Molecular marker, primer group, kit and method for identifying high, low and hybrid oleic acid millet

Abstract

The invention provides a molecular marker, a primer group, a kit and a method for identifying high-oleic-acid millet, low-oleic-acid millet and hybrid-oleic-acid millet, and belongs to the technical field of molecular biology. The molecular marker is obtained by comparing a Yin91 gene of low-oleic-acid millet with a Yin91 gene of high-oleic-acid millet; the primer group comprises Yin91 (59-553) F: 5 '-CATGCATGACCCGTACGAA-3' and Yin91 (59-553) R: 5 '-TCGTCCATACCACCATCT-3', the kit comprises the primer group, and the method is characterized in that the primer group is adopted to carry out PCR (Polymerase Chain Reaction) amplification on the millet to be detected so as to identify the high, low and hybrid oleic acid types of the millet to be detected, so that the high, low and hybrid oleic acid types of the millet can be rapidly identified, and the primer group is suitable for oleic acid type detection, breeding and seed selection.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and relates to a molecular marker, a primer set, a kit and a method for identifying high, low and heterozygous oleic acid millets. Background Art

[0002] Millet originated in the Yellow River Basin of my country. Due to its high water utilization rate and drought and ridge resistance, it is widely adapted to the arid and semi-arid areas in the north and is an important stress-resistant grain crop. After being shelled, millet is called millet, which is rich in various amino acids, fatty acids, vitamins, minerals and dietary fiber. It is nutritious and balanced, and has many health benefits such as lowering blood lipids, blood pressure, blood sugar and anti-oxidation.

[0003] At present, millet is mainly eaten in the form of rice porridge. However, existing millet processed foods are prone to oxidation and deterioration, have a short shelf life, and poor processing adaptability, which limits the diversified consumption of millet and the extension of the industrial chain. The main reason for the short shelf life of millet processed products is the low oleic acid content and high linoleic acid content in existing millet varieties. 85% of the total fatty acids in millet are unsaturated fatty acids, of which oleic acid accounts for about 13.3% and linoleic acid accounts for about 69.9%. The ratio of linoleic acid to oleic acid (linoleic ratio) is 5.25:1, which is much higher than other grain crops. Linoleic acid has two unsaturated bonds, is easily oxidized and has poor thermal stability, while the molecular structure stability of oleic acid is 10 times higher than that of linoleic acid. There are no high oleic acid and low linoleic acid types in the existing millet promotion varieties, which cannot meet the product processing needs.

[0004] Marker Assisted Selection (MAS) is a molecular selection method for target traits using molecular markers that are closely linked to the target traits. It has the advantages of high efficiency, accuracy and economy, and accelerates the process of new germplasm selection. The markers currently used for molecular marker-assisted breeding mainly include SSR, SNP, CAPs, InDel, RFLP, RAPD, AFLP, STS, etc. InDel markers refer to the insertion or deletion of nucleotides with different fragments in the sequence of allele loci. The corresponding specific primers need to be designed according to the gene sequences on both sides of the insertion or deletion site. The genotyping can be identified by simple molecular operations such as PCR amplification and electrophoresis detection. The PCR amplification bands of this marker are stable and easy to observe, and the requirements for instruments and identification technology are relatively low. It has been widely used in crop germplasm resource analysis, map-based cloning, gene localization, and genetic map construction. Using InDel markers, a method for accurately and quickly identifying high, low, and heterozygous oleic acid millet is established, which is of great significance to improving the breeding efficiency of high oleic acid millet and shortening the breeding cycle. Summary of the invention

[0005] In response to the above problems, the present invention provides a molecular marker, a primer set, a kit and a method for identifying high, low and hybrid oleic acid millet. The molecular marker is obtained by comparing the Yin91 gene of low oleic acid millet and the Yin91 gene of high oleic acid millet. The primer set is a primer set designed and verified for specificity of the molecular marker, and is used to identify high, low and hybrid oleic acid types of millet.

[0006] A molecular marker for identifying high, low and heterozygous oleic acid millets, wherein the molecular marker is an InDel marker Yin91 obtained by comparing the Yin91 gene of low oleic acid millet with the Yin91 gene of high oleic acid millet;

[0007] The nucleotide sequence of the Yin91 gene of low oleic millet is shown in SEQ ID NO: 1;

[0008] The nucleotide sequence of the Yin91 gene of high oleic millet is shown in SEQ ID NO:2.

