DNA fingerprint spectrum for identifying feeding oat variety and application of DNA fingerprint spectrum
By constructing DNA fingerprint maps of 21 SNP sites in oat varieties, and combining primers and gene chips, the problems of time-consuming traditional identification methods and difficulty in distinguishing phenotypic similar varieties have been solved, enabling rapid and accurate identification and management of oat varieties.
Patent Information
- Application Number
- CN202511680162.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-11-18
- Filing Date
- 2025-11-17
- Publication Date
- 2026-03-17
AI Technical Summary
Traditional morphological methods for identifying oat varieties are cumbersome, time-consuming, and difficult to distinguish between phenotypic varieties. A fast and accurate identification method is needed.
DNA fingerprinting of oat varieties was constructed using 21 SNP sites based on the OT3098v2 reference genome. Combined with primer combinations, molecular probe combinations, and gene chips, rapid identification was achieved through genomic DNA comparison.
It enables rapid, accurate, and low-cost identification of oat varieties, and is applicable to variety screening, traceability, breeding, and germplasm resource management, improving identification efficiency and accuracy.
Smart Images
Figure CN121674597A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to the field of biological detection technology, and more specifically to SNP combinations for identifying feed oat varieties and their applications. Background Technology
[0002] oat( Avena sativa Oat grass (L.) is an annual herbaceous plant belonging to the Poaceae family. Its seeds are highly nutritious, containing abundant dietary fiber and antioxidants, which help lower postprandial blood sugar. Oat grass can increase milk yield and quality, making it an important component of dairy cow diets. In recent years, oats have gained attention due to their rich nutritional value, and research on the breeding of new oat varieties has developed rapidly. There is an urgent need for a method to quickly identify new oat varieties for timely confirmation and protection. Traditional morphological identification methods, which identify different varieties based on phenotypic differences, are intuitive and reliable, but the process is cumbersome, time-consuming, and difficult to distinguish between varieties with similar phenotypic traits. DNA molecular markers can identify different varieties by utilizing differences at the DNA molecular level. Among them, DNA fingerprinting is widely used for individual identification due to its multi-site nature, high variability, and simple and stable heritability. This is a valuable genetic marker for studying the genetic structure, ecology and evolution, and classification of plant and animal populations.
[0003] In view of this, the present invention provides a DNA fingerprint pattern capable of identifying feed oat varieties. Summary of the Invention
[0004] To meet the current needs for oat variety identification in oat variety research and agricultural production, this invention provides a DNA fingerprint spectrum for identifying feed oat varieties and its application. Using the DNA fingerprint spectrum information provided by this invention, oat varieties can be identified quickly and accurately, and can be used for variety screening, variety tracing, and breeding. It is beneficial for germplasm resource collection, protection, and improvement, and is time-saving, low-cost, and has broad market benefits.
[0005] To achieve the technical objective of this invention, the following technical solution is provided: In a first aspect, the present invention provides a DNA fingerprint for identifying feed oat varieties, which includes 21 SNP loci, the physical locations and polymorphisms of which are shown in Table 1:
[0006] Specifically, the sequences are shown in SEQ ID NO.1-SEQ ID NO.21, where the positions represent specific SNP site information. Table 1. Location and sequence information of the 21 site combinations
[0007]
[0008] This SNP was determined based on the OT3098v2 reference genome, which can be downloaded at: https: / / wheat.pw.usda.gov / GG3 / sites / default / files / data_downloads / PepsiCo_OT3098_V2_panoat_nomenclature.fasta.gz.
[0009] The second aspect provides a method for identifying oat varieties, which compares the genotypes of 21 SNP loci in the genomic DNA of the oat variety to be tested with the fingerprint pattern formed by the 21 SNP loci. The 21 SNP sites are those listed in Table 1.
[0010] The third aspect provides primer combinations for identifying oat varieties, which are capable of amplifying the SNP site sequences described in Table 1 in the sample to be tested.
[0011] The fourth aspect provides a molecular probe combination for identifying oat varieties, which can detect the SNP site combinations as shown in Table 1 in the sample to be tested.
