InDel marker for identifying red pulp character of pear and application of InDel marker

Abstract

The invention discloses an InDel marker for identifying the red pulp character of a pear, a primer and application of the InDel marker. The InDel marker is located at the 4882085-4882277 bp position of a chromosome 5 of a red fragrant pear genome, and the overall length is 182 bp. By utilizing the InDel marker and the method, the pulp color of the hybrid generation of the hybrid combination of the red-peel red-pulp parent and the green-peel white-pulp parent can be quickly and accurately identified in the pear seedling stage through conventional molecular detection, and the green-peel white-pulp pears and the hybrid generation of the red-peel red-pulp pears are efficiently distinguished. The method disclosed by the invention can be used for detecting each tissue, organ and each development stage of the plant, has the advantages of convenience, rapidness, good specificity, high accuracy, no influence of climate environment and the like, can be used for early auxiliary selection of the phenotype of the red flesh of the pear plant, can greatly improve the breeding selection efficiency and accuracy of the red flesh pear variety, and has a wide application prospect. The blank in the field of pear plant red pulp character molecular marker development is filled up, and great promotion significance is achieved for pear germplasm improvement and fruit value improvement.

Description

Technical Field

[0001] The present application belongs to the technical field of artificial cultivation and molecular markers of pears, and specifically relates to an InDel marker, primers, applications thereof, and a kit for identifying the red flesh trait of pears. The molecular markers and primers of the present application can be used for the identification and variety breeding of red flesh pears. Background Art

[0002] The color of pear (Pyrus spp.) flesh is a key trait that determines its nutritional quality and market value. Red flesh, rich in antioxidants such as anthocyanins and flavonoids, is highly valued in fresh consumption, processing, and health food development.

[0003] Currently, pear breeding relies entirely on phenotypic observation after fruit ripening. However, pear trees have a long juvenile stage, typically requiring three to five years or even longer to reach fruiting age. This results in a long breeding cycle, high resource investment, and low selection efficiency. If selection for superior phenotypes could be performed early in the seedling stage, breeding efficiency would be significantly improved and investment reduced.

[0004] Molecular marker-assisted selection (MAS) technology makes it possible to identify traits early. Molecular screening of the red-fleshed phenotype will greatly improve the breeding efficiency of red-fleshed pears. However, due to the scarcity of red-fleshed pear resources, the application of MAS technology in the selection and cultivation of red-fleshed pears is still blank.

[0005] To date, scientists have conducted extensive and in-depth research on the genetic relationship between peel and flesh color. Genetic studies of Rosaceae fruit trees have found that peel and flesh color traits are often regulated by different genetic loci. For example, apple flesh (including that of red-skinned apples) is mostly white, while red-fleshed apples have skins that range from red to yellow and other colors (Cong Peihua; Chinese Apple Varieties, 1st ed., Beijing: China Agriculture Press, 2015). Furthermore, due to the scarcity of red-fleshed pear germplasm resources, the peels of currently discovered red-fleshed pear varieties are mostly red, while white-fleshed pears have a wider range of peel colors, including red, green, yellow, and brown (Li Xiugen and Zhang Shaoling; Chinese Pear Trees, 1st ed., Beijing: China Agriculture Press, 2020). These observations suggest that peel and flesh color are not genetically co-evolved. In view of the above situation, developing InDel markers related to the red flesh trait of pears and using them for the identification and selective breeding of red flesh pears will have great promoting significance for the improvement of pear germplasm and the enhancement of fruit value. Summary of the Invention

[0006] In order to fill the above-mentioned gaps in existing research applications, this application has developed a new and effective InDel marker and related primers for identifying the red flesh trait of pears, and provided an InDel marker detection kit for identifying the red flesh trait of pears. The molecular marker and detection kit of this application can be used for precise, targeted and efficient breeding of red flesh pears.

[0007] The inventors discovered in their long-term research on pear hybrid breeding that in some specific pear hybrid populations (such as green skin and white flesh × red skin and red flesh), a "linked inheritance" phenomenon of red skin and red flesh phenotypes can be observed. Based on this discovery, we carried out selection experiments on the red flesh phenotype of specific hybrid breeding combinations, and innovatively applied the established red skin phenotype molecular markers to the screening of red flesh phenotypes in specific hybrid populations of pears, achieving efficient molecular marker-assisted selection of red flesh phenotypes in Pyrus plants, filling the technical gap in the field of molecular marker development for red flesh traits in Pyrus plants. This scheme can be used for the early selection of red flesh phenotypes in specific hybrid populations of pears, and has high application value for the innovation of variety quality of special fruit varieties.

