Method for identifying authenticity of hybrid offspring of tea tree based on SSR and KASP molecular markers

Abstract

The invention discloses a method for identifying the authenticity of hybrid filial generations of tea trees based on SSR and KASP molecular markers, and belongs to the technical field of molecular biology and tea tree breeding. According to the invention, the authenticity of the hybrid offspring of two tea tree varieties is identified by utilizing a primer group designed by an SSR (Simple Sequence Repeat) marker, a method for reducing the number of tested samples is invented by combining the real offspring with re-sequencing result data of male and female parents, and an SNP (Single Nucleotide Polymorphism) molecular marker for identifying the hybrid offspring of the tea tree is developed. The KASP primer group capable of identifying the authenticity of the hybrid offspring of the tea tree is screened out, and by adopting the primer group disclosed by the invention, the identification of the authenticity of the hybrid offspring of the two tea trees can be realized, and the hybrid offspring polluted by pollen can be excluded; the identification method disclosed by the invention can replace an identification method for screening a specific SSR marker by utilizing a capillary electrophoresis technology, and the identification of the hybrid offspring of the tea tree can be quickly, accurately and effectively realized. Important basis and support are provided for molecular breeding for efficiently creating tea germplasm.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology and tea tree breeding, and in particular to a method for identifying the authenticity of tea tree hybrid progeny based on SSR and KASP molecular markers. Background Art

[0002] Tea trees belong to the genus Camellia of the Theaceae family. They originated in southwest China and have a cultivation history of more than 3,000 years. They have been introduced to more than 50 countries including Japan, India, and Sri Lanka, and have become one of the three major beverage crops in the world. At present, tea tree breeding plays an important role in scientific research and industrial development. As a perennial, evergreen woody, cross-pollinated plant, tea trees are highly heterogeneous in genetic composition. It is particularly important to improve breeding efficiency through early identification and accurate selection of target traits in the early stages of variety breeding. Therefore, conducting authenticity identification of interspecific hybrid progeny is a primary task for breeders and scientific researchers.

[0003] Molecular marker technology is one of the important means of identifying tea germplasm resources. In recent years, with the rapid development of molecular marker technology, variety identification and seed purity identification have entered the DNA level. Because of its high accuracy, stability and repeatability, it provides more accurate and reliable results for crop variety purity analysis. SSR markers are co-dominant markers that can identify homozygous and heterozygous types. They require a small amount of DNA sample, have low requirements for DNA, are simple to operate, have strong reliability, have a large number of allelic differences, and do not require the use of radioactive isotopes. However, the DNA sequences at both ends of the repeating motif must be clearly identified. If they cannot be directly found in the DNA database, they need to be sequenced again, which is costly. At present, SNP molecular markers are the most mainstream third-generation molecular markers. Their markers have the characteristics of wide quantitative distribution, high polymorphism and good stability. They are considered by the International Union for the Protection of New Varieties of Plants (UPOV) to be one of the effective methods for variety identification. Competitive allele specific PCR (KASP) is a new high-throughput SNP typing technology. This technology has high accuracy, high throughput and low cost. It is currently the most ideal genotyping technology. It has been widely used in crops such as rice, wheat, corn, cucumber and grape.

[0004] Regarding the identification of tea hybrid offspring, the Chinese patent application document with publication number CN116814838B discloses the use of capillary electrophoresis technology to screen specific SSR markers, and combines whole genome data to develop specific InDel markers to identify the authenticity of tea hybrid offspring. A total of 4 SSR markers and 1 InDel marker were screened to verify the forward and reverse cross progeny. Yang Jun (2022) et al. used EST-SSR capillary electrophoresis fluorescence labeling technology to study 65 natural hybrid offspring of golden peony, indicating that the selected 28 pairs of SSR marker loci have high polymorphism and high exclusion probability, which are suitable for genetic analysis and individual parentage identification.

