SSR (simple sequence repeat) detection method for grape variety identification and application thereof

Abstract

The invention discloses an SSR (Simple Sequence Repeat) detection method for identifying grape varieties and application of the SSR detection method. The SSR detection kit for identifying the grape varieties comprises SSR primers and a probe, and the SSR primers comprise a VVS2 primer, a VVS16 primer, a VVMD5 primer, a VVMD6 primer, a VVMD7 primer, a VVMD17 primer, a VVMD25 primer, a VVMD27 primer, a VVMD28 primer, a VVMD32 primer, a VrZAG47 primer, a VrZAG62 primer, a VrZAG79 primer, a VrZAG83 primer and a VMC4F3 primer. According to the method, the polymorphic bands with the band size difference of 1bp can be distinguished, automation is easy, operation is simple, the result is stable and reliable, and large-scale and multi-batch data integration and analysis are easy.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to an SSR detection method for grape variety identification and application thereof. Background Art

[0002] SSR, or simple sequence repeat (SSR), is one of the most commonly used microsatellite markers. They are widely distributed in the genomes of various eukaryotic organisms. Since the flanking sequences of a specific microsatellite in the genome of SSR are usually single sequences with strong conservatism, the DNA fragments flanking the microsatellite can be cloned and sequenced, and then primers can be artificially synthesized based on the flanking sequences of the microsatellite for PCR amplification, thereby amplifying a single microsatellite locus. Due to the variation in the number of repeat units at a single microsatellite locus, the change in the length of the individual amplification product produces length polymorphism, which is called simple sequence repeat length polymorphism (SSLP). Each amplification site represents a pair of alleles at this site. Compared with markers such as RFLP, RAPD and AFLP, SSR marker technology has the advantages of high polymorphism, co-dominant inheritance and simple operation. It is used in the fields of variety identification, molecular-assisted breeding of crops such as cereals, beans and fruit trees, and has become an ideal tool for molecular marker screening and identification.

[0003] Grape belongs to the genus Vitis L. of the family Vitaceae (Vltaeeae), a deciduous woody vine, and is one of the world's important economic fruits, accounting for about 25% of the world's fruit production. At present, there are more than 70 species of grape germplasm resources discovered, and 16,000 varieties have been registered. The origin of the genus Vitis is wide, and the influence of artificial introduction, exchange, breeding, and nutrient selection has led to a wide variety of grape varieties and rich and diverse genotypes. Moreover, with the expansion of the cultivation range, the number of varieties has continued to increase, forming many variety groups with local characteristics. However, this phenomenon of rich variation and a wide variety of varieties has caused a large number of homonyms and homonyms in the long-term introduction and cultivation process, making the identification of grape varieties very difficult. At present, my country's grape introduction companies are faced with the problem that the authenticity of the imported grape seedlings cannot be identified, and grapes of different strains of the same variety cannot be distinguished morphologically.

[0004] Traditional morphological identification can no longer effectively meet the needs of rapid identification from a wide variety of grape varieties because it is easily affected by environmental conditions and subjective factors. Molecular marker technology is not affected by the appearance of the plant, the environment, etc., and can accurately identify the authenticity of grape varieties. In addition, SSR molecular markers have the advantages of high polymorphism, genetic stability, and wide distribution throughout the grape genome, and have gradually become the main means of identifying grape varieties. Variety identification is of great significance in protecting the rights of breeders and identifying grape fruit tree varieties. Therefore, the development of grape variety identification detection kits based on SSR molecular markers is very necessary, and the first-generation sequencing platform based on Sanger sequencing has been widely used in molecular breeding, variety identification, and scientific research.

[0005] SSR molecular identification technology is mainly used in agricultural crops and various economic crops and has shown great application potential in the identification of multiple species. Therefore, according to the different genetic composition of different grape varieties, there are differences in the number of repetitions of simple repeat sequences in genomic DNA. This difference can be detected by PCR amplification and electrophoresis using an SSR detection kit, thereby distinguishing different grape varieties. And currently there are few reports on the use of SSR molecular marker technology to identify grape varieties, and the grape fruit tree varieties that can be identified are also limited. Therefore, the present invention independently developed 15 pairs of SSR marker primers, which can be used to quickly identify a variety of common grape varieties at home and abroad through SSR genotyping technology, providing technical support for grape fruit tree variety identification and further development and utilization.

[0006] So far, many SSR primers have been developed for grapes, especially some primers with high polymorphism and good amplification effect, which have been widely used in various analytical studies of grapes. However, some newly developed molecular marker detection reagents on the market are rarely used on grapes. In 2016, the "SSR Molecular Marker Method for Grape Variety Identification" was listed as an agricultural industry standard formulation project. With the support of related projects and the advancement of molecular technology and equipment, the present invention aims to solve and form standardized, accurate and stable grape SSR detection reagents, and screen out a set of SSR molecular identification systems that meet domestic and foreign grape variety identification standards, provide technical support for domestic and foreign grape variety identification, new variety protection, variety registration and market rights protection, and play an important role in future grape variety identification work.

