ISSR-PCR reaction system for identifying kiwi fruit varieties with different colors and method and application thereof

Abstract

The invention provides an ISSR-PCR reaction system for identifying kiwi fruit varieties with different colors and a method and application of the ISSR-PCR reaction system, and belongs to the technical field of molecular markers. The ISSR-PCR reaction system for identifying the kiwi fruit varieties with different colors is as follows: the DNA template is 70ng / mu L, the primer is 1.0 mu mol / L, the Mg < 2 + > is 2mM, the mix is 10mu L, and the remaining ddH2O is prepared into 20 mu L of the system. The amplification procedure is as follows: the pre-denaturation temperature is 94 DEG C, and the time is 5 minutes; the denaturation temperature is 94 DEG C for 30 seconds; the annealing temperature is set according to different primer conditions, and the time is 50s; the extension temperature is 72 DEG C, and the extension time is 1 min; circulation is performed for 35 times; and then extending at 72 DEG C for 7 minutes, and storing at 10 DEG C. The system is used for amplifying 42 kiwi fruit germplasm resources with different colors, the amplification result shows that the established ISSR-PCR reaction system is good in repeatability and high in stability, and the established ISSR molecular fingerprint spectrum can be used for variety identification.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular markers, and particularly relates to an ISSR-PCR reaction system for identifying different color kiwifruit varieties, and a method and application thereof. BACKGROUND

[0002] Kiwifruit is a perennial dioecious deciduous liana, belonging to the Actinidiaceae family and the Actinidia genus. There are 54 species in the Actinidia genus, and 21 sub-species. Kiwifruit is delicious and nutritious, rich in various nutrients, especially vitamin C. It is a fruit with high nutritional value and economic value, and is known as the king of fruits. China is the original country of kiwifruit, and has abundant genetic resources. After decades of development, China has become the largest kiwifruit producer in the world in terms of planting area and yield.

[0003] Kiwifruit has obvious interspecific hybridization in nature, and the chromosome ploidy is complex. The efficiency of genetic research using conventional methods is not high. The development of DNA molecular markers provides an effective way for kiwifruit research. Molecular marker technologies such as RAPD, AFLP, SSR, and ISSR have been applied to the evaluation of genetic diversity of kiwifruit germplasm resources, genetic map construction, sex identification, and trait mapping of kiwifruit. Among them, the ISSR (inter-simple sequence repeat) molecular marker technology is a DNA fingerprint marker that combines the advantages of RAPD and SSR technologies, and has high efficiency in germplasm resource classification and identification. In recent years, some scholars have used ISSR technology to analyze the genetic diversity of kiwifruit germplasm resources from different sources with good results. Zou You et al. used ISSR molecular marker technology to analyze the genetic diversity and genetic relationship of 14 kiwifruit varieties, and studied the relationship between the genetic distance of each variety and its geographical distribution. Xie Hui et al. used ISSR molecular markers to analyze the genetic diversity of 'Hongyang' kiwifruit and its hybrid offspring, and constructed their molecular fingerprint maps. Zhang Anshi et al. used ISSR markers to analyze the genetic diversity of 32 samples of Chinese, delicious, soft jujube, and hairy flower kiwifruit, and constructed DNA fingerprint maps. Liu Jian used ISSR molecular markers to analyze the genetic diversity of 16 male kiwifruit materials.

[0004] Flesh color is one of the important economic traits of kiwifruit, and commercial kiwifruit varieties are divided into three types according to the flesh color: green-fleshed, yellow-fleshed and red-fleshed varieties. At present, different ISSR-PCR reaction systems suitable for different flesh color kiwifruit are different, and there is a lack of an ISSR-PCR reaction system suitable for different flesh color kiwifruit. So far, the research on the optimization of ISSR-PCR reaction system for different color kiwifruit has not been reported.

[0005] Therefore, how to develop an ISSR-PCR reaction system suitable for different color kiwifruit, to provide a theoretical basis for future research on genetic diversity and important economic traits of different color kiwifruit germplasm resources using this technology, and lay a foundation for identifying varieties and breeding excellent new varieties through DNA fingerprinting, has become a technical problem to be solved. SUMMARY

[0006] The purpose of the present application is to solve the above technical problems, and to provide an ISSR-PCR reaction system for identifying different color kiwifruit varieties and its method and application. The molecular marker method provided by the present application can analyze the genetic diversity of different color kiwifruit varieties, and the established ISSR molecular fingerprinting can be well applied to the identification of different color kiwifruit varieties.

