SNPM80, a molecular marker closely linked to soybean aphid resistance gene and application thereof

Abstract

The application provides a molecular marker SNPM80 closely linked with a soybean anti-aphid gene and application, and relates to the technical field of molecular marker assisted breeding. The soybean anti-aphid gene is located between the physical position 30,700,000-30,900,000 on the 13th chromosome of a soybean genome, and a molecular marker and a corresponding molecular marker detection method are designed based on the soybean anti-aphid gene. By using the molecular marker for assisted selection, the screening efficiency is greatly improved, so that the breeding period of the anti-aphid variety is shortened.

Description

[0001] This application is a divisional application of Chinese patent application No. 202210003867.7, entitled "A Soybean Aphid-Resistant Gene and Its Molecular Marker and Application", the original applicant of which was the Institute of Crop Science, Chinese Academy of Agricultural Sciences. Technical Field

[0002] This invention belongs to the field of molecular marker-assisted breeding technology, specifically relating to a soybean aphid-resistant gene, its molecular marker, and its application. Background Technology

[0003] Soybean aphids ( Aphis glycines Soybean aphids (Mass aphids) are one of the major pests of soybeans, widely distributed in major soybean-producing areas of my country. They affect soybean yield and quality by sucking the photosynthetic products of the soybean phloem and transmitting plant viruses. Soybean aphids often congregate on the tender stems and leaves of the plant, causing chlorophyll to disappear at the infested areas, leaves to curl, and severely affected plants to have stunted stems and leaves, reduced branching and pod formation, thus impacting soybean yield. Severe aphid infestations during the seedling stage can kill the entire plant. If large-scale aphid outbreaks are not controlled in time, yield losses can reach 20-40%, and in severe cases, can exceed 50%. Currently, soybean aphid control relies on the use of insecticides, which not only increases production costs but also kills aphid natural enemies and harms the environment. Utilizing host plant resistance is an effective method for aphid control.

[0004] Domestic and international researchers have screened several aphid-resistant soybean resources, discovering resistance sources such as Dowling, Jackson, PI 71506, PI 567543C, and PI 567597C (HILL et al. 2004; MENSAH et al. 2005; MIAN et al. 2008; BHUSAL et al. 2013). Domestically, local varieties such as Qingpi Pingding, Duludou, and wild soybean 85-32 have been identified. However, these resources are difficult to directly apply to breeding due to their poor agronomic traits. Traditional breeding methods are limited by long cycles and low efficiency. Molecular marker-assisted breeding technology utilizes the close linkage between molecular markers and genes determining target traits. By detecting molecular markers, the presence of the target gene can be detected, significantly improving breeding efficiency. However, current domestic research on aphid resistance in soybeans is relatively limited, lacking excellent resistance resources and molecular markers suitable for breeding. The discovered resistance resources cannot be effectively utilized. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a soybean aphid-resistant gene, its molecular markers, and applications, and to develop and fully utilize molecular markers closely linked to the aphid-resistant gene for screening superior aphid-resistant lines and breeding aphid-resistant soybean varieties.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a soybean aphid-resistant gene, which is located at a physical position between 30,700,000 and 30,900,000 on chromosome 13 of the soybean genome.

[0007] The present invention also provides a molecular marker closely linked to the above-mentioned aphid-resistant gene, said molecular marker including a combination of one or more of SNPM20, SNPM80, M1147 and M1151; The SNPM20 is located at positions 30,702,907 on chromosome 13 of soybean. The base of aphid-resistant soybean is T, and the base of aphid-susceptible soybean is G. The SNPM80 is located at position 30,855,712 on chromosome 13 of soybean. The base of aphid-resistant soybean is T, and the base of aphid-susceptible soybean is G. The amplified fragment of M1147 in aphid-resistant soybean is 228 bp, and the amplified fragment in aphid-susceptible soybean is 211 bp. The M1151 amplification fragment is 286 bp in aphid-resistant soybean and 239 bp in aphid-susceptible soybean.