[0007] At present, 9 pairs of SSR primers, namely VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62 and VZAG79, have been used as universal markers for international grape variety identification. However, international grape varieties are mainly Eurasian species. As the concentrated distribution area of ​​the world's East Asian grape population, China has rich and unique germplasm resources. Relying only on these international pairs of markers has limited the ability to distinguish Chinese grape varieties. Therefore, with the increasing number of new grape varieties bred in my country and the increasing demand for grape variety resource management, my country urgently needs to develop a set of grape SSR marker identification system that is more suitable for China.

[0008] Currently, there is a lack of an SSR detection method with high detection efficiency for grape variety identification and its application. Summary of the invention

[0009] The purpose of the present invention is to provide a grape variety identification SSR detection method with high detection efficiency and application thereof.

[0010] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0011] In a first aspect, the present application provides a grape variety identification SSR detection kit.

[0012] In a second aspect, the present application provides an SSR detection method for grape variety identification.

[0013] The invention discloses a grape variety identification SSR detection kit, comprising SSR primers and probes, wherein the SSR primers are VVS2 primers, VVS16 primers, VVMD5 primers, VVMD6 primers, VVMD7 primers, VVMD17 primers, VVMD25 primers, VVMD27 primers, VVMD28 primers, VVMD32 primers, VrZAG47 primers, VrZAG62 primers, VrZAG79 primers, VrZAG83 primers and VMC4F3 primers.

[0014] The VVS2 primers are the upstream primers of VVS2 and the downstream primers of VVS2, and the probe is a 6-FAM probe; the VVS16 primers are the upstream primers of VVS16 and the downstream primers of VVS16, and the probe is a 6-FAM probe; the VVMD5 primers are the upstream primers of VVMD5 and the downstream primers of VVMD5, and the probe is a PET probe; the VVMD6 primers are the upstream primers of VVMD6 and the downstream primers of VVMD6, and the probe is a PET probe; The VVMD7 primers are the upstream primers of VVMD7 and the downstream primers of VVMD7, and the probe is the NED probe; the VVMD17 primers are the upstream primers of VVMD17 and the downstream primers of VVMD17, and the probe is the VIC probe; the VVMD25 primers are the upstream primers of VVMD25 and the downstream primers of VVMD25, and the probe is the PET probe; the VVMD27 primers are the upstream primers of VVMD27 and the downstream primers of VVMD27, and the probe is the The probe is a NED probe; the VVMD28 primer is an upstream primer of VVMD28 and a downstream primer of VVMD28, and the probe is a VIC probe; the VVMD32 primer is an upstream primer of VVMD32 and a downstream primer of VVMD32, and the probe is a 6-FAM probe; the VrZAG47 primer is an upstream primer of VrZAG47 and a downstream primer of VrZAG47, and the probe is a VIC probe; the VrZAG62 primer is an upstream primer of VrZAG62 and a downstream primer of VrZAG62; The upstream primer of VrZAG79 and the downstream primer of VrZAG62, the probe is a 6-FAM probe; the VrZAG79 primer is the upstream primer of VrZAG79 and the downstream primer of VrZAG79, the probe is a VIC probe; the VrZAG83 primer is the upstream primer of VrZAG83 and the downstream primer of VrZAG83, the probe is a PET probe; the VMC4F3 primer is the upstream primer of VMC4F3 and the downstream primer of VMC4F3, the probe is a NED probe.

[0015] Further, the upstream primer of the VVS2 primer has a nucleotide sequence of SEQ ID No. 1;

[0016] The downstream primer of the VVS2 primer has a nucleotide sequence of SEQ ID No. 2;

[0017] The upstream primer of the VVS16 primer has a nucleotide sequence of SEQ ID No. 3;

[0018] The downstream primer of the VVS16 primer has a nucleotide sequence of SEQ ID No. 4;

