CN105385768B - Method for identifying millet variety by adopting SSR molecular marker technology and application - Google Patents

Abstract

The invention discloses a method for identifying millet varieties by adopting an SSR molecular marker technology and application thereof, belonging to the technical field of plant variety identification. The method provided by the invention extracts the millet DNA, utilizes the millet DNA and 5 to carry out PCR amplification reaction on the SSR core primer, and identifies the variety of the millet according to the electrophoresis detection result after the electrophoresis detection; the nucleotide sequences of the 5 pairs of SSR core primers are shown as SEQID NO. 1-10. The method provided by the invention can effectively distinguish the variety of the millet and provides an important way for further identifying a large number of varieties of the millet.

Description

Method for identifying millet variety by adopting SSR molecular marker technology and application

Technical Field

The invention relates to a method for identifying millet varieties by adopting an SSR molecular marker technology and application thereof, belonging to the technical field of plant variety identification.

Background

The foxtail millet belonging to the genus setaria of the family gramineae has the physiological characteristics of short growth period, drought resistance, barren resistance and the like, is widely distributed in temperate zones and tropical zones of the continental areas of Europe, and is one of the main cultivated crops in the middle and upstream areas of the yellow river of China. Meanwhile, the millet has extremely high nutritive value, contains rich amino acids, proteins, vitamins and trace elements such as calcium, copper, iron, zinc, selenium, iodine, magnesium and the like, and has very obvious nutritive and health-care effects. For many years, variety identification has been based on morphological markers. Although the method is simple and economical, the period is long, the cost is high, the method is limited by seasons, and the expression of a plurality of characters is influenced by cultivation measures and environmental factors, so that the accuracy of identification is limited. The DNA molecular marker technology has the advantages of high efficiency, accuracy, no influence of environmental conditions, simple experiment operation and the like, and is widely applied to the authenticity identification and purity detection research of plant varieties. SSR markers are second-generation molecular markers established on the basis of PCR and developed in recent years. The SSR molecular marker has the characteristics of abundant quantity, high polymorphism, genetic codominance, stable band amplification, high precision, short detection time, mature technology and the like, and is applied to crop genetics research.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a method for identifying millet varieties by adopting an SSR molecular marker technology, which adopts the following technical scheme:

the invention aims to provide a method for identifying millet varieties by adopting an SSR molecular marker technology, which comprises the steps of extracting millet DNA, carrying out PCR amplification reaction on SSR core primers by utilizing the millet DNA and 5, and identifying the millet varieties according to an electrophoresis detection result after electrophoresis detection; the nucleotide sequences of the 5 pairs of SSR core primers are shown as SEQID NO. 1-10.

Preferably, the method steps are as follows:

1) extracting millet DNA;

2) carrying out PCR amplification reaction on the SSR core primers by using the millet DNA obtained in the step 1) and 5, and then carrying out 8% non-denaturing polyacrylamide gel electrophoresis detection and silver staining for color development; each pair of SSR core primers comprises a forward primer and a reverse primer, and the nucleotide sequences of 5 pairs of SSR core primers are shown as SEQID No. 1-10;

3) and (3) observing the electrophoresis result in the step 2), counting the stable and easily-distinguished difference bands, recording the band according to a 0 and 1 system, assigning a value of 1 when the band has the difference, assigning a value of 0 when the band has no difference, calculating a genetic similarity coefficient according to the recorded result, and identifying the variety of the millet.

Preferably, the extraction of the millet DNA in the step 1) is to take millet sprouts, store the millet sprouts at-80 ℃ for later use after freezing by liquid nitrogen, and then extract the millet DNA by using a plant genome kit.

More preferably, the millet sprouts are sprouts 10d-15d after the millet sprouts.

