Molecular marker remarkably related to cassava bacterial wilt resistance and application of molecular marker

Abstract

The invention belongs to the technical field of molecular biology, and discloses a molecular marker remarkably related to cassava bacterial wilt resistance and application of the molecular marker. The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to cassava bacterial wilt resistance, which is characterized in that the sequence of the SNP molecular marker is shown as SEQ ID NO: 3, the SNP site is positioned at the 392nd site from the 5'terminal of the sequence shown as SEQ ID NO: 3, and the polymorphism of the SNP molecular marker is A / T. By detecting the genotype of the SNP molecular marker, cassava with strong resistance to bacterial wilt can be accurately selected, and the SNP molecular marker can be effectively used for cassava molecular marker assisted breeding; according to actual breeding requirements, genotype identification can be carried out on cassava, genotype cassava with high bacterial wilt resistance is selected for hybridization, cassava filial generation with high bacterial wilt resistance is obtained, breeding time is saved, cost is low, accuracy is high, and the cassava breeding process is accelerated.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to molecular markers that are significantly associated with resistance to bacterial wilt of cassava and their applications. Background Technology

[0002] Cassava ( Manihot esculenta Cassava (Crantz) provides a staple food for large populations in West Africa, Southeast Asia, and tropical South America. More than one-fifth of the cassava root is composed of starch, and the cost of producing cassava starch is 15%-30% lower per hectare than producing corn starch. This makes it a strategically important energy crop with great development potential, and cassava is also a more cost-effective alternative to corn as livestock feed. However, cassava is susceptible to numerous diseases (approximately 40 types) and pests (over 100 types), severely hindering the development of the cassava industry. Diseases and pests reduce cassava's survival rate, severely impact photosynthetic efficiency, inhibit normal growth, and can even cause root rot, leading to reduced yields. Bacterial wilt of cassava is often one of the major diseases affecting cassava production, causing significant global yield losses, sometimes up to one-third, and reducing starch extraction by nearly half.

[0003] To cope with pathogen infection, plants have evolved a variety of countermeasures: (1) plant cell structure and cell walls provide the first line of defense against pathogen invasion; (2) hypersensitive reactions in infected tissues lead to cell death to prevent further proliferation of pathogens; and (3) systemic acquired resistance (SAR) is triggered in uninfected parts to enhance the overall resistance of the plant. The output of defense strategies depends on the activation of defense pathways, including pathogen recognition and complex downstream signal transduction. Therefore, key genes regulating these processes may be potential targets for improving plant resistance. Cassava germplasm resources have rich natural variability and genetic diversity, so identifying cassava disease resistance loci is key to successful molecular breeding for disease resistance.

[0004] Currently, methods for testing the disease resistance levels of different cassava varieties require the following steps: First, large-scale cultivation of cassava plants of different varieties is conducted. After one month of growth, they are inoculated with bacterial wilt pathogens, and the severity of the disease is then observed to determine the disease resistance of the cassava variety. Overall, this method is time-consuming and labor-intensive. Similarly, methods for selecting highly resistant cassava varieties after breeding require crossbreeding different cassava varieties to obtain progeny cassava, which are then inoculated with bacterial wilt pathogens. The severity of the disease is then observed to determine the disease resistance of the hybrid progeny cassava. This method is generally highly random and incurs high time and labor costs.

[0005] Therefore, there is an urgent need for a simple and efficient method to identify the resistance levels of different cassava varieties to bacterial wilt. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in the prior art. It provides a molecular marker significantly associated with resistance to bacterial wilt of cassava and its application.

[0007] The first aspect of this invention aims to provide SNP molecular markers associated with resistance to bacterial wilt of cassava.

[0008] The second aspect of the present invention aims to provide the application of substances that detect the SNP molecular markers of the first aspect of the present invention in screening cassava with strong resistance to bacterial wilt or in preparing products that screen cassava with strong resistance to bacterial wilt.

[0009] A third aspect of the present invention is to provide a primer set for amplifying the SNP molecular markers of the first aspect of the present invention.

[0010] A fourth aspect of the present invention is to provide a reagent kit.

[0011] The fifth aspect of this invention aims to provide the application of the SNP molecular marker of the first aspect of this invention, the primer set of the third aspect of this invention, and / or the kit of the fourth aspect of this invention.

[0012] The sixth aspect of this invention aims to provide a method for identifying the resistance of cassava varieties to bacterial wilt disease.

[0013] The seventh aspect of this invention aims to provide the application of the method of the sixth aspect of this invention in cassava-assisted breeding.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an SNP molecular marker associated with resistance to bacterial wilt of cassava, the sequence of which is shown in SEQ ID NO:3, the SNP site being located at position 392 from the 5' end of the sequence shown in SEQ ID NO:3, and the polymorphism being A / T.

