Molecular markers, primers, fingerprint and application thereof for identifying acer truncatum germplasm resources
By developing SNP molecular markers and fingerprinting of Acer palmatum germplasm resources, and utilizing high-throughput sequencing and KASP technology, the problem of difficult germplasm resource identification was solved, achieving efficient and accurate germplasm resource identification and breeding support.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU ACAD OF AGRI SCI
- Filing Date
- 2026-05-06
- Publication Date
- 2026-07-21
AI Technical Summary
The identification of Japanese maple germplasm resources in the market is difficult, especially the serious problems of "synonyms" and "different species with the same name", which affect the quality of seedlings and market order.
SNP molecular markers and fingerprints were developed for 78 Acer palmatum germplasm resources. SNP sites were identified using high-throughput sequencing technology. KASP primers and kits were designed for rapid identification using KASP genotyping technology.
It has enabled the efficient and accurate identification and differentiation of 78 species of Japanese maple germplasm resources, providing reliable germplasm resource identification and molecular marker-assisted breeding methods to ensure seedling quality.
Smart Images
Figure CN122428053A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the identification of SNP molecular markers, fingerprinting, and their applications for different Acer palmatum germplasm resources. Background Technology
[0002] Japanese maple ( Acer palmatum Japanese maple (Acer palmatum) is a plant belonging to the genus Acer in the family Sapindaceae. With its graceful shape and rich foliage colors, it is an excellent ornamental tree species for landscaping and garden decoration. Its strong adaptability and outstanding ornamental value make it suitable for both garden planting and urban roadside greening. As a native tree species that meets the requirements of scientific greening—"colorization, preciousness, and economic benefits"—it holds an important position in the construction of colorful plant landscapes in my country. Internationally, the development of germplasm resources and research on the horticultural application of Japanese maple are relatively mature. Europe, America, Japan, and other regions have reached a high level in variety selection, cultivation techniques, and landscape application, and have cultivated many horticultural varieties with excellent ornamental traits. In recent years, many domestic research institutions have also gradually carried out work on the collection, application, and innovation of Japanese maple germplasm resources.
[0003] With breakthroughs in breeding technology, the seedling market is booming. However, the market is rife with cases of "different names for the same thing" and "different things with the same name"; the phenomenon of passing off inferior products as superior ones, or even using other seedlings to "pass off as superior ones," also occurs frequently.
[0004] KASP genotyping technology enables high-throughput, low-cost, rapid, and convenient verification. The screened KASP markers can be applied to germplasm resource identification and marker-assisted breeding. While KASP technology has been widely used in marker-assisted selection breeding of various crops, it has not yet been reported in *Acer palmatum*. By collecting and organizing core germplasm resources of *Acer palmatum*, developing new core SNP molecular markers for germplasm identification, and constructing fingerprint maps, germplasm resource tags can be established for horticultural varieties of *Acer palmatum*. Summary of the Invention
[0005] This invention addresses the aforementioned technical problems by providing a simple molecular marker method for the identification of 78 different germplasm resources of *Acer palmatum*. Specifically, this invention provides the following technical solution: In a first aspect, the present invention provides a combination of SNP molecular markers for identifying or assisting in the identification of Acer palmatum germplasm resources, characterized in that the positions of the SNP molecular markers on the chromosome are arranged in the following order as shown in the table below: .
[0006] This invention utilizes high-throughput sequencing to identify eight SNP loci within the whole genome of Acer palmatum that can be used to identify or assist in the identification of Acer palmatum germplasm resources. These SNP loci can be used to distinguish between specific different germplasm resources, or they can be used to form fingerprint maps for the identification of 78 species of Acer palmatum.
[0007] Furthermore, the sequences of the aforementioned SNP sites are shown in SEQ ID NO. 1 to SEQ ID NO. 8, and the SNP is located at position 251 of any sequence in SEQ ID NO. 1 to SEQ ID NO. 8. .
[0008] On the other hand, a fingerprint spectrum for identifying Acer palmatum germplasm resources is characterized in that the fingerprint spectrum is composed of the aforementioned SNP molecular markers, the sequences of which are shown in SEQ ID NO. 1 to SEQ ID NO. 8, and the SNP is located at position 251 of SEQ ID NO. 1 to SEQ ID NO. 8.
[0009] Those skilled in the art will understand that the fingerprint pattern described in this invention is not composed of continuous bases, but rather of genotypes of SNPs dispersed at different sites on different chromosomes. Therefore, the genotypes of the eight SNP sites of different germplasm resources in this invention can form different genotype barcodes, and the fingerprint pattern can also be considered to be composed of genotype barcodes of different germplasm resources of Acer palmatum.