Preferably, the millet in step 1) is selected from big leaf yellow millet, purple stalk sword handle, Qianjiantig millet, four-inch red root, red root rope head, thin skin white millet, hen mouth, big full millet, dry tip rough, sixty days returning to storehouse, halter rope head, Jinmiahuang, Molin millet, small purple seedling millet, red wine millet, black millet, Qianjianzi, mule tail, bird millet, barley stubble millet, small rope head, red seedling golden rake teeth, bamboo leaf red, pheasant, red seedling rope head, big goose neck, cooked stick, eight channels, dry tip, green bamboo leaf, two green seedlings, two white cereals, garlic skin white cereals, jade yellow cereals, flush red, yellow roughneck, purple stem, white dew yellow, pressed millet car, Zhang pure one, black millet, green cereals, big black cereals, small turnip or white hair cereals.

Preferably, the PCR amplification reaction of step 3) comprises 10 XPCR buffer, 25mM MgCl₂10mM dNTP, 10mM forward primer, 10mM reverse primer, 5U/. mu.L Taq enzyme, 50 ng/. mu.L template DNA and ddH₂O。

More preferably, the PCR amplification reaction in step 3) is performed in a total volume of 25. mu.L, which includes 10 XPCR buffer 2.5. mu.L, 25mM MgCl₂3 μ L, 10mM dNTP 1.25 μ L, 10mM forward primer 1 μ L, 10mM reverse primer 1 μ L, 5U/. mu.L Taq enzyme 0.2 μ L, 50 ng/. mu.L template DNA1 μ L, ddH₂O 16.5μL。

Preferably, the PCR amplification reaction in step 3) is performed by the following procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

Preferably, the method comprises the following specific steps:

1) extraction of millet DNA: taking the germinated millet sprouts of 10-15 days, freezing by liquid nitrogen, storing at-80 ℃ for later use, and extracting the DNA of the millet sprouts by using a plant genome kit to obtain millet DNA;

2) PCR amplification and electrophoresis detection: carrying out PCR amplification reaction on the SSR core primers with nucleotide sequences shown as SEQID No.1-10 by using the millet DNA obtained in the step 1) and 5; then carrying out 8% non-denaturing polyacrylamide gel electrophoresis detection on the PCR product, and carrying out silver staining for color development;

each pair of SSR core primers comprises a forward primer and a reverse primer; the total volume of the PCR amplification reaction system is 25 mu L, wherein the total volume comprises 10 XPCR buffer 2.5 mu L and 25mM MgCl₂3 μ L, 10mM dNTP 1.25 μ L, 10mM forward primer 1 μ L, 10mM reverse primer 1 μ L, 5U/. mu.L Taq enzyme 0.2 μ L, 50 ng/. mu.L template DNA1 μ L, ddH₂O16.5 mu L; the PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃;

3) and (3) observing the electrophoresis result in the step 2), counting the stable and easily-distinguished difference bands, recording the band according to a 0 and 1 system, assigning a value of 1 when the band has the difference, assigning a value of 0 when the band has no difference, calculating a genetic similarity coefficient according to the recorded result, and identifying the variety of the millet.

The application of any one of the methods in identifying the variety of the millet. The invention has the beneficial effects that:

1. the invention utilizes SSR molecular markers to identify 45 millet varieties, and the result shows that: 35 alleles are amplified by 5 pairs of millet SSR primers, and 7 alleles are averagely amplified by each pair of primers; the polymorphic information index of each primer is 0.688-0.846, and the average value is 0.779; calculating genetic similarity coefficients among varieties through NTsys-pc 2.11, wherein the genetic similarity coefficients among the varieties are 0.6571-0.9429, and the average genetic similarity coefficient of 45 millet varieties is 0.7554; the 5 pairs of the selected millet primers have good polymorphism and discrimination ability, can effectively distinguish 45 millet varieties, and provides an important way for further discriminating a large number of millet varieties.

2. The method can effectively identify the millet varieties by only using 5 pairs of millet primers, has the identification accuracy rate of 100 percent, has great potential in the aspect of further identifying a large number of millet varieties, has better effect, saves more time, cost and the like compared with the prior patent.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.

In the following examples, the names of varieties of millet and the numbers of resource pools are from the germplasm information network of Chinese crops.

