SSR molecular marker and application thereof in rosa roxburghii tratt variety identification

Abstract

The invention relates to the technical field of plant breeding, in particular to an SSR molecular marker and application thereof in rosa roxburghii tratt variety identification. The SSR molecular marker comprises nucleotide sequences as shown in SEQ ID NO.1-16, and the nucleotide sequences aim at eight SSR sites in sequence. The SSR molecular marker provided by the invention can be applied to rapid and accurate authenticity identification of a plurality of rosa roxburghii tratt varieties, is not influenced by environment, sampling time, tending management measures and the like, fully improves the identification efficiency of the varieties, has the advantages of convenience and rapidness in operation, accuracy in detection, low cost and the like, and has a wide application prospect. And a scientific theoretical basis is provided for evaluating roxburgh rose resources and protecting legitimate rights and interests of breeders and forest farmers from the DNA level.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to an SSR molecular marker and an application thereof in identification of roxburghii varieties. Background Art

[0002] Rosa roxburghii Tratt. is a wild plant with sweet and sour fruits. It is rich in vitamin C, vitamin P, superoxide dismutase, multiple amino acids, minerals and bioactive substances such as flavonoids. It has broad application prospects in food processing, medicine, health products and cosmetics industries. The rosa roxburghii industry has been promoted in a wide range, but the wild germplasm resources of rosa roxburghii are rich and the resources are unclear. There are many varieties including Guohong, Fentuan, Fenbian, Shaoci, Wuci, Guinong 1, Guinong 5, Guinong 8, Guinong 9, Guinong 10, Guinong 13, Guinong 14, Guinong 15, Guinong 18, Guinong 20, etc. Because these rosa roxburghii varieties have excellent traits, high commodity value, and phenotypic traits are difficult to distinguish from other germplasms, in order to prevent variety infringement, it is urgent to establish a stable and reliable rosa roxburghii variety identification system.

[0003] SSR markers have the advantages of rich polymorphism and strong stability. The molecular ID technology based on SSR markers has the advantages of rapidity, accuracy and low cost, and has been promoted and applied in the industry. At present, there are few molecular markers available for sea buckthorn, and the genome has not yet been published, which has limited the molecular breeding process of sea buckthorn. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an SSR molecular marker and application thereof in the identification of roxburghii varieties.

[0005] The invention downloads the genome of Samantha rose from NCBI, mines SSR markers, designs primers, obtains polymorphic primers after screening, and forms a primer database in combination with polymorphic primers of the roxburghii transcriptome. Further, 8 SSR sites of different repeat types with good polymorphism and high stability are screened out by capillary gel electrophoresis detection of M13 SSRPCR products. The detection results can be applied to the identification of multiple roxburghii varieties.

[0006] In a first aspect, the present invention provides an SSR molecular marker, including: Chin7, Chin17, Chin24, Chin65, Rox27, Rox28, Rox40, Rox44; Chin7 includes: F: AATATACACGAACAACAACCATCG, R: GGAACATGACCCCTTTTCTTATT; Chin17 includes: F: GATGCTTTCATTCTGCTTCAAC, R: ATTTTTACCGTACTCTGGGTGCT; Chin24 includes: F: GAGTCATGTTGAATGATATTGGC, R: TTTCCTCTTTTCTTCTTTTTCCC; Chin65 includes: F: GAGAGAGTAGCCTTTGATGAGGA, R: GAATGTCGTCAGGGAGCAGTAG; Rox27 includes: F: AGTAGTCTAGTGGTTCGACTCTTC, R: GTAGCTCGCAAGGCTCATAAC; Rox28 includes: F: TCTATACATATGACCGGCAATC, R: GTGTAGTCATTTATTTGGTACCTCC; Rox40 includes: F: ATGGATAAAGACGTGTCTAATTGTA, R: ATATATGGAATAACCGTCCTATTA; Rox44 includes: F: AATGAATACTTTGCAAGAGGG, R: GTATACGTTTACTTTGGCTCGAATA.

[0007] The fragment amplified by Chin7 as above includes 4 repeats, and the repeated nucleotide sequence is CTACA; The fragment amplified by Chin17 as above includes 6 repeats, and the repeat nucleotide sequence is TC; The fragment amplified by Chin24 as above includes 13 repeats, and the repeated nucleotide sequence is TGGA; The fragment amplified by Chin65 as above includes 7 repeats, and the repeated nucleotide sequence is CTC; The fragment amplified by Rox27 as above includes 4 repeats, and the repeated nucleotide sequence is TAT; The fragment amplified by Rox28 as above includes 3 repeats, and the repeated nucleotide sequence is ATG; The fragment amplified by Rox40 as above includes 3 repeats, and the repeat nucleotide sequence is CGG; The fragment amplified by Rox44 as above includes 4 repeats, and the repeated nucleotide sequence is AAT.

[0008] In a second aspect, the present invention provides a kit, comprising: the SSR molecular marker.

[0009] In a third aspect, the present invention provides the use of the SSR molecular marker or the kit in the identification of roxburghii varieties.

