Molecular markers, primer sets, kits and methods for identifying high, low and heterozygous oleic acid millets

Abstract

The present invention provides a molecular marker, primer set, kit, and method for identifying high, low, and heterozygous oleic acid millets, belonging to the field of molecular biology technology. The molecular marker is obtained by comparing the Yin91 gene of low-oleic acid millet with the Yin91 gene of high-oleic acid millet; the primer set includes Yin91(59-553)F: 5'-CATGCCATGACCCGTACGAA-3'; Yin91(59-553)R: 5'-TCGTCCACTACCACCACTCT-3'; the kit includes the primer set; and the method uses the primer set to perform PCR amplification on the millet to be tested to identify the high, low, and heterozygous oleic acid types of the millet to be tested. The present invention can quickly identify the high, low, and heterozygous oleic acid types of millet and is suitable for oleic acid type detection and 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 originates in my country's Yellow River Basin. Due to its high water use efficiency and drought and ridge resistance, it is widely adapted to arid and semi-arid regions of northern China, making it an important stress-resistant grain crop. Hulled millet, known as millet, is rich in various amino acids, fatty acids, vitamins, minerals, and dietary fiber, offering a rich and balanced nutritional profile. It has numerous health benefits, including lowering blood lipids, blood pressure, and blood sugar levels, and providing antioxidant benefits.

[0003] At present, millet is mainly consumed 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 that of 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 or low linoleic acid types in the existing promoted millet varieties, which cannot meet the needs of product processing.

[0004] Marker-assisted selection (MAS) utilizes molecular markers closely linked to target traits to conduct molecular-level selection for target traits. It boasts the advantages of high efficiency, accuracy, and cost-effectiveness, accelerating the selection of new germplasm. Currently, the main markers used in MAS include SSR, SNP, CAPs, InDel, RFLP, RAPD, AFLP, and STS. InDel markers are characterized by the insertion or deletion of distinct nucleotide fragments within the sequence of an allelic locus. Specific primers must be designed based on the gene sequence flanking the insertion or deletion site, and genotyping can be achieved through simple molecular manipulations such as PCR amplification and electrophoresis. The PCR amplification bands of these markers are stable and easy to observe, requiring minimal instrumentation and identification techniques. They are currently widely used in crop germplasm analysis, map-based cloning, gene mapping, and genetic map construction. Using InDel markers, a method for accurately and rapidly identifying high-, low-, and heterozygous oleic millet varieties has been established, which is of great significance for improving the efficiency of high-oleic millet breeding and shortening the breeding cycle. Summary of the Invention

[0005] In response to the above problems, the present invention provides a molecular marker, primer set, kit and 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 to be specific for 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, 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 heterozygous 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, comprising using the primer set to perform PCR amplification on the DNA of the millet to be tested 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 millet DNA extraction methods.

[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 based on the electrophoresis detection results, specifically:

[0019] When only a 553bp band appears in the electrophoresis test results, the millet to be tested is of 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 heterozygous oleic acid type;

[0021] When only a 276 bp 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 included: 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 each of the 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 millet is less than 2.44; the linoleic acid ratio of hybrid oleic millet is between 2.44 and 3.18; the linoleic acid ratio of low oleic millet is greater than 3.18;

[0026] Among them, the present invention uses the low oleic acid, high linoleic acid material Jigu 39 as the female parent and the 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; and 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; based on the resource 00014652 with the lowest linolenic acid ratio, the low-oleic and high-linoleic acid variety Jigu 39 from Hebei Province and its F7 progeny material (Jigu 39×00014652), the present invention performed BSA sequencing and developed InDel molecular markers for detecting the oleic acid content of millet, and established a method for accurately and rapidly 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. 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 The phenotypic characteristics of the high oleic acid material screening in Example 1 of the present invention are shown in Figure A; 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 the four screened high oleic acid materials; Figures C to E show the agronomic traits, peeled kernels, and kernels of foxtail millet material 00014652, respectively; Figures F to H show the agronomic traits, peeled kernels, and kernels of Jigu 39 ears, respectively.

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

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

[0033] Figure 4 These are some of the identification results using the InDel marker Yin91 in 223 RIL populations in Example 6 of the present invention, where 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. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments to facilitate understanding by those skilled in the art.

[0035] Example 1 Screening out 4 parts of high oleic acid and low linoleic acid millet materials with a linolenic acid to oil ratio close to 1:1

[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 contents of oleic acid and linoleic acid in 690 millet germplasm resources (i.e. 690 millet seeds) were rapidly determined by gas chromatography. Figure 1 Figure A shows that the oleic acid content ranges from 6.81 to 43.34%, the linoleic acid content ranges from 46.63 to 72.96%, the linoleic acid ratio ranges from 1.08 to 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. Their oleic acid contents were 43.34%, 35.94%, 39.01%, and 42.38%, respectively; 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. 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 High Oleic Acid Regulatory Site in Millet

[0043] The highest oleic acid content of foxtail millet material 00014652 was combined with the Hebei low oleic acid and high linoleic acid variety Jigu 39. The RIL population has reached 223 lines in the F7 generation. 20 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. A 99% confidence level was used as the threshold, and the window above the confidence level was defined as the candidate interval. The candidate region was roughly located within the 32.866M interval between 7118001…39984000 on chromosome 1. The candidate interval was between Seita.1G078700 and Seita.1G344500. SNPs or InDel sites with significant differences in SNP-index or InDel-index between two progenies were selected genome-wide. That is, sites with a SNP-index or InDel-index greater than or equal to 1.0 in progeny 2 (extreme trait B) and a SNP-index or InDel-index less than or equal to 0.0 in progeny 1 (extreme trait A) were selected. This resulted in 14,397 candidate SNP and InDel polymorphic marker sites (see

[14] ). Figure 2 ).