[0010] On the other hand, the present invention provides KASP primers for detecting the aforementioned SNP molecular markers, the primer sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.32.
[0011] On the other hand, the present invention provides a kit for identifying or assisting in the identification of Acer palmatum germplasm resources, the kit comprising primers and / or probes for detecting the aforementioned SNP molecular markers and / or fingerprint patterns.
[0012] On the other hand, another object of the present invention is to provide any of the following applications of the above-mentioned SNP molecular markers, fingerprints, primers, and kits: (1) Application in identifying Acer palmatum germplasm resources; (2) Application in the identification, improvement or molecular marker-assisted breeding of Acer palmatum germplasm resources; (3) Application in screening or creating different Acer palmatum germplasm resources; (4) Application in constructing a DNA fingerprint database of Japanese maple.
[0013] On the other hand, another object of the present invention is to provide a method for identifying or assisting in the identification of Acer palmatum germplasm resources, characterized in that it includes: (1) Extract total DNA from the Acer palmatum sample to be identified; (2) Detect the genotype of the aforementioned SNP markers; (3) Determine the germplasm resources of the Acer palmatum sample to be identified based on the test results.
[0014] Optionally, step 2) can be performed by sequencing or KASP or any other available genotyping technique to detect the genotype of each base of the DNA barcode.
[0015] The specific judgment method for step 3) is as follows: if the genotype of the Acer palmatum sample to be identified is consistent with the genotype of any germplasm resource in the aforementioned fingerprint spectrum, it is judged as the corresponding Acer palmatum germplasm resource.
[0016] On the other hand, the present invention provides any of the following applications of the aforementioned method: (1) Application in identifying Acer palmatum germplasm resources; (2) Application in the identification, improvement or molecular marker-assisted breeding of Acer palmatum germplasm resources; (3) Application in screening or creating different Acer palmatum germplasm resources; (4) Application in constructing a DNA fingerprint database of Japanese maple.
[0017] This invention offers the following advantages: it develops novel SNP molecular markers, which can be used to differentiate between specific germplasm resources and to form fingerprint profiles for the identification of 78 species of Japanese maple. This provides assurance for the identification, resource utilization, and breeding of Japanese maple. It also has significant implications for molecular marker breeding of Japanese maple. Attached Figure Description
[0018] The present invention and its beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.
[0019] Figure 1 This is a fingerprint spectrum plotted from the KASP test results of 78 samples to be tested. Detailed Implementation
[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0021] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The Japanese maple plant samples used in the following embodiments were all obtained from the Jiangsu Academy of Agricultural Sciences' Maple Seed Base; similar samples can also be purchased commercially.
[0022] Example 1 Resequencing Analysis 1. Experimental materials The sources and information of the materials are shown in Table 1: Table 1. Sources and Information of Materials 2. Sample DNA extraction, library construction, and sequencing First, the leaves of each sample were preserved using liquid nitrogen. Genomic DNA was extracted from samples 1 to 78 using a kit and sequenced using whole-genome resequencing technology.
[0023] Specific experimental steps: Library construction was initiated with 1 μg of DNA; High-quality genomic DNA was extracted using the CTAB method; 0.75% agarose gel electrophoresis was used to detect DNA fragment size and the degree of DNA degradation. The NanoDrop One spectrophotometer (Thermo Fisher Scientific) was used to detect DNA purity, with an OD260 / 280 ratio between 1.8 and 2.2, indicating no protein or visible contaminants. The Qubit 3.0 fluorescence analyzer (Life Technologies, Carlsbad, CA, USA) was used to detect DNA concentrations greater than 50 ng / μl and total amounts greater than 2 μg.
[0024] After DNA was broken down by sonication with a Covaris M220, magnetic beads were used for fragment selection, resulting in sample bands concentrated between 200-400 bp. The qualified libraries were then put into the sequencing machine for sequencing.
[0025] 3. Data quality control The raw sequencing reads were filtered using the FastP software (v0.23.4).
[0026] 4. Data comparison and SNP identification In this invention, we use the genome of the Japanese maple 'Jinling Yellow Maple' as the reference genome, use BWA alignment software to align the sequencing fragments back to the reference genome, and then use Picard-tools to remove the sequencing fragments generated by PCR-duplication.
