SNP (Single Nucleotide Polymorphism) molecular marker for early identification of cold resistance of actinidia chinensis tree body in dormancy period

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for early identification of the cold resistance of an actinidia chinensis tree body in the dormancy period, and the SNP molecular marker is located at the Chr69297479 position on the No.6 chromosome of the actinidia chinensis and is closely associated with the cold-resistant phenotype median lethal temperature (LT50) of the actinidia chinensis. The method can be used for identifying the tree body dormancy period cold resistance of natural population and hybrid filial generation of actinidia chinensis. The homozygous genotype (CC) of the SNP locus corresponds to a high-cold-resistance individual in the dormancy period of an actinidia chinensis tree body, and the heterozygous genotype (TC) of the SNP locus corresponds to a low-cold-resistance individual. The cold resistance prediction accuracy in the F1-generation hybrid population reaches 94.94%, and the prediction accuracy in the natural population reaches 96.15%. The molecular marker detection method disclosed by the invention is convenient and rapid, has good specificity and high accuracy, is not influenced by climate, environment and the like, can rapidly and effectively evaluate the cold resistance of natural resources and hybrid filial generations of the actinidia chinensis, and provides powerful technical support for breeding cold-resistant varieties of the actinidia chinensis.

Description

Technical Field

[0001] The present invention relates to the field of kiwifruit breeding and molecular marker technology, and in particular to a SNP molecular marker, primers, applications thereof, and a kit for early identification of cold resistance of Chinese kiwifruit trees during their dormancy period. The SNP molecular marker and primers of the present invention can be used for prediction of cold resistance and variety breeding of Chinese kiwifruit. Background Art

[0002] Kiwifruit (Actinidia Lindl.), a perennial deciduous vine, produces fruit rich in vitamin C, minerals, folic acid, and other nutrients. Its twining vines offer unique advantages in vertical greening and garden landscaping, making it a popular specialty fruit tree that combines both edible and aesthetic value. However, temperature is a key ecological constraint within its cultivation range, and low-temperature stress significantly impacts kiwifruit growth and development. Therefore, breeding broadly adaptable, cold-resistant varieties has become a key goal in kiwifruit breeding.

[0003] Currently, cold-resistance testing for kiwifruit relies primarily on field testing and measurement of physiological cold-resistance indicators (such as relative conductivity). However, these methods have significant limitations. Especially in cold-resistance breeding, field testing of cold-resistance traits in hybrid offspring is labor-intensive, susceptible to interference from factors such as annual climate fluctuations and human management, and results are long and unstable. While physiological indicator testing can reflect short-term cold-resistance status, it requires large numbers of samples and repeated experiments, resulting in low efficiency.

[0004] With the development of high-throughput sequencing technology, quantitative trait loci (QTL) mapping technology has been gradually applied to the genetic analysis of important traits in fruit trees. For example, Li et al. located a QTL interval related to trunk diameter in the Chinese kiwifruit (A. chinesis) population and developed SNP molecular markers; another study developed InDel molecular markers related to cold resistance in the soft-fleshed kiwifruit (Actinidia arguta) population, but they are not applicable to Chinese kiwifruit. To date, SNP molecular markers for cold resistance in Chinese kiwifruit have not been reported. As an important commercial cultivar, Chinese kiwifruit has a great significance in developing cold resistance molecular markers and accelerating the cold resistance breeding process due to the frequent occurrence of extreme weather. Summary of the Invention

[0005] In order to solve the problem of lack of SNP molecular markers for evaluating the cold resistance of Chinese kiwifruit in the existing technology, the present invention provides a new and effective SNP molecular marker and related primers for early identification of the cold resistance of Chinese kiwifruit trees during the dormant period. The SNP molecular markers developed in the present invention can quickly detect the cold resistance of Chinese kiwifruit natural resources and their hybrid populations, quickly evaluate the cold resistance of trees during the dormant period, so as to screen natural resources with high cold resistance and hybrid offspring, and improve their breeding efficiency.

[0006] The present invention uses QTL positioning in diploid hybrid populations to identify a QTL interval closely associated with the semi-lethal temperature (LT50) of the cold resistance trait of Chinese kiwifruit, and successfully develops a SNP molecular marker within this interval. The SNP molecular marker is used to verify the cold resistance of F1 generation populations and natural populations. The prediction accuracy in the F1 generation population is 94.94%, and the prediction accuracy in the natural population is 96.15%.

