Transcription detection method for identifying downy mildew resistance of soybean germplasm

Abstract

A transcription detection method for identifying downy mildew resistance of soybean germplasm. The transcription detection method for identifying the downy mildew resistance of the soybean germplasm comprises using an SAGT1 gene specific primer to measure the expression level of an SAGT1 gene in a soybean, using a PR1 gene specific primer to measure the expression level of a PR1 gene in the soybean, and determining the downy mildew resistance of the soybean on the basis of the expression levels of the SAGT1 and PR1 genes. Experiments prove that the transcription expression levels of the SAGT1 and PR1 genes are highly positively correlated with the soybean downy mildew resistance of soybeans, the consistency of a disease resistance result of soybeans detected by utilizing the expression levels of the SAGT1 and PR1 genes and a field identification result is as high as 70.00%, and the present invention can be used for identifying and screening the downy mildew resistance of soybean germplasm resources.

Description

A transcriptional detection method for identifying downy mildew resistance in soybean germplasm

[0001] Cross-reference to related applications: This application claims priority to the Chinese patent application (application number 202311710053.8) filed on December 13, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The invention relates to the field of biotechnology, and relates to a transcription detection method for identifying resistance of soybean germplasm to downy mildew. Background Art

[0003] In plant pathology research, plant disease resistance is categorized into vertical and horizontal resistance. Vertical resistance, also known as race-specific resistance, refers to a host variety that is immune or highly resistant to one or a few pathogenic races, while being highly susceptible to other races. Vertical resistance is typically controlled by a single or a few major genes. Because vertical resistance is race- or biotype-specific, widespread implementation in production can easily lead to the pathogenic race becoming dominant, causing infection and elimination, resulting in unstable and long-lasting disease resistance. Horizontal resistance, also known as non-race-specific resistance, is typically controlled by multiple genes and protects against multiple or all races, slowing the progression of the disease and minimizing damage to the host population. Horizontal resistance does not exert directional selection pressure on pathogenic races, nor does it cause race variation, thus preventing a loss of resistance within the variety. Consequently, resistance is stable and long-lasting.

[0004] In the past, breeding focused on the discovery and utilization of single dominant disease-resistance genes, achieving significant progress. However, there was a lack of molecular insights into how to improve horizontal resistance to achieve broad-spectrum and durable disease resistance. With the continuous deepening of research on disease-resistance signaling pathways, especially the SA signaling pathway and advances in transcriptome sequencing technology, new clues have been provided to address this issue. It is known that crop resistance to obligate and facultative parasites is regulated by the salicylic acid (SA) signaling pathway. Studies on the molecular mechanisms of SA biosynthesis, metabolism, long-distance transport, and key node genes have fully clarified the important role of the SA signaling pathway in plant disease resistance. Transcriptome sequencing analysis of disease resistance has accumulated a large amount of data on different crops and different diseases, but how to quickly and easily apply this massive amount of data in crop breeding remains an unresolved problem. To this end, based on the latest international progress and the applicant's many years of research and transcriptome analysis on the soybean downy mildew (SDM) resistance gene SAGT1, a new concept of transcriptional breeding (TB) has been proposed.

[0005] The molecular basis of transcriptional breeding is to select key target sites and key target genes that can represent the overall disease resistance level of the SA pathway, detect their transcriptional expression levels, and comprehensively evaluate the disease resistance levels of different materials (high resistance HR, resistance R, moderate resistance MR, susceptible S, and highly susceptible HS). By directly utilizing the innate and endogenous disease resistance mechanisms evolved by plants over a long period of time, and through hybridization, polymerization and accumulation of the effects of various positive disease resistance regulatory factors in the SA pathway, new germplasms and new varieties with broad-spectrum and long-lasting disease resistance can be cultivated.

[0006] Marker-assisted selection (MAS) has achieved significant success in stacking R genes and improving crop disease resistance. However, MAS focuses on a single point or a few, a small segment of DNA sequence. Its hybridization and pyramiding approach only stacks a few R genes, effectively creating a point-by-point stacking. TB, on the other hand, focuses on the SA signaling network encompassing a large number of genes, achieving a network-by-network stacking through hybridization, resulting in a more pronounced effect.

[0007] Invention Disclosure

[0008] The technical problem to be solved by the present invention is how to detect the resistance to downy mildew of soybean.

[0009] To solve the above technical problems, the present invention first provides the use of a substance for detecting the expression level of SAGT1 and / or PR1 genes in detecting or assisting in detecting soybean downy mildew resistance.

[0010] In the above application, the substance for detecting the expression levels of SAGT1 and PR1 genes can be composed of a substance for detecting the expression level of SAGT1 gene (such as a SAGT1 gene-specific primer pair) and a substance for detecting the expression level of PR1 gene (such as a PR1 gene-specific primer pair).

[0011] In the above application, the substance for detecting the expression level of the SAGT1 gene may include (or may be) a SAGT1 gene primer pair composed of two single-stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2 in the sequence list, or a SAGT1 gene primer pair composed of two single-stranded DNAs shown in SEQ ID No.5 and SEQ ID No.6 in the sequence list.

[0012] The material for detecting the expression level of the SAGT1 gene may further include specific primers for an internal reference gene (such as the Actin gene).

[0013] The substance for detecting the expression level of the SAGT1 gene may be composed of a SAGT1 gene primer pair and a specific primer of an internal reference gene.

[0014] The substance for detecting the expression level of the PR1 gene may include (or may be) a PR1 gene primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing or a PR1 gene primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 7 and SEQ ID No. 8 in the sequence listing.

