InDel marker related to browning resistance character of pear pulp and application of InDel marker

Abstract

The invention discloses an InDel marker related to the browning resistance character of pear pulp, a primer and a detection kit, the InDel marker is located at the 22496111-22496306 bp position of a chromosome 2 of a red fragrant pear genome, the overall length is 196 bp, the InDel marker is a deletion or insertion polymorphic site located in a pear Ras related leucine-rich repeat protein coding gene PbrPIRL promoter region, and the InDel marker is located at the deletion or insertion polymorphic site of the pear Ras related leucine-rich repeat protein coding gene PbrPIRL promoter region. The InDel marker is closely linked with the anti-browning character of pear pulp, a pear pulp single plant prone to browning shows that an InDel marker sequence is deleted, and a pear pulp anti-browning single plant shows that the InDel marker sequence is inserted. Based on the molecular marker and the method disclosed by the invention, the anti-browning variety can be quickly and accurately identified through a conventional molecular experiment. Besides, the detection method has the characteristics of simplicity and convenience in operation, high specificity and high accuracy, is not interfered by external factors such as climate and environment, provides reliable technical support for breeding of anti-browning pear varieties, and has important economic value and wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology and fruit tree breeding, in particular to a pear fruit flesh anti-browning trait related InDel marker, primer, kit and application thereof in pear fruit flesh anti-browning breeding. BACKGROUND

[0002] Pear (Pyrus L.) is an important fruit widely planted and consumed globally, and the browning problem of fresh-cut fruit flesh significantly affects the appearance quality, commodity value and consumer acceptance of the fruit. Studies have shown that the core mechanism of pear fruit flesh browning after fresh cutting is that polyphenol oxidase (PPO) in fruit flesh cells catalyzes the oxidation of endogenous phenolic substances to quinone compounds in the presence of oxygen, and quinone further polymerizes to form dark brown pigments. This process is related to the content of antioxidant substances in the fruit flesh, the integrity of the cell structure and environmental factors (such as temperature, humidity, oxygen concentration, etc.).

[0003] In recent years, the rapid development of molecular biology technology has brought great changes to the field of fruit tree breeding, and the molecular marker assisted selection (MAS) breeding technology has become a key means to improve the efficiency of breeding. Compared with traditional phenotype selection methods, MAS technology detects genetic variation at the DNA level, has the advantages of rapidness, precision, and being not affected by the environment, etc., and can effectively shorten the breeding period and improve the accuracy of selection. Insertion / deletion (InDel) molecular markers, as polymorphic markers developed based on short fragment insertion or deletion variation in genome sequences, have been widely used in plant genetic breeding due to their high genetic stability, simple detection operation, strong correlation with target traits, etc., providing important technical support for fruit tree molecular assisted selection and showing broad application prospects.

[0004] However, the current research on molecular markers for pear fruit flesh anti-browning traits is still in its infancy, and existing achievements are mostly focused on phenotype correlation analysis or genome-wide association analysis (GWAS), and there is no report on the development of precise InDel molecular markers for specific anti-browning candidate genes (such as PPO inhibitor encoding protein, antioxidant enzyme or genes regulating browning related signal pathways). Due to the lack of specific molecular markers closely related to the anti-browning trait, traditional breeding still relies on phenotype screening, resulting in long breeding cycle and low efficiency of anti-browning varieties, which is difficult to meet the urgent needs of the industry for high-quality anti-browning pear varieties.

[0005] Therefore, developing a specific InDel molecular marker that is highly associated with genes regulating browning resistance in pear flesh is of great theoretical and practical significance for improving the accuracy and efficiency of browning resistance breeding and accelerating the breeding process of browning-resistant varieties. Summary of the Invention

[0006] To fill the aforementioned gaps in existing research, this application develops a novel and effective InDel marker and its primers related to the browning resistance trait of pear flesh. The InDel marker of this application can efficiently and accurately screen pear progeny plants and superior varieties with browning resistance characteristics, providing an efficient and reliable technical solution for pear browning resistance breeding and fruit quality improvement.

