A SNP molecular marker related to the content of total flavonoids in olea europaea and application thereof

Abstract

This invention provides a SNP molecular marker related to the total flavonoid content of olives and its application, belonging to the field of molecular marker technology. The SNP molecular marker is located at position 36578427 on olive chromosome 2, exhibiting a G / A single nucleotide mutation. The genotype at this site is significantly correlated with the total flavonoid content of olive fruit, with the AA genotype showing the highest flavonoid content, followed by the AG genotype, which in turn shows the highest flavonoid content, followed by the GG genotype. This invention utilizes KASP fluorescence typing technology, requiring only the extraction of genomic DNA from olive leaves to complete seedling genotyping. Based on the typing results, the flavonoid accumulation level in the fruit can be predicted. This overcomes the technical drawback of traditional breeding methods that require 3-5 years after planting and fruiting before chemical determination of flavonoids. High-throughput batch testing can be conducted year-round, significantly shortening the olive breeding cycle. It is suitable for early identification, resource screening, and molecular-assisted breeding of high-flavonoid olive germplasm, and has significant application value in the field of selecting high-quality olive varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and in particular to an SNP molecular marker related to the total flavonoid content of olive oil and its application. Background Technology

[0002] Flavonoids in olive (Olea europaea L.) fruit possess excellent antioxidant activity and health benefits, and are one of the core functional components of olive. Selecting olive germplasm resources with high flavonoid content is an important breeding goal for improving olive varieties, enhancing fruit quality, and increasing application value.

[0003] Currently, the industry generally adopts the conventional "post-fruiting testing" technique for screening high-flavonoid olive germplasm. This process involves: conventional seedling cultivation and field planting and maintenance of the olive germplasm to be tested; waiting for the plants to pass through the juvenile stage and enter the fruiting and reproductive growth stage (the natural growth cycle is usually 3-5 years); harvesting mature fruits; and then using chemical detection methods such as spectrophotometry and high-performance liquid chromatography to determine the flavonoid content of the fruit. Finally, based on the test results, the flavonoid accumulation characteristics of the germplasm are determined, and high-quality, high-content germplasm resources are screened. This traditional screening technique relies on physicochemical testing after fruit maturity, providing intuitive results and clear judgment criteria. However, it has significant technical bottlenecks and limitations in practical breeding applications, severely restricting the efficiency of breeding high-quality olive germplasm. On the one hand, the breeding cycle is lengthy. Traditional screening must rely on fruit samples from the olive fruiting period for testing, requiring waiting for the plants to complete the long juvenile growth stage and enter the reproductive growth stage. The overall screening cycle is about 4 years, significantly delaying the overall process of olive germplasm improvement and new variety breeding, making it difficult to meet the industry's demand for efficient breeding. On the other hand, the screening process is costly and wasteful of resources. This technology requires long-term land occupation, routine field management, and subsequent batch chemical testing and analysis of all germplasm samples to be tested. A large number of inferior germplasm samples with low flavonoid content (below 35 μg / g) need to undergo full-cycle cultivation management before they can be identified and eliminated. The land resources, manpower, materials, maintenance, and testing costs invested in the early stages are all wasted in vain, resulting in low overall cost-effectiveness and poor resource utilization for germplasm screening.

[0004] In summary, how to overcome the technical limitations of traditional fruiting period testing, establish an early, accurate, and efficient high-flavonoid germplasm screening technology system for olive seedlings, effectively shorten the breeding cycle, reduce screening costs, and improve germplasm screening efficiency are key technical challenges that urgently need to be addressed in the field of olive quality breeding. Summary of the Invention

[0005] In view of this, the present invention provides an SNP molecular marker related to the total flavonoid content of olive and its application. The method of the present invention can not only detect olive leaf samples in a high-throughput manner, which is fast and economical, but also can be detected in the early stage of olive growth, shortening the breeding cycle; and is not limited by season, so it can be detected all year round.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of SNP molecular markers in the identification of total flavonoid content in olive oil. The molecular markers are located at position 36578427 on chromosome 2 of olive oil, where there is a G / A single nucleotide polymorphism. The total flavonoid content of olive oil with the AA genotype is greater than that of the AG genotype, which is greater than that of the GG genotype.

[0008] The present invention also provides a primer set for amplifying the molecular marker, including a first upstream primer, a second upstream primer and a universal downstream primer; the first upstream primer is shown in SEQ ID NO.1, the second upstream primer is shown in SEQ ID NO.2, and the universal downstream primer is shown in SEQ ID NO.3.

