A guava ISSR-PCR molecular marker combination and its application

Abstract

The present invention discloses a guava ISSR-PCR molecular marker combination and its application, which mainly realizes the function of identifying guava genetic diversity analysis based on an ISSR primer set composed of SEQ ID NO: 1 to 10. And the primer set in the present invention can achieve high-precision measurement work with a larger sample size compared to the traditional method, and the identifiable polymorphism is as high as 89.68%. Moreover, based on this method, its band resolution is higher, the amplified fragments are clearer, the reaction is more stable, the polymorphism recognition is higher, and the identification of large quantities of experimental materials can be completed in a short time. It can accurately distinguish the relationship between guava germplasm resources, which is of great significance to the identification of guava germplasm resources.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and particularly relates to a guava ISSR-PCR molecular marker combination and application thereof. Background Art

[0002] Guava (Psidium guajava L.) is a tree of the genus Guava in the Myrtaceae family. It is also known as guava, chicken shit fruit, and guava. Guava is native to tropical America and is now widely distributed in tropical and subtropical regions. It has been introduced to China for cultivation for more than 300 years. Guava is mainly cultivated in southern China and is also distributed in the southwest. Guava is rich in protein, vitamins C, A, B, and various trace elements required by the human body, such as calcium, phosphorus, iron, and potassium. It can be eaten fresh, cooked, and made into wine, and can also be processed into juice, jam, canned food, etc. It also has medicinal value such as antibacterial, antidiarrheal, antioxidant, liver protection, blood sugar lowering, blood lipid lowering, blood pressure lowering, anti-cardiovascular disease and anti-tumor. In addition, its seed oil content is 5% to 13%, rich in unsaturated fatty acids, and can be used as a health oil.

[0003] At present, guava germplasm resource nurseries have been established in many provinces and autonomous regions in my country, and the introduction, trial planting and collection of guava are carried out continuously. However, at this stage, the identification of guava germplasm resources is still mainly based on morphological characteristics, and there is a lack of scientific and systematic detection methods, which makes it difficult to correctly judge the genetic relationship of germplasm. There are often phenomena of the same thing having different names, and the same name having different things, which brings great challenges to the identification of guava varieties.

[0004] ISSR (Inter-simple Sequence Repeat) is a common method in the study of species genetic diversity. It is not affected by the environment, has rich binding sites and high sensitivity and accuracy. In terms of variety identification, DNA molecular marker technology has more outstanding advantages than traditional technologies.

[0005] In related technologies, although there are reports on the use of ISSR molecular marker technology to identify guava, its identification accuracy generally cannot meet the high-precision requirements under large sample sizes. Some researchers analyzed the kinship of 36 guava germplasm resources and amplified 165 bands, of which 129 were polymorphic bands, and the polymorphism percentage was only 78.18%. A study conducted a kinship analysis on 16 guava materials and amplified 44 repeatable bands, of which polymorphic bands accounted for only 61.4%, and the similarity coefficients between various germplasms were between 0.689 and 0.981.

[0006] Therefore, developing a guava variety identification method that can be used in larger-scale sample screening and can show a higher polymorphism percentage is of great significance for improving the efficiency and accuracy of guava variety identification. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a guava ISSR-PCR molecular marker combination and its application. The present application discovered a set of primer sets of guava ISSR-PCR molecular markers, which can accurately identify guava samples with a large sample size and accurately classify germplasm, thereby realizing the accurate identification of guava varieties and reducing the difficulty and uncertainty of guava breeding.

[0008] The first aspect of the present invention provides a set of ISSR primers for identifying guava genetic diversity analysis, the primers consisting of SEQ ID NOs: 1-10.

[0009] The specific nucleotide sequences of SEQ ID NOs: 1 to 10 are shown below.

