CAPS molecular marker for identifying soybean restorer gene Rf3 and recessive gene rf3 and application of CAPS molecular marker

Abstract

The invention provides an application of a reagent for detecting an SNP (Single Nucleotide Polymorphism) site in identifying a soybean restorer gene Rf3 and a recessive gene rf3. The SNP site corresponds to T / A base mutation at 41,115 and 564bp of a chromosome 9 of a reference genome G.max Wm82. A6. V1. The invention further provides a method for identifying the soybean restorer gene Rf3 and the recessive gene rf3. A CAPS molecular marker for identifying the soybean restorer gene Rf3 and the recessive gene rf3 is developed according to the SNP sites, the CAPS molecular marker is named as CAPS-564, and the nucleotide sequence of the CAPS-564 is shown as SEQ ID NO.3. The invention also discloses a method for identifying the soybean restorer gene Rf3 and the recessive gene rf3. A primer pair for amplifying the CAPS molecular marker comprises an upstream primer of which the nucleotide sequence is shown as SEQ ID NO.1 and a downstream primer of which the nucleotide sequence is shown as SEQ ID NO.2. The CAPS molecular marker disclosed by the invention can be used for effectively distinguishing a sterile fertility restoring gene Rf3 and a recessive gene rf3 and detecting purity identification of a hybrid seed prepared by a restoring line containing the Rf3 gene.

Description

Technical Field

[0001] The invention belongs to the technical field of hybrid soybean genetic breeding, and specifically relates to a CAPS molecular marker for identifying a soybean restoration gene Rf3 and a recessive gene rf3 and an application thereof. Background Art

[0002] As a native variety in my country, soybean (Glycine max (Linn.) Merr.) has a long history of cultivation. In recent years, my country's demand for soybeans has increased dramatically. According to rough statistics, the annual dependence on foreign soybeans exceeds 80%, which has greatly stimulated my country's food security policy. To this end, based on the limited existing arable land area, increasing the yield per unit area of ​​soybeans to alleviate the pressure of imported soybeans has become the research focus of many scientific researchers, especially after the launch of the soybean revitalization plan. One of the effective ways to increase yield is through hybrid breeding, that is, through parental hybridization to cultivate offspring with super-parent advantages, which are reflected in agronomic traits such as resistance and yield.

[0003] In the process of soybean hybrid breeding, the "three-line method" breeding is often used, namely sterile line, restorer line, and maintainer line. There are three main types of sterile lines reported so far, namely RN type, ZD type, and M type (Sun Huan et al., 1994; Zhao Limei et al., 1998; Zhao Limei et al., 2005; Zhang Lei et al., 1999). However, the discovery of sterile lines took a long time. At the end of the last century, the first sterile line in my country and even the world was cultivated by Sun Huan's research team at Jilin Academy of Agricultural Sciences. The cultivation of restorer lines and maintainer lines was completed one after another, realizing the true "three-line" matching breeding, which greatly accelerated the rapid development of my country's soybean industry. Compared with sterile lines, there are more reports on the breeding of restorer lines. Existing studies have mainly focused on the A1 linkage group, D2 linkage group, J linkage group, M linkage group and O linkage group of soybean (Tang Fuyue et al., 2009; Dong Jiansheng et al., 2008; Dong et al., 2012; Wang et al., 2016; Zhao Limei et al., 2007; Yang et al., 2007; Li Shuguang et al., 2010). However, the molecular marker of the present invention is developed based on the Rf3 restorer gene on chromosome 9 (K linkage group) of the soybean genome, that is, the gene targeted by the present invention is a completely new gene.

[0004] The Rf3 gene was first located by the hybrid soybean research team of Jilin Academy of Agricultural Sciences. The gene is located on chromosome 9 of the restorer line JLR2, between the molecular markers BARCSOYSSR_09_1161 and BARCSOYSSR_09_1184 published on the soybean genome public website (https: / / www.soybase.org), with an interval of 272.7 Kb. Based on this interval, it is temporarily named the fertility restoration segment. For this segment, the ZH13 v2.0 genome (Shen et al., 2019) was used as the reference genome, JLR2 was used as the reference parent, and the sterile line JLCMS5A was used as the comparison parent. The F 2 The isolate population was subjected to BSA sequencing.

[0005] Currently, there are limited CAPS molecular marker primers for the soybean cytoplasmic male sterility restoration gene Rf3. Therefore, it is an urgent problem to provide a CAPS molecular marker primer that can identify the soybean restoration gene Rf3 and its recessive gene rf3 and recessive gene rf3. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a CAPS molecular marker for identifying soybean restoration gene Rf3 and recessive gene rf3 and its application in view of the above-mentioned deficiencies in the prior art. The present invention can effectively distinguish the genotype of hybrids of the Rf3 / rf3 system and identify the purity of hybrids. It can also be used for other related research of the Rf3 / rf3 system, such as the fine positioning of the Rf3 gene.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is: the use of a reagent for detecting SNP sites in identifying the soybean restoration gene Rf3 and the recessive gene rf3, wherein the SNP site corresponds to the T / A base mutation at 41,115,564bp on chromosome 9 of the reference genome G.max Wm82.a6.v1.

