Fresh sweet corn aphid resistance related SNP site combination and application thereof

Abstract

The invention belongs to the technical field of molecular marker assisted breeding, and particularly relates to a fresh sweet corn aphid resistance related SNP site combination and application thereof. The SNP (Single Nucleotide Polymorphism) site combination of the aphid-resistant fresh sweet corn comprises an S4174685152 positioned on a chromosome 4, and an S5207733210 and an S5207734430 positioned on a chromosome 5. The developed aphid-resistant functional breeding marker site combination for the fresh-eating sweet corn can assist in breeding of the fresh-eating corn, rapid detection and early detection are achieved, the resistance of the fresh-eating sweet corn to the corn aphid can be predicted only by detecting the genotype of a specific site, and the aphid-resistant functional breeding marker site combination has the advantages of being clear in selection target, easy and convenient to operate and not affected by the environment. The invention provides an effective way for efficient breeding of fresh sweet corn.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding technology, specifically relating to a combination of SNP sites related to aphid resistance in fresh sweet corn and their application. Background Technology

[0002] Sweet corn is a type of corn consumed during its milk-ripe stage, primarily including three categories: sweet corn, waxy corn, and sweet-waxy corn. However, the corn aphid (Rhopalosiphummaidis), a major corn pest, can significantly reduce the yield and market quality of sweet corn. Currently, pest control mainly relies on chemical pesticides and insect-resistant breeding. However, due to the fresh-eating nature of sweet corn, pesticide residues pose a potential food safety risk. Therefore, genetically modifying and breeding aphid-resistant varieties has become the safest and most effective solution.

[0003] Currently, breeding of sweet maize still mainly relies on traditional phenotypic selection methods, but these methods have inherent drawbacks such as high cost, low efficiency, and long breeding cycles. In contrast, marker-assisted breeding (MAS) technology has significant advantages, including high cost-effectiveness, improved selection efficiency, and shorter breeding cycles. Molecular marker technology has been widely used in maize genetic improvement. Genome-wide association studies (GWAS) are important methods for elucidating the genetic mechanisms of complex traits in crops. As one of the most genetically diverse crops, maize has a large number of rare allelic variations in its genome, providing an ideal research system for using GWAS technology to analyze the genetic structure of aphid resistance traits in sweet maize.

[0004] Therefore, identifying a SNP locus that is significantly associated with aphid resistance in fresh maize to improve the breeding efficiency and accuracy of aphid-resistant fresh maize has become an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the problems of low efficiency and long cycle in existing conventional breeding methods for aphid-resistant sweet corn varieties, this invention provides a combination of SNP loci related to aphid resistance in sweet corn and its application, specifically including the following technical solution:

[0006] This invention provides a combination of SNP sites for aphid-resistant fresh sweet corn, wherein the combination of SNP sites includes one or more of S4_174685152, S5_207733210 and S5_207734430;

[0007] The S4_174685152 is located on chromosome 4, at position 174,685,152 bp, and contains a G / A single nucleotide variant.

[0008] The S5_207733210 is located on chromosome 5 at position 207,733,210 bp, and exhibits a C / T single nucleotide variant.

[0009] The S5_207734430 is located on chromosome 5 at position 207,734,430 bp, and exhibits a T / C single nucleotide variant.

[0010] Preferably, the haplotype of the SNP site combination is GCC, GCT, or GTT.

[0011] This invention also provides the application of the SNP locus combinations described above in one or more of the following: genetic breeding of fresh sweet corn, genetic diversity analysis, and population structure analysis.

[0012] This invention also provides the application of the SNP site combinations described above in the screening of core germplasm for fresh sweet corn.

[0013] Preferably, the associated trait of the SNP site combination is aphid resistance.

[0014] The present invention also provides a method for identifying or assisting in the identification of aphid-resistant fresh sweet corn, using the SNP site combination described above for identification or assistance.

[0015] Preferably, the method steps include:

[0016] Using the genomic DNA of the fresh sweet corn to be tested as a template, primers were used to amplify the genomic DNA to obtain the amplification product;

[0017] The haplotype of the amplified product was detected, and the aphid resistance of the fresh sweet corn to be tested was determined based on the haplotype results.