[0010] UBC807:5'-AGAGAGAGAGAGAGAGT-3'(SEQ ID NO:1)

[0011] UBC808: 5'-AGAGAGAGAGAGAGAGC-3' (SEQ ID NO: 2)

[0012] UBC809: 5'-AGAGAGAGAGAGAGAGG-3' (SEQ ID NO: 3)

[0013] UBC810:5'-GAGAGAGAGAGAGAGAT-3'(SEQ ID NO:4)

[0014] UBC835:5'-AGAGAGAGAGAGAGAGYC-3'(SEQ ID NO:5)

[0015] UBC841:5'-GAGAGAGAGAGAGAGAYC-3'(SEQ ID NO:6)

[0016] UBC842:5'-GAGAGAGAGAGAGAGAYG-3'(SEQ ID NO:7)

[0017] UBC880:5'-GGAGAGGAGAGGAGA-3'(SEQ ID NO:8)

[0018] UBC881:5'-GGGTGGGGTGGGGGTG-3'(SEQ ID NO:9)

[0019] UBC888: 5'-BDBCACACACACACACA-3' (SEQ ID NO: 10)

[0020] In the present invention, the inventors found that the above primer set can achieve higher-precision polymorphism detection with a larger sample size compared to the guava ISSR primer set in the prior art, providing favorable technical support for improving guava germplasm and variety identification.

[0021] The second aspect of the present invention provides a detection reagent for identifying guava genetic diversity analysis, wherein the detection reagent contains the ISSR primer set described in the first aspect of the present invention.

[0022] In the present invention, compared with the prior art, the detection reagent of the present invention can achieve higher-precision polymorphism detection with a larger sample size, providing favorable technical support for improving guava germplasm and variety identification.

[0023] The third aspect of the present invention provides the use of the ISSR primer set described in the first aspect of the present invention in preparing a guava genetic diversity detection product.

[0024] In some embodiments of the present invention, the product includes a detection reagent, a detection kit, and a detection chip.

[0025] In some embodiments of the present invention, the product further contains a PCR amplification reagent.

[0026] In some embodiments of the present invention, the PCR amplification reagents include but are not limited to PCR premix, buffer, and system solvent.

[0027] Of course, those skilled in the art can also reasonably add other auxiliary reagents for amplification according to actual use requirements to achieve the purpose of directional amplification.

[0028] The fourth aspect of the present invention provides the use of the ISSR primer set described in the first aspect of the present invention in guava germplasm breeding.

[0029] In the present invention, the inventors amplified the guava sample DNA based on the ISSR primer set, and analyzed the germplasm information of the guava sample according to the band information of the amplified product in the gel imaging, and the measured polymorphism percentage reached 89.68%, far exceeding the measured rate in the prior art. And for all 59 guava samples, accurate classification was achieved with extremely high accuracy, thereby effectively realizing the high-quality guava planting screening and providing data support for subsequent breeding work.

[0030] A fifth aspect of the present invention provides a method for identifying the genetic diversity of guava varieties, comprising the following steps:

[0031] (1) extracting guava sample DNA, and independently amplifying based on at least one primer in SEQ ID NOs: 1 to 10 to obtain an amplified product;

[0032] (2) Separating the amplified products by electrophoresis and performing gel imaging to obtain band information;

[0033] (3) Cluster analysis was performed based on the band information, and a DNA fingerprint was established to identify the guava varieties.

[0034] In some embodiments of the present invention, the guava sample DNA is extracted based on a commercial kit. Of course, those skilled in the art can also select other conventional DNA extraction methods in the art to extract DNA according to actual detection requirements.

[0035] In some embodiments of the present invention, the amplification reaction system in step (1) is:

[0036]

[0037] In some embodiments of the present invention, the amplification reaction system in step (1) is:

[0038]

[0039] In some embodiments of the present invention, the reaction procedure of the amplification in step (1) is: pre-denaturation at 94-95°C for 2-5 min; denaturation at 94-95°C for 25-30 s, annealing at 50°C for 42-45 s, 40-45 cycles; extension at 72°C for 9-10 min.

[0040] In some embodiments of the present invention, the reaction procedure of the amplification in step (1) is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 50°C for 45 s, 40 cycles; and extension at 72°C for 10 min.

