SNP, KASP marker closely linked to the number of tassel branches of maize and its application

Abstract

The application discloses a SNP (Single Nucleotide Polymorphism) closely linked with the number of tassel branches of corn, a KASP (Kompetitive Allele Specific PCR) marker and application thereof, and belongs to the technical field of molecular marker assisted breeding.The application provides a SNP and a KASP marker closely linked with the number of tassel branches of corn; the SNP is located at 24904636bp of a No.3 chromosome of a V4 version of a B73 genome; the forward primer of the KASP marker is 1TBN23-KASP-106F1 and 1TBN23-KASP-106F2, and the reverse primer is 1TBN23-KASP-106R; the provided SNP and KASP marker can be used for good typing of the number of tassel branches of corn, and can be used for identification of the number of tassel branches of corn and molecular marker assisted breeding, and has important theoretical and practical guiding significance for accelerating the genetic breeding and improvement process of corn varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, and in particular to SNP and KASP markers closely linked to the number of branches in maize tassels and their applications. Background Technology

[0002] As one of the world's major food crops, maize is not only an important food source but also widely used in feed, industrial raw materials, and bioenergy production. The number of tassel branches in maize is a key trait determining pollination ability and pollen quantity, directly affecting maize's density tolerance, hybrid seed production efficiency, and yield performance. Therefore, molecular markers developed around the number of tassel branches have broad application prospects in improving maize breeding efficiency and achieving precise aggregation of target traits, and have become an important technological breakthrough direction in maize molecular breeding.

[0003] Single nucleotide polymorphisms (SNPs) are DNA sequence polymorphisms caused by variations in a single nucleotide base at the genomic level. They are numerous, widely distributed, and genetically stable. With the development of gene sequencing technology and the reduction in sequencing costs, direct sequencing and sequence comparison of the same gene or gene fragment in different individuals can determine the presence of SNP variations. Therefore, SNP detection is beneficial for genotyping and is suitable for rapid and large-scale screening of the relationship between unknown or known SNPs and a certain genetic trait. Kompetitive allele-specific PCR (KASP) is a gel-free fluorescent polymerase chain reaction genotyping technique based on SNPs and has been widely used in animal and plant genetic breeding research. Summary of the Invention

[0004] The purpose of this invention is to provide SNPs and KASP markers closely linked to the number of branches on maize tassels and their applications, so as to provide a major-effect SNP related to the number of branches on maize tassels, and to assist in the identification of the trait of the number of branches on maize tassels and molecular marker-assisted breeding.

[0005] To achieve the above objectives, the present invention provides a SNP that is closely linked to the number of branches of the maize tassel, the SNP being located at 24904636 bp on chromosome 3 of the B73 genome V4 version.

[0006] Preferably, the SNP site of maize plants with fewer tassel branches is AA, and the SNP site of maize plants with more tassel branches is GG.

[0007] Preferably, the number of branches on the corn tassel is less than 6, and the number of branches on the corn tassel is greater than 6.

[0008] KASP markers for identifying the number of branches in maize tassels, wherein the KASP markers are SNPs closely linked to the number of branches in maize tassels, and include two forward primers and one reverse primer.

[0009] Preferably, the two forward primers are 1TBN23-KASP-106F1 and 1TBN23-KASP-106F2, and the one reverse primer is 1TBN23-KASP-106R; the sequence of 1TBN23-KASP-106F1 is shown in SEQ ID NO.1, the sequence of 1TBN23-KASP-106F2 is shown in SEQ ID NO.2, and the sequence of 1TBN23-KASP-106R is shown in SEQ ID NO.3.

[0010] The application of SNPs closely linked to the number of branches on maize tassels, as described above, in the identification of the number of branches on maize tassels.

[0011] The application of SNPs closely linked to the number of branches on maize tassels, as described above, in maize breeding.

[0012] The application of the KASP marker, as described above, for identifying the number of branches on maize tassels in the identification of the number of branches on maize tassels.

[0013] The application of the KASP marker for identifying the number of male branches in maize, as described above, in maize breeding.

