SNP site and dCAPS molecular marker for screening early spring bud sprouting tea tree resources and application thereof

Abstract

The present application relates to a kind of screening spring bud germination early tea tree resources SNP site, dCAPS molecular marker, primer pair, method and its application, the SNP site of the screening spring bud germination early tea tree resources is located at the base of tea tree 4th chromosome 227699536 position, base polymorphism is C / G, the SNP site is named as Chr04:227699536_C / G.The present application obtains 1 in the coding region of TGY040725 Significant correlation SNP site (Chr04:227699536_C / G) of early growth trait, develops the SNP-dCAPS molecular marker that is closely linked with early growth trait of tea tree, has higher accuracy to screening spring bud germination early tea tree resources, accuracy can reach 93%, and identification cost is lower.

Description

Technical Field

[0001] The present invention relates to a SNP site and dCAPS molecular marker for screening early spring bud sprouting tea tree resources and applications thereof, and in particular to the screening and identification of a tightly linked SNP site for an extra-early-growing trait of tea trees and the development and application of a dCAPS molecular marker thereof, belonging to the field of molecular botany. Background Art

[0002] Tea, a leaf-producing cash crop, has a crucial trait for its spring shoot germination, closely linked to its economic value. However, unlike other simple traits, tea germination timing is not static. Different varieties and growing environments can affect germination timing, leading to varying degrees of early and late germination. Traditional breeding methods are time-consuming and labor-intensive, necessitating a simple, intuitive, and rapid method for identifying early and late germination in tea plants.

[0003] With the development of science and technology, various DNA molecular markers have been applied to tea tree research during the screening of early spring budding tea resources, such as restriction endonuclease fragment length polymorphism (RFLP), random amplified polymorphic DNA markers (RAPD), simple sequence repeats (SSR), and single nucleotide polymorphisms (SNP). With the application of new sequencing technologies, SNP, as a molecular marker technology centered on DNA sequences, has gained widespread recognition in the industry. In addition to the advantages of other previously used molecular markers, it also has the advantages of high resolution, large amounts, and high genetic stability. The use of SNP molecular marker technology is beneficial to the study of various aspects of tea trees and has a significant impact on the understanding, protection, and development of tea germplasm, and can greatly promote the selection and breeding of tea varieties. Converting SNPs into CAPS markers or dCAPS markers is low-cost and suitable for conventional molecular biology experiments, making it an effective means of detecting SNPs.

[0004] CAPS (cleaved amplified polymorphic sequence) markers combine PCR and restriction enzyme digestion. Primers are designed based on known sequences, and PCR amplification is performed on the SNP mutation site. The PCR product is digested with a specific restriction enzyme, and the digested fragments are detected by agarose gel electrophoresis for single nucleotide polymorphism analysis. Derived cleaved amplified polymorphic sequence (dCAPS) markers are a modified version of cleaved amplified polymorphic sequence markers. CAPS markers are restricted to mutations occurring within the restriction enzyme recognition site, although a significant portion of mutations do not occur within these sites. dCAPS markers introduce mismatched bases near the mutation site, thereby creating or removing a restriction enzyme recognition site. This marker offers advantages such as codominance, site specificity, ease of use, and low cost, making it widely used in variety classification and marker-assisted breeding.

[0005] Chinese invention patent application CN117568511A discloses a molecular marker for rapidly identifying the early-budding trait of tea plants. The patent involves a single-nucleotide polymorphism (SNP) marker site, Chr04:227558248_C / G, which is closely linked to the budding period trait of tea plants. This allows for marker-assisted breeding of early-budding tea varieties. However, the restriction endonuclease BstNI used in this patent application is relatively expensive, and the accuracy of a single molecular marker is limited, so there is room for improvement in both cost and accuracy. Summary of the Invention

[0006] In view of the deficiencies in the prior art, one of the objectives of the present invention is to provide a SNP site for screening early tea tree resources that sprout in spring; a second objective of the present invention is to provide a dCAPS molecular marker for screening early tea tree resources that sprout in spring; a third objective of the present invention is to provide a primer pair for amplifying the dCAPS molecular marker; a fourth objective of the present invention is to provide a method for screening early tea tree resources that sprout in spring, so as to reduce identification costs and improve identification accuracy; a fourth objective of the present invention is to provide the application of the SNP site, dCAPS molecular marker, and primer pair in screening early tea tree resources that sprout in spring and assisted selection breeding.