[0041] According to the fifth aspect of the present invention, in some embodiments of the present invention, the primers are a combination of SEQ ID NOs: 2, 4 to 8 and 10.

[0042] In this embodiment of the present invention, in order to save workload, the inventors randomly selected only 7 primers with clear bands, high polymorphism and good repeatability from the above 10 primers for the preparation of guava DNA fingerprint map, and finally obtained the guava germplasm resource fingerprint map coding that was effective for 59 guava samples, realizing the efficient classification of guava varieties.

[0043] In some embodiments of the present invention, the primers are a combination of SEQ ID NOs: 1-10.

[0044] In the present invention, the inventors have found that the ISSR-PCR reaction system can be well used for germplasm identification of guava samples, so that the amplified polymorphism can reach more than 89%, thereby obtaining the corresponding germplasm relationship (kinship) and specific DNA molecular fingerprint map, which provides a reference for the application of ISSR molecular markers in guava classification, and also provides effective technical support and theoretical basis for the germplasm development and breeding identification of guava samples.

[0045] In some embodiments of the present invention, the specific operation of cluster analysis is as follows: Specifically, the amplified band information is read, and the clear and repeated bands at the same position within the range of 100 to 5000 bp on the electrophoresis map are recorded as "1", and the bands without bands at the same position are recorded as "0", thereby generating a 0 and 1 band matrix. The SimQual program in the NT-SYSpc2.0 software is used to calculate the similarity coefficient matrix, and the SHNN in the Clustering program is used for cluster analysis; the Tree Plot module is used to generate a cluster diagram, and a molecular evolution tree is constructed.

[0046] In some embodiments of the present invention, the DNA fingerprint is constructed using TBtools software, and the drawing information is based on the 0, 1 data matrix generated by the band information.

[0047] The beneficial effects of the present invention are:

[0048] (1) The present invention provides a set of ISSR primers for identifying guava genetic diversity analysis, which can achieve high-precision measurement work with a larger sample size, and the identifiable polymorphism is as high as 89.68%, far exceeding traditional technologies, and realizes accurate classification of guava varieties.

[0049] (2) The present invention combines multiple steps such as guava DNA extraction method, guava ISSR-PCR reaction, and separation of ISSR-PCR amplification products by agarose gel. Through this combination, large-scale high-accuracy analysis of guava variety genetic diversity is achieved. Moreover, based on this method, the band resolution is higher, the amplified fragments are clearer, the reaction is more stable, and the polymorphism recognition is higher, so that the identification of large quantities of experimental materials can be completed in a short time.

[0050] (3) The identification method of the present invention has low identification cost, effectively shortens the sample detection time, has stable and reliable results, and can accurately distinguish the relationship between guava germplasm resources, which is of great significance for the identification of guava germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The figure is an agarose gel electrophoresis diagram of some guava samples obtained in the examples of the present invention.

[0052] Figure 2 The figure shows the amplification results of some guava samples using UBC880 primers.

[0053] Figure 3 The figure shows the amplification results of some guava samples using UBC888 primers.

[0054] Figure 4 This is a UPGMA cluster diagram of different guava samples based on ISSR genetic distance in an embodiment of the present invention.

[0055] Figure 5 This is a UPGMA cluster diagram of different guava samples based on ISSR genetic distance in an embodiment of the present invention.

[0056] Figure 6 This is a UPGMA cluster diagram of different guava samples based on ISSR genetic distance in an embodiment of the present invention.

[0057] Figure 7 This is the original image of the DNA fingerprint coding map obtained in the example of the present invention.

[0058] Figure 8 for Figure 7 A partial enlarged view of the DNA fingerprinting map is shown.

[0059] Fig. 9 for Figure 7 A partial enlarged view of the DNA fingerprinting map is shown. DETAILED DESCRIPTION

[0060] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0061] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[0062] Guava total DNA extraction

[0063] In the following examples, the guava samples used were collected from various places in Guangdong Province by the Fruit Research Institute of Guangdong Academy of Agricultural Sciences, with a total of 59 guava samples (young leaves).