[0008] The present invention also provides a primer pair for identifying the above-mentioned molecular marker. The primer pair designed for SNPM20 includes SNPM20-F and SNPM20-R. The nucleotide sequence of SNPM20-F is shown in SEQ ID NO.1, and the nucleotide sequence of SNPM20-R is shown in SEQ ID NO.2. The primer pairs designed for SNPM80 include SNPM80-F and SNPM80-R, the nucleotide sequence of SNPM80-F is shown in SEQ ID NO.3, and the nucleotide sequence of SNPM80-R is shown in SEQ ID NO.4; The primer pairs designed for M1147 include M1147-F and M1147-R, the nucleotide sequence of M1147-F is shown in SEQ ID NO.5, and the nucleotide sequence of M1147-R is shown in SEQ ID NO.6; The primer pairs designed for M1151 include M1151-F and M1151-R, the nucleotide sequence of M1151-F is shown in SEQ ID NO.7, and the nucleotide sequence of M1151-R is shown in SEQ ID NO.8.

[0009] This invention also provides the application of the above-mentioned aphid-resistant gene, molecular marker, or primer pair in marker-assisted breeding of soybean.

[0010] The present invention also provides a method for detecting molecular markers against soybean aphids, comprising the following steps: using soybean genomic DNA as a template, mixing it with the above primer pair to prepare a PCR reaction system, and performing PCR amplification.

[0011] Preferably, the PCR amplification program includes: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 25 s, 58℃ annealing for 25 s, 72℃ extension for 10 s, 34 cycles; 72℃ extension for 5 min.

[0012] Preferably, the PCR reaction system, in 20 μL, comprises: 1 μL template, 2 μL each of upstream and downstream primers, 10 μL 2x M5HiPer plus Taq HiFi PCR Mix, and the remainder ddH2O.

[0013] The present invention also provides a method for screening aphid-resistant soybeans, comprising the following steps: using soybean genomic DNA as a template, mixing it with the above primer pairs to prepare a PCR reaction system, and performing PCR amplification; PCR products were analyzed, and varieties or lines exhibiting any of the following characteristics were identified as aphid-resistant soybeans: (1) Molecular marker SNPM20 was amplified into T at positions 30, 702, 907 on soybean chromosome 13; (2) The molecular marker SNPM80 was amplified into T at position 30,855,712 on soybean chromosome 13; (3) A 228 bp DNA fragment was amplified by the molecular marker M1147; (4) Molecular marker M1151 amplified a 286 bp DNA fragment.

[0014] Beneficial Effects: This invention utilizes a population resulting from a cross between the aphid-resistant soybean germplasm Fangzheng Moshi soybean and the aphid-susceptible soybean variety Beifeng 9 to locate a novel aphid-resistant gene locus using map-based cloning. Genetic analysis revealed that the aphid resistance trait in Fangzheng Moshi soybean is controlled by a dominant single gene. Fine mapping precisely mapped the novel aphid-resistant gene to a 152.8 kb region. Two molecular markers within this region, SNPM20 and SNPM80, are linked to this gene, and using these markers to screen for materials containing the target gene demonstrates high reliability. Furthermore, other closely linked molecular markers, M1147 and M1151, can also be used to screen for materials containing the target gene. Using these molecular markers for marker-assisted selection significantly improves screening efficiency, thereby shortening the breeding cycle for aphid-resistant varieties. Attached Figure Description

[0015] Figure 1 A comparison of aphid resistance between Fangzheng Moshi Bean (A) and Beifeng No. 9 (B); Figure 2 This is a diagram showing the results of fine gene mapping. Figure 3 Genotyping of the M1147 molecular marker in the population is shown in the figure, with lane numbers corresponding to Table 4, and the same applies below. Figure 4 Genotyping of the M1151 molecular marker in a population. Detailed Implementation

[0016] This invention provides a soybean aphid-resistant gene, which is located on chromosome 13 of the soybean genome at a physical position between 30,700,000 and 30,900,000 bp.

