CAPS molecular markers for identifying soybean restorer gene Rf3 and recessive gene rf3 and their applications
By developing CAPS molecular markers and enzyme digestion electrophoresis technology based on SNP sites, the problem of identifying soybean restorer gene Rf3 and recessive gene rf3 has been solved, enabling accurate identification of genotype and purity in soybean breeding, simplifying the operation process and improving the reliability of the test results.
Patent Information
- Application Number
- CN202510038863.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-10
- Publication Date
- 2025-10-31
- Estimated Expiration
- 2045-01-10
AI Technical Summary
The lack of effective CAPS molecular markers in existing technologies for identifying soybean restorer gene Rf3 and recessive gene rf3 makes it difficult to accurately distinguish the genotype and hybrid purity of Rf3/rf3 hybrids.
CAPS molecular markers based on SNP sites were developed. Using EcoNI enzyme digestion and agarose gel electrophoresis, the soybean restorer gene Rf3 and recessive gene rf3 were identified by primer pair PCR amplification and enzyme digestion product electrophoretic analysis.
It enables accurate identification of the soybean restorer gene Rf3 and recessive gene rf3, simplifies the operation process, and improves the reliability and accuracy of the test results. It is applicable to genotype and purity identification in soybean breeding.
Smart Images
Figure CN119570975B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of hybrid soybean genetic breeding technology, specifically involving a CAPS molecular marker for identifying soybean restorer gene Rf3 and recessive gene rf3 and its application. Background Technology
[0002] Soybean (Glycine max (Linn.) Merr.), as a native species of my country, has a long history of cultivation. One effective way to increase yield is through hybridization breeding, that is, to breed offspring with superior parental advantages through parental crossbreeding. These advantages are reflected in agronomic traits such as resistance and yield.
[0003] In soybean hybridization breeding, the "three-line method" is commonly used, consisting of a sterile line, a restorer line, and a maintainer line. Currently, three main types of sterile lines have been reported: RN, ZD, and M (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 considerable amount of time. At the end of the last century, the research team of Sun Huan at the Jilin Academy of Agricultural Sciences successfully cultivated a truly sterile line in my country and even globally. They subsequently completed the cultivation of restorer and maintainer lines, achieving a truly comprehensive "three-line" breeding system, which greatly accelerated the rapid development of my country's soybean industry. Compared to sterile lines, there are more reports on the breeding of restorer lines. Existing studies mainly focus on linkage groups A1, D2, J, M, and O in 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 this invention is based on the Rf3 restorer gene on chromosome 9 (K linkage group) of the soybean genome, that is, the gene targeted by this invention is a completely new gene.
[0004] The Rf3 gene was first mapped by the hybrid soybean research team at the Jilin Academy of Agricultural Sciences. This gene is located on chromosome 9 of the restorer line JLR2, specifically between the molecular markers BARCSOYSSR_09_1161 and BARCSOYSSR_09_1184 published on the soybean genome website (https: / / www.soybase.org), with a range of 272.7 kb. Based on this range, it was tentatively named the fertility restorer region. Using the ZH13 v2.0 genome (Shen et al., 2019) as the reference genome, JLR2 as the reference parent, and the sterile line JLCMS5A as the comparison parent, BSA sequencing was performed on the segregating F2 population of the hybrids.
[0005] Currently, there are limited CAPS molecular marker primers targeting the soybean cytoplasmic male sterility restorer gene Rf3. Therefore, providing a CAPS molecular marker primer capable of identifying the soybean restorer gene Rf3 and its recessive gene rf3 is an urgent problem to be solved. Summary of the Invention
[0006] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a CAPS molecular marker for identifying the soybean restorer gene Rf3 and the recessive gene rf3, and its application. This invention can effectively distinguish the genotypes of Rf3 / rf3 hybrids and identify the purity of hybrids. It can also be used for other related research on the Rf3 / rf3 system, such as the fine mapping of the Rf3 gene.
[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the application of reagents for detecting SNP sites in the identification of soybean restorer gene Rf3 and recessive gene rf3, wherein the SNP sites correspond to the T / A base mutation at 41,115,564 bp 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-mentioned SNP sites for identifying soybean restorer gene Rf3 and recessive gene rf3, the CAPS molecular marker being named CAPS-564, the nucleotide sequence of which is shown in SEQ ID NO.3.