[0019] The upstream primer of the VVMD5 primer has a nucleotide sequence of SEQ ID No.5; the downstream primer of the VVMD5 primer has a nucleotide sequence of SEQ ID No.6; the upstream primer of the VVMD6 primer has a nucleotide sequence of SEQ ID No.7; the downstream primer of the VVMD6 primer has a nucleotide sequence of SEQ ID No.8; the upstream primer of the VVMD7 primer has a nucleotide sequence of SEQ ID No.9; the downstream primer of the VVMD7 primer has a nucleotide sequence of SEQ ID No.10; the upstream primer of the VVMD17 primer has a nucleotide sequence of SEQ ID No.11; the downstream primer of the VVMD17 primer has a nucleotide sequence of SEQ ID No.12; the upstream primer of the VVMD25 primer has a nucleotide sequence of SEQ ID No.13; the downstream primer of the VVMD25 primer has a nucleotide sequence of SEQ ID No.14; the upstream primer of the VVMD27 primer has a nucleotide sequence of SEQ ID No.15; the downstream primer of the VVMD27 primer has a nucleotide sequence of SEQ ID The upstream primer of the VVMD28 primer has the nucleotide sequence of SEQ ID No.17; the downstream primer of the VVMD28 primer has the nucleotide sequence of SEQ ID No.18; the upstream primer of the VVMD32 primer has the nucleotide sequence of SEQ ID No.19; the downstream primer of the VVMD32 primer has the nucleotide sequence of SEQ ID No.20; the upstream primer of the VrZAG47 primer has the nucleotide sequence of SEQ ID No.21; the downstream primer of the VrZAG47 primer has the nucleotide sequence of SEQ ID No.22; the upstream primer of the VrZAG62 primer has the nucleotide sequence of SEQ ID No.23; the downstream primer of the VrZAG62 primer has the nucleotide sequence of SEQ ID No.24; the upstream primer of the VrZAG79 primer has the nucleotide sequence of SEQ ID No.25;

[0020] The downstream primer of the VrZAG79 primer has a nucleotide sequence of SEQ ID No.26;

[0021] The upstream primer of the VrZAG83 primer has a nucleotide sequence of SEQ ID No.27;

[0022] The downstream primer of the VrZAG83 primer has a nucleotide sequence of SEQ ID No.28;

[0023] The upstream primer of the VMC4F3 primer has a nucleotide sequence of SEQ ID No.29;

[0024] The downstream primer of the VMC4F3 primer has a nucleotide sequence of SEQ ID No.30.

[0025] Furthermore, the kit includes primers, 6-FAM probe, PET probe, NED probe, PCR buffer, hot start Taq enzyme, dNTP, FRET-PCR standard, PCR negative control; the PCR negative control is double distilled water.

[0026] Furthermore, the FRET-PCR standard is a recombinant plasmid constructed by inserting the amplified nucleotide fragment obtained by amplifying the plasmid standard DNA template using the FRET-PCR primers and probes into the pUC57 vector.

[0027] The PCR amplification system of the grape variety identification SSR detection kit of the present invention uses a 25uL amplification system for the PCR reaction (the kit components include): 2.5×PCR Mix (5mM MgCl2, 125mM PH8.3Tris-HCl, 125mMKCl, 0.5mM dNTPs), 10.0uL, 10umol / L composite primer 15Primers 5.0uL, 5U / uL Taq DNA polymerase 1.0uL, 20-50ng / ul DNA template 1uL, PCR Grade Water 8.0uL; the PCR reaction program is: 94°C pre-denaturation for 4min, 94°C denaturation for 30s, 58°C annealing for 30s, 72°C extension for 45s, a total of 35 cycles, and 72°C extension for 10min.

[0028] A grape variety identification SSR detection method of the present invention comprises the following steps:

[0029] (1) Capillary electrophoresis-SSR technology is used to quickly identify grape varieties. The specific steps of the identification method are as follows:

[0030] (2) DNA extraction: DNA from fresh leaves / plant tissues of grape varieties is extracted using a DNA extraction kit or the CTAB method;

[0031] (3) DNA quality assessment: Use agarose gel electrophoresis to check the quality of nucleic acid. The bands should be clear, without nucleic acid degradation and nucleic acid contamination. Use a spectrophotometer to assess the purity of deoxyribonucleic acid. The DNA concentration should be ≥20 ng / uL.

[0032] (4) Multiplex PCR amplification: Multiplex PCR amplification of grape varieties was performed using 15 SSR primer loci, including VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG79, VZAG62, VVS16, VVMD6, VVMD17, VMC4F3, VrZAG47, and VrZAG83;

[0033] (5) SSR genotyping: With the help of the first-generation Sanger sequencing platform technology, the fully automatic gene analyzer performs capillary electrophoresis-fragment analysis to detect PCR products; before detection, 1.0uL of PCR product is added to each sample well, and the volume ratio of molecular weight standard and formamide mixture is 0.5:8.5, 9uL. After the sample is mixed, it is denatured at 95℃ for 4min and immediately placed on ice for 4min. After electrophoresis, the original SSR data of different grape varieties are obtained, and the typing results are compared and analyzed by fragment analysis software to calculate the fragment length, the number of SSR alleles and site analysis;

[0034] (6) Grape variety identification: Different grape varieties can be effectively identified based on the different SSR gene typing of different grape varieties using the same primers.