[0007] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0008] A SNP site for screening early spring bud sprouting tea tree resources, the SNP site is located at base 227699536 of chromosome 4 of the tea tree, the base polymorphism is C / G, and the SNP site is named Chr04:227699536_C / G.

[0009] The inventors' team conducted genome-wide association analysis on 126 tea plant materials and found a SNP site on chromosome 4 that is associated with the early-maturing trait of tea plants. They also identified the candidate gene TGY040725 (Protein SIEL-like) and found a SNP site Chr04:227699536_C / G on this gene that is highly correlated with the early-maturing trait of tea plants.

[0010] Based on the same inventive concept, the present invention also provides: a dCAPS molecular marker for screening spring bud sprouting early tea tree resources, the nucleotide sequence of the dCAPS molecular marker is shown in SEQ ID NO. 1, and the nucleotide sequence contains the SNP site described above. The SNP site is located at position 26 of the nucleotide sequence.

[0011] Therefore, the present invention uses genome-wide association technology to provide a dCAPS molecular marker for identifying the early spring shoot trait of tea trees, which provides partial theoretical support for the identification of tea trees in terms of early or late germination and provides a new molecular marker for tea tree breeding.

[0012] The applicant's research found that when the SNP site of the present invention is the reference genome type, that is, only carrying the C allele, it can be digested with the restriction endonuclease XhoI, whose cleavage sequence is CTCGAG, and the cleavage product is a lower main band of 142 bp in length. When the amplified fragment of a variety carrying only the G allele is digested with XhoI, it cannot be digested, and the band remains 163 bp. However, the cleavage product of a variety with the heterozygous C / G mutation is two main bands of 163 bp and 142 bp in length. Therefore, it can be used to identify the early-maturing trait of tea plants.

[0013] Based on the same inventive concept, the present invention also provides: a primer pair for amplifying the dCAPS molecular marker described above, the sequence of the primer pair is as follows:

[0014] Primer F: 5′-CCATCAGTAGAGAAACCAATCTCGA-3′ (SEQ ID NO. 2);

[0015] Primer R: 5′-GGTTGTCCAGTGTTGAAGTCTACT-3′ (SEQ ID NO. 3).

[0016] Based on the same inventive concept, the present invention also provides: a method for screening spring bud sprouting early tea tree resources, comprising the following steps:

[0017] (1) Extracting DNA from tea tree samples to be tested;

[0018] (2) using the tea plant sample DNA to be tested as a template, performing PCR amplification using the primer pair described above to obtain an amplified product;

[0019] (3) digesting the amplified product with restriction endonuclease XhoI, and performing electrophoresis to obtain an electrophoretic band pattern of the tea tree sample to be tested;

[0020] (4) Band pattern comparison and identification: If the electrophoresis band pattern is a 163bp main band, the genotype of the SNP site of the tea tree sample to be tested is GG, and the tea tree sample to be tested is identified as an early-maturing variety; if the electrophoresis band pattern is two main bands of 163bp and 142bp, the genotype of the SNP site of the tea tree sample to be tested is CG, and the tea tree sample to be tested is identified as a medium-maturing variety; if the electrophoresis band pattern is a 142bp main band, the genotype of the SNP site of the tea tree sample to be tested is CC, and the tea tree to be tested is identified as a late-maturing variety. In this way, early tea tree resources with spring buds can be screened. Further, in each step (2), the PCR amplification reaction system includes: primer F 0.5μL, primer R 0.5μL, template DNA 1μL, 2×Rapid Taq Master Mix 5μL and ddH2O 3μL.

