAATAGTAAGTAAAAGAGCAATGTGAGAGAGAAGACAAGAGAATTAAGAGAACAAAATCGCTTGAATTCTAATGAGGTAAAAATTTCTAGCTTAATGGTTGCATATGAAGAAGCAACTCTAAAACTAGCT[G / T]ACAGTTCATTAAATCACAGCTGTGTAGGCTTTCTATGTATATGGTCAACTAACCTATCTCAATCTCGCAGCAGAACAAACGTTGGGCTTGATATGTTGAAGAACTTGGGAACCCATAAGCAAAAGAGTAAATGTATGACTTGGTCAAGAGGCTACGTATGAAATGTGCATTAACAAACCGTAGAAAATATCACTAGCGGT。

[0069] SNP3 is a G-to-A mutation at position 11,654,518 on chromosome 8 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.3: ATTGCACGTTACGAGAATAGCCCATCTCAATCAGTCCCGAGACCTGAGCTTCTTTGTATGGACAAGGATTGTCTGCCCTTTTTGTTCTTGTGTCCTTTCATGTCTTTCTATTTATGTGGTTACGATTAAATCATGTCAATATTTTATATTACTATTCATTTTTGTCTTATTATCTCAATAAAAAACAATATAAAATATT[G / A]ACGTGACTTAACCGTGACCACACAGGAGGGCACGGGAGGGCAGACAATCTTTGTCCTCTTTGTATATATGAAACTGTCAACCTTTTGATAGGGTTGTTACATAACTTTTTATATATATATATATATATATATCGAATTAGTTTATATAGAAAAATGTATATTAAATATTAATCTTAAGGTCCTCAACCACTATAAATTGA。

[0070] SNP4 is a mutation from C to A at position 1855043 on chromosome 15 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.4: CTGGTGGGTTGGCCTTTAGCCAACATAGGTTGTCGAAAACACATAGAGGGACTTTCTCATTAACCTAATCTGTTGGCCCATCTGTGGTTTCGCTTATTCAGTCATACAGTGTCATCAGCCTACAAATTATAGGCATGATTACCTTTATAATGTGCCTGCCTCTCATCTCTTATTGCCATTGTCTCTTACACAAACATGTTA[C / A]ATTTATTGTATGAATTACTCATAACTACACTGACATTTCATCTTTAAAAGAATTGATTGTTATTTCTATGCTTTAGGAAAATGGATATCAATTGCTTTTTTTGAGTGCACGTGCCATTTCTCAAGCCCTGTCACCAGACAATTTTTATTCAACCTTAAGCAGGTTATGCTTAACCCATGCTAAATCCTTGATTTTCATT.

[0071] SNP5 is a mutation from G to A at position 3179027 on chromosome 16 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.5: ATTCAGAGAGCTGGGGACCTGCAGTACGTCCCAAGGAATACGAAGATCATGGGCATTTTGTCGATAAAGACAACGAAAATAAACAGGTGGTGATCGATTCTCTCAGTGAATATTGGGATTCTCAGAATCCAAGCACGACTGCTGATCCTTCCAATCAACAATGGATATCTACAGAAAGTC TTTTAAACGACCAATATCATG[G / A]CCACGCCTCTCATTCAACGCGCACGCTGTGCAGCAGGAACCTGAAGAATCAAAAGTTTACGAAAGCGCCAGCTCCTTATCTTTTGGCAACATTTGCATCGCTTCTGATTGAGTTTGTGGCTCGGCTTCAAAATCTGGTGGATGAATTCGAAGAG.

[0072] InDel1 contains a T insertion mutation at position 7674347 on chromosome 8 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.6: GTAGAATTTTAGATAACGGATATATGTGAGTGTCGCAATCAAAGCAATCACATTTTATCAATAACGCACTAATATAATATGATGAGACAAAATAATCTCAAAATCTTAAAATAATAGTACGAACATCACGTAACAGTACGACAACCGCACTCGTTAAACTTTGTTGGATAACTCTTATCGTCTTTATCGCAAAAAAAAAA[- / T]AATAATAATAAATAAATTGAAATTTTGGAAAATGAAATTGGATTAAATTAACAGGAAGGTGTACGGTATGGTTTAGTTGGGAGGTGGGGTCGGAAATGGATGGGGAGCGTAAGCAAACGGATGACTTTGACCAAAAGCCAGGCTGGCGAGCATTTTGGGGCGGGGGAGAAAATGATTTTTTTAATTGAAATAGAAAACG. The position of InDel1 is marked with square brackets "[ ]", where the non-mutant is indicated by "-" first, and the mutant base "T" is indicated by " / " second.