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

[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 an InDel marker based on a large chromosomal deletion. Design primers within 300 bp above and below the deletion site. Extract DNA from Jigu 39 and foxtail millet material 00014652 and use them as templates for PCR amplification. Perform electrophoresis to observe whether there is any change in band size. If there is a change in the parental band, it is confirmed 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 constant temperature metal bath at 65°C 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 thoroughly by inversion, and let it stand at room temperature for 15 minutes. Centrifuge at 10,000 rpm for 15 minutes.

[0053] (5) After centrifugation, transfer the supernatant to another clean centrifuge tube (generally take 400 μL), add an equal volume of isopropanol, gently invert, and place in a -20℃ 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 minutes 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 25 μL <![CDATA[ddH2O]]> 19 μL Total 50 μL

[0063] The PCR amplification procedure is:

[0064] Table 3 PCR amplification program

[0065]

[0066] 3) The types of different markers were determined in 223 progeny varieties, and the InDel marker Yin91 was finally located and found to be tightly 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 identical to the reference genome Yugu 1;

[0068] The nucleotide sequence of the Yin91 gene of the high oleic millet foxtail millet 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 , obtained InDel marker Yin91, which is 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 sequences are 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 amplified 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] In order to enhance the applicability and sensitivity of the primers, the primers were designed to be between 18 and 25 bp in length and to prevent interference between the primers. The two primers mentioned above 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-mentioned 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 Identifying High, Low, and Hybrid Oleic Millets

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

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

[0081] S2. The primer set Yin91(59-553)F / Yin91(59-553)R in Example 4 was used to perform PCR amplification on the foxtail millet DNA to be tested using 2×Taq Master Mix from Jinan 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 25 μL <![CDATA[ddH2O]]> 19 μL Total volume 50 μL

[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 insulation at 72°C for 5 min.

[0086] S3, performing electrophoresis detection on the obtained PCR amplification products, and then determining the high, low, or hybrid oleic acid type 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 heterozygous 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 types of millet, namely, foxtail millet material 00014652, Jigu39, and offspring 20FY901 and 20FY952 of Jigu39×00014652, was identified. The amplified size of Jigu39 was about 553 bp, indicating that it was a low oleic acid type millet; the amplified size of foxtail millet material 00014652 was about 276 bp, indicating that it 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 a band of about 276 bp, indicating that they were heterozygous oleic acid type millet; it can be seen that the conclusion of the identification by this method is consistent with the results of 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 was detected in 223 strains of the "Jigu 39×00014652" F7 recombinant inbred line population. 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; and the linoleic acid ratio of low-oleic millet was greater than 3.18. The test results in this example were: there were 124 high-oleic millet strains with a linoleic acid ratio between 1.08 and 2.42; there were 12 hybrid oleic millet strains with a linoleic acid ratio between 2.44 and 3.18; and there were 87 low-oleic millet strains 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 identification results are as follows: Figure 4 As shown in Table 3, it was found that the results of the 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 for shortening the breeding cycle for high-quality millet. This study screened four semi-wild high-oleic and low-linoleic foxtail millet materials from 690 resource materials. These materials had linolenic acid ratios of 1.08 to 1.51, but their agronomic traits were poor and precluded direct use as parents. BSA sequencing was performed on foxtail millet material 00014652, which has the lowest linolenic acid ratio; the Hebei low-oleic and high-linoleic variety Jigu 39; and the 0014652×Jigu 39 F7 progeny. InDel molecular markers for detecting high and low oleic acid content in millet were developed, establishing a method for accurately and rapidly identifying high-, low-, and heterozygous oleic millet varieties. This method provides an effective theoretical basis for molecular marker-assisted breeding of high-oleic millet. This molecular marker-assisted breeding can accelerate the development of new high-oleic millet varieties, shorten the breeding cycle, and improve breeding efficiency.

[0098] Other parts not described in detail are all prior art. Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Those skilled in the art can also derive other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection 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 and 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 includes: 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 heterozygous oleic acid millets, characterized in that: The kit includes the primer set according to claim 2.

4. The kit according to claim 3, wherein 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, wherein: The identification of the high, low and mixed oleic acid types of the millet to be tested is to perform electrophoresis on the PCR amplification products, and judge the high, low and mixed oleic acid types of the millet to be tested based on the electrophoresis test results, specifically: When only a 553 bp band appears in the electrophoresis test results, the millet to be tested is of 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 heterozygous oleic acid type; When only a 276 bp 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, wherein: 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, wherein: 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 insulation at 72°C for 5 min.

10. The method for identifying high, low, or heterozygous oleic acid millets according to claim 6, 7, or 9, wherein: 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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