A method for identifying the authenticity of tea tree hybrid offspring based on SSR and KASP molecular markers

Abstract

The present invention discloses a method for identifying the authenticity of tea tree hybrid offspring based on SSR and KASP molecular markers, belonging to the technical fields of molecular biology and tea tree breeding. The present invention uses a primer set designed by SSR markers to identify the authenticity of the hybrid offspring of two tea tree varieties, combines the resequencing result data of the true offspring and the parents to invent a method for reducing the number of samples to be tested, develops SNP molecular markers for identifying tea tree hybrid offspring, and screens out a KASP primer set capable of identifying the authenticity of tea tree hybrid offspring. Using the primer set of the present invention can not only identify the authenticity of the hybrid offspring of two tea tree varieties, but also exclude the hybrid offspring contaminated by pollen; the identification method of the present invention can replace the identification method of screening specific SSR markers by capillary electrophoresis technology, and quickly, accurately and effectively realize the identification of tea tree hybrid offspring. It provides an important basis and support for the molecular breeding of efficiently creating tea tree germplasms.

Description

Technical Field

[0001] The present invention relates to the technical fields of molecular biology and tea tree breeding, and particularly relates to a method for identifying the authenticity of tea tree hybrid offspring based on SSR and KASP molecular markers. Background Art

[0002] The tea tree belongs to the genus Camellia of the family Theaceae, originated in the southwestern region of China, and has a cultivation history of more than 3,000 years. It has been introduced into more than 50 countries such as Japan, India, and Sri Lanka, and has 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, and cross-pollinated plant, the tea tree is highly heterogeneous and heterozygous in genetic composition. Therefore, it is particularly important to improve the breeding efficiency by early identification and accurate selection of target traits at the initial stage of variety breeding. Therefore, carrying out the authenticity identification of interspecific hybrid offspring is a primary task for breeders and scientific researchers.

[0003] Molecular marker technology is one of the important means for the identification of tea tree 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 distinguish homozygous and heterozygous types. They require a small amount of DNA samples, have low requirements for DNA, are easy to operate, have strong reliability, and have a large number of allelic differences. They do not require the use of radioactive isotopes, but the DNA sequences at both ends of the repetitive motif must be clear. If they cannot be directly found in the DNA database, sequencing needs to be carried out again, and the cost is high. At present, as the most mainstream third-generation molecular marker, SNP molecular markers have the characteristics of wide quantity distribution, high polymorphism, and good stability, and are considered by the International Union for the Protection of New Varieties of Plants (UPOV) as one of the effective methods for variety identification. Kompetitive allele specific PCR (KASP) is a new type of high-throughput SNP genotyping technology. This technology has high accuracy, large throughput, and low cost, and is currently the most ideal genotyping technology. At present, it has been widely applied to crops such as rice, wheat, corn, cucumber, and grape.

[0004] Regarding the identification of tea tree hybrid offspring, the Chinese patent application document with the publication number CN116814838B discloses the use of capillary electrophoresis technology to screen specific SSR markers and the development of specific InDel markers in combination with whole-genome data to identify the authenticity of tea tree hybrid offspring. A total of 4 SSR markers and 1 InDel marker were screened to verify the reciprocal cross offspring. Yang Jun (2022) et al. studied 65 natural hybrid offspring of Jinmudan using EST-SSR capillary electrophoresis fluorescence labeling technology, indicating that the 28 pairs of SSR marker loci selected had high polymorphism and a high exclusion probability, suitable for genetic analysis and individual paternity testing.

[0005] Due to the large genome, a large number of repetitive sequences, and the highly heterozygous characteristics caused by self-incompatibility of tea trees, it is extremely difficult to develop SNP loci in tea trees using the KASP technology. In the results of identifying tea tree hybrid offspring using SSR marker technology, there are usually many false positives. Therefore, based on the results of SSR marker identification and resequencing data, combined with the KASP technology, the problem of difficult development of SNP loci in tea trees can be solved, and core SNP loci capable of identifying the authenticity of tea tree hybrid offspring can be developed, further reducing the false positives in the identification results and improving the identification accuracy. However, there are no maturely applied KASP markers and primers for identifying the authenticity of tea tree hybrid offspring in the existing technology. 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 tea tree hybrid offspring.

[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 identifying the authenticity of tea tree hybrid offspring. The SSR marker combination includes 5 SSR markers, namely Cs-SSR1 to 5,

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

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

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

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

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

[0014] The second aspect of the present invention provides a primer combination for amplifying the above SSR marker combination. The primers for 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.

[0015] The third aspect of the present invention provides the application of the above SSR marker combination or the above primer combination in identifying the authenticity of the hybrid offspring of 'Dan Gui' and 'Jin Xuan'.

[0016] The fourth aspect of the present invention provides a core SNP molecular marker combination for identifying the authenticity of tea tree hybrid offspring, including 4 KASP markers, namely Cs-KASP1 to 4. The specific information is as follows:

[0017] Marker 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 described in claim 4. The primer set includes any one of the following primer sets (1)-(4):

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

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

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

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

[0023] The sixth aspect of the present invention provides the application of the above SNP molecular marker combination or KASP primer set in identifying the authenticity of tea tree hybrid offspring.