[0021] Furthermore, in step (2), the reaction conditions for PCR amplification are: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 15 s, for a total of 35 cycles; termination extension at 72°C for 5 min; and storage at 4°C.

[0022] Furthermore, in each step (3), 10 μL of the enzyme digestion system includes 3 μL of amplified product, 0.4 μL of restriction endonuclease XhoI, 1 μL of 10×FastDigest Green Buffer, and 5.6 μL of ddH2O; the enzyme digestion conditions are incubation at 37°C for 30 min.

[0023] Furthermore, in step (3), 3% agarose gel is used for electrophoresis detection.

[0024] Furthermore, in step (4), the dCAPS molecular marker is divided into three band types. If the electrophoresis band type shows a main band with a length of 163 bp at the top, it cannot be cut by XhoI. At this time, the genotype of the SNP site of the tea tree sample to be tested is a homozygous mutant type (GG), and the tea tree sample to be tested is identified as an early-maturing variety; if the electrophoresis band type is two main bands with lengths of 163 bp and 142 bp, the genotype of the SNP site of the tea tree sample to be tested is a heterozygous mutation (CG), and the tea tree sample to be tested is identified as a medium-maturing variety; if the electrophoresis band type is a main band with a length of 142 bp at the bottom, the genotype of the SNP site of the tea tree sample to be tested is a wild type (CC), and the tea tree to be tested is identified as a late-maturing variety.

[0025] Based on the same inventive concept, the present invention also provides: the use of the above-mentioned SNP site or the dCAPS molecular marker or the primer pair in screening early spring bud sprouting tea tree resources and / or assisting tea tree variety selection and breeding.

[0026] Based on the same inventive concept, the present invention also provides: application of the above method in screening early spring bud sprouting tea tree resources and / or assisting in tea tree variety selection and breeding.

[0027] The present invention relates to a SNP site, dCAPS molecular marker, and application thereof for the early-germination trait of tea plants, belonging to the technical field of molecular markers. Based on genome-wide association analysis data of tea plants, the present invention identifies a candidate gene TGY040725 associated with the early-germination trait of tea plants. A SNP site significantly associated with the target trait is located on this gene, and a related dCAPS primer pair is designed for application.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention obtained a SNP site (Chr04:227699536_C / G) in the TGY040725 coding region that is significantly associated with the early-maturing trait, and developed a SNP-dCAPS molecular marker that is tightly linked to the early-maturing trait of tea plants. It has higher accuracy in screening early tea tree resources that sprout in spring, with an accuracy of up to 93%, which is further improved compared to the accuracy of existing technologies such as CN117568511A.

[0030] The dCAPS molecular marker of the present invention was used to verify polymorphism in 12 tea varieties with different spring shoot germination timings. Further verification of the remaining 32 tea germplasm resources showed that the molecular marker was significantly or highly significantly associated with the early sprouting trait. This demonstrates that the dCAPS molecular marker of the present invention is convenient, rapid, and highly accurate for identifying early sprouting.

[0031] The present invention converts screened SNP sites for tea plant early-budding traits into dCAPS molecular markers, combines PCR technology with restriction enzyme digestion, and then performs agarose electrophoresis. This method has the advantages of being rapid, low-cost, short-cycle, and easy to operate. This method can quickly and effectively distinguish between early and late germination times of tea varieties.

[0032] The restriction endonuclease XhoI used in the present invention is only half the price of the BstNI endonuclease used in the prior art CN117568511A, and the accuracy of trait identification can be increased to over 93%, thus achieving higher accuracy and lower detection costs. Furthermore, if used in conjunction with other molecular markers, accuracy can be further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram showing the association between the mutation site of gene TGY040725 and the early-maturing traits of tea trees.

[0034] Figure 2 This is the electrophoresis diagram of tea plant genomic DNA.