[0012] The fifth aspect provides a gene chip for identifying oat varieties, said gene chip being loaded with the aforementioned primer combination or molecular probe combination.
[0013] The sixth aspect provides a kit for identifying oat varieties, which has the aforementioned primer combination or molecular probe combination or gene chip.
[0014] The seventh aspect provides a method for identifying oat varieties, using the aforementioned primer combinations, molecular probe combinations, gene chips, or kits to detect the samples to be tested.
[0015] The eighth aspect provides the application of DNA fingerprinting for identifying feed oat varieties in any of the following: (A1) Identification or auxiliary identification of feed oat varieties; (A2) Identification or auxiliary identification of oat germplasm resources for feed; (A3) Breeding of feed oats.
[0016] Beneficial effects The DNA fingerprinting method for identifying feed oats provided by this invention enables the identification of multiple varieties with minimal markers, achieving a simple, efficient, and economical approach. It plays a crucial role in areas such as oat crop pedigree and genetic analysis, variety approval, identification and management of counterfeit seeds, genetic testing, and crop mapping. Attached Figure Description
[0017] Figure 1 This refers to the discriminative power of the various quantity gradient marker sets provided in Embodiment 1 of the present invention; Figure 2 This invention provides a DNA fingerprint for identifying feed oats. Detailed Implementation
[0018] The present invention will be further illustrated below with reference to a detailed description of specific embodiments. However, these embodiments are merely illustrative and should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and can be referred to the third edition of the original book "Bioinformatics and Functional Genomics" or related books. The bioinformatics software and products used are also commercially available. Various processes and methods not described in detail are conventional methods known in the art. The source of materials used, trade names, and components that need to be listed are indicated upon their first appearance. Unless otherwise specified, the same reagents used thereafter are considered the same as those initially indicated.
[0019] Furthermore, it should be noted that the site combinations and applications provided by this invention are the result of the inventors' arduous creative work and optimization efforts.
[0020] Example 1: Obtaining the fingerprint spectrum of oats 1. Test materials
[0021] To achieve more comprehensive coverage of oat varieties both domestically and internationally, this invention conducts genotyping and gene analysis on 80 oat varieties (lines) worldwide to construct a fingerprint database of oat varieties (lines). The varieties are shown in Table 2: Table 2. Oat varieties (lines) for feed
[0022]
[0023] It should be noted that some of the above varieties do not yet have Chinese translations.
[0024] Of course, in one embodiment of the present invention, more feed oat varieties can be collected for fingerprint mapping. As long as the fingerprint map is obtained using the method and idea of the present invention, it is within the protection scope of the present invention.
[0025] 2. DNA extraction, detection, and sequencing DNA was extracted from 80 oat varieties in step 1, and the DNA sequence of each oat variety was analyzed. Sequencing methods used were all conventional in the art, and this invention does not impose any limitations.
[0026] 3. Sample screening and data quality control After basic quality control and removal of redundant sites from the genotype data, the IBS distance between two samples was calculated using Plink (V1.90) software, and highly similar sample pairs were identified based on the calculation results. The criteria for determining highly similar samples are as follows: when the DST between samples is ≥0.99, they are judged as highly similar samples, where DST is the probability of homomorphism between two individuals at the genomic level. When highly similar samples are found, only one sample from each group is retained for subsequent analysis. No highly similar samples were found in this embodiment of the invention.
[0027] It should be noted that sample screening and data quality control are conventional methods in the field, and this invention does not impose any limitations.
[0028] To ensure the quality of the genotype data used for analysis and the validity of the results, Plink (V1.90) software was used to perform quality control on the samples and SNP loci before analysis. The quality control criteria were as follows: a. Sample detection rate >= 0.9; b. Only loci on chromosomes were retained; c. Only loci without deletions were retained; d. Only SNP loci were retained; e. Minimum allele frequency >= 0.3. After quality control, 80 samples and 1,200,269 loci remained. Based on this, Plink (V1.90) software was used to remove strongly linked loci, resulting in a final number of 65,716 loci.