[0015] The material for detecting the expression level of the PR1 gene may further include specific primers for an internal reference gene (such as the Actin gene).

[0016] The substance for detecting the expression level of the PR1 gene may be composed of a PR1 gene primer pair and a specific primer for an internal reference gene.

[0017] In the above, the specific primers for the internal reference gene can be an Actin gene primer pair consisting of two single-stranded DNAs shown in SEQ ID No.9 and SEQ ID No.10, or an Actin gene primer pair consisting of two single-stranded DNAs shown in SEQ ID No.11 and SEQ ID No.12.

[0018] The present invention also provides a method for detecting or assisting in detecting downy mildew resistance in soybeans, the method comprising: using the substance for detecting the expression levels of SAGT1 and PR1 genes to be tested to detect the expression levels of SAGT1 and PR1 genes relative to the Actin gene in the tested soybeans, and determining the downy mildew resistance of the tested soybeans according to the following IV based on the relative expression levels of the two genes:

[0019] I: The tested soybean with the relative expression level of SAGT1 gene>10 and the relative expression level of PR1 gene>3 is or is a candidate for high-resistance soybean;

[0020] II:II-1 or II-2:

[0021] II-1: The soybeans to be tested with a relative expression level of SAGT1 gene >10 and a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being disease-resistant soybeans;

[0022] II-2: The tested soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) and a relative expression level of PR1 gene>3 are or are candidates for disease-resistant soybeans;

[0023] III:III-1 or III-2 or III-3:

[0024] III-1: The tested soybeans with a relative expression level of SAGT1 gene of 0-5 (including 5) and a relative expression level of PR1 gene>3 are or are candidates for medium-resistant soybeans;

[0025] III-2: The soybean to be tested with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) and a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) is or is a candidate for medium-resistant soybean;

[0026] III-3: The tested soybeans with a relative expression level of SAGT1 gene >10 and a relative expression level of PR1 gene of 0-0.5 (including 0.5) are or are candidates for medium-resistant soybeans;

[0027] IV: IV-1 or IV-2:

[0028] IV-1: The soybeans to be tested with a relative expression level of SAGT1 gene of 0-5 (including 5) and a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being susceptible soybeans;

[0029] IV-2: The soybeans to be tested with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) and a relative expression level of PR1 gene of 0-0.5 (including 0.5) are or are candidates for being susceptible soybeans;

[0030] V: The soybean to be tested with a relative expression level of SAGT1 gene of 0-5 (including 5) and a relative expression level of PR1 gene of 0-0.5 (including 0.5) is or is a candidate for being a highly susceptible soybean.

[0031] In the above method, the soybean SAGT1 and PR1 gene expression levels may be the expression levels of SAGT1 and PR1 genes in soybean leaves.

[0032] The present invention also provides any of the following methods:

[0033] X1) A method for detecting or assisting in detecting soybean downy mildew resistance, comprising: using the substance for detecting SAGT1 gene expression level to detect the expression level of the soybean SAGT1 gene relative to the Actin gene, and determining soybean downy mildew resistance based on the relative expression level of the SAGT1 gene;

[0034] The soybeans to be tested with a relative expression level of SAGT1 gene >10 are or are candidates for high-resistance soybeans; the soybeans to be tested with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) are or are candidates for disease-resistant or moderate-resistance soybeans; the soybeans to be tested with a relative expression level of SAGT1 gene of 0-5 (including 5) are or are candidates for highly susceptible or susceptible soybeans;

[0035] X2) A method for detecting or assisting in detecting soybean downy mildew resistance, comprising: using the substance for detecting PR1 gene expression level to detect the expression level of the soybean PR1 gene relative to the Actin gene, and determining soybean downy mildew resistance based on the relative expression level of the PR1 gene;

[0036] Soybeans with a PR1 gene relative expression level >3 are or are candidates for high-resistant soybeans; soybeans with a PR1 gene relative expression level of 0.5-3 (excluding 0.5 and including 3) are or are candidates for disease-resistant or moderate-resistant soybeans; soybeans with a PR1 gene relative expression level of 0-0.5 (including 0.5) are or are candidates for highly susceptible or susceptible soybeans.

[0037] The present invention also provides a method for screening soybeans resistant to downy mildew, the method comprising: using the substance for detecting the expression levels of SAGT1 and PR1 genes to detect the expression levels of soybean SAGT1 and PR1 genes relative to the Actin gene, and screening soybeans resistant to downy mildew according to the following IV based on the relative expression levels of the two genes:

[0038] I: The soybean with SAGT1 gene relative expression level>10 and PR1 gene relative expression level>3 is or is a candidate for high-resistance soybean;

[0039] II:II-1 or II-2:

[0040] II-1: Soybeans with a SAGT1 gene relative expression level > 10 and a PR1 gene relative expression level of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being disease-resistant soybeans;

[0041] II-2: Soybeans with a SAGT1 gene relative expression level of 5-10 (excluding 5 and including 10) and a PR1 gene relative expression level >3 are or are candidates for disease-resistant soybeans;

[0042] III:III-1 or III-2 or III-3:

[0043] III-1: Soybeans with a SAGT1 gene relative expression level of 0-5 (including 5) and a PR1 gene relative expression level >3 are or are candidates for medium-resistant soybeans;

[0044] III-2: Soybeans with a SAGT1 gene relative expression level of 5-10 (excluding 5 and including 10) and a PR1 gene relative expression level of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being medium-resistant soybeans;

[0045] III-3: Soybeans with a relative expression level of SAGT1 gene >10 and a relative expression level of PR1 gene of 0-0.5 (including 0.5) are or are candidates for being medium-resistant soybeans;

[0046] IV: IV-1 or IV-2:

[0047] IV-1: Soybeans with a SAGT1 gene relative expression level of 0-5 (including 5) and a PR1 gene relative expression level of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being susceptible soybeans;

[0048] IV-2: Soybeans with a SAGT1 gene relative expression level of 5-10 (excluding 5 and including 10) and a PR1 gene relative expression level of 0-0.5 (including 0.5) are or are candidates for being susceptible soybeans;

[0049] V: Soybeans with a SAGT1 gene relative expression level of 0-5 (including 5) and a PR1 gene relative expression level of 0-0.5 (including 0.5) are or are candidates for being highly susceptible soybeans.