[0005] Due to the large genome, a large number of repetitive sequences and the highly heterozygous characteristics caused by self-incompatibility, it is extremely difficult to develop SNP sites in tea trees using KASP technology, and the results of identifying tea tree hybrid offspring using SSR marker technology usually have many false positives. Therefore, based on the results of SSR marker identification and resequencing data, the KASP technology can be combined to solve the problem of the difficulty in developing SNP sites in tea trees, develop core SNP sites that can identify the authenticity of tea tree hybrid offspring, further reduce false positives in identification results, and improve the authenticity of identification. However, there are no mature KASP markers and primers for authenticity identification of tea tree hybrid offspring in the prior art. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to provide a method for efficiently identifying the authenticity of hybrid offspring of tea trees.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The first aspect of the present invention provides an SSR marker combination for authenticity identification of tea plant hybrid progeny, wherein the SSR marker combination includes 5 SSR markers, namely Cs-SSR1 to 5,

[0009] The repeating unit of the Cs-SSR1 is GA, and the number of repetitions is 5;

[0010] The repeating unit of Cs-SSR2 is CAGCAC, and the number of repetitions is 4;

[0011] The repeating unit of the Cs-SSR3 is CACCAT, and the number of repetitions is 6;

[0012] The repeating unit of the Cs-SSR4 is TC, and the number of repetitions is 18;

[0013] The repeating unit of the Cs-SSR5 is AT, and the number of repetitions is 6.

[0014] The second aspect of the present invention proposes a primer combination for amplifying the above-mentioned SSR marker combination, and the primers of Cs-SSR1~5 are shown as SEQ ID NO:1-2, SEQ ID NO:3-4, SEQ ID NO:5-6, SEQ ID NO:7-8, and SEQ ID NO:9-10 respectively.

[0015] The third aspect of the present invention proposes the use of the above-mentioned SSR marker combination or the above-mentioned primer combination in identifying the authenticity of the hybrid progeny of 'Dangui' and 'Jinxuan'.

[0016] The fourth aspect of the present invention proposes a core SNP molecular marker combination for authenticity identification of tea plant hybrid progeny, including four KASP markers, namely Cs-KASP1 to 4, and the specific information is as follows:

[0017] Tag Name chromosome SNP physical location Allele Cs-KASP1 Chr1 208677931 [A / G] Cs-KASP2 Chr9 139154846 [A / C] Cs-KASP3 Chr6 203935933 [G / A] Cs-KASP4 Chr8 204502469 [A / G] .

[0018] The fifth aspect of the present invention provides a KASP primer set for amplifying the SNP molecular marker combination according to claim 4, wherein the primer set comprises any one of the following primer sets (1)-(4):

[0019] The primer set (1) includes the forward primers shown in SEQ ID NO.11-12 and the reverse universal primer shown in SEQ ID NO.13;

[0020] The primer set (2) includes forward primers shown in SEQ ID NO.14-15 and reverse universal primer shown in SEQ ID NO.16;

[0021] The primer set (3) includes forward primers shown in SEQ ID NO.17-18 and reverse universal primer shown in SEQ ID NO.19;

[0022] The primer set (4) includes the forward primers shown in SEQ ID NO.20-21 and the reverse universal primer shown in SEQ ID NO.22.

[0023] The sixth aspect of the present invention proposes the use of the above-mentioned SNP molecular marker combination or KASP primer set in identifying the authenticity of tea plant hybrid progeny.

[0024] The seventh aspect of the present invention is a method for identifying the authenticity of tea plant hybrid progeny based on KASP technology, comprising the following steps:

[0025] S1: The genomic DNA of the tea plant to be tested is used as a template;

[0026] S2: using the above KASP primer set to perform PCR amplification reaction to obtain an amplified product;

[0027] S3: The fluorescence detection platform is then used to scan the fluorescence signal and perform genotyping on the amplified product;

[0028] S4: Read the genotyping data and obtain the genotyping graph.