[0008] More specifically, when screening hybrid populations using existing peel color markers, the inventors discovered that the genotyping results for marker InDel-48-6 were highly consistent with the flesh color of individual fruits within the population. Further verification revealed that the InDel variant of this marker co-segregated with the flesh color phenotype, with a discrimination accuracy of 100%. This discovery, for the first time, reveals the cross-trait potential of peel color markers for flesh color identification, providing a breakthrough tool for establishing early molecular identification of pear flesh color and effectively shortening the breeding time for red-fleshed varieties. Based on this discovery, this application proposes a novel InDel marker and related primers for identifying the red flesh trait in pears, as well as a detection kit for this InDel marker.

[0009] Specifically, in the first aspect, the present application provides an InDel marker (InDel-48-6 marker) for identifying the red flesh trait of pears. The InDel marker is located at 4882085-4882277bp on chromosome 5 of the red fragrant and crispy pear genome, with a total length of 182bp. Its nucleotide sequence is shown in SEQ ID NO.1. The InDel marker is used to identify the flesh color of the hybrid offspring of a hybrid combination of a red-skinned, red-flesh parent and a green-skinned, white-flesh parent.

[0010] Furthermore, the sequence of the forward primer (upstream primer) of the above-mentioned InDel marker is shown in SEQ ID NO.2, which is 5'-TCTATATTGCTTGCACCGAA-3', and the sequence of the reverse primer (downstream primer) is shown in SEQ ID NO.3, which is 5'-TTAATGGTGGTGGAGTCAAG-3'; when used for screening the red flesh trait of pears, this pair of primers can amplify two clear bands in both red flesh and white flesh individual plants, among which the double band combination of 172bp and 178bp is for red flesh individuals, and the double band combination of 172bp and 182bp is for white flesh individuals.

[0011] In a second aspect, the present application also relates to the use of the above-mentioned InDel-labeled detection reagent in the breeding of red-fleshed pear varieties.

[0012] In a third aspect, the present application provides an InDel marker detection kit for identifying the red flesh trait of pears, wherein the detection kit comprises a detection reagent for the above-mentioned InDel marker.

[0013] Furthermore, the above detection kit contains the following primer pairs:

[0014] Forward primer: 5'-TCTATATTGCTTGCACCGAA-3',

[0015] Reverse primer: 5′-TTAATGGTGGTGGAGTCAAG-3′.

[0016] In a fourth aspect, the present application provides a method for detecting an InDel marker for identifying the red flesh trait of pears, comprising the following steps:

[0017] (1) Extracting genomic DNA of the pear material to be tested

[0018] The genomic DNA was extracted from the young leaf tissue of the pear to be tested using a genomic DNA extraction kit;

[0019] (2) PCR amplification of the genomic DNA of the pear material to be tested

[0020] a. The reaction system includes:

[0021] Genster (Mix), 10 μL;

[0022] 10 μM forward primer: 5′-TCTATATTGCTTGCACCGAA-3′, 0.8 μL;

[0023] 10 μM reverse primer: 5′-TTAATGGTGGTGGAGTCAAG-3′, 0.8 μL;

[0024] 1 μL of template DNA with a DNA concentration of 50 ng / μL;

[0025] ddH2O, 7.4 μL;

[0026] b. The reaction procedure is:

[0027] Pre-denaturation at 94°C for 2 min;

[0028] Denaturation at 94°C for 30 seconds; annealing at 50°C for 30 seconds; extension at 72°C for 30 seconds; 30 cycles in total;

[0029] Final extension at 72°C for 5 min;

[0030] After the PCR amplification process is completed, store it in a 4°C environment;

[0031] (3) Amplification product detection

[0032] Polypropylene gel electrophoresis technology was used to detect PCR amplification products. 8% gel was used and electrophoresis was performed at a constant voltage of 145V for 150 minutes. After the electrophoresis, the gel was rinsed for 5 seconds and silver staining was performed. This pair of primers can amplify two clear bands in both red meat and white meat plants. The double band combination of 172bp and 178bp belongs to red meat individuals, and the double band combination of 172bp and 182bp belongs to white meat individuals.

[0033] In addition, the present application also provides an InDel marker detection method for identifying the red flesh trait of pears, which includes the step of detecting the above-mentioned InDel marker.

[0034] In summary, the InDel markers and related primers for identifying the red flesh trait of pears proposed in this application are suitable for identifying the flesh color of hybrid offspring of a cross combination of a red-skinned, red-flesh parent and a green-skinned, white-flesh parent, and can be used for early auxiliary selection of the red flesh phenotype of Pyrus plants. This method is accurate and efficient, and can effectively shorten the breeding time of red flesh pear varieties, filling the technical gap in the field of molecular marker development for red flesh traits in Pyrus plants, and has great promoting significance for the improvement of pear germplasm and the enhancement of fruit value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 This is a diagram showing the phenotypic identification results of pear fruit in an experiment according to an embodiment of the present application.