[0014] Therefore, the specific technical effects of the SNP and KASP markers closely linked to the number of branches in maize tassels provided by this invention, and their applications, are as follows:

[0015] (1) This invention provides an SNP and KASP marker that are closely linked to the number of branches of maize tassels; the SNP is located at 24904636bp on chromosome 3 of B73 genome V4 version; the forward primers for the KASP marker are 1TBN23-KASP-106F1 and 1TBN23-KASP-106F2, and the reverse primer is 1TBN23-KASP-106R;

[0016] (2) The SNP and KASP markers provided by this invention can effectively classify the number of branches of maize tassels and can be used for the identification of the number of branches of maize tassels and molecular marker-assisted breeding. They have important theoretical and practical guiding significance for accelerating the genetic selection and improvement of maize varieties.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a normal distribution diagram of the number of male tassel branches in 1149 maize inbred lines in Example 1 of the present invention; where A is a histogram of the frequency distribution of the number of male tassel branches in 2023; B is a histogram of the frequency distribution of the number of male tassel branches in 2024; C is a histogram of the frequency distribution of the number of male tassel branches in BLUP-TBN; and D is a box plot of the TBN frequency. This indicates a significant difference. P <0.05; This indicates that the difference is extremely significant. P <0.001; ns indicates no significant difference;

[0020] Figure 2 This is the SNP marker chromosome distribution map obtained in Embodiment 3 of the present invention;

[0021] Figure 3 These are the QQ plot and Manhattan plot of the genome-wide association analysis of the number of male tassel branches in maize in Example 3 of this invention; where A is the QQ plot of the number of male tassel branches in 2023; B is the Manhattan plot of the number of male tassel branches in 2023; C is the QQ plot of the number of male tassel branches in 2024; D is the Manhattan plot of the number of male tassel branches in 2023; E is the QQ plot of the number of male tassel branches based on BLUP values; and F is the Manhattan plot of the number of male tassel branches based on BLUP values.

[0022] Figure 4 These are the KASP molecular marker typing results of 195 maize tassel branch number samples in Example 4 of this invention; where orange-red represents GG, blue represents AA, and gray represents NTC control;

[0023] Figure 5 This is the analysis result of the male spike branch number effect value of the KASP haplotype in Example 4 of the present invention. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0027] Example 1

[0028] Using 1149 high-quality maize inbred lines bred over the past 15 years from the Shenzhen Genome Institute of the Chinese Academy of Agricultural Sciences, these lines were sown in Acheng area of ​​Harbin City, Heilongjiang Province (126°53′8.73″E, 45°29′39.88″N) in 2023 and 2024, and grown under natural field conditions. A randomized block design was used, with each inbred line planted in two rows, each row 3m long, with a row spacing of 0.65m and a plant spacing of 0.2m. Fertilization and irrigation were managed as usual in the field. At maturity, the number of tassel branches was recorded. Five representative plants of each maize variety were selected and the number of tassel branches was counted and compiled by visual inspection. The phenotypic data of maize under two environments were statistically analyzed using Microsoft Excel 2022 and IBM SPSS Statistics V27.0. The normality was evaluated based on the coefficient of variation, skewness, and kurtosis. Finally, the frequency distribution histogram was plotted using Origin2021 software to test the normality of the phenotypic data.

[0029] The results are as follows Figure 1 As shown, statistical analysis of the number of male tassel branches in maize revealed that the mean number of branches ranged from 6.53 to 7.67 under both environments, with phenotypic variation ranging from 1 to 26 and a coefficient of variation from 32.5% to 44.3%. The coefficient of variation exceeded 10% in all environments, indicating rich phenotypic variation in the number of male tassel branches in the maize inbred line population, making it suitable for GWAS analysis. The skewness (0.91) and kurtosis (4.69) of the number of male tassel branches in 2024 were relatively high, and the frequency histogram showed a significant right skewness, reflecting the substantial influence of environmental factors on this trait.

[0030] Example 2

[0031] Maize genomic DNA extraction, library construction, and sequencing were performed. The specific library construction method is as follows:

[0032] (1) Take 1.0g of fresh young leaves, cut them into small pieces and put them into a mortar. Grind them with liquid nitrogen and then add 3mL of 1.5×CTAB. Grind them into a homogenate and transfer it into a 15mL centrifuge tube. Then add 1mL of 1.5×CTAB to the mortar to rinse and transfer it into the centrifuge tube. Mix well and incubate in a 65℃ water bath for 30min, shaking slowly from time to time.

[0033] The 1.5×CTAB formulation is shown in Table 1.

[0034] Table 1. 1.5×CTAB formulation (1L)

[0035]

[0036] Add deionized water to a final volume of 1L, and add mercaptoethanol to a final concentration of 0.2% (2mL) before use.

[0037] (2) After cooling to room temperature, add an equal volume of chloroform / isoamyl alcohol (volume ratio of 24:1), mix gently until the lower layer turns dark green.

[0038] (3) Centrifuge at 4200 rpm for 10 min, transfer the upper aqueous phase to a new 15 mL centrifuge tube, add 2 volumes of pre-cooled anhydrous ethanol, mix and let stand for 5 min. Incubate at -20℃ for 30 min to precipitate DNA.