[0024] The seventh aspect of the present invention provides a method for identifying the authenticity of tea tree hybrid offspring based on KASP technology, including the following steps:

[0025] S1: Using the genomic DNA of the tea tree to be tested as a template;

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

[0027] S3: Then, use a fluorescence detection platform to perform fluorescence signal scanning and genotyping on the amplification products;

[0028] S4: Read the genotyping data to obtain a genotyping map.

[0029] Furthermore, the genotyping is as follows:

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

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

[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, the points on the diagonal of the coordinate axis represent heterozygous genotypes, representing hybrid offspring.

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

[0035] Furthermore, the PCR amplification program is as follows: Pre-denaturation: temperature 94 °C, 15 min, 1 cycle; Denaturation: temperature 94 °C, 20 sec; Annealing / Extension: temperature 61 - 55 °C, 60 sec (-0.6 °C / cycle); 10 cycles for the second step; Denaturation: temperature 94 °C, 20 sec; Annealing / Extension: temperature 55 °C, 60 sec, 26 cycles for the third step. Finally, read the genotyping data at 25 °C for 30 sec.

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

[0037] 1. The present invention uses the results of SSR marker identification and re-sequencing data to develop SNP molecular markers for identifying tea plant hybrid offspring, and a method for quickly identifying the authenticity of tea plant hybrid offspring based on the KASP technology.

[0038] 2. Based on the identification results of the SSR primer set and combined with the re-sequencing 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 tea tree hybrid offspring, and screens out a specific primer set capable of identifying the authenticity of tea tree hybrid offspring. Using the primer set of the present invention can not only identify the authenticity of two tea tree hybrid offspring, but also exclude the hybrid offspring contaminated by pollen;

[0039] 3. The identification method of the present invention can replace the identification method of screening specific SSR markers by capillary electrophoresis technology, quickly, accurately and effectively realize the identification of tea tree hybrid offspring, and reduce the sequencing cost. It provides an important basis and support for the molecular breeding of efficiently creating tea tree germplasms. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a line graph of the number of SNPs screened under different numbers of true offspring in the third aspect of the present invention;

[0041] Figure 2 It is the peak graph of 'Dan Gui' in the capillary electrophoresis of the marker Cs-SSR2 in Example 1 of the present invention;

[0042] Figure 3 It is the peak graph of 'Jin Xuan' in the capillary electrophoresis of the marker Cs-SSR2 in Example 1 of the present invention;

[0043] Figure 4 It is the peak graph identified as true offspring in the capillary electrophoresis of the marker Cs-SSR2 in Example 1 of the present invention;

[0044] Figure 5 It is the capillary identification result graph of 10 offspring in Example 1 of the present invention;

[0045] Figure 6 It is the genotyping graph of the KASP marker Cs-KASP4 in 32 hybrid offspring in Example 3: The scatter plot of the X-axis and Y-axis represents the marker identification of 'Dan Gui', 'Jin Xuan' and hybrid offspring; The blue dots, orange dots and green dots represent the homozygotes of the female parent 'Dan Gui', the homozygotes of the male parent 'Jin Xuan' and the heterozygotes of the hybrid offspring respectively;

[0046] Figure 7 It is the genotyping graph of the KASP marker Cs-KASP4 in 32 hybrid offspring in Example 3: The scatter plot of the X-axis and Y-axis represents the marker identification of 'Dan Gui', 'Jin Xuan' and hybrid offspring; The blue dots, orange dots and green dots represent the homozygotes of the male parent 'Dan Gui', the homozygotes of the female parent 'Jin Xuan' and the heterozygotes of the hybrid offspring respectively. DETAILED DESCRIPTION OF THE INVENTION

[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: Use a NanoDrop 2000 (Thermo Scientific) nucleic acid detector to detect and confirm the quality and concentration of DNA, and then uniformly dilute it to 35 - 40 ng / μL.

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

[0060] For the 5 SSR markers, namely Cs-SSR1 to 5, the repeat unit of Cs-SSR1 is GA with 5 repeats; the repeat unit of Cs-SSR2 is CAGCAC with 4 repeats; the repeat unit of Cs-SSR3 is CACCAT with 6 repeats; the repeat unit of Cs-SSR4 is TC with 18 repeats; the repeat unit of Cs-SSR5 is AT with 6 repeats.

[0061] Select these 5 pairs of SSR marker primers to verify the reciprocal cross progeny. The PCR amplification system (Table 1) is as follows:

[0062] Table 1 PCR amplification reaction system

[0063]

[0064] Among them, 2×Taq Master Mix is from Beijing ComWin Biotech Co., Ltd., product number CW2849M. Since this experiment uses a 96-well plate for PCR amplification, 20 μL of mineral oil needs to be added for sealing after adding the reaction system and centrifuging to prevent evaporation.