[0007] Specifically, in the first aspect, this application provides an InDel marker related to the browning resistance trait of pear flesh. The InDel marker is located at 22496111-22496306bp on chromosome 2 of the Red Fragrant Pear genome, with a total length of 196bp. Its nucleotide sequence is shown in SEQ ID NO.1. The InDel marker is used to identify the browning resistance trait of pear flesh.

[0008] Furthermore, the aforementioned InDel marker is a polymorphic site located in the promoter region of the PbrPIRL gene encoding the pear Ras-related leucine-rich repeat protein, indicating deletion or insertion. Individual pear plants prone to browning exhibit the deletion of the InDel marker sequence, while individual pear plants resistant to browning exhibit the insertion of the InDel marker sequence.

[0009] Furthermore, the amplification primer sequences for the InDel-labeled components are as follows:

[0010] Forward primer: 5'-AGAGTCAGACCCTAAATG-3' (SEQ ID NO.2);

[0011] Reverse primer: 5'-ACTACCCGCCCTCATAAA-3' (SEQ ID NO.3).

[0012] Secondly, this application also relates to the application of the above-mentioned InDel-marked detection reagent in the breeding of pear pulp resistant to browning.

[0013] Thirdly, this application provides a test kit for identifying the browning resistance trait of pear pulp, the test kit containing a test reagent for the aforementioned InDel marker.

[0014] Furthermore, the above-mentioned test kit contains the following primer pairs:

[0015] Forward primer: 5'-AGAGTCAGACCCTAAATG-3';

[0016] Reverse primer: 5'-ACTACCCGCCCTCATAAA-3'.

[0017] Fourthly, this application provides a method for identifying the browning resistance trait of pear pulp, the method comprising the following steps:

[0018] (1) Extracting genomic DNA from the pear material to be tested

[0019] Genomic DNA was extracted from the pear leaves to be tested;

[0020] (2) PCR amplification of genomic DNA from the pear material to be tested.

[0021] a. The reaction system includes:

[0022] Genestar (Mix), 10 μL;

[0023] 10 μM forward primer: 5'-AGAGTCAGACCCTAAATG-3', 0.8 μL;

[0024] 10 μM reverse primer: 5'-ACTACCCGCCCTCATAAA-3', 0.8 μL;

[0025] 1 μL of template DNA with a DNA concentration of 50 ng / μL;

[0026] ddH2O, 7.4 μL;

[0027] b. The reaction procedure is as follows:

[0028] Pre-denaturation: 94℃, 2 min;

[0029] Denaturation at 94℃ for 30 seconds; Annealing at 60℃ for 30 seconds; Extension at 72℃ for 30 seconds; 30 cycles in total;

[0030] Final extension at 72℃ for 5 minutes;

[0031] After the PCR amplification program is completed, store it at 4°C.

[0032] (3) Detection of amplification products

[0033] PCR amplification products were detected by polyacrylamide gel electrophoresis. An 8% gel was used, and electrophoresis was performed at a constant voltage of 160V. After electrophoresis, the gel was rinsed for 5 seconds and then silver stained for color development. The PCR amplification products of browning-resistant pear varieties showed one band at 196bp on the electrophoresis pattern, while the PCR amplification products of browning-prone pear varieties showed two bands at 196bp and 176bp on the electrophoresis pattern.

[0034] In addition, this application also provides a method for detecting InDel markers to identify the browning resistance trait of pear pulp, the method including the step of detecting the InDel markers.