[0010] The present invention further provides the use of the SSR molecular marker or the kit in molecular marker-assisted breeding of roxburghii, cultivating excellent quality or improving germplasm resources.

[0011] In a fourth aspect, the present invention provides a method for identifying a variety of roxburghii, comprising: The genomic DNA of the roxburghii sample to be tested is extracted, and PCR amplification is performed using the SSR molecular markers, and the variety of the roxburghii sample to be tested is determined according to the PCR amplification result.

[0012] Furthermore, the procedure of the PCR amplification includes: First step PCR reaction: 93~95℃ 5~10min; 93-95°C for 30-60s, 55-65°C for 30-60s, 70-74°C for 20-40s, 25-35 cycles; 70~74℃ 5~10min; After adding fluorescent markers, the second step of PCR reaction is carried out: 93~95℃ 5~10min; 93-95°C for 30-60s, 55-65°C for 30-60s, 70-74°C for 20-40s, 13-20 cycles; 70~74℃ 5~10min.

[0013] Furthermore, the variety of the roxburghii sample to be tested is determined according to the PCR amplification result, including: The PCR amplification result and the DNA fingerprint are compared, and the variety of the tested roxburghii sample is determined according to the comparison result; the DNA fingerprint is constructed based on the amplified fragment size of the SSR molecular markers of different varieties of roxburghii.

[0014] Furthermore, the varieties include: Pink ball, Guohong, few thorns, no thorns, pink edges, Guinong No. 1, Guinong No. 5, Guinong No. 8, Guinong No. 9, Guinong No. 10, Guinong No. 13, Guinong No. 14, Guinong No. 18 or Guinong No. 20.

[0015] The present invention has the following beneficial effects: The present invention obtains 8 SSR loci through research and screening, and develops corresponding SSR molecular markers (including 8 SSR primer pairs), and the detection results based on these SSR molecular markers can realize accurate definition of roxburghii varieties. The SSR molecular markers provided by the present invention can quickly and accurately realize the identification of multiple roxburghii varieties, and have important application value in the field of roxburghii breeding. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. 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.

[0017] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0018] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0019] Example 1 1. Experimental Materials The invention adopts roxburgh varieties to carry out experiments with roxburgh varieties that have been approved and named in China as reference varieties, including Guohong, Fentuan, Fenbian, No Thorn, Less Thorn, Guinong No. 1, Guinong No. 5, Guinong No. 8, Guinong No. 9, Guinong No. 10, Guinong No. 13, Guinong No. 14, Guinong No. 18 and Guinong No. 20. The roxburgh material of the invention is derived from the roxburgh leaves required by the roxburgh germplasm resource bank in Qiannan Prefecture, Guizhou Province, and the healthy and tender young leaves are collected.

[0020] 2. Experimental process (1) Genomic DNA extraction and detection: The Tiangen kit (DP305) was used to extract the sample DNA. The integrity of the DNA was detected by 1% agarose gel electrophoresis. The purity and concentration of the DNA were detected by UV spectrophotometer. Finally, the DNA was diluted to 10 ng / µL with ddH2O for later use.

[0021] (2) SSR primer design The EST-SSR primers of Rosa roxburghii were derived from the published primer sequences. The 60 pairs of SSR primers of closely related species were designed based on the SSR loci screened from the transcriptome sequence of Samantha rose downloaded from the NCBI website. (The SSR locus screening parameters are: the repeating motif is 1~6 bp, and the minimum number of repeats of mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide are 10, 6, 3, 3, 3, 3 respectively).

[0022] (3) Polymorphic primer synthesis and screening TP-M13-SSR primers include three primers: an M13 tail (TGTAAAACGACGGCCAGT) added to the 5' end of the forward primer; a common reverse primer; and a primer with three M13 fluorescent labels: ROX (red), HEX (green), and FAM (blue).

[0023] 70 pairs of synthesized primers were used to detect the M13-SSR-PCR product capillary gel electrophoresis, and PCR amplification was performed using DNA from eight roxburghii varieties (Guohong, Fentuan, Fenbian, Wuci, Shaoci, Guinong 1, and Guinong 8) as templates using the optimized PCR program (Table 3). The amplification results were detected by 3.5% agarose gel electrophoresis, and finally 8 pairs of optimal primers (primers corresponding to Table 2) were obtained, which had the richest polymorphism and no impurity peaks.

[0024] Table 1 List of experimental synthetic primers

[0025] PCR system: PCR reaction system (25.5µL) includes: DNA 1µL, 2*Taq PCR Mix 12.5µL, F-primer 1 µL (10µM), R-primer 1µL (10µM), ddH2O 9.5µL.

[0026] The PCR program is as follows: Table 2 PCR reaction process

[0027] (4) PCR amplification and capillary electrophoresis detection The polymorphic primers obtained according to (3) are used for the second round of PCR amplification and fluorescent labels are added.

[0028] The PCR amplification system was as follows: 15 μl of the first round PCR product, 0.5 μl of 2× Taq DNA enzyme (2.5 U / μL), 0.5 μl of M13 fluorescent primer (10 μmol / L), 10× PCR Buffer (Mg 2+ free) 5μL.