[0050] The present invention also provides any of the following methods:

[0051] X1) A method for screening soybeans resistant to downy mildew, comprising: using the substance for detecting the expression level of the SAGT1 gene to detect the expression level of the soybean SAGT1 gene relative to the Actin gene, and screening soybeans resistant to downy mildew based on the relative expression level of the SAGT1 gene:

[0052] Soybeans with a relative expression level of SAGT1 gene >10 are or are candidates for high-resistance soybeans; soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) are or are candidates for disease-resistant or moderate-resistance soybeans; soybeans with a relative expression level of SAGT1 gene of 0-5 (including 5) are or are candidates for highly susceptible or susceptible soybeans;

[0053] X2) A method for screening soybeans resistant to downy mildew, comprising: using the substance for detecting PR1 gene expression level to detect the expression level of the soybean PR1 gene relative to the Actin gene, and screening the soybeans resistant to downy mildew based on the relative expression levels of the PR1 gene:

[0054] The soybeans to be tested with a relative expression level of PR1 gene >3 are or are candidates for high-resistant soybeans; the soybeans to be tested with a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) are or are candidates for disease-resistant or moderate-resistant soybeans; the soybeans to be tested with a relative expression level of PR1 gene of 0-0.5 (including 0.5) are or are candidates for highly susceptible or susceptible soybeans.

[0055] The substance for detecting the expression level of SAGT1 and / or PR1 gene also falls within the protection scope of the present invention.

[0056] The use of the substance for detecting the expression level of SAGT1 and / or PR1 genes in the preparation of a product for detecting or assisting in detecting soybean downy mildew resistance also falls within the scope of protection of the present invention.

[0057] In the present invention, the soybean is Jilin Xiaoli No. 1, Kefeng No. 1, Kefeng No. 14, Zaofeng No. 5, Jingshanpu, Amsoy, Tiefeng No. 18, Dongnong 49, Tiefeng No. 31, Wuxing No. 1, Qihuang 25, Wenfeng No. 7, Binhai 13-1, Guanshui Tiejiaoqing, Heinong 10 or the soybeans in Table 6, or their offspring.

[0058] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 shows the correlation analysis of six genes associated with downy mildew resistance.

[0060] Figure 2 shows the evaluation of soybean downy mildew resistance using the nine-square grid method. Modes for Carrying Out the Invention

[0061] The experimental methods in the following examples, unless otherwise specified, are conventional methods. The materials, reagents, and instruments used in the following examples, unless otherwise specified, are commercially available. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0062] The Jingshanpu (Wang Ruixia, Breeding of soybean variety Chengdou No. 5 and its supporting cultivation technology, Hebei Agricultural Science, 2009, 13(6): 62-63 143) in the following examples is a biological material that the public can obtain from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0063] The Qihuang 25 in the following examples (Hao Xinxian et al., Breeding of a new soybean cyst nematode resistant variety Qihuang 25, Soybean Bulletin, Issue 3, 1996) is a biological material that the public can obtain from the applicant. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0064] The public can obtain the biomaterial Binhai 13-1 in the following examples (Wang Lan et al., Research on Agronomic and Quality Traits of Cultivated and Wild Soybeans, Soybean Science and Technology, Agricultural Production, 2014) from the applicant. The biomaterial is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0065] The Heilongjiang Agricultural Sciences, 1988) biomaterial Heilongjiang Agricultural Sciences, 10 (Wang Binru et al., New High-Yield, Disease-Resistant, and High-Quality Soybean Varieties: Heilongjiang Agricultural Sciences, 1988) in the following examples is available to the public from the applicant. This biomaterial is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0066] Plant total RNA extraction kit: Coolaber, product number: RE600; reverse transcription kit HiScript III 1st Strand cDNA Synthesis kit, Nanjing Vazyme, product number R312-02; real-time fluorescence kit Taq Pro Universal SYBR qPCR Master Mix, Nanjing Vazyme, product number: Q712-02; 2× Rapid Tap Master Mix (Vazyme), product number R222-03.

[0067] Example 1: Identification of soybean downy mildew-resistant germplasm resources using detection of key target genes

[0068] 1. Identification of downy mildew resistance in different soybean varieties in the field

[0069] Soybean downy mildew thrives in high humidity and cool temperatures (20-25°C). Sow 20-30 seeds of each soybean variety in the experimental fields of Shenyang Agricultural University to ensure that 15 plants were available for disease resistance testing.

[0070] 2. Inoculate soybean downy mildew pathogen (Peronospora manshurica) 40 days after soybean sowing and before flowering:

[0071] Diseased soybean leaves inoculated with soybean downy mildew pathogen (Peronospora manshurica) were picked, rinsed with tap water, disinfected with 75% ethanol for 0.5 min, and then kept moist at 18-20°C for 15 hours. A large number of spores were produced after washing and collecting the spores. The spore concentration was prepared to 1×10 5 The suspension of 100mg / mL is ready for use;

[0072] Use a sprayer to inoculate the spore suspension onto the plants of the material to be identified, focusing on spraying the back of the leaves. The inoculation amount should be controlled at 4ml / plant to 6mL / plant. Keep the plants moist for 24 hours after inoculation. Spraying or irrigation can be used to maintain humidity.