Example 1: establishment of the method

1. Preparing a millet material: planting 15 kinds of millet materials including Guanzhou red, duck beak, big yellow, big white hair, old rice, red sticky rice, knife handle, gougen red, Qianjiang, big young seedling, yellow sticky rice, red millet, yellow millet, duck beak, big yellow and red seedling grain in a greenhouse; freezing the germinated millet sprouts of about 10-15 days in liquid nitrogen, and storing in an ultra-low temperature refrigerator at-80 deg.C for use; the information of 15 varieties of millet is shown in table 1.

Table 115 variety information of millet

Serial number	Resource pool number	Experiment number	Name (R)	Serial number	Resource pool number	Experiment number	Name (R)
								1	00001318	CS01	Gaizhou red	9	00000090	FS05	Big young seedling
2	00001319	CS02	Duck mouth	10	00000098	FS06	Yellow sticky rice
								3	00001320	CS03	Root of large-headed yellow	11	00000122	FS07	Red millet
4	00001533	CS04	White hair	12	00000136	FS08	Yellow rice
								5	00000011	FS01	Changes from old to old	13	00000198	FS09	Duck mouth
6	00000053	FS02	Red glutinous rice	14	00000523	FS10	Big yellow particle
								7	00000058	FS03	The knife handle is neat	15	00000529	FS11	Red seedling grain
8	00000070	FS04	Cash string

2. Extraction of millet DNA: extracting DNA of various millet materials by using a plant genome kit, and detecting the purity of the DNA by using 1% agarose gel electrophoresis; diluting the DNA concentration of each material to 50 ng/mu L, and placing the diluted DNA in a refrigerator at the temperature of-20 ℃ for later use;

3. PCR amplification reaction and electrophoresis detection: the total volume of the PCR reaction was 25. mu.L, including 10 XPCR buffer 2.5. mu.L, MgCl2(25mM) 3. mu.L, dNTP (10mM) 1.25. mu.L, forward primer (10mM)1. mu.L, reverse primer (10mM)1. mu.L, Taq enzyme (5U/. mu.L) 0.2. mu.L, template DNA (50 ng/. mu.L) 1. mu.L, ddH2O 16.5.5. mu.L; the reaction procedure for PCR was: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃; adding 4 mu L of 6 times Loading buffer into the PCR amplification product, taking 1.5 mu L of sample, detecting by 8% non-denatured polyacrylamide gel electrophoresis, and carrying out silver staining and color development.

4. Selecting 4 reference germplasms (CS 01-CS 04) from different genome millet, primarily screening 130 pairs of SSR primers synthesized in the experiment, and selecting 45 pairs of primers which are different among different genome millet according to polymorphism of amplification products; then, re-screening the polymorphic primers by using 11 reference germplasm materials (FS 01-FS 11), wherein the polymorphism information of the SSR re-screened primers of the millet is shown in a table 2; finally, 5 pairs of primers which are clear in band and convenient to count and have good polymorphism among different millet qualities are selected as SSR core primers, and the results of the amplification of the millet SSR core primers are shown in Table 3. The non-core primer information is shown in Table 4.

TABLE 2 amplification results of millet SSR rescreened primers

TABLE 3 amplification results of millet SSR core primers

TABLE 4 non-core primer information

5. Statistical analysis: observing the electrophoresis result of the PCR product, counting the stable and easily-distinguished difference bands by Gel-Pro analyzer software, recording the band according to a 0 and 1 system, assigning a value of 1 when the band exists, assigning a value of 0 when the band does not exist, and constructing corresponding 0 and 1 characters of each variety; the results were recorded and the genetic similarity coefficient was calculated using NTsys-pc 2.11.

6. Results and analysis:

6A, extracting by adopting a kitThe millet DNA strip is clear and bright, has no degradation phenomenon and good DNA integrity, and the detection of a spectrophotometer finds that A_260/280The ratio is within 1.8-2.0, and the PCR requirements can be met.