[0073] Among them, SNP1 had an effect value of 5.07 mg / g FW on malic acid content in the pulp; SNP2 had an effect value of 2.36 mg / g FW on malic acid content in the pulp; SNP3 had an effect value of 4.85 mg / g FW on malic acid content; SNP4 had an effect value of 6.41 mg / g FW on malic acid content; SNP5 had an effect value of 4.76 mg / g FW on malic acid content; and InDel1 had an effect value of 5.07 mg / g FW on malic acid content in the pulp.

[0074] (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.

[0075] (3) Calculation of the number of positive effect sites of apple fruit acidity by the combination of SNP1 to SNP5 and InDel1 (probe combination of molecular markers for auxiliary selection of apple fruit acidity trait).

[0076] The T allele variant of SNP1 A / T has a positive effect on malic acid content. The number of alleles with positive effects in the three test sample genotypes, AA, AT and TT, are 0, 1 and 2, respectively.

[0077] The insT allelic variant of InDel1 has a positive effect on malic acid content. The number of alleles with positive effects in the three test sample genotypes, namely AA, A / insT, and insT / insT, are 0, 1, and 2, respectively.

[0078] The T allele variation of SNP2 G / T has a positive effect on malic acid content. When SNP5 is not AA, the number of alleles with positive effects in the three test sample genotypes GG, GT and TT are 0, 1 and 2, respectively; when SNP5 is AA, the number of alleles with positive effects in the three test sample genotypes GG, GT and TT are 0, 0.5 and 1, respectively.

[0079] The A allele variation of SNP3 G / A has a positive effect on malic acid content. When SNP5 is not AA, the number of alleles with positive effects in the three test sample genotypes GG, GA and AA are 0, 1 and 2, respectively; when SNP5 is AA, the number of alleles with positive effects in the three test sample genotypes GG, GA and AA are 0, 0.5 and 1, respectively.

[0080] The A allele variation of SNP4 C / A has a positive effect on malic acid content. When SNP5 is not AA, the number of alleles with positive effects in the three test sample genotypes CC, CA and AA are 0, 1.5 and 3, respectively; when SNP5 is AA, the number of alleles with positive effects in the three test sample genotypes CC, CA and AA are 0, 0.5 and 1, respectively.

[0081] The G allele variant of SNP5 G / A has a positive effect on malic acid content. The number of alleles with positive effects in the three test sample genotypes, AA, GA and GG, are 0, 1 and 2, respectively.

[0082] (4) Auxiliary selection of apple fruit acidity traits Genomic DNA was extracted from the apple species to be tested.

[0083] The extracted genomic DNA was precisely quantified.

[0084] Enzymatic digestion was used to fragment the quantified genomic DNA, and a DNA library was constructed.

[0085] Adapter sequences were added to both ends of the DNA library fragments for sample labeling.

[0086] The target sequence is captured using the probe combination described above.

[0087] 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.

[0088] Based on the genotyping data, the acidity trait of the fruit of the tested Malus species was identified.

[0089] The identification process is based on genotyping data from SNP1 to SNP5 and InDel1. Based on the obtained genotyping data, the sum of the number of alleles with a positive effect on fruit acidity in the SNP1 to SNP5 genotypes is calculated and denoted as NPA.

[0090] NPA was used as an indicator of the acidity of the fruit of the tested *Malus* species. A higher NPA value indicates higher acidity in the fruit. The specific identification criteria are as follows:

[0091] If the NPA is less than 3.5, the fruit of the tested *Malus* species has very low acidity, and the malic acid content is less than 4.0 mg / g fresh weight; if the NPA is 4.0~6.5, the fruit of the tested *Malus* species has low acidity, and the malic acid content is between 4.0 mg / g and 7.5 mg / g fresh weight; if the NPA is 8.0~9.0, the fruit of the tested *Malus* species has high acidity, and the malic acid content is between 8.0 mg / g and 10.0 mg / g fresh weight; if the NPA is greater than 10.0, the fruit of the tested *Malus* species has very high acidity, and the malic acid content is 10.0 mg / g fresh weight.

