Specific molecular marker of malus hallings 1 thornless strain and application thereof

Abstract

The application provides a specific molecular marker of Malus sieversii non-stinging No.1 non-stinging strain and application thereof, and belongs to the technical field of crop molecular breeding. The application can simply and quickly identify the non-stinging No.1 non-stinging strain in Malus sieversii materials by simultaneously detecting two marker sites through one double-PCR, and using two InDel markers, and meanwhile, the simultaneous detection of the two marker sites can ensure the accuracy, effectiveness and reliability of the detection results by mutual verification and mutual testing, so that the application has great application value in the aspects of distinguishing Malus sieversii varieties and identifying interspecific hybrid seeds, and can provide technical support for innovative large-fruit non-stinging fresh-eating Malus sieversii varieties. In addition, the application can shorten the breeding cycle of non-stinging Malus sieversii and accelerate the breeding process by extracting leaf genomic DNA of the seedling of the breeding material at the seedling stage for double-PCR amplification, and identifying whether it is the non-stinging No.1 non-stinging strain by observing electrophoresis bands.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of crop molecular breeding, and particularly relates to a specific molecular marker of a Rosa roxburghii Tratt. thornless No. 1 thornless line and application thereof. BACKGROUND

[0002] Rosa roxburghii Tratt. Rosa roxburghii Tratt. is a new fruit tree unique to China and is being vigorously developed. It is commercially planted and product-processed in Guizhou, Sichuan, Henan and Guangxi Zhuang Autonomous Region. The cultivation area in Guizhou Province has reached more than 2.3 million mu, becoming a characteristic industry developed in the province. Rosa roxburghii Tratt. has extremely high nutritional, health care and medicinal values, but due to the fact that the fresh consumption population is limited due to the dense thorns on the fruit, more than 90% of the fresh fruit is used for processing, and less than 10% is used for fresh consumption. At the same time, the thorns of Rosa roxburghii Tratt. also bring many inconveniences to cultivation management, fresh fruit harvesting, transportation and processing. Therefore, the collection and research of thornless Rosa roxburghii Tratt. germplasm resources become the key to improving the quality of fresh fruit of Rosa roxburghii Tratt. R. roxburghii Tratt. f. esetesa Ku

[0003] Due to long-term seedling propagation, natural variation and interspecific hybridization, the traits of Rosa roxburghii Tratt. are uneven, and the varieties are mixed, and multiple variation types appear. At the same time, the thornless variation of Rosa roxburghii Tratt. and the ordinary Rosa roxburghii Tratt. only have one difference in appearance, that is, the fruit is thornless, and there is no difference in leaf morphology. Therefore, it is difficult to distinguish them by using morphological methods. Moreover, the method for identifying the thornless trait by using inDel markers has not been involved in the research on specific molecular markers of Rosa roxburghii Tratt. SUMMARY

[0004] In order to solve the above technical problems, the application provides a specific molecular marker of a Rosa roxburghii Tratt. thornless No. 1 thornless line and application thereof. The application can simply, quickly, accurately and effectively identify the Rosa roxburghii Tratt. thornless No. 1 thornless line in the Rosa roxburghii Tratt. material by detecting two marker sites at one time by using two InDel markers, has great application value in the aspects of distinguishing the Rosa roxburghii Tratt. varieties and identifying interspecific hybrids, can provide technical support for innovating large-fruit thornless fresh-eating Rosa roxburghii Tratt. varieties, and can shorten the breeding period of the thornless Rosa roxburghii Tratt. and accelerate the breeding process.

[0005] In order to achieve the above purpose, the application adopts the following technical scheme:

[0006] The application provides a molecular marker, which is composed of a molecular marker 153.61 and a molecular marker 341.112; the upstream primer sequence of the molecular marker 153.61 is shown in SEQ ID No. 1, and the downstream primer sequence is shown in SEQ ID No. 2; the upstream primer sequence of the molecular marker 341.112 is shown in SEQ ID No. 3, and the downstream primer sequence is shown in SEQ ID No. 4.

[0007] The application also provides a specific primer combination, which contains primers with nucleotide sequences shown in SEQ ID No. 1 to SEQ ID No. 4.

