A PCR kit for identifying southern highbush blueberry varieties and its application

Abstract

The invention discloses a PCR kit for identifying southern highbush blueberry varieties and application thereof, belonging to the technical field of molecular marker development and application. The present invention uses the genome data of the evergreen blueberry variety (Vaccinium darrowii) for the first time to independently develop SSR (Simple Sequence Repeats) molecular markers. Combined with the SSR molecular markers previously independently developed using the genome data of the blueberry variety Drapper, a PCR kit for identifying southern highbush blueberry varieties is obtained. The PCR kit includes an SSR primer set, namely SSR1-8+SSR4-3+SSR7-7+SSR1551. This primer set can effectively distinguish and identify 48 southern highbush blueberry varieties, has the advantages of high sensitivity, economy, rapidity, and simple operation, and can be used for the identification of common varieties in the current blueberry industry. At the same time, DNA fingerprints of the 48 varieties are constructed, providing an important scientific basis for solving the problem of variety identification in the blueberry seedling market. At the same time, the genetic diversity of the 48 varieties is systematically analyzed, which is of great significance for optimizing southern highbush blueberry breeding combinations and broadening their genetic basis in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker development and application, and particularly relates to a PCR kit for identifying southern highbush blueberry varieties and an application thereof. Background Art

[0002] Blueberries are native to North America and belong to the genus Vaccinium (Vaccinium L.) in the Ericaceae family. Due to the high nutritional value of their fruit, they have been domesticated and cultivated worldwide since the early 20th century. Over the past century, my country's blueberry industry has grown rapidly as cultivation techniques have continuously improved, establishing four major production areas: Northeast China, the Shandong Peninsula, the Yangtze River Basin, and the Yunnan-Guizhou Plateau.

[0003] Blueberry cultivars can be divided into five types based on tree size and chilling requirements: northern highbush, southern highbush, semi-highbush, lowbush, and rabbiteye. Accurate identification and phylogenetic analysis of blueberry varieties are crucial prerequisites for the healthy development, scientific exploitation, and rational utilization of the blueberry industry. However, my country's blueberry introductions are multifaceted, leading to disorganized cultivar information management. During production, transliterated and translated cultivar names often coexist with self-created trade names, leading to confusion in seedlings and the frequent passing of counterfeit or inferior goods as genuine products, resulting in significant economic losses for blueberry growers and companies.

[0004] Traditional variety identification is based on blueberry leaves, flowers, fruits and tree posture. However, due to different judgment standards for morphological traits by different scholars, the traits between varieties cross and overlap, and the strong plasticity in different environments, this identification method has low accuracy and is very difficult, resulting in confusion in variety names, and identification errors such as the same thing having different names, and different things having the same name often occur.

[0005] The genetic diversity of plant varieties is primarily reflected through the polymorphism of genetic markers. Molecular markers are genetic markers based on nucleic acid polymorphism. Unaffected by tissue type, developmental stage, or environmental conditions, they offer advantages such as high abundance and high polymorphism, making them ideal genetic markers. Molecular markers have been widely used in germplasm resource research, hybrid identification, genetic map construction, target gene localization, genetic diversity, kinship studies, and marker-assisted selection breeding. Among them, microsatellites (also known as simple sequence repeats (SSRs)) are simple sequence repeats consisting of tandemly repeated fragments of 2-6 nucleotides, uniformly distributed throughout the genome. They possess advantages such as high polymorphism, good stability, multiple alleles, co-dominance, abundance, good genome coverage, and ease of use. This technique has been applied to studies of plant relationships, genetic diversity analysis, cultivar fingerprinting, cultivar identification, and DNA fingerprinting for species such as Chinese plum (Prunus salicina), peach (Prunus persica), black wolfberry (Lycium ruthenicum), and loquat (Eriobotryajaponica). Our research group previously successfully identified 48 northern highbush blueberry varieties using three pairs of SSR molecular markers developed from Drapper genomic data. This significant advancement in identifying 48 blueberry varieties using only three pairs of primers is a significant step forward. However, given the large variety of blueberry varieties, some varieties remain difficult to distinguish due to the limited number of primers used and the relatively low resolution. Summary of the Invention