[0009] Preferably, the 5' end of the first upstream primer is linked to a FAM fluorescent tag sequence, and the second upstream primer contains a HEX fluorescent tag sequence.

[0010] The present invention also provides the application of the primer set described above in the preparation of a kit for identifying the total flavonoid content of olive.

[0011] The present invention also provides a kit for identifying the total flavonoid content of olive oil, comprising the aforementioned primer set.

[0012] This invention also provides a method for identifying the total flavonoid content of olive oil, comprising the following steps:

[0013] S1. Extract genomic DNA from the olive samples to be tested;

[0014] S2. Using the genomic DNA of the olive sample to be tested as a template, KASP-PCR amplification was performed using the primer set described in claim 2;

[0015] S3. Genotyping and identification of total flavonoid content in olives were performed based on the fluorescence read from the KASP-PCR amplification products: if red fluorescence was read, the genotype was AA; if blue fluorescence was read, the genotype was GG; if green fluorescence was read, the genotype was AG; the total flavonoid content of olives in AA genotype was > that in AG genotype was > that in GG genotype.

[0016] This invention also provides the application of the aforementioned SNP molecular markers in the identification, screening, or breeding of olive germplasm with high total flavonoid content.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects: The SNP molecular marker of the present invention is located at position 36578427 on chromosome 2 of olive, and there is a G / A single nucleotide mutation. The genotype at this site is significantly correlated with the total flavonoid content of olive fruit, with the AA genotype having the highest flavonoid content, followed by the AG genotype, which in turn has the highest flavonoid content. The present invention utilizes KASP fluorescence typing technology, which only requires the collection of genomic DNA from olive leaves to complete the seedling genotype identification. Based on the typing results, the flavonoid accumulation level in the fruit can be predicted. This overcomes the technical drawback of traditional breeding, which requires 3-5 years after planting and fruiting before chemical determination of flavonoids. High-throughput batch testing can be carried out throughout the year, significantly shortening the olive breeding cycle. It is suitable for early identification, resource screening, and molecular-assisted breeding of high-flavonoid olive germplasm, and has important application value in the field of breeding high-quality olive varieties. Attached Figure Description

[0018] Figure 1 Box plot of genotyping at locus SNP36578427 for 150 olive resources.

[0019] Figure 2 The scatter plot shows the fluorescence typing of olive resources at site SNP36578427. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1. Development of SNP molecular markers for olives with high flavonoid content

[0022] 1. Construction of test materials

[0023] The test materials came from a natural population of 150 olive germplasm resources, which were introduced from countries such as Spain, Italy, and Greece. The trees were 8 to 12 years old and were planted in the olive germplasm resource nursery of Zhejiang Academy of Agricultural Sciences under the same cultivation and management methods.

[0024] 2. Development of molecular markers

[0025] Transcriptome sequencing was performed on leaves from 18 olive trees, and flavonoid content was measured in mature fruits. Association analysis revealed two genes significantly associated with flavonoid content: EVM0020954 and EVM0027514 (olive reference genome: https: / / ngdc.cncb.ac.cn / gwh / Assembly / 10300 / show). SNP calling of these two genes identified nine non-synonymous SNPs in exons, located at positions 36578120, 36578343, 36578427, 36578504, and 36579197 on chromosome 2, positions 36579270 and 36579414 on chromosome 21, and positions 931889 and 932864 on chromosome 21. These SNPs were selected as candidate SNPs affecting flavonoid content. Flanking sequences of 100 bp upstream and downstream of each candidate SNP were extracted for KASP primer design. The 5' end contains FAM and HEX fluorescent linker sequences, and the reverse primer is a universal primer.

[0026] 3. KASP molecular marker primer design

[0027] Nine SNP sites with good polymorphism were selected from the screened sites for KASP marker development. Flanking sequences of 100 bp upstream and downstream of the target sites were extracted for KASP primer design. The 5' end contains FAM and HEX fluorescent linker sequences. Universal primers were used for the reverse primers. The nine SNP sites and their primers are as follows:

[0028] 1. SNP-36578120 (C / A)

[0029] First upstream primer: 5'-GCATAACAGGAAGTTAGCAAGCACC-3' (SEQ ID NO.1);

[0030] Second upstream primer: 5'-CATAACAGGAAGTTAGCAAGCACA-3' (SEQ ID NO.2);

[0031] Universal downstream primer: 5'-CTCTCCCTTCTGTTGAGCATCCAAA-3' (SEQ ID NO.3);

[0032] 2. SNP-36578343 (A / T)