[0008] The application also provides a kit containing the primer combination.

[0009] The application also provides an application of the molecular marker or the primer combination or the kit in the breeding of non-stinging roxburgh rose.

[0010] The application also provides a method for molecular marker, which mixes primers of the molecular marker 153.61 and the molecular marker 341.112, and then amplifies roxburgh rose breeding materials, if 500bp and 100bp amplification fragments can be amplified at the same time, it is marked that the breeding material is non-stinging roxburgh rose No. 1 non-stinging strain; the upstream primer sequence of the molecular marker 153.61 is shown in SEQ ID No. 1, and the downstream primer sequence is shown in SEQ ID No. 2; the upstream primer sequence of the molecular marker 341.112 is shown in SEQ ID No. 3, and the downstream primer sequence is shown in SEQ ID No. 4.

[0011] The application also provides a method for detecting non-stinging roxburgh rose No. 1 non-stinging strain, which uses primers with specific sequences shown in SEQ ID No. 1 to SEQ ID No. 4 to perform PCR amplification on extracted roxburgh rose genomic DNA; and then observes the amplification results to draw a conclusion.

[0012] Preferably, the amplification is double-PCR amplification, and the total volume of the amplification reaction is 17.5-22.5 muL, which contains 0.9-1.1 muL DNA, 0.4-0.6 muL of the upstream primer and 0.4-0.6 muL of the downstream primer of the molecular marker 153.61, 0.4-0.6 muL of the upstream primer and 0.4-0.6 muL of the downstream primer of the molecular marker 341.112, 6-8 muL ddH2O and 9-11 muL Taq DNA polymerase.

[0013] Preferably, the process of the amplification reaction is as follows: 93-95 DEG C pre-denaturation for 3-5 min, 93-95 DEG C denaturation for 25-35 s, 45-55 DEG C annealing for 25-35 s, 70-74 DEG C extension for 35-45 s, 25-35 cycles, and finally total extension for 5-8 min and 3-5 DEG C incubation.

[0014] Preferably, the method for drawing conclusions is as follows: if two specific 500bp and 100bp amplification fragments of different sizes can be amplified simultaneously, it indicates that the tested breeding material is the thornless prickly pear strain No. 1.

[0015] Compared with the prior art, the present invention has the following technical effects:

[0016] (1) This invention uses two InDel markers to detect two marker sites simultaneously in a single double PCR test. This allows for simple and rapid identification of the thornless No. 1 thornless variety in prickly pear material. Moreover, the simultaneous detection of the two marker sites allows for mutual verification and validation, ensuring the accuracy, effectiveness and reliability of the detection results.

[0017] (2) This invention has significant application value in distinguishing prickly pear varieties and identifying interspecific hybrids.

[0018] (3) This invention can provide technical support for the innovation of large-fruited, thornless fresh prickly pear varieties.

[0019] (4) Using the present invention, the genomic DNA of the leaves of the breeding material is extracted during the seedling stage and double PCR amplification is performed. By observing the electrophoretic bands, it can be identified whether it is the thornless No. 1 thornless variety. Therefore, the breeding cycle of thornless prickly pear can be greatly shortened and the breeding process can be accelerated. Attached Figure Description

[0020] Figure 1 This is a map of the duplex PCR amplification products of the two primer pairs in Example 6;

[0021] Figure 2 The images show the amplification products of the primers SEQ ID No. 1~2 in Example 6 using conventional PCR.

[0022] Figure 3 The images show the amplification products of the primers SEQ ID No. 3~4 in Example 6 using conventional PCR. Detailed Implementation

[0023] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, kits, and instruments used in the following examples are commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0024] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0025] Example 1: Obtaining Thornless No. 1

[0026] The single plant of the thornless prickly pear No. 1 germplasm resource was collected from Qianxi County, Bijie City, Guizhou Province, and is now planted in the prickly pear germplasm resource nursery of Guizhou University.

[0027] Example 2: Obtaining the offspring of the thornless strain No. 1

[0028] After natural pollination of individual plants of the thornless No. 1 germplasm resource, healthy seeds were collected and sown to propagate F1 generation individual plants. After natural pollination of F1 generation individual plants, healthy seeds were collected and sown to propagate F2 generation individual plants.

