InDel molecular marker closely linked with grapefruit juice sac granulation and application thereof

Abstract

The invention belongs to the technical field of molecular marker development and breeding, and particularly relates to an InDel molecular marker closely linked with grapefruit juice sac granulation and application of the InDel molecular marker. The nucleotide sequence of the InDel molecular marker is shown as SEQ ID NO: 1, the InDel molecular marker is closely linked with grapefruit juice sac granulation, the grapefruit juice sac granulation character can be identified, meanwhile, a filial generation single plant with the character can be removed in the seedling stage, the citrus hybridization seed selection cost is reduced, and the new variety breeding efficiency of citrus hybridization breeding is improved. A primer pair is developed based on the InDel molecular marker, PCR amplification is carried out through the primer pair, the genetic juice sac granulation phenotype of the pomelo fruit filial generation can be rapidly and accurately identified, the cost is low, and the accuracy rate is high.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker development and breeding technology, specifically involving an InDel molecular marker closely linked to the granulation of grapefruit juice vesicles and its application. Background Technology

[0002] Juice vesicles are the basic building blocks of the pulp tissue in citrus fruits. Juice vesicle granulation significantly reduces the flavor, internal quality, and economic value of citrus fruits. Traditionally, juice vesicle granulation occurs during the ripening or post-harvest storage of citrus fruits and is caused by the deposition of lignin in the juice vesicles, making it a typical physiological disorder.

[0003] Currently, methods for controlling granulation of citrus fruits mainly include: breeding new varieties, selecting appropriate rootstocks, balancing fertilization and irrigation, and using plant growth regulators. However, in practical applications, existing control measures have poor stability, are greatly affected by the environment, and are not suitable for large-scale promotion and application, failing to achieve the goal of cost reduction and efficiency improvement. Furthermore, most measures primarily target granulation in post-harvest stored fruits, and cannot effectively reduce granulation of the pulp during the citrus plant's growth period or fruit ripening, thus their control effect has certain limitations.

[0004] Molecular marker-assisted breeding (MMR) technology offers advantages such as ease of operation, high accuracy, and good reproducibility, making it an effective means to address this problem. This technology can not only identify the target trait at the seedling stage and eliminate hybrid offspring that do not conform to the target trait, but also significantly reduce the human, material, and financial resources required in pomelo breeding, thereby improving the efficiency of citrus variety selection. However, currently, there is no effective molecular marker to identify this trait. Developing a molecular marker that can rapidly identify the hereditary granulation trait in citrus fruits and applying it to hybridization breeding of citrus fruits is of great significance for improving the efficiency and reducing the cost of breeding superior varieties. Summary of the Invention

[0005] The purpose of this invention is to provide an InDel molecular marker closely linked to the granulation of juice vesicles in pomelo fruit and its application, to distinguish between pomelos with normal juice vesicles and those with granulated juice vesicles, to quickly eliminate granulated juice vesicles in seedlings, thereby reducing the cost of breeding new pomelo varieties and significantly improving the efficiency of breeding new pomelo varieties.

[0006] This invention provides an InDel molecular marker that is closely linked to the granulation of grapefruit juice vesicles, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0007] Preferably, the InDel molecular marker is a double-stranded DNA molecule, wherein the nucleotide sequence of one strand is shown as positions 361-415 of SEQ ID NO:5.

[0008] The present invention also provides primer pairs for detecting the InDel molecular markers described in the above technical solutions, wherein the primer pairs include a forward primer and a reverse primer;

[0009] The forward primer comprises the nucleotide sequence shown in SEQ ID NO:2;

[0010] The reverse primer comprises the nucleotide sequence shown in SEQ ID NO:3.

[0011] The present invention also provides a kit for detecting the InDel molecular marker described in the above-described technical solution, the kit comprising the primer pair described in the above-described technical solution.