[0018] When the haplotype is GCC, fresh sweet corn is highly susceptible;

[0019] When the haplotype is GCT, fresh sweet corn exhibits moderate resistance;

[0020] When the haplotype is GTT, fresh sweet corn exhibits high resistance.

[0021] This invention also provides the application of the SNP site combinations or methods described above in the breeding of fresh sweet corn.

[0022] The present invention also provides a breeding method for aphid-resistant fresh sweet corn, which involves detecting the haplotype of the fresh sweet corn at the SNP site combination as described above, and selecting fresh sweet corn with haplotypes of GCT or GTT for breeding.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention provides a combination of SNP sites for aphid-resistant fresh sweet corn, comprising one or more of S4_174685152, S5_207733210, and S5_207734430; S4_174685152 is located on chromosome 4 at position 174,685,152 bp, exhibiting a G / A single nucleotide variant; S5_207733210 is located on chromosome 5 at position 207,733,210 bp, exhibiting a C / T single nucleotide variant; and S5_207734430 is located on chromosome 5 at position 207,734,430 bp, exhibiting a T / C single nucleotide variant. The aphid-resistant functional breeding markers for sweet corn developed by this invention can assist in the breeding of sweet corn. By simply detecting the genotype at a specific locus, the resistance of sweet corn to corn aphids can be predicted. This method has the advantages of targeted selection and independence from environmental influences. Furthermore, since corn aphid outbreaks mainly occur in the later stages of the sweet corn growth cycle, the method of this invention allows for early detection. The molecular markers disclosed in this invention can also be used for molecular aggregation breeding of multiple traits in sweet corn, providing an effective approach for efficient breeding of sweet corn. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0026] Figure 1 This is a distribution diagram of SNPs on the ten chromosomes of maize in Example 2;

[0027] Figure 2 This is the QQ diagram of the genome-wide association analysis of maize in Example 3;

[0028] Figure 3 The Manhattan plot shows the results of the genome-wide association analysis of maize in Example 3;

[0029] Figure 4 For the haplotype results statistics in Example 4;

[0030] Figure 5 This is a box plot of the haplotype in Example 4. Detailed Implementation

[0031] This invention provides a combination of SNP sites for aphid-resistant fresh sweet corn, comprising one or more of S4_174685152, S5_207733210, and S5_207734430; S4_174685152 is located on chromosome 4 at position 174,685,152 bp, exhibiting a G / A single nucleotide variant; S5_207733210 is located on chromosome 5 at position 207,733,210 bp, exhibiting a C / T single nucleotide variant; and S5_207734430 is located on chromosome 5 at position 207,734,430 bp, exhibiting a T / C single nucleotide variant.

[0032] In one embodiment, the genome version referenced for the SNP site combination of the present invention is B73v4. In another embodiment, the haplotype of the SNP site combination is GCC, GCT, or GTT.

[0033] This invention also provides the application of the SNP locus combinations described above in one or more of the following: genetic breeding of fresh sweet corn, genetic diversity analysis, and population structure analysis.

[0034] This invention also provides the application of the SNP site combinations described above in the screening of core germplasm for fresh sweet corn.

[0035] In one implementation, in the application described above, the associated trait of the SNP locus combination is the aphid resistance trait. The association between the haplotype of the SNP locus and the aphid resistance trait is as previously described and will not be repeated here.

[0036] The present invention also provides a method for identifying or assisting in the identification of aphid-resistant fresh sweet corn, which uses the SNP site combination described above for identification or assistance in identification.

[0037] As one implementation method, the method steps include:

[0038] Using the genomic DNA of fresh sweet corn as a template, primers are used to amplify the genomic DNA, obtaining amplification products. The haplotype of the amplification products is detected, and the aphid resistance of the fresh sweet corn is determined based on the haplotype results. When the haplotype is GCC, the fresh sweet corn is highly susceptible; when the haplotype is GCT, the fresh sweet corn is moderately resistant; and when the haplotype is GTT, the fresh sweet corn is highly resistant. As one implementation method, the primers are used to amplify the genomic DNA of fresh sweet corn.

[0039] This invention also provides the application of the SNP site combinations or methods described above in the breeding of fresh sweet corn.