[0065] The PCR amplification program is as follows:

[0066]

[0067] Table 2 Primer sequences for amplifying 5 markers

[0068]

[0069] 4. Fragment Analyzer TM Capillary electrophoresis:

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

[0071] 5. Analysis of capillary electrophoresis results: Use PROSize TMUse 2.0 software to display the capillary electrophoresis results. If a pair of markers show the parental peak patterns simultaneously in the peak map obtained after amplifying 5 markers in the hybrid offspring ( Figure 3 ), and there are no other obvious main peaks, it is identified as a hybrid; if the obtained peak map is inconsistent with the parents or only shows the peak map of one parent, it is identified as a non-hybrid, and the corresponding plants should be removed or transplanted in the later stage.

[0072] Example 2:

[0073] This example provides a method for developing core SNP molecular markers for identifying tea tree hybrid offspring based on KASP technology. The specific steps are as follows:

[0074] 1. Determination of core SNP loci: Based on the results described in Example 1, re-sequencing was performed on 1 each of the male and female parents and 8 true offspring. According to the VCF file in the re-sequencing data, SNP screening was carried out through a Linux server, and the vcftools command was used for filtering. The specific parameter settings are as follows:

[0075] (1) Retain bi-allelic loci;

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

[0077] (3) Set the minor allele frequency to be less than 0.05;

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

[0079] Use the bcftools command to extract the SNP locus information where the male and female parents are homozygous and the true offspring are heterozygous in the filtered VCF file. The specific command needs to output the chromosome name, SNP physical position, reference allele, alternative allele, and genotype; according to the principle that there are no other variant sites within 100 bp before and after the SNP locus. Finally, 4 core SNP markers were selected, and the specific information is as described in Table 3 below.

[0080] Marker 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] Convert the developed SNP loci into KASP primers. Align with the reference genome, extract the flanking conserved sequences of 100 bp before and after the 4 SNP loci. For each SNP locus, design two forward primers upstream and one reverse primer downstream. Each KASP primer pair combination is used to amplify the corresponding SNP marker. A total of 12 primers, and their nucleotide sequences are shown in Table 4:

[0083]

[0084]

[0085] Example 3:

[0086] In this example, the Cs-KASP4 primer pair designed in Example 2 was used. The genomic DNA of the tea tree varieties 'Dan Gui' and 'Jin Xuan' and their reciprocal cross F1 generation seedlings in Example 1 was used as the template, and double-distilled water was used as the control to perform KASP genotyping to identify the authenticity of the offspring, including the following steps:

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

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

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

[0090] Table 5 PCR amplification reaction system

[0091]

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

[0093] Table 6 PCR amplification reaction program

[0094]

[0095] (3) Fluorescence data reading and analysis

[0096] After the PCR reaction, the fluorescence data was read and analyzed using a BIO-RAD real-time quantitative instrument, and the true hybrid offspring were determined according to the genotyping results. (If the tested tea tree shows a blue signal or an orange signal, then the tea tree variety is a homozygous parent; if the tested tea tree shows a green signal, then the tea tree variety is a hybrid offspring.)

[0097] (3) If a well-defined genotype cluster is not obtained after the initial KASP thermal cycling program, the conditions detailed in Table 7 should be used for an additional 3 thermal cycles. Then the reaction plate should be read again and the results analyzed.

[0098] Table 7 PCR amplification reaction program

[0099]

[0100]

[0101] The results are as follows Figure 4 shown. The results show that the PCR primers designed using the SNP combination of the present invention, combined with Kompetitive allele specific PCR (KASP), can distinguish tea plants 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-cross progeny, and the points on the diagonal of the coordinate axis represent heterozygous genotypes, representing the true hybrid progeny.

[0102] The KASP genotyping technology of the present invention performs accurate biallelic genotyping on the target SNP, and can be used for the identification of the authenticity of tea plant hybrid progeny according to the developed SNP loci and the designed primers.

[0103] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A primer combination of SSR markers for authenticity identification of hybrid progeny of Dangui and Jinxuan tea varieties, characterized in that: The SSR markers include Cs-SSR1~5; the primer sequences of Cs-SSR1~5 are shown in 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.

2. Use of the primer combination described in claim 1 in identifying the authenticity of hybrid progeny of Dangui and Jinxuan tea tree varieties.

3. A KASP primer set for amplifying SNP molecular markers for authenticity identification of hybrid progeny of Dangui and Jinxuan tea varieties, characterized in that: The primer set includes forward primers shown in SEQ ID NO.20-21 and reverse universal primers shown in SEQ ID NO.

22.

4. Use of the KASP primer set described in claim 3 in identifying the authenticity of hybrid progeny of Dangui and Jinxuan tea tree varieties.

5. A method for identifying the authenticity of hybrid offspring of Dangui and Jinxuan tea trees 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: performing a PCR amplification reaction using the KASP primer set described in claim 3 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.

6. The identification method according to claim 5, 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.

7. The identification method according to claim 5, 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.

8. The identification method according to claim 5, characterized in that: The PCR amplification program is: pre-denaturation: temperature 94°C, 15 min, 1 cycle; denaturation: temperature 94°C, 20 sec; annealing / extension: temperature 61-55°C, 60 sec, the temperature decreases by 0.6°C each cycle; step 2, 10 cycles; denaturation: temperature 94°C, 20 sec; annealing / extension: temperature 55°C, 60 sec, step 3, 26 cycles.

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