[0029] Furthermore, the genotyping is:

[0030] If the tea tree to be tested shows a blue signal or an orange signal, the tea tree variety is a homozygous parent;

[0031] If the tea tree to be tested shows a green signal, then the tea tree variety is a hybrid progeny.

[0032] Furthermore, the points close to the Y-axis and the points close to the X-axis represent two different homozygous genotypes, representing two different parents.

[0033] Furthermore, points on the diagonal line of the coordinate axis represent heterozygous genotypes and represent hybrid offspring.

[0034] Furthermore, the PCR amplification system is 10 μL: 5 μL 2×KASP Master Mix, 4.8 μL 20 ng / μL DNA template, 0.024 μL 100 μM of one forward primer, 0.024 μL 100 μM of another forward primer, 0.06 μL 100 μM of a reverse primer, and the volume is supplemented to 10 μL with ddH2O.

[0035] Furthermore, the PCR amplification program is: pre-denaturation: temperature 94°C, 15min, 1 cycle; denaturation: temperature 94°C, 20sec; annealing / extension: temperature 61-55°C, 60sec (-0.6°C / cycle); step 2, 10 cycles; denaturation: temperature 94°C, 20sec; annealing / extension: temperature 55°C, 60sec, step 3, 26 cycles. Finally, the genotyping data was read at 25°C, 30sec.

[0036] The beneficial effects of the present invention are:

[0037] 1. The present invention utilizes the results of SSR marker identification and resequencing data to develop SNP molecular markers for tea tree hybrid progeny identification, and a method for rapidly identifying the authenticity of tea tree hybrid progeny based on KASP technology.

[0038] 2. Based on the identification results of the SSR primer set and combined with the resequencing data, the present invention proposes a method for reducing the number of samples to be tested, develops a set of core KASP markers for identifying hybrid progeny of tea trees, and screens out a specific primer set that can identify the authenticity of hybrid progeny of tea trees. The primer set of the present invention can not only identify the authenticity of the hybrid progeny of two tea trees, but also exclude the hybrid progeny contaminated by pollen;

[0039] 3. The identification method of the present invention can replace the identification method of using capillary electrophoresis technology to screen specific SSR markers, quickly, accurately and effectively identify tea hybrid progeny, and reduce sequencing costs, providing important basis and support for the molecular breeding of efficient creation of tea germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 A line graph showing the number of SNPs screened under different numbers of real progeny in the third aspect of the present invention;

[0041] Figure 2 This is the peak diagram of 'Dangui' in capillary electrophoresis of labeled Cs-SSR2 in Example 1 of the present invention;

[0042] Figure 3 This is the peak diagram of 'Jinxuan' in capillary electrophoresis of labeled Cs-SSR2 in Example 1 of the present invention;

[0043] Figure 4 The peak diagram of the real progeny identified in the capillary electrophoresis of the labeled Cs-SSR2 in Example 1 of the present invention;

[0044] Figure 5 This is a graph showing the capillary identification results of 10 progeny in Example 1 of the present invention;

[0045] Figure 6 The genotype diagram of KASP marker Cs-KASP4 in 32 hybrid progenies in Example 3: the scatter plots on the X-axis and Y-axis represent the marker identification of 'Dangui', 'Jinxuan' and hybrid progenies; the blue dots, orange dots and green dots represent the homozygous female parent 'Dangui', the homozygous male parent 'Jinxuan' and the heterozygous hybrid progeny, respectively;

[0046] Figure 7 This is the genotype diagram of the KASP marker Cs-KASP4 in 32 hybrid progenies in Example 3: the scatter plots on the X-axis and Y-axis represent the marker identification of 'Dangui', 'Jinxuan' and hybrid progenies; the blue dots, orange dots and green dots represent the homozygous paternal 'Dangui', the homozygous maternal 'Jinxuan' and the heterozygous hybrid progeny, respectively. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0049] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0050] Embodiment 1:

