Molecular marker primer group for resisting soybean mosaic virus, kit and application of molecular marker primer group

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-soybean mosaic virus molecular marker primer group, a kit and application of the anti-soybean mosaic virus molecular marker primer group, the anti-soybean mosaic virus molecular marker primer group comprises a primer group designed based on an RSC3K genome sequence; the RSC3K genome sequence is formed by the following steps: two alleles LOC100526921 and LOC100812666 of a susceptible variety are respectively subjected to a segment of deletion, and then are recombined to form the RSC3K genome sequence. The product obtained by amplification of the molecular marker primer group shows a codominant result, no contrast is needed, the cost is low, genotype detection can be carried out on a breeding material in a short time and with high throughput, and time and energy are saved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a molecular marker primer set and a kit for resisting soybean mosaic virus and applications thereof. Background Art

[0002] Soybean mosaic virus (SMV) is a plant virus that poses a serious threat to the global soybean industry. It mainly infects soybean plants, causing them to develop obvious symptoms such as mosaic and deformity. These symptoms directly affect photosynthesis. In severe cases, the yield of infected soybean fields may drop by 30% to 50%, resulting in significant economic losses.

[0003] Traditional breeding methods are time-consuming and inefficient. Currently, Marker-Assisted Selection (MAS) is a relatively effective and convenient technology for identifying and screening resistant materials. This technology uses molecular markers to assist in the selection and breeding of crops. Through the corresponding molecular markers, breeders can determine the disease resistance of the material at an early stage, saving a lot of time and resources. Currently, the more commonly used types of molecular markers are SSR, SNP, CAPS, gene chips, etc., but the detection methods of these markers are cumbersome or costly, and are difficult to use for the screening of large quantities of breeding materials. Summary of the invention

[0004] The purpose of the present invention is to provide a molecular marker primer set, a kit and an application thereof for resistance to soybean mosaic virus. The product amplified using the molecular marker primer set of the present invention shows a co-dominant result, does not require the setting of a control, has low cost, and can perform high-throughput genotype detection on breeding materials in a short time, saving time and energy.

[0005] The present invention provides a molecular marker primer set for resisting soybean mosaic virus, the molecular marker primer set comprising: SC3 Primer sets designed based on K genome sequence;

[0006] The R SC3 The K genome sequence includes: the two alleles LOC100526921 and LOC100812666 of the susceptible variety each underwent a deletion and then recombined.

[0007] As a preferred solution, the molecular marker primer set includes: an upstream primer JT1229-F, and two downstream primers JT1229-R1 and JT1229-R2;

[0008] The gene sequence of JT1229-F is shown in SEQ ID NO.1;

[0009] The gene sequence of JT1229-R1 is shown in SEQ ID NO.2;

[0010] The gene sequence of JT1229-R2 is shown in SEQ ID NO.3.

[0011] The present invention provides the use of the above-mentioned molecular marker primer set in preparing a resistance identification kit.

[0012] As a preferred embodiment, the resistance includes: resistance to soybean mosaic virus.

[0013] The invention provides a resistance identification kit, comprising the above-mentioned molecular marker primer set and conventional PCR reagents.

[0014] The present invention provides a resistance identification method, comprising the following steps: extracting genomic DNA of a sample to be tested, performing PCR amplification using the above-mentioned molecular marker primer set or the molecular marker primer set in the above-mentioned kit, and performing resistance identification according to the amplification result.

[0015] As a preferred solution, when the molecular marker primer set is used to amplify a 1.5 kb product, the sample to be tested is a product containing R SC3 K type;

[0016] When the molecular marker primer set is used to amplify a product of 1.0 kb, the sample to be tested does not contain R SC3 K-type.

[0017] As a preferred solution, the final concentration of each primer in the molecular marker primer set is 0.20-0.25 μM / strip.

[0018] As a preferred solution, the PCR amplification reaction system is 50 μL, including: 1 μL of upstream primer, 0.5 μL of each downstream primer, 1 μL of DNA template, 25 μL of 2×Taq DNA polymerase and the remainder of ultrapure water.

[0019] The present invention also provides the use of the above molecular marker primer set or the above kit or the above resistance identification method in resistance breeding.