[0029] Example 3: Collection of test materials

[0030] Thirty-four leaf samples of *Pyracantha fortuneana* were collected from the *Pyracantha fortuneana* germplasm resource nursery of Guizhou University in Guiyang, Guizhou Province (26°42.408'N, 106°67.353'E), with three replicates for each sample. Specific information is shown in Table 1. The collected leaf samples were then rinsed with distilled water, dried, flash-frozen in liquid nitrogen, and stored in an ultra-low temperature freezer for later use.

[0031] Table 1 List of Test Material Samples

[0032]

[0033]

[0034]

[0035] Example 4: Extraction of genomic DNA from the prickly pear material sample to be tested

[0036] Total DNA was extracted from the sample using the BioTake Universal Plant DNA Extraction Kit (centrifuge column type). The procedure is as follows:

[0037] (1) Column equilibration step: Add 500 μL of equilibration solution to the adsorption column AC (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm for 1 minute, discard the filtrate in the collection tube, and put the adsorption column back into the collection tube.

[0038] (2) Take an appropriate amount of plant tissue and grind it into a fine powder in a mortar with liquid nitrogen.

[0039] (3) Transfer the fine powder (100 mg of fresh plant tissue or 30 mg of dry tissue) to a 1.5 mL centrifuge tube, do not thaw, add 550 μL of 65℃ preheated Buffer P1 and 4 μL of RNase A, vortex vigorously for 1 minute to mix, and let stand at room temperature for 10 minutes.

[0040] (4) Add 130 μL of Buffer P2, vortex vigorously for 1 minute, and centrifuge at 12000 rpm for 3 minutes;

[0041] (5) Carefully aspirate the supernatant into separation column A, being careful not to aspirate interfacial substances. Centrifuge at 12,000 rpm for 1 minute and collect the filtrate.

[0042] (6) Transfer the filtrate to a new 2 mL centrifuge tube, add 1.5 times the volume of Buffer P3 and immediately vortex gently to mix thoroughly;

[0043] (7) Add the mixture obtained in the previous step (including any possible precipitate) to an adsorption column AC (place the adsorption column in the collection tube), centrifuge at 12000 rpm for 1 minute, and discard the waste liquid in the collection tube (the maximum sample loading volume of the adsorption column AC is 700 μL at a time; if the sample volume is greater than 700 μL, it needs to be loaded onto the column multiple times).

[0044] (8) Add 700 μL of washing buffer WB, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid;

[0045] (9) Add 500 μL of washing buffer WB, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid;

[0046] (10) Place the adsorption column AC back into the empty collection tube and centrifuge at 13000 rpm for 3-5 minutes to remove as much of the washing liquid as possible, so as to avoid the residual ethanol in the washing liquid inhibiting the downstream reaction.

[0047] (11) Take out the adsorption column AC and put it into a clean centrifuge tube. Add 50 μL of elution buffer EB (preheat the elution buffer in a 65°C water bath beforehand) to the middle of the adsorption membrane. Let it stand at room temperature for 3-5 minutes, then centrifuge at 12000 rpm for 1 minute to collect the DNA.

[0048] (12) Store the DNA at ~20℃ for later use.

[0049] Example 5: Design of InDel-labeled primers

[0050] (1) Preparation of template sequence

[0051] The InDel site was located by comparing the resequencing data with the reference genome. Based on the found InDel site, primers were designed with a length of 601 bp, consisting of 300 bp on each side of the InDel site, based on the prickly pear genome sequence.

[0052] (2) Design of marker primers

[0053] Based on the above template sequence, PCR primers were designed for the target sequence that met the requirements using SnapGene software. The primer design principles were: Tm value of 50–60℃, primer GC content of 45–55%, primers designed around the locus, amplified product size of 100–300 bp, and primer length of 18–24 bp. There should be no complementary bases at the 3' end of the primers, and the primers should not exhibit hairpin structures or primer dimers. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and primer information is shown in Table 2.

