Molecular marker method for auxiliary identification of low-nitrogen-resistant millet and application

Abstract

The invention discloses a molecular marker method for assisting in identifying low-nitrogen-resistant millet and application. The molecular marker method comprises the following steps: step 1, extracting DNA (Deoxyribose Nucleic Acid) of to-be-detected millet; 2, carrying out PCR (Polymerase Chain Reaction) amplification on the millet DNA; 3, PCR amplification product detection is conducted, and if a specific fragment with the size of 501 bp is obtained through amplification, the millet variety is the low-nitrogen-resistant millet variety. Experiments prove that the SiM65737 disclosed by the invention is a molecular marker related to low-nitrogen resistance of the millet, can be used for early screening and prediction of nitrogen utilization of the millet, and can be used for molecular marker-assisted breeding and low-nitrogen-resistant millet variety breeding. The SiM65737 molecular marker can be detected in different tissues and developmental stages of millet, so that the nitrogen absorption and utilization efficiency of millet can be screened and detected more quickly.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a molecular marker method for assisting identification of low-nitrogen resistant millet and its application. Background Art

[0002] Millet has strong adaptability and good mineral nutrient absorption characteristics. It is often used as a cultivated crop in soil-poor areas and has gradually developed into an important coarse grain crop in my country. Nitrogen is an important element necessary for crop growth and development, and it is also the most lacking nutrient element in the soil (Lu Haidong et al., Journal of Applied Ecology, 2010). Applying nitrogen fertilizer is one of the important ways to increase crop yields (JU C, Field Crops Research, 2015).

[0003] However, in recent years, in order to achieve high yields over large areas, people have blindly applied excessive amounts of nitrogen fertilizers, resulting in serious waste of resources and soil pollution. The main reasons are the lack of low-nitrogen tolerant varieties and low nitrogen fertilizer utilization rates.

[0004] Therefore, breeding low-nitrogen tolerant varieties and improving the nitrogen absorption and utilization efficiency of crops become the key to solving this series of problems.

[0005] my country's millet planting area accounts for about 80% of the world's millet planting area, and its output accounts for 90% of the world's total millet output (Diao Xianmin, China Agricultural Science and Technology Press, 2007). Through the selection of excellent varieties and the improvement of cultivation systems, the production capacity of millet has been improved to a certain extent, but it is difficult to make major breakthroughs in terms of yield, quality and resistance, which has caused the production level of millet to stagnate. Rangel and Tanksley pointed out that the narrow breeding resources are one of the fundamental reasons for this phenomenon (Rangel PH, Pesq Agropec Bras, 1996; Tanksley SD, Science, 1997).

[0006] Therefore, current crop breeding urgently needs to discover new genes and develop new molecular markers to broaden the genetic basis and shorten the breeding period. Summary of the invention

[0007] In view of this, the present invention provides a molecular marker method and application for assisting in identifying low-nitrogen tolerant millet.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A molecular marker method for assisting identification of low nitrogen tolerance millet comprises the following steps:

[0010] Step 1: Extract the millet DNA to be tested;

[0011] Step 2: PCR amplification of millet DNA;

[0012] Step 3: Detect the PCR amplification product. If a specific fragment of 501 bp is amplified, it is a low-nitrogen tolerant millet variety.

[0013] Preferably, in step 2, PCR amplification is performed using the primers in the following table:

[0014]

[0015] Preferably, in step 3, the PCR reaction system is:

[0016] 10μl: Master PCRMix 5μl, 10mM primers F and R 0.5μl each, template DNA 10ng 2μl, add ddH2O to 10μl.

[0017] Preferably, in step 3, the PCR reaction conditions are:

[0018] Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 30 s, for a total of 36 cycles, and insulation at 12°C.

[0019] Application of a molecular marker method to assist in identifying low-nitrogen tolerance millet in millet breeding.

[0020] Preferably, the millet breeding application method comprises:

[0021] The above molecular marker SiM65737 was used to amplify millet genomic DNA, and millet with a PCR amplification length of 501 bp was selected as breeding material.

[0022] Preferably, the millet breeding is to cultivate low-nitrogen tolerant millet varieties.