[0033] First upstream primer: 5'-AAGCACRAAGAGCATGGACAACAA-3' (SEQ ID NO.4);

[0034] Second downstream primer: 5'-GCACRAAGAGCATGGACAACAT-3' (SEQ ID NO.5);

[0035] Universal downstream primer: 5'-GTTGTCCTCGTTGATCTCTCCCTTY-3' (SEQ ID NO.6);

[0036] 3. SNP-36578427 (G / A)

[0037] First upstream primer: 5'-GATTGATCATATTTTGGATGCTCAACAG-3' (SEQ ID NO.7);

[0038] Second upstream primer: 5'-CGATTGATCATATTTTGGATGCTCAACAA-3' (SEQ ID NO.8);

[0039] Universal downstream primer: 5'-CAGCGACGTTAATGTTCTCSACGAT-3' (SEQ ID NO.9);

[0040] 4. SNP-36578504 (C / G)

[0041] First upstream primer: 5'-AATACCAGCGACGTTAATGTTCTCC-3' (SEQ ID NO.10);

[0042] Second upstream primer: 5'-AATACCAGCGACGTTAATGTTCTCG-3' (SEQ ID NO.11);

[0043] Common downstream primer: 5'-GAGAGATCAACGAGGACAACGTTCTT-3' (SEQ ID NO.12);

[0044] 5. SNP-36579197 (C / A)

[0045] First upstream primer: 5'-CTTGACACCATGCTTGGCCC-3' (SEQ ID NO.13);

[0046] Second upstream primer: 5'-AACTTGACACCATGCTTGGCCA-3' (SEQ ID NO.14);

[0047] Common downstream primer: 5'-TTGTAGGTGTCTGGCTCGGTGATTT-3' (SEQ ID NO.15);

[0048] 6. SNP-36579270 (A / T)

[0049] First upstream primer: 5'-CAGAACGATCTTGGTTTCAGAACA-3' (SEQ ID NO.16);

[0050] Second upstream primer: 5'-AAATCAGAACGATCTTGGTTTCAGAACT-3' (SEQ ID NO.17);

[0051] Common downstream primer: 5'-GGGGGACAGTTCAGCTTGCATATTT-3' (SEQ ID NO.18);

[0052] 7. SNP-36579414 (G / A)

[0053] First upstream primer: 5'-TCTGAATTGTCCTTGCACATCAAG-3' (SEQ ID NO.19);

[0054] Second upstream primer: 5'-AACTTCTGAATTGTCCTTGCACATCAAA-3' (SEQ ID NO.20);

[0055] Common downstream primer: 5'-GGTTGAGAAGGAATTGGGGAGATGTT-3' (SEQ ID NO.21);

[0056] 8. SNP-931889 (G / A)

[0057] First upstream primer: 5'-AACTGGTAATCACATACAGAGACAG-3' (SEQ ID NO.22);

[0058] Second upstream primer: 5'-ACTGGTAATCACATACAGAGACAA-3' (SEQ ID NO.23);

[0059] Common downstream primer: 5'-GGACAATTCAGAAGTTTGCAAGGAGTATT-3' (SEQ ID NO.24);

[0060] 9. SNP-932864 (G / A)

[0061] First upstream primer: 5'-CTCTCTCTCTCTCAATGGAGG-3' (SEQ ID NO.25);

[0062] Second upstream primer: 5'-TCTCTCTCTCTCTCAATGGAGA-3' (SEQ ID NO.26);

[0063] Common downstream primer: 5'-GGAGTGGCAAGTGATTTGGGATGTA-3' (SEQ ID NO.27).

[0064] KASP PCR molecular marker detection was performed by linking the FAM fluorescent linker sequence to the 5' end of the first upstream primer and the HEX fluorescent linker sequence to the 5' end of the second upstream primer.

[0065] Example 2. Detection of SNP molecular markers in olives with high flavonoid content

[0066] This study used 150 olive germplasm resources (numbered SPM001-SPM150) as experimental materials to determine the flavonoid content of mature fruits. Combined with the genotyping results of nine molecular markers at positions 36578120, 36578343, 36578427, 36578504, 36579197, 36579270, 36579414, 931889, and 932864 on chromosome 2, the stability, completeness, and phenotypic suitability of each marker were comprehensively analyzed to screen for highly efficient molecular markers suitable for assisted breeding of olive flavonoid content.

