Optimization and development and application of molecular marker system for identification of germplasm specificity and diversity of Parsley chinensis

Abstract

The present invention relates to the technical field of molecular biology, and in particular to a molecular marker system for identifying the specificity and diversity of Pleurotus eryngii germplasm and its application. The optimal ISSR-PCR reaction system is: each 10 μL reaction system contains 1 μL (50 ng / μL) of template DNA, 0.2 μL (10 μmol / L) of primers, 5 μL of 2×Tap Master Mix ((Tap DNA polymerase, elongation promoting factor, dNTPs)), dd H 2 O is 3.8 μL, and the primer is any one of UBC824, UBC834, UBC835, UBC844, UBC864, UBC866, UBC873, UBC874, UBC878, and UBC881. The reaction system detects the seven-leaf floribunda variety (line) by ISSR-PCR molecular markers, and the results are stable and reliable.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a molecular marker system for identifying the specificity and diversity of Parsley germplasm and an application thereof. Background Art

[0002] Paris polyphylla Smith is a perennial herbaceous plant of the genus Paris in the Liliaceae family according to the Engler system. It is also known as Chinese Paris. It is one of the two original plants of Paris in the Chinese Pharmacopoeia and is a commonly used traditional Chinese medicine in my country. It is distributed in Yunnan, Zhejiang, Guizhou, Fujian, Chongqing, Guangxi, Hunan, Hubei and other regions in my country. Paris polyphylla Smith has the effects of clearing away heat and detoxifying, reducing swelling and relieving pain, cooling the liver and calming the nerves, anti-tumor, antibacterial, anti-inflammatory, analgesic, hemostatic, and cardiovascular protection. It is one of the main raw materials for more than 80 Chinese patent medicines with national medicine standards, such as Yunnan Baiyao, Pien Tze Huang, Gongxuening Capsules, Ji Desheng Snake Medicine, and Tongxuekang.

[0003] The essence of genetic diversity is the variation of genetic material. Exploring genetic diversity is the basis for studying plant evolution and kinship. Inter-Simple Sequence Repeat (ISSR) is a type of molecular marker. ISSR technology is widely used in the detection of genetic diversity of different types of plants. ISSR is a molecular marker method based on PCR technology. The PCR reaction system has a significant impact on the test results due to different group allocation ratios, and the optimal PCR reaction system varies significantly among different species. In summary, establishing and optimizing the ISSR-PCR system for Asterix septempunctata is a key step in the utilization of Asterix septempunctata germplasm resources, but there has been no related research or methods reported on this species before. Summary of the invention

[0004] In view of this, the present invention provides a molecular marker system for identification of germplasm specificity and diversity of seven-leaf gentian and its application. The molecular marker system for identification of germplasm specificity and diversity of seven-leaf gentian is as follows: each 10 μL reaction system contains 1 μL (50 ng / μL) of template DNA, 0.2 μL (10 μmol / L) of primers, 5 μL of 2×Tap MasterMix (Tap DNA polymerase, extension promoting factor, dNTPs), dd H 2 O is 3.8 μL, the primer is any one of UBC824, UBC834, UBC835, UBC844, UBC864, UBC866, UBC873, UBC874, UBC878, and UBC881, and the reaction system performs ISSR-PCR molecular marker detection on the seven-leaf floribunda variety (line), and the result is stable and reliable.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of an ISSR-PCR primer set in genetic diversity analysis, germplasm resource identification and molecular breeding of Parsley septempunctata. The ISSR-PCR primer set has:

[0007] (I) any one or more of the nucleotide sequences shown in SEQ ID NOs. 1 to 10; or

[0008] (II) having a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but differing from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0009] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0010] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III).

[0011] The present invention also provides a molecular marker system for identifying the specificity and diversity of the germplasm of Pseudomonas aeruginosa, comprising: each 10 μL reaction system contains 5 ng / μL of template DNA, 0.2 μmol / L of primers, 5 μL of 2×Tap MasterMix, dd H 2 O is supplemented to 10 μL; the primers have:

[0012] (I) any one or more of the nucleotide sequences shown in SEQ ID NOs. 1 to 10; or

[0013] (II) having a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but differing from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0014] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0015] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III).

