DNA fingerprint spectrum for identifying feeding oat variety and application of DNA fingerprint spectrum

Abstract

The invention discloses a DNA (Deoxyribose Nucleic Acid) fingerprint spectrum for identifying a feeding oat variety and application thereof, relates to the technical field of biology, and provides 21 molecular markers capable of analyzing and identifying the feeding oat variety. A molecular probe combination, a gene chip and a kit prepared from the 21 SNP molecular markers can play an important role in the aspects of the blood relationship and genetic basis of oat crops, variety certification and determination, counterfeit and shoddy seed identification, management and detection of genetic breeding and crop maps and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the field of biological detection technology, and more particularly to a SNP combination for identifying fodder oat varieties and an application thereof. Background Art

[0002] Oats (Avena sativa L.) are an annual herb belonging to the Poaceae family. Its seeds are of high nutritional value, rich in dietary fiber and antioxidants, and help to reduce postprandial blood sugar. Oat grass can increase milk production and milk quality, and is an important dietary component of dairy cows. In recent years, oats have been valued for their rich nutrition, and the breeding research of new oat varieties has made rapid progress. There is an urgent need for a method that can quickly identify new oat varieties so that varieties can be confirmed and protected in a timely manner. The traditional morphological identification method identifies different varieties by the differences in phenotypic traits between different varieties. Although it is intuitive and reliable, the identification process is cumbersome and laborious, takes a long time, and it is difficult to identify varieties with similar phenotypic traits; DNA molecular markers can use the differences in different varieties at the DNA molecular level to identify different varieties. Among them, DNA fingerprints are widely used to identify individuals due to their multi-site, high variability, simple and stable inheritance. This is a valuable genetic marker for studying the genetic structure, ecology and evolution, and classification of animal and plant populations.

[0003] In view of this, the present invention provides a DNA fingerprint capable of identifying fodder oat varieties. Summary of the invention

[0004] In order to meet the needs of current oat variety research and agricultural production for oat variety identification, the present invention provides a DNA fingerprint for identifying fodder oat varieties and its application. The DNA fingerprint information provided by the present invention can be used to quickly and accurately identify oat varieties, and can be used for variety screening, variety tracing, and breeding, which is beneficial to the collection, protection and improvement of germplasm resources, and is time-saving, low-cost, and has broad market benefits.

[0005] In order to achieve the technical purpose of the present invention, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a DNA fingerprint for identifying forage oat varieties, which includes 21 SNP sites, whose physical locations and sequence information are shown in Table 1. Specifically, the sequences are shown in SEQ ID NO.1-SEQ ID NO.21:

[0007] Table 1 Location and sequence information of 21 loci combinations

[0008]

[0009]

[0010] A second aspect provides a method for identifying oat varieties, wherein the genotype of 21 SNP sites of the genomic DNA of the oat variety to be tested is compared with a fingerprint map formed by the 21 SNP sites;

[0011] Among them, the 21 SNP sites are the 21 SNP sites described in Table 1.

[0012] The third aspect provides a primer combination for identifying oat varieties, wherein the primer combination can amplify the SNP site sequence described in Table 1 in the sample to be tested.

[0013] The fourth aspect provides a molecular probe combination for identifying oat varieties, wherein the molecular probe combination is capable of detecting the SNP site combination as described in Table 1 in the sample to be tested.

[0014] The fifth aspect provides a gene chip for identifying oat varieties, wherein the gene chip is loaded with the above-mentioned primer combination or molecular probe combination.

[0015] The sixth aspect provides a kit for identifying oat varieties, which has the above-mentioned primer combination or molecular probe combination or gene chip.

[0016] The seventh aspect provides a method for identifying oat varieties, using the above-mentioned primer combination or molecular probe combination or gene chip or kit to detect the sample to be tested.

[0017] The eighth aspect provides the use of a DNA fingerprint for identifying fodder oat varieties in any of the following:

[0018] (A1) Identify or assist in identifying fodder oat varieties;

[0019] (A2) Identify or assist in identifying forage oat germplasm resources;

[0020] (A3) Breeding of forage oats.

[0021] Beneficial effects:

[0022] The DNA fingerprint for identifying fodder oats provided by the present invention can achieve the purpose of identifying multiple varieties with as few markers as possible, so as to achieve the purpose of simplicity, high efficiency and economy. It can play an important role in oat crop blood relationship and genetic basis, variety approval, fake and inferior seed identification, management and detection, genetic breeding and crop map, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the discrimination power of each quantity gradient label set provided in Example 1 of the present invention;

[0024] Figure 2The invention provides a DNA fingerprint for identifying fodder oats. DETAILED DESCRIPTION

[0025] The present invention is further explained below with reference to the detailed description of specific embodiments, but these embodiments are merely illustrative and cannot be construed as limiting the present invention. If not otherwise specified, the technical means adopted in the embodiments are conventional means well known to those skilled in the art, and can be carried out with reference to the third edition of the original work "Bioinformatics and Functional Genomics" or related books, and the bioinformatics software and products adopted are also commercially available. Various processes and methods not described in detail are conventional methods known in the art, and the sources of materials used, trade names, and those that are necessary to list their components are all indicated when they first appear, and the same reagents used thereafter, if not otherwise specified, are all identical with the content indicated for the first time.