6B, 104 effective amplification primers in 130 pairs of primers are used, and account for 77.0 percent of the total amplification primers; 45 pairs of primers with polymorphism between different genome foxtail millet occupy 43.3% of the effective amplification primers. According to the amplification results of 11 millet varieties (table 2), the allele factors of SiG 01-SiG 05 are high, the polymorphism is good, and the PIC value is more than 0.55; the polymorphic information content of SiG 06-SiG 45 is low, the identification effect on reference germplasm materials is not ideal, and the varieties of millet can not be effectively identified by using SiG 06-SiG 45. Therefore, SiG 01-SiG 05 are selected as SSR core primers for identifying the germ plasm resources of the millet, only the 5 pairs of primers can be effectively identified, and the identification effect of other primers is not ideal.

6C, obtaining 22 alleles in total by 5 pairs of millet SSR primers according to an amplification result, and averaging 4.4 alleles of each pair of primers; the characters of 0 and 1 of 11 millet varieties are shown in a table 5; the polymorphic information index of each primer is 0.581-0.708, the average value is 0.652, and the primers with the best amplification polymorphism are SiG02 and SiG 03; the results show that the selected primers all have good polymorphism.

0, 1 characters of table 511 millet varieties

Genetic similarity coefficient between 611 millet varieties in table

	FS01	FS02	FS03	FS04	FS05	FS06	FS07	FS08	FS09	FS10	FS11
												FS01	1.0000
FS02	0.5909	1.0000
												FS03	0.9091	0.5909	1.0000
FS04	0.5909	0.5455	0.5909	1.0000
												FS05	0.6818	0.6364	0.6818	0.5455	1.0000
FS06	0.7273	0.5000	0.6364	0.6818	0.5909	1.0000
												FS07	0.6364	0.6818	0.7273	0.4091	0.6818	0.3636	1.0000
FS08	0.5909	0.5455	0.5000	0.8182	0.6364	0.7727	0.3182	1.0000
												FS09	0.6364	0.5000	0.5455	0.4091	0.7727	0.5455	0.6364	0.5909	1.0000
FS10	0.8182	0.5909	0.7273	0.5909	0.5000	0.5455	0.5455	0.5909	0.5455	1.0000
												FS11	0.5909	0.5455	0.5909	0.4545	0.7273	0.4091	0.7727	0.4545	0.7277	0.5000	1.0000

6D, calculating genetic similarity coefficients (table 6) among 11 millet varieties by using NTsys-pc 2.11 according to characters 0 and 1 of each variety obtained by SSR analysis, wherein the genetic similarity coefficients are 1, the same variety is obtained, and the genetic similarity coefficients are not 1, and the variety is not one variety. The genetic similarity coefficient among the varieties is 0.3182-0.9091, the genetic similarity coefficient between FS07 and FS08 is minimum, the genetic similarity coefficient between FS01 and FS03 is maximum, and the average genetic similarity coefficient of 11 varieties is 0.6008; it can be seen that the 5 pairs of primers can effectively distinguish 11 millet varieties.

Example 2

The embodiment provides a method for identifying millet varieties by adopting an SSR molecular marker technology, which comprises the following steps:

1. preparing a millet material: planting 45 kinds of millet materials such as big-leaf yellow millet, purple peduncle, Qianjiang tight millet, four-inch red root, red root rope head, thin-skinned white millet, hen mouth and the like in a greenhouse; freezing the germinated millet sprouts of about 10-12 days in liquid nitrogen, and storing in an ultra-low temperature refrigerator at-80 deg.C for use; the information of the 45 varieties of millet is shown in table 7.

Table 745 variety information

2. Extraction of millet DNA: extracting DNA of various millet materials by using a plant genome kit, and detecting the purity of the DNA by using 1% agarose gel electrophoresis; diluting the DNA concentration of each material to 50 ng/mu L, and placing the diluted DNA in a refrigerator at the temperature of-20 ℃ for later use;

3. SSR primers: 5 pairs of SSR core primers suitable for identifying the variety of the millet are screened again in the example 1;

4. PCR amplification reaction and electrophoresis detection: the total volume of the PCR reaction was 25. mu.L, including 10 XPCR buffer 2.5. mu.L, MgCl2(25mM) 3. mu.L, dNTP (10mM) 1.25. mu.L, forward primer (10mM)1. mu.L, reverse primer (10mM)1. mu.L, Taq enzyme (5U/. mu.L) 0.2. mu.L, template DNA (50 ng/. mu.L) 1. mu.L, ddH2O 16.5.5. mu.L; the reaction procedure for PCR was: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃; adding 4 mu L of 6 times Loading buffer into the PCR amplification product, taking 1.5 mu L of sample, detecting by 8% non-denatured polyacrylamide gel electrophoresis, and carrying out silver staining and color development.