[0016] In some specific embodiments of the present invention, the amplification procedure of the molecular marker includes: pre-denaturation at 94°C for 5 minutes, denaturation at 95°C for 15 seconds, annealing at 48.5-63.2°C for 15 seconds, extension at 72°C for 30 seconds, a total of 35 cycles, and finally extension at 72°C for 6 minutes.

[0017] In some specific embodiments of the present invention, the annealing temperature is determined according to the selected primers, including:

[0018]

[0019] The invention also provides a kit, comprising the molecular marker system.

[0020] The present invention also provides the use of any of the following items in genetic diversity analysis of Parsley septempunctata:

[0021] (I), the molecular marker system; and / or

[0022] (II), the kit.

[0023] The present invention also provides a method for ISSR-PCR labeling of Parsley septempunctata, comprising the following steps:

[0024] Step 1, extracting genomic DNA from the sample of Pleurotus eryngii;

[0025] Step 2: Using the molecular marker system or the kit, perform ISSR-PCR amplification on the genomic DNA in step 1.

[0026] The present invention also provides a method for analyzing genetic diversity of Parsley septempunctata, comprising the following steps:

[0027] Step 1, extracting genomic DNA from the sample of Pleurotus eryngii;

[0028] Step 2: using the molecular marker system or the kit to perform ISSR-PCR amplification on the genomic DNA in step 1 to obtain a PCR amplification product;

[0029] Step 3, performing electrophoresis separation on the PCR amplification product described in step 2 to obtain electrophoresis band information;

[0030] Step 4: Calculate the genetic similarity coefficient and construct a phylogenetic tree.

[0031] In some specific embodiments of the present invention, the Paridis japonica sample in step 1 comprises young leaves of Paridis japonica.

[0032] In some specific embodiments of the present invention, the electrophoretic separation in step 3 comprises using 1.5% agarose gel electrophoresis.

[0033] In some specific embodiments of the present invention, step 4 specifically includes: using NTSYSpc 2.10e to calculate the genetic similarity coefficient between individuals; and using UPGMA to construct a phylogenetic tree.

[0034] The present invention includes but is not limited to providing the following beneficial effects:

[0035] The present invention uses the optimized amplification reaction system to screen the annealing temperature of different primers, explore the optimal annealing temperature of the primers, and determine the optimal reaction system of seven-leaf clematis: 10 μL reaction system contains 1 μL (50 ng / μL) of template DNA, 0.2 μL (10 μmol / L) of primers, 5 μL of 2×Tap MasterMix (Tap DNA polymerase, extension promoting factor, dNTPs), ddH 2 O is 3.8μL. The optimal annealing temperatures of 10 primers (UBC824, UBC834, UBC835, UBC844, UBC864, UBC866, UBC873, UBC874, UBC878, UBC881) were screened by the optimized reaction system, which were 53.0℃, 55.8℃, 56.3℃, 52.3℃, 51.2℃, 63.2℃, 51.9℃, 60.8℃, 48.5℃, and 54.3℃, respectively. The optimized reaction system was used to detect ISSR-PCR molecular markers in 15 varieties (lines) of Asteraceae. The results showed that the above reaction system was stable and reliable. By establishing and optimizing the ISSR-PCR system of Asteraceae, a stable system of its molecular markers was obtained, which laid a foundation for the subsequent protection and utilization of Asteraceae resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0037] Figure 1 The figure shows the results of DNA quality test of seven-leaf Polygonum multiflorum samples; wherein, lanes Q1 to Q15 correspond to 15 seven-leaf Polygonum multiflorum germplasm samples respectively;

[0038] Figure 2 The figure shows the electrophoresis amplification results of the optimization experiment of ISSR-PCR reaction system of Pseudocerma septempunctata; where M represents Marker;

[0039] FIG3 shows the results of ISSR primer screening; wherein each lane represents a different primer amplification result, Figure 3A The figure shows the results of ISSR-PCR electrophoresis amplification using primers UBC824. Figure 3BThe figure shows the ISSR-PCR electrophoresis amplification results of primers UBC834 and UBC835. Figure 3C The figure shows the results of ISSR-PCR electrophoresis amplification using primers UBC844. Figure 3D The figure shows the ISSR-PCR electrophoresis amplification results of primers UBC864 and UBC866. Figure 3E The results of ISSR-PCR electrophoresis amplification using primers UBC873 and UBC874 are shown. Figure 3F The figure shows the ISSR-PCR electrophoresis amplification results of UBC878 primer and UBC881 primer;