[0035] Figure 3 These are the PCR amplification products of 12 tea tree resources, M, marker D2000, and the length of the PCR amplification product is 163bp.

[0036] Figure 4 This is the gel electrophoresis diagram of the PCR amplification products of 12 tea tree resources digested with restriction endonuclease XhoI.

[0037] Figure 5 shows the PCR amplification products of 32 tea tree resources, M, marker D2000, and the length of the PCR amplification product is 163 bp.

[0038] Figure 6 is a gel electrophoresis diagram of 32 tea tree resource PCR amplification products digested with restriction endonuclease XhoI, wherein: Figure 6a The relevant data of 16 tea tree resources are provided. Figure 6b The following are the relevant data for another 16 tea tree resources. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below with reference to the examples. It should be noted that, unless otherwise specified, the features of the embodiments and examples of the present invention may be combined with each other. Unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0040] Example 1 Screening and Identification of SNP Sites in Tea Plant Germination Trait-Related Genes

[0041] The genome-wide association data of tea tree spring shoot germination were analyzed. The results showed that TGY040725 had a significant correlation with tea tree spring shoot germination. Therefore, TGY040725 was identified as a candidate gene related to tea tree germination traits. Using R language software, the molecular variation data of gene TGY040725 were associated with the phenotypic data of spring shoot germination of tea varieties. A SNP site (Chr04:227699536_C / G) significantly associated with tea tree germination traits was screened in the coding region of the gene (such as Figure 1 The wild-type and mutant nucleotide sequences were compared using DNAMAN7 software, and the SNP site was verified. It was finally found that when the non-synonymous SNP site had a C base, the amplified fragment could be digested by XhoI, but when the SNP site mutated to a G base, it could not be digested by XhoI.

[0042] Example 2 Gene SNP site conversion dCAPS molecular markers and related primers

[0043] Restriction endonuclease XhoI was selected for digestion, and mismatched bases were introduced by designing primers to construct a restriction endonuclease recognition site. The sequence length was approximately 150 bp. The nucleotide sequence of the dCAPS molecular marker located at the XhoI digestion site was selected as shown in SEQ ID No. 1. The primer sequence pair for the dCAPS molecular marker was designed using dCAPS Finder 2.0 and SnapGene software. The markers used in the positioning process and their primer information are shown in Table 1.

[0044] Table 1 dCAPS molecular markers and primer sequence information related to Chr04:227558248_C / G

[0045]

[0046] The sequence used above is the reverse complementary sequence of the wild-type gene. The underlined portion in the dCAPS molecular marker sequence (SEQ ID No. 1) is the restriction enzyme cleavage site of XhoI. The bold base T will be introduced into the mismatch base A when designing the primer, and the bold base G is the mutant base.

[0047] Example 3: Verification of polymorphism of dCAPS molecular marker enzyme-digested banding patterns and their association with early-birth traits

[0048] 1. Observation of phenotypic traits of spring shoots of tea trees

[0049] The tea plant materials used in the present application were collected from a tea plantation in Hunan Province (113°E, 28°N). The tea plant materials were divided into three groups according to the sprouting index (SPI) of the tea shoots: early-sprouting tea plants (SPI≥3), denoted as E; late-sprouting tea plants (SPI<1.9), denoted as L; and medium-sprouting tea plants (1.9≤H<3), denoted as M.

[0050] 2. Test tea plant materials

[0051] The tea plant materials were collected from a tea plantation in Hunan Province (113°E, 28°N). The tea plant materials were divided into three groups according to the sprouting index (SPI) of the tea shoots: early-sprouting tea plants (SPI≥3), denoted as E; late-sprouting tea plants (SPI<1.9), denoted as L; and medium-sprouting tea plants (1.9≤H<3), denoted as M.