[0029] 4.3 Tag Filtering Based on the core marker screening method proposed by Yang et al. (2022) (see https: / / doi.org / 10.1186 / s12870-022-03920-2), core SNP marker combinations were screened. While ensuring that chromosomes were evenly distributed, different numbers of marker sets were randomly selected from the remaining loci, and this was repeated several times to generate SNP marker combinations. The discriminative power of each marker combination was then calculated, which is the ratio of the number of samples with unique genotypes to the total number of samples. In this study, marker sets ranging from 1 to 21 were selected, each 100 times. The marker combination with the highest discriminative power from each set was then selected to represent the discriminative power for that number of samples. The discriminative power of marker sets at each size gradient is shown in Table 2. Figure 1 .
[0030] Table 2. Discriminative power of different numbers of labeled sets
[0031] Although 12 marker sets are sufficient to completely distinguish these 80 materials, it is recommended to retain one marker on each chromosome to increase the discriminative power between samples, i.e., use 21 marker sets as core SNP markers.
[0032] 4.4 Fingerprint Map Construction The combination with the highest discriminative power in the 21-marker set was used as the core SNP marker for fingerprint mapping. The construction results are as follows: Figure 2 As shown in the figure, one row represents one sample and one column represents one label. The position and sequence of each label are shown in Table 1 of the instruction manual, and will not be repeated here.
[0033] Genetic similarity analysis of fingerprint profiles of experimental cases Based on 21 core SNP markers, the pairwise genetic similarity between 80 materials was calculated. The formula for calculating genetic similarity is as follows: GS = nij / Nij Where nij represents the number of markers detected in both materials and with different genotypes in the two materials, and Nij represents the number of markers detected in both materials. See Table 3 for the genetic similarity calculation results between some materials: Table 3. Results of genetic similarity analysis of some materials
[0034] Note: ID1: Sample name of the first material; ID2: Sample name of the second material; Identical_count: Number of markers with the same genotype; Total_count: Number of markers examined in both materials; GS: Genetic similarity.
[0035] As can be seen from the analysis results in Table 3, no two materials are similar, indicating that the fingerprint spectrum provided by this invention has a high recognition rate.
[0036] Example 2: Combining SNP sites from fingerprint patterns to prepare primer and probe combinations. Those skilled in the art can design primers based on the sequence information of the 21 sites in Table 1 provided by the present invention, and conduct secondary structure evaluation and Tm value evaluation on the designed primers to finally obtain primers with good specificity, high sensitivity, and the ability to achieve the detection purpose under the same reaction conditions.
[0037] The secondary structure assessment and Tm value assessment can be performed using any method commonly used in the field. For example, the secondary structure can be assessed using DNA folding form (see http: / / unafold.rna.albany.edu / ?q=mfold / DNA-Folding-Form), and the Tm value can be assessed using the software RaW-Probe.
[0038] The above methods are all conventional methods. Based on the site information in the SNP site combination provided in this application, they can be obtained without any creative effort. Therefore, the primers obtained by the SNP site combination provided in this invention also fall within the protection scope of this invention.
[0039] Similarly, probes prepared using the SNP site combinations provided by this invention, such as the Tanqman probe, also fall within the scope of protection of this invention.
[0040] Example 3: Combining SNP sites from fingerprint patterns for gene chip preparation The SNP gene chip of the present invention uses conventional methods to fix the primers or probes obtained in Example 2 onto a polymer substrate, such as a nylon membrane, nitrocellulose membrane, plastic, silicone wafer, micro magnetic beads, etc., or fix the probes onto a glass plate, or directly synthesize the primers or probes obtained in Example 2 on a hard surface such as glass. The method of using the SNP gene chip of this application is the same as the conventional method.
[0041] It should be noted that those skilled in the art can prepare SNP detection gene chips using any of the methods described herein, or they can entrust a biotechnology company to prepare them. However, gene chips prepared based on the SNP site combinations in the fingerprint pattern provided in this application are all within the protection scope of this invention.
