A molecular marker method for assisting in the identification of low-nitrogen tolerant millet and its application

Abstract

This invention discloses a molecular marker method and its application for assisting in the identification of low-nitrogen-tolerant millet, comprising the following steps: Step 1: Extracting DNA from the millet seed to be tested; Step 2: Performing PCR amplification on the millet DNA; Step 3: Detecting the PCR amplification product. If a specific fragment of 501 bp is amplified, it is a low-nitrogen-tolerant millet variety. Experiments have demonstrated that SiM65737 of this invention is a molecular marker related to low-nitrogen tolerance in millet, which can be used for early screening and prediction of nitrogen utilization in millet, and can be used for marker-assisted breeding and the selection of low-nitrogen-tolerant millet varieties. The SiM65737 molecular marker can be detected in different tissues and developmental stages of millet, which facilitates faster screening and detection of nitrogen absorption and utilization efficiency in millet.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a method and application of molecular markers for assisting in the identification of low-nitrogen-tolerant millet. Background Technology

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

[0003] However, in recent years, in order to achieve high yields on a large scale, people have blindly applied excessive amounts of nitrogen fertilizer, resulting in serious waste of resources and soil pollution problems. The main reasons are the scarcity of low-nitrogen tolerant varieties and the low utilization rate of nitrogen fertilizer.

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

[0005] my country accounts for approximately 80% of the world's millet planting area and 90% of the world's total millet production (Diao Xianmin, China Agricultural Science and Technology Press, 2007). While the production capacity of millet has been improved to some extent through the breeding of superior varieties and the improvement of cultivation systems, significant breakthroughs in yield, quality, and resistance have been difficult to achieve, resulting in stagnant growth in millet production. Rangel and Tanksley et al. pointed out that the limited 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 base and shorten the breeding cycle. Summary of the Invention

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

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A molecular marker method for assisting in the identification of low-nitrogen tolerant millet includes the following steps:

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

[0011] Step 2: Perform PCR amplification on millet DNA;

[0012] Step 3: Perform PCR amplification product detection. 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 listed in the table below:

[0014] .

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

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

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

[0018] Pre-degradation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 55℃ for 40 s, extension at 72℃ for 30 s, for a total of 36 cycles, followed by holding at 12℃.

[0019] Application of a molecular marker method for assisting in the identification of low-nitrogen-tolerant millet in millet breeding.

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

[0021] The genomic DNA of millet was amplified using the molecular marker SiM65737, and millet with a PCR amplification length of 501 bp was selected as breeding material.

[0022] Preferably, the millet breeding is for developing low-nitrogen-tolerant millet varieties.

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

[0024] The present invention achieves the following technical effects compared to the prior art:

[0025] Experiments have shown that amplifying millet genomic DNA using the molecular marker SiM65737 resulted in yield reductions of less than 20% for 87.5% of millet varieties with PCR amplification products of 501 bp after nitrogen stress treatment, while yield reductions of more than 30% for 88.9% of millet varieties with no PCR bands after nitrogen stress treatment. This indicates that SiM65737 of the present invention is a molecular marker related to low nitrogen tolerance in millet, which can be used for early screening and prediction of nitrogen utilization in millet, and can be used for marker-assisted breeding and selection of low nitrogen-tolerant millet varieties. The SiM65737 molecular marker can be detected in different tissues and developmental stages of millet, which is beneficial for more rapid screening and detection of nitrogen absorption and utilization efficiency in millet. Attached Figure Description

[0026] Figure 1 Electrophoretic images of various test materials detected by the molecular marker SiM65737 of this invention;

[0027] Where M: molecular weight marker; 1-41 represent different millet germplasms, and the arrow indicates the 501bp target band. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] This invention discloses a method for assisting in the identification of molecular markers for low-nitrogen-tolerant millet, comprising the following steps:

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

[0031] Step 2: Use the primers in the table below to perform PCR amplification of millet DNA;

[0032] ;

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

[0034] The PCR reaction system is as follows:

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

[0036] The PCR reaction conditions are as follows:

[0037] Pre-degradation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 55℃ for 40 s, extension at 72℃ for 30 s, for a total of 36 cycles, followed by holding at 12℃.

[0038] This invention discloses the application of a molecular marker method for assisting in the identification of low-nitrogen-tolerant millet in millet breeding.

[0039] Millet breeding application methods include:

[0040] The genomic DNA of millet was amplified using the molecular marker SiM65737, and millet with a PCR amplification length of 501 bp was selected as breeding material.

[0041] Millet breeding aims to develop low-nitrogen-tolerant millet varieties.