[0017] The aphid-resistant gene described in this invention is preferably obtained by mapping the offspring population of a cross between the aphid-resistant soybean germplasm Fangzheng Moshidou and the aphid-susceptible soybean variety Beifeng 9 using map-based cloning. In this invention, the allele at this locus in the aphid-resistant variety Fangzheng Moshidou significantly enhances the aphid resistance of soybeans.

[0018] The present invention also provides a molecular marker closely linked to the above-mentioned aphid-resistant gene, said molecular marker including a combination of one or more of SNPM20, SNPM80, M1147 and M1151; The SNPM20 is located at positions 30,702,907 on chromosome 13 of soybean. The base of aphid-resistant soybean is T, and the base of aphid-susceptible soybean is G. The SNPM80 is located at position 30,855,712 on chromosome 13 of soybean. The base of aphid-resistant soybean is T, and the base of aphid-susceptible soybean is G. The amplified fragment of M1147 in aphid-resistant soybean is 228 bp, and the amplified fragment in aphid-susceptible soybean is 211 bp. The M1151 amplification fragment is 286 bp in aphid-resistant soybean and 239 bp in aphid-susceptible soybean.

[0019] The molecular markers SNPM20 and SNPM80 described in this invention are linked to the aphid-resistant gene, and using these two markers to screen materials containing the target gene is highly reliable. Therefore, the resistance of the test material to aphids can be predicted using any one or more molecular markers of this invention.

[0020] The present invention also provides a primer pair for identifying the above-mentioned molecular marker. The primer pair designed for SNPM20 includes SNPM20-F and SNPM20-R. The nucleotide sequence of SNPM20-F is shown in SEQ ID NO.1, and the nucleotide sequence of SNPM20-R is shown in SEQ ID NO.2. The primer pairs designed for SNPM80 include SNPM80-F and SNPM80-R, the nucleotide sequence of SNPM80-F is shown in SEQ ID NO.3, and the nucleotide sequence of SNPM80-R is shown in SEQ ID NO.4; The primer pairs designed for M1147 include M1147-F and M1147-R, the nucleotide sequence of M1147-F is shown in SEQ ID NO.5, and the nucleotide sequence of M1147-R is shown in SEQ ID NO.6; The primer pairs designed for M1151 include M1151-F and M1151-R, the nucleotide sequence of M1151-F is shown in SEQ ID NO.7, and the nucleotide sequence of M1151-R is shown in SEQ ID NO.8.

[0021] The primer pairs designed based on different molecular markers in this invention are listed in Table 1.

[0022] Table 1 Primer pairs designed for molecular markers

[0023] This invention also provides the application of the above-mentioned aphid-resistant gene, molecular marker, or primer pair in marker-assisted breeding of soybean.

[0024] The aphid-resistant gene, molecular marker, or primer pair described in this invention can be used to determine the aphid resistance of soybean varieties (lines). The presence of the aphid-resistant gene can be determined when any of the following conditions are met, thus enabling its application in molecular marker-assisted breeding of aphid-resistant soybeans and shortening the breeding cycle: The amplification of T at positions 30,702,907 on soybean chromosome 13 using marker SNPM20, the amplification of T at positions 30,855,712 on soybean chromosome 13 using marker SNPM80, the amplification of a 228 bp DNA fragment using marker M1147, and the amplification of a 286 bp DNA fragment using marker M1151 all indicate the existence of a new aphid resistance gene locus in soybean varieties.

[0025] The present invention also provides a method for detecting molecular markers against soybean aphids, comprising the following steps: using soybean genomic DNA as a template, mixing it with the above primer pair to prepare a PCR reaction system, and performing PCR amplification.

[0026] This invention does not specifically limit the method for extracting soybean genomic DNA; however, the CTAB method is preferred in the embodiments. The present invention uses the genomic DNA and the primer pairs described above to prepare a PCR reaction system. The PCR reaction system, in 20 μL increments, preferably includes: 1 μL template, 2 μL each of upstream and downstream primers, 10 μL of 2×M5 HiPer plus Taq HiFiPCR Mix, and the remainder ddH2O. The concentrations of the upstream and downstream primers in this invention are preferably both 2 mmol·L⁻¹. -1 The present invention utilizes the PCR reaction system for PCR amplification, and the preferred PCR amplification program includes: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 25 s, 58℃ annealing for 25 s, 72℃ extension for 10 s, 34 cycles; 72℃ extension for 5 min.

