CN118600102A - A ZmB4FMV1 gene, locus and application thereof for detecting alpha-tocopherol content in sweet corn kernels - Google Patents

Abstract

The invention discloses a ZmB4FMV1 gene, a site and an application thereof for detecting the alpha-tocopherol content of sweet corn kernels, and identifies the genotype of the SNP sites at positions 172026809, 171865153, 171866110, 171863018, 171863558, 171864361, 171864389, 171864453, 171864496, 171864722 and 171866207 of chromosome 8 of the sweet corn genome, and selects the sweet corn with the genotype of the variable site for breeding and planting, so as to realize the breeding of sweet corn with high alpha-tocopherol content. Genotype detection can be completed quickly, with low cost, simple operation and suitable for high-throughput detection. The site will greatly promote the bio-fortification breeding of sweet corn with high alpha-tocopherol content. The present invention can quickly complete genotype detection based on membrane protein gene loci, has low cost, simple operation, is suitable for high-throughput detection, and promotes the biofortification breeding of sweet corn with high alpha-tocopherol content.

Description

A ZmB4FMV1 gene, locus and application thereof for detecting alpha-tocopherol content in sweet corn kernels

Technical Field

The invention belongs to the field of genetic engineering, and in particular relates to a ZmB4FMV1 gene for detecting the alpha-tocopherol content of sweet corn kernels, a site and an application thereof.

Background Art

Sweet corn is a fruit and vegetable crop and an important dietary source of micronutrients. The structures of all vitamin E compounds differ only in the position and number of methyl substituents on the 2,3-dihydrobenzofuran ring, but their biological activities vary greatly. Vitamin E includes eight fat-soluble isomers (including tocopherol and tocotrienol), of which alpha-tocopherol has the highest content among the eight vitamin E components, accounting for about 38% of the total vitamin E content, which is three times the content of alpha-tocopherol. Studies have shown that although tocopherol and tocotrienol are similar in structure, there are huge differences in their biological functions. Compared with tocopherol, tocotrienol has stronger antioxidant and lipid oxidation inhibition. It also has physiological functions such as lowering cholesterol and anti-tumor. For sweet corn seeds, these physiological functions of alpha-tocopherol can not only ensure the normal development of sweet corn seeds, but also increase the nutrition and added value of sweet corn. It is of great significance to increase the content of alpha-tocopherol in sweet corn kernel tissue through genetic breeding.

Methods based on genomics and population genetics provide important opportunities for analyzing the genetic basis of vitamin E synthesis. The rich genetic variation of sweet corn populations provides favorable conditions for the utilization of key genes. It creates conditions for the targeted manipulation of the levels and types of tocopherols and tocotrienols in crops. In previous studies, linkage and association analyses of tocopherol and tocotrienol content in corn kernels provided a lot of important information for the genetic regulation of vitamin E synthesis and metabolism. However, most of the major quantitative trait genetic loci affecting tocopherol composition usually point to VTE4, that is, VTE4 is the key locus for vitamin E synthesis in corn, and there are fewer studies on other loci. However, except for VTE4, other genes/locus for screening high tocopherol and high tocotrienol content are rarely reported. Therefore, a ZmB4FMV1 gene, locus and its application for detecting the alpha-tocopherol content in sweet corn kernels are needed.

Summary of the invention

The purpose of the present invention is to provide a ZmB4FMV1 gene and site for detecting the alpha-tocopherol content of sweet corn kernels and applications thereof.

The present invention is achieved through the following technical solutions:

The application of the SNP site of chromosome 8 of the sweet corn genome of the present invention, the sweet corn genome is genome B73, v4 version, ID is Zm00001d012284, including the following SNP sites:

1) The SNP site at position 172026809 on chromosome 8 of the sweet corn genome has a polymorphism of G/C;

2) The SNP site at position 171865153 on chromosome 8 of the sweet corn genome has an A/T polymorphism;

3) The SNP site at position 171866110 on chromosome 8 of the sweet corn genome has a polymorphism of G/T;

4) The SNP site at position 171863018 on chromosome 8 of the sweet corn genome has a polymorphism of C/T;

5) The SNP site at position 171863558 on chromosome 8 of the sweet corn genome has a polymorphism of C/A;

6) The SNP site at position 171864361 on chromosome 8 of the sweet corn genome has a polymorphism of C/T;

7) The SNP site at position 171864389 on chromosome 8 of the sweet corn genome has a polymorphism of C/T;

8) The SNP site at position 171864453 on chromosome 8 of the sweet corn genome has a polymorphism of G/T;

9) The SNP site at position 171864496 on chromosome 8 of the sweet corn genome has a polymorphism of G/A;

10) The SNP site at position 171864722 of chromosome 8 of the sweet corn genome has a polymorphism of T/C;

11) The SNP site at position 171866207 on chromosome 8 of the sweet corn genome has a polymorphism of G/A;

The SNP site is related to the expression level of the gene ZmB4FMV1 and thus affects the alpha-tocopherol content.