[0040] Figure 4 The figure shows the ISSR-PCR electrophoresis amplification results of UBC824 primers at different annealing temperatures; among them, the electrophoresis bands of lanes 1 to 8 with annealing temperatures of 59.6℃, 58.9℃, 57.7℃, 55.9℃, 53.0℃, 51.9℃, 50.5℃, and 49.6℃ respectively;

[0041] Figure 5 The ISSR-PCR electrophoresis amplification results of UBC834 primers at different annealing temperatures are shown; among them, the electrophoresis bands of lanes 1 to 8 with annealing temperatures of 57.7°C, 56.9°C, 55.8°C, 53.9°C, 51.6°C, 49.6°C, 47.7°C, and 50°C, respectively;

[0042] Figure 6 The ISSR-PCR electrophoresis amplification results of UBC844 primers at different annealing temperatures are shown; among them, the electrophoresis bands of lanes 1 to 8 with annealing temperatures of 60°C, 59.3°C, 58.1°C, 56.3°C, 54°C, 52.3°C, 50.9°C, and 50°C respectively;

[0043] Figure 7 The figure shows the ISSR-PCR electrophoresis amplification results of primers UBC848, UBC849, UBC850, and UBC851;

[0044] Figure 8 The ISSR-PCR electrophoresis amplification results of primers UBC801, UBC802, UBC803, UBC804, UBC805, and UBC806 are shown;

[0045] Fig. 9 The figure shows the ISSR-PCR electrophoresis amplification results of UBC820 primers at different annealing temperatures;

[0046] Fig.10 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC824; among them, lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0047] Fig.11 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC834; wherein lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0048] Fig.12 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC835; wherein lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0049] Fig.13 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC844; wherein lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0050] Fig.14 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC864; wherein lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0051] Fig.15 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC866; among them, lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0052] Fig.16 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC873; among them, lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0053] Fig.17 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC874; wherein lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0054] Fig.18 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC878; wherein lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0055] Fig.19 The figure shows the results of ISSR-PCR electrophoresis amplification using primer UBC881; wherein lanes Q1 to Q15 correspond to 15 samples of Pleurotus eryngii germplasm;

[0056] Fig. 20 Shown is the phylogenetic tree of 15 samples of Pleurotus eryngii constructed based on ISSR-PCR. DETAILED DESCRIPTION

[0057] The present invention discloses a molecular marker system for specificity and diversity identification of seven-leaf floribunda germplasm and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0058] The purpose of the present invention is to provide a seven-leaf thorn worm ISSR-PCR reaction system and a labeling method and application, taking seven-leaf thorn worm as the research object, through orthogonal test, to establish and optimize the seven-leaf thorn worm ISSR-PCR reaction system, and provide a reference for the study of genetic diversity of seven-leaf thorn worm. To achieve the above purpose, the present invention adopts the following technical scheme:

[0059] The invention provides a seven-leaf parsley ISSR-PCR reaction system, wherein each 10 μL reaction system contains 1 μL (50 ng / μL) of template DNA, 0.2 μL (10 μmol / L) of primers, 5 μL ((Tap DNA polymerase, elongation promoting factor, dNTPs)), dd H 2 O is 3.8 μL, and the primer is any one of UBC824, UBC834, UBC835, UBC844, UBC864, UBC866, UBC873, UBC874, UBC878, and UBC881;

[0060] The UBC824 primer sequence is TCTCTCTCTCTCTCTCTG (SEQ ID NO. 1);

[0061] The UBC834 primer sequence is AGAGAGAGAGAGAGAGYT (SEQ ID NO. 2);

[0062] The UBC835 primer sequence is AGAGAGAGAGAGAGAGYC (SEQ ID NO. 3);

[0063] The UBC844 primer sequence is CTCTCTCTCTCTCTCTRC (SEQ ID NO. 4);

[0064] The UBC864 primer sequence is ATGATGATGATGATGATG (SEQ ID NO. 5);

[0065] The UBC866 primer sequence is CTCCTCCTCCTCCTCCTC (SEQ ID NO. 6);

[0066] The UBC873 primer sequence is GACAGACAGACAGACA (SEQ ID NO. 7);

[0067] The UBC874 primer sequence is CCCTCCCTCCCTCCCT (SEQ ID NO. 8);

[0068] The UBC878 primer sequence is GGATGGATGGATGGAT (SEQ ID NO.9);

[0069] The UBC881 primer sequence is GGGTGGGGTGGGGTG (SEQ ID NO. 10).