[0067] Genotyping was performed on 150 olive resources using the LGC SNP line platform. The PCR amplification reaction system consisted of 2 μL of KASP HiGeno 2×Probe Mix, 1 μL of SNP Primer Mix (4×), and 2 μL of DNA sample. The amplification program was as shown in Table 2.

[0068] Table 2 KASP PCR amplification procedure

[0069]

[0070] After the reaction was completed, the endpoint fluorescence signal was read using an LGC OMEGA fluorescence detector, and the genotype was analyzed using Kluster Caller software. The genotyping results are shown in Table 3.

[0071] Meanwhile, the total flavonoid content of the 150 mature olive fruits was determined using rutin as a standard and the aluminum chloride colorimetric method. After pitting the olives, they were crushed. 100 mg of olive pomace was placed in a 15 mL test tube, and 10 mL of 70% ethanol solution was added. The mixture was ultrasonically extracted for 30 min at a power of 500 W to obtain the extract. After centrifugation, 9 mL of the supernatant was collected in a 15 mL test tube, and 1 mL of 5% sodium nitrite solution was added. The mixture was vortexed and allowed to stand for 5 min. 1 mL of 10% aluminum nitrate solution was added to the mixture, vortexed, and allowed to stand for 5 min. The mixture was then transferred to a 25 mL volumetric flask, and 5 mL of 1.2 mol / L sodium hydroxide solution was added. The volume was adjusted to 70% ethanol solution. After 15 min, the absorbance was measured at a wavelength of 510 nm. The total flavonoid content of the mature olive fruits was calculated based on the absorbance values. The results are shown in Table 3.

[0072] Table 3. Total flavonoid content and genotypes in mature fruits of different olive varieties.