[0006] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide a PCR kit for identifying southern highbush blueberry varieties and its application. In view of the current situation that there is no efficient SSR marker technology system for southern highbush blueberry and the varieties are still under identification, 48 common southern highbush blueberry varieties were collected for genetic diversity research. SSR molecular markers were independently developed using the genome data of the evergreen blueberry variety (Vaccinium darrowii). Combined with the SSR molecular markers previously independently developed using the genome data of the blueberry variety Drapper and the selected polymorphic primers, these 48 southern highbush blueberry varieties were identified, the optimal variety identification primer combination was screened, and the blueberry variety SSR characteristic fingerprint was constructed, providing an important basis for blueberry variety identification and molecular marker-assisted breeding.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The first object of the present invention is to provide a blueberry SSR molecular marker primer set, which includes four pairs of SSR molecular marker primer pairs, and the nucleotide sequences of the primer pairs are shown in SEQ ID NO.1-8.

[0009] The second object of the present invention is to provide a PCR kit for identifying southern highbush blueberry varieties, comprising the above-mentioned blueberry SSR molecular marker primer set.

[0010] Based on the above technical solution, the PCR kit further includes Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction enhancer and optimizer, stabilizer, and deionized water.

[0011] A third object of the present invention is to provide a method for identifying southern highbush blueberry varieties, comprising the steps of:

[0012] (1) Extracting genomic DNA from known southern highbush blueberry varieties;

[0013] (2) performing PCR amplification on the genomic DNA obtained in step (1) using the primer pair;

[0014] (3) detecting the PCR amplification product obtained in step (2) using polyacrylamide gel electrophoresis, and reading and analyzing the data;

[0015] (4) Constructing SSR fingerprints of southern highbush blueberry varieties;

[0016] (5) Using steps (1) to (3), the amplification results of the corresponding sites of the southern highbush blueberry variety to be detected are obtained. If the amplification results are completely consistent with the base sizes in the fingerprint map of the known southern highbush blueberry variety, the variety of the unknown southern highbush blueberry is obtained.

[0017] Based on the above technical solution, further, the varieties that can be identified by the method include Bladen, jewelry

[0018] Jewel, Jubilee, O'Neal, Reveille, Sharpblue, Star, Crisp Blue

[0019] Bluecrisp, Blue Ridge, Gulfcoast, Avonblue, Duplin, Suziblue, Sapphire, Georgiagem, Pearl River, Southmoon, Summit, Magnolia, Ozarkblue, Rebel, Craven, Lenoir, Arlen, Topblue SX Topblue, Diana SX Blue, Aiblue SX Ai

[0020] Blue, Emerald, Guputon, Naitive Blue, Dixie Blue, Carteret, Beaufort, Primadonna, Meadowlark, Farthing, Norman, Eureka, EB9-12 Magnifica, Sunshine Blue, Bluerain, Scintilla, Springwide, Abundance, Flicher, Palmetto, Round, and EB8-42 Julieta.

[0021] Based on the above technical solution, further, the volume of the PCR amplification system in step (2) is 10 μL, including 0.5 μL each of forward and reverse primers (10 μmol / L), 4 μL of 2*Taq PCR Master Mix, 100 ng of template, and the balance is deionized water.

[0022] Based on the above technical solution, the PCR amplification program in step (2) is further as follows: pre-denaturation at 94°C for 6 to 10 min; denaturation at 94°C for 35 to 40 s, annealing at the annealing temperature for 30 to 70 s, extension at 72°C for 36 s to 2 min, 35 to 38 cycles, and extension at 72°C for 10 min.

[0023] Based on the above technical solution, further, the annealing temperature of the molecular marker primer pair shown in SEQ ID NO.1-2 is 56°C; the annealing temperature of the molecular marker primer pair shown in SEQ ID NO.3-4 is 57°C; the annealing temperature of the molecular marker primer pair shown in SEQ ID NO.5-6 is 56°C; and the annealing temperature of the molecular marker primer pair shown in SEQ ID NO.7-8 is 61°C.

[0024] Based on the above technical solution, further, the detection in step (3) is silver staining detection.

