SNP marker set and core KASP primer combination for industrial hemp variety and resource identification and application

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker set and a core KASP primer combination for industrial cannabis sativa variety and resource identification and application, which are developed by screening and verifying industrial cannabis sativa genome re-sequencing data. The identification efficiency of a single KASP marker in the KASP markers developed by applying the SNP marker set on a selected variety is only 5%, and the judgment efficiency can reach 100% by judging the selected variety by using seven KASP markers at the same time. The SNP marker set provided by the invention provides a basis for the development of functional KASP markers, the core KASP combined marker developed on the basis of 7 SNP loci has the characteristics of simplicity, convenience, rapidness, high specificity, good accuracy and the like, and different industrial hemp varieties can be distinguished on a detection platform in a relatively short time.

Description

Technical Field

[0001] The present invention belongs to the field of industrial hemp variety protection and resource identification, and specifically relates to a functional SNP marker set, a core KASP primer combination and their applications for industrial hemp variety and resource identification. Background Art

[0002] As an environmentally friendly and low-carbon cash crop, industrial hemp not only has strong environmental adaptability but also plays a certain role in improving the ecological environment. Using its stalks, flowers, leaves and fruits as raw materials, various products can be made, and it has unique application values in the fields of textile, materials, medicine and food. However, at present, problems such as mixed seed sources in the industrial hemp seed market are relatively serious, which restricts the sound development of the industrial hemp industry. In order to protect and utilize existing varieties and resources, it is urgent to efficiently and accurately identify existing germplasm resources and varieties and distinguish different varieties. At present, the identification of crop germplasm resources and varieties has gradually upgraded from phenotypic index identification to molecular identification. Reported molecular markers applied to industrial hemp molecular identification include RAPD, AFLP, SCAR, SSR and KASP, etc. KASP, full name Kompetitive Allele-Specific PCR, is a gene typing technology mainly based on SNP developed in recent years and is a molecular marker applicable to high, medium and low throughput detections. The KASP technology can accurately judge the molecular marker type in a short time, and has a relatively low average gene analysis error, with extremely high analysis stability and flexibility, high accuracy, and low reaction cost. It has obvious advantages especially in large-scale marker detection work and can realize high-throughput breeding platform detection. However, the number of KASP markers developed for industrial hemp variety and resource identification is very limited at present, and there is an urgent need to increase KASP markers available for industrial hemp variety and resource identification. Summary of the Invention

[0003] To solve the above problems in the background art, the present invention discloses an SNP marker set, a core KASP primer combination and their applications for industrial hemp variety and resource identification.

[0004] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0005] An SNP marker set for industrial hemp variety and resource identification, the SNP marker set includes 15 SNP marker sites, and the site numbers are Cs_SNP1 to Cs_SNP15;

[0006] The Cs_SNP1 marker is base C / G and is located at position 12619395 of chromosome NC_044370.1 of the hemp reference genome CS10;

[0007] The Cs_SNP2 marker is the base T / C, located at position 75046096 on chromosome CS10 (NC_044370.1) of the cannabis reference genome;

[0008] The Cs_SNP3 marker is the base T / A, located at position 36149128 on chromosome CS10 (NC_044371.1) of the cannabis reference genome;

[0009] The Cs_SNP4 marker is the base G / A, located at position 39155949 on chromosome CS10 (NC_044375.1) of the cannabis reference genome;

[0010] The Cs_SNP5 marker is the base T / A, located at position 90229301 on chromosome CS10 (NC_044373.1) of the cannabis reference genome;

[0011] The Cs_SNP6 marker is the base G / A, located at position 2802125 on chromosome CS10 (NC_044373.1) of the cannabis reference genome;

[0012] The Cs_SNP7 marker is the base C / T, located at position 49646705 on chromosome CS10 (NC_044379.1) of the cannabis reference genome;

[0013] The Cs_SNP8 marker is the base G / C, located at position 29342982 on chromosome CS10 (NC_044373.1) of the cannabis reference genome;

[0014] The Cs_SNP9 marker is the base C / T, located at position 899163 on chromosome CS10 (NC_044374.1) of the cannabis reference genome;

[0015] The Cs_SNP10 marker is the base A / T, located at position 101754099 on chromosome CS10 (NC_044370.1) of the cannabis reference genome;