[0070] The mixed solution formed by the ISSR-PCR reaction system of Pleurotus eryngii was further amplified according to the following procedure: pre-denaturation at 94°C for 5 min, denaturation at 95°C for 15 s, annealing at 48.5-63.2°C for 15 s, extension at 72°C for 30 s, a total of 35 cycles, and finally extension at 72°C for 6 min and storage at 4°C.

[0071] Preferably, the annealing temperature of the UBC824 primer sequence is 53.0°C; the annealing temperature of the UBC834 primer sequence is 55.8°C; the annealing temperature of the UBC835 primer sequence is 56.3°C; the annealing temperature of the UBC844 primer sequence is 52.3°C; the annealing temperature of the UBC864 primer sequence is 51.2°C; the annealing temperature of the UBC866 primer sequence is 63.2°C; the annealing temperature of the UBC873 primer sequence is 51.9°C; the annealing temperature of the UBC874 primer sequence is 60.8°C; the annealing temperature of the UBC878 primer sequence is 48.5°C; and the annealing temperature of the UBC881 primer sequence is 54.3°C.

[0072] Furthermore, the present invention provides a method for ISSR-PCR labeling of Parsley septempunctata, comprising the following steps:

[0073] Extract the genomic DNA of Pleurotus eryngii and perform quality inspection;

[0074] The ISSR-PCR reaction system is used to perform PCR amplification on the extracted genomic DNA.

[0075] The ISSR-PCR reaction system of Parsley septempunctata provided by the invention can be applied to the genetic diversity analysis of Parsley septempunctata.

[0076] The technical effect achieved by the present invention: the present invention uses the optimized amplification reaction system to screen the annealing temperatures of different primers, explores the optimal annealing temperature of the primers, and determines the optimal reaction system of seven-leaf parsley: 10 μL of the reaction system contains 1 μL (50 ng / μL) of template DNA, 0.2 μL (10 μmol / L) of primers, 5 μL of 2×Tap MasterMix (Tap DNA polymerase, elongation promoting factor, dNTPs), and 3.8 μL of ddH2O. The optimized reaction system was used to screen out the best annealing temperatures of 10 primers (UBC824, UBC834, UBC835, UBC844, UBC864, UBC866, UBC873, UBC874, UBC878, and UBC881), which were 53.0℃, 55.8℃, 56.3℃, 52.3℃, 51.2℃, 63.2℃, 51.9℃, 60.8℃, 48.5℃, and 54.3℃, respectively. The optimized reaction system was used to detect ISSR-PCR molecular markers in 15 varieties (lines) of P. septempunctata, and the results showed that the above reaction system was stable and reliable. By establishing and optimizing the ISSR-PCR system of P. septempunctata, a stable system of its molecular markers was obtained, which laid a foundation for the subsequent protection and utilization of P. septempunctata resources.

[0077] Unless otherwise specified, the molecular marker system for identifying the germplasm specificity and diversity of Parsley chinensis provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0078] The present invention will be further described below in conjunction with embodiments:

[0079] Example

[0080] 1 Materials

[0081] Fifteen samples of Pleurotus eryngii germplasm were collected from Yichang City, Hubei Province (2 samples, Q1 and Q2), Anqing City, Anhui Province (2 samples, Q3 and Q4), Dazhou City, Sichuan Province (1 sample, Q5), Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province (1 sample, Q6), Ji'an City, Jiangxi Province (1 sample, Q7), Ankang City, Shaanxi Province (1 sample, Q8), Nanping City, Fujian Province (5 samples, Q10, Q11, Q13, Q14, Q15), and Wenshan City, Yunnan Province (2 samples, Q9 and Q12), and were planted in the greenhouse of Fujian Agriculture and Forestry University after collection.