[0037] Figure 2 Schematic diagram of the position of the InDel-48-6 marker in this application on the genetic map and physical map.

[0038] Figure 3 This is the electrophoresis result (development) of the product amplified by the primer labeled InDel-48-6 of this application.

[0039] Figure 4 This is the electrophoresis result (application) of the amplification product labeled with the primer InDel-48-6 of this application. DETAILED DESCRIPTION

[0040] In order to make the purpose and technical solution of this application more clearly understood, the following further describes this application in detail with reference to specific embodiments. Those skilled in the art can easily understand other advantages of this application from the contents disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of this application.

[0041] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present application are for describing specific specific embodiments, rather than for limiting the scope of protection of the present application.

[0042] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by those skilled in the art. In this application, unless otherwise specified, all instruments, reagents, and raw materials are commercially available or commonly used in the art. The methods in the following examples, unless otherwise specified, are conventional methods in the art.

[0043] Example 1

[0044] This example provides the development of InDel markers related to the red flesh trait of pears

[0045] 1. Phenotypic identification

[0046] The flesh color of individual fruit from the F1 strain of 'Zhongli 5' x 'Invalid' (eight red-fleshed and seven white-fleshed) was determined. All fruit were grown at the Zhengzhou Fruit Research Institute of the Chinese Academy of Agricultural Sciences. Three healthy, uniformly sized pear fruits were randomly selected at maturity for phenotypic evaluation of flesh color. Figure 1 ).

[0047] 2. Development of pear flesh color marking

[0048] In the early development of the red skin marker, we constructed a genetic population related to the skin, and through investigation and statistical analysis of the skin color traits, we screened out molecular markers related to the red skin trait, and used these markers to construct genetic maps and physical maps ( Figure 2 ). During the phenotypic identification of the red meat population, it was found that all red meat individuals had red skin. This phenomenon suggests that there is a potential genetic linkage between the red skin and red meat traits. Based on the above association hypothesis, 22 pairs of markers with a high degree of co-segregation with the red skin trait (≥90%) were preferentially selected from the candidate markers for the red skin trait developed in the early stage for screening and verification of flesh color. After statistical analysis, only one pair of markers (InDel-48-6) among the 22 pairs of markers had a co-segregation of the amplified fragment length with the flesh color. The phenotypic discrimination accuracy of this marker in the verification set was 100%, and the amplification stability was high ( Figure 3 ).

[0049] The sequence of the tag is:

[0050] TCTATATTGCTTGCACCGAAACTTTTTTTTTTTCAATTTTTTCTTAATCAACATTGCATTTCAACACAAAAGATATAATAACACATAAGAGCGGAACTATTAAGGAACCAAAGTGATCCGAGTCCATACACATATGAAAGAACCCTTCCTCACATAAAACTTGACTCCACCACCATTAA (SEQ ID NO. 1).

[0051] The specific primer sequences for this marker are:

[0052] Forward primer (upstream primer): 5'-TCTATATTGCTTGCACCGAA-3' (SEQ ID NO. 2);

[0053] Reverse primer (downstream primer): 5'-TTAATGGTGGTGGAGTCAAG-3' (SEQ ID NO. 3).

[0054] Example 2

[0055] This example is the application of the InDel marker provided in Example 1 in the identification of the red flesh trait of pears

[0056] Extract DNA from the leaves of all F1 plants, and use PCR amplification and molecular marker verification to predict and identify the fruit-setting phenotype of the offspring. The specific steps are as follows:

[0057] (1) DNA extraction

[0058] Young leaf tissue, pre-frozen at -80°C, was ground using a high-throughput grinder and then added to a preheated lysis buffer containing 2% β-mercaptoethanol. Lysis was performed in a 65°C water bath for 20-30 minutes. Chloroform was then added for mixing and centrifugation. The supernatant was adsorbed with a binding buffer and then purified using an inhibitor removal buffer and a rinse buffer (containing anhydrous ethanol). Finally, the DNA product was obtained using an elution buffer. All centrifugation steps were performed using a refrigerated centrifuge (12,700 × g).

[0059] (2) PCR amplification

[0060] a. The reaction system includes:

[0061] Genster (Mix) 10 μL;

[0062] 10 μM forward primer 0.8 μL;

[0063] 10 μM reverse primer 0.8 μL;

[0064] Template DNA 1 μL (DNA concentration 50 ng / μL);

[0065] ddH2O 7.4μL (Up to 20μL);

[0066] A total of 20 μL.