[0035] In summary, this invention is the first to discover a 20bp DNA sequence with a heterozygous deletion in the promoter region of the PbrPIRL gene in browning-prone pear varieties. This sequence can be used as an InDel molecular marker for rapid identification of browning-resistant and browning-prone pear varieties. PCR amplification products of browning-resistant progeny or pear varieties show a clear band at 196bp on the electrophoresis pattern, while PCR amplification products of browning-prone pear varieties show two bands at 196bp and 176bp on the electrophoresis pattern. This can be used for large-scale screening of browning-resistant pear varieties. Based on the molecular markers and methods of this invention, browning-resistant varieties can be rapidly identified through routine molecular experiments. Furthermore, the detection method of this invention is simple to operate, highly specific, and accurate, and is unaffected by external factors such as climate and environment, providing reliable technical support for the breeding of browning-resistant pear varieties, and has significant economic value and broad application prospects. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 QTL mapping of the main effect of pear pulp against browning.

[0038] Figure 2 This is a diagram showing the structural variation analysis of IGV in candidate genes during resequencing from different germplasm resources.

[0039] Figure 3 The image shows the polyacrylamide gel electrophoresis results of InDel markers in the 'Mantianhong' (prone to browning) × 'Hongxiangsu' (resistant to browning) genetic population.

[0040] Figure 4 The image shows the results of polyacrylamide gel electrophoresis verification of InDel markers in different brown pear germplasm resources. Detailed Implementation

[0041] To make the objectives and technical solutions of this application clearer, the following detailed description is provided in conjunction with specific embodiments. Those skilled in the art can easily understand other advantages of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application.

[0042] Before further describing the specific implementation of this application, it should be understood that the scope of protection of this application is not limited to the specific implementation schemes described below; it should also be understood that the terminology used in the embodiments of this application is for describing specific implementation schemes, and not for limiting the scope of protection of this application.

[0043] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all instruments, reagents, and materials used in this application are commercially available or commonly used in the art. The methods in the following embodiments are conventional methods in the art, unless otherwise specified.

[0044] Example 1: Development of InDel markers for browning resistance in pear pulp

[0045] 1. Test materials

[0046] The research materials for this invention all came from the pear resource nursery of the Zhengzhou Fruit Research Institute of the Chinese Academy of Agricultural Sciences, including 339 individual plants of the F1 generation of the cross between 'Mantianhong' (prone to browning) and 'Hongxiangsu' (resistant to browning); as well as 34 resistant natural varieties and 31 prone germplasm resources.

[0047] 2. Test methods

[0048] 2.1 Development of molecular markers for anti-browning of pear pulp

[0049] Based on previous interval mapping (IM) analysis using MapQTL software, this study located the major QTL for browning resistance on chromosome 2 of the 'Dangshan Su' genome, with a QTL locus spanning 49.292 cM (from 43.476 cM to 92.768 cM). This interval was then mapped to the Chr2:12.99 Mb-28.53 Mb physical region of the reference genome of the browning-resistant variety 'Hongxiang Su'. Figure 1In the F1 generation of the cross between 'Mantianhong' (prone to browning) and 'Hongxiangsu' (resistant to browning), extreme browning-resistant and extreme browning-prone single plants, identified through multiple years of continuous browning identification, were selected for molecular marker and phenotypic concordance verification. Based on resequencing data from over 300 pear germplasm resources, high-density InDel molecular markers were designed in the localization region using Primer 5.0 software. The primer design principles were: primer length 18-30 bp, TM value 55-65℃, and product length 80-300 bp.

[0050] 2.1.1 PCR amplification reaction

[0051] PCR amplification was performed using a 2×Taq premixed PCR reaction kit, following the manufacturer's instructions. The kit consisted of 10 μL Genestar (Mix); 0.8 μL forward primer; 0.8 μL reverse primer; 7.4 μL ddH2O; and 1 μL DNA template. The PCR reaction program included five steps: pre-denaturation, denaturation, annealing, extension, and final extension. The program parameters were set as follows: 94℃-2 min; 94℃-30 s, 60℃-30 s, 72℃-30 s, 30 cycles; 72℃-5 min. The amplified products were stored at 4℃.