[0020] Beneficial effect: The present invention provides a molecular marker primer set for resisting soybean mosaic virus, the molecular marker primer set comprising: SC3 The primer set designed based on the K genome sequence; SC3The K genome sequence includes: two alleles LOC100526921 and LOC100812666 of the susceptible variety are recombined after each deletion. The molecular marker type involved in the present invention is an insertion-deletion (InDel) marker, and the genotype of the sample can be detected by agarose gel electrophoresis. Compared with other types of molecular markers such as simple sequence repeats (Simple Sequence Repeat, SSR), single nucleotide polymorphisms (Single nucleotide polymorphism, SNP), and enzyme-cut amplified polymorphic sequences (C1eavedAmplifiedPolymorphicSequences, CAPS), the operation is simple, the product is co-dominant, no control is required, the cost is low, and the genotype detection of breeding materials can be carried out in a short time with high throughput, saving time and energy.

[0021] The products amplified by SSR primers often require polyacrylamide gel electrophoresis, which is time-consuming and cumbersome; the sequences identified by SNP require sequencing reactions to complete, and the sequences identified by CAPS require the participation of endonucleases, both of which are relatively expensive and not suitable for the screening of breeding materials. There is a large length difference between the products amplified by the InDel primers of the present invention, and agarose gel electrophoresis can be used to screen a large number of materials, which is currently an ideal choice for auxiliary selection breeding. The molecular markers of the present invention omit the step of internal reference control, saving costs and improving detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 For the SMV resistance gene R SC3 Comparison of sequence composition differences between K resistant and susceptible varieties;

[0023] Figure 2 The banding results of soybean varieties amplified by primers F and R1 and R2 are shown in Figure 1. Bands 1 to 10 from left to right are 21-250 (ɑ deletion), Weidou 8, Zhonghuang 348, A2234, OAC Tablot, UPBS 9018-74, Bert, Weidou 7, OAC Dorado and OlymPus.

[0024] Figure 3 The results of amplifying the banding patterns of soybean varieties using primers F and R1; bands 1 to 10 are the same Figure 2 ;

[0025] Figure 4 The results of amplifying the banding patterns of soybean varieties using primers F and R2; bands 1 to 10 are the same Figure 2 ;

[0026] Figure 5 This is the result of the sample genotype detection using molecular marker JT0212; bands 1-8 from left to right are Kefeng No. 1, Williams 82, Zhudou 42, Handou 26, Shidou 25, Shidou 27, Yidou 13, and Qihuang No. 1. DETAILED DESCRIPTION

[0027] The present invention provides a molecular marker primer set for resisting soybean mosaic virus, the molecular marker primer set comprising: SC3 Primer sets designed based on K genome sequence;

[0028] The R SC3 The K genome sequence includes: the two alleles LOC100526921 and LOC100812666 of the susceptible variety each underwent a deletion and then recombined.

[0029] In the present invention, the SC3 strain of soybean mosaic virus (SMV) is a dominant strain in the Huanghuaihai and middle and lower reaches of the Yangtze River, and has a wide range of infection on soybeans. SC3 K(R SC3 K sequence is the same as R SV4 The sequence is consistent with that of LC325439, see: Ishibashi K, Saruta M, Shimizu T, Shu M, Anai T, Komatsu K, Yamada N, Katayose Y, Ishikawa M, Ishimoto M, Kaga A. Soybean antiviral immunity conferred by dsRNase targets the viral replication complex. Nat Commun. 2019, 10(1): 4033.) is formed by the deletion and recombination of two alleles of the susceptible variety. As a specific embodiment, Kefeng No. 1 is resistant to SMV-SC3, Williams 82 is susceptible to SMV-SC3, R SC3 K is formed by recombination of alleles LOC100526921 and LOC100812666 after a deletion. Figure 1 As mentioned above, R SC3K is composed of the 5' end of LOC100526921 (gray area) and the 3' end of LOC100812666 (black area), that is, LOC100526921 has a 1153 bp deletion at the 3' end, and LOC100812666 has a 879 bp deletion at the 5' end, recombining into a new gene of about 2.0 kb in length; the upstream primer F (yellow arrow) of the molecular marker JT1229 is designed in the 5' non-coding region (gray area) of LOC100526921, the downstream primer R1 (green arrow) is designed in the 3' coding region (gray area) of LOC100526921, and the downstream primer R2 (green arrow) is designed in the 3' non-coding region (black area) of LOC100812666.