[0009] The present invention also provides a primer pair for amplifying the above-mentioned 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] This invention also provides a method for identifying the soybean restorer gene Rf3 and the recessive gene rf3, comprising the following steps:
[0011] S1. Extract DNA from the soybean sample to be tested, and use the soybean sample DNA to be tested as a template to perform PCR amplification using the primer pair in claim 3 to obtain PCR products.
[0012] S2. The PCR product obtained in S1 is digested with EcoNⅠ enzyme to obtain the digested product;
[0013] S3. The enzyme digestion products obtained in S2 are detected by agarose gel electrophoresis. If the enzyme digestion products show a 1623bp electrophoretic band, they contain the restorer gene Rf3; if the enzyme digestion products show a 1925bp electrophoretic band, they contain the recessive gene rf3.
[0014] Preferably, the PCR amplification reaction system described in S1 is: 10 μL of 2×Super Pfx MasterMix, 7.8 μL of ddH2O, 0.6 μL of upstream primer, 0.6 μL of downstream primer, and 1 μL of DNA template;
[0015] The PCR amplification reaction conditions were as follows: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 s, annealing at 61 °C; 72 °C extension 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 restriction enzyme digestion reaction system in S2 is as follows: 4 μL of the PCR product, 13.6 μL of ddH2O, 0.4 μL of EcoNⅠ restriction endonuclease, and 2 μL of enzyme digestion buffer; the EcoNⅠ restriction enzyme digestion reaction conditions are: reaction at 37 °C for 30 min.
[0017] Preferably, the reaction system for the agarose gel electrophoresis detection described in S3 is: agarose gel with a concentration of 1%, and the reaction conditions are: 150 V, 40 min.
[0018] This invention also provides the application of the above-mentioned CAPS molecular markers and primer pairs in distinguishing between the soybean restorer gene Rf3 and the recessive gene rf3.
[0019] Compared with the prior art, the present invention has the following advantages:
[0020] 1. In this invention, the linkage molecular marker of the soybean Rf3 restorer gene is located on the Rf3 gene, and therefore is closer to the Rf3 gene, which can more accurately identify the fertility of individual plants in the population.
[0021] 2. The identification method of this invention is simple and easy to operate, and the detection results are reliable: the restriction endonuclease used is a commonly used enzyme, readily available in the market, and the enzyme digestion operation is convenient and can be completed in a regular laboratory with no strict requirements on the experimental environment. Because the enzyme digestion recognition is specific, the detection results are highly reliable.
[0022] 3. Currently, the understanding of restorer genes in the soybean industry is not comprehensive. The Rf3 gene is a newly disclosed gene in the last two years, making this marker a valuable tool for screening materials containing the Rf3 gene. It holds significant value for future breeding and increasing the abundance of restorer line germplasm resources.
[0023] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description
[0024] Figure 1 These are the identification results of materials containing different restoration genes. Detailed Implementation
[0025] Example 1
[0026] This embodiment provides the application of a reagent for detecting SNP sites in identifying the soybean restorer gene Rf3 and the recessive gene rf3. The SNP sites correspond to the T / A base mutation at 41,115,564 bp on chromosome 9 of the reference genome G.max Wm82.a6.v1.
[0027] This embodiment also provides a CAPS molecular marker developed based on the above-mentioned SNP sites for identifying the soybean restorer gene Rf3 and the 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-mentioned CAPS molecular marker, wherein the primer pair includes 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.
[0029] Example 2
[0030] This embodiment provides a method for identifying the soybean restorer gene Rf3 and the recessive gene rf3, including the following steps:
[0031] S1. Extract DNA from the soybean sample to be tested using a plant genomic DNA extraction kit (purchased from Kangwei Century); using the soybean sample DNA as a template, perform PCR amplification using the primer pairs in Example 1 to obtain PCR products.