[0007] In the first aspect, the present invention provides a SNP molecular marker for early identification of the cold resistance of Chinese kiwifruit trees during the dormancy period. The SNP molecular marker is located at Chr6_9297479 on chromosome 6 (Chr6) of the Chinese kiwifruit 'Hongyang' V3 genome, and is closely associated with the semi-lethal temperature (LT50) of the cold resistance phenotype of Chinese kiwifruit. It can be used to identify the cold resistance of Chinese kiwifruit trees during the dormancy period in natural populations and hybrid offspring.

[0008] Furthermore, the SNP molecular marker Chr6_9297479 of the present invention has a homozygous genotype (CC) corresponding to individuals with high cold resistance during the dormant period of Chinese kiwifruit trees, and a heterozygous genotype (TC) corresponding to individuals with low cold resistance during the dormant period of Chinese kiwifruit trees.

[0009] Furthermore, a specific primer pair was designed for the Chr6_9297479 site described in the present invention, and the primer sequences are as follows:

[0010] Forward primer: 5′-CTCAGTCAATGGAGAATCCAAATG-3′ (SEQ ID NO: 1);

[0011] Reverse primer: 5'-CTTCTACGCCAATGAAACAAGAATT-3' (SEQ ID NO: 2).

[0012] In a second aspect, the present invention provides a SNP molecular marker detection kit that is closely linked to the cold resistance of Chinese kiwifruit trees during dormancy, wherein the detection kit comprises a detection reagent for the above-mentioned SNP molecular marker.

[0013] Furthermore, the detection kit of the present invention comprises the following primer pairs:

[0014] Forward primer: 5′-CTCAGTCAATGGAGAATCCAAATG-3′;

[0015] Reverse primer: 5′-CTTCTACGCCAATGAAACAAGAATT-3′.

[0016] Furthermore, the detection kit of the present invention also includes:

[0017] Reagents for extracting genomic DNA from Actinidia chinensis;

[0018] Reagents for performing PCR amplification;

[0019] Reagents for agarose gel electrophoresis.

[0020] In addition, the present invention also relates to the use of the above-mentioned SNP molecular marker and detection kit in the following operations:

[0021] (1) Evaluation of cold resistance of natural resources of Actinidia chinensis;

[0022] (2) Early detection of cold resistance of hybrid progeny of Actinidia chinensis;

[0023] (3) Breeding of cold-resistant Chinese kiwifruit varieties.

[0024] In a third aspect, the present invention also provides a method for detecting the cold resistance of Chinese kiwifruit trees during their dormancy period, the method comprising the step of detecting the above-mentioned SNP molecular markers.

[0025] Furthermore, the method of the present invention for detecting the cold resistance of Chinese kiwifruit trees during dormancy period comprises the following steps:

[0026] (1) Extracting genomic DNA from the tested Actinidia chinensis samples (including hybrid progeny or natural populations);

[0027] (2) performing PCR amplification using the primer pair described in claim 3, performing Sanger sequencing on the amplified product, and detecting the genotype of the SNP site of Chr6_9297479;

[0028] (3) The cold resistance of the sample to be tested was determined based on the genotype of the above-mentioned SNP sites. The full length of the sequencing fragment was 301 bp. The homozygous site CC phenotype at 151 bp indicated that the tree had high cold resistance during the dormant period, and the heterozygous site TC phenotype indicated that the peach tree had low cold resistance during the dormant period.

[0029] In summary, the present invention, for the first time, uses a high-density genetic linkage map to locate QTLs for cold-resistance phenotypes in Actinidia chinensis, screens and identifies SNPs associated with cold-resistance traits, and develops corresponding SNP molecular markers. Using the molecular markers and methods of the present invention, it is possible to rapidly detect cold-resistance in natural Actinidia chinensis resources and their hybrid populations, rapidly evaluate cold-resistance during dormancy, screen for highly cold-resistance natural resources and hybrid offspring, and improve breeding efficiency. The SNP molecular markers of the present invention were used to verify cold-resistance in F1 and natural populations, with a prediction accuracy of 94.94% in the F1 population and 96.15% in the natural population. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 This is a diagram showing the QTL mapping results for the cold-resistant phenotype in an embodiment of the present invention.

[0032] Figure 2 The phenotypic data corresponding to different SNP genotypes in the embodiment of the present invention ('Hongyang'×'Boshan Biyu'♂F1 generation population), and * in the figure indicates significant difference (p<0.05).