[0092] Specifically, the T allele variation at SNP1 has a positive effect on fruit acidity; if the genotype at SNP1 is AA, then the number of alleles in the SNP1 genotype that have a positive effect on fruit acidity is 0; if the genotype at SNP1 is AT, then the number of alleles in the SNP1 genotype that have a positive effect on fruit acidity is 1; if the genotype at SNP1 is TT, then the number of alleles in the SNP1 genotype that have a positive effect on fruit acidity is 2.

[0093] The insT allele variation at InDel1 has a positive effect on fruit acidity; if the genotype at InDel1 is AA (without insT), then the number of alleles in the InDel1 genotype that have a positive effect on fruit acidity is 0; if the genotype at InDel1 is A / insT, then the number of alleles in the InDel1 genotype that have a positive effect on fruit acidity is 1; if the genotype at InDel1 is insT / insT, then the number of alleles in the InDel1 genotype that have a positive effect on fruit acidity is 2.

[0094] The T allele at SNP2 has a positive effect on fruit acidity; when the genotype at SNP5 is not AA and the genotype at SNP2 is GG, the number of alleles in the SNP2 genotype that have a positive effect on fruit acidity is 0; when the genotype at SNP5 is not AA and the genotype at SNP2 is GT, the number of alleles in the SNP2 genotype that have a positive effect on fruit acidity is 1; when the genotype at SNP5 is not AA and the genotype at SNP2 is TT, the number of alleles in the SNP2 genotype that have a positive effect on fruit acidity is... The number of alleles with a positive effect on fruit acidity is 2; when the genotype at SNP5 is AA and the genotype at SNP2 is GG, the number of alleles with a positive effect on fruit acidity in the genotype of SNP2 is 0; when the genotype at SNP5 is AA and the genotype at SNP2 is GT, the number of alleles with a positive effect on fruit acidity in the genotype of SNP2 is 0.5; when the genotype at SNP5 is AA and the genotype at SNP2 is TT, the number of alleles with a positive effect on fruit acidity in the genotype of SNP2 is 1.

[0095] The A allele at SNP3 has a positive effect on fruit acidity. When the genotype at SNP5 is not AA and the genotype at SNP3 is GG, the number of alleles in the SNP3 genotype that have a positive effect on fruit acidity is 0. When the genotype at SNP5 is not AA and the genotype at SNP3 is GA, the number of alleles in the SNP3 genotype that have a positive effect on fruit acidity is 1. When the genotype at SNP5 is not AA and the genotype at SNP3 is AA, the number of alleles in the SNP3 genotype that have a positive effect on fruit acidity is... The number of alleles with a positive effect on fruit acidity is 2; when the genotype at SNP5 is AA and the genotype at SNP3 is GG, the number of alleles with a positive effect on fruit acidity in the SNP3 genotype is 0; when the genotype at SNP5 is AA and the genotype at SNP3 is GA, the number of alleles with a positive effect on fruit acidity in the SNP3 genotype is 0.5; when the genotype at SNP5 is AA and the genotype at SNP3 is AA, the number of alleles with a positive effect on fruit acidity in the SNP3 genotype is 1.

[0096] The A allele at SNP4 has a positive effect on fruit acidity; when the genotype at SNP5 is not AA and the genotype at SNP4 is CC, the number of alleles in the SNP4 genotype that have a positive effect on fruit acidity is 0; when the genotype at SNP5 is not AA and the genotype at SNP4 is CA, the number of alleles in the SNP4 genotype that have a positive effect on fruit acidity is 1.5; when the genotype at SNP5 is not AA and the genotype at SNP4 is AA, the number of alleles in the SNP4 genotype that have a positive effect on fruit acidity is... The number of alleles with a positive effect is 3; when the genotype at SNP5 is AA and the genotype at SNP4 is CC, the number of alleles with a positive effect on fruit acidity in the SNP4 genotype is 0; when the genotype at SNP5 is AA and the genotype at SNP4 is CA, the number of alleles with a positive effect on fruit acidity in the SNP4 genotype is 0.5; when the genotype at SNP5 is AA and the genotype at SNP4 is AA, the number of alleles with a positive effect on fruit acidity in the SNP4 genotype is 1.

[0097] The G allele at SNP5 has a positive effect on fruit acidity; when the genotype at SNP5 is AA, the number of alleles with a positive effect on fruit acidity in the SNP5 genotype is 0; when the genotype at SNP5 is GA, the number of alleles with a positive effect on fruit acidity in the SNP5 genotype is 1; when the genotype at SNP5 is GG, the number of alleles with a positive effect on fruit acidity in the SNP5 genotype is 2.