[0015] In some embodiments of the present invention, when the genotype of the SNP locus is AA or AT, it is a cassava with strong resistance to bacterial wilt, and when the genotype of the SNP locus is TT, it is a cassava with weak resistance to bacterial wilt.

[0016] This invention, through extensive screening and identification, identified a key locus significantly correlated with cassava disease resistance. This locus exhibits polymorphisms of AA, AT, or TT. When the polymorphism is AA or AT, the cassava variety shows strong resistance to bacterial wilt; when the polymorphism is TT, the cassava variety shows weak resistance to bacterial wilt. By detecting this locus, it is possible to accurately identify whether unknown cassava samples are disease-resistant varieties, and batch identification can be performed. This method is simple, efficient, and widely applicable in cassava breeding practices.

[0017] A second aspect of the invention provides the use of substances that detect the SNP molecular markers of the first aspect of the invention in screening cassava resistant to bacterial wilt or in preparing products that screen cassava resistant to bacterial wilt.

[0018] In some embodiments of the present invention, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: Northern blotting, PCR, gene chip method, and nucleic acid sequencing.

[0019] In some embodiments of the present invention, PCR is used to detect the SNP molecular markers of the first aspect of the present invention.

[0020] In some embodiments of the present invention, the substance includes primers for PCR. For example, an upstream primer with the nucleotide sequence 5'-ATGGCCCCTTCCTTGCTCG-3' (SEQ ID NO:1) and a downstream primer with the nucleotide sequence 5'-TCAGCCTGCTGAATTCTCGTG-3' (SEQ ID NO:2).

[0021] In some embodiments of the present invention, the product includes at least one of reagents, reagent kits, test strips, chips, and systems.

[0022] A third aspect of the invention provides a primer set for amplifying the SNP molecular markers of the first aspect of the invention.

[0023] In some embodiments of the present invention, the primer set includes an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is 5'-ATGGCCCCTTCCTTGCTCG-3' (SEQ ID NO:1), and the nucleotide sequence of the downstream primer is 5'-TCAGCCTGCTGAATTCTCGTG-3' (SEQ ID NO:2).

[0024] A fourth aspect of the present invention provides a kit comprising the primer set of the third aspect of the present invention.

[0025] In some embodiments of the present invention, the kit further includes a buffer used in PCR.

[0026] In some embodiments of the present invention, the buffer used in the PCR is any reagent required for PCR amplification, such as DNA polymerase, dNTP, Taq enzyme, MgCl2, etc.

[0027] A fifth aspect of the present invention provides the use of the SNP molecular marker of the first aspect of the present invention, the primer set of the third aspect of the present invention, and / or the kit of the fourth aspect of the present invention in any one of (1)-(4): (1) To identify or assist in the identification of resistance to bacterial wilt of cassava; (2) To prepare products for identification or to assist in the identification of resistance to bacterial wilt of cassava; (3) Cassava-assisted breeding; (4) Prepare products for cassava-assisted breeding.

[0028] A sixth aspect of the present invention provides a method for identifying bacterial wilt resistance in cassava varieties, wherein the bacterial wilt resistance of the cassava variety is determined by detecting the genotype of the SNP molecular marker of the first aspect of the present invention in the genome of the cassava variety to be tested, based on the genotype.

[0029] In some embodiments of the present invention, the method includes the following steps: using the genome of the cassava variety to be tested as a template, performing PCR amplification using the primer set of the third aspect of the present invention or the kit of the fourth aspect of the present invention to obtain PCR amplification products; performing sequencing analysis on the PCR amplification products to determine the genotype of the SNP molecular marker of the first aspect of the present invention in the genome of the cassava variety to be tested.

[0030] In some embodiments of the present invention, when the genotype of the SNP locus is AA or AT, it is a cassava with strong resistance to bacterial wilt, and when the genotype of the SNP locus is TT, it is a cassava with weak resistance to bacterial wilt.

[0031] In some embodiments of the present invention, the cassava genome can be obtained using conventional methods in this technical field, including the phenol-chloroform method and various extraction kits for extracting cassava RNA, which is then reverse transcribed into cDNA.

[0032] In some embodiments of the present invention, the PCR amplification reaction program is as follows: pre-denaturation at 94-97℃ for 2-4 min; denaturation at 94-97℃ for 20-35 s, annealing at 54-56℃ for 20-35 s, extension at 70-72℃ for 50-70 s, for 34-36 cycles; and extension at 70-72℃ for 8-10 min.

[0033] In some embodiments of the present invention, the method for sequencing the PCR amplification products is not particularly limited, as long as the sequence of the PCR amplification product, i.e., the fragment containing the SNP marker, can be effectively obtained. The PCR amplification products can be detected using at least one method selected from first-generation gene sequencing, second-generation high-throughput gene sequencing, or third-generation high-throughput gene sequencing, or any other feasible method for determining the genotype. Thus, the genotype results can be obtained rapidly, efficiently, and accurately.