[0012] This invention also provides the application of reagents for detecting the InDel molecular markers described in the above-described technical solutions, or primer pairs described in the above-described technical solutions, or kits described in the above-described technical solutions, in one or more of the following:

[0013] (1) To identify or assist in the identification of granulation of pomelo fruit juice cells;

[0014] (2) To differentiate or assist in the differentiation of grapefruit with normal juice vesicles and granulated juice vesicles;

[0015] (3) Screening or assisting in screening grapefruits with normal juice vesicles;

[0016] (4) Assisted breeding of grapefruit with normal fruit juice vesicles;

[0017] The double-stranded DNA of the pomelo contains the nucleotide sequence shown in SEQ ID NO:1 in both strands, and the genotype is AA; the double-stranded DNA of the pomelo contains only one strand of the nucleotide sequence shown in SEQ ID NO:1 in one strand, and the genotype is Aa; the double-stranded DNA of the pomelo does not contain the nucleotide sequence shown in SEQ ID NO:1 in either strand, and the genotype is aa.

[0018] Grapefruit with genotype AA or Aa has normal juice vesicles, while grapefruit with genotype aa has granulated juice vesicles.

[0019] Preferably, the grapefruit is any one or more of the following:

[0020] (1) Guanxi Honey Pomelo;

[0021] (2) Pingshan pomelo;

[0022] (3) Hybrid offspring of Guanxi honey pomelo and Pingshan pomelo;

[0023] (4) Backcross offspring of Guanxi honey pomelo and the hybrid offspring of Guanxi honey pomelo and Pingshan pomelo.

[0024] The present invention also provides a method for identifying or assisting in the identification of granulation of juice vesicles in pomelo fruit, comprising the following steps:

[0025] The genomic DNA of the grapefruit to be tested was amplified by PCR using the primer pairs described in the above technical solution or the primer pairs in the kit described in the above technical solution to obtain the amplification product;

[0026] The amplification product was analyzed by gel electrophoresis. If the amplification product was a double band of 812 bp and 757 bp, the pomelo fruit juice vesicles were normal; if the amplification product was a single band of 812 bp, the pomelo fruit juice vesicles were normal; if the amplification product was a single band of 757 bp, the pomelo fruit juice vesicles were granulated.

[0027] Preferably, the PCR amplification system, in 20 μL, comprises: 10 μL PCR Mix, 100 ng genomic DNA, 1 μM forward primer, 1 μM reverse primer, and the remainder ddH2O.

[0028] Preferably, the PCR amplification program includes: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 55℃ annealing for 20 sec, 72℃ extension for 30 sec, 35 cycles; 72℃ further extension for 5 min, and storage at 12℃.

[0029] The present invention also provides a breeding method for grapefruit with normal fruit juice sacs, the breeding method comprising selecting grapefruits whose genomic DNA does not contain the InDel molecular marker described in the above technical solution as parents for breeding.

[0030] Beneficial effects:

[0031] The nucleotide sequence of the InDel molecular marker provided by this invention, as shown in SEQ ID NO:1, is closely linked to granulation of juice vesicles in pomelo fruit. This not only allows for the identification of granulation phenotype in pomelo fruit but also enables the removal of hybrid offspring plants with this trait during the seedling stage, reducing the cost of citrus hybridization and improving the efficiency of new citrus hybridization breeding. Primer pairs developed based on the InDel molecular marker, PCR amplification using these primer pairs, can rapidly and accurately identify the heritable granulation phenotype in pomelo fruit hybrid offspring, with low cost and high accuracy. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0033] Figure 1 The phenotypes of juice vesicles in pomelo fruit are shown; where A represents the granulated juice vesicle phenotype and B represents the normal juice vesicle phenotype.

[0034] Figure 2 The genetic mapping results for the granulation trait in grapefruit juice cells;

[0035] Figure 3 The results of comparing the genomic DNA sequences of normal juice sac plants with juice sac granulation plants;

[0036] Figure 4 The results show the PCR amplification of primer pairs in Guanxi honey pomelo, Pingshan pomelo, and 15 progeny plants; where GX represents Guanxi honey pomelo and PS represents Pingshan pomelo.

[0037] Figure 5 The results show the PCR amplification of primer pairs in Guanxi pomelo, Pingshan pomelo, and 186 progeny plants; where GX represents Guanxi pomelo and PS represents Pingshan pomelo; A is a long band, B is a double band, and C is a short band.