[0016] The Cs_SNP11 marker is the base G / A, located at position 63182464 on chromosome CS10 (NC_044375.1) of the cannabis reference genome;

[0017] The Cs_SNP12 marker is the base A / C, located at position 8647140 on chromosome CS10 (NC_044376.1) of the cannabis reference genome;

[0018] The Cs_SNP13 marker is the base T / C, located at position 13975553 on chromosome CS10 (NC_044376.1) of the cannabis reference genome;

[0019] The Cs_SNP14 marker is base A / G and is located at position 8563942 of chromosome CS10 (NC_044378.1) of the cannabis reference genome;

[0020] The Cs_SNP15 marker is base G / A and is located at position 1449271 of chromosome CS10 (NC_044377.1) of the cannabis reference genome.

[0021] A core KASP marker for industrial cannabis variety identification is developed based on the above SNP marker set. The core KASP marker includes 7 primer combinations, which contain sequences with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.21. The 7 primer combinations are numbered Cs_KASP1 to Cs_KASP7, and the development bases are Cs_SNP14, Cs_SNP3, Cs_SNP15, Cs_SNP7, Cs_SNP11, Cs_SNP8, and Cs_SNP4 in sequence.

[0022] Furthermore, the primer combination includes a first forward primer, a second forward primer, and a reverse primer.

[0023] Furthermore,

[0024] The nucleotide sequence of the first forward primer of the primer combination Cs_KASP1 is as shown in SEQ ID NO.1, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.3;

[0025] The nucleotide sequence of the first forward primer of the primer combination Cs_KASP2 is as shown in SEQ ID NO.4, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.6;

[0026] The nucleotide sequence of the first forward primer of the primer combination Cs_KASP3 is as shown in SEQ ID NO.7, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.8, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.9;

[0027] The nucleotide sequence of the first forward primer of the primer combination Cs_KASP4 is as shown in SEQ ID NO.10, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.11, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.12;

[0028] The nucleotide sequence of the first forward primer of the primer combination Cs_KASP5 is shown in SEQ ID NO.13, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.14, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.15;

[0029] The nucleotide sequence of the first forward primer of the primer combination Cs_KASP6 is shown in SEQ ID NO.16, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.18;

[0030] The nucleotide sequence of the first forward primer of the primer combination Cs_KASP7 is shown in SEQ ID NO.19, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.20, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.21.

[0031] An application of the above KASP marker in the identification of industrial hemp varieties and resources, and the application is as follows:

[0032] S1: Extract the genomic DNA of the industrial hemp sample;

[0033] S2: Using the DNA in S1 as a template, perform PCR amplification with KASP primers, and obtain the fluorescence signal value of the corresponding product and complete genotyping through a fluorescence quantitative PCR instrument.

[0034] Furthermore, the total reaction system for the PCR amplification is 5 μl: including 1.25 μl of a DNA sample at 50 - 100 ng / μL, 1.25 μl of a primer mixture, and 2.5 μl of KASP 2× MasterMix.

[0035] Furthermore, the primer mixture is a mixture of the first forward primer, the second forward primer, and the reverse primer, and the ratio is 1:1:8.

[0036] Furthermore, the reaction conditions for the PCR amplification are: activation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 61°C - 55°C for 60 s, repeat 10 cycles, with a decrease of 0.6°C for each cycle; denaturation at 94°C for 20 s, annealing and extension at 55°C for 60 s, repeat 30 cycles, and reading at 25°C for 30 s.

[0037] The beneficial effects of the present invention compared with the prior art are as follows: The SNP sites in the provided SNP marker set are all located in the exon regions of the genome, which helps to develop functional KASP markers. The provided core KASP primer combination containing 7 KASP markers can effectively distinguish the selected 42 varieties, further expanding the KASP marker library for industrial hemp resources and variety identification, and providing a reference for the protection and utilization of industrial hemp variety resources.

[0038] The present invention utilizes the industrial hemp genome resequencing data, and through screening and verification, develops an SNP marker set and a core KASP primer combination for industrial hemp variety identification. Among the KASP markers developed using this SNP marker set, the identification efficiency of a single KASP marker for the selected varieties is only 5%. Meanwhile, when judging the selected varieties with 7 KASP markers, the judgment efficiency can reach 100%. The SNP marker set provided by the present invention provides a basis for the development of functional KASP markers. The core KASP combined markers developed based on 7 SNP sites have the characteristics of simplicity, rapidity, high specificity, and good accuracy, and can distinguish different industrial hemp varieties on the detection platform in a relatively short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a genotyping result diagram of 42 varieties by the KASP marker combination.