[0034] The method provided by this invention improves the identification efficiency of disease-resistant cassava varieties. This molecular marker method for identifying the resistance of cassava varieties to Fusarium wilt is not limited by the developmental stage of cassava, and can be used for early detection, thereby predicting the level of disease resistance of cassava. It saves time and effort and is of great significance for the breeding of disease-resistant cassava varieties.

[0035] A seventh aspect of the present invention provides the application of the method of the sixth aspect of the present invention in cassava-assisted breeding.

[0036] The beneficial effects of this invention are: This invention identifies SNP molecular markers associated with the resistance of different cassava varieties to bacterial wilt by performing correlation analysis. The SNP marker, as shown in SEQ ID NO:3, is located at position 392 and contains three genotypes: TT, AT, and AA. Cassava with the AT and AA genotypes exhibits significantly stronger resistance to bacterial wilt than the TT genotype. By detecting the genotype of this SNP marker, cassava varieties with strong resistance to bacterial wilt can be accurately selected, which is effective for marker-assisted breeding of cassava. Furthermore, it allows for genotyping of cassava according to actual breeding needs, enabling the selection of cassava with strong resistance to bacterial wilt for hybridization to obtain cassava progeny with strong resistance to bacterial wilt. This approach not only saves breeding time and is cost-effective and highly accurate but also accelerates the cassava breeding process.

[0037] The present invention further provides primer sets, kits and detection methods for detecting SNP molecular markers, which can be used for rapid identification and screening of cassava with strong resistance to bacterial wilt.

[0038] Compared with existing technologies (methods for screening disease-resistant cassava varieties after cassava breeding), this invention can accurately identify whether unknown cassava samples are disease-resistant varieties by detecting SNP molecular markers, and can be used for batch identification. These co-dominant molecular markers that are significantly associated with resistance to bacterial wilt of cassava are conducive to the establishment of a germplasm screening system that is significantly associated with resistance content to bacterial wilt of cassava. This system can be applied simply, efficiently, and widely in the cassava breeding practice.

[0039] Compared with conventional methods for identifying the disease resistance of cassava varieties and cassava molecular marker-assisted breeding, the method provided by this invention allows for early detection and prediction of cassava disease resistance levels, saving time and effort and playing an important role in the breeding of disease-resistant cassava varieties. Attached Figure Description

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This diagram identifies loci significantly associated with cassava resistance to bacterial wilt. A shows a Manhattan plot of molecular markers significantly associated with cassava resistance to bacterial wilt and their correlation with cassava resistance to the disease; B shows the results of significant linkage disequilibrium relationships between molecular markers significantly associated with cassava resistance to bacterial wilt and other loci (see below); C shows the changes in encoded amino acids caused by different molecular marker types; and D shows the results of resistance to bacterial wilt in different cassava varieties. . Detailed Implementation

[0041] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0042] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0043] The term "molecular marker" refers to a specific DNA segment that reflects a certain difference in the genome of an individual or population.

[0044] The term "polymorphism" refers to the diversity of DNA molecules formed by the existence of more than two alleles at a certain locus within a population.

[0045] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0046] Example 1 The discovery of molecular markers significantly associated with resistance to bacterial wilt of cassava is detailed below: Genome-wide association analysis (GWIA) was performed on resistance to bacterial wilt and population resequencing data from different cassava varieties. The results showed a significant association between a genotype on chromosome 3 and disease resistance, indicating that the natural variation in cassava bacterial wilt resistance levels is mainly regulated by a locus on chromosome 3. Figure 1(As shown in A).

[0047] Comparative analysis of the genotypes of different cassava varieties revealed a SNP polymorphism at position 25542952 on chromosome 3 of cassava, which was significantly associated with resistance to bacterial wilt in cassava. It is preliminarily concluded that this polymorphism leads to differences in disease resistance among different cassava varieties. Figure 1 (A)

[0048] Further linkage disequilibrium analysis revealed a significant linkage disequilibrium between this SNP polymorphism site and other sites. Figure 1 (B) A single-base mutation was found at this SNP site. Figure 1 As shown in Figure C); the box plot shows that when the genotype is AA / AT, the tested cassava variety has strong resistance to bacterial wilt; when the genotype is TT, the tested cassava variety has weak resistance to bacterial wilt. Figure 1 Therefore, the SNP polymorphism at Chr3_25542952 can represent the functional polymorphism of this genotype, which is the difference in bacterial wilt resistance among different cassava varieties caused by natural genetic variation at this locus.