5. Statistical analysis: observing the electrophoresis result of the PCR product, counting the stable and easily-distinguished difference bands by Gel-Pro analyzer software, recording the band according to a 0 and 1 system, assigning a value of 1 when the band exists, assigning a value of 0 when the band does not exist, and constructing corresponding 0 and 1 characters of each variety; recording results, and calculating a genetic similarity coefficient by using NTsys-pc 2.11; the recorded results were used to calculate genetic diversity data such as Polymorphic Information Content (PIC), Shannon's index (I), effective gene (Ne), and the like, using POPGENE 32. 6. Results and analysis:

6A, the millet DNA strips extracted by the kit are clear and bright, have no degradation phenomenon and have good DNA integrity, and the ratio of A260 to 280 detected by a spectrophotometer is within 1.8-2.0, so that the PCR requirements can be met;

6B, according to the SSR result, 35 alleles are obtained from 5 pairs of millet SSR primers, and 7 alleles are obtained from each pair of primers on average; characters of 0 and 1 of 45 millet varieties are shown in a table 8; the polymorphic information index of each primer is 0.688-0.846, the average value is 0.779, wherein the primer with the best amplification polymorphism is SiG04, and the table 9 shows. The selected core primers have polymorphic information indexes larger than 0.65, so that the primers have good polymorphism and can effectively identify 45 millet varieties.

TABLE 845 characters of millet variety 0, 1

TABLE 9 amplification results of core primer for variety identification of millet

6C, calculating genetic similarity coefficients (table 10) among 45 millet varieties by using NTsys-pc 2.11 according to characters 0 and 1 of each variety obtained by SSR analysis, wherein the genetic similarity coefficients among the varieties are 0.6571-0.9429, and the average genetic similarity coefficient of the 45 millet varieties is 0.7554; therefore, the selected primers have good polymorphism and identification energy, and can effectively distinguish 45 millet varieties. The identification accuracy is up to 100%.

Genetic similarity coefficients between table 1045 millet varieties

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (9)

1. A method for identifying millet varieties by adopting an SSR molecular marker technology is characterized in that millet DNA is extracted, PCR amplification reaction is carried out on the millet DNA and an SSR core primer by using 5, and the millet varieties are identified according to electrophoresis detection results after electrophoresis detection; the nucleotide sequences of the 5 pairs of SSR core primers are shown as SEQID NO. 1-10.

2. The method of claim 1, comprising the steps of:

1) extracting millet DNA;

2) carrying out PCR amplification reaction on the SSR core primers by using the millet DNA obtained in the step 1) and 5, and then carrying out 8% non-denaturing polyacrylamide gel electrophoresis detection and silver staining for color development; each pair of SSR core primers comprises a forward primer and a reverse primer, and the nucleotide sequences of 5 pairs of SSR core primers are shown as SEQID No. 1-10;

3) and (3) observing the electrophoresis result in the step 2), counting the stable and easily-distinguished difference bands, recording the band according to a 0 and 1 system, assigning a value of 1 when the band has the difference, assigning a value of 0 when the band has no difference, calculating a genetic similarity coefficient according to the recorded result, and identifying the variety of the millet.

3. The method as claimed in claim 2, wherein the step 1) of extracting the millet DNA is to take millet sprouts, store the millet sprouts at-80 ℃ after freezing by liquid nitrogen for later use, and then extract the millet DNA by using a plant genome kit.