[0038] Figure 6 The results are PCR amplification of primer pairs in Guanxi pomelo, Pingshan pomelo, and 222 backcross progeny single plants; where GX represents Guanxi pomelo and PS represents Pingshan pomelo; A is a short band and B is a double band. Detailed Implementation

[0039] This invention provides an InDel molecular marker that is closely linked to the granulation of grapefruit juice vesicles, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0040] In one embodiment, the InDel molecular marker of the present invention is a double-stranded DNA molecule, wherein the nucleotide sequence of one strand is shown as positions 361-415 of SEQ ID NO:5.

[0041] This invention evaluates the granulation phenotype of offspring from a hybrid population of Guanxi Honey Pomelo (with normal fruit juice vesicles) × Pingshan Pomelo (with normal fruit juice vesicles), and combines whole-genome resequencing and bioinformatics analysis to screen out a sequence (SEQ ID NO:1) that is most strongly associated with the granulation phenotype of pomelo fruit juice vesicles: the 55bp InDel sequence shows significant differences between juice vesicle granulation and normal juice vesicle materials, and can be used as a candidate site for developing molecular markers, serving as a linkage marker for granulation status predicted based on sequencing results.

[0042] The present invention also provides primer pairs for detecting the InDel molecular marker described in the above technical solutions, wherein the primer pairs include a forward primer and a reverse primer; the forward primer includes the nucleotide sequence shown in SEQ ID NO:2; and the reverse primer includes the nucleotide sequence shown in SEQ ID NO:3.

[0043] The present invention also provides a kit for detecting the InDel molecular marker described in the above-described technical solution, the kit comprising the primer pair described in the above-described technical solution.

[0044] As one implementation, the kit of the present invention also includes PCRMix.

[0045] The present invention also provides the application of reagents for detecting the InDel molecular markers described in the above technical solutions, or primer pairs described in the above technical solutions, or kits described in the above technical solutions in one or more of the following: (1) identifying or assisting in identifying the granulation status of grapefruit juice vesicles; (2) distinguishing or assisting in distinguishing between grapefruits with normal juice vesicles and those with granulated juice vesicles; (3) screening or assisting in screening grapefruits with normal juice vesicles; (4) assisting in the breeding of grapefruits with normal juice vesicles.

[0046] The grapefruit double-stranded DNA has two strands containing the nucleotide sequence shown in SEQ ID NO:1, and the genotype is AA; only one strand of the grapefruit double-stranded DNA contains the nucleotide sequence shown in SEQ ID NO:1, and the genotype is Aa; neither strand of the grapefruit double-stranded DNA contains the nucleotide sequence shown in SEQ ID NO:1, and the genotype is aa; grapefruits with genotype AA or Aa have normal juice vesicles, while grapefruits with genotype aa have granulated juice vesicles.

[0047] In one embodiment, the pomelo of the present invention is any one or more of the following: (1) Guanxi honey pomelo; (2) Pingshan pomelo; (3) hybrid offspring of Guanxi honey pomelo and Pingshan pomelo; (4) backcross offspring of Guanxi honey pomelo and the hybrid offspring of Guanxi honey pomelo and Pingshan pomelo.

[0048] The present invention also provides a method for identifying or assisting in the identification of granulation of juice vesicles in pomelo fruit, comprising the following steps:

[0049] The genomic DNA of the grapefruit to be tested was amplified by PCR using the primer pairs described in the above technical solution or the primer pairs in the kit described in the above technical solution to obtain the amplification product;

[0050] If the amplification product is a double band of 812bp and 757bp, the pomelo fruit juice vesicles are normal; if the amplification product is a single band of 812bp, the pomelo fruit juice vesicles are normal; if the amplification product is a single band of 757bp, the pomelo fruit juice vesicles are granulated.

[0051] In one embodiment, the pomelo described in this invention is a pomelo in its seedling stage.