[0007] In order to achieve the above technical purpose, the technical scheme adopted by the present application is as follows:

[0008] The present application first provides an ISSR-PCR reaction system for identifying different color kiwifruit varieties, which comprises: the DNA template is 70 ng / μL, the primer is 1.0 μmol / L, the Mg 2+ is 2 mM, the mix is 10 uL, and the rest is prepared into a system of 20 uL with ddH2O; wherein, the primer sequence and annealing temperature are as follows:

[0009]

[0010] The second purpose of the present application is to provide an ISSR-PCR reaction system for identifying different color kiwifruit varieties as described above, which comprises the following steps:

[0011] (1) Selecting different color kiwifruit germplasm resources materials, picking young leaves, grinding with a tissue grinder, and extracting DNA with a kit;

[0012] (2) Establishing an ISSR-PCR amplification system according to the ISSR-PCR reaction system as described above, using the DNA obtained in step (1) as the PCR template, and performing PCR amplification;

[0013] (3) agarose gel electrophoresis is used to detect the amplification product obtained in step (2), and whether the ISSR-PCR amplification primer appears on the same position on the agarose gel is counted, an ISSR amplification band matrix database is established, and the polymorphic bands are analyzed.

[0014] Further, the PCR amplification procedure in step (2) is as follows:

[0015] 1) pre-denaturation temperature 94℃, time 5min;

[0016] 2) denaturation temperature 94℃, time 30s;

[0017] 3) annealing temperature is set according to different primers in Table 1, time 50s;

[0018] 4) extension temperature 72℃, time 1min; 35 cycles;

[0019] 5) re-extension temperature 72℃, time 7min, 10℃ storage.

[0020] Further, the gel electrophoresis detection step in step (3) is as follows: 2% agarose gel electrophoresis is used, and the electrophoresis instrument parameters are set as voltage 130V, and the running gel time is 45min.

[0021] Further, the DNA extraction method in step (1) is that the BIOFIT plant genomic DNA extraction kit is used for extraction.

[0022] The third object of the present application is to provide the application of the ISSR-PCR reaction system or the method as described above in identifying different color kiwifruit varieties.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The present application provides a set of ISSR-PCR reaction system and method for identifying genetic diversity analysis of different color kiwifruit varieties, which can be well used for genetic diversity analysis of different color kiwifruit varieties, and establishes the ISSR molecular fingerprint of each variety for variety identification. 2+Single factor experiment and orthogonal optimization design experiment were carried out in concentration, mix volume and other aspects, and the system suitable for amplification of 42 different color kiwifruit materials was found, and the suitable amplification system and program parameters for amplification of other kiwifruit varieties were also found. The system is used for amplification of 42 different color kiwifruit germplasm resources, and the amplification results show that the established ISSR-PCR reaction system has good repeatability and strong stability. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Part of kiwifruit DNA gel electrophoresis detection.

[0026] Figure 2 Part of primer screening amplification results.

[0027] Figure 3 Primer 880 annealing temperature gradient results.

[0028] Figure 4 The results of the influence of DNA concentration on the PCR system.

[0029] Figure 5 The results of the influence of primer concentration (primer 879) on the PCR system.

[0030] Figure 6 The results of the influence of Mg 2+ on the PCR system.

[0031] Figure 7 The results of the influence of Taq mix on the PCR system.

[0032] Figure 8 ISSR-PCR amplification system optimization results; wherein, M is DL 2000 DNA marker, 1-16 are 16 kinds of PCR amplification results after orthogonal design.

[0033] Figure 9 Amplification results of primer 862 on 42 materials; wherein, M is DL 2000 DNA marker, 1-42 are 42 kiwifruit young leaf test materials; the ISSR-PCR reaction system suitable for 'Hongyang' kiwifruit is: total volume 20 μL, 10×PCR buffer 2.0 μL, Mg 2+ 0.75 mmol / L, Taq enzyme 1.5 U, template DNA 60 ng, primer 1.25 μmol / L, dNTPs 0.15 mmol / L; amplification program: 94℃ pre-denaturation for 7 min, 94℃ denaturation for 30 s, 58℃ annealing for 45 s, 72℃ extension for 2 min, 45 cycles, 72℃ extension for 7 min; the optimal annealing temperature of primer UBC841 is 58.5℃. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is described in detail below with examples. It is necessary to point out that the following examples are only used to explain and describe the present application, and do not limit the present application. Some non-essential improvements and adjustments made by those skilled in the art according to the above description still belong to the protection scope of the present application.