Furthermore, when the expression of ZmB4FMV1 in the sweet corn kernels containing the SNP site is increased, it is identified as sweet corn with high alpha-tocopherol content; when the expression of ZmB4FMV1 in the sweet corn kernels is reduced, it is identified as sweet corn with low alpha-tocopherol content.

In another aspect, a variant site detection primer set is provided, wherein the detection site primer set comprises a forward primer as shown in SEQ ID NO: 1; a reverse primer as shown in SEQ ID NO: 2; and a universal primer as shown in SEQ ID NO: 3.

In another aspect, a kit for detecting high alpha-tocopherol content in sweet corn comprises the variable site detection primer set.

In another aspect, a method for screening sweet corn with high alpha-tocopherol content comprises the following steps:

In terms of molecular marker-assisted selection breeding (MAS), based on the primer combination described in claim 3, the genotypes of the SNP sites at positions 172026809, 171865153, 171866110, 171863018, 171863558, 171864361, 171864389, 171864453, 171864496, 171864722, and 171866207 of chromosome 8 of the sweet corn genome are identified, and sweet corn with the genotype of the variable site is selected for breeding and planting, so as to achieve the breeding of sweet corn with high alpha-tocopherol content.

Compared with the prior art, the present invention adopts the above technical solution, and the biggest feature is:

Compared with the prior art, the present invention has the following effects:

1. The present invention uses association analysis technology to newly explore and identify SNP sites related to the control of alpha-tocopherol content in sweet corn kernels and the related ZmB4FMV1 (Zm00001d012284), supplementing and enriching the genetic sites for molecular identification of alpha-tocopherol content in sweet corn kernels.

2. The KASP molecular marker based on SNP detection developed by the present invention based on the membrane protein gene (ZmB4FMV1) site can quickly complete genotype detection, has low cost, simple operation, and is suitable for high-throughput detection. This site will greatly promote the biofortification breeding of sweet corn with high alpha-tocopherol content.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram of the GWAS of the present invention identifying ZmB4FMV1 as a candidate gene for alpha-tocopherol mutation;

Figure 1 (a) is a local Manhattan plot including chromosome 8; Figure 1 (b) is a LD heat map; Figure 1 (a) is the location of the candidate gene ZmB4FMV1;

FIG2 is a schematic diagram showing the significant correlation between alpha-tocopherol content and ZmB4FMV1 gene expression in a sweet corn population according to the present invention;

FIG3 is a schematic diagram showing the comparison of alpha-tocopherol content between different alleles of the significant SNP (S8_171864496) of the present invention;

The P value was calculated using one-way ANOVA. n is the number of genotypes for each allele;

FIG. 4 is a distribution diagram of fluorescence values after amplification using the KASP primers of the present invention.

DETAILED DESCRIPTION

The technical scheme of the present invention is further described below in conjunction with embodiments and comparative examples, but they should not be construed as limiting the present invention:

The objective of the present invention is achieved through the following technical solutions:

Example 1 Mining and analysis of alpha-tocopherol related gene ZmB4FMV1

1. Sweet corn association analysis group:

295 sweet corn inbred line materials were selected from widely representative inbred line materials collected from the world's major sweet corn growing areas (including China, the United States, Thailand, Japan, Argentina, etc.) to form a sweet corn association analysis population. Leaf tissues were collected at the seedling stage, and whole genome resequencing was performed on the llumina Hiseq 2500 platform after genomic DNA extraction, library construction and other steps. The average sequencing depth was 11X, and about 9.86 million high-quality SNP data sets were identified on the entire genome. Secondly, the kernel tissues of the sweet corn materials in the population were sampled 15 days after pollination, and a transcriptome sequencing library with an insert size of 300-500 bp was constructed. Transcriptome sequencing was performed using 150bp double-end paired RNA sequencing technology, and transcriptome sequencing of the whole kernel tissue was performed on the llumina Hiseq 2500 sequencing platform, generating a total of 31.27 million read bases. After bioinformatics analysis, the expression data of each gene in each material in the sweet corn population were obtained. This completes the expression results of kernel tissue in the sweet corn population.