[0073] 3. 15 days after inoculation, when soybean disease is prevalent, investigate the middle leaves of each plant based on the lesion reaction type and number of lesions. Disease resistance was evaluated using a six-level grading system. Specific grading standards and resistance levels are shown in Tables 1 and 2.

[0074] Table 1. Grading standards for soybean downy mildew disease

[0075] Calculation method of disease index:

[0076] Disease index DI = Σ(disease level value × number of leaves with corresponding disease level) / (total number of leaves surveyed × highest disease level) × 100%.

[0077] Table 2. Resistance level classification

[0078] 4. Through field indirect bacterial identification, the highly resistant (HR) varieties among the above materials are: Jilin Xiaoli No. 1, Donnong 49 and Guanshui Tiejiaoqing; the resistant (R) varieties are: Zaofeng No. 5, Amsoy, Tiefeng No. 18 and Tiefeng No. 31; the moderately resistant (MR) varieties are: Kefeng No. 14 and Wuxing No. 1; the susceptible (S) varieties are: Jingshanpu, Qihuang No. 25, Wenfeng No. 7, and Heinong No. 10; the highly susceptible (HS) varieties are: Kefeng No. 1 and Binhai 13-1.

[0079] 2. Screening of key target genes

[0080] 1. Select 6 soybean plants of Jilin Xiaoli No. 1, a highly resistant variety to downy mildew, and Kefeng No. 1, a highly susceptible variety, both 40 days old and with the same growth conditions. Divide each variety into 2 groups (one group is inoculated with fungi, and the other group is not inoculated as a control CK). Inoculate the third healthy leaf from the top as the material:

[0081] The diseased leaves of soybean downy mildew pathogen (Peronospora manshurica) were removed from the diseased plants and the spores on the back of the diseased leaves were washed into a clean beaker with distilled water to prepare a spore suspension with a spore concentration of 1×10 per ml. 5 Use a brush to lightly dip the spore suspension and evenly brush it on the back of the soybean leaves; keep the inoculated soybean plants in an environment of 20-25℃ and high humidity.

[0082] 2. 72 hours after inoculation, diseased leaves were observed and collected. Total RNA from soybean leaves was extracted using a plant total RNA extraction kit. RNA quality was assessed by electrophoresis on a 1.5% agarose gel. Clear bands were observed, with the 28S band significantly brighter than the 18S band and no noticeable tailing, demonstrating no significant RNA degradation. RNA purity was further assessed using a NanoDrop analyzer, with an A260 / A280 ratio between 2.0 and 2.2. These results demonstrate that the extracted RNA was of high integrity and met the requirements for transcriptome sequencing.

[0083] 3. The RNA samples (Jilin Xiaoli 1CK, Kefeng No. 1 CK, Jilin Xiaoli 1 inoculated with bacteria, Kefeng No. 1 inoculated with bacteria, 3 biological replicates, a total of 12 RNA samples, RNA concentration of 400-500 ng / μL) were provided to Beijing Aovison Gene Technology Co., Ltd. for transcriptome sequencing.

[0084] 4. Based on the transcriptome sequencing results, genes related to resistance to soybean downy mildew pathogens were obtained, as follows:

[0085] A1(Glyma13g194400), ALD1(Glyma08g180600), CBP60D(Glyma03g39170), EDS5(Glyma11g112100), SARD1(Glyma08g04920), NDR1b(Glyma12g214100) , GR(Glyma16g27210), GST(Glyma07g16855), GST(Glyma14g03470), GST(Glyma20g23420), GT(Glyma03g34480), GT(Glyma08g26830), GT(Glyma16g29 330), GT(Glyma16g29400), NPR1(Glyma02g283300), NPR1(Glyma09g07440), PAL1.1(Glyma19g36620), PAL1.3(Glyma03g181600), PAL2.1(Glyma10g0 6600), PAL2.3 (Glyma13g20800), POD (Glyma08g19170), POD (Glyma13g20170), PR1 (Glyma15g06790), SAGT1 (Glyma08g41460) and UGT4 (Glyma16g29370).

[0086] 3. Detection of the correlation between key target gene expression levels and soybean downy mildew resistance

[0087] 1. Semi-quantitative RT-PCR was used to preliminarily analyze the expression of differentially expressed genes in five soybean varieties: the highly resistant variety Jilin Xiaoli 1 (JLXL1), the resistant variety Amsoy, the moderately resistant variety Kefeng 14 (KF14), the susceptible variety Qihuang 25 (QH25), and the highly susceptible variety Kefeng 1 (KF1). Total RNA was extracted from leaves using a plant total RNA extraction kit. RNA quality was assessed by electrophoresis on a 1.5% agarose gel, revealing clear bands and no significant RNA degradation. RNA was converted to cDNA using the HiScript IIQ RT SuperMix for qPCR kit (Vazyme, Cat. No. R222-01).

[0088] 2. Based on the gene sequences published by NCBI, Actin (Glyma18g290800) was used as the internal reference gene. PCR primers were designed for 25 genes (see Table 3). The expression of differentially expressed genes in five soybean varieties was preliminarily analyzed by RT-PCR.