[0008] The present invention also provides a CAPS molecular marker developed based on the above SNP site for identifying soybean restoration gene Rf3 and recessive gene rf3. The CAPS molecular marker is named CAPS-564, and the nucleotide sequence of CAPS-564 is shown in SEQ ID NO.3.

[0009] The present invention also provides a primer pair for amplifying the CAPS molecular marker, the primer pair comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0010] The present invention also provides a method for identifying soybean restoration gene Rf3 and recessive gene rf3, comprising the following steps:

[0011] S1. Extracting the soybean sample DNA to be tested, using the soybean sample DNA to be tested as a template, and performing PCR amplification using the primer pair in claim 3 to obtain a PCR product;

[0012] S2, digesting the PCR product obtained in S1 with EcoNI to obtain a digestion product;

[0013] S3. Perform agarose gel electrophoresis on the enzyme cleavage product obtained in S2. If a 1623 bp electrophoresis band appears in the enzyme cleavage product, it contains the restoration gene Rf3; if a 1925 bp electrophoresis band appears in the enzyme cleavage product, it contains the recessive gene rf3.

[0014] Preferably, the reaction system for PCR amplification in S1 is: 2×Super Pfx MasterMix 10 μL, ddH 2 O7.8 μL, upstream primer 0.6 μL, downstream primer 0.6 μL and DNA template 1 μL;

[0015] The reaction conditions of the PCR amplification are as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing temperature at 61°C; extension at 72°C for 1 min, for a total of 35 cycles; final extension at 72°C for 10 min; and storage at 4°C.

[0016] Preferably, the EcoNI digestion reaction system in S2 is: 4 μL of the PCR product, 13.6 μL of ddHO 2 O, 0.4 μL of EcoNI restriction endonuclease, and 2 μL of enzyme digestion buffer; the reaction conditions of the EcoNI digestion are: reacting at 37° C. for 30 minutes.

[0017] Preferably, the reaction system for agarose gel electrophoresis detection in S3 is: agarose gel with a concentration of 1%, and the reaction conditions are: 150V, 40min.

[0018] The present invention also provides the application of the CAPS molecular marker and primer pair in distinguishing the soybean restoration gene Rf3 and the recessive gene rf3.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. The soybean Rf3 restoration gene linkage molecular marker in the present invention exists on the Rf3 gene, and is therefore closer to the Rf3 gene, and can more accurately identify the fertility of individual plants in the population.

[0021] 2. The identification method of the present invention is simple and easy to operate, and the test results are reliable: the restriction endonuclease used is a common enzyme, which is common in the market, and the enzyme cutting operation is convenient, and the operation can be completed in an ordinary laboratory, and the experimental environment requirements are not strict. Since the enzyme cutting recognition is specific, the test results are highly reliable.

[0022] 3. Currently, the soybean industry does not have a comprehensive understanding of the restorer gene. The Rf3 gene is a newly announced gene in the past two years, so this marker is a good auxiliary tool for screening materials containing the Rf3 gene. It is of great value for future breeding and increasing the abundance of restorer line germplasm resources.

[0023] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the result of identification of materials containing different restored genes. DETAILED DESCRIPTION

[0025] Example 1

[0026] This embodiment provides an application of a reagent for detecting a SNP site in identifying a soybean restorer gene Rf3 and a recessive gene rf3, wherein the SNP site corresponds to a T / A base mutation at 41,115,564 bp on chromosome 9 of a reference genome G.max Wm82.a6.v1.

[0027] This embodiment also provides a CAPS molecular marker developed based on the above SNP site for identifying soybean restoration gene Rf3 and recessive gene rf3. The CAPS molecular marker is named CAPS-564, and the nucleotide sequence of CAPS-564 is shown in SEQ ID NO.3.

[0028] This embodiment also provides a primer pair for amplifying the above CAPS molecular marker, wherein the primer pair comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2.