[0025] The fourth object of the present invention is to provide the use of the above-mentioned blueberry SSR molecular marker primer set in any one of the following ①-⑥:

[0026] ①, Application of the SSR molecular marker primer pair in constructing a blueberry genetic map;

[0027] ②. Application of the SSR molecular marker primer pair in blueberry germplasm identification;

[0028] ③. Application of the SSR molecular marker primer pair in blueberry breeding;

[0029] ④. Application of the SSR molecular marker primer pair in blueberry genetic diversity analysis;

[0030] ⑤. Application of the SSR molecular marker primer pair in blueberry genetic relationship analysis;

[0031] ⑥. Application of the SSR molecular marker primer pair in blueberry molecular marker-assisted breeding.

[0032] The beneficial effects of the present invention compared to the prior art are:

[0033] The existing technology often requires many pairs of primers to distinguish blueberry varieties, and even then, it can only distinguish a relatively small number of blueberry varieties (such as within 50), resulting in low identification efficiency and high cost. The present invention independently developed molecular marker primers related to SSR of blueberry varieties from blueberry (Drapper + Darrow) genome data, studied the genetic diversity of 48 common southern highbush blueberry varieties, and screened out the optimal variety identification primer combination SSR1-8+SSR4-3+SSR7-7+SSR1551, and constructed the SSR characteristic fingerprint of southern highbush blueberry varieties, providing an important basis for southern highbush blueberry variety identification and molecular marker-assisted breeding, enriching the types of molecular marker identification of southern highbush blueberry varieties, significantly improving identification efficiency, and saving costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0035] Figure 1 Figure 2: Electrophoresis results of PCR amplification products of marker SSR1-8 in 48 southern highbush blueberry varieties. M is a 100 bp DNA ladder (TIANGEN, Beijing). From top to bottom, the lengths of standard DNA bands are 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp, respectively. The concentration is 50 ng·μL. -1 ; Samples 1 to 48 are described in Table 2.

[0036] Figure 2 The electrophoresis results of PCR amplification products of marker SSR4-3 in 48 southern highbush blueberry varieties are shown. M is a 100 bp DNA ladder (TIANGEN, Beijing). From top to bottom, the lengths of standard DNA bands are 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp, respectively. The concentration is 50 ng·μL -1 ; Samples 1 to 48 are described in Table 2.

[0037] Figure 3 The electrophoresis results of PCR amplification products of marker SSR7-7 in 48 southern highbush blueberry varieties are shown. M is a 100 bp DNA ladder (TIANGEN, Beijing). From top to bottom, the lengths of standard DNA bands are 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp, respectively. The concentration is 50 ng·μL -1 ; Samples 1 to 48 are described in Table 2.

[0038] Figure 4 The electrophoresis results of PCR amplification products of marker SSR1551 in 48 southern highbush blueberry varieties are shown. M is a 100 bp DNA ladder (TIANGEN, Beijing). From top to bottom, the lengths of standard DNA bands are 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp, respectively. The concentration is 50 ng·μL -1 ; Samples 1 to 48 are described in Table 2.

[0039] Figure 5 This is a phylogenetic cluster tree of different southern highbush blueberry varieties. DETAILED DESCRIPTION

[0040] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0041] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

[0042] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only. Experimental methods not specifically noted may be carried out in accordance with conventional methods or in accordance with the instructions of the manufacturer of the products used. The materials, reagents, etc. used in the following examples, unless otherwise specified, may be obtained commercially. SSR marker primers were commissioned to be synthesized by BGI.

[0043] Example 1 Determination of blueberry SSR molecular markers and primers

[0044] Based on the genome sequences of the developed blueberry varieties Darrow and Drapper, SSR loci were searched and 220 pairs of SSR primers were designed. DNA was extracted from 12 common blueberry varieties (Duke, Draper, Huron, Liberty, Blue Ribbon, Top, Coda, Lark, Faxin, Eureka, Leybold, and Elu). PCR and polyacrylamide gel electrophoresis were performed on these twelve varieties using these 220 primer pairs. Parameters such as annealing temperature, amplification capability, band specificity, clarity, and whether stable bands could be obtained in all twelve varieties were analyzed and compared for each primer pair, and 60 primer pairs were preliminarily screened. Based on the molecular weight range and PIC value of the alleles of each primer pair, four pairs of SSR primers were selected that could accurately read the genotype data of 48 southern highbush blueberry varieties (see Table 2) and distinguish the corresponding variety combinations.