[0027] This invention can sequence the PCR products after the PCR reaction to determine the polymorphism of a specified site, or determine the polymorphism of molecular marker sites by polyacrylamide gel electrophoresis or amplification electrophoresis, thereby determining the aphid resistance of soybean varieties (lines).

[0028] The present invention also provides a method for screening aphid-resistant soybeans, comprising the following steps: using soybean genomic DNA as a template, mixing it with the above primer pairs to prepare a PCR reaction system, and performing PCR amplification; PCR products were analyzed, and varieties or lines exhibiting any of the following characteristics were identified as aphid-resistant soybeans: (1) Molecular marker SNPM20 was amplified into T at positions 30, 702, 907 on soybean chromosome 13; (2) The molecular marker SNPM80 was amplified into T at position 30,855,712 on soybean chromosome 13; (3) A 228 bp DNA fragment was amplified by the molecular marker M1147; (4) Molecular marker M1151 amplified a 286 bp DNA fragment.

[0029] The PCR reaction system and PCR amplification procedure described in this invention are preferably the same as those described above, and will not be repeated here.

[0030] The following detailed description, in conjunction with embodiments, illustrates a soybean aphid-resistant gene, its molecular markers, and its applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 Population construction and phenotypic identification 1. Raw materials: Fangzheng Moshi soybean (sourced from the National Crop Germplasm Bank, unified number ZDD00236), which is classified as Grade 1 aphid resistance according to the resistance grading standard in Table 2, and the resistance does not depend on the number of soybean aphids.

[0032] Table 2 Grading Standards for Soybean Aphid Resistance Identification

[0033] 2. Using Fangzheng Moshi bean to construct a population, and using map-based cloning to locate aphid-resistant genes: Fangzheng Moshi bean was crossed with the highly aphid-susceptible variety Beifeng 9 (…). Figure 1 Then, a genetic segregating population resistant to aphids was constructed through self-pollination. The resistance identification results showed that all F1 plants showed resistance to aphids, and the ratio of aphid-resistant plants to aphid-susceptible plants in the F2 generation population was 3:1, indicating that the aphid resistance of Fangzheng Moshi Bean is controlled by a dominant single gene.

[0034] Example 2 Location of resistance genes and determination of linkage molecular markers Using the BSA (Block Segregation Analysis) method, a mixed pool was constructed by mixing DNA from aphid-resistant and aphid-susceptible plants. Primers exhibiting polymorphism between parents and conforming to extreme pooling patterns were screened from 580 pairs of SSR primers covering the entire soybean genome in the SoyBase database. Three markers, Satt114, Sct_033, and Satt_335, were identified as resistance-linked markers. These three linked markers, along with other encrypted markers, were used to identify the genotypes of F2 generation plants and F5 generation plants formed by multiple generations of self-pollination from F2 generation plants. Preliminary mapping was performed based on phenotype, and the initial mapping interval was located between markers M1133 and M1184, totaling 1.01 Mb. To further narrow down the interval, encrypted markers were used to identify F5 generation plants. 5:6 The genotypes of the successor plants, combined with phenotypic data, were used to screen exchanged individual plants, narrowing the interval to between markers SNPM20 and SNPM80, totaling 152.8 kb. Figure 2 This region does not overlap with any region of any aphid-resistant gene currently published in soybeans, indicating that it is a new aphid-resistant gene.

[0035] To better utilize the aphid resistance loci carried by Fangzheng soybean, SNP markers SNPM20 and SNPM80, which are closely linked to the localization interval, and SSR markers M1147 and M1151 are used as linkage markers for aphid resistance loci, and are used for molecular marker-assisted selection of aphid resistance in progeny populations and other soybean materials.