[0052] In one embodiment, the amplification products are analyzed by gel electrophoresis. In one embodiment, the nucleotide sequence of the 812 bp band is shown in SEQ ID NO:5. In one embodiment, the nucleotide sequence of the 757 bp band is shown in SEQ ID NO:4.

[0053] As one embodiment, the PCR amplification system of the present invention, in 20 μL, includes: 10 μL PCR Mix, 100 ng genomic DNA, 1 μM forward primer, 1 μM reverse primer, and the remainder ddH2O.

[0054] As one implementation method, the PCR amplification program of the present invention includes: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 sec, annealing at 55°C for 20 sec, extension at 72°C for 30 sec, 35 cycles; further extension at 72°C for 5 min, and storage at 12°C.

[0055] This invention also provides a breeding method for pomelos with normal fruit juice vesicles. The breeding method includes selecting pomelos whose genomic DNA does not contain the InDel molecular marker described in the above-mentioned technical solution as parents for breeding. This invention does not impose strict requirements on the specific breeding method; after parent selection, conventional steps in the art can be used.

[0056] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an InDel molecular marker closely linked to the granulation of grapefruit juice sacs and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] The InDel molecular marker design and preparation method includes the following steps:

[0059] 1. Granulation trait of fruit juice vesicles in the offspring of 231 F1 generation hybrid populations of Guanxi Honey Pomelo (normal fruit juice vesicles) × Pingshan Pomelo (normal fruit juice vesicles) Figure 1 The results of the evaluation are shown in Table 1.

[0060] Table 1. Phenotypic characteristics of juice vesicle granulation in single offspring of Guanxi Honey Pomelo × Pingshan Pomelo hybrids

[0061]

[0062]

[0063]

[0064]

[0065] As can be seen from Table 1, the granulation trait of the offspring of the Guanxi Honey Pomelo × Pingshan Pomelo hybrid showed obvious segregation, approximately 3:1, suggesting that this trait is a quality trait controlled by a single gene.

[0066] 2. To identify the gene for this trait, extreme single-plant pool sequencing was performed on the offspring of this hybrid population. The genetic mapping results are as follows: Figure 2 As shown, the region of 13.90Mb to 17.60Mb on chromosome 6 was identified as a candidate region.

[0067] 3. To further screen for variant sites closely linked to the granulation phenotype within candidate regions, Beijing Novogene Technology Co., Ltd. was commissioned to complete whole-genome resequencing (sequencing depth 30×) of the progeny of 6 hybrid populations with normal fruit juice vesicles and 6 hybrid populations with granulated fruit juice vesicles, obtaining a total of more than 300G of data.

[0068] 4. Based on the pomelo category to which the materials belong, the sequencing reads of the 12 materials were aligned to the Late White Pomelo genome (http: / / citrus.hzau.edu.cn / data / Genome_info / HWB.v1.0 / HWB.v1.0.genome.fa) using BWA (0.7.17-r1188) software to obtain a BAM file containing all variation information within the candidate segment.

[0069] 5. The variant sites within the candidate region were randomly visualized using iGV software. Sequence alignment results for different granulation states were compared and examined one by one. Based on the material's phenotypic characteristics, the ratios of various bases / monomers at each site were statistically analyzed. The sequence polymorphisms with the highest phenotypic association within the candidate region were manually reviewed and screened as candidate sites for molecular marker development. A 55bp InDel was found to show significant differences between normal and granulated grapefruit juice vesicles. Figure 3 This result is a linkage marker for the granulation status of grapefruit juice cells predicted based on sequencing results.

[0070] 6. Based on the above sequence information, an experiment was designed for verification. The InDel sequence (SEQ ID NO:1: 5'-CACAACAGCAACGGACGCTGTTCTCAGTATTTAATTTTTTTTTTATAAAA CAAAA-3') was developed as a PCR marker. Based on the pomelo genome sequence, a forward primer (SEQ ID NO:2: 5'-TCATCAAGCGATTGAACCTAG-3') was designed upstream of the InDel, and a reverse primer (SEQ ID NO:3: 5'-CAGAAACTGGCAAGACGGA-3') was designed downstream to form a primer pair. When the amplification product of the pomelo seedling is a double band of 812bp (SEQ ID NO:5) and a band of 757bp (SEQ ID NO:4), or a single band of 812bp (SEQ ID NO:5), it indicates that the pomelo fruit juice vesicles are normal; when the amplification product is only a single band of 757bp (SEQ ID NO:4), it indicates that the pomelo fruit juice vesicles are granulated. Based on the above experimental design, the genotype of each material at this locus can be directly determined from the gel image.