[0029] The reaction procedure is as follows: Table 3 PCR reaction program

[0030] After obtaining the PCR product, 6 µL of each M13 fluorescent product with different colors was mixed into the same well for capillary electrophoresis detection. Capillary electrophoresis method: After formamide and molecular weight internal standard were mixed at a volume ratio of 100:1, 9 µL was added to the sample plate, and then 1 µL of 10-fold diluted PCR product (PCR product of 8 sets of primers mixed in the present invention) was added, and capillary electrophoresis was performed using a sequencer.

[0031] (5) Data reading and statistics The raw data exported from the capillary electrophoresis sequencer were read using GeneMarker V2.2.0 fragment analysis software, and the position of the molecular weight internal standard in each lane was compared with the position of the peak of each sample to obtain the fragment size. To ensure the accuracy of the data, it is recommended to use manual reading or manual rechecking for all experimental data. The data were read using the method recommended in Table 3. For single peak and multi-peak peak types, the main peak and the direction of numerical rounding were selected.

[0032] (6) Construction of DNA fingerprint GeneMarker V2.2.0 was used to read 8 pairs of primers for 25 roxburghii varieties of germplasm resources, and the sizes of allele fragments at different sites and the corresponding capillary electrophoresis patterns were obtained. The allele fragment length values ​​were read in sequence according to different allele sites, and the different allele variations at the corresponding sites of different varieties were recorded. The recording method is as follows: at the same allele site, the short fragment is in front and the long fragment is in the back, separated by " / ". Record in the order of sites Chin7, Chin17, Chin24, Chin65, Rox27, Rox28, Rox40, and Rox44, and the recording results of different sites are separated by spaces, and the "molecular ID card" code of roxburghii is obtained.

[0033] (7) Construction of fingerprint QR code Through the online tool of forage QR code generator, the above fingerprint coding information and the basic information of the sea buckthorn variety are generated into a QR information code, as shown below.

[0034] Table 4 Fingerprint

[0035] From the above results, it can be seen that the above 14 varieties of sea buckthorn can be accurately distinguished by the SSR primer combination provided by the present invention.

[0036] The present invention further uses the above DNA fingerprint to analyze 261 roxburghii materials (56 germplasms), and the results show that it is sufficient to correctly distinguish all roxburghii materials.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An SSR molecular marker, characterized in that: include: Chin7, Chin17, Chin24, Chin65, Rox27, Rox28, Rox40, Rox44; Chin7 includes: F: AATATACACGAACAACAACCATCG, R: GGAACATGACCCCTTTTCTTATT; Chin17 includes: F: GATGCTTTCATTCTGCTTCAAC, R: ATTTTTACCGTACTCTGGGTGCT; Chin24 includes: F: GAGTCATGTTGAATGATATTGGC, R: TTTCCTCTTTTCTTCTTTTTCCC; Chin65 includes: F: GAGAGAGTAGCCTTTGATGAGGA, R: GAATGTCGTCAGGGAGCAGTAG; Rox27 includes: F: AGTAGTCTAGTGGTTCGACTCTTC, R: GTAGCTCGCAAGGCTCATAAC; Rox28 includes: F: TCTATACATATGACCGGCAATC, R: GTGTAGTCATTTATTTGGTACCTCC; Rox40 includes: F: ATGGATAAAGACGTGTCTAATTGTA, R: ATATATGGAATAACCGTCCTATTA; Rox44 includes: F: AATGAATACTTTGCAAGAGGG, R: GTATACGTTTACTTTGGCTCGAATA.

2. A kit, characterized in that: include: The SSR molecular marker according to claim 1.

3. Use of the SSR molecular marker described in claim 1 or the kit described in claim 2 in the identification of roxburghii varieties.

4. Use of the SSR molecular marker described in claim 1 or the kit described in claim 2 in molecular marker-assisted breeding, cultivation of excellent quality or germplasm resource improvement of Rosa roxburghii.

5. A method for identifying varieties of roxburghii, characterized in that: include: Extract genomic DNA of the roxburghii sample to be tested, perform PCR amplification using the SSR molecular marker described in claim 1, and determine the variety of the roxburghii sample to be tested according to the PCR amplification result.

6. The identification method according to claim 7, characterized in that: The procedure of the PCR amplification includes: First step PCR reaction: 93~95℃ 5~10min; 93-95°C for 30-60s, 55-65°C for 30-60s, 70-74°C for 20-40s, 25-35 cycles; 70~74℃ 5~10min; After adding fluorescent markers, the second step of PCR reaction is carried out: 93~95℃ 5~10min; 93-95°C for 30-60s, 55-65°C for 30-60s, 70-74°C for 20-40s, 13-20 cycles; 70~74℃ 5~10min.

7. The identification method according to claim 5 or 6, characterized in that: The method of determining the variety of the roxburghii sample to be tested according to the PCR amplification result includes: The PCR amplification result and the DNA fingerprint are compared, and the variety of the tested sea buckthorn sample is determined according to the comparison result; the DNA fingerprint is constructed based on the amplified fragment size of the SSR molecular marker of different varieties of sea buckthorn according to claim 1.

Images