[0009] A primer set for identifying high, low and heterozygous oleic acid millets, the primer set comprising:

[0010] The sequence of the forward primer was Yin91(59-553)F: 5′-CATGCCATGACCCGTACGAA-3′;

[0011] The sequence of the reverse primer was Yin91(59-553)R: 5′-TCGTCCACTACCACCACTCT-3′;

[0012] The primer set is a specific primer set designed based on the above-mentioned InDel marker Yin91.

[0013] A kit for identifying high, low and hybrid oleic acid millets, comprising the above primer set.

[0014] Furthermore, the kit also includes conventional reagents for PCR amplification.

[0015] Furthermore, conventional reagents used for PCR amplification include: 2×Taq Master Mix and ddH2O.

[0016] A method for identifying high, low and hybrid oleic acid millets, wherein the method comprises using the above primer set to perform PCR amplification on the DNA of the millet to be tested, so as to identify the high, low and hybrid oleic acid types of the millet to be tested;

[0017] The millet DNA to be tested can be obtained by conventional methods for extracting millet DNA.

[0018] Furthermore, the high, low, and hybrid oleic acid types of the millet to be tested are identified by electrophoresis of the PCR amplification products, and the high, low, and hybrid oleic acid types of the millet to be tested are determined according to the electrophoresis detection results, specifically:

[0019] When the electrophoresis test results show only a 553 bp band, the millet to be tested is low-oleic acid type;

[0020] When the electrophoresis test results show both a 553 bp band and a 276 bp band, the millet to be tested is a hybrid oleic acid type;

[0021] When only a 276bp band appears in the electrophoresis test results, the millet to be tested is of the high oleic acid type.

[0022] Furthermore, the PCR amplification system includes: 2× Taq Master Mix, primer set Yin91(59-553)F / Yin91(59-553)R, DNA template of millet to be tested, and ddH2O;

[0023] Specifically, the PCR amplification system includes: 25 μL of 2×Taq Master Mix, 2 μL of primer set Yin91(59-553)F / Yin91(59-553)R, 2 μL of the millet DNA template to be tested, and 19 μL of ddH2O.

[0024] Furthermore, the PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 35 s, 35 cycles, and incubation at 72°C for 5 min.

[0025] Furthermore, the linoleic acid ratio of high oleic acid millet is less than 2.44; the linoleic acid ratio of hybrid oleic acid millet is between 2.44 and 3.18; the linoleic acid ratio of low oleic acid millet is greater than 3.18;

[0026] Among them, the present invention uses low oleic acid, high linoleic acid material Jigu 39 as the female parent and high oleic acid, low linoleic acid material 00014652 as the male parent to carry out hybridization F7 generation, with a total of 223 offspring, and the linolenic acid ratio is between 1.08 and 5.49, among which the linolenic acid ratio of high oleic acid millet is between 1.08 and 2.42; the linolenic acid ratio of hybrid oleic acid millet is between 2.44 and 3.18; the linolenic acid ratio of low oleic acid millet is between 3.32 and 5.49.

[0027] The molecular markers, primer sets, kits and methods for identifying high, low and heterozygous oleic acid millets of the present invention have the following beneficial effects:

[0028] Since the cultivation of high-oleic millet varieties is of great significance for extending the shelf life of millet processed products and shortening the breeding cycle of high-quality millet; the present invention screened out 4 semi-wild high-oleic and low-linoleic foxtail millet materials from 690 resource materials, whose linolenic acid ratios were between 1.08 and 1.51, but due to poor agronomic traits, they could not be directly used as parents; the present invention was based on the resource 00014652 with the lowest linolenic acid ratio, the low-oleic and high-linoleic acid variety Jigu 39 in Hebei Province and its F7 progeny material (Jigu 39×00014652), performed BSA sequencing, developed InDel molecular markers for detecting the high and low oleic acid content of millet, and established a method for accurately and quickly identifying high, low and heterozygous oleic millets, providing a more effective theoretical and practical basis for molecular marker-assisted cultivation of high-oleic millet;