[0026] In addition, it should be noted that the site combination and application provided by the present invention were completed by the inventors through arduous creative labor and optimization work.

[0027] Example 1 Obtaining oat fingerprint

[0028] 1. Test materials

[0029] In order to achieve more comprehensive coverage of oat varieties at home and abroad, the present invention conducts gene analysis and typing on 80 oat varieties (lines) worldwide to construct a fingerprint database of oat varieties (lines). Each variety is shown in Table 2:

[0030] Table 2 Varieties (lines) of oats for feeding

[0031]

[0032]

[0033]

[0034] Of course, in one embodiment of the present invention, more varieties of fodder oats can be collected to construct fingerprints. As long as the fingerprints are obtained using the methods and ideas of the present invention, they are within the protection scope of the present invention.

[0035] 2. DNA extraction, detection and sequencing

[0036] The DNA of the 80 oat varieties in step 1 was extracted, and the DNA sequence of each oat variety was detected. The sequencing method was a conventional method in the art, and the present invention is not limited thereto.

[0037] 3. Sample screening and data quality control

[0038] After basic quality control and removal of redundant sites for the genotype data, the IBS distance between the two samples was calculated using Plink (V1.90) 1 software, and the extremely similar sample pairs were identified based on the calculation results. The criteria for determining extremely similar samples are as follows: when DST between samples is ≥ 0.99, they are judged to be extremely similar samples, and DST is the probability that two individuals are isomorphic at the genome level. When extremely similar samples are found, only one extremely similar sample in each group is retained for subsequent analysis. In the embodiment of the present invention, no extremely similar samples were found.

[0039] It should be noted that sample screening and data quality control are conventional methods in the art and are not limited by the present invention.

[0040] To ensure the quality of the genotype data used for analysis and the validity of the analysis results, Plink (V1.90)1 software was used to perform quality control on samples and SNP sites before analysis. The quality control standards were as follows: a. Sample detection rate >= 0.9, b. Only sites on chromosomes were retained, c. Only sites without deletions were retained, d. Only SNP sites were retained, and e. The minimum allele frequency was >= 0.3

[0041] After quality control, 80 samples and 1200269 loci remained. On this basis, Plink (V1.90)1 software was used to remove strongly linked loci, and the final number of remaining loci was 65716.

[0042] 4.3 Marker screening

[0043] Based on the core marker screening method proposed by Yang et al. (2022) (see https: / / doi.org / 10.1186 / s12870-022-03920-2), the core SNP marker combination was screened. Under the premise of ensuring that all chromosomes are distributed as much as possible, different numbers of marker sets are randomly selected from the remaining loci, and the samples are extracted several times to generate SNP marker combinations. Then the discrimination power of each marker combination is calculated, that is, the number of samples with unique genotypes and the ratio of the number of samples with unique genotypes to the total number of samples. This time, 1 to 21 marker sets were extracted 100 times for each marker set, and then the marker combination with the highest discrimination in each marker set was selected to represent the discrimination of that quantity. The discrimination power of each quantitative gradient marker set is shown in Tables 2 and Figure 1 .

[0044] Table 2. Discrimination power of different number of marker sets

[0045] Number of markers Discernment Number of markers Discernment Number of markers Discernment 1 0.0375 8 0.95 15 1 2 0.1125 9 0.95 16 1 3 0.2875 10 0.95 17 1 4 0.4125 11 0.9875 18 1 5 0.6875 12 1 19 1 6 0.8 13 1 20 1 7 0.8625 14 1 21 1

[0046] Although the 80 materials can be completely distinguished using a set of 12 markers, in order to increase the differentiation between samples, it is recommended to retain one marker on each chromosome, that is, to use a set of 21 markers as core SNP markers.

[0047] 4.4 Fingerprint construction

[0048] The combination with the highest discrimination power among the 21 markers was used as the core SNP marker to construct the fingerprint map. The construction results are shown in the figure. Figure 2 As shown in the figure, one row is one sample and one column is one marker. The position and sequence of each marker are shown in Table 1 in the specification, and will not be repeated here.

[0049] Genetic similarity analysis of fingerprints of experimental cases

[0050] Based on 21 core SNP markers, the genetic similarity between the 80 materials was calculated. The genetic similarity calculation formula is as follows:

[0051] GS=nij / Nij

[0052] Among them, nij is the number of markers detected in both materials and with different typing in the two materials, and Nij is the number of markers detected in both materials. The calculation results of genetic similarity between some materials are shown in Table 3:

[0053] Table 3 Genetic similarity analysis results of some materials

[0054]

[0055]

[0056] Note: ID1: sample name of the first material; ID2: sample name of the second material; Identical_count: number of markers with consistent genotypes; Total_count: number of markers checked for both materials; GS: genetic similarity.

