Method for constructing ginger molecular identity card and variety molecular identification based on SSR (Simple Sequence Repeat)
Through SSR primer-based PCR amplification and fluorescence capillary electrophoresis analysis, a ginger molecular identity card was constructed, which solved the problem of ginger variety identification, achieved accurate variety differentiation and clarified genetic relationships, and supported the protection of new varieties.
Patent Information
- Application Number
- CN202510780961.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-12
- Publication Date
- 2025-10-14
AI Technical Summary
Existing technologies make it difficult to effectively distinguish and identify ginger varieties, especially due to the difficulty in variety identification and the mixing of germplasm resources due to the characteristics of asexual reproduction and insufficient in-depth genome research.
Five pairs of fluorescently labeled SSR primers were used to amplify ginger genomic DNA by PCR, and the allele information was analyzed by fluorescence capillary electrophoresis to construct a ginger molecular identity card, and digital sequences and QR codes were used for variety identification.
It has achieved accurate identification of ginger varieties, can significantly distinguish the genetic differences between different varieties, provide a theoretical basis for the protection of new varieties, and improve the efficiency and accuracy of variety identification.
Smart Images
Figure CN120776027A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biotechnology, in particular to a method for constructing a ginger molecular identity card and molecular identification of varieties based on SSR. BACKGROUND
[0002] Ginger (Zingiber officinale Roscoe) originates from tropical rainforest areas and is a perennial plant of Zingiberaceae and Zingiber. There are many varieties of ginger, and typical local varieties include 'Laizhou large ginger', 'Laizhou small ginger', 'Sichuan bamboo root ginger' and 'Zhangliang ginger'. However, since ginger is a vegetative propagation crop, some local varieties formed by introduction and breeding in different regions may be the same variety. Molecular marker assisted selection can select the genotype of the target trait through molecular markers closely linked to the target trait. SSR (simple sequence repeat) has become the preferred marker for genetic diversity research, variety identification, molecular mapping, marker assisted selection and QTL analysis due to its co-dominant inheritance, high genome coverage and non-coding region that remains conservative during evolution. At present, there are few reports on SSR markers of ginger, which is related to the characteristics of vegetative propagation and insufficient research on the genome sequencing. Therefore, it is an important prerequisite for molecular marker assisted breeding to use ginger genome data to screen and identify SSR markers with high polymorphism of ginger and establish a molecular identity card for ginger. Therefore, it is necessary to develop a method for rapidly identifying ginger varieties and construct a molecular identity card for ginger varieties with different regional characteristics. SUMMARY
[0003] The present application aims to provide a method for constructing a ginger molecular identity card and molecular identification of varieties based on SSR, which can solve the problems of great difficulty in identifying existing ginger varieties and mixed germplasm resources.
[0004] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a method for constructing a ginger molecular identity card based on SSR, comprising the following steps:
[0005] extracting ginger genomic DNA;
[0006] performing PCR amplification on the ginger genomic DNA using 5 pairs of fluorescently labeled SSR primers, wherein the 5 pairs of SSR primers are G101, G102, G103, G104 and G105;
[0007] adopting fluorescent capillary electrophoresis to analyze the allelic information of each SSR site and convert it into a digital sequence;
[0008] constructing a ginger molecular identity card based on the digital sequence.
[0009] Preferably, the PCR amplification system is: 60 ng muL-1 template DNA 1 muL, 2x SanTaq PCR Mix 10 muL, 0.5 muL of each forward and reverse primer, and 8 muL of ddH2O.
[0010] Preferably, the PCR reaction conditions are: 95 DEG C pre-denaturation for 5 min; 95 DEG C pre-denaturation for 30 s, 60 DEG C annealing for 30 s, 72 DEG C extension for 1 min, a total of 30 cycles.
[0011] Preferably, if the length difference of the PCR products is large, the same fluorescent label is used; if the length difference of the products is small, different fluorescent labels are used.
[0012] Preferably, 5 pairs of SSR markers are arranged in a fixed order, a string is generated based on the coded digital sequence, and the string is converted into an identity two-dimensional code through two-dimensional code generation software.
[0013] Preferably, the SSR primer pair can further include G104R and G104F.
[0014] Most preferably, the SSR primer pair can further include G105R and G105F.
[0015] The average PIC value of 5 pairs of ginger SSR primers is 0.818, and the polymorphism is high.
[0016] Furthermore, different SSR sites are arranged according to molecular weight, and the same site is marked as 1 with a peak and 0 without a peak.