[0029] The present invention provides an effective theoretical basis for molecular marker-assisted breeding of high-oleic millet by establishing a method for accurately and rapidly identifying high, low and hybrid oleic acid types of millet; utilizing the molecular marker-assisted breeding technology can accelerate the breeding of new high-oleic millet varieties, shorten the breeding cycle and improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the screening and phenotypic traits of high oleic acid materials in Example 1 of the present invention; wherein, Figure A shows the oleic acid and linoleic acid contents of 690 resource materials; Figure B shows the oleic acid and linoleic acid contents of 4 screened high oleic acid materials; Figures C to E show the plant agronomic traits, peeled grains, and grains of foxtail millet material 00014652, respectively; Figures F to H show the plant agronomic traits, peeled grains, and grains of Jigu 39 ears, respectively;

[0031] Figure 2 Figure 2 is the frequency distribution of differential sites in the high linoleic acid ratio mixed pool and the low linoleic acid ratio mixed pool in Example 2 of the present invention; wherein, Figure A is the frequency distribution of SNP sites in the high oleic acid mixed pool; Figure B is the frequency distribution of SNP sites in the low oleic acid mixed pool; Figure C is the annotation of candidate SNP polymorphism marker sites; Figure D is the annotation of candidate InDel sites;

[0032] Figure 3 It is the comparison result of Yin91 gene sequence of Jigu 39 and Yin91 gene sequence of foxtail millet material 00014652 in Example 3 of the present invention;

[0033] Figure 4 This is a partial identification result of using InDel marker Yin91 in 223 RIL populations in Example 6 of the present invention, wherein 1 to 28 represent 28 millet samples in the 223 RIL populations, and M represents the molecular weight marker DL2000. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0035] Example 1: 4 high-oleic and low-linoleic millet materials with a linolenic acid to oil ratio close to 1:1 were screened out

[0036] The existing millet variety resources have low oleic acid content and high linoleic acid content (see Table 1). The linoleic acid ratio varies from 3.3-11.3%, with an average of 5.5. 2 / 3 of the linoleic acid ratio is between 5 and 7. There is a lack of high oleic acid and low linoleic acid resource materials.

[0037] Table 1 Oleic acid and linoleic acid content of existing millet varieties

[0038]

[0039]

[0040] The content of oleic acid and linoleic acid in 690 millet germplasm resources (i.e. 690 millet seeds) was rapidly determined by gas chromatography. Figure 1 Figure A shows that the oleic acid content is between 6.81 and 43.34%, the linoleic acid content is between 46.63 and 72.96%, the linoleic acid ratio is between 1.08 and 11.25, and the average linoleic acid ratio is 5.5.

[0041] By analyzing the linoleic acid ratio, four semi-wild foxtail millet materials with high oleic acid and low linoleic acid were screened out, 00014652, 00014627, 00014628, and 00014629, whose oleic acid contents were 43.34%, 35.94%, 39.01%, and 42.38%, respectively, and their linoleic acid contents were 46.63%, 54.31%, 49.59%, and 47.48%, respectively. Their linoleic acid ratios were between 1.08 and 1.51, which were 30% lower in linoleic acid content and 2 times higher in oleic acid content than other millet materials. Figure 1 Figure B in Figure 1. Among them, the foxtail millet material 00014652 with the highest oleic acid content has a stalk height of 240 cm, is late-maturing, has a growth period of 120 days (Shijiazhuang), has poor agronomic traits, and its grains are similar to those of foxtail grass. Figure 1 Figures C to E in the figure. The characteristics of the ears of Jigu 39, which has a higher linoleic acid content, are just the opposite. Figure 1 Figures F to H in the figure.

[0042] Example 2: Locating the regulatory site for high oleic acid in millet

[0043] The combination of the foxtail millet material 00014652 with the highest oleic acid content and the Hebei low-oleic and high-linoleic acid variety Jigu 39 has resulted in 223 strains in the F7 generation. Twenty extreme materials with high and low linoleic acid ratios were selected to construct gene pools for BSA analysis. The v2.2 genome database (https: / / phytozome-next.jgi.doe.gov / info / Sitalica_v2_2) was used as the reference genome, and the 99% confidence level was used as the threshold line. The window above the confidence level was the candidate interval. The candidate region was roughly located in the 32.866M interval between 7118001…39984000 on chromosome 1. The candidate interval was between Seita.1G078700 and Seita.1G344500. SNP or InDel sites with significant differences in SNP-index or InDel-index between two offspring were selected in the whole genome, that is, sites with SNP-index or InDel-index greater than or equal to 1.0 for offspring 2 (extreme trait B) and SNP-index or InDel-index less than or equal to 0.0 for offspring 1 (extreme trait A) were selected, and 14,397 candidate SNP and InDel polymorphic marker sites were obtained (see Figure 2 ).