[0035] Furthermore, in step (4), the PCR reaction program is: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 45 s, for a total of 35 cycles; extension at 72°C for 10 min.

[0036] Application of the grape variety identification SSR detection kit of the present invention in grape SSR marker identification.

[0037] The invention discloses an application of the grape variety identification SSR detection kit in grape fingerprint construction, variety identification and genetic diversity analysis.

[0038] Beneficial effects: The present invention can not only distinguish polymorphic bands with a band size difference of 1 bp, but also is easy to automate, simple to operate, with stable and reliable results, high detection efficiency, and easy to integrate and analyze large-scale and multi-batch data, and is suitable for the construction or identification of grape variety fingerprints, etc. The kit of the present invention combines fluorescence capillary electrophoresis, has the advantages of automation, standardization, reduction of human errors, a large number of allele detections, the ability to distinguish samples with small variety differences, accurate and stable results, etc., can achieve high-throughput sample and site high-throughput detection, and shorten the time for variety identification.

[0039] Compared with the prior art, the present invention has the following advantages: (1) Multiplex PCR is a PCR reaction in which two or more pairs of PCR primers are added to a reaction system (single multiplex reaction) to simultaneously amplify multiple nucleic acid fragments. Rapid amplification can be achieved within 2 hours, and it has the advantages of simple method and high amplification efficiency.

[0040] (2) The amplified products with different fluorescent labels of the present invention can be electrophoresed in the same lane, and the signal is clear. The size of the amplified fragments is obviously different, and the fragment size can be accurately calculated. The electrophoresis peak of each DNA sample is different, which is easy to judge. It has the advantages of high sensitivity, good resolution, accurate and reliable results, high efficiency and rapidity.

[0041] (3) The present invention selects 9 pairs of SSR molecular marker primers for international grape variety identification, counts the commonly used grape SSR primers in China, and selects 6 pairs (VVS16, VVMD6, VVMD17, VMC4F3, VrZAG47, VrZAG83) of primers with the highest frequency of use, high polymorphism and good amplification effect for analysis. The primer combination provided by the present invention can distinguish different grape varieties conveniently and quickly, achieving the advantages of cost saving, improved efficiency, convenient operation and accurate results.

[0042] (4) The present invention uses a multiplex PCR reaction system to amplify polymorphic fragments, counts commonly used grape SSR primers, and screens out 15 pairs of primers with the highest frequency of use, high polymorphism, and good amplification effect for analysis. The present invention is based on an SSR detection kit for domestic and foreign grape variety identification, using a total of 15 pairs of SSR primers, screening out a stable and consistent PCR annealing temperature, and amplifying polymorphic fragments through a multiplex PCR reaction system, applying the polymorphism of SSR molecular markers and the high-resolution characteristics of capillary gel electrophoresis technology. The set of primers provided by the present invention can be used for grape fingerprint construction, variety identification, genetic diversity analysis, etc., and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 The SSR capillary electrophoresis diagram of the Kyoho grape variety of the present invention;

[0045] Figure 2 The SSR capillary electrophoresis diagram of the Sunshine Rose grape variety of the present invention;

[0046] Figure 3 The SSR capillary electrophoresis diagram of the summer black grape variety of the present invention;

[0047] Figure 4 The SSR capillary electrophoresis diagram of the Meirenzhi grape variety of the present invention;

[0048] Figure 5 The SSR capillary electrophoresis diagram of the Moonlight Seedless Grape variety of the present invention;

[0049] Figure 6 This is the SSR capillary electrophoresis diagram of the Zhengzhou Zaoyu grape variety of the present invention. DETAILED DESCRIPTION

[0050] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0051] Example 1

[0052] The invention discloses a grape variety identification SSR detection kit, comprising SSR primers and probes, wherein the SSR primers are VVS2 primers, VVS16 primers, VVMD5 primers, VVMD6 primers, VVMD7 primers, VVMD17 primers, VVMD25 primers, VVMD27 primers, VVMD28 primers, VVMD32 primers, VrZAG47 primers, VrZAG62 primers, VrZAG79 primers, VrZAG83 primers and VMC4F3 primers.