[0040] Figure 2 It is a data introduction diagram of 39 industrial hemp varieties. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The following further illustrates the solutions of the present invention in conjunction with the attached tables and embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall be covered within the protection scope of the present invention.

[0042] The SNP marker sites of the present invention are screened by the following method:

[0043] Based on the whole-genome resequencing data of 46 industrial hemp resources, screening is carried out according to the following criteria:

[0044] (1) Retain the SNP sites where the sequences before and after on the DNA strand of the chromosome are greater than 50bp and are conservative SNP sites;

[0045] (2) Retain the sites with an average depth of 5X or more, a quality value greater than 30, a minimum integrity greater than 0.9, a minimum allele frequency greater than 0.05, and the SNP being a biallelic site;

[0046] (3) 100 bp of sequence upstream and downstream of the SNP marker were intercepted, and then the sequence was aligned to the reference genome using blast software (version: 2.10.1+), and markers at multiple positions in the alignment were removed;

[0047] (4) retaining markers with polymorphic information content (PIC) greater than 0.35;

[0048] (5) Primers are designed for SNP markers and converted into KASP markers. When designing KASP primers, other mutation sites need to be avoided.

[0049] Randomly select 2-3 SNP sites on each chromosome for designing KASP primers and perform first-generation sequencing verification in varieties with different genotypes. The SNP marker set described in the present invention is consistent with the sequencing results. Of the 46 samples of industrial hemp, 39 data are from the NCBI (National Center for Biotechnology Information) public database, such as Figure 2 As shown, another 7 are from resources of the applicant’s unit.

[0050] Embodiment 1:

[0051] (1) 42 core accessions of industrial hemp were selected from the resource library and planted in an artificial climate chamber. When they grew to the three-leaf stage, the young leaves were quickly frozen in liquid nitrogen and ground, and DNA was extracted from the leaves using a universal plant genomic DNA rapid extraction kit;

[0052] (2) Use Qubit3.0 fluorescent nucleic acid concentration detector to detect the concentration of each sample.

[0053] (3) Template DNA with a concentration of 50-100 ng / μL was taken and PCR amplified in a 96-well plate. The primers used were shown in Table 1 and the reaction system was shown in Table 2. The primer mixture was a mixture of the first forward primer, the second forward primer and the reverse primer in a ratio of 1:1:8. KASP Master Mix was a kit specifically used for KASP labeling. The main brand used was KASP Master Mix v4.0 (latest version) of LGC Biosearch Technologies. The reaction conditions were shown in Table 3. In step 2, denaturation at 95°C for 20 s, annealing at 61°C-55°C and extension for 60 s constituted one cycle; decreasing 0.6°C in each cycle means starting from 61°C and decreasing 0.6°C in each cycle until decreasing from 61°C to 55°C.

[0054] (4) Use a fluorescent quantitative PCR instrument to read the signal of the PCR amplification product, and use software to collect the signal to distinguish the allele type.Figure 1 Show the genotyping results of 42 different varieties, and the genotyping results of each variety are different, so as to distinguish different varieties.

[0055] Table 1 Information on the primer sequences of the core KASP combination

[0056]

[0057]

[0058] Table 2 PCR amplification system

[0059]

[0060]

[0061] Table 3 PCR amplification conditions

[0062]