[0033] Figure 3 The phenotypic data corresponding to different SNP genotypes in the embodiment of the present invention (natural population of diploid kiwifruit) are shown. In the figure, * indicates significant difference (p<0.05). DETAILED DESCRIPTION

[0034] In order to make the purpose and technical solution of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments. Those skilled in the art can easily understand other advantages of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0035] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. In the present invention, unless otherwise specified, all instruments, reagents, and raw materials are commercially available or commonly used in the art. The methods described in the following examples are conventional methods in the art unless otherwise specified.

[0037] Example:

[0038] 1. Test materials

[0039] The diploid Chinese kiwifruit 'Hongyang'×'Boshan Biyu'♂F1 hybrid population plants were used, with 79 plants corresponding to LT50.

[0040] 2. Identification of cold resistance of hybrid F1 generation of Actinidia chinensis

[0041] This study uses a cold-resistant F1 hybrid population of Actinidia chinensis 'Hongyang' x 'Boshan Biyu', previously constructed by our research group, and its parents. 140 F1 hybrids were planted at the Comprehensive Experimental Base of the Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences (Xinxiang, Henan Province), while the parents were planted at the Kiwifruit Branch of the National Horticultural Germplasm Resource Bank, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences.

[0042] Method for determining relative electrical conductivity (REL) in shoots: One-year-old shoots from the F1 hybrid population of the diploid Actinidia chinensis 'Hongyang' × 'Boshan Biyu'♂ and the parental lines 'Hongyang' and 'Boshan Biyu'♂ were collected in late December 2023 and late December 2024, respectively. Three biological replicates were used for each plant. The collected shoots were evenly placed in a low-temperature incubator (Zhihe, Suzhou, China), tightly wrapped with plastic wrap, and then incubated at -5°C, -10°C, -15°C, -20°C, and -25°C, respectively. The incubator was cooled at a rate of 1°C / min and maintained at the set temperature for 8 hours. The low-temperature-treated shoots were then removed and thawed at room temperature at 20°C for 1 hour.

[0043] (1) Thoroughly wash the branches with deionized water to ensure that the surface is clean and free of impurities; then, avoid the bud area, cut the middle section of the branch into thin slices with a thickness of 1 to 2 mm and mix them thoroughly.

[0044] (2) Accurately weigh 0.2 g of sample and place it in a 50 mL graduated centrifuge tube. Then add 10 mL of deionized water. When treating branches from each plant, take three replicates of each sample to improve the reliability of the experimental data.

[0045] (3) The sample was placed in a constant temperature water bath shaker and shaken at 200 r / min for 2 h at a constant temperature of 25°C. The initial conductivity value (R1) was then measured using a Leici DDSJ-308F conductivity meter (Leci, Shanghai, China).

[0046] (4) Place the centrifuge tube in a boiling water bath for 30 minutes. Then, transfer it to a shaker and shake it for 30 minutes to lower the temperature inside the tube to approximately 20°C. The final conductivity value (R2) is then measured using a conductivity meter. The relative conductivity is calculated using the following formula: relative conductivity = (R1 / R2) × 100%.

[0047] (5) Calculation of half-lethal temperature (LT50): The measured relative conductivity data were used to calculate the LT50 of the F1 hybrid population using the 'readxl', 'magritter', 'dplyr', 'ggplot2', and 'plyr' packages of the R / QTL (ver 1.50) software. Data with an R2 ≥ 0.5 were retained.

[0048] (6) The BLUE values ​​of LT50 in two years (2023 and 2024) were calculated using the ‘lme4’, ‘magritter’, and ‘lsmeans’ packages in R / QTL (ver 1.50).

[0049] 3. QTL mapping of cold resistance in hybrid populations of Actinidia chinensis

[0050] BLUE values ​​for LT50 in two years (2023 and 2024) were calculated using the 'lme4', 'magritter', and 'lsmeans' packages in R / QTL (ver 1.50). Valid data were obtained from the cold resistance of 79 progeny plants. QTL mapping was performed using R / QTL (ver 1.50) using a high-density genetic linkage map of the 'Hongyang' × 'Boshan Biyu' F1 hybrid population, previously constructed by our research group. Component interval mapping (CIM) was performed using the "CIM()" function with a LOD threshold of 3 and a mapping step size of 1 cm. All other parameters remained default. The peak of each QTL was identified at the highest LOD value, and the boundaries of each QTL were determined by a 1.5 LOD drop support interval. After QTL identification, multi-QTL analysis was performed using the "makeQTL()" and "fitQTL()" functions in R / QTL to estimate the percentage of phenotypic variance explained (PVE) for each QTL.