[0042] Example 4 Analytical kit for identifying feed oat varieties The detection kit for identifying feed oat varieties provided in this application includes primers, probes, or gene chips obtained based on the SNP site combinations obtained in Example 2. Depending on the type of use, it also includes corresponding detection reagents. For example, when the SNP site combination obtained based on Example 1 is a TaqMan probe, it also includes buffers, ligases, AceQ Universal U+ Probe Master Mix V2, TaqMan Probe, etc., commonly used in real-time PCR reactions.
[0043] Those skilled in the art can configure different test kits for identifying feed oat varieties depending on the usage method, but all test kits configured based on the SNP site combinations provided in this application are within the protection scope of this invention.
[0044] Example 5: Identification of feed oat varieties Based on the SNP locus combination in the fingerprint spectrum for identifying feed oat varieties provided in Example 1 of this application, known feed oat varieties are detected, specifically as follows:
[0045] DNA from the sample variety was collected using conventional methods. The primers or probes provided in Example 2 were used to detect the DNA in the sample variety, yielding detection results for 21 loci. The detection results were then compared with... Figure 2 The provided fingerprint patterns were compared to obtain the oat variety analysis results.
[0046] Specifically: Samples 1, 2, 3, 4, and 5 are known to be the varieties Titan, Shooter, Charism, Haymaker, and Everleaf26, respectively. DNA was collected from seeds of samples 1-5, and the SNP combinations provided in this invention were used to detect the collected DNA samples. The genotypes of samples 1, 2, 3, 4, and 5 are shown in Table 4. Table 4. Typing results of the tested samples
[0047] The typing results show that samples 1, 2, 3, 4, and 5 have different SNPs at 21 loci, indicating that samples 1-5 are different oat varieties. Further comparison with the fingerprint information provided in this application confirmed that sample 1 is the Titan variety, sample 2 is the Shooter variety, sample 3 is the Charism variety, sample 4 is the Haymaker variety, and sample 5 is the Everleaf26 variety.
[0048] It should be noted that, given that this application has already disclosed specific varieties and SNP polymorphisms, those skilled in the art can easily obtain the specific information of the fingerprint spectrum of this application, and this invention will not elaborate further.
[0049] Industrial applications Those skilled in the art can use the fingerprint profile of feed oat varieties, consisting of only 21 SNP loci provided in this application, to analyze oat varieties at the genomic level, screen varieties, identify varieties, and also apply it to the tracing of feed oat varieties, as well as the collection, protection, and improvement of germplasm resources.
[0050] The above description is merely a preferred embodiment to aid in understanding the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any alterations or modifications made by those skilled in the art based on this description without departing from the spirit of the present invention should also fall within the scope of the present invention.
Claims
1. A DNA fingerprint of a forage oat variety, comprising a combination of 21 SNP loci, the physical positions of which are shown in the table below, the SNP being determined based on the OT3098v2 reference genome; 2. A method for identifying a forage oat variety, comprising comparing the genotypes of 21 SNP loci of the genomic DNA of the forage oat variety to be tested with the fingerprint formed by the 21 SNP loci; wherein the 21 SNP loci are the 21 SNP loci of claim 1.
3. A primer combination for identifying a forage oat variety, the primer combination being capable of amplifying the sequences of the SNP loci as described in claim 1 in a sample to be tested.
4. A molecular probe combination for identifying a forage oat variety, the molecular probe combination being capable of detecting the combination of SNP loci as described in claim 1 in a sample to be tested.
5. A gene chip for identifying a forage oat variety, the gene chip being loaded with the primer combination of claim 3 or the molecular probe combination of claim 4.
6. A kit for identifying a forage oat variety, the kit comprising the primer combination of claim 3 or the molecular probe combination of claim 4 or the gene chip of claim 5.
7. A method for identifying a forage oat variety, the method comprising using the primer combination of claim 3 or the molecular probe combination of claim 4 or the gene chip of claim 5 or the kit of claim 6 to test a sample to be tested.
8. Use of the DNA fingerprint of a forage oat variety in any one of the following: (A1) identifying or assisting in identifying a forage oat variety; (A2) identifying or assisting in identifying a forage oat germplasm resource; (A3) forage oat breeding.