[0023] Preferably, the low-nitrogen tolerant millet variety is a variety whose grain yield reduction under low-nitrogen stress is less than 20%.

[0024] Compared with the prior art, the present invention has achieved the following technical effects:

[0025] Experiments have shown that when the genomic DNA of millet is amplified using the molecular marker SiM65737, the yield reduction of millet varieties with a PCR amplification product size of 501 bp after low nitrogen stress treatment is less than 20%, while the yield reduction of millet varieties with no PCR product bands of 88.9% after nitrogen stress treatment is greater than 30%, indicating that the SiM65737 of the present invention is a molecular marker related to millet tolerance to low nitrogen, which can be used for early screening and prediction of millet nitrogen utilization, molecular marker-assisted breeding and low nitrogen tolerance millet variety selection. The SiM65737 molecular marker can be detected in different millet tissues and developmental stages, which is conducive to faster screening and detection of millet nitrogen absorption and utilization efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Electrophoresis diagram of each test material detected by the molecular marker SiM65737 of the present invention;

[0027] Wherein, M: molecular weight marker; 1-41 represent different millet germplasms, and the arrow indicates the target band of 501 bp. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention discloses a molecular marker method for assisting identification of low-nitrogen tolerant millet, comprising the following steps:

[0030] Step 1: Extract the millet DNA to be tested;

[0031] Step 2: Perform PCR amplification of millet DNA using the primers in the table below;

[0032]

[0033] Step 3: Detect the PCR amplification product. If a specific fragment of 501 bp is amplified, it is a low-nitrogen tolerant millet variety;

[0034] The PCR reaction system is:

[0035] 10μl: Master PCRMix 5μl, 10mM primers F and R 0.5μl each, template DNA 10ng 2μl, add ddH2O to 10μl;

[0036] The PCR reaction conditions were:

[0037] Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 30 s, for a total of 36 cycles, and insulation at 12°C.

[0038] The invention discloses application of a molecular marker method for assisting identification of low-nitrogen resistant millets in millet breeding.

[0039] Millet breeding application methods include:

[0040] The above molecular marker SiM65737 was used to amplify millet genomic DNA, and millet with a PCR amplification length of 501 bp was selected as breeding material.

[0041] Millet breeding is to cultivate low nitrogen tolerant millet varieties.

[0042] Low nitrogen tolerant millet varieties are varieties whose grain yield reduction under low nitrogen stress is less than 20%.

[0043] Example 1: SiM65737 is a molecular marker associated with low nitrogen tolerance in millet

[0044] 1. Development of SiM65737

[0045] The low nitrogen tolerant variety Yugu 28 was crossed with the low nitrogen sensitive germplasm Qiyehuang to obtain recombinant inbred lines (RILs). 120 RIL lines were subjected to field low nitrogen stress treatment, and the root system, plant height, biomass, nitrogen use efficiency and grain yield of each line were investigated, and QTL positioning and analysis were performed. Combined with the genome and transcriptome sequencing of the RIL lines, SiLNR1 was successfully identified, and no related research has been reported on this gene. SiM65737 is an Indel molecular marker linked to SiLNR1.

[0046] 2. Low nitrogen stress treatment of 32 millet germplasms

[0047] 22 promoted varieties, 16 stable lines and 3 germplasm resources were used as materials to carry out low nitrogen stress treatment in soil potted plants in the field. Soil potted plant treatment conditions: soil organic matter 2.05g / kg, total nitrogen 0.50g / kg, available nitrogen 75.5mg / kg, available potassium 35.22mg / kg, available phosphorus 17.35mg / kg. Starting from the 5-leaf stage, 1000mL of modified Hoagland nutrient solution was irrigated every 3 days per pot, low nitrogen stress treatment (nitrogen concentration of Hoagland nutrient solution was 0.5mmol / L), normal nitrogen treatment (nitrogen concentration of Hoagland nutrient solution was 2.5mmol / L), 6 plants were planted per pot for each variety, and 3 replicates were set. At maturity, 9 plants of each variety and each treatment were taken to investigate plant height, root biomass, grain yield and nitrogen utilization efficiency. The results are shown in Table 1. Under low nitrogen stress, the higher the nitrogen utilization efficiency, the less the grain yield and plant height will decrease.