[0017] A molecular identification method of a ginger variety, comprising the following steps:
[0018] First, the genomic DNA of the ginger to be identified is extracted;
[0019] 5 pairs of SSR primers are used for PCR amplification and fluorescent capillary electrophoresis analysis;
[0020] The obtained allele coding sequence is compared with the molecular identity card of the known variety to determine the identity of the variety to be identified.
[0021] The obtained molecular identity card of the known ginger variety is compared, if there is a consistent case, it is determined as the same variety, if it is inconsistent, it is determined as different varieties; the molecular identity card obtained for the unknown variety is compared with the known variety to determine whether it is a known variety, if it is a new variety, it can be named and preserved.
[0022] Compared with the prior art, the present application has the following advantages:
[0023] 1) Ginger varieties are often classified based on morphological differences, but the same variety can grow differently in different regions, leading to the possibility that known local varieties may be the same. Molecular IDs, based on DNA, carry genetic information and are unaffected by environmental factors, allowing accurate identification of ginger varieties.
[0024] 2) The ginger molecular ID constructed based on SSR can significantly distinguish the genetic differences between different ginger varieties, and at the same time clarify the genetic evolutionary relationship of ginger; the obtained ginger molecular ID is accurate and specific, which can provide a theoretical basis and technical support for the protection of new varieties.
[0025] 3) Converting the digital code of the ginger molecular ID card into a QR code is conducive to improving the efficiency of machine acquisition and thus realizing the rapid identification of different ginger varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 The amplification site map of 'Laiwu ginger' on primer G101 in Example 1 of the present invention
[0027] Figure 2 This is a map of the amplification sites of the five pairs of primers used in Example 2 of the present invention for 'Myanmar Ginger': DETAILED DESCRIPTION
[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.
[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0030] Example 1 takes the construction of molecular identification cards for 10 varieties including 'Laiwu ginger' as an example to demonstrate the efficient identification effect of the three pairs of SSR primers provided by the present invention.
[0031] The specific steps are as follows:
[0032] 1) Genomes of 10 ginger varieties, including 'Laiwu Big Ginger', 'Laiwu Small Ginger', 'Shannong No. 1', 'Zhangliang Ginger', 'Zhugen Ginger', 'Qianwei Small Yellow Ginger', 'Guizhou Small Yellow Ginger', 'Luoping Small Yellow Ginger', 'Myanmar Ginger', and 'Henan Huai Ginger', were extracted using the NoviZan Plant Genome Extraction Kit (DC104). The genomic DNA quality was tested by 1% agarose gel electrophoresis and the concentration of the nucleic acid was determined using a Nanodrop one nucleic acid analyzer (Thermo Scientific).TM )Detecting DNA concentration, diluting DNA concentration to 60 ng / μL with ddH2O -1 , and storing in -20℃ refrigerator.
[0033] 2) 3 pairs of fluorescently labeled SSR primers including G101R, G101F; G102R, R102F; G103R, G103F were synthesized by Shanghai Biotech Co., Ltd. Primer sequences and SSR types are shown in Table 1.
[0034] Table 1 5 pairs of SSR primer types
[0035]
[0036] 3) Using 3730XL DNA analyzer for PCR amplification. Using 20 μL PCR amplification system to amplify SSR sites: 60 ng / μL -1 template DNA 1 μL, 2x SanTaq PCR Mix (TRAN, AS122-11) 10 μL, 0.5 μL of forward and reverse primers respectively, and 8 μL of ddH2O.
[0037] 4) PCR reaction conditions are as follows: 95℃ pre-denaturation for 5 min; 95℃ pre-denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles.
[0038] 5) Constructing ginger molecular identity card, see Table 2.
[0039] Table 2 Molecular identity cards of 10 varieties constructed using 3 pairs of primers
[0040]
[0041]
[0042] The above results show that using 3 pairs of primers in the first group, 10 varieties have differences at different sites, and using 3 pairs of primers can achieve the purpose of identifying part of the varieties.
[0043] Example 2
[0044] Using 5 groups of phenotypically similar ginger varieties to construct SSR molecular identity cards and perform variety identification, and exhibiting the distinguishing effect of the molecular identity card of the application.
[0045] The specific steps are as follows:
[0046] 1) Extract the genomes of 10 varieties of ginger, including 'Myanmar ginger', 'Fat child', 'Flat top four', 'Anqiu big ginger', and 'Laiwu big ginger', using the plant genome extraction kit (DC104) of Novozyme. Use 1% agarose gel electrophoresis to detect the quality of genomic DNA, use the Nanodrop one nucleic acid concentration meter (Thermo Scientific TM ) to detect the DNA concentration, and use ddH2O to dilute the DNA concentration to 60 ng / μL -1 , and store it in a -20℃ refrigerator.