[0051] This embodiment provides a method for identifying the authenticity of tea plant hybrid progeny using SSR-labeled primer pairs combined with capillary electrophoresis technology, comprising the following steps:

[0052] 1. Genomic DNA extraction: The genomic DNA was extracted from the seedling tissues of 'Dangui' and 'Jinxuan' as well as their direct and reverse cross F1 generation in the Guohe Tea Variety and Resource Garden of Anhui Agricultural University (31°25′N, 117°09′E) using the modified CTAB method:

[0053] (1) Weigh 0.1 g of tea tree tissue and place it in a 2 mL centrifuge tube pre-cooled with liquid nitrogen and grind it using a ball mill (add 2 steel balls and an appropriate amount of PVPP to the centrifuge tube in advance).

[0054] (2) Add 700 μL C TAAB extract (preheated to 65°C) into the centrifuge tube and incubate in a 65°C water bath for 15 min. Shake up and down 6-8 times every 5 min.

[0055] (3) Add 600 μL of nucleic acid extraction solution and centrifuge at 12,000 rpm for 10 min. Take 500 μL of the supernatant and place it in a new 1.5 mL centrifuge tube, add 500 μL of isopropanol, shake gently 6-8 times, centrifuge at 12,000 rpm for 5 min, and discard the supernatant.

[0056] (4) Add 500 μL of 70% ethanol, mix the bottom precipitate by pipetting, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and repeat step (4) once.

[0057] (5) Open the centrifuge tube cap and place it in a fume hood to dry the remaining ethanol. Add 100 μL of sterile water and mix well for later use.

[0058] 2. DNA sample detection and dilution: The quality and concentration of DNA were detected and confirmed using NanoDrop 2000 (ThermoScientific) nucleic acid analyzer, and then uniformly diluted to 35-40 ng / μL.

[0059] 3. PCR amplification: In the early stage of the laboratory, a total of 5 SSR marker primers were screened (Table 2).

[0060] There are 5 SSR markers, namely Cs-SSR1~5. The repeating unit of Cs-SSR1 is GA, and the number of repeats is 5; the repeating unit of Cs-SSR2 is CAGCAC, and the number of repeats is 4; the repeating unit of Cs-SSR3 is CACCAT, and the number of repeats is 6; the repeating unit of Cs-SSR4 is TC, and the number of repeats is 18; the repeating unit of Cs-SSR5 is AT, and the number of repeats is 6.

[0061] These five pairs of SSR marker primers were selected to verify the forward and reverse cross progenies. The PCR amplification system (Table 1) is as follows:

[0062] Table 1 PCR amplification reaction system

[0063]

[0064] Among them, 2×Taq Master Mix comes from Beijing Kangwei Century Biotechnology Co., Ltd., with the catalog number CW2849M. Since this experiment uses a 96-well plate for PCR amplification, 20 μL of mineral oil should be added to seal the plate after adding the reaction system and centrifuging to prevent evaporation.

[0065] The PCR amplification procedure is as follows:

[0066]

[0067] Table 2 Primer sequences used to amplify 5 markers

[0068]

[0069] 4. Fragment Analyzer TM Capillary electrophoresis:

[0070] All reagents used in the capillary electrophoresis experiment are from the DNF-900 35-500bp kit. Place all prepared reagents into the instrument (Fragment Analyzer TM 96, USA) designated position, run the corresponding program, and the electrophoresis results of 95 samples can be obtained each time.

[0071] 5. Capillary electrophoresis result analysis: using PROSize TM2.0 software to display the capillary electrophoresis results. If the peak graph obtained after the hybrid progeny is amplified with 5 markers has a pair of markers showing that it appears in both parental peak graphs ( Figure 3 ) and there are no other obvious main peaks, it is identified as a hybrid; if the peak graph obtained is inconsistent with the parents or only shows the peak graph of one parent, it is identified as a non-hybrid and the corresponding plants should be removed or transplanted later.