[0049] The SNP site is located at base 25542952 on chromosome 3 of cassava, specifically at position 392 from the 5' end of the sequence shown in SEQ ID NO:3, and its polymorphism is A / T.

[0050] Example 2 A molecular marker method for predicting and identifying resistance to bacterial wilt in different cassava varieties includes the following steps: S1. Extract RNA from the cassava variety to be identified and reverse transcribe it into cDNA; design upstream and downstream primers F and R based on the flanking nucleotide sequences of the molecular markers obtained in Example 1; Upstream primer F: 5'-ATGGCCCCTTCCTTGCTCG-3' (SEQ ID NO:1); Downstream primer R: 5'-TCAGCCTGCTGAATTCTCGTG-3' (SEQ ID NO:2); S2. Using the primers shown as upstream and downstream primers respectively, PCR amplification was performed on the cDNA reverse transcribed from cassava RNA. The PCR amplification yielded a target fragment of 1359 bp. The PCR amplification reaction system and procedure are shown in Table 1 and Table 2, respectively. Table 1 PCR amplification reaction system

[0051] Table 2 PCR amplification reaction procedure

[0052] S3. Sequencing analysis of the target fragment. The target fragment (nucleotide sequence shown in SEQ ID NO:3) contained AA at the 392nd base, and the genotype of the corresponding SNP site was a disease-resistant cassava variety. The polymorphism at this site was confirmed to be AA, AT, or TT. AA indicates that the amplification and sequencing results showed only A at this site; AT indicates that the amplification and sequencing results showed both A and T sequences at this site; and TT indicates that the amplification and sequencing results showed only T at this site.

[0053]

[0054] Example 3 The application of molecular markers significantly associated with resistance to bacterial wilt in cassava in marker-assisted breeding of cassava specifically includes: Molecular testing was performed on cassava varieties, and individuals with AA or AT bases at position 25542952 on cassava chromosome 3 (i.e., position 392 from the 5' end of the sequence shown in SEQ ID NO:3) were selected as varieties with relatively strong disease resistance.

[0055] In summary, the SNP molecular markers obtained by screening for resistance to bacterial wilt in cassava according to this invention can be effectively used for the molecular identification of resistance to bacterial wilt in different cassava varieties. This method is simple, efficient, and has a wide range of applications. The development of these molecular markers is of great significance for the breeding and screening of disease-resistant cassava varieties.

[0056] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A SNP molecular marker associated with bacterial wilt resistance in cassava, characterized in that, The sequence of the SNP molecular marker is shown as SEQ ID NO: 3, and the SNP site is located at the 392th site from the 5' end of SEQ ID NO: 3, and the polymorphism is A / T.

2. The SNP molecular marker of claim 1, wherein, When the genotype of the SNP site is AA or AT, it is a cassava with strong bacterial wilt resistance, and when the genotype of the SNP site is TT, it is a cassava with weak bacterial wilt resistance.

3. Use of a substance for detecting the SNP molecular marker of claim 1 or 2 in screening a cassava with strong bacterial wilt resistance or in preparing a product for screening a cassava with strong bacterial wilt resistance. Preferably, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of Northern blotting, PCR, gene chip method, nucleic acid sequencing method; Preferably, the product comprises at least one of reagents, kits, test papers, chips, systems.

4. A primer set for amplifying the SNP molecular marker of claim 1 or 2; Preferably, the nucleotide sequence of the primer set is as follows: F: 5'-ATGGCCCCTTCCTTGCTCG-3'; R: 5'-TCAGCCTGCTGAATTCTCGTG-3'.

5. A kit comprising the primer set of claim 4.

6. The kit of claim 5, wherein The kit further comprises a buffer used in PCR.

7. Use of the SNP molecular marker of claim 1 or 2, the primer set of claim 4, and / or the kit of claim 5 or 6 in any one of (1)-(4): (1) identifying or assisting in identifying bacterial wilt resistance of cassava; (2) preparing a product for identifying or assisting in identifying bacterial wilt resistance of cassava; (3) cassava assisted breeding; (4) preparing a product for cassava assisted breeding.

8. A method for identifying bacterial wilt resistance of a cassava variety, by detecting the genotype of the SNP molecular marker of claim 1 or 2 in the genome of a cassava variety to be tested, and determining the bacterial wilt resistance of the cassava variety to be tested according to the genotype.

9. The method of claim 8, wherein, The method comprises the following steps: using the primer set of claim 4 or the kit of claim 5 or 6 to perform PCR amplification with the genome of the cassava variety to be tested as a template to obtain a PCR amplification product; and performing sequencing analysis on the PCR amplification product to determine the genotype of the SNP molecular marker of claim 1 or 2 in the genome of the cassava variety to be tested.

10. Use of the method of claim 8 or 9 in cassava assisted breeding.

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