[0057] According to the analysis results in Table 3, it can be seen that no two materials are similar, which shows that the fingerprint provided by the present invention has a high recognition degree.

[0058] Example 2: Using the SNP site combination in the fingerprint map to prepare the primer combination and probe combination

[0059] A person skilled in the art designs primers based on the sequence information of the 21 sites in Table 1 provided by the present invention, and performs secondary structure evaluation and Tm value evaluation on the designed primers, and finally obtains primers with good specificity and high sensitivity, and can achieve the detection purpose under the same reaction conditions.

[0060] The secondary structure evaluation and Tm value evaluation can be performed by any method commonly used in the art, such as using DNA folding form to evaluate its secondary structure, see (http: / / unafold.rna.albany.edu / ?q=mfold / DNA-Folding-Form) for details, and then using the software RaW-Probe to evaluate its Tm value.

[0061] The above methods are all conventional methods. According to the site information in the SNP site combination provided in the present application, they can be obtained without any creative labor. Therefore, the primers obtained according to the SNP site combination provided by the present invention also fall within the protection scope of the present invention.

[0062] Likewise, the use of the SNP site combination provided by the present invention to prepare probes, such as tanqman probes, also falls within the protection scope of the present invention.

[0063] Example 3: Using the SNP site combination in the fingerprint map to prepare a gene chip

[0064] The SNP gene chip of the present invention is obtained by fixing the primers or probes obtained in Example 2 on a polymer substrate, such as a nylon membrane, a nitrocellulose membrane, plastic, a silica gel wafer, micro magnetic beads, etc., by conventional methods, or fixing the probes on a glass plate, or directly synthesizing the primers or probes obtained in Example 2 on a hard surface such as glass. The method of using the SNP gene chip of the present application is the same as the conventional method.

[0065] It should be noted that those skilled in the art can prepare SNP detection gene chips in any manner, and can also entrust a biological company to prepare them, but gene chips prepared based on the SNP site combination in the fingerprint map provided in this application all fall within the scope of protection of the present invention.

[0066] Example 4 Analytical kit for identifying fodder oat varieties

[0067] The detection kit for identifying forage oat varieties provided in the present application includes primers or probes or gene chips obtained based on the SNP site combination obtained in Example 2. Depending on the type of use, corresponding detection reagents are also included. For example, when the Taqman probe is obtained based on the SNP site combination obtained in Example 1, the conventionally used buffer, ligase, AceQUniversal U+Probe Master Mix V2, TaqMan Probe, etc. for fluorescent quantitative PCR reactions are also included.

[0068] Those skilled in the art configure different detection kits for identifying forage oat varieties according to different usage modes, but the detection kits configured based on the SNP site combination provided in this application all fall within the scope of protection of the present invention.

[0069] Example 5 Identification of fodder oat varieties

[0070] Based on the SNP site combination in the fingerprint map for identifying forage oat varieties provided in Example 1 of the present application, known forage oat varieties are tested, specifically:

[0071] The DNA of the sample variety was collected by conventional methods, and the primers or probes provided in Example 2 were used to detect the DNA of the sample variety to obtain the detection results of 21 sites of the sample. Figure 2 The fingerprint map provided is compared to obtain the oat variety analysis results.

[0072] Industrial Applications

[0073] Based on the fingerprint map for identifying forage oat varieties consisting of only 21 SNP sites provided in the present application, those skilled in the art can analyze oat varieties at the genome level, screen and identify varieties, and can also apply it to the tracing of forage oat varieties, and the collection, protection and improvement of germplasm resources.

[0074] The above description is only to help understand the preferred examples of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Without violating the concept of the present invention, various changes or modifications made to the present invention by those skilled in the art on this basis should also fall within the scope of the present invention.

Claims

1. DNA fingerprints for identifying forage oat varieties include 21 SNP loci combinations, whose physical locations are shown in the following table, and whose sequences are shown in SEQ ID NO.1-SEQ ID NO.21:

2. A method for identifying oat varieties, comprising comparing the genotype of 21 SNP sites of the genomic DNA of the oat variety to be tested with the fingerprint map formed by the 21 SNP sites; in, The 21 SNP sites are the 21 SNP sites described in claim 1.

3. A primer combination for identifying oat varieties, wherein the primer combination can amplify the SNP site sequence as described in claim 1 in the sample to be tested.

4. A molecular probe combination for identifying oat varieties, wherein the molecular probe combination can detect the SNP site combination as described in claim 1 in a sample to be tested.

5. A gene chip for identifying oat varieties, wherein the gene chip is loaded with the primer combination according to claim 3 or the molecular probe combination according to claim 4.

6. A kit for identifying oat varieties, comprising the primer combination according to claim 3 or the molecular probe combination according to claim 4 or the gene chip according to claim 5.

7. A method for identifying oat varieties, comprising detecting a sample 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.

8. Application of DNA fingerprint for identification of fodder oat varieties in any of the following: (A1) Identify or assist in identifying fodder oat varieties; (A2) Identify or assist in identifying forage oat germplasm resources; (A3) Breeding of forage oats.

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