[0071] SEQ ID NO:4:5'-TCATCAAGCGATTGAACCTAGGCCCTTGCATAGATAA TGTAAATGCGTCGGAATATTCAAAGATTTACAATATATGCTTGCTTCAGCTACATTAGGATAAAAAGAAATTCAATGACGGTAAAATCACCTCTCTTTTTTTTCTTTTTTTTTGAACTCAAAGTTAATTTAAAATTTGAGGGGAATACATTTAAAACTGTTGGGGTTGATTTTGTTTTATTATATTGTCCACTCATTCGGAGTCCACAATATTTACAATAAAAAATTACTTCAATTTTATCCAAATATATTTTTGAACTGTGATATCAAAAAATCTGAAACGCAAATAATATTAAGAAAAAAAAAACATAAATTCTAAATGAAAACAAAAACAAAACAAACAAAACAAAAACAAAACCTTCCAAGTAAATGTTGGTCGTTCACCTCAAGTTCAAAACCTTTTAAACAAATTAGCATTGTTCAATTGAGGCGCGTCACGTGTACATCAACAGCTCACAGCCTTGGTTTCATCTTCACTCTCCCGCTGCTCACACGGTCATGAGCGCCATTTAGCGCCTTGTTTTGATCATAATCAAATCATTCATCAATAATCCTCACTTTCAAATTTCAACAAAGAAAAAAACGAAATGGCCACGTCAGCGGAAGCCAATAAGCCGGAGCCGGCGACTGGCTACCCGGTCACCTACGCCTACGCGGCCCCACCACCTCAGCCGCAAGTGGCACCCACCAACCGATCTCCATTCCGTCTTGCCAGTTTCTG-3';

[0072] SEQ ID NO:5: 5'-TCATCAAGCGATTGAACCTAGGCCCTTGCATAGATAA TGTAAATGCGTCGGAATATTCAAAGATTTACAATATATGCTTGCTTCAGCTACATTAGGATAAAAAGAAATTCAATGACGGTAAAATCACCTCTCTTTTTTTTCTTTTTTTTTGAACTCAAAGTTAATTTAAAATTTGAGGGGAATACATTTAAAACTGTTGGGGTTGATTTTGTTTTATTATATTGTCCACTCATTCGGAGTCCACAATATTTACAATAAAAAATTACTTCAATTTTATCCAAATATATTTTTGAACTGTGATATCAAAAAATCTGAAACGCAAATAATATTAAGAAAAAAAAAACATAAATTCTAAATGAACACAACAGCAACGGACGCTGTTCTCAGTATTTAATTTTTTTTTTATAAAACAAAAAACAAAAACAAAACAAACAAAACAAAAACAAAACCTTCCAAGTAAATGTTGGTCGTTCACCTCAAGTTCAAAACCTTTTAAACAAATTAGCATTGTTCAATTGAGGCGCGTCACGTGTACATCAACAGCTCACAGCCTTGGTTTCATCTTCACTCTCCCGCTGCTCACACGGTCATGAGCGCCATTTAGCGCCTTGTTTTGATCATAATCAAATCATTCATCAATAATCCTCACTTTCAAATTTCAACAAAGAAAAAAACGAAATGGCCACGTCAGCGGAAGCCAATAAGCCGGAGCCGGCGACTGGCTACCCGGTCACCTACGCCTACGCGGCCCCACCACCTCAGCCGCAAGTGGCACCCACCAACCGATCTCCATTCCGTCTTGCCAGTTTCTG-3'。

[0073] Example 2

[0074] Molecular marker identification of the granulation phenotype of fruit juice vesicles

[0075] 1. Preparation of genomic DNA

[0076] Genomic DNA was extracted from the parental plants (Guanxi Honey Pomelo × Pingshan Pomelo) and 15 F1 generation hybrid seedlings using a modified CTAB method.