[0040] The present invention also provides a breeding method for aphid-resistant fresh sweet corn, which involves detecting the haplotype of the fresh sweet corn at the SNP site combination as described above, and selecting fresh sweet corn with haplotypes of GCT or GTT for breeding.

[0041] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a combination of SNP sites related to resistance to aphids in fresh sweet corn and their application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1: Determination of aphid resistance in fresh sweet corn

[0043] In cornfields infested with aphids, randomly select an affected corn plant and collect a wingless adult corn aphid from the plant for later use.

[0044] Wingless adult corn aphids were collected and inoculated onto seedlings of the highly aphid-susceptible corn inbred line CML322. The seedlings were then placed in an incubator for monoclonal family propagation. The specific method was as follows: Aphids were cultured under suitable environmental conditions for aphid growth and reproduction. Every two weeks, the CML322 seedlings in the incubator were replaced with new CML322 seedlings. The replacement method involved cutting off old leaves containing a small number of aphids and transferring them to the new seedlings. Following the above method, a sufficient number of aphids were cultured according to experimental requirements.

[0045] Twenty-four core inbred lines of sweet corn were used as a population and planted in a greenhouse. Eight seeds were sown in each material, with the corn inbred line CML322 serving as a susceptible control and the American inbred line Mo17 as a resistant control. When the corn seedlings reached the three-leaf stage, one-day-old corn aphids were inoculated onto the leaves. The occurrence and distribution of aphids were observed and recorded daily until the susceptible control CML322 plants withered. The survival rate of each material was then counted, and aphid resistance was assessed based on aphid population dynamics data. Aphid resistance was divided into five levels: level 1, level 3, level 5, level 7, and level 9 (level 1: highly susceptible, level 9: highly resistant). CML322 and Mo17 served as the susceptible control (level 1) and the resistant control (level 9), respectively.

[0046] Example 2: Genotyping of Associated Populations

[0047] This study used the CTAB method to extract genomic DNA from the young leaves of 234 fresh sweet corn populations described in Example 1, and then performed 5× coverage depth whole-genome resequencing using the Illumina sequencing platform. The data processing workflow is as follows:

[0048] (1) Use FASTP software to perform quality control filtering on the raw sequencing data based on the Q20 standard;

[0049] (2) The filtered data were aligned to the maize reference genome B73v4 using BWA software;

[0050] (3) Use the Picard software package to label repetitive sequences in sequencing data;

[0051] (4) Use GATK software to detect variant sites;

[0052] (5) Use bcftools software to filter genotype data. The screening criteria include:

[0053] ① Only biallelic SNP sites are retained;

[0054] ② Minor allele frequency (MAF) > 0.05;

[0055] ③ Missing rate <5%;

[0056] ④ The chain imbalance strength of adjacent SNPs is >0.5.

[0057] A total of 2,547,399 SNPs were retained after filtering, as shown in Table 1 and [other tables]. Figure 1 As shown, this is used for subsequent genome-wide association analysis.

[0058] Table 1. SNP statistics after filtering

[0059] chromosome SNP number Density (SNP / Mb) 1 362,718 1,181.33 2 291,820 1,193.82 3 284,977 1,209.23 4 346,406 1,402.48 5 275,048 1,228.43 6 188,417 1,082.65 7 223,629 1,226.16 8 223,410 1,233.47 9 170,827 1,069.21 10 187,832 1,244.07 total 2,555,084 1,213.05

[0060] Example 3: Genome-wide association analysis of maize aphid resistance

[0061] Genome-wide association analysis (GWAS) was performed on the aphid resistance trait of the 234 sweet corn populations described in Example 1 using GEMMA software and a mixed linear model (with kinship matrix and population structure matrix as covariates). The reference sweet corn genome version was B73v4. The significance threshold for the GWAS analysis was set to 1×10⁻⁶. -6 The analysis identified three SNP loci significantly associated with aphid resistance, as shown in Table 2. Figures 2-3 As shown, the three SNP loci significantly associated with aphid resistance are S4_174685152 on chromosome 4, S5_207733210 on chromosome 5, and S5_207734430 on chromosome 5.