[0045] Table 1 Developed blueberry polymorphic SSR primers

[0046]

[0047] Example 2: Using the SSR molecular marker primers provided by the present invention to distinguish southern highbush blueberry varieties

[0048] 1. Rapid DNA extraction and detection

[0049] The genomic DNA of 48 test materials (see Table 2) was extracted using the Plant Genome Extraction Kit (Model DP320-03) produced by Tiangen Biochemical Technology Co., Ltd. The DNA concentration and the presence of RNA contamination, protein or phenol contamination were detected by agarose gel electrophoresis and Nano Drop. (Pure sample DNA OD 260 / OD 280 The ratio should be between 1.7-1.9) and diluted to 50 ng·μL -1 , and stored in a -20℃ refrigerator for future use.

[0050] 2. Core primer selection

[0051] By analyzing the distribution of SSR molecular markers on chromosomes, polymorphism levels, PCR amplification stability and amplification product banding patterns, primers that meet the conditions determined in Example 1 were selected, as shown in Table 1.

[0052] 3. PCR amplification

[0053] The PCR amplification system consisted of 20 μL of 1 μL each of forward and reverse primers (10 μmol / L), 8 μL of 2*Taq PCR Master Mix (Tiangen Biochemical Technology Co., Ltd.), 100 ng of template, and 8 μL of ddH2O. The PCR amplification reaction was performed on a Bio-Rad PTC-200 thermal cycler (USA) with the following procedure: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 40 s, annealing for 70 s (annealing temperature for the SSR1-8 molecular marker primer pair was 56°C; annealing temperature for the SSR4-3 molecular marker primer pair was 57°C; annealing temperature for the SSR7-7 molecular marker primer pair was 56°C; annealing temperature for the SSR1551 molecular marker primer pair was 61°C), and extension at 72°C for 2 min, for 35 cycles, and extension at 72°C for 10 min.

[0054] 4. Amplification product detection

[0055] PCR amplification products were detected by 12% polyacrylamide gel and silver staining as follows:

[0056] (1) 10% polyacrylamide gel electrophoresis at 120 V for 180 min;

[0057] (2) Remove the gel and fix it for 20 min (fixative solution: add 10 mL of glacial acetic acid and 200 mL of anhydrous ethanol to 1000 mL of distilled water and dilute to 2 L);

[0058] (3) Discard the fixative, rinse with ddH2O, and immerse in staining solution for 10 min (staining solution: 6 g of silver nitrate, 10 mL of glacial acetic acid, and 200 mL of anhydrous ethanol in 2 L of distilled water);

[0059] (4) Discard the staining solution, wash with ddH2O, and develop until the bands are clear (developer: add 60 g of NaOH to 2 L of ultrapure water, cool in an ice bath, and add 10 mL of 37% formaldehyde before use);

[0060] (5) Discard the developer, clean with ddH2O, seal with plastic wrap, take photos and record the results. Figure 1-4 shown.

[0061] 5. Analysis of genetic relationships among southern highbush blueberry varieties based on SSR markers

[0062] The data were analyzed by combining manual reading with Gel-pro32 software. The genetic similarity coefficient was obtained by NTSYS-pc (Version 2.1) software. Cluster analysis was performed using the Neighbor-Joining (NJ) method. A cluster diagram was constructed based on the genetic similarity coefficient. Figure 5 ), which can distinguish 48 southern highbush blueberry varieties.

[0063] 6. SSR fingerprinting and variety identification of southern highbush blueberry varieties

[0064] Electrophoresis results were recorded using a 0,1 system, with the presence of a band in an amplified band being marked as 1 and the absence of a band being marked as 0. A total of 48 distinct genotypes were detected in 48 southern highbush blueberry varieties using the primer combination SSR1-8+SSR4-3+SSR7-7+SSR1551. A preliminary fingerprint of 48 southern highbush blueberry varieties was constructed, and the fingerprint coding results are shown in Table 2. The size of each band amplified by each SSR primer pair is recorded. If the SSR primers are polymorphic within the same variety, the band sizes are sorted in ascending order and separated by "-". This provides a molecular basis for the identification of individual southern highbush blueberry varieties.