[0077] 2. PCR amplification of the genomic DNA of all the pomelos described above was performed using the primer pairs developed in Example 1; wherein,

[0078] The PCR reaction system was as follows: 10 μL PCRMix (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), 100 ng DNA, 1 μM each of forward and reverse primers (synthesized from Wuhan Tianyi Huayu Gene Technology Co., Ltd.), and ddH2O to a final volume of 15 μL.

[0079] The PCR reaction thermal cycling parameters were: 98℃ for 3 min; 98℃ for 10 sec, 55℃ for 20 sec, 72℃ for 30 sec, 35 cycles; 72℃ for 5 min, and storage at 12℃. The reaction was performed on a BIOCENER instrument.

[0080] 3. The amplified products were detected by 2.0% agarose gel electrophoresis on a horizontal electrophoresis tank of the Beijing 61 Electrophoresis System. 1×TAE buffer (0.04M Tris-acetate, 0.001M EDTA, pH 8.0) was used at 8V / cm for 30 min. After electrophoresis, images were taken and stored using a BIO-RAD gel imaging system (UVP).

[0081] 4. Through amplification of the maternal parent Guanxi pomelo, the paternal parent Pingshan pomelo, and 15 typical hybrid offspring, it was found that, except for offspring No. 07 which did not produce a band and could not be matched with the phenotypic results, the results of the other 14 hybrid offspring were consistent. Figure 4 The overall calculated match rate of the granulated molecular markers in typical progeny is (14 / 15) × 100% = 93.33%.

[0082] 5. Further statistical analysis of the amplification results of 186 hybrid progeny revealed that the molecular marker bands in 175 progeny pairs matched the phenotypic expression, while those in 11 progeny pairs did not. Overall, the marker concordance rate reached (175 / 186) × 100% = 94.09%. Figure 5 (and Table 2).

[0083] Table 2. Molecular marker validation results of 186 hybrid progeny of Guanxi honey pomelo and Pingshan pomelo.

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] Note: "√" means it meets the requirements, and "×" means it does not meet the requirements.

[0091] Example 3

[0092] Prediction of granulation status in 222 backcross population progeny (with Guanxi pomelo as the female parent and juice granulation plants from the Guanxi pomelo × Pingshan pomelo hybrid progeny as the male parent).

[0093] 1. Preparation of genomic DNA

[0094] Genomic DNA was extracted from the progeny of 222 grapefruit backcross populations using a modified CTAB method.

[0095] 2. The genomic DNA of all the above-mentioned grapefruits was amplified by PCR using the primer pair developed in Example 1. The PCR reaction system was as follows: 10 μL PCR Mix (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), 100 ng DNA, 1 μM each of forward and reverse primers (synthesized by Hantian Yihua Yu Gene Technology Co., Ltd.), and ddH2O was added to a final volume of 15 μL.

[0096] The PCR reaction thermal cycling parameters were: 98℃ for 3 min; 98℃ for 10 sec, 55℃ for 20 sec, 72℃ for 30 sec, 35 cycles; 72℃ for 5 min, and storage at 12℃. The reaction was performed on a BIOCENER instrument.

[0097] 3. The amplified products were detected by 2.0% agarose gel electrophoresis on a horizontal electrophoresis tank of the Beijing 61 Electrophoresis System. 1×TAE buffer (0.04M Tris-acetate, 0.001M EDTA, pH 8.0) was used at 8V / cm for 30 min. After electrophoresis, images were taken and stored using a BIO-RAD gel imaging system (UVP).

[0098] 4. Statistical analysis of the granulation of 222 grapefruit backcross progeny revealed that 108 backcross progeny exhibited a single band of 757 bp (SEQ ID NO:4), and 114 backcross progeny exhibited double bands of both 757 bp (SEQ ID NO:4) and 812 bp (SEQ ID NO:5). The statistical result was 108:114, a ratio approximately 1:1. Figure 6 (and Table 3).