[0036] Using Williams 82 from the Phytozome database as the reference genome, specific primers containing two SNPs, SNPM20 and SNPM80, were designed using Primer 5.0 software (Table 1). At bases 30, 702, and 907 of the reference genome, the genotype of the corresponding base was either T or G. When the nucleotide at the SNP site was T, the identified material was consistent with the genotype of Fangzheng Moshi Dou, indicating it was either a soybean aphid-resistant material or a candidate soybean aphid-resistant material. When the nucleotide at the SNP site was G, the identified material was consistent with the genotype of Beifeng 9, indicating it was either a soybean aphid-susceptible material or a candidate soybean aphid-susceptible material. At base position 30,855,712 of the reference genome, the genotype of this base is T or G. When the nucleotide at the SNP site is T, the identified material is consistent with the genotype of Fangzheng Moshi Bean, and is a material resistant to soybean aphids or a candidate material resistant to soybean aphids; when the nucleotide at the SNP site is G, the identified material is consistent with the genotype of Beifeng 9, and is a material susceptible to soybean aphids or a candidate material susceptible to soybean aphids.

[0037] Within the localization interval, the molecular markers M1147 and M1151 have different genotypes. When the amplified band of M1147 is 228 bp, the identified material has the same genotype as Fangzheng Moshidou, indicating it is either resistant to soybean aphids or a candidate for resistant soybean aphids. When the amplified band of M1147 is 211 bp, the identified material has the same genotype as Beifeng 9, indicating it is either susceptible to soybean aphids or a candidate for susceptible soybean aphids. When the amplified band of M1151 is 286 bp, the identified material has the same genotype as Fangzheng Moshidou, indicating it is either resistant to soybean aphids or a candidate for resistant soybean aphids. When the amplified band of M1151 is 239 bp, the identified material has the same genotype as Beifeng 9, indicating it is either susceptible to soybean aphids or a candidate for susceptible soybean aphids.

[0038] Table 3 Marker site information

[0039] Example 3 Validation of molecular markers in offspring populations Genomic DNA was extracted from leaves of Fangzheng Moshi Bean, Beifeng 9, and their progeny populations using the CTAB method and then amplified by PCR. The reaction mixture consisted of 20 μL of genomic DNA, with a concentration of 1 μL and a concentration of 2 mmol / L. -1Each of the forward and reverse primers was 2 μl, ddH2O 4 μl, and 2x M5 HiPer plus Taq HiFi PCR Mix (with blue dye) 10 μl (Beijing Jumei Biotechnology Co., Ltd., catalog number MF002-plus-01). The PCR program was as follows: 95℃ denaturation for 3 min, 95℃ denaturation for 25 s, 58℃ annealing for 25 s, 72℃ extension for 10 s, 34 cycles; 72℃ extension for 5 min, and finally storage at 4℃. The above PCR reactions were amplified on an ABI (Applied Biosystems, USA) PCR thermal cycler.

[0040] The sequences amplified by SNPM20 and SNPM80 were detected by 1% agarose gel electrophoresis. After obtaining bands of the same size as the target bands, the PCR products were sequenced by Beijing Bomeide Gene Technology Co., Ltd.

[0041] The amplified bands labeled M1147 and M1151 were detected by 6% deformable polyacrylamide gel electrophoresis. After electrophoresis, silver nitrate staining was performed, and the genotypes could be read after staining.