In 2019, 295 sweet corn lines were planted at the Field Experiment Station of Guangdong Academy of Agricultural Sciences (E113°224, N23°093) with a planting pattern of about 12 plants per row. The experiment adopted a randomized block design, with 3 replicates for each inbred line. All sweet corn lines in each plot were self-pollinated, and pollination data were recorded. 20 days after pollination, an appropriate amount of grains were harvested from 3 plants in each row. Immediately after harvesting, the grains were frozen in liquid nitrogen until transferred to a -80°C refrigerator for storage.

2. Determination of alpha-tocopherol:

Vitamin E extraction: Sweet corn kernels were precooled with liquid nitrogen and then ball-milled for 60 seconds at 30 Hz using a mill (MM400; Retsch). 2 g of fresh sweet corn flour was accurately weighed in a frozen state. The sample was saponified with 2 mL of ethanol (95%). Antioxidants (1 mL of sodium chloride, 176 mg/mL; 4 mL of pyrogallol ethanol, 63.055 g/L; 1 mL of ascorbic acid, 176 mg/mL) and 2 mL of potassium hydroxide (600 g/L) were then added and mixed using a vortex mixer. The mixture was then extracted with n-hexane/ethyl acetate (9:1, v/v). The liquid phase of the organic layer was collected and concentrated to dryness using a concentrator under nitrogen. The residue was dissolved in 1% isopropanol in n-hexane for HPLC analysis.

Quantitative detection of vitamin E: The quantitative detection of vitamin E was performed by normal phase high performance liquid chromatography (NP-HPLC). The NP-HPLC system included a Waters 515 HPLC pump and a Waters 2475 multi-λ fluorescence detector. The chromatographic column was Agilent (ZORBAX RX-SIL, 250 mm × 4.6 mm) with a flow rate of 1 mL/min. The mobile phase was 0.85% n-hexane-2-propanol, and 20 µL was injected. The excitation wavelengths for detection were 290 nm and 330 nm. The content of each vitamin E component was calculated using a standard curve constructed using 8 external standards.

3. Sweet corn population SNP profile and expression level identification

Leaf tissues were collected at the seedling stage, and after genomic DNA extraction and library construction, whole genome resequencing was performed on the llumina Hiseq2500 platform. The average sequencing depth was 11X, and about 9.86 million high-quality SNP maps were identified on the entire genome. Secondly, the grain tissues were sampled 15 days after pollination, and a transcriptome sequencing library with an insert size of 300-500 bp was constructed. Transcriptome sequencing was performed using 150bp double-end paired RNA sequencing technology. Transcriptome sequencing of the whole grain tissue was performed on the llumina Hiseq 2500 sequencing platform, generating a total of 31.27 million read bases. Thus, the expression results of genes in grain tissues of each individual in the sweet corn population were completed.

4. Genome-wide association analysis of alpha-tocopherol

Association analysis was performed using ultra-high density molecular markers (SNPs, 9.86 million), and genome-wide association analysis was performed on alpha-tocopherol content in grain tissues of sweet corn population materials using mixed linear models (MLM), and genetic loci controlling alpha-tocopherol content were identified. The number of effective SNPs in the whole genome was estimated using GEC (Genetic Type I error calculator) software. P-value=1/n was used as the significance threshold of genome-wide association analysis (n, the number of effective SNPs in the whole genome). The membrane protein gene ZmB4FMV1 (gene ID: Zm00001d012284, Chr8: 171862718-171867456; reference genome version: B73 V4) locus was identified by genome-wide association analysis as a gene/locus significantly associated with alpha-tocopherol. This gene/locus is located at SNP site S8_172026809 of the maize reference genome (B73, V4) and is closely linked to the membrane protein (ZmB4FMV1) gene. The variant base of this site is C/G, among which C is the favorable allele with an allele frequency of 0.055. This site is significantly correlated with the alpha-tocopherol content in sweet corn kernels.

TASSEL (3.0) was used for genome-wide association analysis, and mixed linear models were used to control population structure and kinship. To determine the genome-wide threshold for GWAS results, the number of effective SNPs in the genome was estimated using GEC (Genetic Type I errorcalculator) software with default parameters. Then, P=5.08×10 ^-7 (P=1/n, n=number of effective SNP markers) was selected as the significance threshold for genome-wide association analysis. To determine the independence of significant SNPs, conditional GWAS was performed using the most significant SNP in the QTL region as a covariate. Candidate genes for GWAS loci were located within the confidence interval or within 100 kb for further subsequent analysis. Based on the results of the alpha-tocopherol genome-wide association analysis, 11 SNP loci significantly associated with alpha-tocopherol were identified in the lead SNP (S8_172026809) and ZmB4FMV1 gene region (Figure 1). These 11 SNP loci were 100% linked and could be used to screen materials with high alpha-tocopherol content.