[0072] Embodiment 2:

[0073] This embodiment provides a method for core SNP molecular markers for tea plant hybrid progeny identification based on KASP technology, and the specific steps are as follows:

[0074] 1. Determination of core SNP sites: Based on the results described in Example 1, one parent and eight real offspring were selected for resequencing. SNP screening was performed on a Linux server according to the VCF file in the resequencing data. The vcftools command was used for filtering. The specific parameters were set as follows:

[0075] (1) retaining biallelic loci;

[0076] (2) setting the genotype missing rate to less than 1;

[0077] (3) setting the minimum allele frequency to less than 0.05;

[0078] (4) Set the average sequencing depth to 5×.

[0079] The bcftools command was used to extract the SNP loci information of the parents who were homozygous and the real offspring who were heterozygous in the filtered VCF file. The specific command needed to output the chromosome name, SNP physical position, reference allele, alternative allele and genotype; according to the principle that there were no other variable sites 100bp before and after the SNP site. Finally, 4 core SNP markers were selected, and the specific information is described in Table 3 below.

[0080] Tag Name chromosome SNP physical location Allele Cs-KASP1 Chr1 208677931 [A / G] Cs-KASP2 Chr9 139154846 [A / C] Cs-KASP3 Chr6 203935933 [G / A] Cs-KASP4 Chr8 204502469 [A / G]

[0081] 2. Synthesis of KASP primer pairs:

[0082] The developed SNP sites were converted into KASP primers, compared with the reference genome, and the 100bp flanking conservative sequences before and after the four SNP sites were extracted. For each SNP site, two forward primers were designed upstream and one reverse primer was designed downstream. Each KASP primer pair combination was used to amplify the corresponding SNP marker. A total of 12 primers, whose nucleotide sequences are shown in Table 4:

[0083]

[0084]

[0085] Embodiment 3:

[0086] This example uses the Cs-KASP4 primer pair designed in Example 2, uses the genomic DNA of the F1 seedling tissue of the tea varieties 'Dangui' and 'Jinxuan' and their forward and reverse crosses in Example 1 as templates, and uses double distilled water as a control to perform KASP genotyping to identify the authenticity of the progeny, including the following steps:

[0087] 1. DNA sample detection and dilution: The quality and concentration of DNA were detected and confirmed using NanoDrop 2000 (ThermoScientific) nucleic acid analyzer, and then uniformly diluted to 10-20 ng / μL.

[0088] 2. PCR amplification: PCR amplification was performed on a BIO-RAD real-time quantitative instrument using the KASP primer pair designed in Example 2, and the product was scanned for fluorescence signals and genotyped;

[0089] (1) The PCR amplification system is as follows:

[0090] Table 5 PCR amplification reaction system

[0091]

[0092] (2) The PCR amplification procedure is as follows:

[0093] Table 6 PCR amplification reaction program

[0094]

[0095] (3) Fluorescence data reading and analysis

[0096] After the PCR reaction is completed, the fluorescence data is read and analyzed using the BIO-RAD real-time quantitative instrument, and the true hybrid progeny is determined based on the typing results. (If the tea tree to be tested shows a blue signal or an orange signal, the tea tree variety is a homozygous parent; if the tea tree to be tested shows a green signal, the tea tree variety is a hybrid progeny.)

[0097] (3) If well-defined genotype clusters are not obtained after the initial KASP thermal cycling program, the plate should be re-thermal cycled three times using the conditions detailed in Table 7. The plate should then be re-read and the results analyzed.

[0098] Table 7 PCR amplification reaction program

[0099]

[0100]

[0101] The results are as follows Figure 4 As shown, the results show that the PCR primers designed by the SNP combination of the present invention, combined with competitive allele specific PCR (KASP), can distinguish tea trees with different genotypes. The points close to the Y axis and the points close to the X axis represent two different homozygous genotypes, representing two different parents or self-pollinated offspring, and the points on the diagonal of the coordinate axis represent heterozygous genotypes, representing real hybrid offspring.