[0064] The specific test number information and variety source are shown in Table 1.

[0065] Table 1 Guava sample test number information and variety source

[0066]

[0067]

[0068] The specific extraction steps are as follows:

[0069] The pipette tips, centrifuge tubes, mortars, etc. used in the guava total DNA extraction process were sterilized in advance by high pressure sterilization for 30 minutes.

[0070] Take the lysate in the plant genomic DNA extraction kit (M5 HiPer Plant Genomic DNA Kit, purchased from Beijing Polymer), add 10% polyvinyl pyrrolidone and 0.1% mercaptoethanol by mass ratio, and preheat in a 65℃ water bath. Grind 0.1g of guava young leaves to be tested in liquid nitrogen, add the above lysate mixture, mix well, pour into a sterilized centrifuge tube, and place in a 65℃ water bath for 45min to 1h. During this period, shake once every 10min to help complete lysis. Add an equal volume of chloroform, mix well, and centrifuge at 12000rpm for 8min at room temperature. Take the supernatant and repeat the centrifugation once. Take the supernatant, add the binding solution in the kit at a volume ratio of 1:1.5, mix well; aspirate the supernatant and transfer it to the DNA purification column. Centrifuge at 12000rpm for 30s at room temperature, discard the liquid in the collection tube, and repeat this step until the solution is completely transferred. Add 700 μL of rinse solution (from the above-mentioned plant genomic DNA extraction kit) to the DNA purification column, centrifuge at 12000rpm at room temperature for 30s, discard the liquid in the collection tube, and then repeat this step once; centrifuge again at 12000rpm for 2min. Take out the DNA purification column and dry it until there is no ethanol residue. Then place the DNA purification column in a new 1.5ml centrifuge tube, add 50-100 μL of ddH2O to the center of the purification column, let it stand for 2min, and centrifuge at 12000rpm for 1min. Add 50-100 μL of ddH2O again, let it stand for 2min, and centrifuge at 12000rpm for 1min. Discard the purification column, collect the liquid in the centrifuge tube, and obtain the purified guava total DNA.

[0071] The purity of the obtained guava total DNA was detected by 1.0% agarose gel electrophoresis and stored at -20°C for future use.

[0072] Among them, the agarose gel electrophoresis diagram of some samples is as follows Figure 1 shown.

[0073] ISSR primer combination and PCR reaction system setup

[0074] In this example, the inventors used a total of 10 primers to construct a detection primer combination for detecting guava variety polymorphism.

[0075] The specific primer sequences are shown in Table 2.

[0076] Table 2 Primers and their nucleotide sequences

[0077] Primer number Primer sequence (5'→3') UBC807 AGAGAGAGAGAGAGAGT(SEQ ID NO:1) UBC808 AGAGAGAGAGAGAGAGC(SEQ ID NO:2) UBC809 AGAGAGAGAGAGAGAGG(SEQ ID NO:3) UBC810 GAGAGAGAGAGAGAGAT(SEQ ID NO:4) UBC835 AGAGAGAGAGAGAGAGYC(SEQ ID NO:5) UBC841 GAGAGAGAGAGAGAGAYC(SEQ ID NO:6) UBC842 GAGAGAGAGAGAGAGAYG(SEQ ID NO:7) UBC880 GGAGAGGAGAGGAGA (SEQ ID NO: 8) UBC881 GGGTGGGGTGGGGTG (SEQ ID NO: 9) UBC888 BDBCACACACACACACA(SEQ ID NO:10)

[0078] Based on the above 10 primers, PCR detection was performed on guava total DNA, and the specific reaction system is shown in Table 3.

[0079] Table 3 PCR reaction system

[0080] Components content Guava total DNA 2μL SEQ ID NO: 1 to 10 any one of the primers (10 μmol / L) 1μL MasterMix 15μL Deionized water 12μL total 30μL

[0081] The reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 50°C for 45 s, 40 cycles; and extension at 72°C for 10 min.