[0089] The RT-PCR reaction system was as follows: 2× Rapid Tap Master Mix 25 μl; 1 μl cDNA template; 1 μl each of primers AC-F / AC-R (10 μM) and differential gene F / R (10 μM); and sterile ddH2O to make up to 50 μl.

[0090] The RT-PCR reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, annealing at the annealing temperature for 15 s, 72°C for 30 min (number of cycles shown in Table 3); 72°C for 5 min.

[0091] Table 3. Primers and their sequences used in semi-quantitative RT-PCR analysis

[0092] 3. The RT-PCR products were subjected to electrophoresis, and the expression level of soybean Actin was used as the internal standard to analyze the relative expression levels of the differentially expressed genes using Image J. The experimental results showed that no expression was detected in 11 genes, including A1 (Glyma13g194400), ALD1 (Glyma08g180600), CBP60D (Glyma03g39170), EDS5 (Glyma11g112100), NDR1b (Glyma12g214100), NPR1 (Glyma02g283300), NPR1 (Glyma09g07440), PAL1.3 (Glyma03g181600), POD (Glyma13g20170), SARD1 (Glyma08g04920), and UGT4 (Glyma16g29370). Although the expression of the remaining 14 genes was detected, only the expression of six genes, SAGT1 (Glyma08g41460), POD (Glyma08g19170), PAL2.1 (Glyma10g06600), PAL2.3 (Glyma13g20800), GST (Glyma20g23420), and PR1 (Glyma15g06790), was correlated with the disease resistance of the varieties (Table 4).

[0093] Table 4. Semi-quantitative RT-PCR analysis of the transcriptional expression of candidate target genes in soybeans with different resistances

[0094] 4. RNA was extracted from leaves of Jilin Xiaoli 1, Dongnong 49, Guanshui Tiejiaoqing, Zaofeng 5, Amsoy, Tiefeng 31, Tiefeng 18, Kefeng 14, Wuxing 1, Jingshanpu, Qihuang 25, Wenfeng 7, Kefeng 1, Binhai 13-1, and Heinong 10. RNA quality was assessed by 1.5% agarose gel electrophoresis, revealing clear bands, demonstrating no significant RNA degradation. RNA was then converted to cDNA.

[0095] 5. qRT-PCR was performed using a real-time fluorescence kit using real-time fluorescence PCR primers for SAGT1 (Glyma08g41460), PR1 (Glyma15g06790), POD (Glyma08g19170), PAL2.1 (Glyma10g06600), PAL2.3 (Glyma13g20800), GST (Glyma20g23420) and the internal reference gene Actin (Glyma18g290800) (Table 5).

[0096] The qRT-PCR reaction system was as follows: Taq Pro Universal SYBR qPCR Master Mix 10 μl, cDNA template 1 μl, primer F / primer R (10 μM) 0.4 μl each, and sterile ddH2O to make up to 20 μl.

[0097] qRT-PCR reaction program: 95°C for 30 s; 95°C for 10 s, 60°C for 15 s, 40 cycles; 95°C for 15 s, 60°C for 60 s, 95°C for 15 s.

[0098] Each sample was biologically replicated three times, and relative gene expression was calculated using the 2-ΔCT method. qRT-PCR was performed using an ABI 7500 fast PCR instrument.

[0099] Table 5. qRT-PCR primers for 6 candidate target genes

[0100] 6. qRT-PCR was used to detect the transcriptional expression levels of six target candidate genes, SAGT1 (Glyma08g41460), POD (Glyma08g19170), PAL2.1 (Glyma10g06600), PAL2.3 (Glyma13g20800), GST (Glyma20g23420) and PR1 (Glyma15g06790), in 15 different resistant materials (see Table 6). Among them, the correlation coefficient between the SAGT1 gene and downy mildew resistance was R 2 =0.7981; the correlation coefficient of PR1 is R 2 =0.7475; POD correlation coefficient R 2 =0.5315; correlation coefficient R of PAL2.1 2 =0.4263; correlation coefficient R of PAL2.3 2 =0.4857; the correlation coefficient of GST is R 2 =0.4761 (Figure 1). The results showed that all six genes were correlated with soybean downy mildew resistance. Among them, the transcription levels of the SAGT1 gene (XM_028390010.1; March 12, 2019) and the PR1 gene (NM_001371207.1; July 1, 2019) were relatively high and highly positively correlated with soybean downy mildew resistance. They can be used as detection target genes for the identification and screening of soybean germplasm resources for downy mildew resistance.

[0101] Table 6. qRT-PCR analysis of candidate target genes in soybeans with different SDM resistance

[0102] In Table 6: JL1: Jilin Xiaoli No. 1; DN49: Dongnong 49; GSTJQ: Guanshui Tiejiaoqing; ZF5: Zaofeng No. 5; TF31: Tiefeng No. 31; TF18: Tiefeng No. 18; KF14: Kefeng No. 14; WX1: Wuxing No. 1; JSP: Jingshanpu; QH25: Qihuang No. 25; WF7: Wenfeng No. 7; KF1: Kefeng No. 1; BH13-1: Binhai 13-1; HN10: Heinong 10.

[0103] 7. Method for detecting soybean downy mildew resistance using SAGT1 and PR1 gene expression levels

[0104] Based on the SAGT1 / Actin ratio (i.e., the relative expression level of SAGT1) of 15 soybean varieties with known disease resistance levels, the relative expression level of SAGT1 was divided into three intervals. The soybean downy mildew resistance can be determined according to the three intervals:

[0105] When the relative expression level of SAGT1 is >10, the soybean is classified as high-resistance soybean (HR);

[0106] When the relative expression level of SAGT1 is 5-10 (excluding 5 and including 10), the soybean is classified as disease-resistant or moderately resistant (R / MR).