[0052] 2.1.2 Polyacrylamide gel electrophoresis

[0053] Prepare an 8% polyacrylamide gel: Add 30.7 mL of ddH2O, 14 mL of 30% gel preparation solution, 5 mL of 10×TBE buffer, 350 μL of 10% APS, and 33 μL of TEMED in sequence, and mix thoroughly. Then, slowly inject the mixture into the gap between two glass plates using a syringe, avoiding air bubbles. Insert the sample comb and let it stand for approximately 30 minutes to allow the gel to solidify. After solidification, mount the glass plates into the electrophoresis tank, add 0.5×TBE electrophoresis buffer until it covers the short glass plate, remove the comb, and clean any residual gel and air bubbles from the sample wells. Add a 600 bp DNA marker to the first sample well, and then add 0.7 μL of PCR product to the subsequent wells. Perform electrophoresis at a constant voltage of 160 V. Rinse: After electrophoresis, remove the glass plates and recover the electrophoresis buffer for future use. Carefully separate the glass plates, transfer the gel to a tray containing water, and rinse for 5 seconds. Silver staining: Immerse the gel in a pre-prepared 0.5 L of 0.2% silver nitrate solution and stain on a shaker for 8 min. Then, rinse the gel with deionized water for 1-2 min, and transfer it to a sodium hydroxide solution containing formaldehyde (8 g sodium hydroxide and 4 mL formaldehyde dissolved in 0.5 L of deionized water). Shake on a shaker until the bands are clear. Finally, observe the gel under a light box, record the results, and photograph for storage.

[0054] After three rounds of nested primer screening, a total of 237 primer pairs were screened, and 13 pairs with high concordance rates were finally obtained, narrowing the candidate regions to two regions on chromosome 2: 20.4-22.5 Mb and 24.1-26.1 Mb. Within the candidate regions, there were 421 genes. Based on resequencing data from the parents 'Mantianhong' and 'Hongxiangsu', as well as extreme browning phenotype materials in natural populations (including browning-resistant germplasm resources 'Korla Fragrant Pear', 'Eli', and 'Danxia Red', and browning-prone germplasm resources 'Late-maturing Torch Pear', 'Xiangyun Torch Pear', and 'Meirensu'), genes with structural variations (deletion or insertion ≥5 bp) in promoters or coding regions were screened using IGV, and primers were designed at the structural variation sites using Primer 5.0 software.

[0055] 2.2 Validation of InDel molecular markers in different germplasm resources

[0056] The InDel molecular markers were used to further identify genotypes in pear germplasm resources that were prone to and resistant to browning in natural populations, and to analyze the accuracy of genotype and phenotype in natural populations.

[0057] 3. Test Results

[0058] 3.1 Development of molecular markers for anti-browning regions in pear pulp

[0059] Using the 'Hongxiangsu' genome as a reference, and based on resequencing data from the parents 'Mantianhong', 'Hongxiangsu', and extreme phenotypes of pulp browning in natural populations, IGV analysis was used to identify genes with structural variations in their promoters or coding regions, initially yielding 88 genes with structural variations. Figure 2 Specific primers were designed and selected using Premier5 software to verify candidate genes for pear pulp resistance to browning. Using the developed InDel marker, 20 browning-resistant and 16 browning-prone individual plants (Table 1) were selected from 339 F1 plants of 'Hongxiangsu' (♂) × 'Mantianhong' (♀) for verification, and the concordance rate between genotype and phenotype in the genetic population was analyzed. Finally, 24 genes with high concordance rates between genotype and phenotype were selected (Table 2). These 24 molecular markers were all located in the promoter regions of their corresponding genes. The primer amplification products of the InDel molecular markers are shown below. Figure 3 As shown, the concordance rate between its phenotype and genotype reached 94.4%.

[0060] Table 1. Genetic population lines used in the validation experiment

[0061]

[0062] Table 2 High-concordance molecular markers

[0063]

[0064]

[0065] 3.2 Validation of InDel molecular markers in natural populations

[0066] Using the aforementioned InDel molecular markers, based on natural population germplasm resources, 34 browning-resistant germplasm resources and 31 browning-prone germplasm resources were selected (Table 3, including two parents, Hongxiangsu and Mantianhong, totaling 67 materials) for population validation of the molecular markers. Genotype-phenotype association analysis identified three candidate genes highly associated with the browning phenotype: Pear_GLEAN_10026027, Pear_GLEAN_10026072, and Pear_GLEAN_10007734. Their genotype and phenotypic variations were highly consistent. Figure 4 ).