[0029] Example 2

[0030] This embodiment provides a method for identifying soybean restoration gene Rf3 and recessive gene rf3, comprising the following steps:

[0031] S1. Extracting the soybean sample DNA to be tested by using a plant genomic DNA extraction kit (purchased from Kangwei Century); using the soybean sample DNA to be tested as a template, performing PCR amplification using the primer pair in Example 1 to obtain a PCR product;

[0032] The PCR amplification reaction system was 20 μL, including 10 μL 2×Super Pfx MasterMix, 7.8 μL ddHO 2 O, 0.6 μL of upstream primer and downstream primer, 1 μL of DNA template; the reaction procedure of the PCR amplification: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing temperature at 61°C; extension at 72°C for 1 min, a total of 35 cycles; final extension at 72°C for 10 min; storage at 4°C;

[0033] S2, digesting the PCR product obtained in S2 with EcoNI to obtain a digestion product;

[0034] The EcoNI digestion system 20 μL, including 4 μL of the PCR product, 13.6 μL of ddHO 2 O, 0.4 μL EcoNI restriction endonuclease, 2 μL enzyme digestion buffer; the reaction conditions of the EcoNI digestion are: 30 min at 37°C;

[0035] S3. Perform agarose gel electrophoresis on the enzyme cleavage product obtained in S3, prepare agarose gel electrophoresis with a concentration of 1%, and perform electrophoresis at a voltage of 150V for 40 minutes; if the enzyme cleavage product shows an electrophoresis band of 1623bp, it contains the recovery gene Rf3; if the enzyme cleavage product shows an electrophoresis band of 1925bp, it contains the recessive gene rf3.

[0036] 35 different species of materials were selected (as shown in Table 1) and identified using the method in Example 2. The results are as follows: Figure 1 shown.

[0037] Table 135 List of species and resources

[0038]

[0039]

[0040] Figure 1 These are the identification results of materials containing different restoration genes, among which lanes 23, 24, and 25 contain the restoration Rf3 gene, while lanes 33, 34, and 35 do not contain the restoration Rf3 gene, indicating that the CAPS molecular marker provided by the present invention has a wide range of applicability and can be used to detect the restoration gene Rf3 of introduced germplasm and resources. Figure 1 A and B are electrophoresis diagrams of PCR products of CAPS-564, M: MakerⅢ, 1-35 are PCR amplification products of 35 varieties and resources; C and D are electrophoresis diagrams of PCR products of CAPS-564 after restriction digestion, M: M: MakerⅢ, 1-35 are PCR amplification products of 35 varieties and resources after EcoNⅠ digestion.

[0041] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Application of a reagent for detecting SNP sites in identifying soybean restoration gene Rf3 and recessive gene rf3, characterized in that: The SNP site corresponds to the T / A base mutation at 41,115,564 bp on chromosome 9 of the reference genome G.max Wm82.a6.v1.

2. A CAPS molecular marker developed according to the SNP locus of claim 1 for identifying soybean restoration gene Rf3 and recessive gene rf3, characterized in that: The CAPS molecular marker is named CAPS-564, and the nucleotide sequence of CAPS-564 is shown in SEQ ID NO.

3.

3. A primer pair for amplifying the CAPS molecular marker according to claim 2, characterized in that: The primer pair includes an upstream primer whose nucleotide sequence is shown as SEQ ID NO.1 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO.

2.

4. A method for identifying soybean restoration gene Rf3 and recessive gene rf3, characterized in that: The following steps are involved: S1. Extracting the soybean sample DNA to be tested, using the soybean sample DNA to be tested as a template, and performing PCR amplification using the primer pair in claim 3 to obtain a PCR product; S2, digesting the PCR product obtained in S1 with EcoNI to obtain a digestion product; S3. Perform agarose gel electrophoresis on the enzyme cleavage product obtained in S2. If a 1623 bp electrophoresis band appears in the enzyme cleavage product, it contains the restoration gene Rf3; if a 1925 bp electrophoresis band appears in the enzyme cleavage product, it contains the recessive gene rf3.

5. A method for identifying soybean restoration gene Rf3 and recessive gene rf3 according to claim 4, characterized in that: The reaction system for PCR amplification described in S1 was: 2× Super Pfx MasterMix 10 μL, ddH2O 7.8 μL, upstream primer 0.6 μL, downstream primer 0.6 μL and DNA template 1 μL; The reaction conditions of the PCR amplification are as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing temperature at 61°C; extension at 72°C for 1 min, for a total of 35 cycles; final extension at 72°C for 10 min; and storage at 4°C.

6. A method for identifying soybean restoration gene Rf3 and recessive gene rf3 according to claim 4, characterized in that: The reaction system of EcoNI digestion in S2 is: 4 μL of the PCR product, 13.6 μL of ddH2O, 0.4 μL of EcoNI restriction endonuclease, and 2 μL of digestion buffer; the reaction conditions of EcoNI digestion are: reaction at 37°C for 30 minutes.

7. A method for identifying soybean restoration gene Rf3 and recessive gene rf3 according to claim 4, characterized in that: The reaction system of agarose gel electrophoresis detection in S3 is: agarose gel with a concentration of 1%, and the reaction conditions are: 150V, 40min.

8. Use of the CAPS molecular marker according to claim 2 and the primer pair according to claim 3 in distinguishing the soybean restoration gene Rf3 and the recessive gene rf3.

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