[0053] The VVS2 primers are the upstream primers of VVS2 and the downstream primers of VVS2, and the probe is a 6-FAM probe; the VVS16 primers are the upstream primers of VVS16 and the downstream primers of VVS16, and the probe is a 6-FAM probe; the VVMD5 primers are the upstream primers of VVMD5 and the downstream primers of VVMD5, and the probe is a PET probe; the VVMD6 primers are the upstream primers of VVMD6 and the downstream primers of VVMD6, and the probe is a PET probe; The VVMD7 primers are the upstream primers of VVMD7 and the downstream primers of VVMD7, and the probe is the NED probe; the VVMD17 primers are the upstream primers of VVMD17 and the downstream primers of VVMD17, and the probe is the VIC probe; the VVMD25 primers are the upstream primers of VVMD25 and the downstream primers of VVMD25, and the probe is the PET probe; the VVMD27 primers are the upstream primers of VVMD27 and the downstream primers of VVMD27, and the probe is the The probe is a NED probe; the VVMD28 primer is an upstream primer of VVMD28 and a downstream primer of VVMD28, and the probe is a VIC probe; the VVMD32 primer is an upstream primer of VVMD32 and a downstream primer of VVMD32, and the probe is a 6-FAM probe; the VrZAG47 primer is an upstream primer of VrZAG47 and a downstream primer of VrZAG47, and the probe is a VIC probe; the VrZAG62 primer is an upstream primer of VrZAG62 and a downstream primer of VrZAG62; The upstream primer of VrZAG79 and the downstream primer of VrZAG62, the probe is a 6-FAM probe; the VrZAG79 primer is the upstream primer of VrZAG79 and the downstream primer of VrZAG79, the probe is a VIC probe; the VrZAG83 primer is the upstream primer of VrZAG83 and the downstream primer of VrZAG83, the probe is a PET probe; the VMC4F3 primer is the upstream primer of VMC4F3 and the downstream primer of VMC4F3, the probe is a NED probe.

[0054] Further, the upstream primer of the VVS2 primer has a nucleotide sequence of SEQ ID No. 1;

[0055] The downstream primer of the VVS2 primer has a nucleotide sequence of SEQ ID No. 2;

[0056] The upstream primer of the VVS16 primer has a nucleotide sequence of SEQ ID No. 3;

[0057] The downstream primer of the VVS16 primer has a nucleotide sequence of SEQ ID No. 4;

[0058] The upstream primer of the VVMD5 primer has a nucleotide sequence of SEQ ID No.5; the downstream primer of the VVMD5 primer has a nucleotide sequence of SEQ ID No.6; the upstream primer of the VVMD6 primer has a nucleotide sequence of SEQ ID No.7; the downstream primer of the VVMD6 primer has a nucleotide sequence of SEQ ID No.8; the upstream primer of the VVMD7 primer has a nucleotide sequence of SEQ ID No.9; the downstream primer of the VVMD7 primer has a nucleotide sequence of SEQ ID No.10; the upstream primer of the VVMD17 primer has a nucleotide sequence of SEQ ID No.11; the downstream primer of the VVMD17 primer has a nucleotide sequence of SEQ ID No.12; the upstream primer of the VVMD25 primer has a nucleotide sequence of SEQ ID No.13; the downstream primer of the VVMD25 primer has a nucleotide sequence of SEQ ID No.14; the upstream primer of the VVMD27 primer has a nucleotide sequence of SEQ ID No.15; the downstream primer of the VVMD27 primer has a nucleotide sequence of SEQ ID The upstream primer of the VVMD28 primer has the nucleotide sequence of SEQ ID No.17; the downstream primer of the VVMD28 primer has the nucleotide sequence of SEQ ID No.18; the upstream primer of the VVMD32 primer has the nucleotide sequence of SEQ ID No.19; the downstream primer of the VVMD32 primer has the nucleotide sequence of SEQ ID No.20; the upstream primer of the VrZAG47 primer has the nucleotide sequence of SEQ ID No.21; the downstream primer of the VrZAG47 primer has the nucleotide sequence of SEQ ID No.22; the upstream primer of the VrZAG62 primer has the nucleotide sequence of SEQ ID No.23; the downstream primer of the VrZAG62 primer has the nucleotide sequence of SEQ ID No.24; the upstream primer of the VrZAG79 primer has the nucleotide sequence of SEQ ID No.25; the downstream primer of the VrZAG79 primer has the nucleotide sequence of SEQ ID No.26;

[0059] The upstream primer of the VrZAG83 primer has a nucleotide sequence of SEQ ID No.27;

[0060] The downstream primer of the VrZAG83 primer has a nucleotide sequence of SEQ ID No.28;

[0061] The upstream primer of the VMC4F3 primer has a nucleotide sequence of SEQ ID No.29;

[0062] The downstream primer of the VMC4F3 primer has a nucleotide sequence of SEQ ID No.30.

[0063] The kit includes primers, 6-FAM probe, PET probe, NED probe, PCR buffer, hot start Taq enzyme, dNTP, FRET-PCR standard, and PCR negative control; the PCR negative control is double distilled water.

[0064] The FRET-PCR standard is a recombinant plasmid constructed by inserting the amplified nucleotide fragment obtained by amplifying the plasmid standard DNA template using the FRET-PCR primers and probes into the pUC57 vector.