[0035] Example 1

[0036] I. Test materials and methods

[0037] 1. Plant materials

[0038] Forty-two kiwifruit germplasm resources of different colors (Table 1) were selected in Guangji kiwifruit base in Shifang City, Sichuan Province as test materials. The young leaves of kiwifruit were collected in April to May, and the leaves without diseases and pests and physical and mechanical damage were used as the collection standard. After the leaves were quickly frozen with liquid nitrogen, they were stored in a -80℃ ultra-low temperature refrigerator for standby.

[0039] Table 1: Forty-two kiwifruit test materials

[0040]

[0041] 2. Main reagents and instruments

[0042] (1) Main reagents

[0043] Table 2: Main reagents

[0044]

[0045] (2) Main instruments

[0046] Table 3: Main instruments

[0047]

[0048] 3. Test methods

[0049] (1) DNA extraction

[0050] The DNA of the young leaves of kiwifruit was extracted according to the BIOFIT plant genomic DNA extraction kit. The tissue grinder was used for grinding, and the kit was used for extraction. The specific steps were referred to the instruction manual.

[0051] (2) Detection of DNA quality and purity

[0052] The extracted DNA was detected by agarose gel electrophoresis and ultraviolet spectrophotometer.

[0053] DNA integrity detection: 1% agarose electrophoresis, and then observe the situation by imaging light instrument; DNA concentration and content detection: use ultraviolet spectrophotometer Nanodrop2000 to detect, record the absorbance value of DNA at 260nm, 280nm, and calculate their ratio. If the ratio is close to 1.8, it means that the extracted DNA is relatively pure.

[0054] (3) Primer design and synthesis

[0055] Select 100 ISSR primers published by Columbia University, and entrust Genkey Biological Company to synthesize according to the primer sequence.

[0056] (4) Primer screening and temperature gradient setting

[0057] 1) Select two templates with large differences from the test materials, and use the two templates to preliminarily screen the primers;

[0058] 2) Select three templates for secondary screening, select primers with good amplification effect, clear bands, high polymorphism and good repeatability in all samples for subsequent amplification of all samples;

[0059] 3) According to the given primer annealing temperature of the manufacturer, set several gradients, repeat the test twice, and select the stable annealing temperature as the optimal temperature of the primer according to the results of the two tests.

[0060] (5) Establish ISSR-PCR amplification system

[0061] Single factor test on DNA template concentration, enzyme content, upstream and downstream primer concentration, Mg 2+ concentration, etc. in the amplification system, the test factor level design table is shown in Table 4, and then further optimize the amplification conditions by orthogonal method using the single factor test results, and the orthogonal design table is shown in Table 5. Amplification procedure: pre-denaturation temperature 94℃, time 5min; denaturation temperature 94℃, time 30s; annealing temperature is set according to different primers, time 50s; extension temperature 72℃, time 1min; 35 cycles; then extension temperature 72℃, time 7min, 10℃ storage.

[0062] Table 4 Test factor level design table

[0063]

[0064] Table 5 Orthogonal test design table

[0065]

[0066] The orthogonal optimization results were scored by intuitive analysis method. The amplification results of 16 combinations were scored from high to low according to 1-16 points. The amplification results with more, clear and bright bands were scored. The best result was 16 points and the worst result was 1 point.

[0067] (6) Detection of amplification products

[0068] First, prepare 2% agarose gel; then set the electrophoresis instrument parameters to voltage 130V and running time 45min; after running, expose the gel in the imaging instrument, and finally record the experimental results and save them.