[0102] The KASP genotyping technology of the present invention performs accurate biallelic genotyping of target SNPs, and can be used to identify the authenticity of tea tree hybrid progeny based on the developed SNP sites and designed primers.

[0103] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SSR marker combination for authenticity identification of tea plant hybrid progeny, characterized in that: The SSR marker combination includes 5 SSR markers, namely Cs-SSR1 to 5; The repeating unit of the Cs-SSR1 is GA, and the number of repetitions is 5; The repeating unit of Cs-SSR2 is CAGCAC, and the number of repetitions is 4; The repeating unit of the Cs-SSR3 is CACCAT, and the number of repetitions is 6; The repeating unit of the Cs-SSR4 is TC, and the number of repetitions is 18; The repeating unit of the Cs-SSR5 is AT, and the number of repetitions is 6.

2. A primer combination for amplifying the SSR marker combination according to claim 1, characterized in that: The primers of Cs-SSR1 to 5 are shown as SEQ ID NO: 1-2, SEQ ID NO: 3-4, SEQ ID NO: 5-6, SEQ ID NO: 7-8, and SEQ ID NO: 9-10, respectively.

3. Use of the SSR marker combination of claim 1 or the primer combination of claim 2 in identifying the authenticity of hybrid progeny of 'Dangui' and 'Jinxuan'.

4. A core SNP molecular marker combination for authenticity identification of tea plant hybrid progeny, characterized in that: It includes 4 KASP markers, namely Cs-KASP1~4, and the core marker information is as follows: 。 5. A KASP primer set for amplifying the SNP molecular marker combination according to claim 4, characterized in that: The primer set includes any one of the following primer sets (1)-(4): The primer set (1) includes the forward primers shown in SEQ ID NO.11-12 and the reverse universal primer shown in SEQ ID NO.13; The primer set (2) includes forward primers shown in SEQ ID NO.14-15 and reverse universal primer shown in SEQ ID NO.16; The primer set (3) includes forward primers shown in SEQ ID NO.17-18 and reverse universal primer shown in SEQ ID NO.19; The primer set (4) includes the forward primers shown in SEQ ID NO.20-21 and the reverse universal primer shown in SEQ ID NO.

22.

6. Use of the SNP molecular marker combination according to claim 4 or the KASP primer set according to claim 5 in identifying the authenticity of hybrid progeny of tea plants.

7. A method for identifying the authenticity of tea plant hybrid progeny based on KASP technology, characterized in that: The following steps are involved: S1: The genomic DNA of the tea plant to be tested is used as a template; S2: using the above KASP primer set to perform PCR amplification reaction to obtain an amplified product; S3: The fluorescence detection platform is then used to scan the fluorescence signal and perform genotyping on the amplified product; S4: Read the genotyping data and obtain the genotyping graph.

8. The identification method according to claim 7, characterized in that: The genotyping is: If the tea tree to be tested shows a blue signal or an orange signal, the tea tree variety is a homozygous parent; If the tea tree to be tested shows a green signal, then the tea tree variety is a hybrid progeny.

9. The identification method according to claim 7, characterized in that: The PCR amplification system is 10 μL: 5 μL 2×KASP Master Mix, 4.8 μL 20 ng / μL DNA template, 0.024 μL 100 μM of one forward primer, 0.024 μL 100 μM of another forward primer, 0.06 μL 100 μM of a reverse primer, and the volume is supplemented to 10 μL with ddH2O.

10. The identification method according to claim 7, characterized in that: The PCR amplification program is: pre-denaturation: temperature 94°C, 15min, 1 cycle; denaturation: temperature 94°C, 20sec; annealing / extension: temperature 61-55°C, 60sec (-0.6°C / cycle); step 2, 10 cycles; denaturation: temperature 94°C, 20sec; annealing / extension: temperature 55°C, 60sec, step 3, 26 cycles.

Images