[0082] 2. Genomic DNA extraction and detection

[0083] The young leaves of the plant were used as materials, ground with liquid nitrogen for 4-5 minutes, and then the plant genomic DNA was extracted using a kit method (Tiangen Biotechnology Co., Ltd.; catalog number Dp305-03). The quality of DNA extraction was tested by 1.5% agarose gel electrophoresis and micro-ultraviolet spectrophotometer. The results are shown in the attached Figure 1 The DNA bands were clear and bright, with no obvious tailing, indicating that the integrity of the genomic DNA was good; the OD of DNA was detected by a micro-UV spectrophotometer. 260 / OD 280 The values ​​were all between 1.70 and 1.90, indicating that the purity of genomic DNA was good.

[0084] 3ISSR reaction system optimization

[0085] Using the genomic DNA of the leaves of Polygonum multiflorum (Q4) as the template and UBC844 as the primer, the L25 (53) orthogonal design was used to optimize the 2×Tap MasterMix (Tap DNA polymerase, elongation facilitating factor, dNTPs, Novozymes Biotech Co., Ltd., catalog number p222-02-AA) content, primer volume (concentration 10μM), and DNA template (50ng / μL) volume. The composition of the PCR amplification system is shown in Table 1, and the reaction system is 10μL. ISSR-PCR amplification reaction procedure: pre-denaturation at 95℃ for 5min; denaturation at 95℃ for 15s; annealing at 52.3℃ for 15s; extension at 72℃ for 30s; cycle 35 times; extension at 72℃ for 6min, and storage at 4℃. The PCR amplification products were screened by 2% agarose gel electrophoresis. The results are shown in Figure 2 , according to the amplification efficiency, band clarity, background quality, etc., the optimization was carried out. In the figure, except for lanes 1, 2, 6, 11, 12, and 21, all other treatments have bands; among them, the bands amplified in lanes 9, 13, and 18 are clear and bright, without tailing, which is an ideal treatment. Considering the effect and cost, condition No. 9 (experimental group) is the best reaction system: 2×TaqPCRMasterMixⅡ5μL, primer volume 0.2μL, template DNA volume 1μL, ddH 2 Add 0.1% HO to make up to 10 μL.

[0086] Table 1 Optimization of ISSR-PCR reaction system

[0087]

[0088] 4ISSR primer screening

[0089] The genomic DNA of Q4 was used as a template, and 100 pairs of ISSR primers published by Columbia University were used. The primers were synthesized by Fuzhou Shangya Biotechnology Co., Ltd. for PCR amplification. The primer screening criteria were clear amplification bands, no mixed bands, and no background. Finally, 10 effective primers were screened (Figure 3). The specific primer sequences are shown in Table 2.

[0090] Table 2 10 pairs of ISSR primers for Pseudocephala septempunctata

[0091]

[0092] 5. Determination of the optimal primer annealing temperature

[0093] The optimal annealing temperature of 10 pairs of UBC primers was determined by the optimized ISSR-PCR system. Each primer was tested at a gradient temperature of (Tm±5)℃ according to the predicted theoretical annealing temperature Tm (Tm=62.3+41×GC%-500 / total base number), and 8 temperature gradients were set to screen the bands with good stability and high clarity to determine the optimal annealing temperature. The results showed that different annealing temperatures had a significant effect on the amplification of ISSR-PCR. The band diagrams of some amplification primers are shown in Figures 3 to 5. When the annealing temperature was not optimal, no bands or blurred bands appeared. The optimal annealing temperatures of 10 primers (UBC824, UBC834, UBC835, UBC844, UBC864, UBC866, UBC873, UBC874, UBC878, and UBC881) were 53.0°C, 55.8°C, 56.3°C, 52.3°C, 51.2°C, 63.2°C, 51.9°C, 60.8°C, 48.5°C, and 54.3°C, respectively (Table 3).

[0094] Table 3 Optimal annealing temperature determined for each primer

[0095]

[0096]

[0097] Comparative Example

[0098] Using the primer sequences shown in Table 4, the genomic DNA of the seven-leafed gentian (Q4) in the embodiment was used as a template, and the amplification program in the 3ISSR reaction system was optimized for amplification. The results are as follows Figure 6 to Figure 8 shown. Figure 6 The bands of primers 848, 849, 850, and 851 were tailing, indicating that these four primers were not suitable; Figure 7 For primers 801, 802, 803, 804, 805, and 806, no amplification products were observed, indicating that these six primers were not suitable for this variety; Figure 8For the annealing temperature amplification of primer 820, 8 annealing gradients were set, and primer dimers appeared in all of them, indicating that this primer is not suitable for this variety.