[0107] The relative expression level of SAGT1 is 0-5 (including 5), and the soybean at this time is highly susceptible or susceptible soybean (HS / S).

[0108] Based on the PR1 / Actin ratio (i.e., the relative expression level of PR1) of 15 soybean varieties with known disease resistance levels, the relative expression level of PR1 is divided into three intervals. The downy mildew resistance of soybean can be determined based on the three intervals:

[0109] When the relative expression level of PR1 is >3, the soybean is classified as high-resistant soybean (HR);

[0110] The relative expression level of PR1 is 0.5-3 (excluding 0.5 and including 3), and the soybeans at this time are disease-resistant or moderately resistant soybeans (R / MR);

[0111] The relative expression level of PR1 is 0-0.5 (including 0.5), and the soybean at this time is highly susceptible or susceptible soybean (HS / S).

[0112] Based on the relative expression levels of SAGT1 and PR1 in 15 soybean varieties with known disease resistance levels, the relative expression levels of the two genes were divided into 5 levels to comprehensively determine soybean downy mildew resistance (Figure 2):

[0113] I: SAGT1 relative expression level > 10, and PR1 relative expression level > 3, at this time the soybean is high resistance soybean (HR);

[0114] II: II-1 or II-2, the soybeans at this time are disease-resistant soybeans (R):

[0115] II-1: SAGT1 relative expression level >10, and PR1 relative expression level 0.5-3 (excluding 0.5 and including 3);

[0116] II-2: SAGT1 relative expression level 5-10 (excluding 5 and including 10), and PR1 relative expression level >3;

[0117] III: III-1 or III-2 or III-3, the soybeans at this time are medium-resistant soybeans (HR):

[0118] III-1: SAGT1 relative expression level 0-5 (including 5), and PR1 relative expression level >3;

[0119] III-2: SAGT1 relative expression level 5-10 (excluding 5 and including 10), and PR1 relative expression level 0.5-3 (excluding 0.5 and including 3);

[0120] III-3: SAGT1 relative expression level >10, PR1 relative expression level 0-0.5 (including 0.5);

[0121] IV: IV-1 or IV-2, the soybeans at this time are susceptible soybeans (S):

[0122] IV-1: SAGT1 relative expression level 0-5 (including 5), and PR1 relative expression level 0.5-3 (excluding 0.5 and including 3);

[0123] IV-2: SAGT1 relative expression level 5-10 (excluding 5 and including 10), PR1 relative expression level 0-0.5 (including 0.5);

[0124] V: SAGT1 relative expression level is 0-5 (including 5), and PR1 relative expression level is 0-0.5 (including 0.5). At this time, the soybean is highly susceptible soybean (HS).

[0125] 4. Detection of soybean downy mildew resistance using SAGT1 and PR1 gene expression levels

[0126] 30 accessions were used to measure the transcript expression levels of the SAGT1 and PR1 genes in leaves using the qRT-PCR method described in step 3 (5). Three biological replicates were performed for each accession. Disease resistance achieved was determined based on these two genes alone or in combination, as described in step 2 (7). The results are shown in Table 7.

[0127] Thirty soybean varieties were sown in experimental fields at Shenyang Agricultural University for field inoculation testing. 20-30 seeds were seeded for each soybean variety, ensuring that 15 plants would be available for disease resistance testing. 20-30 seeds were seeded for each soybean variety, ensuring that 15 plants would be available for disease resistance testing. Inoculation testing was performed 40 days after sowing, before flowering. The testing method is described in Step 1. Specific disease resistance testing results are shown in Table 7.

[0128] Table 7. Transcriptional expression levels and disease resistance results of SAGT1 / Actin and PR1 / Actin in 30 soybean germplasm resources

[0129] Among them, Huaidou No. 10:

[0130] http: / / www.chinaseed114.com / seed / 2 / seed_7623.html;

[0131] Jidou No. 12: Literature: Research on the breeding of high-yield and high-protein soybean Jidou No. 12;

[0132] Ha No. 53: http: / / soy.cropdb.cn / variety / varis / 600178.htm;

[0133] LN92-7369: Literature: Phenolic Content and Antioxidant Properties of Soybean(Glycine max(L.)Merr.)Seeds;

[0134] M85-610W: Literature: ALPHA SOYBEANS;

[0135] Guixia No. 3: Literature: A new high-quality summer soybean variety - Guixia No. 3;

[0136] Shahe green beans: National Germplasm Resources Bank (https: / / www.cgris.net / query / croplist.php;

[0137] Dulu beans:

[0138] https: / / xueshu.baidu.com / usercenter / paper / show?paperid=1a5x0tg07t040tt0kx5p0430fg154841&site=xueshu_se%EF%BC%9B%20https: / / www.docin.com / p-781471305.html;

[0139] Harvest 22: https: / / www.doc88.com / p-0979787841113.html;

[0140] Putou 506: Literature: A new vegetable soybean variety - Quandou 8473 Qing;

[0141] Longdou No. 7:

[0142] http: / / www.a-seed.cn / html / 2020 / shendingpinzhong_0716 / 51841.html;

[0143] Kennong No. 2: https: / / www.1633.com / tec / otpupbzt.shtml;

[0144] Dongnong 64-286: Literature: Effect of sodium sulfite on soybean yield increase;

[0145] Longdou No. 7: National Germplasm Resources Bank (https: / / www.cgris.net / query / croplist.php;