[0069] II. Experimental results and analysis

[0070] 1. DNA extraction and quality detection

[0071] After exposure with the gel imaging instrument, as shown in Figure 1 , the genomic conditions were bright and clear with clear boundaries and no RNA display. Then, the genomic DNA was detected by the nucleic acid detector, as shown in Table 6. The concentration range was between 60.815-360.091 ng / μL, and the OD 260nm / OD 280n m ratio was almost close to 1.80, indicating that the DNA sample extracted by the plant DNA kit had less impurity content, meeting the requirements of DNA purity and concentration for molecular experiments, and could be used for subsequent experiments.

[0072] Table 6. OD values and concentrations of some kiwi DNA

[0073]

[0074] 2. Primer screening and temperature gradient setting

[0075] (1) Primer screening

[0076] The synthesized 100 ISSR primers were screened, and the results are shown in Figure 2 . After repeated experiments and preliminary screening and rescreening, 12 ISSR primers with good stability, high polymorphism and clear bands were obtained, which were used for subsequent ISSR-PCR system optimization.

[0077] (2) Temperature gradient setting

[0078] The 12 screened ISSR primers were selected for temperature gradient selection, as shown in Figure 3 . Each primer was set to 12 temperature gradients, which were 48.0℃, 48.3℃, 49.0℃, 50.2℃, 51.7℃, 53.3℃, 54.7℃, 56.3℃, 57.8℃, 59.0℃, 59.7℃ and 60.0℃, respectively.Figure 3 In the middle, it can be seen that the primer 880 at 51.7℃, the number of bands and bright. By screening 12 ISSR primer suitable annealing temperature, as shown in Table 7. The results of analysis, annealing temperature on the amplification results have certain influence, the results of different annealing temperature exist certain difference, find the suitable annealing temperature, more conducive to the amplification results, also facilitate the subsequent ISSR-PCR optimization.

[0079] Table 7 primer sequence

[0080]

[0081] 3, ISSR-PCR amplification system optimization

[0082] (1) single factor experiment on the influence of PCR system

[0083] 1) the influence of DNA concentration on PCR system

[0084] According to the scoring criteria of the single factor results are scored statistics. As Figure 4 shown, when the amplification system of DNA concentration is 70ng / uL, the average score of the highest result is 14 points, the DNA concentration of 30ng / uL, the average score of the lowest is 5 points, combined with the original concentration of kiwi genome, determine the DNA concentration of 70ng / uL is the best DNA concentration level;

[0085] 2) the influence of primer concentration on PCR system

[0086] As Figure 5 shown, the average score increases with the increase of primer concentration, when the primer concentration reaches 1.0umol / L, the average score of the highest is 9 points, according to the results and consumables cost situation of many aspects, determine the primer concentration of 1.0umol / L is the best primer concentration level;

[0087] 3) the influence of Mg 2+ on PCR system

[0088] As Figure 6 shown, when the Mg 2+ concentration is 2mM, the average score of the results reaches the highest is 16 points, when the Mg 2+ concentration increases to 4mM, the score standard is the lowest is 4 points, with the increase of Mg 2+ concentration, the average score of the experiment shows the trend of rising to decline, too much Mg 2+ may be the system of salt ions too much, thus affecting the amplification effect, according to the results of analysis, determine the Mg 2 + concentration of 4mM is the best concentration level;

[0089] 4)mix on PCR system

[0090] As Figure 7 shown, when the volume of mix reaches 10uL, the average score is the highest, which is 11, and when the volume is 7.5uL, the average score is the lowest, which is 3, with the increase of the volume of mix, the average score presents the trend of first increasing, then decreasing and then increasing, considering the cost-effectiveness and amplification effect, the volume of mix is selected as 10uL as the best volume level.

[0091] (2) intuitive analysis scoring method

[0092] The orthogonal optimization results are scored by using the intuitive analysis method, and the results are shown in Figure 8 , which are the amplification results of 16 combinations, and are scored from high to low according to 1-16, the amplification results with more, clear and bright bands are scored, the best result is 16, the worst is 1, and then Figure 8 the scoring results from left to right are: 1, 11, 4, 12, 13, 14, 2, 7, 9, 10, 5, 16, 15, 8, 3, 6.