[0073] SPM001 Olea europaea 'ZN01' 26.35 AC AA GG GG CA AT GG GA GG SPM002 Olea europaea 'ZN02' 24.53 CC AA GG GG AA AT GG GA GG SPM003 Olea europaea 'ZN03' 28.95 AC TA GG CC CA AT GA GA GG SPM004 Olea europaea 'ZN04' 29.31 CC AA GG GG CA AT GG GA GG SPM005 Olea europaea 'ZN05' 28.14 CC AA GG CC CA AT GG Uncallable AG SPM006 Olea europaea 'ZN06' 32.45 AC AA AG CC AA TT GG Uncallable GG SPM007 Olea europaea 'ZN07' 30.25 CC AA AG GG AA TT GG Uncallable GG SPM008 Olea europaea 'ZN08' 27.85 AC TA GG CC CC AA GG GG GG SPM009 Olea europaea 'ZN09' 26.54 CC AA GG GG ? AA GG GA AG SPM010 Olea europaea 'ZN10' 24.58 CC AA GG GG CC AA GG GG GG SPM011 Olea europaea 'ZN11' 32.54 AC AA AG CC CA AT GG GA GG SPM012 Olea europaea 'ZN12' 32.51 AC TA AG CC CA AT GG GA GG SPM013 Olea europaea 'ZN13' 33.21 CC AA AG CC CA AT GA Uncallable AG SPM014 Olea europaea 'ZN14' 29.35 AC AA GG CC CA AT GG GG GG SPM015 Olea europaea 'ZN15' 28.65 CC AA GG GG CA AT GG GA GG SPM016 Olea europaea 'ZN16' 29.31 AC AA GG GG CA AT GG Uncallable AG SPM017 Olea europaea 'ZN17' 25.42 AC AA GG CC CA AT GG GA GG SPM018 Olea europaea 'ZN18' 26.82 AC TA GG CC CA AT GA GA GG SPM019 Olea europaea 'ZN19' 29.35 AA TA GG GG CA AT GG GA GG SPM020 Olea europaea 'ZN20' 30.25 CC AA GG GG CA AT GA GG GG SPM021 Olea europaea 'ZN21' 35.64 CC AA AG CC CA AT GG GA AG SPM022 Olea europaea 'ZN22' ‌ 24.56 AC AA GG GG CC AA GG Uncallable AA SPM023 Olea europaea 'ZN23' ‌ 35.25 AC AA AG GG CC AA GG Uncallable GG SPM024 Olea europaea 'ZN24' ‌ 26.35 CC AA GG GG CA AT GG GA GG SPM025 Olea europaea 'ZN25' ‌ 30.25 AC AA GG GG CC AA GG GG GG SPM026 Olea europaea 'ZN26' ‌ 28.52 AC TA GG CC CA AT GG GA GG SPM027 Olea europaea 'ZN27' ‌ 30.25 AC TA GG CC CA AT GA GA GG SPM028 Olea europaea 'ZN28' ‌ 28.52 CC AA GG CC CA AT GG GA GG SPM029 Olea europaea 'ZN29' ‌ 25.64 AC TA GG CC CC AA GG GA GG SPM030 Olea europaea 'ZN30' ‌ 33.54 AC AA AG GG CA AT GG GA GG SPM031 Olea europaea 'ZN31' ‌ 31.25 CC AA AG CC AA TT GA GA GG SPM032 Olea europaea 'ZN32' ‌ 26.25 AC AA GG CC CC AA GG Uncallable GG SPM033 Olea europaea 'ZN33' ‌ 31.02 CC AA AG CC CC AA GG GG GG SPM034 Olea europaea 'ZN34' ‌ 29.25 CC AA GG GG CA AT GG GA GG SPM035 Olea europaea 'ZN35' ‌ 24.61 CC AA GG CC CC AA GG GA GG SPM036 Olea europaea 'ZN36' ‌ 34.25 AC TA AG CG CC AA GG GA AG SPM037 Olea europaea 'ZN37' ‌ 30.56 AC TA AG CC CC AA GG Uncallable AG SPM038 Olea europaea 'ZN38' ‌ 26.35 AC TA GG CC AA TT GA GA GG SPM039 Olea europaea 'ZN39' ‌ 30.65 CC AA AG CC CC AA GG Uncallable AA SPM040 Olea europaea 'ZN40' ‌ 30.52 AA TT AG CC CA AT GG GA GG SPM041 Olea europaea 'ZN41' ‌ 26.58 CC AA GG CC CC AA GG GA AG SPM042 Olea europaea 'ZN42' ‌ 30.52 CC AA AG CC CA AT GA Uncallable ? SPM043 Olea europaea 'ZN43' ‌ 24.62 CC AA GG CC CC AA GG GA AG SPM044 Olea europaea 'ZN44' ‌ 36.52 AA TA AA GG CC AA GG GA AG SPM045 Olea europaea 'ZN45' ‌ 28.56 AC TA GG CC CC AA GG GA AG SPM046 Olea europaea 'ZN46' ‌ 28.52 CC AA AG CC CC AA GG GG GG SPM047 Olea europaea 'ZN47' ‌ 24.63 AC TA GG GG CC AA GG GA AG SPM048 Olea europaea 'ZN48' ‌ 26.56 AC TA GG CC CC AA GG Uncallable AG SPM049 Olea europaea 'ZN49' ‌ 30.52 CC AA AG CC CA AT GG Uncallable AG SPM050 Olea europaea 'ZN50' ‌ 26.52 CC AA GG CC CC AA GG GA GG SPM051 Olea europaea 'ZN51' ‌ 29.65 