[0065] The PCR amplification system of the grape variety identification SSR detection kit of the present invention uses a 25uL amplification system for the PCR reaction (the kit components include): 2.5×PCR Mix (5mM MgCl2, 125mM PH8.3Tris-HCl, 125mMKCl, 0.5mM dNTPs), 10.0uL, 10umol / L composite primer 15Primers 5.0uL, 5U / uL Taq DNA polymerase 1.0uL, 20-50ng / ul DNA template 1uL, PCR Grade Water 8.0uL; the PCR reaction program is: 94°C pre-denaturation for 4min, 94°C denaturation for 30s, 58°C annealing for 30s, 72°C extension for 45s, a total of 35 cycles, and 72°C extension for 10min.

[0066] A grape variety identification SSR detection method of the present invention comprises the following steps:

[0067] (1) Capillary electrophoresis-SSR technology is used to quickly identify grape varieties. The specific steps of the identification method are as follows:

[0068] (2) DNA extraction: DNA from fresh leaves / plant tissues of grape varieties is extracted using a DNA extraction kit or the CTAB method;

[0069] (3) DNA quality assessment: Use agarose gel electrophoresis to check the quality of nucleic acid. The bands should be clear, without nucleic acid degradation and nucleic acid contamination. Use a spectrophotometer to assess the purity of deoxyribonucleic acid. The DNA concentration should be ≥20 ng / uL.

[0070] (4) Multiplex PCR amplification: Multiplex PCR amplification of grape varieties was performed using 15 SSR primer loci, including VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG79, VZAG62, VVS16, VVMD6, VVMD17, VMC4F3, VrZAG47, and VrZAG83. The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 45 s, for a total of 35 cycles; extension at 72°C for 10 min.

[0071] (5) SSR genotyping: With the help of the first-generation Sanger sequencing platform technology, the fully automatic gene analyzer performs capillary electrophoresis-fragment analysis to detect PCR products; before detection, 1.0uL of PCR product is added to each sample well, and the volume ratio of molecular weight standard and formamide mixture is 0.5:8.5, 9uL. After the sample is mixed, it is denatured at 95℃ for 4min and immediately placed on ice for 4min. After electrophoresis, the original SSR data of different grape varieties are obtained, and the typing results are compared and analyzed by fragment analysis software to calculate the fragment length, the number of SSR alleles and site analysis;

[0072] (6) Grape variety identification: Different grape varieties can be effectively identified based on the different SSR gene typing of different grape varieties using the same primers.

[0073] Application of the grape variety identification SSR detection kit of the present invention in grape SSR marker identification.

[0074] The invention discloses an application of the grape variety identification SSR detection kit in grape fingerprint construction, variety identification and genetic diversity analysis.

[0075] Test Example 1

[0076] The SSR detection and application method for identifying grape varieties of the present invention is specifically carried out according to the following steps:

[0077] Experimental materials: A total of 6 different grape variety materials were collected in this experiment, and the grape varieties were Kyoho, Sunshine Rose, Summer Black, Beauty Finger, Moonlight Seedless, and Zhengzhou Early Jade as shown in Table 1. They were derived from European and American hybrids, Eurasian species, and Chinese varieties.

[0078] Table 1

[0079] serial number Grape Varieties 1 Jufeng European and American hybrid 2 Sunshine Rose European and American hybrid 3 Summer Black European and American hybrid 4 Beauty Finger Eurasian species 5 Moonlight Seedless China 6 Zhengzhou Early Jade China

[0080] DNA extraction: Take 6 fresh leaves of different grape varieties and use the CTAB method to extract DNA from the 6 fresh leaves of grape varieties.

[0081] DNA quality assessment: Agarose gel electrophoresis was used to check the quality of nucleic acids, with clear bands and no nucleic acid degradation or nucleic acid contamination. A spectrophotometer was used to assess the purity of deoxyribonucleic acid, and the DNA concentration was ≥20 ng / uL.

[0082] Multiplex PCR amplification: Multiplex PCR amplification of grape varieties was performed using 15 SSR primer loci, including VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG79, VZAG62, VVS16, VVMD6, VVMD17, VMC4F3, VrZAG47, and VrZAG83;

[0083] The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 45 s, for a total of 35 cycles; and extension at 72°C for 10 min.

[0084] SSR genotyping: With the help of the first-generation Sanger sequencing platform technology, the fully automatic gene analyzer was used for electrophoresis through capillary electrophoresis-fragment analysis to detect PCR products. Before the test, 1.0uL of PCR product, molecular weight standard (internal standard orange-500) and formamide mixture were added to each sample well at a volume ratio of 0.5:8.5, 9uL. After the sample was mixed, it was denatured at 95℃ for 4min and immediately placed on ice for 4min. After electrophoresis, the original data of grape variety SSR was obtained, and the typing results were analyzed by fragment analysis software.