[0030] In the present invention, based on R SC3 K genome sequence may include: an upstream primer JT1229-F, two downstream primers JT1229-R1 and JT1229-R2; the gene sequence of JT1229-F is shown in SEQ ID NO.1: 5'-CCTCATCAAACCAAGACATAGC-3'; the gene sequence of JT1229-R1 is shown in SEQ ID NO.2: 5'-CTCACTTCCCAGTCAA AACG-3'; the gene sequence of JT1229-R2 is shown in SEQ ID NO.3: 5'-ACTTCCC AAAAGACAACCCA-3'.

[0031] The present invention provides a resistance identification kit, comprising the above-mentioned molecular marker primer set and conventional PCR reagents. As a specific embodiment, the conventional PCR reagents of the present invention include: DNA polymerase or Taq DNA polymerase.

[0032] The present invention provides a resistance identification method, comprising the following steps: extracting genomic DNA of a sample to be tested, performing PCR amplification using the above-mentioned molecular marker primer set or the molecular marker primer set in the above-mentioned kit, and performing resistance identification according to the amplification result.

[0033] As a specific implementation, three primers are added to the PCR system at the same time, and the final concentration of each primer is: 0.20-0.25 μM / strip. The DNA of soybean plants is used as a template, and a high-fidelity DNA polymerase or Taq DNA polymerase can be used for amplification. The PCR reaction system is added according to the instructions of the amplification enzyme (taking a 50 μL system as an example, 1 μL of upstream primer, 0.5 μL of downstream primer each, 1 μL of DNA template, 25 μL of 2×Taq DNA polymerase, and 22 μL of ultrapure water). In the present invention, the PCR reaction time can be calculated according to the extension time of the polymerase (the extension length does not exceed 2 kb, such as the extension speed of the polymerase is 1 kb / minute, and the extension time set by the reaction program does not exceed 2 minutes).

[0034] In the present invention, R SC3 In soybeans with K genotype, a product of about 1.5 kb can be amplified by primers F and R2 (in the defined PCR program), while in soybeans containing LOC100526921 and LOC100812666 genotypes, a fragment of about 1.0 kb can be amplified by primers F and R1 (in the defined PCR program).

[0035] To further illustrate the present invention, a molecular marker primer set, a kit and applications thereof for resisting soybean mosaic virus provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] The primer information of the molecular marker is one upstream primer and two downstream primers, and the specific information is: upstream primer JT1229-F (SEQ ID NO.1): 5'-CCTCATCAAACCAAGACATAGC-3'; downstream primer JT1229-R1 (SEQ ID NO.2): 5'-CTCACTTCCCAGTCAAAACG-3', JT1229-R2 (SEQ ID NO.3): 5'-ACTTCCCAAAAGACAACCCA-3'.

[0038] The method for using the molecular marker is as follows: three primers are added to the PCR system at the same time, and the final concentration of each primer is: 0.20-0.25 μM / strip. The DNA of soybean plants is used as a template, and a high-fidelity DNA polymerase or Taq DNA polymerase can be used for amplification. The PCR reaction system is added according to the polymerase instructions (taking a 50 μL system as an example, 1 μL of upstream primers, 0.5 μL of downstream primers each, 1 μL of DNA template, 25 μL of 2×Taq DNA polymerase, and 22 μL of ultrapure water). Taking 3GTaqMasterMixforPAGE(Red Dye) (Cat. No.: P115-02) of Novozymes as an example, the PCR reaction conditions are: 95°C (pre-denaturation) for 3 min; 95°C (denaturation) for 15 s, 58°C (annealing) for 15 s, 72°C (extension) for 2 min, 35 cycles; 72°C (final extension) for 5 min. The PCR reaction time is calculated according to the extension time of the polymerase (the extension length does not exceed 2 kb. For example, if the extension speed of the polymerase is 1 kb / min, the extension time set by the reaction program does not exceed 2 minutes).

[0039] Test Example 1 Calculation method of genotype detection rate and resistance consistency rate of molecular markers

[0040] 1. SMV inoculation and resistance identification of soybean plants

[0041] The friction inoculation method is usually used to inoculate soybean plants with soybean mosaic virus. The leaves of plants infected with SMV-SC3 are ground into a suspension in a mortar or juicer, and healthy soybean plants with newly unfolded true leaves are selected as recipients. The venom is dipped in a brush and rubbed on the leaves of the recipient plants 3 to 5 times to allow the virus to enter the plant. To ensure the success of the inoculation, the plants need to be managed under appropriate temperature and humidity conditions after inoculation to promote the spread of the virus.