[0146] Pingdou No. 2: Literature: Breeding and high-yield cultivation techniques of a new early-maturing and high-yielding soybean variety Pingdou No. 2;

[0147] Jiufeng No. 6:

[0148] https: / / baike.sogou.com / v168851705.htm?ch=frombaikevr&fromTitle=%E4%B9%9D%E4%B8%B06%E5%8F%B7;

[0149] Hao Dou 2000:

[0150] https: / / baike.sogou.com / v202497357.htm?fromTitle=%E9%83%9D%E8%B1%862000%E5%8F%B7&ch=frombaikevr;

[0151] Shidou No. 2: https: / / www.chinaseed114.com / seed / 1 / seed_4620.html;

[0152] Qi Huang 27: https: / / baike.sogou.com / v128451407.htm? ch=frombaikevr&fromTitle=%E9%BD%90%E9%BB%8427

[0153] Lingyin No. 2: https: / / www.sohu.com / a / 49502278_119777;

[0154] Tiefeng 35: Literature: Breeding of a new soybean variety Tiefeng 35;

[0155] Four-grain yellow (Nong 27): National Germplasm Resources Bank (https: / / www.cgris.net / query / croplist.php;

[0156] Dongnong 54:

[0157] http: / / www.360doc.com / content / 20 / 0405 / 08 / 65958933_903948432.shtml;

[0158] Tiefeng 35:

[0159] https: / / baike.baidu.com / item / %E9%93%81%E4%B8%B035 / 8863803?fr=ge_ala;

[0160] NNU 99-6:

[0161] https: / / baike.baidu.com / item / %E5%8D%97%E5%86%9C99-6 / 5465966?fr=ge_ala;

[0162] Lindou No. 1: Literature: Breeding and cultivation of a new high-quality soybean variety Lindou No. 1;

[0163] Rongxian winter beans: https: / / www.gengzhongbang.com / article-28784-1.html;

[0164] Jindou 22: Literature: Jindou No. 22, Shanxi Agricultural Sciences, Vol. 27, No. 2, 1999;

[0165] Medium bean 35:

[0166] https: / / baike.baidu.com / item / %E4%B8%AD%E8%B1%8635 / 8502022?fr=ge_ala;

[0167] North Bean 30:

[0168] https: / / baike.baidu.com / item / %E5%8C%97%E8%B1%8630 / 1036341?fr=ge_ala.

[0169] The results showed that the consistency between the results of soybean disease resistance detection using SAGT1 expression levels and field identification results was 20 / 30 × 100% = 66.7%; the consistency between the results of soybean disease resistance detection using PR1 expression levels and field identification results was 18 / 30 × 100% = 60%; and the consistency between the results of soybean disease resistance detection using SAGT1 and PR1 expression levels and field identification results was 21 / 30 × 100% = 70.00%. This indicates that the use of SAGT1 and PR1 expression levels to detect soybean downy mildew resistance is highly accurate and can be used to detect soybean downy mildew resistance.

[0170] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art. Industrial Applicability

[0171] This study, based on early transcript sequencing and inoculation identification of resistant and susceptible varieties, identified target genes highly correlated with soybean downy mildew resistance. This study, for the first time, proposed using qRT-PCR to detect the transcriptional expression levels of target genes for precise antifungal identification of soybean germplasm resources. Experiments demonstrated that the transcriptional expression levels of the SAGT1 and PR1 genes were highly positively correlated with soybean downy mildew resistance. The consistency between the results of soybean disease resistance detection using SAGT1 and PR1 expression levels and field identification results was as high as 70.00%. SAGT1 and PR1 can be used as target genes for identification and screening of soybean germplasm resources for downy mildew resistance.

Claims

1. Application of a substance for detecting the expression level of SAGT1 and / or PR1 gene in detecting or assisting in detecting soybean downy mildew resistance.

2. The use according to claim 1, characterized in that: The substance for detecting the expression level of SAGT1 and PR1 genes consists of a substance for detecting the expression level of SAGT1 gene and a substance for detecting the expression level of PR1 gene.

3. The use according to claim 2, characterized in that: The substance for detecting the expression level of the SAGT1 gene includes a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 1 and SEQ ID No. 2 in the sequence list or a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 5 and SEQ ID No. 6 in the sequence list; The substance for detecting the expression level of PR1 gene comprises a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 3 and SEQ ID No. 4 in the sequence list or a primer pair consisting of two single-stranded DNAs shown in SEQ ID No. 7 and SEQ ID No. 8 in the sequence list.

4. A method for detecting or assisting in detecting soybean downy mildew resistance, comprising: The expression levels of SAGT1 and PR1 genes of the soybean to be tested relative to the Actin gene are detected using the substance for detecting the expression levels of SAGT1 and PR1 genes as described in any one of claims 1 to 3, and the downy mildew resistance of the soybean to be tested is determined according to the following IV based on the relative expression levels of the two genes: I: The tested soybean with relative expression level of SAGT1 gene>10 and relative expression level of PR1 gene>3 is or is a candidate for high-resistance soybean; II:II-1 or II-2: II-1: The tested soybeans with a relative expression level of SAGT1 gene>10 and a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being disease-resistant soybeans; II-2: The tested soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) and a relative expression level of PR1 gene>3 are or are candidates for being disease-resistant soybeans; III: III-1 or III-2 or III-3: III-1: The tested soybeans with a relative expression level of SAGT1 gene of 0-5 (including 5) and a relative expression level of PR1 gene>3 are or are candidates for medium-resistant soybeans; III-2: The tested soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) and a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being medium-resistant soybeans; III-3: The tested soybeans with a relative expression level of SAGT1 gene>10 and a relative expression level of PR1 gene of 0-0.5 (including 0.5) are or are candidates for being medium-resistant soybeans; IV: IV-1 or IV-2: IV-1: The tested soybeans with a relative expression level of SAGT1 gene of 0-5 (including 5) and a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being susceptible soybeans; IV-2: The tested soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) and a relative expression level of PR1 gene of 0-0.5 (including 0.5) are or are candidates for being susceptible soybeans; V: The soybean to be tested with a relative expression level of SAGT1 gene of 0-5 (including 5) and a relative expression level of PR1 gene of 0-0.5 (including 0.5) is or is a candidate for being a highly susceptible soybean.