[0044] Example 3 Development of Millet High Oleic Acid InDel Marker

[0045] Fine positioning of InDel markers was performed, and the candidate interval was further located to the range of 1.5M between markers Yin42 (35417784) and Yin53 (36972637). Further positioning revealed that InDel marker Yin91 was closely linked to the high oleic acid trait.

[0046] 1) Detect the oleic acid and linoleic acid contents in 223 progeny seeds and calculate the linoleic acid ratio.

[0047] 2) Design InDel markers based on large chromosome deletions, design primers within 300bp above and below the deletion site, extract DNA from Jigu 39 and foxtail millet material 00014652, and use them as templates for PCR amplification and electrophoresis detection to observe whether there are changes in band size. If there are changes in the parental bands, it can be determined that the primers can separate the two parents.

[0048] The process of extracting DNA from Jigu39 and 00014652 is as follows:

[0049] (1) Preheat CTAB (sterilized) to 65°C.

[0050] (2) Take 1-2 portions of fresh tissues of Jigu39 and 00014652 and grind them in 1.5 mL EP tubes.

[0051] (3) Add 600 μL of CTAB to the EP tube and keep it in a 65°C constant temperature metal bath for 1 to 2 h, stirring gently 2 to 3 times.

[0052] (4) Remove the centrifuge tube and cool it to room temperature. Add an equal volume of chloroform / isoamyl alcohol (24:1), mix by inversion, leave at room temperature for 15 minutes, and centrifuge at 10,000 rpm for 15 minutes.

[0053] (5) After centrifugation, transfer the supernatant to another clean centrifuge tube (generally 400 μL), add an equal volume of isopropanol, gently invert, and place in a -20°C refrigerator to freeze for more than 30 minutes.

[0054] (6) Take out the centrifuge tube and place it in a centrifuge. Centrifuge at 10,000 rpm for 10 min and discard the supernatant.

[0055] (7) Take out the centrifuge tube and wash it twice with 75% ethanol and once with 95% ethanol.

[0056] (8) After the ethanol has evaporated, add 20 μL of TE to the EP tube containing the DNA sample to dissolve the DNA and store it in a -20°C refrigerator.

[0057] The primers designed for InDel markers based on large chromosome deletions are:

[0058] The sequence of the forward primer was Yin91(59-553)F: 5′-CATGCCATGACCCGTACGAA-3′;

[0059] The sequence of the reverse primer Yin91(59-553)R: 5'-TCGTCCACTACCACCACTCT-3'.

[0060] The PCR amplification system is as follows:

[0061] Table 2 PCR amplification reaction system

[0062] DNA template 2μL Yin91(59-553)F 2μL Yin91(59-553)R 2μL 2×Taq Master Mix 25μL <![CDATA[ddH2O]]> 19μL Total 50μL

[0063] The procedure for PCR amplification is:

[0064] Table 3 PCR amplification program

[0065]

[0066] 3) The types of different markers were determined in 223 progeny varieties, and it was finally located that the InDel marker Yin91 was closely linked to the high oleic acid trait.

[0067] Among them, the nucleotide sequence of the Yin91 gene of the low oleic millet Jigu 39 is shown in SEQ ID NO: 1, which is the same as the reference genome Yugu 1;

[0068] The nucleotide sequence of the Yin91 gene of the high oleic millet Coryta tenuifolia material 00014652 is shown in SEQ ID NO: 2;

[0069] The Yin91 gene of Jigu 39 was compared with the Yin91 gene of foxtail millet material 00014652. The comparison results are shown in Figure 3 , InDel marker Yin91 was obtained, which was located in the upstream promoter region of Seita.1G290800, with the start and end sites being 35983511 and 35983723, respectively. 277 bp were deleted in this region in foxtail millet material 00014652, and the sequence of Jigu39 was identical to that of the reference genome Yugu1.

[0070] Example 4 Primer Sets and Kits for Identifying High, Low, and Hybrid Oleic Millets

[0071] A specific amplification primer set was designed for InDel marker Yin91. The specific primer set sequence is as follows:

[0072] The sequence of the forward primer was Yin91(59-553)F: 5′-CATGCCATGACCCGTACGAA-3′;

[0073] The sequence of the reverse primer Yin91(59-553)R: 5'-TCGTCCACTACCACCACTCT-3'.