Table 1 Results of association analysis of alpha-tocopherol

aCandidate genes are candidate genes related to alpha-tocopherol content within the significant loci.

b Location information of the major SNP, including the chromosome and specific base position. The base position was determined with reference to the maize B73 reference genome V4 sequence.

The c contribution rate was determined by mixed linear model in tassel (3.0) software.

d The non-favorable allele type present for this SNP.

e favorable allele type for alpha-tocopherol content.

f is the frequency of favorable alleles in the population.

g is the number of alleles in the population after removing the missing genotypes.

Example 2 Verification of the effect of gene ZmB4FMV1 on alpha-tocopherol content

Alpha-tocopherol was precisely quantified in fresh kernel tissue of sweet corn 20 days after pollination. The content of alpha-tocopherol was determined by normal phase HPLC (NP-HPLC), the liquid phase infusion pump model was Waters 515, the fluorescence detector model was Waters 2475, the chromatographic column model was Agilent ZORBAX RX-SIL, 250 mm × 4.6 mm, the liquid phase contained 0.85% n-hexane, and the flow rate was 1 ml/min. Dual wavelength inspection was used, and the detection wavelengths were 290 and 330 nm.

Combined with the transcriptome data of grain tissue 15 days after pollination, the alpha-tocopherol content in the grain tissue was significantly correlated with the expression level of the gene ZmB4FMV1, with a correlation coefficient of 0.225 and a P value of 9.41×10-5, reaching an extremely significant level (Figure 2), indicating that the expression level of ZmB4FMV1 was significantly correlated with the alpha-tocopherol content.

In order to verify that there are significant differences in alpha-tocopherol content between different alleles, since the above 11 SNP sites are 100% linked, one of the SNPs, S8_171864496, was selected and a one-way ANOVA was performed on the phenotypic differences under different allele types. The results showed that there were extremely significant differences between the alleles GG/AA, and the favorable allele could increase the average alpha-tocopherol content by 81% (Figure 3).

Example 3 Molecular marker development

1. Development of alpha-tocopherol-related KASP markers

Association analysis and transcriptome analysis verified the function of gene ZmB4FMV1 in the alpha-tocopherol content of sweet corn kernel tissue. Combined with the 11 SNP sites that were 100% linked and significantly associated with alpha-tocopherol identified by association analysis, any SNP site can be developed for markers to screen sweet corn materials with high alpha-tocopherol content. In this example, KASP markers were developed for S8_171864496 (GG/AA) in the above-mentioned SNP site (Figure 4). KASP primer design was completed by PRIMER-BLAST, a web primer design tool provided by NCBI. Primer synthesis was performed by LGC (Laboratory of the Government Chemist, Hoddeston, UK).

For the above alpha-tocopherol-related membrane protein gene (ZmB4FMV1) locus, marker screening primers based on KASP technology were developed for S8_171864496 (GG/AA) in the SNP locus for screening of favorable allele genetic loci with high alpha-tocopherol content. The design of KASP primers was completed using the NCBI-BLAST program. Three primers were designed for each detection site, including two forward primers for genotype screening and one universal reverse primer. The primers were purified after synthesis by UPLC method.

Primer information is as follows:

KASP primer sequence F1: gaaggtgaccaagttcatgctCTCTAGGAACAAAGCACTCG

(SEQ ID NO: 1)

KASP primer sequence F2: gaaggtcggagtcaacggattCTCTAGGAACAAAGCACTCA

(SEQ ID NO: 2)

KASP universal primer sequence: CTCCCAAACATGCCCTTACT (SEQ ID NO: 3)

The PCR amplification reaction system is shown in Table 2 (96-well white opaque PCR plate, 10.14 μL reaction system). The KASP amplification reaction procedure is shown in Table 3.