[0067] b. The reaction procedure is:

[0068] Pre-denaturation at 94°C for 2 minutes,

[0069] Denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 30 cycles.

[0070] Final extension at 72°C for 5 min.

[0071] After the PCR amplification process is completed, store it in a 4°C environment.

[0072] (3) Detection of product amplification results

[0073] Polypropylene gel electrophoresis (PAGE) technology was used, using 8% gel and a constant voltage of 145V for electrophoresis for 150 minutes. After the electrophoresis, the gel was rinsed for 5 seconds and silver staining was performed. This pair of primers can amplify two clear bands in both red meat and white meat plants. The double band combination of 172bp and 178bp belongs to red meat individuals, and the double band combination of 172bp and 182bp belongs to white meat individuals.

[0074] According to the method described above, the PAGE results were judged and 38 red meat plants were screened out of 67 plants in the F1 population by InDel-48-6 marker ( Figure 4). Among them, those marked with numbers are individual plants predicted to be red flesh, and those marked in red font have passed phenotypic identification. In the second year after molecular identification, phenotypic identification was performed on the newly produced individual plants, and it was found that 9 individual plants, including No. 14, No. 21, No. 26, No. 29, No. 33, No. 48, No. 51, No. 53 and No. 56, were red flesh individual plants, and 10 individual plants, including No. 1, No. 2, No. 3, No. 18, No. 19, No. 25, No. 31, No. 40, No. 44 and No. 54, were white flesh individual plants. The phenotypic identification results are completely consistent with the predicted results. It can be seen that using the InDel-48-6 molecular marker of the present application, the individual plants with red and white flesh colors can be quickly distinguished during the seedling stage.

[0075] The preferred specific implementation methods and embodiments of the present application are described in detail above, but the present application is not limited to the above implementation methods and embodiments. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the concept of the present application.

Claims

1. An InDel marker for identifying the red flesh trait of pear, characterized in that: The InDel marker is located at 4882085-4882277bp on chromosome 5 of the red fragrant and crispy pear genome, with a total length of 182bp. Its nucleotide sequence is shown in SEQ ID NO.

1. The InDel marker is used to identify the flesh color of the hybrid offspring of a hybrid combination of a red-skin, red-flesh parent and a green-skin, white-flesh parent.

2. The InDel marker according to claim 1, characterized in that The forward primer sequence of the InDel tag is 5'-TCTATATTGCTTGCACCGAA-3', and the reverse primer sequence is 5'-TTAATGGTGGTGGAGTCAAG-3'.

3. Use of the InDel-labeled detection reagent according to claim 1 or 2 in the breeding of red-fleshed pear varieties.

4. An InDel marker detection kit for identifying the red flesh trait of pear, characterized in that: The detection kit comprises a detection reagent for the InDel marker according to claim 1.

5. The detection kit according to claim 4, characterized in that The detection kit contains the following primer pairs: Forward primer: 5'-TCTATATTGCTTGCACCGAA-3', Reverse primer: 5′-TTAATGGTGGTGGAGTCAAG-3′.

6. A method for detecting an InDel marker for identifying the red flesh trait of pear, characterized in that: The method comprises the following steps: (1) Extracting genomic DNA of the pear material to be tested The genomic DNA was extracted from the young leaf tissue of the pear to be tested using a genomic DNA extraction kit; (2) PCR amplification of the genomic DNA of the pear material to be tested a. The reaction system includes: Genster (Mix), 10 μL; 10 μM forward primer: 5′-TCTATATTGCTTGCACCGAA-3′, 0.8 μL; 10 μM reverse primer: 5′-TTAATGGTGGTGGAGTCAAG-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: Pre-denaturation at 94°C for 2 min; Denaturation at 94°C for 30 seconds; annealing at 50°C for 30 seconds; extension at 72°C for 30 seconds; 30 cycles in total; Final extension at 72°C for 5 min; After the PCR amplification process is completed, store it in a 4°C environment; (3) Amplification product detection Polypropylene gel electrophoresis technology was used to detect PCR amplification products. 8% gel was used and electrophoresis was performed at a constant voltage of 145V for 150 minutes. After the electrophoresis, the gel was rinsed for 5 seconds and silver staining was performed. This pair of primers can amplify two clear bands in both red meat and white meat plants. The double band combination of 172bp and 178bp belongs to red meat individuals, and the double band combination of 172bp and 182bp belongs to white meat individuals.

7. A method for detecting an InDel marker for identifying the red flesh trait of pear, characterized in that: The method comprises the step of detecting the InDel marker according to claim 1.

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