[0062] Table 2. SNP information significantly associated with resistance to maize aphids.

[0063] SNPID chromosome Location (bp) p-value S4_174685152 4 174,685,152 <![CDATA[8.44×10 -7 ]]> S5_207733210 5 207,733,210 <![CDATA[6.51×10 -7 ]]> S5_207734430 5 207,734,430 <![CDATA[9.05×10 -7 ]]>

[0064] Example 4: Haplotype Effect Analysis

[0065] Among 189 aphid-resistant sweet corn breeding core lines, three haplotypes were found at the S4_174685152, S5_207733210, and S5_207734430 loci: GCC, GCT, and GTT. Figure 4 Among them, the GCC haplotype had the highest frequency in the population (85 inbred lines), followed by the GCT haplotype (59 inbred lines), and the GTT haplotype had the lowest frequency (45 inbred lines).

[0066] A t-test was performed on the three haplotypes mentioned above, and the results are as follows: Figure 5 As shown, there are significant differences in aphid resistance among these three haplotypes. Specifically, the GCC haplotype indicates high susceptibility, the GCT haplotype indicates moderate resistance, and the GTT haplotype indicates high resistance. Based on this, molecular markers targeting these three loci can be developed for aphid-resistant breeding of fresh sweet corn, enabling the screening of breeding materials with excellent aphid resistance.

[0067] In summary, this invention provides the application of SNP loci in aphid-resistant maize. The aphid-resistant functional breeding markers for sweet maize developed by this invention can assist in the breeding of sweet maize. By simply detecting the genotype of a specific locus, the resistance of sweet maize to maize aphids can be predicted. It has the advantages of clear selection targets and being unaffected by environmental factors. Furthermore, since maize aphid outbreaks mainly occur in the later stages of the sweet maize growth cycle, the method of this invention allows for early detection. The molecular markers disclosed in this invention can also be used for molecular aggregation breeding of multiple traits in sweet maize. This invention provides an effective approach for efficient breeding of sweet maize.

[0068] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A combination of SNP sites for aphid-resistant fresh sweet corn, characterized in that, The SNP locus combination includes one or more of S4_174685152, S5_207733210 and S5_207734430; The S4_174685152 is located on chromosome 4, at position 174,685,152 bp, and contains a G / A single nucleotide variant. The S5_207733210 is located on chromosome 5 at position 207,733,210 bp, and exhibits a C / T single nucleotide variant. The S5_207734430 is located on chromosome 5 at position 207,734,430 bp, and exhibits a T / C single nucleotide variant.

2. The SNP site combination according to claim 1, characterized in that, The haplotypes of the SNP site combinations are GCC, GCT, or GTT.

3. The application of the SNP locus combination described in claim 1 or 2 in one or more of the genetic breeding, genetic diversity analysis, and population structure analysis of fresh sweet corn.

4. The application of the SNP site combination as described in claim 1 or 2 in the screening of core germplasm of fresh sweet corn.

5. The application according to claim 3 or 4, characterized in that, The associated trait of the SNP site combination is aphid resistance.

6. A method for identifying or assisting in the identification of aphid-resistant fresh sweet corn, characterized in that, Identification or auxiliary identification can be performed using the SNP site combinations described in claim 1 or 2.

7. The method as described in claim 6, characterized in that, The method steps include: Using the genomic DNA of the fresh sweet corn to be tested as a template, primers were used to amplify the genomic DNA to obtain the amplification product; The haplotype of the amplified product was detected, and the aphid resistance of the fresh sweet corn to be tested was determined based on the haplotype results. When the haplotype is GCC, fresh sweet corn is highly susceptible; When the haplotype is GCT, fresh sweet corn exhibits moderate resistance; When the haplotype is GTT, fresh sweet corn exhibits high resistance.

8. The application of the SNP site combination as described in claim 1 or 2 or the method as described in claim 6 or 7 in the breeding of fresh sweet corn.

9. A breeding method for aphid-resistant sweet corn for fresh consumption, characterized in that, The haplotype of the fresh sweet corn at the SNP site combination as described in claim 1 or 2 is detected, and fresh sweet corn with haplotypes of GCT or GTT is selected for breeding.

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