[0052] 3. Verification of the correlation between the dCAPS molecular marker and the sprouting time of tea plants

[0053] (1) Extraction of tea plant DNA;

[0054] The DNA of the tea plant materials was extracted using the Plant DNA Isolation Mini Kit (Nanjing Nvigan Biotech Co., Ltd.) and the quality of the extracted DNA was verified by 1% agarose gel electrophoresis. Figure 2 The results are shown in FIG. 1.

[0055] (2) The DNA of 12 tea plants with different spring shoot sprouting traits was used as a template for specific PCR amplification using the dCAPS molecular marker primers provided in Example 2, and the amplification products were obtained.

[0056] The reaction system for PCR amplification included 10 uL of the amplification products, 0.5 uL of the forward primer (227699536-F), 0.5 uL of the reverse primer (227699536-R), 1 uL of the template DNA (wild-type or mutant gene sequence as shown in Table 1), 5 uL of 2x Rapid Taq Master Mix, and 3 uL of ddH2O.

[0057] The reaction conditions for PCR amplification were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 15 s, for a total of 35 cycles, and final extension at 72°C for 5 min.

[0058] After the amplification results were verified to be accurate, 4 uL of the PCR amplification products were electrophoresed on a 3% agarose gel for detection, and the products were stored at 4°C for later use.​

[0059] (3) digesting the amplified products of the 12 tea plant genomic DNAs with the restriction endonuclease XhoI, and electrophoresing the resulting digestion products on a 3% agarose gel. Observing the electrophoretic band patterns on a gel imager and preserving the experimental results;

[0060] The 10 μL enzyme digestion system includes 3 μL of the amplified product in step (2), 0.4 μL of restriction endonuclease XhoI, 1 μL of 10×FastDigest Green Buffer, and 5.6 μL of ddH2O; the enzyme digestion conditions are incubation at 37°C for 30 min.

[0061] 4. Results Analysis

[0062] Figure 3 This is the electrophoresis diagram of 12 tea tree resource amplifications in this example. Figure 4 The following is an electrophoresis diagram of the enzyme-digested fragments obtained by dCAPS molecular marker detection of 12 tea tree resources in this embodiment. The enzyme-digested products of 2 tea tree materials are a main band at the top with a band length of 163bp, which is consistent with the band length before enzyme digestion, and the corresponding tea tree varieties are early-growing; the enzyme-digested products of 6 tea tree materials contain 2 main bands with band lengths of 163bp and 142bp, respectively, and the corresponding tea tree varieties are medium-growing; the enzyme-digested products of 4 tea tree materials are a main band at the bottom with a band length of 142bp, and the corresponding tea tree varieties are late-growing. This shows that the marker enzyme digestion polymorphism is good, and the enzyme digestion band type has a good correspondence with the tea tree spring shoot germination phenotype, indicating that the dCAPS molecular marker method can be used to effectively identify tea varieties with different spring shoot germination times.

[0063] Example 4 Further verification of the association between enzyme-cut banding patterns of tea germplasm resources and tea spring shoot germination phenotypes

[0064] This example uses the dCAPS molecular markers and primers provided in Example 2, and further verifies the association between the enzyme-cut banding patterns and early-maturing traits of the remaining 32 tea tree resources through the detection method provided in Example 3, and verifies the reliability of the detection results by analyzing the relationship between the mutation site and the spring shoot germination phenotype of the tea tree under different genetic models.

[0065] 1. Tea tree materials for testing

[0066] The source is consistent with that of Example 3.

[0067] 2. Detection method

[0068] The steps are consistent with the detection method in Example 3.

[0069] 3. Results Analysis

[0070] Figure 5a-5b This is the electrophoresis diagram of 32 tea tree resource amplifications in this example. Figure 6a-6b This is the electrophoresis diagram of the enzyme-digested fragments obtained by dCAPS molecular marker detection of the amplified fragments. Nine samples showed a single upper band, indicating the wild type; eight showed two bands, indicating heterozygous mutants; and 15 showed a single lower band, indicating pure and neutral mutants. The early-maturing traits of the 32 tea varieties recorded above were marked on the electrophoresis diagram and divided into three types: early-maturing varieties (E), medium-maturing varieties (M), and late-maturing varieties (L). The wild type corresponds to the late-maturing varieties, the heterozygous mutant corresponds to the medium-maturing varieties, and the pure and neutral mutant corresponds to the early-maturing varieties. The identification accuracy rate reached 93.75%, indicating that this dCAPS locus can relatively accurately distinguish the early and late germination time of tea plants.