[0051] 4. DNA extraction from kiwifruit leaves

[0052] DNA was extracted from leaves of 79 plants corresponding to the LT50 QTL mapping interval for SNP identification within the LT50 mapping interval. Young leaves from 26 natural diploid kiwifruit plants were collected for validation of all SNP markers in the natural population. All leaves were frozen in liquid nitrogen and stored at -80°C until further use.

[0053] The DNA extraction process was performed according to the instructions of the DNA kit provided by Tiangen Biotechnology Co., Ltd. (Beijing, China). All samples were stored in a -20°C refrigerator for future use.

[0054] 5. SNP extraction within the QTLs interval

[0055] Molecular markers (SNPs) were extracted from the Chr6 mapping interval, and the phenotypic data corresponding to the extracted SNPs were analyzed for significance using IBM SPSS Statistics (ver27), and finally SNPs with significant differences were screened out (p<0.05).

[0056] 6. Primer design and synthesis

[0057] The complete amino acid sequence of chromosome 6 was extracted from the 'Hongyang v3' gene bank (https: / / kiwifruitgenome.org / organism / 5) using TBtools (ver 2.136). The target sequence was then extended 150 bp before and after the SNP site using BioEdit (ver 7.0.9.0), and SNP primers were designed using Primer (ver 5.0). All SNP primers were synthesized by Henan Shangya Biotechnology Co., Ltd. (Zhengzhou, China).

[0058] 7. PCR amplification of SNP target fragments

[0059] SNPs were amplified by PCR and sequenced using Novozymes (Nanjing, China) ( Table 1 ).

[0060] Table 1 PCR reaction system and amplification conditions

[0061]

[0062] The products were verified by agarose gel electrophoresis (200 V, 200 mA, 15 min), and the unpurified PCR products of the correct bands were entrusted to Henan Shangya Biotechnology Co., Ltd. (Zhengzhou, China) for sequencing.

[0063] 8. Results and Analysis

[0064] 8.1 QTL mapping for cold resistance in hybrid populations of Actinidia chinensis

[0065] Based on the constructed high-density genetic linkage map of the 'Hongyang' × 'Boshan Biyu'♂ hybrid population (Li et al., 2024), QTL mapping was performed using the interval mapping method (CIM) combined with the BLUE values ​​of the cold resistance phenotype (LT50) of the hybrid populations in 2023 and 2024. A total of one QTL for the cold resistance phenotype LT50 trait was identified ( Figure 1 ), located on chromosome 6 (Chr6), with a LOD value of 8.42, a phenotypic variance explained (PVE) of 2.22, and a genetic position distance (Low.cM-Up.cM) of 19.13cM-65.96cM (Table 2).

[0066] Table 2 Summary of QTL detection results for half-lethal temperature (LT50)

[0067]

[0068] 8.2 Validation of Cold-Tolerance Molecular Marker SNPs in Hybrid Progenies

[0069] The QTL for LT50 was located on chromosome 6, with a total of 138 SNPs within the Chr6 interval. Combined with the LT50 phenotypic analysis of each individual plant's progeny, a total of 8 significant SNPs were screened. PCR identification of the 8 SNPs in a 79-plant F1 hybrid population revealed that Chr6_9297479 had a validation accuracy of 94.94%. Among them, the homozygous genotype (CC) progeny had a mean LT50 of -17.59°C in 41 plants, while the heterozygous genotype (TC) progeny had a mean LT50 of -16.03°C in 34 plants. The corresponding phenotypic cold resistance difference between the two was 1.56°C ( Figure 2 ). Thus, in the SNP molecular marker Chr6_9297479, the phenotype of the homozygous genotype (CC) in the F1 progeny of the hybrid population is higher than that of the heterozygous genotype (TC).

[0070] 8.3 Validation of Cold-Tolerance SNP Molecular Markers in Kiwifruit Germplasm Resources

[0071] The BLUE values ​​of the cold-resistance phenotype LT50 of 26 diploid kiwifruit natural populations ranged from -10.84℃ to -22.19℃, with an average of -14.91℃.

[0072] The molecular marker Chr6_9297479, selected based on the LT50 QTL mapping results, had a validation accuracy of 96.15% in a natural population of 26 plants. Among them, there were 21 homozygous genotype (CC) progeny plants with an average LT50 of -15.33°C, while there were 4 heterozygous genotype (TC) progeny plants with an average LT50 of -12.95°C. The corresponding phenotypic cold resistance difference between the two was 2.38°C ( Figure 3 ). Therefore, in the SNP molecular marker Chr6_9297479, the phenotype of the homozygous genotype (CC) in the natural population is higher than that of the heterozygous genotype (TC).