[0065] Table 2 SSR fingerprints of 48 southern highbush blueberry varieties

[0066]

[0067]

[0068]

[0069] 7. Detection of unknown southern highbush blueberry varieties

[0070] Using this method, the genome of the southern highbush blueberry variety to be tested is extracted and PCR amplified to obtain the amplification status of the corresponding loci. If the test results are completely consistent with the base lengths in the southern highbush blueberry variety fingerprint, the unknown southern highbush blueberry variety is identified. This test can also be performed on multiple unknown southern highbush blueberry varieties, and the genetic relationships between the samples can be confirmed based on the typing results.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A blueberry SSR molecular marker primer set, characterized in that: The primer set includes 4 pairs of SSR molecular marker primer pairs, and the nucleotide sequences of the primer pairs are shown in SEQ ID NO.1-8.

2. A PCR kit for identifying southern highbush blueberry varieties, characterized in that: It comprises the blueberry SSR molecular marker primer set according to claim 1.

3. The PCR kit according to claim 2, characterized in that The PCR kit further comprises Taq DNA polymerase, dNTPs, MgCl2, a reaction buffer, a PCR reaction enhancer and optimizer, a stabilizer, and deionized water.

4. A method for identifying southern highbush blueberry varieties, characterized in that: The following steps are involved: (1) Extracting genomic DNA from known southern highbush blueberry varieties; (2) performing PCR amplification on the genomic DNA obtained in step (1) using the primer pair described in claim 1; (3) detecting the PCR amplification product obtained in step (2) using polyacrylamide gel electrophoresis, and reading and analyzing the data; (4) Constructing SSR fingerprints of southern highbush blueberry varieties; (5) using steps (1) to (3) to obtain the amplification results of the corresponding sites of the southern highbush blueberry variety to be tested. If the amplification results are completely consistent with the base sizes in the fingerprint of the known southern highbush blueberry variety, the variety of the southern highbush blueberry to be tested is obtained; The varieties that can be identified by the method described include Bladen, Jewel, Jubilee, O'Neal, Reveille, Sharpblue, Star, Bluecrisp, Blue Ridge, Gulfcoast, Avonblue, Duplin, Suziblue, Sapphire, Georgegem, Pearl River, Southmoon, Summit, Magnolia, Ozarkblue, Rebel, Craven, Lenoir, Arlen, Upper Blue, Diana, Love Blue, Emerald, Guputon, Naitive Blue, and more. Blue), Dixie blue, Carteret, Beaufort, Primadonna, Meadowlark, Farthing, Norman, Eureka, Juliet, Sunshine Blue, Bluerain, Scintilla, Springwide, Abundance, Flicher, Palmetto, Round, and Julietta.

5. The method according to claim 4, characterized in that The volume of the PCR amplification system in step (2) is 10 μL, including 0.5 μL each of 10 μmol / L forward and reverse primers, 4 μL of 2*Taq PCR Master Mix, 100 ng of template, and the balance is deionized water.

6. The method according to claim 4, characterized in that The PCR amplification program in step (2) is as follows: pre-denaturation at 94°C for 6-10 min; denaturation at 94°C for 35-40 s, annealing at annealing temperature for 30-70 s, extension at 72°C for 36 s-2 min, 35-38 cycles, and extension at 72°C for 10 min.

7. The method according to claim 6, characterized in that The annealing temperature of the molecular marker primer pair shown in SEQ ID NO.1-2 is 56°C; the annealing temperature of the molecular marker primer pair shown in SEQ ID NO.3-4 is 57°C; the annealing temperature of the molecular marker primer pair shown in SEQ ID NO.5-6 is 56°C; and the annealing temperature of the molecular marker primer pair shown in SEQ ID NO.7-8 is 61°C.

8. The method according to claim 4, characterized in that The detection in step (3) is silver staining detection.

9. Use of the blueberry SSR molecular marker primer set according to claim 1 in any one of the following ①-⑤: ①, Application of the SSR molecular marker primer pair in constructing a blueberry genetic map; ②. Application of the SSR molecular marker primer pair in blueberry germplasm identification; ③. Application of the SSR molecular marker primer pair in blueberry breeding; ④. Application of the SSR molecular marker primer pair in blueberry genetic diversity analysis; ⑤. Application of the SSR molecular marker primer pair in blueberry genetic relationship analysis.

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