[0098] The target sequence was 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 of China Agricultural University in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0099] The acidity of the fruit is determined by the generational sum of the number of alleles with a positive effect on fruit acidity in each marker genotype within the SNP1 to SNP5 ring and InDel1 marker combinations of the tested sample. A higher generational sum indicates higher fruit acidity in the tested sample. Figure 1 .

[0100] Depend on Figure 1 It can be seen that by using the above 5 SNPs and 1 InDel marker (a combination of molecular markers for auxiliary selection of apple fruit acidity traits), the acidity of apple fruit can be predicted comprehensively and accurately.

[0101] Example 2 Using 335 germplasm resources from 25 species of *Malus* with 2-5 years of fruit acidity phenotypic data, 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 were used to genotype molecular markers for apple fruit acidity trait-assisted selection. The generational sum of alleles with positive effects on fruit acidity (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 individual fruit acidity. The results are shown below. Figure 2 .

[0102] Among them, 335 germplasm resources of 25 species originated from the apple breeding base of China Agricultural University in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0103] 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 of China Agricultural University in Beidaihe New District, Qinhuangdao City, Hebei Province.

[0104] The coefficient of determination (R²) of the linear regression equation between NPA value and fruit acidity is 0.682. This indicates that the combination of molecular markers and probes for acid-assisted selection in apple fruit is feasible.

[0105] The experimental results above show that the molecular marker combination provided by the present invention has comprehensive and excellent alleles related to fruit acidity performance in wild species and closely related species of the genus Malus, with accurate prediction and coverage of multiple allele loci. It can integrate excellent alleles related to fruit acidity 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.

[0106] 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.

[0107] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A probe array of molecular markers for the selection of apple fruit acidity traits, characterized in that, Including five probes for assisting selection of apple fruit acidity traits; The nucleotide sequences of the probes used for selection of five apple fruit acidity traits are shown in SEQ ID NO.7~SEQ ID NO.11, respectively.

2. The probe combination of molecular markers for the auxiliary selection of apple fruit acidity traits according to claim 1, characterized in that, The probes used for auxiliary selection of five apple fruit acidity traits were probes for detecting five SNP markers and one InDel marker; the five SNP markers included SNP1 to SNP5, and the one InDel marker was InDel1; the nucleotide sequences of SNP1 to SNP5 are as follows: SEQ ID NO.1 to SEQ ID As shown in NO.5; SNP1 is an A to T mutation at position 7674347 on chromosome 8 of the apple reference genome GDDH13.1; SNP2 is a G to T mutation at position 8728049 on chromosome 8 of the apple reference genome GDDH13.1; SNP3 is a G to A mutation at position 11654518 on chromosome 8 of the apple reference genome GDDH13.1; SNP4 is a C to A mutation at position 1855043 on chromosome 15 of the apple reference genome GDDH13.1; SNP5 is a G to A mutation at position 3179027 on chromosome 16 of the apple reference genome GDDH13.

1. InDel1 is a T insertion mutation at position 7674347 on chromosome 8 of the apple reference genome GDDH13.1, and its nucleotide sequence is shown in SEQ ID NO.

6.

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

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

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

6. The application according to claim 5, characterized in that, Based on the obtained genotyping data, the sum of the number of alleles with a positive effect on fruit acidity in the genotypes SNP1 to SNP5 and InDel1 is calculated and denoted as NPA. The NPA (Neural Protein Acidity) was used as an indicator of the acidity of the fruit of the tested *Malus* species; the higher the NPA value, the higher the acidity of the fruit of the tested *Malus* species.

7. The application according to claim 6, characterized in that, If the NPA is less than 3.5, the fruit of the tested *Malus* species has very low acidity, and the malic acid content is less than 4.0 mg / g fresh weight; if the NPA is 4.0~6.5, the fruit of the tested *Malus* species has low acidity, and the malic acid content is between 4.0 mg / g and 7.5 mg / g fresh weight; if the NPA is 8.0~9.0, the fruit of the tested *Malus* species has high acidity, and the malic acid content is between 8.0 mg / g and 10.0 mg / g fresh weight; if the NPA is greater than 10.0, the fruit of the tested *Malus* species has very high acidity, and the malic acid content is 10.0 mg / g fresh weight.