[0085] Grape variety identification: Different grape varieties can be effectively identified based on the different SSR gene typing of different grape varieties using the same primers.

[0086] Figure 1 The SSR capillary electrophoresis diagram of the Kyoho grape variety of the present invention;

[0087] Figure 2 The SSR capillary electrophoresis diagram of the Sunshine Rose grape variety of the present invention;

[0088] Figure 3 The SSR capillary electrophoresis diagram of the summer black grape variety of the present invention;

[0089] Figure 4 The SSR capillary electrophoresis diagram of the Meirenzhi grape variety of the present invention;

[0090] Figure 5 The SSR capillary electrophoresis diagram of the Moonlight Seedless Grape variety of the present invention;

[0091] Figure 6 This is the SSR capillary electrophoresis diagram of the Zhengzhou Zaoyu grape variety of the present invention.

[0092] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above test examples, and the above test examples and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A grape variety identification SSR detection kit, comprising SSR primers and probes, characterized in that: The SSR primers are VVS2 primer, VVS16 primer, VVMD5 primer, VVMD6 primer, VVMD7 primer, VVMD17 primer, VVMD25 primer, VVMD27 primer, VVMD28 primer, VVMD32 primer, VrZAG47 primer, VrZAG62 primer, VrZAG79 primer, VrZAG83 primer and VMC4F3 primer, The VVS2 primers are the upstream primers of VVS2 and the downstream primers of VVS2, and the probe is a 6-FAM probe; the VVS16 primers are the upstream primers of VVS16 and the downstream primers of VVS16, and the probe is a 6-FAM probe; the VVMD5 primers are the upstream primers of VVMD5 and the downstream primers of VVMD5, and the probe is a PET probe; the VVMD6 primers are the upstream primers of VVMD6 and the downstream primers of VVMD6, and the probe is a PET probe; The VVMD7 primers are the upstream primers of VVMD7 and the downstream primers of VVMD7, and the probe is the NED probe; the VVMD17 primers are the upstream primers of VVMD17 and the downstream primers of VVMD17, and the probe is the VIC probe; the VVMD25 primers are the upstream primers of VVMD25 and the downstream primers of VVMD25, and the probe is the PET probe; the VVMD27 primers are the upstream primers of VVMD27 and the downstream primers of VVMD27, and the probe is the The probe is a NED probe; the VVMD28 primer is an upstream primer of VVMD28 and a downstream primer of VVMD28, and the probe is a VIC probe; the VVMD32 primer is an upstream primer of VVMD32 and a downstream primer of VVMD32, and the probe is a 6-FAM probe; the VrZAG47 primer is an upstream primer of VrZAG47 and a downstream primer of VrZAG47, and the probe is a VIC probe; the VrZAG62 primer is an upstream primer of VrZAG62 and a downstream primer of VrZAG62; The upstream primer of VrZAG79 and the downstream primer of VrZAG62, the probe is a 6-FAM probe; the VrZAG79 primer is the upstream primer of VrZAG79 and the downstream primer of VrZAG79, the probe is a VIC probe; the VrZAG83 primer is the upstream primer of VrZAG83 and the downstream primer of VrZAG83, the probe is a PET probe; the VMC4F3 primer is the upstream primer of VMC4F3 and the downstream primer of VMC4F3, the probe is a NED probe.

2. The grape variety identification SSR detection kit according to claim 1, characterized in that: The upstream primer of the VVS2 primer has a nucleotide sequence of SEQ ID No. 1; The downstream primer of the VVS2 primer has a nucleotide sequence of SEQ ID No. 2; The upstream primer of the VVS16 primer has a nucleotide sequence of SEQ ID No. 3; The downstream primer of the VVS16 primer has a nucleotide sequence of SEQ ID No. 4; The upstream primer of the VVMD5 primer has a nucleotide sequence of SEQ ID No.5; The downstream primer of the VVMD5 primer has a nucleotide sequence of SEQ ID No.6; The upstream primer of the VVMD6 primer has a nucleotide sequence of SEQ ID No.7; The downstream primer of the VVMD6 primer has a nucleotide sequence of SEQ ID No.8; The upstream primer of the VVMD7 primer has a nucleotide sequence of SEQ ID No.9; The downstream primer of the VVMD7 primer has a nucleotide sequence of SEQ ID No.10; The upstream primer of the VVMD17 primer has a nucleotide sequence of SEQ ID No.11; The downstream primer of the VVMD17 primer has a nucleotide sequence of SEQ ID No.12; The upstream primer of the VVMD25 primer has a nucleotide sequence of SEQ ID No.13; The downstream primer of the VVMD25 primer has a nucleotide sequence of SEQ ID No.14; The upstream primer of the VVMD27 primer has a nucleotide sequence of SEQ ID No.15; The downstream primer of the VVMD27 primer has a nucleotide sequence of SEQ ID No.