5. Any of the following methods: X1) A method for detecting or assisting in detecting soybean downy mildew resistance, comprising: The expression level of soybean SAGT1 gene relative to Actin gene is detected by using the substance for detecting the expression level of SAGT1 gene according to any one of claims 1 to 3, and the resistance to downy mildew of soybean is determined according to the relative expression level of SAGT1 gene: The tested soybeans with a relative expression level of SAGT1 gene>10 are or are candidates for high-resistance soybeans; the tested soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) are or are candidates for disease-resistant or medium-resistant soybeans; the tested soybeans with a relative expression level of SAGT1 gene of 0-5 (including 5) are or are candidates for highly susceptible or susceptible soybeans; X2) A method for detecting or assisting in detecting soybean downy mildew resistance, comprising: using the substance for detecting PR1 gene expression level according to any one of claims 1 to 3 to detect the expression level of soybean PR1 gene relative to Actin gene, and determining soybean downy mildew resistance according to the relative expression level of PR1 gene: Soybeans with a PR1 gene relative expression level >3 are or are candidates for being highly resistant soybeans; soybeans with a PR1 gene relative expression level of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being disease-resistant or moderately resistant soybeans; soybeans with a PR1 gene relative expression level of 0-0.5 (including 0.5) are or are candidates for being highly susceptible or susceptible soybeans.

6. A method for screening soybeans resistant to downy mildew, comprising: The expression levels of soybean SAGT1 and PR1 genes relative to the Actin gene are detected using the substance for detecting the expression levels of SAGT1 and PR1 genes as described in any one of claims 1 to 3, and soybeans resistant to downy mildew are screened according to the following IV based on the relative expression levels of the two genes: I: The soybeans with relative expression level of SAGT1 gene>10 and relative expression level of PR1 gene>3 are or are candidates for high-resistance soybeans; II:II-1 or II-2: II-1: The soybeans with a relative expression level of SAGT1 gene>10 and a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being disease-resistant soybeans; II-2: The soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) and a relative expression level of PR1 gene>3 are or are candidates for being disease-resistant soybeans; III: III-1 or III-2 or III-3: III-1: The soybeans with SAGT1 gene relative expression level of 0-5 (including 5) and PR1 gene relative expression level>3 are or are candidates for medium-resistant soybeans; III-2: The soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) and a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being medium-resistant soybeans; III-3: The soybeans with the relative expression level of SAGT1 gene>10 and the relative expression level of PR1 gene 0-0.5 (including 0.5) are or are candidates for being medium-resistant soybeans; IV: IV-1 or IV-2: IV-1: soybeans with a relative expression level of SAGT1 gene of 0-5 (including 5) and a relative expression level of PR1 gene of 0.5-3 (excluding 0.5 and including 3) are or are candidates for being susceptible soybeans; IV-2: The soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) and a relative expression level of PR1 gene of 0-0.5 (including 0.5) are or are candidates for being susceptible soybeans; V: Soybeans with a relative expression level of SAGT1 gene of 0-5 (including 5) and a relative expression level of PR1 gene of 0-0.5 (including 0.5) are or are candidates for being highly susceptible soybeans.

7. Any of the following methods: X1) A method for screening soybeans resistant to downy mildew, comprising: Using the substance for detecting the expression level of the SAGT1 gene according to any one of claims 1 to 3 to detect the expression level of the soybean SAGT1 gene relative to the Actin gene, and screening soybeans resistant to downy mildew according to the relative expression level of the SAGT1 gene: The soybeans with a relative expression level of SAGT1 gene>10 are or are candidates for high resistance soybeans; the soybeans with a relative expression level of SAGT1 gene of 5-10 (excluding 5 and including 10) are or are candidates for disease resistance or medium resistance soybeans; the soybeans with a relative expression level of SAGT1 gene of 0-5 (including 5) are or are candidates for highly susceptible or susceptible soybeans; X2) A method for screening soybeans resistant to downy mildew, comprising: using the substance for detecting the expression level of PR1 gene according to any one of claims 1 to 3 to detect the expression level of soybean PR1 gene relative to Actin gene, and screening soybeans resistant to downy mildew according to the relative expression level of PR1 gene: The soybeans to be tested whose relative expression level of PR1 gene is >3 are or are candidates for being highly resistant soybeans; the soybeans to be tested whose relative expression level of PR1 gene is 0.5-3 (excluding 0.5 and including 3) are or are candidates for being disease-resistant or moderately resistant soybeans; the soybeans to be tested whose relative expression level of PR1 gene is 0-0.5 (including 0.5) are or are candidates for being highly susceptible or susceptible soybeans.

8. The substance for detecting the expression level of SAGT1 and / or PR1 gene according to any one of claims 1 to 3.

9. Use of the substance for detecting the expression level of SAGT1 and / or PR1 gene according to any one of claims 1 to 3 in the preparation of a product for detecting or assisting in detecting soybean downy mildew resistance.

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