[0067] Table 3. Varieties from natural populations in the verification experiment.

[0068]

[0069]

[0070] The above experimental results show that the PCR amplification products of browning-resistant pear varieties show a clear band at 196 bp on the electrophoresis pattern, while the PCR amplification products of easily browning pear varieties show two bands at 196 bp and 176 bp on the electrophoresis pattern. This can be used for large-scale screening of browning-resistant pear varieties. Based on the molecular markers and methods of this invention, browning-resistant varieties can be rapidly identified through routine molecular experiments.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. An InDel marker associated with the browning resistance trait of pear flesh, characterized in that, The InDel marker is located at 22496111-22496306bp on chromosome 2 of the Red Fragrant Pear genome, with a total length of 196bp. Its nucleotide sequence is shown in SEQ ID NO.

1. The InDel marker is used to identify the browning resistance trait of pear flesh.

2. The InDel mark according to claim 1, characterized in that, The InDel marker is a polymorphic site located in the promoter region of the PbrPIRL gene, which encodes a pear Ras-related leucine-rich repeat protein. Individual pear plants prone to browning exhibit the deletion of the InDel marker sequence, while individual pear plants resistant to browning exhibit the insertion of the InDel marker sequence.

3. The InDel mark according to claim 1, characterized in that, The amplification primer sequences for the InDel-labeled markers are as follows: Forward primer: 5'-AGAGTCAGACCCTAAATG-3'; Reverse primer: 5'-ACTACCCGCCCTCATAAA-3'.

4. The application of the InDel-labeled detection reagent according to any one of claims 1-3 in the breeding of pear flesh resistant to browning.

5. A test kit for identifying the browning resistance trait of pear pulp, characterized in that, The test kit contains a test reagent for the InDel label as described in claim 1.

6. The detection kit according to claim 5, characterized in that, The detection kit contains the following primer pairs: Forward primer: 5'-AGAGTCAGACCCTAAATG-3'; Reverse primer: 5'-ACTACCCGCCCTCATAAA-3'.

7. A method for identifying the browning resistance trait of pear pulp, characterized in that, The method includes the following steps: (1) Extracting genomic DNA from the pear material to be tested Genomic DNA was extracted from the pear leaves to be tested; (2) PCR amplification of genomic DNA from the pear material to be tested. a. The reaction system includes: Genestar (Mix), 10 μL; 10 μM forward primer: 5'-AGAGTCAGACCCTAAATG-3', 0.8 μL; 10 μM reverse primer: 5'-ACTACCCGCCCTCATAAA-3', 0.8 μL; 1 μL of template DNA with a DNA concentration of 50 ng / μL; ddH2O, 7.4 μL; b. The reaction procedure is as follows: Pre-denaturation: 94℃, 2 min; Denaturation at 94℃ for 30 seconds; Annealing at 60℃ for 30 seconds; Extension at 72℃ for 30 seconds; 30 cycles in total; Final extension at 72℃ for 5 minutes; After the PCR amplification program is completed, store it at 4°C. (3) Detection of amplification products PCR amplification products were detected by polyacrylamide gel electrophoresis. An 8% gel was used, and electrophoresis was performed at a constant voltage of 160V. After electrophoresis, the gel was rinsed for 5 seconds and then silver stained for color development. The PCR amplification products of browning-resistant pear varieties showed one band at 196bp on the electrophoresis pattern, while the PCR amplification products of browning-prone pear varieties showed two bands at 196bp and 176bp on the electrophoresis pattern.

8. A method for detecting InDel markers to identify the browning resistance trait of pear pulp, characterized in that, The method includes the step of detecting the InDel marker as described in claim 1.

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