[0099] Table 4 Primer sequences

[0100]

[0101] Validation of the ISSR-PCR system optimized for Parsley japonica

[0102] In order to verify the effectiveness of the primers screened in the embodiment and the effect of establishing an optimized ISSR system, 15 samples of seven-leaf gentiana germplasm resources were tested for polymorphism using the 10 screened UBC primers, as shown in Table 5. A total of 55 sites were amplified by the 10 primers, of which 48 were polymorphic sites and the percentage of polymorphic bands was 86.66%. The number of amplification sites of the primers was 5 to 6, of which UBC844, UBC864 and UBC874 had the most amplification sites, and UBC878 had the least. The polymorphism ratio of the primers was 60.0% to 100.0%, with an average of 86.66%, of which UBC844, UBC864, UBC866 and UBC874 had a polymorphism ratio of 100%, and UBC878 had a minimum of 60.0%. The genetic distance between individuals was calculated based on allele frequency, the genetic similarity coefficient between individuals was calculated using NTSYSpc2.10e, and the phylogenetic tree was constructed using UPGMA. The primer amplification bands are attached. Figures 9 to 18 The primers screened have many amplification sites and polymorphic sites. The phylogenetic tree is attached. Fig.19 When the similarity coefficient was 0.786, the 15 resources were divided into three categories. The first category included Q1 and Q2, the second category included Q3, Q4, Q5, Q6, and Q7, and the third category included Q8, Q9, Q10, Q11, Q12, Q13, Q14, and Q15. The above results show that the 10 selected primers and the established ISSR-PCR system can well distinguish the samples of P. septempunctata from different sources.

[0103] Table 5 Band numbers and polymorphisms amplified by primers

[0104]

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of ISSR-PCR primer sets in genetic diversity analysis, germplasm resource identification and molecular breeding of Parsley chinensis, wherein the ISSR-PCR primer sets consist of nucleotide sequences shown in SEQ ID NOs. 1 to 10.

2. A molecular marker system for identification of germplasm specificity and diversity of Parsley quinquefolia, characterized in that: include: Each 10 μL reaction system contains template DNA 5 ng / μL, primer 0.2 μmol / L, 2×Tap Master Mix 5 μL, and dd H2O to make up to 10 μL; the primer consists of the nucleotide sequence shown in SEQ ID NO.1~10.

3. The molecular marker system according to claim 2, characterized in that The amplification procedure of the molecular marker includes: pre-denaturation at 94°C for 5 min, denaturation at 95°C for 15 s, annealing at 48.5-63.2°C for 15 s, extension at 72°C for 30 s, a total of 35 cycles, and finally extension at 72°C for 6 min.

4. The molecular marker system according to claim 3, characterized in that The annealing temperature is determined according to the selected primers, including: 。 5. A kit, characterized in that: Comprising the molecular marker system as described in any one of claims 2 to 4.

6. Application of any of the following in genetic diversity analysis of Pleurotus eryngii: (I) The molecular marker system according to any one of claims 2 to 4; and / or (II) The kit according to claim 5.

7. The ISSR-PCR marker method of Parsley septempunctata, characterized in that: The steps include: Step 1, extracting genomic DNA from the sample of Pleurotus eryngii; Step 2: Using the molecular marker system according to any one of claims 2 to 4 or the kit according to claim 5, perform ISSR-PCR amplification on the genomic DNA in step 1.

8. A method for analyzing genetic diversity of Parsley quinquefolia, characterized in that: The steps include: Step 1, extracting genomic DNA from the sample of Pleurotus eryngii; Step 2, using the molecular marker system according to any one of claims 2 to 4 or the kit according to claim 5, performing ISSR-PCR amplification on the genomic DNA in step 1 to obtain a PCR amplification product; Step 3, performing electrophoresis separation on the PCR amplification product described in step 2 to obtain electrophoresis band information; Step 4: Calculate the genetic similarity coefficient and construct a phylogenetic tree.

9. The method according to claim 8, characterized in that The electrophoretic separation in step 3 includes using 1.5% agarose gel electrophoresis.

10. The method according to claim 8 or 9, characterized in that Step 4 specifically includes: using NTSYSpc 2.10e to calculate the genetic similarity coefficient between individuals; using UPGMA to construct a phylogenetic tree.

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