[0082] The obtained PCR amplification products were tested by 1.5% agarose gel electrophoresis, with the loading volume being 8 μL / sample.

[0083] Among them, taking the amplification results using UBC880 primers and UBC888 primers as an example, the results are as follows Figures 2-3 As shown, it can be found that it excellently displays the amplified bands of different samples with high clarity, which is very easy for technicians to distinguish.

[0084] By using the above primer set to amplify 59 guava samples, the polymorphism amplification results of each primer are shown in Table 4.

[0085] Table 4 Amplification results of primer sets for 59 guava samples

[0086] Primer number Number of amplified bands Number of polymorphic bands Polymorphism percentage / % UBC807 14 13 92.86 UBC808 12 10 83.33 UBC809 9 6 66.67 UBC810 10 7 70.00 UBC835 12 11 91.67 UBC841 13 13 100.00 UBC842 14 12 85.71 UBC880 17 17 100.00 UBC881 10 9 90.00 UBC888 15 15 100.00 total 126 113 89.68

[0087] It can be found that the total number of amplified bands of 10 primers is 126, of which 113 are polymorphic bands, and the measured polymorphism percentage is 89.68%. On average, 12.60 bands are amplified by each primer, and 11.30 polymorphic bands are amplified by each primer on average, and the polymorphic band ratio of each primer is higher than 60%. The above primer amplification results show that the information amount of the bands amplified by the primer set constructed in the embodiment of the present invention is moderate, the polymorphism is rich, and it can be used for subsequent genetic diversity analysis and fingerprint map construction.

[0088] Application of the above ISSR-PCR reaction system in genetic diversity analysis of guava varieties

[0089] Based on the detection results of the above ISSR-PCR reaction system, cluster analysis of guava samples can be performed.

[0090] The specific steps are:

[0091] The clear and repeated bands at the same site within the range of 100 to 5000 bp on the obtained electrophoresis map were recorded as "1", and the bands without bands at the same position were recorded as "0", and the 0 and 1 data matrices of each primer pair for different guava varieties were obtained respectively. The total number of amplified bands and the total number of polymorphisms for each primer were counted. The similarity coefficient matrix was calculated using the SimQual program in the NT-SYSpc2.0 software, and cluster analysis was performed using the SHNN in the Clustering program; the clustering diagram was generated using the Tree Plot module to construct a molecular evolutionary tree.

[0092] Based on the ISSR molecular markers measured by the above ISSR-PCR reaction system, the 59 guava samples were clustered using NTSYSpc2.0 software. The 59 guava samples were divided into different groups according to the degree of genetic relationship, with a genetic similarity coefficient of 0.50-0.96 and an average genetic similarity coefficient of 0.73. There were certain genetic differences between varieties. Among the 59 samples, the varieties C43'Brazil No. 2' and C46'Brazil No. 5' had the closest genetic distance, with a genetic coefficient of 0.96. The varieties C26'Strawberry Guava' and C46'Brazil No. 5' had the farthest genetic distance, with a genetic coefficient of 0.50, indicating that the two varieties had a high degree of genetic differentiation. At the genetic coefficient threshold of 0.708, the 59 germplasm resources can be divided into 4 groups. Group 1 contains 34 germplasms, numbered: C1-C9, C11, C16-C20, C22-C25 and C27-C41. Group 2 contains 6 germplasms, numbered C10-C15 and C21. Group 3 contains 18 germplasms, numbered C42-C59. Sample numbered C26 'Strawberry Guava' is the fourth group alone, indicating that this sample has obvious genetic variation compared with the other samples. The specific UMPGA cluster diagram in this example is as follows Figures 4 to 6 shown.