4. The method according to claim 3, wherein the millet sprouts are sprouts of 10-15 days after the millet sprouts.

5. The method as claimed in claim 2, wherein the variety of said millet in step 1) is selected from the group consisting of yellow rice grain with resource pool number 00008364, black petiole hilted broadsword with resource pool number 00009383, cleaver tight rice with resource pool number 00012203, red root with resource pool number 00012209, red root rope head with resource pool number 00012213, thin skinned white rice with resource pool number 00012215, hen mouth with resource pool number 00022002, plumepoppy rice with resource pool number 00009701, dry sharp rough rice with resource pool number 00009702, sixty days returning to warehouse with resource pool number 00009703, halter head with resource pool number 00009704, golden sprout yellow with resource pool number 00009705, mule with resource pool number 00009706, small purple sprout rice with resource pool number 00009707, red wine rice with resource pool number 00009834, black rice pool number 00020449, cuckoo with resource pool number 6866, cuckoo string with resource pool number 00011262, bird pool number 00011274, and wheat tail pool number 00011274, Small rope head with resource library number 00011276, red seedling golden rake with resource library number 00011277, bamboo leaf red with resource library number 00011279, pheasant with resource library number 00013056, red seedling rope head with resource library number 00013951, goose neck with resource library number 00013964, cooked stick with resource library number 00001571, eight-channel with resource library number 00001572, dry tip with resource library number 00001573, bamboo leaf green with resource library number 00001574, young sprout with resource library number 00001631, white valley with resource library number 00003008, garlic skin white valley with resource library number 00003017, jade yellow valley with resource library number 00003021, flush red with resource library number 00015493, yellow roughneck with resource library number 00004584, purple bar valley with resource library number 00004591, white dew yellow with library number 00004609, pressing vehicle valley with resource library number 00004610, first green with resource library number 00004611, Zhang black grain with resource library number 5928, black grain with resource library number 599, Small lupine with the resource pool number of 00018210 or white wool grain with the resource pool number of 00018211; the resource library number is from the Chinese crop germplasm information network.

6. The method of claim 2, wherein the PCR amplification reaction of step 2) comprises 10 XPCR buffer, 25mM MgCl₂10mM dNTP, 10mM forward primer, 10mM reverse primer, 5U/. mu.L Taq enzyme, 50 ng/. mu.L template DNA and ddH₂O。

7. The method according to claim 2, wherein the PCR amplification reaction of step 2) is performed in a total volume of 25 μ L, which comprises 10 XPCR buffer 2.5 μ L, 25mM MgCl₂3 μ L, 10mM dNTP 1.25 μ L, 10mM forward primer 1 μ L, 10mM reverse primer 1 μ L, 5U/. mu.L Taq enzyme 0.2 μ L, 50 ng/. mu.L template DNA1 μ L, ddH₂O 16.5μL。

8. The method of claim 2, wherein the PCR amplification reaction of step 2) is performed by: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

9. The method according to claim 2, characterized by the specific steps of:

1) extraction of millet DNA: taking the germinated millet sprouts of 10-15 days, freezing by liquid nitrogen, storing at-80 ℃ for later use, and extracting the DNA of the millet sprouts by using a plant genome kit to obtain millet DNA;

2) PCR amplification and electrophoresis detection: carrying out PCR amplification reaction on the SSR core primers with nucleotide sequences shown as SEQID No.1-10 by using the millet DNA obtained in the step 1) and 5; then carrying out 8% non-denaturing polyacrylamide gel electrophoresis detection on the PCR product, and carrying out silver staining for color development;

each pair of SSR core primers comprises a forward primer and a reverse primer; the total volume of the PCR amplification reaction system is 25 mu L, wherein the total volume comprises 10 XPCR buffer 2.5 mu L and 25mM MgCl₂3 μ L, 10mM dNTP 1.25 μ L, 10mM forward primer 1 μ L, 10mM reverse primer 1 μ L, 5U/. mu.L Taq enzyme 0.2 μ L, 50 ng/. mu.L template DNA1 μ L, ddH₂O16.5 mu L; the PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 45s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃;

3) and (3) observing the electrophoresis result in the step 2), counting the stable and easily-distinguished difference bands, recording the band according to a 0 and 1 system, assigning a value of 1 when the band has the difference, assigning a value of 0 when the band has no difference, calculating a genetic similarity coefficient according to the recorded result, and identifying the variety of the millet.