CC AA GG CC CC AA GG GA AG SPM052 Olea europaea 'ZN52' ‌ 29.65 AA AA GG CC AA AT GG GA GG SPM053 Olea europaea 'ZN53' ‌ 30.25 CC AA AG CC CC AA GG Uncallable GG SPM054 Olea europaea 'ZN54' ‌ 28.65 AC TA AG CC CC AA GG GA AG SPM055 Olea europaea 'ZN55' ‌ 28.65 AC TA GG GG CC AA GG GA AG SPM056 Olea europaea 'ZN56' ‌ 30.25 AC AA AG CC CC AA GG GA AG SPM057 Olea europaea 'ZN57' ‌ 29.65 AC TA AG GG CC AA GG GA AG SPM058 Olea europaea 'ZN58' ‌ 26.58 CC AA GG CC CA AT GA Uncallable AG SPM059 Olea europaea 'ZN59' ‌ 25.62 AC AA GG CC ? AA ? GG GG SPM060 Olea europaea 'ZN60' ‌ 30.52 CC AA AG CC CA AT GG GG GG SPM061 Olea europaea 'ZN61' ‌ 32.24 AA AA AG CC CA AT GA GA GG SPM062 Olea europaea 'ZN62' ‌ 26.52 AC TA GG CC CC AA GG GA AG SPM063 Olea europaea 'ZN63' ‌ 28.56 AC AA GG ? CC AA GG GG GG SPM064 Olea europaea 'ZN64' ‌ 25.62 AC TA GG CC CA AT GG Uncallable GG SPM065 Olea europaea 'ZN65' ‌ 28.65 CC AA GG GG CA AT GG Uncallable AG SPM066 Olea europaea 'ZN66' ‌ 26.52 CC AA AG GG CA AT GG Uncallable AG SPM067 Olea europaea 'ZN67' ‌ 25.6 AC TA GG GG CC AA GG GG GG SPM068 Olea europaea 'ZN68' ‌ 30.25 CC AA GG CC CC AA GG GG GG SPM069 Olea europaea 'ZN69' ‌ 26.52 CC AA GG GG CA AT GG GA GG SPM070 Olea europaea 'ZN70' ‌ 32.52 CC AA AG CC CA AT GG GG GG SPM071 Olea europaea 'ZN71' ‌ 50.92 AC AA AA CC AA TT AA Uncallable GG SPM072 Olea europaea 'ZN72' ‌ 29.52 AC TA GG GG CC AA GG GA GG SPM073 Olea europaea 'ZN73' ‌ 39.9 CC AA AA CC AA AA GG GA GG SPM074 Olea europaea 'ZN74' ‌ 28.56 AA AA AA CC AA TT GA GG GG SPM075 Olea europaea 'ZN75' ‌ 45.54 CC AA AA CC CA AT GA GA GG SPM076 Olea europaea 'ZN76' ‌ 28.65 CC AA AG CC ? AA ? GG GG SPM077 Olea europaea 'ZN77' ‌ 29.35 AC AA GG CC AA AT GG GG GG SPM078 Olea europaea 'ZN78' ‌ 28.56 CC AA AG GG AA TT GG GA GG SPM079 Olea europaea 'ZN79' ‌ 30.25 CC AA AG CC AA TT GA Uncallable GG SPM080 Olea europaea 'ZN80' ‌ 31.62 AA AA AG CC AA AT AA GA AG SPM081 Olea europaea 'ZN81' ‌ 51.36 CC AA AA CC AA TT AA Uncallable AG SPM082 Olea europaea 'ZN82' ‌ 30.65 CC AA AG CC CA AA GG GA GG SPM083 Olea europaea 'ZN83' ‌ 32.64 CC AA AG CC CA AT GA GA GG SPM084 Olea europaea 'ZN84' ‌ 29.65 CC AA GG CC CA AT GA GA GG SPM085 Olea europaea 'ZN85' ‌ 62.3 CC AA AA CC AA AT GA GA AG SPM086 Olea europaea 'ZN86' ‌ 53.96 CC AA AA CC CA AT GA Uncallable GG SPM087 Olea europaea 'ZN87' ‌ 28.53 CC AA AG GG AA AT GA GA GG SPM088 Olea europaea 'ZN88' ‌ 52.4 CC AA AA CC CA AT GA GA GG SPM089 Olea europaea 'ZN89' ‌ 31.25 CC AA AG GG CA AT GA GG GG SPM090 Olea europaea 'ZN90' ‌ 25.65 CC AA GG CC CC AA GG GG GG SPM091 Olea europaea 'ZN91' ‌ 29.62 CC AA GG GG CC AT GG GG GG SPM092 Olea europaea 'ZN92' ‌ 25.63 CC AA GG GG CA AA GG GG GG SPM093 Olea europaea 'ZN93' ‌ 28.52 CC AA GG GG CA AA GG GG GG SPM094 Olea europaea 'ZN94' ‌ 30.25 CC AA GG GG CA AT GA GG GG SPM095 Olea europaea 'ZN95' ‌ 28.54 CC AA GG GG CA AA GG GG GG SPM096 Olea europaea 'ZN96' ‌ 30.25 AC TA AG GG CC AA GG GG GG SPM097 Olea europaea 'ZN97' ‌ 25.62 CC AA GG CC CA AA GG GG GG SPM098 Olea europaea 'ZN98' ‌ 32.51 CC AA AG CC CA AT GA GA GG SPM099 Olea europaea 'ZN99' ‌ 26.53 AC TA GG CC CA AT GA GA AG SPM100 Olea europaea 'ZN100' ‌ 32.21 AC AA AG CC CC AA GG GA GG SPM101 Olea europaea 'ZN101' ‌ 30.25 AC TA GG CC CA AT GA GA AG SPM102 Olea