[0074] Among them, primers Yin91(59-553)F / Yin91(59-553)R were used to amplify the millet to be tested. The amplified size of low-oleic millet was about 553bp, the amplified size of high-oleic millet was about 276bp, and the amplified size of heterozygous oleic millet included a band of about 553bp and two parts of about 276bp.

[0075] When designing primers, in order to enhance the applicability and sensitivity of the primers, the designed primer length is between 18 and 25 bp, and the primers do not interfere with each other. The above two primers can be obtained from Sangon Biotech (Shanghai) Co., Ltd.

[0076] This embodiment also provides a kit for identifying high, low and heterozygous oleic acid millets. The kit includes the above primer set.

[0077] The kit also includes common reagents for PCR amplification, such as 2× Taq Master Mix and ddH2O.

[0078] Example 5 Method for Identification of High, Low and Hybrid Oleic Millets

[0079] This embodiment provides a method for identifying high, low, and hybrid oleic acid millets, and the specific method comprises the following steps:

[0080] S1. The method for extracting DNA in Example 3 is used to extract the millet DNA to be tested as a DNA template.

[0081] S2. Use the primer set Yin91(59-553)F / Yin91(59-553)R in Example 4 to perform PCR amplification on the millet DNA to be tested using 2×Taq Master Mix of Nearshore Protein Technology Co., Ltd. to obtain a PCR amplification product.

[0082] The reaction system for PCR amplification is as follows:

[0083] Table 4 PCR amplification reaction system

[0084] DNA template 2μL Yin91(59-553)F 2μL Yin91(59-553)R 2μL 2×Taq Master Mix 25μL <![CDATA[ddH2O]]> 19μL Total volume 50μL

[0085] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 35 s, 35 cycles, and incubation at 72°C for 5 min.

[0086] S3, performing electrophoresis detection on the obtained PCR amplification products, and then judging the high, low, and hybrid oleic acid types of the millet to be tested according to the electrophoresis detection results;

[0087] Among them, when the electrophoresis test results show only a band of about 553bp, the millet to be tested is low-oleic acid type;

[0088] When the electrophoresis test results show a band of about 553 bp and a band of about 276 bp at the same time, the millet to be tested is a hybrid oleic acid type;

[0089] When the electrophoresis test results show only a band of about 276 bp, the millet to be tested is of high oleic acid type.

[0090] In this example, DNA of four kinds of millets, namely, foxtail millet material 00014652, Jigu39, and offspring 20FY901 and 20FY952 of Jigu39×00014652, were identified respectively. The amplified size of Jigu39 was about 553 bp, which was a low oleic acid type millet; the amplified size of foxtail millet material 00014652 was about 276 bp, which was a high oleic acid type millet; the amplified sizes of the hybrid offspring 20FY901 and 20FY952 included a band of about 553 bp and about 276 bp, which were heterozygous oleic acid type millets; it can be seen that the conclusion of the identification by this method is consistent with the results of the direct identification of the oleic acid content of foxtail millet material 00014652 and Jigu39, indicating that this method can be used for the identification of high, low and heterozygous oleic acid types of millet.

[0091] Example 6 Detection and verification of oleic acid in RIL population

[0092] Using gas chromatography, oleic acid and linoleic acid content of 223 strains of the "Jigu 39×00014652" F7 recombinant inbred line population were detected. The oleic acid content ranged from 12.4 to 42.6%, the linoleic acid content ranged from 46.16 to 70.6%, and the linoleic acid ratio ranged from 1.08 to 5.49. Among them, the linoleic acid ratio of high oleic millet was less than 2.44; the linoleic acid ratio of hybrid oleic millet was between 2.44 and 3.18; the linoleic acid ratio of low oleic millet was greater than 3.18. The test results in this embodiment are: there are 124 high oleic millet with a linoleic acid ratio between 1.08 and 2.42; there are 12 hybrid oleic millet with a linoleic acid ratio between 2.44 and 3.18, and there are 87 low oleic millet with a linoleic acid ratio between 3.32 and 5.49. Then, the method in Example 5 was used to identify the above 223 RIL populations. Some of the results of the identification are as follows: Figure 4 As shown in Table 3, it was found that the results of identification by the method of the present invention corresponded to the measured linoleic acid ratio, indicating that the marker of the present invention can be applied to molecular marker-assisted breeding in the field.