Claims

1. SNP marker set for industrial hemp variety and resource identification, characterized in that: The SNP marker set includes 15 SNP marker loci, numbered Cs_SNP1 to Cs_SNP15; The Cs_SNP1 marker is base C / G, located at position 12619395 of chromosome NC_044370.1 of the cannabis reference genome CS10; The Cs_SNP2 marker is base T / C, located at position 75046096 of chromosome NC_044370.1 of the cannabis reference genome CS10; The Cs_SNP3 marker is base T / A, located at position 36149128 of chromosome NC_044371.1 of the cannabis reference genome CS10; The Cs_SNP4 marker is base G / A, located at position 39155949 of chromosome NC_044375.1 of the cannabis reference genome CS10; The Cs_SNP5 marker is base T / A, located at position 90229301 of chromosome NC_044373.1 of the cannabis reference genome CS10; The Cs_SNP6 marker is base G / A, located at position 2802125 of chromosome NC_044373.1 of the cannabis reference genome CS10; The Cs_SNP7 marker is base C / T, located at position 49646705 of chromosome NC_044379.1 of the cannabis reference genome CS10; The Cs_SNP8 marker is base G / C, located at position 29342982 of chromosome NC_044373.1 of the cannabis reference genome CS10; The Cs_SNP9 marker is base C / T, located at position 899163 of chromosome NC_044374.1 of the cannabis reference genome CS10; The Cs_SNP10 marker is base A / T, located at position 101754099 of chromosome NC_044370.1 of the cannabis reference genome CS10; The Cs_SNP11 marker is base G / A, located at position 63182464 of chromosome NC_044375.1 of the cannabis reference genome CS10; The Cs_SNP12 marker is base A / C, located at position 8647140 of chromosome NC_044376.1 of the cannabis reference genome CS10; The Cs_SNP13 marker is base T / C, located at position 13975553 of chromosome NC_044376.1 of the cannabis reference genome CS10; The Cs_SNP14 marker is base A / G, located at position 8563942 of chromosome NC_044378.1 of the cannabis reference genome CS10; The Cs_SNP15 marker is base G / A, located at position 1449271 of chromosome NC_044377.1 of the cannabis reference genome CS10.

2. A core KASP marker for industrial hemp variety identification is developed based on the SNP marker set described in claim 1, characterized in that: The core KASP markers include 7 primer combinations, which contain sequences with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.

21. The 7 primer combinations are numbered Cs_KASP1 to Cs_KASP7, and the development bases are Cs_SNP14, Cs_SNP3, Cs_SNP15, Cs_SNP7, Cs_SNP11, Cs_SNP8, and Cs_SNP4 in sequence.

3. The KASP marker according to claim 2, wherein: The primer combination includes a first forward primer, a second forward primer, and a reverse primer.

4. The KASP marker according to claim 3, wherein: For the primer combination Cs_KASP1, the nucleotide sequence of the first forward primer is as shown in SEQ ID NO.1, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.3; For the primer combination Cs_KASP2, the nucleotide sequence of the first forward primer is as shown in SEQ ID NO.4, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.6; For the primer combination Cs_KASP3, the nucleotide sequence of the first forward primer is as shown in SEQ ID NO.7, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.8, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.9; For the primer combination Cs_KASP4, the nucleotide sequence of the first forward primer is as shown in SEQ ID NO.10, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.11, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.12; For the primer combination Cs_KASP5, the nucleotide sequence of the first forward primer is as shown in SEQ ID NO.13, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.14, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.15; For the primer combination Cs_KASP6, the nucleotide sequence of the first forward primer is as shown in SEQ ID NO.16, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.18; For the primer combination Cs_KASP7, the nucleotide sequence of the first forward primer is as shown in SEQ ID NO.19, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO.20, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.

21.

5. Use of the KASP marker according to any one of claims 2 to 4 in the identification of industrial hemp varieties and resources, characterized in that: The application is as follows: S1: Extract the genomic DNA of the industrial hemp sample; S2: Using the DNA in S1 as a template, perform PCR amplification with KASP primers, and obtain the fluorescence signal value of the corresponding product and complete genotyping through a fluorescence quantitative PCR instrument.

6. The application according to claim 5, wherein: The total reaction system for the PCR amplification is 5 μl, including 1.25 μl of a DNA sample at 50 - 100 ng / μL, 1.25 μl of a primer mixture, and 2.5 μl of KASP 2× MasterMix.

7. The application according to claim 6, wherein: The primer mixture is a mixture of a first forward primer, a second forward primer, and a reverse primer, with a ratio of 1:1:

8.

8. The application according to claim 5, characterized in that: The reaction conditions for the PCR amplification are as follows: activation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 61°C - 55°C for 60 s, repeating 10 cycles with a decrease of 0.6°C in each cycle; denaturation at 94°C for 20 s, annealing and extension at 55°C for 60 s, repeating 30 cycles, and reading at 25°C for 30 s.

Images