europaea 'ZN102' ‌ 26.53 ? AA GG GG AA ? GG Uncallable GG SPM103 Olea europaea 'ZN103' ‌ 29.65 AC TA GG CC CC AA GG GG GG SPM104 Olea europaea 'ZN104' ‌ 26.35 CC AA AG CC CC AA GG GG GG SPM105 Olea europaea 'ZN105' ‌ 28.52 AA TT GG CC CA AT GA GA GG SPM106 Olea europaea 'ZN106' ‌ 28.54 AC TA GG CC CA AT GA Uncallable GG SPM107 Olea europaea 'ZN107' ‌ 30.25 CC AA GG CC CA AT GG GA GG SPM108 Olea europaea 'ZN108' ‌ 28 CC AA AG CC CA AT GG Uncallable AG SPM109 Olea europaea 'ZN109' ‌ 27.52 AC TA GG GG CC AA GG GA AG SPM110 Olea europaea 'ZN110' ‌ 29.65 AC AA AG CC CC AA GG GA AG SPM111 Olea europaea 'ZN111' ‌ 28.54 CC AA GG GG CA AT GG Uncallable GG SPM112 Olea europaea 'ZN112' ‌ 24.52 CC AA GG CC CC AA GG GA AG SPM113 Olea europaea 'ZN113' ‌ 29.65 CC AA AG CC CC AA GG Uncallable AA SPM114 Olea europaea 'ZN114' ‌ 24.52 AC TA GG CC CC AA GG GA AG SPM115 Olea europaea 'ZN115' ‌ 28.56 AC TA GG CC CC AA GG GA AG SPM116 Olea europaea 'ZN116' ‌ 34.21 AC AA AG CC CA AT GG GA GG SPM117 Olea europaea 'ZN117' ‌ 25.63 AC TA GG CC CC AA GG GA GG SPM118 Olea europaea 'ZN118' ‌ 24.32 AC AA GG CC CC AA GG GG GG SPM119 Olea europaea 'ZN119' ‌ 26.85 CC AA AG GG CA AT GG GA GG SPM120 Olea europaea 'ZN120' ‌ 26.53 AC TA GG GG CA AT GG Uncallable AG SPM121 Olea europaea 'ZN121' ‌ 29.86 AA TA GG GG CA AT GG GA GG SPM122 Olea europaea 'ZN122' ‌ 24.52 AC AA GG CC CC AA GG GG GG SPM123 Olea europaea 'ZN123' ‌ 26.35 AC AA GG CC CA AT GG GA GG SPM124 Olea europaea 'ZN124' ‌ 24.32 CC AA GG CC CC AA GG GA AG SPM125 Olea europaea 'ZN125' ‌ 26.53 ? AA GG CC CC AA ? GG GG SPM126 Olea europaea 'ZN126' ‌ 26.35 CC AA GG GG CA AT GA GA GG SPM127 Olea europaea 'ZN127' ‌ 28.56 CC AA GG CC CC AA GG GG GG SPM128 Olea europaea 'ZN128' ‌ 24.35 AC AA GG CC AA TT GA GG GG SPM129 Olea europaea 'ZN129' ‌ 28.65 CC AA GG CC CC AA GG GA AG SPM130 Olea europaea 'ZN130' ‌ 25.31 AC TA GG CC CA AT GA Uncallable AG SPM131 Olea europaea 'ZN131' ‌ 26.35 AC TA GG CC CA AT GA Uncallable AG SPM132 Olea europaea 'ZN132' ‌ 27.52 AC AA GG CC CC AA GG Uncallable AG SPM133 Olea europaea 'ZN133' ‌ 24.63 AC AA GG GG CC AA GG GA AG SPM134 Olea europaea 'ZN134' ‌ 29.65 AC TA GG CC CC AA GG GA AG SPM135 Olea europaea 'ZN135' ‌ 25.32 AC TA GG CC CA AT GG Uncallable AG SPM136 Olea europaea 'ZN136' ‌ 29.35 AC TA GG CC CC AA GG GG GG SPM137 Olea europaea 'ZN137' ‌ 22.35 CC AA GG CC CC AA GG GA GG SPM138 Olea europaea 'ZN138' ‌ 28.65 AC AA GG CC AA AT AA GA GG SPM139 Olea europaea 'ZN139' ‌ 21.35 AC AA GG CC CC AA GG GG GG SPM140 Olea europaea 'ZN140' ‌ 26.52 AC AA GG CC Uncallable AT ? GG AG SPM141 Olea europaea 'ZN141' ‌ 24.32 AC AA GG CC AA ? GG Uncallable GG SPM142 Olea europaea 'ZN142' ‌ 26.85 CC AA AG GG CA Uncallable GG GA GG SPM143 Olea europaea 'ZN143' ‌ 38.25 CC AA AA CC CA AT GA Uncallable AG SPM144 Olea europaea 'ZN144' ‌ 28.54 CC AA AG CC CC AA GG GA AG SPM145 Olea europaea 'ZN145' 29.56 AC TA AG GG CA AT GA GA GG SPM146 Olea europaea 'ZN146' 28.8 AC TA AG GG CC AA GG GA AG SPM147 Olea europaea 'ZN147' 26.56 AC TA GG GG CA AT GA Uncallable AG SPM148 Olea europaea 'ZN148' 48.19 AC TA AA CC CA AT GA Uncallable AG SPM149 Olea europaea 'ZN149' 26.35 AC TA GG GG Uncallable AT GA Uncallable AG SPM150 Olea europaea 'ZN150' 28.96 CC AA AG CC CC AA GG Uncallable AA