16. The upstream primer of the VVMD28 primer has a nucleotide sequence of SEQ ID No.17; The downstream primer of the VVMD28 primer has a nucleotide sequence of SEQ ID No.18; The upstream primer of the VVMD32 primer has a nucleotide sequence of SEQ ID No.19; The downstream primer of the VVMD32 primer has a nucleotide sequence of SEQ ID No.20; The upstream primer of the VrZAG47 primer has a nucleotide sequence of SEQ ID No.21; The downstream primer of the VrZAG47 primer has a nucleotide sequence of SEQ ID No.22; The upstream primer of the VrZAG62 primer has a nucleotide sequence of SEQ ID No. 23; The downstream primer of the VrZAG62 primer has a nucleotide sequence of SEQ ID No. 24; The upstream primer of the VrZAG79 primer has a nucleotide sequence of SEQ ID No.25; The downstream primer of the VrZAG79 primer has a nucleotide sequence of SEQ ID No.26; The upstream primer of the VrZAG83 primer has a nucleotide sequence of SEQ ID No.27; The downstream primer of the VrZAG83 primer has a nucleotide sequence of SEQ ID No.28; The upstream primer of the VMC4F3 primer has a nucleotide sequence of SEQ ID No.29; The downstream primer of the VMC4F3 primer has a nucleotide sequence of SEQ ID No.

30.

3. The grape variety identification SSR detection kit according to claim 1, characterized in that: The kit includes primers, 6-FAM probe, PET probe, NED probe, PCR buffer, hot start Taq enzyme, dNTP, FRET-PCR standard, and PCR negative control; the PCR negative control is double distilled water.

4. The grape variety identification SSR detection kit according to claim 1, characterized in that: The FRET-PCR standard is a recombinant plasmid constructed by inserting the amplified nucleotide fragment obtained by amplifying the plasmid standard DNA template using the FRET-PCR primers and probes into the pUC57 vector.

5. A PCR amplification system of the grape variety identification SSR detection kit according to claim 1, characterized in that: The PCR reaction used a 25uL amplification system (kit components included): 2.5×PCR Mix (5mM MgCl2, 125mM PH8.3Tris-HCl, 125mM KCl, 0.5mM dNTPs), 10.0uL, 10umol / L composite primer 15Primers 5.0uL, 5U / uL Taq DNA polymerase 1.0uL, 20-50ng / ul DNA template 1uL, PCR Grade Water 8.0uL; the PCR reaction program was: 94°C pre-denaturation for 4min, 94°C denaturation for 30s, 58°C annealing for 30s, 72°C extension for 45s, a total of 35 cycles, and 72°C extension for 10min.

6. A grape variety identification SSR detection method, characterized in that The steps include: (1) Capillary electrophoresis-SSR technology is used to quickly identify grape varieties. The specific steps of the identification method are as follows: (2) DNA extraction: DNA from fresh leaves / plant tissues of grape varieties is extracted using a DNA extraction kit or the CTAB method; (3) DNA quality assessment: Use agarose gel electrophoresis to check the quality of nucleic acid. The bands should be clear, without nucleic acid degradation and nucleic acid contamination. Use a spectrophotometer to assess the purity of deoxyribonucleic acid. The DNA concentration should be ≥20 ng / uL. (4) Multiplex PCR amplification: Multiplex PCR amplification of grape varieties was performed using 15 SSR primer loci, including VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG79, VZAG62, VVS16, VVMD6, VVMD17, VMC4F3, VrZAG47, and VrZAG83; (5) SSR genotyping: With the help of the first-generation Sanger sequencing platform technology, the fully automatic gene analyzer performs capillary electrophoresis-fragment analysis to detect PCR products; before detection, 1.0uL of PCR product is added to each sample well, and the volume ratio of molecular weight standard and formamide mixture is 0.5:8.5, 9uL. After the sample is mixed, it is denatured at 95℃ for 4min and immediately placed on ice for 4min. After electrophoresis, the original SSR data of different grape varieties are obtained, and the typing results are compared and analyzed by fragment analysis software to calculate the fragment length, the number of SSR alleles and site analysis; (6) Grape variety identification: Different grape varieties can be effectively identified based on the different SSR gene typing of different grape varieties using the same primers.

7. The SSR detection method for grape variety identification according to claim 6, characterized in that: In step (4), the PCR reaction program is: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 45 s, for a total of 35 cycles; extension at 72°C for 10 min.

8. Use of the grape variety identification SSR detection kit according to any one of claims 1 to 7 in grape SSR marker identification.

9. Use of the grape variety identification SSR detection kit according to any one of claims 1 to 7 in grape fingerprint construction, variety identification and genetic diversity analysis.

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