[0071] The genotypes of 32 tea accessions were collected and the statistical results were analyzed using R language software for association analysis with tea spring shoot germination phenotypes under different genetic models. The results showed that the genotype at this locus was highly significantly associated with the tea spring shoot germination phenotype. As shown in Table 3, under the codominant model, the C / C genotype at the Chr04:227699536_C / G locus was highly significantly associated with the tea spring shoot germination phenotype (P < 0.01); under the dominant model, the C / C genotype at the Chr04:227699536_C / G locus was highly significantly associated with the tea spring shoot germination phenotype (P < 0.01); and under the recessive model, the C / C and C / G genotypes at the Chr04:227699536_C / G locus were highly significantly associated with the tea spring shoot germination phenotype (P < 0.01).

[0072] Table 2 Relationship between mutation sites and spring shoot germination traits of tea varieties under different genetic models

[0073]

[0074]

[0075] It can be seen that the dCAPS molecular marker designed for this SNP site in the present invention can be effectively used for the molecular identification of early-maturing traits of tea trees, and can more accurately classify different tea tree resources into three types: early-maturing varieties, medium-maturing varieties, and late-maturing varieties, which is of great significance in assisting tea tree selection and breeding.

[0076] The contents illustrated in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art shall fall within the scope defined by the claims attached to this application.

Claims

1. A method for screening spring bud sprouting early tea tree resources, characterized in that: The following steps are involved: (1) Extract DNA from tea tree samples to be tested; (2) Using the DNA of the tea tree sample to be tested as a template, PCR amplification is performed using the primer pair to obtain the amplified product; Wherein, the sequence of the primer pair is as follows: Primer F: 5′-CCATCAGTAGAGAAACCAATCTCGA-3′; Primer R: 5′-GGTTGTCCAGTGTTGAAGTCTACT-3′; (3) digesting the amplified product with restriction endonuclease XhoI, and performing electrophoresis to obtain an electrophoretic band pattern of the tea tree sample to be tested; (4) Band type comparison and identification: If the electrophoresis band type is a main band of 163 bp, the genotype of the SNP site of the tea tree sample to be tested is GG, and the tea tree sample to be tested is identified as an early-maturing variety; if the electrophoresis band type is two main bands of 163 bp and 142 bp, the genotype of the SNP site of the tea tree sample to be tested is CG, and the tea tree sample to be tested is identified as a medium-maturing variety; if the electrophoresis band type is a main band of 142 bp, the genotype of the SNP site of the tea tree sample to be tested is CC, and the tea tree to be tested is identified as a late-maturing variety.

2. The method according to claim 1, characterized in that The PCR amplification reaction system in step (2) includes: primer F 0.5 μL, primer R 0.5 μL, template DNA 1 μL, 2× Rapid Taq Master Mix 5 μL and ddH2O 3 μL.

3. The method according to claim 1, characterized in that In step (2), the reaction conditions of the PCR amplification are: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 15 s, for a total of 35 cycles; termination extension at 72°C for 5 min; and storage at 4°C.

4. The method according to claim 1, wherein In step (3), 10 μL of the enzyme digestion system includes 3 μL of amplified product, 0.4 μL of restriction endonuclease XhoI, 1 μL of 10× FastDigest Green Buffer, and 5.6 μL of ddH2O; the enzyme digestion conditions are incubation at 37°C for 30 min.

5. The application of the method according to any one of claims 1 to 4 in screening early tea tree resources for spring bud sprouting and / or in auxiliary tea variety spring shoot sprouting time selection breeding.

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