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

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

[0044] I. Development of SiM65737

[0045] Recombinant inbred lines (RILs) were obtained by crossing the low-nitrogen-tolerant variety Yugu 28 with the low-nitrogen-sensitive variety Qiyehuang. 120 RIL lines were subjected to low-nitrogen stress in the field, and the root system, plant height, biomass, nitrogen use efficiency, and grain yield of each line were examined. QTL mapping and analysis were performed. Based on the genome and transcriptome sequencing of the RIL lines, SiLNR1 was successfully identified; this gene has not been previously reported. SiM65737 is an Indel molecular marker linked to SiLNR1.

[0046] II. Low nitrogen stress treatment of 32 millet germplasm samples

[0047] Using 22 promoted varieties, 16 stable lines, and 3 germplasm resources as materials, low nitrogen stress treatments were conducted in soil-cultivated pots. Soil organic matter concentrations were 2.05 g / kg, total nitrogen 0.50 g / kg, available nitrogen 75.5 mg / kg, available potassium 35.22 mg / kg, and available phosphorus 17.35 mg / kg. Starting at the 5-leaf stage, each pot was irrigated with 1000 mL of modified Hoagland nutrient solution every 3 days. Low nitrogen stress treatment (Hoagland nutrient solution nitrogen concentration: 0.5 mmol / L) and normal nitrogen treatment (Hoagland nutrient solution nitrogen concentration: 2.5 mmol / L) were used. Six plants were planted per pot for each variety, with three replicates. At maturity, nine plants from each treatment of each variety were collected to investigate plant height, root biomass, grain yield, and nitrogen use efficiency. The results are shown in Table 1. Under low nitrogen stress, higher nitrogen use efficiency resulted in less reduction in grain yield and plant height.

[0048] like Figure 1 The image shown is an electrophoresis diagram of various test materials detected using the molecular marker SiM65737 in an embodiment of the present invention. Wherein, M: molecular weight marker; 1-41 represent different millet germplasms, and the arrow indicates the 501bp target band.

[0049] PCR products of 41 millet germplasms were detected using the molecular marker SiM65737 in Table 1.

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

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

[0052] This invention demonstrates that SiM65737 is a molecular marker for identifying low-nitrogen tolerance in millet, and can be used for early prediction and screening of low-nitrogen tolerant millet.

[0053] Table 1 shows the 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 41 millet varieties (lines) SiM65737

[0055]

[0056]

[0057]

[0058] Experiments have shown that amplifying millet genomic DNA using the molecular marker SiM65737 resulted in yield reductions of less than 20% for 87.5% of millet varieties with PCR amplification products of 501 bp after nitrogen stress treatment, while 88.9% of millet varieties with no PCR bands experienced yield reductions of more than 30% after nitrogen stress treatment. This indicates that SiM65737 of the present invention is a molecular marker related to low nitrogen tolerance in millet, which can be used for early screening and prediction of nitrogen utilization in millet, and can be used for marker-assisted breeding and the selection of low-nitrogen-tolerant millet varieties. The SiM65737 molecular marker can be detected in different tissues and developmental stages of millet, which is beneficial for more rapid screening and detection of nitrogen absorption and utilization efficiency in millet.

[0059] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for assisting in the identification of molecular markers for low-nitrogen-tolerant millet, characterized in that, Includes the following steps: Step 1: Extract DNA from the millet seeds to be tested; Step 2: Perform PCR amplification on millet DNA; Step 3: Perform PCR amplification product detection. If a specific fragment of 501 bp is amplified, it is a low-nitrogen tolerant millet variety. In step 2, PCR amplification is performed using the primers listed in the table below: 。 2. The method for auxiliary identification of molecular markers for low-nitrogen-tolerant millet according to claim 1, characterized in that, In step 3, the PCR reaction system is as follows: 10µl: Master PCR Mix 5µl, 10mM primers F and R 0.5µl each, template DNA 10ng 2µl, add ddH2O to 10µl.

3. The method for auxiliary identification of molecular markers for low-nitrogen-tolerant millet according to claim 1, characterized in that, In step 3, the PCR reaction conditions are as follows: Pre-degradation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 55℃ for 40 s, extension at 72℃ for 30 s, for a total of 36 cycles, followed by holding at 12℃.

4. The application of the molecular marker method for auxiliary identification of low-nitrogen-tolerant millet according to any one of claims 1-3 in millet breeding, characterized in that, The millet breeding mentioned above is for developing low-nitrogen-tolerant millet varieties.

5. The application according to claim 4, characterized in that, The millet breeding application method includes: The genomic DNA of millet was amplified using the primers described in claim 1, and millet with a PCR amplification length of 501 bp was selected as breeding material.

6. In the application according to claim 4, the low-nitrogen tolerant millet variety is a variety whose grain yield reduction is less than 20% under low nitrogen stress.

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