[0099] Table 3. Prediction results of molecular markers in 222 grapefruit backcross progeny

[0100]

[0101]

[0102]

[0103]

[0104] As can be seen from the above, the molecular markers provided by this invention are closely linked to the granulation phenotype of grapefruit juice cells, providing a simple, rapid, and effective screening method for early identification of grapefruit granulation and hybridization breeding, thereby improving breeding efficiency.

[0105] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An InDel molecular marker closely linked to granulation of grapefruit juice vesicles, characterized in that, The nucleotide sequence of the InDel molecular marker is shown in SEQ ID NO:

1.

2. The InDel molecular marker according to claim 1, characterized in that, The InDel molecular marker is a double-stranded DNA molecule, one strand of which has a nucleotide sequence as shown in positions 361-415 of SEQ ID NO:

5.

3. The primer pair for detecting the InDel molecular marker as described in claim 1 or 2, characterized in that, The primer pair includes a forward primer and a reverse primer; The forward primer comprises the nucleotide sequence shown in SEQ ID NO:2; The reverse primer comprises the nucleotide sequence shown in SEQ ID NO:

3.

4. A kit for detecting the InDel molecular marker as described in claim 1 or 2, characterized in that, The kit includes the primer pair as described in claim 3.

5. The use of the reagent for detecting the InDel molecular marker as described in claim 1 or 2, or the primer pair as described in claim 3, or the kit as described in claim 4, in one or more of the following: (1) To identify or assist in the identification of granulation of pomelo fruit juice cells; (2) To differentiate or assist in the differentiation of grapefruit with normal juice vesicles and granulated juice vesicles; (3) Screening or assisting in screening grapefruits with normal juice vesicles; (4) Assisted breeding of grapefruit with normal fruit juice vesicles; The double-stranded DNA of the pomelo contains the nucleotide sequence shown in SEQ ID NO:1 in both strands, and the genotype is AA; the double-stranded DNA of the pomelo contains only one strand of the nucleotide sequence shown in SEQ ID NO:1 in one strand, and the genotype is Aa; the double-stranded DNA of the pomelo does not contain the nucleotide sequence shown in SEQ ID NO:1 in either strand, and the genotype is aa. Grapefruit with genotype AA or Aa has normal juice vesicles, while grapefruit with genotype aa has granulated juice vesicles.

6. The application according to claim 5, characterized in that, The pomelo can be any one or more of the following: (1) Guanxi Honey Pomelo; (2) Pingshan pomelo; (3) Hybrid offspring of Guanxi honey pomelo and Pingshan pomelo; (4) Backcross offspring of Guanxi honey pomelo and the hybrid offspring of Guanxi honey pomelo and Pingshan pomelo.

7. A method for identifying or assisting in the identification of granulation of juice vesicles in pomelo fruit, characterized in that, Includes the following steps: The genomic DNA of the grapefruit to be tested was amplified by PCR using the primer pair described in claim 3 or the primer pair in the kit described in claim 4 to obtain the amplification product; If the amplification product is a double band of 812bp and 757bp, the pomelo fruit juice vesicles are normal; if the amplification product is a single band of 812bp, the pomelo fruit juice vesicles are normal; if the amplification product is a single band of 757bp, the pomelo fruit juice vesicles are granulated.

8. The method according to claim 7, characterized in that, The PCR amplification system, in 20 μL increments, includes: 10 μL PCR Mix, 100 ng genomic DNA, 1 μM forward primer, 1 μM reverse primer, and the remainder ddH2O.

9. The method according to claim 7 or 8, characterized in that, The PCR amplification program includes: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 55℃ annealing for 20 sec, 72℃ extension for 30 sec, 35 cycles; 72℃ further extension for 5 min, and storage at 12℃.

10. A breeding method for a pomelo with normal fruit juice vesicles, characterized in that, The breeding method includes selecting grapefruits whose genomic DNA does not contain the InDel molecular marker as described in claim 1 or 2 as parents for breeding.

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