[0074] Note: "?" indicates no signal or weak signal, "Uncallable" indicates a signal but no clear type.

[0075] ANOVA (analysis of variance) was used to compare the total flavonoid content in different genotypes, with a threshold of p < 0.05. SNP-36578427 showed a highly significant correlation with total flavonoid content. The homogeneity of variance test and ANOVA results for total flavonoid content are shown in Tables 4 and 5, respectively. The genotyping results of this locus in 150 olive germplasm resources are shown below. Figure 1 As shown.

[0076] Table 4 Homogeneity of Variance Test

[0077] 0.471 2 147 0.625

[0078] Table 5 ANOVA Results

[0079] Between groups 183.270 2 91.635 5.962 0.003 Within groups 2259.312 147 15.369 -- -- Total 2442.581 149 -- -- --

[0080] Example 3. Validation of the SNP molecular marker SNP-36578427 in olive oil with high flavonoid content

[0081] In Example 2, there were 11 olive plants with genotype AA (SPM044, SPM071, SPM073, SPM074, SPM075, SPM081, SPM085, SPM086, SPM088, SPM143, SPM148), and the flavonoid content of their mature olive fruits was 28.56~62.30 μg / g.

[0082] In Example 2, there were 47 olive plants with the AG genotype. The flavonoid content of their mature olive fruits was 26.35~35.64 μg / g, and the average value was significantly lower than that of the AA type.

[0083] In Example 2, 92 olive trees of the GG genotype (SPM001, SPM002, SPM003, SPM004, SPM005, SPM008, SPM009, SPM010, SPM014, SPM015) were analyzed. The flavonoid content of their mature olive fruits ranged from 21.35 to 30.25 μg / g, with an average content significantly lower than that of the AA genotype. The flavonoid content of mature fruits corresponding to the three genotypes (AA, AG, and GG) is shown below. Figure 2 As shown.

[0084] As can be seen from the above embodiments, the present invention provides an SNP molecular marker related to the total flavonoid content of olives and its application, which can predict the flavonoid accumulation level of the fruit based on the typing results, and significantly shorten the breeding cycle of olives.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of SNP molecular markers in the identification of total flavonoid content in olive oil, characterized in that, The molecular marker is located at position 36578427 on chromosome 2 of olive, where a G / A single nucleotide polymorphism exists. The total flavonoid content of olives with the AA genotype is greater than that of the AG genotype, which is greater than that of the GG genotype.

2. A primer set for amplifying the molecular marker of claim 1, characterized in that, It includes a first upstream primer, a second upstream primer, and a universal downstream primer; the first upstream primer is shown in SEQ ID NO.7, the second upstream primer is shown in SEQ ID NO.8, and the universal downstream primer is shown in SEQ ID NO.

9.

3. The primer set according to claim 2, characterized in that, The first upstream primer has a FAM fluorescent tag sequence attached to its 5' end, and the second upstream primer contains a HEX fluorescent tag sequence.

4. The use of the primer set according to claim 2 or 3 in the preparation of a kit for identifying the total flavonoid content of olive oil.

5. A reagent kit for identifying the total flavonoid content of olive oil, characterized in that, It includes the primer set as described in claim 2 or 3.

6. A method for identifying the total flavonoid content of olive oil, characterized in that, Includes the following steps: S1. Extract genomic DNA from the olive samples to be tested; S2. Using the genomic DNA of the olive sample to be tested as a template, KASP-PCR amplification was performed using the primer set described in claim 2; S3. Genotyping and identification of total flavonoid content in olives were performed based on the fluorescence read from the KASP-PCR amplification products: if red fluorescence was read, the genotype was AA; if blue fluorescence was read, the genotype was GG; if green fluorescence was read, the genotype was AG; the total flavonoid content of olives in AA genotype was > that in AG genotype was > that in GG genotype.

7. The application of the SNP molecular marker as described in claim 1 in the identification, screening, or breeding of olive germplasm with high total flavonoid content.

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