[0054] Table 2 List of InDel-labeled primers

[0055]

[0056] Example 6: Amplification of 34 materials using two methods

[0057] (1) PCR amplification with a single primer pair

[0058] Using the sample DNA from Example 3 as a template, conventional PCR amplification and agarose gel electrophoresis detection were performed using the specific primers synthesized in Example 5, SEQ ID No. 1~2 and SEQ ID No. 3~4, respectively.

[0059] The total volume of the PCR amplification reaction was 20 μL, which included 1 μL of DNA, 0.5 μL each of forward and reverse primers, 8 μL of ddH2O, and 10 μL of Taq DNA polymerase.

[0060] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 40 s, 30 cycles, and a final total extension of 7 min, followed by incubation at 4℃.

[0061] (2) Simultaneous duplex PCR amplification using two pairs of primers

[0062] Using the sample DNA from Example 3 as a template, double PCR amplification and agarose gel electrophoresis detection were performed using the two pairs of specific primers synthesized in Example 5 (SEQ ID No. 1 to SEQ ID No. 4).

[0063] The total volume of the duplex PCR amplification reaction was 20 μL, which included 1 μL of DNA, 0.5 μL each of 153.61 labeled upstream and downstream primers, 0.5 μL each of 341.112 labeled upstream and downstream primers, 7 μL of ddH2O, and 10 μL of Taq DNA polymerase.

[0064] The reaction program for duplex PCR amplification was as follows: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 40 s, 30 cycles, and a final total extension of 7 min, followed by incubation at 4℃.

[0065] (3) Detection of PCR products from the two amplification methods

[0066] The products of conventional PCR and duplex PCR were detected by electrophoresis using 0.9% agarose gel. 5 μL of conventional PCR amplification products from primers SEQ ID No. 1-2 and SEQ ID No. 3-4, as well as the duplex PCR amplification products from primers SEQ ID No. 1-4, were directly loaded onto the gel. Simultaneously, 5 μL of DNA marker was directly loaded as a reference. The electrophoresis conditions were: U = 110 V, I = 100 A, T = 30 min, and the electrophoresis buffer was 0.5 × TAE. After electrophoresis, the results were observed and photographed using a UV gel imaging system.

[0067] Example 7: Dual PCR Product Detection of All Test Materials

[0068] All test materials from Example 3 were amplified using the two PCR amplification methods described in Example 6, and then detected using electrophoresis. The results are as follows: Figures 1-3 As shown, where Figure 1 This is a map of the duplex PCR amplification products using two pairs of primers. Figure 2 The images show the amplification products of conventional PCR using primers SEQ ID No. 1~2. Figure 3 This is a chromatogram of the products amplified by conventional PCR using primers SEQ ID No. 3~4.

[0069] Depend on Figure 1 It can be seen that the electrophoretic bands of the doublet PCR products of the 34 materials showed four band patterns. Among them: materials showing lane 1 band pattern: 18, 19, and 22; materials showing lane 2 band pattern: F, 15, and 16; materials showing lane 3 band pattern: 1, 3, 4, 5, 7, CD, 17, 23, 24, 25, 26, 27, 28, 30, 31, 33, 36, 38, 39, and 40; and materials showing lane 4 band pattern: 14, 20, 21, 29, 32, 34, 35, and 37.

[0070] In presentation Figure 1 Among the three materials in the middle lane 2 type, the information listed in the sample collection information list for F and 16 is "Stingless No. 1", and the information listed in the sample collection information list for 15 is "F2 generation of the Stingless No. 1 strain". All of them are stingless materials. The electrophoresis results of the double PCR amplification products are consistent with the original recorded information, indicating that the detection method of the present invention is accurate and effective.

[0071] exist Figure 2 In the amplification results of primers SEQ ID No. 1~2 on 34 materials, two different band patterns were observed. The materials showing the band pattern shown in lane 1 were: F, 15, 16, 18, 19, and 22. The band patterns of the remaining materials were all shown in lane 2. This indicates that the primers cannot distinguish materials F, 15, and 16 on their own.