[0042] DNA from 34 lines of Fangzheng Mashi soybean and Beifeng 9 and their derivatives was amplified by PCR. The PCR products of SNPM20 and SNPM80 were sequenced and their genotypes were analyzed. The PCR products of M1147 and M1151 were amplified and their genotypes were detected by denaturing polyacrylamide gel electrophoresis. Figure 3 Electrophoresis diagram of M1147 amplification, Figure 4 The M1151 amplification electrophoresis diagram is used to label the genotypes, which are listed in Table 4. The results showed that the genotypes of four markers in the 11 lines were consistent with those of Fangzheng Moshi Bean: the genotypes of SNPM20 and SNPM80 were both TT; the PCR product amplified by M1147 was 228 bp; and the PCR product amplified by M1151 was 286 bp. The genotypes of four markers in the 11 lines were also consistent with those of Beifeng 9: the genotypes of SNPM20 and SNPM80 were both GG; the PCR product amplified by M1147 was 211 bp; and the PCR product amplified by M1151 was 239 bp. Furthermore, the genotypes of four markers in the remaining 12 lines were heterozygous: the genotypes of SNPM20 and SNPM80 were both GT; and the PCR product amplified by M1147 showed two bands, one 228 bp and the other 211 bp. The PCR product amplified by M1151 consists of two bands, one 286 bp and the other 239 bp.

[0043] Further phenotypic identification of these strains was performed, and the phenotype of each strain is recorded in Table 4. The material name / numbering rule is L-row number-single plant number, such as L-827-2 representing the second single plant in row 827. The results showed that strains with the same genotype as Fangzheng Moshi bean were aphid-resistant, strains with the same genotype as Beifeng 9 were aphid-susceptible, and heterozygous strains were aphid-resistant. This indicates that the SNPs and SSRs mentioned above can be used to identify aphid resistance in the Fangzheng Moshi bean population and its derived progeny.

[0044] Table 4 Validation of molecular markers

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of an SNP marker SNPM80, tightly linked to a soybean aphid resistance gene, in the identification and screening of aphid-resistant soybeans, characterized in that, The aphid-resistant gene is located at physical position 30,700,000 to 30,900,000 on chromosome 13 of the soybean genome. The SNPM80 gene is located at position 30,855,712 on chromosome 13 of soybean. The base of aphid-resistant soybean is T, and the base of aphid-susceptible soybean is G. Williams 82 is used as the reference genome.

2. A method for detecting molecular markers of soybean aphid resistance, characterized in that, The steps include: using soybean genomic DNA as a template, adding SNPM80 amplification primer pairs, preparing a PCR reaction system, performing PCR amplification, and amplifying T at position 30,855,712 on soybean chromosome 13, indicating the presence of an aphid-resistant gene in the soybean genome; The aphid-resistant gene is located at physical position 30,700,000 to 30,900,000 on chromosome 13 of the soybean genome. The SNPM80 gene is located at position 30,855,712 on chromosome 13 of the soybean genome. The base of aphid-resistant soybean is T, and the base of aphid-susceptible soybean is G. Williams 82 is used as the reference genome. The primer pair includes SNPM80-F and SNPM80-R, the nucleotide sequence of SNPM80-F is shown in SEQ ID NO.3, and the nucleotide sequence of SNPM80-R is shown in SEQ ID NO.

4.

3. The molecular marker detection method according to claim 2, characterized in that, The PCR amplification program includes: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 25 s, 58℃ annealing for 25 s, 72℃ extension for 10 s, 34 cycles; 72℃ extension for 5 min.

4. The molecular marker detection method according to claim 2 or 3, characterized in that, The PCR reaction system, in 20 μL volumes, includes: 1 μL template, 2 μL each of forward and reverse primers, 10 μL of 2 x M5 HiPer plus Taq HiFi PCR Mix, and the remainder ddH2O.

5. A method for screening aphid-resistant soybeans, characterized in that, The steps include: using soybean genomic DNA as a template, adding SNPM80 amplification primer pairs, preparing a PCR reaction system, and performing PCR amplification; PCR products were detected, and the variety / line that amplified T at position 30,855,712 on chromosome 13 of soybean was identified as aphid-resistant soybean. Using Williams 82 as a reference genome, SNPM80 is located at position 30,855,712 on chromosome 13 of soybean. The base of aphid-resistant soybean is T, and the base of aphid-susceptible soybean is G. The primer pair includes SNPM80-F and SNPM80-R, the nucleotide sequence of SNPM80-F is shown in SEQ ID NO.3, and the nucleotide sequence of SNPM80-R is shown in SEQ ID NO.4.

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