[0042] The main methods for detecting soybean plant resistance are phenotypic observation and serum detection. The phenotypic observation method is to evaluate the resistance level of the plant by monitoring whether the plant has typical symptoms such as mosaic, deformity or necrosis 3 weeks after inoculation. However, there may be errors in judging only by visual symptoms. Therefore, serum detection methods such as enzyme-linked immunosorbent assay (ELISA) are used to determine the virus content in the leaves of the plant. Combining the two methods can more accurately evaluate the resistance of soybean varieties. The results of phenotypic, serum identification and resistance identification of 100 soybean varieties inoculated with SMV-SC3 using the method of Example 1 are shown in Table 1, where the band type with a fragment length of about 1.5 kb is recorded as 1 (R SC3 K genotype), the band type with a fragment length of about 1.0 kb was recorded as 2 (non-R SC3 K genotype).

[0043] 2. Identification of resistance compliance rate by molecular markers

[0044] Spot 5 μL of the amplified PCR product in a 1% agarose gel well, add a marker that can display 1.0-2.0 kb, and run the electrophoresis for 10 minutes at 200 V and 200 mA. Observe the fragment size under ultraviolet light and take photos. The banding results of 100 soybean varieties are shown in Table 1. Combine the results of resistance identification with the banding data to calculate the disease resistance compliance rate of the molecular marker. The formula is:

[0045] Disease resistance compliance rate = (band type is R SC3 K and the number of resistant varieties / band type is R SC3 The number of varieties of K) × 100%.

[0046] Table 1 Evaluation of resistance of 1100 soybean varieties inoculated with SMV-SC3

[0047]

[0048]

[0049]

[0050]

[0051] As can be seen from Table 1, 50 varieties are resistant to SMV-SC3, and 50 varieties are sensitive to SMV-SC3, indicating that SMV-resistant soybeans are relatively evenly distributed in natural populations. In breeding, resistant parents should be selected as much as possible for hybrid combination preparation to enhance the disease resistance of breeding materials from the source.

[0052] Through molecular markers, 1.0 kb or 1.5 kb products were detected in all 100 soybean varieties, so R SC3 The genotype detection rate of K is 100%. Among them, 23 varieties (such as Weidou No. 8) showed a banding type of 1, of which 17 varieties were resistant to the disease and 6 varieties (Bert, ChaPman, Thorne, Zhoudou 23, Yidou 13, Shengdou 125) were susceptible to the disease. The disease resistance compliance rate of this marker in these 100 soybean varieties was 74%, which was a high compliance rate and could be used for molecular assisted selection of resistance breeding.

[0053] Comparative Example 1

[0054] The method of Example 1 was followed, except that primers F and R1 were used to amplify the R SC3 K genotype.

[0055] Comparative Example 2

[0056] The method of Example 1 was followed, except that primers F and R2 were used to amplify the R SC3 K genotype.

[0057] Comparative Example 3

[0058] The method of Example 1 was followed, except that one reaction was amplified using primer JT0212, and the other reaction was amplified using primer TUBB3 of the internal reference gene [for primer information, see: Jin TT, Karthikeyan A, Wang LQ, et al. Digs out and characterization of the resistance gene accountable to soybean mosaic virus in soybean (Glycine max (L.) Merrill). Theoretical and applied genetics. Theoretische und angewandte Genetik, 2022, 135(12): 4217-4232.].

[0059] Test Example 2

[0060] Using the methods of Example 1, Comparative Examples 1 and 2, the resistance of the varieties numbered 1 to 10 in Table 1 was identified, that is, the amplification products of 21-250 (ɑ deletion), Weidou No. 8, Zhonghuang 348, A2234, OAC Tablot, UPBS 9018-74, Bert, Weidou No. 7, OAC Dorado and OlymPus were obtained. The PCR amplification results of Example 1 are as follows: Figure 2 As shown, the PCR amplification results of Comparative Example 1 are as follows Figure 3 As shown, the PCR amplification results of Comparative Example 2 are as follows Figure 4 The resistance of Kefeng No. 1, Williams 82, Zhudou 42, Handou 26, Shidou 25, Shidou 27, Yidou 13 and Qihuang No. 1 in Table 1 was identified by PCR amplification using the method of Comparative Example 3. The PCR amplification results are shown in Figure 5 The resistance verification results of each variety are shown in Table 1.

[0061] from Figure 2 It can be seen that without R SC3 K soybean can amplify a 1.0 kb product containing R SC3 K soybean can amplify a 1.5kb product. All samples can amplify products, and the genotypes of 10 varieties can be distinguished without the need for control experiments, and the test results do not need to be verified by control experiments.