[0093] The results of various analyses obtain different color kiwifruit ISSR-PCR amplification reaction systems: the template is 70ng / μL, the primer is 1.0μmol / L, Mg 2+ is 2mM, mix is 10μL, and the rest is prepared to the system 20μL with ddH2O. The above system parameters are used for amplification of 29 hybrid F1 generations and 13 different varieties of kiwifruit, and as shown in Figure 9 , the amplification results of ISSR primer 862 on the 42 materials show that the number of bands is rich and clear, which indicates that the system reaction is stable, the repeatability is high, the polymorphism is good, and can be used for ISSR reaction of different color kiwifruit.

[0094] The application establishes the ISSR-PCR reaction system suitable for 'Hongyang' kiwifruit: the total volume is 20μL, 10×PCRbuffer is 2.0μL, Mg 2+ is 0.75mmol / L, Taq enzyme is 1.5U, template DNA is 60ng, primer is 1.25μmol / L, and dNTPs is 0.15mmol / L. The amplification procedure is: 94℃ pre-denaturation for 7min, 94℃ denaturation for 30s, 50-58℃ annealing for 45s, 72℃ extension for 2min, 45 cycles, and 72℃ extension for 7min. The optimal annealing temperature of primer UBC841 is 58.5℃.

[0095] III. Conclusion

[0096] The contents of polyphenol and polysaccharide in the leaves of kiwifruit were high. In order to extract DNA from the leaves of kiwifruit quickly and efficiently, the tender leaves should be chosen as sample, and the tissue grinder should be used instead of the traditional mortar and pestle. The DNA extracted by the improved CTAB method and SDS method in the early stage of the experiment contained many impurities. After several tests, the plant DNA kit was used to extract DNA, and high purity DNA was obtained.

[0097] Twelve ISSR primers were selected as the amplification primers in this experiment by the methods of preliminary screening and rescreening. These primers could produce many bands and high polymorphism, and there were differences among different materials. The most suitable annealing temperature of each ISSR primer was selected by temperature gradient setting, and the ISSR-PCR amplification system was optimized from the aspects of template concentration, primer concentration, Mg 2+ The amplification system for 42 different color kiwifruit materials was found by single factor experiment and orthogonal optimization design experiment, and the suitable amplification system and program parameters for other kiwifruit varieties were also found. This reaction system and method could be used for genetic diversity analysis of different color kiwifruit varieties, and the ISSR molecular fingerprint of each variety was established for variety identification.

Claims

1. An ISSR-PCR reaction system for identifying different colored kiwifruit varieties, characterized in that, The ISSR-PCR reaction system includes: DNA template of 70 ng / μL, primers of 1.0 μmol / L, and Mg... 2+ The initial concentration was 2 mM, the mix volume was 10 μL, and the remainder was diluted with ddH2O to a final volume of 20 μL. The primer sequences and annealing temperatures are as follows:

2. A method for identifying different colored kiwifruit varieties using the ISSR-PCR reaction system described in claim 1, characterized in that, Includes the following steps: (1) Select kiwifruit germplasm resources of different colors and extract DNA from tender leaves; (2) Establish an ISSR-PCR amplification system according to the ISSR-PCR reaction system described in claim 1, and perform PCR amplification using the DNA obtained in step (1) as a PCR template; (3) Detect the amplification products obtained in step (2) by agarose gel electrophoresis, count whether the ISSR-PCR amplification primers appear at the same position on the agarose gel, establish an ISSR amplification band matrix database, and analyze polymorphic bands.

3. The method according to claim 2, characterized in that, The PCR amplification procedure described in step (2) is as follows: 1) Pre-denaturation temperature: 94℃, time: 5min; 2) Denaturation temperature 94℃, time 30s; 3) The annealing temperature should be set according to different primers, referring to Table 1, and the time should be 50 seconds; 4) Extend the temperature to 72℃ for 1 minute; repeat 35 times. 5) Extend the temperature to 72℃ for 7 minutes, and store at 10℃.

4. The method according to claim 2, characterized in that, The gel electrophoresis detection steps in step (3) are as follows: 2% agarose gel electrophoresis is performed with the electrophoresis apparatus parameters set to voltage 130V and running time 45min.

5. The method according to claim 2, characterized in that, The DNA extraction method described in step (1) is to use the BIOFIT plant genomic DNA extraction kit for extraction.

6. The ISSR-PCR reaction system of claim 1 or the method of any one of claims 2-5 is used for the identification of different colored kiwifruit varieties by establishing ISSR molecular fingerprinting.

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