[0072] exist Figure 3 In the study, the amplification results of primers SEQ ID No. 3~4 for 34 samples showed a pattern similar to... Figure 2 The materials exhibiting slightly different band patterns are as follows: materials showing the band pattern shown in lane 1 are: 1, 3, 4, 5, 7, CD, 17, 18, 19, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 36, 38, 39, and 40; materials showing the band pattern shown in lane 2 are: F, 14, 15, 16, 20, 21, 29, 32, 34, 35, and 37. This indicates that the primer cannot distinguish materials F, 15, and 16 on its own. However, the electrophoresis results show that materials F, 15, and 16 have different band patterns from materials 18, 19, and 22. Furthermore, materials F, 15, 16, 18, 19, and 22... Figure 1 Electrophoretic bands in Figure 2 and Figure 3 The electrophoretic bands overlapped in the same way.

[0073] Therefore, neither primer pair alone can distinguish the thornless material No. 1; only when two primer pairs are used simultaneously can the thornless material No. 1 be accurately distinguished.

[0074] In summary, this invention identifies the thornless strain 1 in prickly pear materials by simultaneously detecting two marker sites using a single duplex PCR. The simultaneous detection of the two marker sites allows for mutual verification and validation, ensuring the accuracy and reliability of the detection. Furthermore, this invention utilizes the extraction of genomic DNA from the leaves of seedlings for duplex PCR amplification, and the identification of whether the material is the thornless strain 1 can be determined by observing the electrophoretic bands, thus shortening the breeding cycle and accelerating the breeding process.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention. sequence list <110> Guizhou University <120> A specific molecular marker for a thornless prickly pear variety No. 1 and its application <130> 2022.01.18 <141> 2022-03-02 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial synthesis <400> 1 gcaaccgaaa tccaatgtg 19 <210> 2 <211> 19 <212> DNA <213> Artificial synthesis <400> 2 caagcttact cagtcctcc 19 <210> 3 <211> 18 <212> DNA <213> Artificial synthesis <400> 3 aagtgttgcg tgttaacg 18 <210> 4 <211> twenty four <212> DNA <213> Artificial synthesis <400> 4 gttgtcagta tagaataagt atgg 24

Claims

1. A molecular marker, characterized in that, It consists of molecular marker 153.61 and molecular marker 341.112; the upstream primer sequence of molecular marker 153.61 is shown in SEQ ID No.1 and the downstream primer sequence is shown in SEQ ID No.2; the upstream primer sequence of molecular marker 341.112 is shown in SEQ ID No.3 and the downstream primer sequence is shown in SEQ ID No.

4.

2. A specific primer combination, characterized in that, Primers containing nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No.

4.

3. A kit containing the primer combination as described in claim 2.

4. The application of the molecular marker of claim 1, the primer combination of claim 2, or the kit of claim 3 in the breeding of thornless prickly pear.

5. A method for detecting the thornless variety No. 1 of prickly pear, characterized in that, The primers for molecular markers 153.61 and 341.112 were mixed to amplify the prickly pear breeding material. If two specific amplified fragments of different sizes, 500 bp and 100 bp, could be amplified simultaneously, it would indicate that the tested breeding material was the thornless prickly pear strain 1. The upstream primer sequence for molecular marker 153.61 is shown in SEQ ID No. 1, and the downstream primer sequence is shown in SEQ ID No.

2. The upstream primer sequence for molecular marker 341.112 is shown in SEQ ID No. 3, and the downstream primer sequence is shown in SEQ ID No.

4.

6. The method as described in claim 5, characterized in that, The amplification was a duplex PCR amplification, and the total volume of the amplification reaction was 17.5–22.5 μL, including 0.9–1.1 μL of DNA, 0.4–0.6 μL each of the upstream and downstream primers for molecular marker 153.61, 0.4–0.6 μL each of the upstream and downstream primers for molecular marker 341.112, 6–8 μL of ddH2O, and 9–11 μL of Taq DNA polymerase.

7. The method as described in claim 6, characterized in that, The amplification reaction process is as follows: pre-denaturation at 93-95℃ for 3-5 min, denaturation at 93-95℃ for 25-35 s, annealing at 45-55℃ for 25-35 s, extension at 70-74℃ for 35-45 s, 25-35 cycles, and finally total extension for 5-8 min, followed by holding at 3-5℃.

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