8. The application according to claim 6, characterized in that, The T allele at SNP1 has a positive effect on fruit acidity; if the genotype at SNP1 is AA, then the number of alleles in the SNP1 genotype that have a positive effect on fruit acidity is 0; if the genotype at SNP1 is AT, then the number of alleles in the SNP1 genotype that have a positive effect on fruit acidity is 1; if the genotype at SNP1 is TT, then the number of alleles in the SNP1 genotype that have a positive effect on fruit acidity is 2. The insT allele variation at InDel1 has a positive effect on fruit acidity; if the genotype at InDel1 is AA, then the number of alleles with a positive effect on fruit acidity in the InDel1 genotype is 0; if the genotype at InDel1 is A / insT, then the number of alleles with a positive effect on fruit acidity in the InDel1 genotype is 1; if the genotype at InDel1 is insT / insT, then the number of alleles with a positive effect on fruit acidity in the InDel1 genotype is 2. The T allele at SNP2 has a positive effect on fruit acidity; when the genotype at SNP5 is not AA and the genotype at SNP2 is GG, the number of alleles in the SNP2 genotype that have a positive effect on fruit acidity is 0; when the genotype at SNP5 is not AA and the genotype at SNP2 is GT, the number of alleles in the SNP2 genotype that have a positive effect on fruit acidity is 1; when the genotype at SNP5 is not AA and the genotype at SNP2 is TT, the number of alleles in the SNP2 genotype that have a positive effect on fruit acidity is... The number of alleles with a positive effect on fruit acidity is 2; when the genotype at SNP5 is AA and the genotype at SNP2 is GG, the number of alleles with a positive effect on fruit acidity in the SNP2 genotype is 0; when the genotype at SNP5 is AA and the genotype at SNP2 is GT, the number of alleles with a positive effect on fruit acidity in the SNP2 genotype is 0.5; when the genotype at SNP5 is AA and the genotype at SNP2 is TT, the number of alleles with a positive effect on fruit acidity in the SNP2 genotype is 1. The A allele at SNP3 has a positive effect on fruit acidity. When the genotype at SNP5 is not AA and the genotype at SNP3 is GG, the number of alleles in the SNP3 genotype that have a positive effect on fruit acidity is 0. When the genotype at SNP5 is not AA and the genotype at SNP3 is GA, the number of alleles in the SNP3 genotype that have a positive effect on fruit acidity is 1. When the genotype at SNP5 is not AA and the genotype at SNP3 is AA, the number of alleles in the SNP3 genotype that have a positive effect on fruit acidity is... The number of alleles with a positive effect on fruit acidity is 2; when the genotype at SNP5 is AA and the genotype at SNP3 is GG, the number of alleles with a positive effect on fruit acidity in the SNP3 genotype is 0; when the genotype at SNP5 is AA and the genotype at SNP3 is GA, the number of alleles with a positive effect on fruit acidity in the SNP3 genotype is 0.5; when the genotype at SNP5 is AA and the genotype at SNP3 is AA, the number of alleles with a positive effect on fruit acidity in the SNP3 genotype is 1. The A allele at SNP4 has a positive effect on fruit acidity; when the genotype at SNP5 is not AA and the genotype at SNP4 is CC, the number of alleles in the SNP4 genotype that have a positive effect on fruit acidity is 0; when the genotype at SNP5 is not AA and the genotype at SNP4 is CA, the number of alleles in the SNP4 genotype that have a positive effect on fruit acidity is 1.5; when the genotype at SNP5 is not AA and the genotype at SNP4 is AA, the number of alleles in the SNP4 genotype that have a positive effect on fruit acidity is... The number of alleles with a positive effect is 3; when the genotype at SNP5 is AA and the genotype at SNP4 is CC, the number of alleles with a positive effect on fruit acidity in the SNP4 genotype is 0; when the genotype at SNP5 is AA and the genotype at SNP4 is CA, the number of alleles with a positive effect on fruit acidity in the SNP4 genotype is 0.5; when the genotype at SNP5 is AA and the genotype at SNP4 is AA, the number of alleles with a positive effect on fruit acidity in the SNP4 genotype is 1. The G allele at SNP5 has a positive effect on fruit acidity; when the genotype at SNP5 is AA, the number of alleles with a positive effect on fruit acidity in the SNP5 genotype is 0; when the genotype at SNP5 is GA, the number of alleles with a positive effect on fruit acidity in the SNP5 genotype is 1; when the genotype at SNP5 is GG, the number of alleles with a positive effect on fruit acidity in the SNP5 genotype is 2.

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