Table 2 KASP PCR reaction system

Table 3 KASP amplification reaction procedure

The result of KASP amplification reaction is shown in FIG4 . The SNP AA allele of KASP primers has a high alpha-tocopherol content in the sweet corn material to excite HEX fluorescence, and the SNP GG allele has a low alpha-tocopherol content in the sweet corn material to excite FAM fluorescence. The allele AA and the allele GG can be distinguished by fluorescence quantitative PCR detection. The KASP primers developed by the present invention are highly flexible and can quickly complete the detection of a small amount and a large amount of sweet corn material genotypes, and can quickly screen sweet corn materials with high alpha-tocopherol content. As shown in FIG4 , the genotyping results of this KASP marker are completely consistent with the high-throughput sequencing results. It has high stability and universality. The application of this marker in the improvement of sweet corn alpha-tocopherol will play an important role in promoting the high-quality breeding of sweet corn.

2. KASP marker genotype detection method

As shown in the sequence, the lowercase character sequence gaaggtgaccaagttcatgct is the FAM fluorescent group linker sequence, and the lowercase character sequence gaaggtcggagtcaacggatt is the HEX fluorescent group linker sequence. The products amplified by the primers containing these two fluorescent group sequences and the universal reverse primer carry fluorescent groups that can excite FAM and HEX, respectively. After the KASP reaction system and reaction procedure, the PCR reaction of each material is completed. The KASP reaction system is fluorescently scanned using a PCR instrument or an enzyme marker with a fluorescence scanning function to complete the typing analysis of the sample, obtain the genotype information of the sweet corn material, and then complete the allele type determination and predict the alpha-tocopherol content.

The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims (5)

1. Application of gene ZmB4FMV1 site on chromosome 8 of sweet corn genome, wherein the sweet corn genome is genome B73, v4 version, ID is Zm00001d012284, characterized in that it includes the following SNP sites: 1) The SNP site at position 172026809 on chromosome 8 of the sweet corn genome has a polymorphism of G/C and a favorable allele of C; 2) The SNP site at position 171865153 on chromosome 8 of the sweet corn genome has a polymorphism of A/T and a favorable allele of T; 3) The SNP site at position 171866110 on chromosome 8 of the sweet corn genome has a polymorphism of G/T and a favorable allele of T; 4) The SNP site at position 171863018 on chromosome 8 of the sweet corn genome has a polymorphism of C/T and a favorable allele of T; 5) The SNP site at position 171863558 on chromosome 8 of the sweet corn genome has a polymorphism of C/A and the favorable allele is A; 6) The SNP site at position 171864361 on chromosome 8 of the sweet corn genome has a polymorphism of C/T and a favorable allele of T; 7) The SNP site at position 171864389 on chromosome 8 of the sweet corn genome has a polymorphism of C/T and a favorable allele of T; 8) The SNP site at position 171864453 on chromosome 8 of the sweet corn genome has a polymorphism of G/T and a favorable allele of T; 9) The SNP site at position 171864496 on chromosome 8 of the sweet corn genome has a polymorphism of G/A and a favorable allele of A; 10) The SNP site at position 171864722 of chromosome 8 of the sweet corn genome has a polymorphism of T/C and a favorable allele of C; 11) The SNP site at position 171866207 on chromosome 8 of the sweet corn genome has a polymorphism of G/A and a favorable allele of A; The SNP site is highly linked and significantly correlated with the expression of gene ZmB4FMV1, thereby affecting the alpha-tocopherol content. 2. The use according to claim 1, characterized in that: the expression of ZmB4FMV1 in the sweet corn kernels containing the favorable allele of the SNP site is increased, and the sweet corn is identified as having a high alpha-tocopherol content; the expression of ZmB4FMV1 in the sweet corn kernels containing the unfavorable allele of the SNP site is reduced, and the sweet corn is identified as having a low alpha-tocopherol content. 3. A variant site detection primer set, characterized in that: the detection site primer set includes a forward primer as shown in SEQ ID NO: 1; a reverse primer as shown in SEQ ID NO: 2; and a universal primer as shown in SEQ ID NO: 3. 4. A kit for detecting high alpha-tocopherol content in sweet corn, characterized in that it comprises the variant site detection primer set according to claim 3. 5. A method for screening sweet corn with high alpha-tocopherol content, characterized in that it comprises the following steps: In the early stage of molecular marker-assisted selection breeding (MAS), based on the primer combination described in claim 3, the genotypes of the SNP sites at positions 172026809, 171865153, 171866110, 171863018, 171863558, 171864361, 171864389, 171864453, 171864496, 171864722, and 171866207 of chromosome 8 of the sweet corn genome are identified, and sweet corn with a genotype having a favorable variable site is selected for breeding and planting, thereby achieving the breeding of sweet corn with a high alpha-tocopherol content.