[0093] Construction of guava DNA fingerprint:

[0094] Based on the band information presented by each primer, the DNA fingerprint of the guava to be tested can be further established. The specific steps are as follows:

[0095] In this embodiment, in order to save workload, the inventors randomly selected only 7 primers with clear bands, high polymorphism and good repeatability from the above 10 primers for the preparation of guava DNA fingerprint map. Of course, those skilled in the art can also reasonably select different numbers of primers' band information as reference to draw DNA fingerprint map according to actual use requirements.

[0096] In this embodiment, the primers selected by the inventors are the band information obtained by UBC808, UBC810, UBC835, UBC841, UBC842, UBC880, and UBC888 among the above 10 primers. The clear, typical, and polymorphic polymorphic bands in these 7 primers were assigned values, and the typical band assignment information of the primers is shown in Table 5.

[0097] Table 5 Value assignment of typical bands amplified by 7 ISSR primers

[0098]

[0099]

[0100] Among them, * represents the variety characteristic band.

[0101] According to the polymorphic band assignment standard in Table 5, DNA fingerprint coding was performed on 59 guava test samples, and the fingerprint coding is shown in Table 6. The fingerprint is generated in TBtools based on the 0, 1 data matrix obtained by amplification. Figures 7 to 9 shown.

[0102] Table 6 Fingerprint codes of 59 guava germplasm resources

[0103]

[0104]

[0105] In summary, it can be found that the above-mentioned ISSR-PCR reaction system can be well used for germplasm identification of guava samples, thereby obtaining the corresponding germplasm relationship and specific DNA map, thereby providing effective technical support for the germplasm development and breeding identification of guava samples.

[0106] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. SEQUENCE LISTING <110> Fruit Research Institute, Guangdong Academy of Agricultural Sciences <120> A guava ISSR-PCR molecular marker combination and its application <130> <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> DNA <213> Artificial sequence <400> 1 agagagag agagagt 17 <210> 2 <211> 17 <212> DNA <213> Artificial sequence <400> 2 agagagag agagc 17 <210> 3 <211> 17 <212> DNA <213> Artificial sequence <400> 3 agagagag agagagg 17 <210> 4 <211> 17 <212> DNA <213> Artificial sequence <400> 4 gagagagaga gagagat 17 <210> 5 <211> 18 <212> DNA <213> Artificial sequence <400> 5 agagagag agagagyc 18 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <400> 6 gagagagaga gagagayc 18 <210> 7 <211> 18 <212> DNA <213> Artificial sequence <400> 7 gagagagaga gagagayg 18 <210> 8 <211> 15 <212> DNA <213> Artificial sequence <400> 8 ggagaggaga ggaga 15 <210> 9 <211> 15 <212> DNA <213> Artificial sequence <400> 9 gggtggggtg gggtg 15 <210> 10 <211> 17 <212> DNA <213> Artificial sequence <400> 10 bdbcacacac acacaca 17

Claims

1. Application of ISSR primer sets in the preparation of guava genetic diversity detection products; The primer set consists of SEQ ID NOs: 1-10.

2. The use according to claim 1, characterized in that: The product includes a detection reagent.

3. Application of ISSR primer sets in guava germplasm breeding; The primer set consists of SEQ ID NOs: 1-10.

4. A method for identifying the genetic diversity of guava varieties, comprising the following steps: (1) extracting guava sample DNA, and amplifying based on each primer in the primer set independently to obtain an amplification product; (2) Separating the amplified products by electrophoresis and performing gel imaging to obtain band information; (3) Cluster analysis was performed based on the band information, and a DNA fingerprint was established to identify the guava varieties; The primer set is a combination of SEQ ID NOs: 1 to 10 or a combination of SEQ ID NOs: 2, 4 to 8 and 10; Wherein, the reaction system for amplification in step (1) is: 。 5. The method according to claim 4, characterized in that The amplification reaction procedure in step (1) is: pre-denaturation at 94-95°C for 2-5 min; denaturation at 94-95°C for 25-30 s, annealing at 50°C for 42-45 s, 40-45 cycles; and extension at 72°C for 9-10 min.

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