[0073] In this example, the following primers were designed for the SNP molecular marker Chr6_9297479:

[0074] Forward primer: 5′-CTCAGTCAATGGAGAATCCAAATG-3′ (SEQ ID NO: 1);

[0075] Reverse primer: 5'-CTTCTACGCCAATGAAACAAGAATT-3' (SEQ ID NO: 2).

[0076] In this embodiment, a method for applying the above-mentioned SNP molecular marker is also provided, comprising the following steps:

[0077] (1) Extracting DNA from offspring or wild resources of Actinidia chinensis that need to be identified;

[0078] (2) PCR amplification of the extracted genomic DNA of Actinidia chinensis was performed using the above-mentioned SNP primers, followed by agarose gel electrophoresis, and then the electrophoresis bands were directly subjected to Sanger sequencing to identify the genotype of the SNP site;

[0079] (3) The cold resistance of the identified resources was determined based on the genotype of the SNP locus. The SNP marker Chr6_9297479 was a homozygous locus CC for cold-resistant individuals, and a heterozygous locus TC for non-cold-resistant individuals.

[0080] The preferred specific implementation modes and embodiments of the present invention are described in detail above, but the present invention is not limited to the above implementation modes and embodiments. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the concept of the present invention.

Claims

1. A SNP molecular marker for early identification of cold resistance of Chinese kiwifruit trees during dormancy, characterized in that: The SNP molecular marker is located at Chr6_9297479 on chromosome 6 of Actinidia chinensis and is closely associated with the semi-lethal temperature of the cold resistance phenotype of Actinidia chinensis. It can be used to identify the cold resistance of natural populations and hybrid offspring of Actinidia chinensis during the dormancy period.

2. The SNP molecular marker according to claim 1, characterized in that The SNP molecular marker Chr6_9297479, its homozygous genotype CC corresponds to individuals with high cold resistance during the dormant period of Chinese kiwifruit trees, and its heterozygous genotype TC corresponds to individuals with low cold resistance during the dormant period of Chinese kiwifruit trees.

3. The SNP molecular marker according to claim 1, characterized in that A specific primer pair was designed for the Chr6_9297479 site, and the primer sequences are as follows: Forward primer: 5′-CTCAGTCAATGGAGAATCCAAATG-3′; Reverse primer: 5′-CTTCTACGCCAATGAAACAAGAATT-3′.

4. A SNP molecular marker detection kit closely linked to the cold resistance of Chinese kiwifruit trees during dormancy, characterized in that: The detection kit comprises a detection reagent for the SNP molecular marker according to claim 1.

5. The detection kit according to claim 4, characterized in that The detection kit contains the following primer pairs: Forward primer: 5′-CTCAGTCAATGGAGAATCCAAATG-3′; Reverse primer: 5′-CTTCTACGCCAATGAAACAAGAATT-3′.

6. The detection kit according to claim 4, characterized in that The detection kit also includes: Reagents for extracting genomic DNA from Actinidia chinensis; Reagents for performing PCR amplification; Reagents for agarose gel electrophoresis.

7. Use of the SNP molecular marker according to any one of claims 1 to 3, or the detection kit according to any one of claims 4 to 6 in the following operations: (1) Evaluation of cold resistance of natural resources of Actinidia chinensis; (2) Early detection of cold resistance of hybrid progeny of Actinidia chinensis; (3) Breeding of cold-resistant Chinese kiwifruit varieties.

8. A method for detecting the cold resistance of Chinese kiwifruit trees during dormancy, characterized in that: The method comprises the step of detecting the SNP molecular marker according to claim 1.

9. A method for detecting the cold resistance of Chinese kiwifruit trees during dormancy, characterized in that: The following steps are involved: (1) Extracting genomic DNA from the tested kiwifruit sample; (2) performing PCR amplification using the primer pair described in claim 3, performing Sanger sequencing on the amplified product, and detecting the genotype of the SNP site of Chr6_9297479; (3) The cold resistance of the sample to be tested was determined based on the genotype of the above-mentioned SNP sites. The full length of the sequencing fragment was 301 bp. The homozygous site CC phenotype at 151 bp indicated that the tree had high cold resistance during the dormant period, and the heterozygous site TC phenotype indicated that the peach tree had low cold resistance during the dormant period.

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