[0027] For each sample, a bwa (version: 0.7.17; parameter: mem) alignment analysis was performed. The filtered clean reads were aligned to the reference genome, and the alignment results were statistically analyzed.
[0028] Genetic variations such as SNPs and InDel were identified using the HaplotypeCaller module of the genome analysis tool GATK (v4.2.2.0).
[0029] Example 2: Development of core SNPs for identifying 78 Acer palmatum germplasm resources 1. Identification of core SNPs Based on the grouping of 78 Acer palmatum accessions, SNP combinations that distinguish the 78 Acer palmatum germplasm resources were selected. To reduce false positives for SNPs, the following analytical criteria were adopted: 1. Locus detection rate = 100%; 2. MAF > 0.15; 3. Heterozygosity < 0.4; 4. Redundant markers removed; 5. No other SNPs within 30 bp before and after the selected SNP; 6. GC content: GC content analysis of SNP loci to avoid selecting loci with excessively high or low GC content. The GC content within 150 bp before and after the selected SNP locus should be 40%-60%; 7. PIC >= 0.2; 8. Locus polymorphism >= 0.4; 9. No more than 8 consecutive single-base repeats within 30 bp before and after the SNP locus; 10. No homology between the SNP locus and other locations in the genome within 50 bp before and after the SNP locus.
[0030] Finally, eight SNPs were selected. The genotype combinations of these different SNPs constituted the fingerprint pattern, as shown in Table 2 below (NN indicates that there is no detection signal at this locus).
[0031] Table 2 Genotypes of 78 Japanese maple samples Example 3: KASP primer design and molecular marker verification 1) Extract total DNA from the sample to be tested; 2) Primer and probe design; Based on the SNP sites identified in Example 1, KASP primers were designed based on chromosome sequences. The primer sequences are shown in Table 3. 3) Using the primers and probes designed in step 2), the qualified DNA extracted in step 1) is subjected to KASP genotyping. The primers and combinations are then prepared into a detection kit.
[0032] 4) Determine the germplasm resources of the sample based on the mass spectrometry results.
[0033] Table 3 KASP primers See results Figure 1 , Figure 1 The fingerprint profiles showed that the genotypes of the 78 germplasm resources were completely different, and the 8 SNP markers could completely distinguish the 78 samples.
[0034] In summary, the fingerprint spectrum prepared by this invention can be used for the accurate identification of 78 germplasm resources.
[0035] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A combination of SNP molecular markers for the identification of Acer palmatum germplasm resources, characterized in that, The sequence of the SNP is shown in SEQ ID NO. 1 to SEQ ID NO. 8, and the SNP is located at position 251 of any sequence in SEQ ID NO. 1 to SEQ ID NO.
8.
2. A fingerprint pattern for identifying Acer palmatum germplasm resources, characterized in that, The fingerprint spectrum is composed of the SNP molecular marker combination described in claim 1.
3. A KASP primer set for detecting the SNP molecular marker combination of claim 1 or constructing the fingerprint spectrum of claim 2, characterized in that the sequence of the primer set is shown in SEQ ID NO.9~SEQ ID NO.
32.
4. A kit for identifying Acer palmatum germplasm resources, the kit comprising the primer set as described in claim 3.
5. Any of the following applications of the SNP molecular marker combination of claim 1, the fingerprint spectrum of claim 2, the primer set of claim 3, and / or the kit of claim 4: (1) Application in the identification of Acer palmatum germplasm resources; (2) Application in the improvement of Acer palmatum germplasm resources or molecular marker-assisted breeding; (3) Application in screening or creating different Acer palmatum germplasm resources; (4) Application in constructing a DNA fingerprint database of Japanese maple.
6. A method for identifying germplasm resources of Acer palmatum, characterized in that, include: (1) Extract total DNA from the Acer palmatum sample to be identified; (2) Detect the genotype of the SNP molecular marker combination described in claim 1; (3) Determine the germplasm resources of the Acer palmatum sample to be identified based on the test results.
7. The method as described in claim 6, characterized in that, Step 2) The genotype of the SNP molecular marker described in claim 1 can be detected by sequencing or KASP or any other available genotyping technique.
8. Any of the following applications of the method according to any one of claims 6 to 7: (1) Application in the identification of Acer palmatum germplasm resources; (2) Application in the improvement of Acer palmatum germplasm resources or molecular marker-assisted breeding; (3) Application in screening or creating different Acer palmatum germplasm resources; (4) Application in constructing a DNA fingerprint database of Japanese maple.