[0048] like Figure 1 The electrophoresis diagram of each test material detected by using molecular marker SiM65737 in the embodiment of the present invention is shown, wherein M: molecular weight marker; 1-41 are different millet germplasms, and the arrow indicates the target band of 501 bp.

[0049] Detection of PCR products of 41 millet germplasms in Table 1 by molecular marker SiM65737

[0050] Genomic DNA was extracted from the 41 millet varieties (lines) in Table 1, and PCR amplification was performed using the primer sequences of SiM65737-F and SiM65737-R, and the PCR products were detected by 5.0% agarose gel electrophoresis. The detection results of the PCR products of the 41 varieties (lines) are shown in Table 1. The PCR amplification products of 32 millet varieties (lines) were 501 bp in size, of which 28 varieties (lines) had a yield reduction of less than 20% after low nitrogen stress treatment; the amplification products of the other 9 varieties (lines) had no bands, of which 8 varieties (lines) had a yield reduction of more than 30% after low nitrogen stress treatment.

[0051] Therefore, 87.5% of millet varieties (lines) with a PCR amplification product size of 501 bp are low-nitrogen tolerant varieties, while 88.9% of millet varieties (lines) with no PCR product bands are low-nitrogen sensitive varieties.

[0052] It is shown that SiM65737 of the present invention is a molecular marker for identifying millet tolerance to low nitrogen, and can be used for early prediction and screening of millet tolerance to low nitrogen.

[0053] Table 1 shows the results of amplification products, plant height, root biomass, grain yield per plant and nitrogen use efficiency of 41 millet varieties (lines) SiM65737.

[0054] Table 1. Amplification products, plant height, root biomass, grain yield and nitrogen use efficiency of SiM65737 in 41 millet varieties (lines)

[0055]

[0056]

[0057]

[0058] Experiments have shown that when the molecular marker SiM65737 is used to amplify millet genomic DNA, 87.5% of the millet varieties with a PCR amplification product size of 501bp have a yield reduction of less than 20% after low nitrogen stress treatment, while 88.9% of the millet varieties with no PCR product bands have a yield reduction of more than 30% after nitrogen stress treatment, indicating that the SiM65737 of the present invention is a molecular marker related to millet tolerance to low nitrogen, which can be used for early screening and prediction of millet nitrogen utilization, molecular marker-assisted breeding and low nitrogen tolerance millet variety breeding. The SiM65737 molecular marker can be detected in different millet tissues and developmental stages, which is conducive to faster screening and detection of millet nitrogen absorption and utilization efficiency.

[0059] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A molecular marker method for assisting identification of low nitrogen tolerance millet, characterized in that: The steps include: Step 1: Extract the millet DNA to be tested; Step 2: PCR amplification of millet DNA; Step 3: Detect the PCR amplification product. If a specific fragment of 501 bp is amplified, it is a millet variety that is tolerant to low nitrogen.

2. The molecular marker method for assisting identification of low nitrogen tolerance millet according to claim 1, characterized in that: In step 2, PCR amplification was performed using the primers in the following table:

3. The molecular marker method for assisting identification of low nitrogen tolerance millet according to claim 1, characterized in that: In step 3, the PCR reaction system is: 10μl: Master PCRMix 5μl, 10mM primers F and R 0.5μl each, template DNA 10ng 2μl, add ddH2O to 10μl.

4. The molecular marker method for assisting identification of low nitrogen tolerance millet according to claim 1, characterized in that: In step 3, the PCR reaction conditions are: Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 30 s, for a total of 36 cycles, and insulation at 12°C.

5. Application of a molecular marker method for assisting identification of low-nitrogen tolerant millet according to claims 1-4 in millet breeding.

6. The use according to claim 5, wherein the millet breeding application method comprises: The above molecular marker SiM65737 was used to amplify millet genomic DNA, and millet with a PCR amplification length of 501 bp was selected as breeding material.

7. The use according to claim 5, wherein the millet breeding is to cultivate low-nitrogen tolerant millet varieties.

8. The use according to claim 7, wherein the low nitrogen tolerant millet variety is a variety whose grain yield reduction under low nitrogen stress is less than 20%.

Images