[0093] Table 3 Summary of identification results of linolenic acid ratio of some millets in 223 RIL populations

[0094]

[0095]

[0096] Note: In Table 3, 1 is high oleic acid millet, 2 is low oleic acid millet, and 3 is hybrid millet.

[0097] Cultivating high oleic millet varieties and improving the shelf life of millet processed products are of great significance to shortening the breeding cycle of high-quality millet. The present invention screened out 4 semi-wild high oleic and low linoleic foxtail millet materials from 690 resource materials, with a linoleic ratio of 1.08 to 1.51, but the agronomic traits are poor and cannot be directly used as parents. BSA sequencing was performed on foxtail millet material 00014652 with the lowest linoleic ratio, Hebei Province low oleic and high linoleic varieties Jigu 39 and 0014652×Jigu 39F7 offspring materials, and InDel molecular markers for detecting high and low oleic acid content in millet were developed, and a method for accurately and quickly identifying high, low, and heterozygous oleic millet was established, providing an effective theoretical basis for molecular marker-assisted breeding of high oleic millet. Through the molecular marker-assisted breeding, the cultivation of new high oleic millet varieties can be accelerated, the breeding cycle can be shortened, and the breeding efficiency can be improved.

[0098] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all the embodiments. A person skilled in the art can also obtain other embodiments based on the embodiments without inventiveness, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A molecular marker for identifying high, low and heterozygous oleic acid millets, characterized in that: The molecular marker is an InDel marker Yin91 obtained by comparing the Yin91 gene of low oleic millet with the Yin91 gene of high oleic millet; The nucleotide sequence of the Yin91 gene of low oleic millet is shown in SEQ ID NO: 1; The nucleotide sequence of the Yin91 gene of high oleic millet is shown in SEQ ID NO:

2.

2. A primer set for identifying high, low and heterozygous oleic acid millets, characterized in that: The primer set comprises: The sequence of the forward primer was Yin91(59-553)F: 5′-CATGCCATGACCCGTACGAA-3′; The sequence of the reverse primer Yin91(59-553)R: 5'-TCGTCCACTACCACCACTCT-3'.

3. A kit for identifying high, low and hybrid oleic acid millets, characterized in that: The kit comprises the primer set according to claim 2.

4. The kit according to claim 3, characterized in that The kit also includes conventional reagents for PCR amplification.

5. The kit according to claim 3 or 4, characterized in that Conventional reagents used for PCR amplification include: 2×Taq Master Mix and ddH2O.

6. A method for identifying high, low and hybrid oleic acid millets, characterized in that: The method comprises using the primer set described in claim 2 to perform PCR amplification on the DNA of the millet to be tested, so as to identify the high, low and hybrid oleic acid types of the millet to be tested.

7. The method for identifying high, low and hybrid oleic acid millets according to claim 6, characterized in that: The identification of the high, low and hybrid oleic acid types of the millet to be tested is to perform electrophoresis on the PCR amplification products, and judge the high, low and hybrid oleic acid types of the millet to be tested according to the electrophoresis detection results, specifically: When the electrophoresis test results show only a 553 bp band, the millet to be tested is low-oleic acid type; When the electrophoresis test results show both a 553 bp band and a 276 bp band, the millet to be tested is a hybrid oleic acid type; When only a 276bp band appears in the electrophoresis test results, the millet to be tested is of the high oleic acid type.

8. The method for identifying high, low, and hybrid oleic acid millets according to claim 6 or 7, characterized in that: The PCR amplification system includes: 2×Taq Master Mix, primer set Yin91(59-553)F / Yin91(59-553)R, DNA template of millet to be tested and ddH2O.

9. The method for identifying high, low and hybrid oleic acid millets according to claim 8, characterized in that: The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 35 s, 35 cycles, and incubation at 72°C for 5 min.

10. The method for identifying high, low, and hybrid oleic acid millets according to claim 6, 7, or 9, characterized in that: The linoleic acid ratio of high oleic acid millet is less than 2.44; the linoleic acid ratio of hybrid oleic acid millet is between 2.44 and 3.18; and the linoleic acid ratio of low oleic acid millet is greater than 3.18.

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