[0047] 2) Five pairs of fluorescently labeled SSR primers, including G101R, G101F; G102R, R102F; G103R, G103F; G104R, G104F; and G105R, G105F, were synthesized by Shanghai Sangon Biotech Co., Ltd.
[0048] 3) PCR amplification was performed using a 3730XL DNA analyzer. A 20 μL PCR amplification system was used to amplify the SSR site: 60 ng / μL -1 template DNA 1 μL, 2×SanTaq PCR Mix (TRAN, AS122-11) 10 μL, 0.5 μL of forward and reverse primers, and 8 μL of ddH2O.
[0049] 4) The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ pre-denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles.
[0050] 5) Construct a molecular identity card for ginger, as shown in Table 3.
[0051] Table 3 Comparison of molecular identity cards of ginger varieties with similar phenotypes
[0052] Variety Molecular ID Laiwu ginger 110100010010110010100111 Myanmar ginger 011110101100111010010000 Fat child 110110000110111010110000 Flat top four 111100010010111010100011 Anqiu ginger 011000010011111010110011
[0053] The five varieties with similar phenotypes were identified, and the five pairs of primers could significantly distinguish each variety. The constructed molecular identity card had strong distinguishing ability for varieties with similar phenotypes.
[0054] A molecular identification method for a ginger variety, which first extracts the genomic DNA of the ginger to be identified;
[0055] PCR amplification and fluorescence capillary electrophoresis analysis are carried out by using 5 pairs of SSR primers; the obtained allele coding sequence is compared with the molecular identity card of known varieties, and the identity of the variety to be identified is judged. The obtained molecular identity card of known ginger varieties is compared, if there is a consistent situation, it is judged as the same variety, if it is not consistent, it is judged as different varieties; the obtained molecular identity card of unknown varieties is compared with known varieties, and whether it is a known variety is judged, if it is a new variety, it can be named and preserved. The ginger molecular identity card constructed based on SSR can significantly distinguish the genetic differences of different ginger varieties, and at the same time, the genetic evolution relationship of ginger can be determined; the obtained ginger molecular identity card has the characteristics of accuracy and specificity, and can provide theoretical basis and technical support for new variety protection; the digital coding of the ginger molecular identity card is converted into a two-dimensional code, which is conducive to improving the efficiency of machine acquisition, and then realizing the rapid identification of different ginger varieties.
[0056] Although the embodiments of the present application are described in conjunction with the drawings, various modifications and changes can be made by those skilled in the art without departing from the spirit and scope of the present application, and such modifications and changes fall within the scope defined by the appended claims.
Claims
1. A method for constructing a ginger molecular ID card based on SSR, characterized in that, The following steps are involved: Extract ginger genomic DNA; PCR amplification of the ginger genomic DNA was performed using 5 pairs of fluorescently labeled SSR primers, wherein the 5 pairs of SSR primers were G101, G102, G103, G104, and G105; Fluorescence capillary electrophoresis was used to analyze the allele information of each SSR locus and convert its code into a digital sequence; A ginger molecular identity card is constructed based on the digital sequence.
2. A method for constructing a ginger molecular ID card based on SSR according to claim 1, characterized in that, The PCR amplification system is: 60ngμL -1 Template DNA 1 μL, 2× SanTaq PCR Mix 10 μL, forward and reverse primers 0.5 μL each, ddH2O 8 μL.
3. A method for constructing a ginger molecular ID card based on SSR according to claim 1, characterized in that, The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; pre-denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, for a total of 30 cycles.
4. A method for constructing a ginger molecular ID card based on SSR according to claim 1, characterized in that: If the PCR products have large differences in length, use the same fluorescent marker; if the products have small differences in length, use different fluorescent markers.
5. A method for constructing a ginger molecular ID card based on SSR according to claim 1, characterized in that: Arrange 5 pairs of SSR markers in a fixed order, generate a character string based on the encoded digital sequence, and convert the character string into an identity QR code using QR code generation software.
6. A molecular identification method for ginger varieties, characterized in that: The following steps are involved: First, the genomic DNA of the ginger to be identified was extracted; PCR amplification and fluorescence capillary electrophoresis analysis were performed using five pairs of SSR primers; The obtained allele coding sequence is compared with the molecular ID of the known variety to determine the identity of the variety to be identified.
7. The molecular identification method for ginger varieties according to claim 6, characterized in that: When three pairs of SSR primers G101, G102, and G103 are used, 66 ginger varieties can be distinguished; when four pairs of primers are used, 78 varieties can be distinguished; and when five pairs of primers are used, 112 varieties can be distinguished.