[0062] from Figure 3It can be seen that without R SC3 K soybean can amplify a 1.0 kb product containing R SC3 K soybeans cannot be amplified to products. However, this scheme has a disadvantage, that is, the test results are not co-dominant. The phenomenon that the sample to be tested cannot amplify the product may be caused by multiple factors (such as DNA degradation) leading to the failure of product amplification. Control experiments are also needed to assist in the detection of R SC3 Whether K exists.

[0063] from Figure 4 It can be seen that the R SC3 K soybean can amplify a 1.5kb product without R SC3 K soybeans cannot amplify products. However, this scheme is similar to Scheme 2, and the test results are not co-dominant. The phenomenon that the sample to be tested cannot amplify products may be caused by multiple factors (such as DNA degradation) leading to product amplification failure. Control experiments are also needed to assist in the detection of R SC3 Whether K exists.

[0064] from Figure 5 As can be seen from the figure, the molecular marker JT0212 requires two PCR reactions (one reaction is amplified with JT0212 primers, and the other reaction is amplified with the internal reference gene TUBB3 primers) to determine whether the sample has R SC3 The genotype of K, that is, all varieties can be amplified with the internal reference gene to indicate that there is no problem with the DNA quality and Taq enzyme system as an experimental control; while JT0212 amplifies R SC3 K gene, only the varieties containing this gene can amplify the 1.7kb band, indicating that the variety is a resistant variety; SC3 No band was amplified for the K variety, indicating that this variety may be a susceptible variety (the failure to amplify the band may also be due to problems with the DNA or PCR reaction conditions, but the existence of the TUBB3 control band proves that there are no problems with the DNA or other conditions). Therefore, the product amplified by JT0212 can be used to identify the genotype of the variety.

[0065] It can be seen that the present invention utilizes R SC3 The InDel molecular marker primer set was developed based on the sequence difference characteristics of K. After the primers amplified the sample, two products of different sizes could be obtained, which could be identified by agarose gel electrophoresis, thus realizing the genotype identification of a large number of materials, and was an ideal choice for resistance breeding.

[0066] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A molecular marker primer set for resistance to soybean mosaic virus, characterized in that: The molecular marker primer set includes: based on R SC3 Primer sets designed based on K genome sequence; The R SC3 The K genome sequence includes: the two alleles LOC100526921 and LOC100812666 of the susceptible variety each underwent a deletion and then recombined.

2. The molecular marker primer set according to claim 1, characterized in that: The molecular marker primer set includes: an upstream primer JT1229-F, and two downstream primers JT1229-R1 and JT1229-R2; The gene sequence of JT1229-F is shown in SEQ ID NO.1; The gene sequence of JT1229-R1 is shown in SEQ ID NO.2; The gene sequence of JT1229-R2 is shown in SEQ ID NO.

3.

3. Use of the molecular marker primer set according to claim 1 or 2 in the preparation of a resistance identification kit.

4. The use according to claim 3, characterized in that: The resistance includes: resistance to soybean mosaic virus.

5. A resistance identification kit, characterized in that: It comprises the molecular marker primer set according to claim 1 or 2 and conventional PCR reagents.

6. A method for identifying resistance, characterized in that: The following steps are involved: Extract genomic DNA of the sample to be tested, perform PCR amplification using the molecular marker primer set described in claim 1 or 2 or the molecular marker primer set in the kit described in claim 5, and perform resistance identification based on the amplification results.

7. The method for identifying resistance according to claim 6, characterized in that: When the molecular marker primer set is used to amplify a 1.5 kb product, the sample to be tested contains R SC3 K type; When the molecular marker primer set is used to amplify a product of 1.0 kb, the sample to be tested does not contain R SC3 K-type.

8. The method for identifying resistance according to claim 6, characterized in that: The final concentration of each primer in the molecular marker primer set is 0.20-0.25 μM / strip.

9. The method for identifying resistance according to claim 6, characterized in that: The PCR amplification reaction system is 50 μL, including: 1 μL of upstream primer, 0.5 μL of each downstream primer, 1 μL of DNA template, 25 μL of 2×Taq DNA polymerase and the balance of ultrapure water.

10. Use of the molecular marker primer set according to claim 1 or 2, the kit according to claim 5, or the resistance identification method according to any one of claims 6 to 9 in resistance breeding.

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