[0032] The PCR amplification reaction system consisted of 20 μL, including 10 μL of 2×Super Pfx MasterMix, 7.8 μL of ddH2O, 0.6 μL each of the upstream and downstream primers, and 1 μL of DNA template. The PCR amplification reaction program was as follows: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 s, annealing at 61 °C; 72 °C extension for 1 min, for a total of 35 cycles; final extension at 72 °C for 10 min; and storage at 4 °C.
[0033] S2. The PCR product obtained in S2 is digested with EcoNⅠ enzyme to obtain the digested product;
[0034] The EcoNⅠ digestion system consists of 20 μL of the PCR product, 13.6 μL of ddH2O, 0.4 μL of EcoNⅠ restriction endonuclease, and 2 μL of digestion buffer. The EcoNⅠ digestion reaction conditions are: 37 °C for 30 min.
[0035] S3. The enzyme digestion products obtained in S3 are subjected to agarose gel electrophoresis. A 1% agarose gel electrophoresis solution is prepared and electrophoresed at 150 V for 40 min. If the enzyme digestion product shows a 1623 bp electrophoresis band, it contains the restorer gene Rf3. If the enzyme digestion product shows a 1925 bp electrophoresis band, it contains the recessive gene rf3.
[0036] Thirty-five different varieties of materials were selected (as shown in Table 1) and identified using the method described in Example 2. The results are as follows: Figure 1 As shown.
[0037] Table 1. List of 35 varieties and resources
[0038]
[0039] Figure 1 The results show the identification of materials containing different restorer genes. Lanes 23, 24, and 25 contain the restorer Rf3 gene, while lanes 33, 34, and 35 do not. This indicates that the CAPS molecular marker provided by this invention has a wide range of applicability and can be used to detect the restorer gene Rf3 in introduced germplasm and resources. Figure 1 In the image, A and B are electrophoresis images of PCR products of CAPS-564, M: Maker III, and 1-35 are PCR amplification products of 35 varieties and resources; C and D are electrophoresis images of PCR products of CAPS-564 after enzyme digestion, M: Maker III, and 1-35 are PCR amplification products of 35 varieties and resources digested with EcoNⅠ.
[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.
Claims
1. The application of reagents for detecting SNP sites in identifying soybean restorer 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 method for identifying the soybean restorer gene Rf3 and the recessive gene rf3, characterized in that, Includes the following steps: S1. Extract DNA from the soybean sample to be tested. Using the soybean sample DNA as a template, perform PCR amplification using the upstream primer with the nucleotide sequence shown in SEQ ID NO.1 and the downstream primer with the nucleotide sequence shown in SEQ ID NO.2 to obtain the PCR product. S2. The PCR product obtained in S1 is digested with EcoNⅠ enzyme to obtain the digested product; S3. The enzyme digestion products obtained in S2 are detected by agarose gel electrophoresis. If the enzyme digestion products show a 1623 bp electrophoretic band, they contain the restorer gene Rf3; if the enzyme digestion products show a 1925 bp electrophoretic band, they contain the recessive gene rf3.
3. The method for identifying the soybean restorer gene Rf3 and the recessive gene rf3 according to claim 2, characterized in that, The PCR amplification reaction system described in S1 is as follows: 10 μL of 2×Super Pfx MasterMix, 7.8 μL of ddH2O, 0.6 μL of upstream primer, 0.6 μL of downstream primer, and 1 μL of DNA template; The PCR amplification reaction conditions were as follows: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 s, annealing at 61 °C; 72 °C extension for 1 min, for a total of 35 cycles; final extension at 72 °C for 10 min; and storage at 4 °C.
4. The method for identifying the soybean restorer gene Rf3 and the recessive gene rf3 according to claim 2, characterized in that, The EcoNⅠ restriction enzyme digestion reaction system described in S2 is as follows: 4 μL of the PCR product, 13.6 μL of ddH2O, 0.4 μL of EcoNⅠ restriction endonuclease, and 2 μL of enzyme digestion buffer; the EcoNⅠ restriction enzyme digestion reaction conditions are as follows: reaction at 37 ℃ for 30 min.
5. The method for identifying the soybean restorer gene Rf3 and the recessive gene rf3 according to claim 2, characterized in that, The reaction system for agarose gel electrophoresis detection described in S3 is: 1% agarose gel, and the reaction conditions are: 150 V, 40 min.