[0039] (4) Centrifuge at 4200 rpm for 10 min, discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate once, invert the centrifuge tube to dry the DNA, and add 50 μL of TE to dissolve the DNA.

[0040] (5) Detect the concentration of DNA and adjust it with water to 20 ng / μL.

[0041] (6) Library construction and sequencing were performed using the TAIL-PCR-seq method (see Zhao S, Wang Y, Zhu Z, et al. Streamlined whole-genome genotyping through NGS-enhanced thermalasymmetric interlaced (TAIL)-PCR[J]. Plant Communications[2025-10-16].DOI:10.1016 / j.xplc.2024.100983.) and simplified whole-genome resequencing was completed for 1149 maize materials.

[0042] Example 3

[0043] GWAS analysis of the number of branches on maize tassels yielded significant SNPs, as detailed below:

[0044] The acquired sequencing data were processed and analyzed using a high-performance computer server. Raw data processing: After quality assessment of the raw PE (Pair-end) sequencing data using FastQC, quality control was performed using BWA, and sequencing reads were aligned to a reference genome (B73 V4 version). SNP detection was then performed using GATK. After quality control filtering at both the sample and variant levels, 57,849 high-quality SNP markers (minimum allele frequency >0.05, missing data <20%) were identified, ensuring the accuracy and reliability of the analysis results.

[0045] To better understand the population structure and genetic background, a phylogenetic tree was constructed using iqTree software. Principal component analysis (PCA) was performed on the genome-wide SNP data using Plink software, and population structure analysis was conducted using Faststructure software to clarify the genetic structure within the population. Genome-wide association analysis was performed on the number of male branches and their BLUP values ​​using the high-quality SNPs selected in the earlier stage.

[0046] Association analysis between SNP markers and various traits was performed using a mixed linear model of genotype, phenotype, population structure, and kinship matrix in GEMMA. All SNPs satisfying p < 1.7286e-5 were extracted from the GWAS results file using awk and converted to BED format files (Chr, Start, End). Based on two years of tassel branch number data, GWAS analysis identified one major SNP associated with this trait, located at 24904636 bp on chromosome 3 of the B73 genome V4 version. Results are shown below. Figures 2-3 . Figure 2 This is the SNP marker chromosome distribution map obtained in Embodiment 3 of the present invention. Figure 3 Examples 3 of this invention show the QQ plot and Manhattan plot of the genome-wide association analysis of the number of male tassel branches in maize; where A is the QQ plot of the number of male tassel branches in 2023; B is the Manhattan plot of the number of male tassel branches in 2023; C is the QQ plot of the number of male tassel branches in 2024; D is the Manhattan plot of the number of male tassel branches in 2023; E is the QQ plot of the number of male tassel branches based on BLUP values; and F is the Manhattan plot of the number of male tassel branches based on BLUP values. The QQ plot shows the relationship between the observed statistic (p-value) and the expected statistic; the Manhattan plot shows the significance level of the association between each SNP in the whole genome and the trait of the number of male tassel branches (statistical significance is expressed as −log10 (p-value), the smaller the p-value, the stronger the significance).

[0047] Example 4

[0048] The KASP tagging development and verification of the principal SNPs obtained in Example 3 are as follows:

[0049] (1) DNA was extracted from 195 maize inbred lines using the CTAB method (the method was the same as steps (1)-(4)) in Example 2). KASP primers were designed upstream and downstream of the major SNP sites obtained in Example 3, and the primer sequences are shown in Table 2.

[0050] Table 2 Primer sequences for KASP markers

[0051]

[0052] (2) Using DNA as a template, fluorescent polymerase chain reaction (PCR) was performed for genotyping using the primers shown in Table 2. The PCR amplification reaction system was calculated in 10 μL as follows: 2 μL of 4-50 ng / μL genomic DNA, 0.14 μL of primer mix (prepared by mixing 6 μL of forward primer 1 + 6 μL of forward primer 2 + 15 μL of reverse primer + 23 μL of ddH2O), 5 μL of 2×Probe Mix A solution, and 3 μL of ddH2O.

[0053] The PCR amplification program was as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, 61℃ annealing for 40 s, 10 cycles; 95℃ denaturation for 20 s, 55℃ annealing for 40 s, 31 cycles; 25℃ for 10 min; 4℃ for storage.

[0054] After amplification, KASP detection was performed according to the AQP genotyping system operating instructions. The PCR program on the ABI 7500 qPCR instrument was set to 35℃ for 30 seconds. The results file was exported, and the genotypes were further determined based on the sample clusters. Results analysis was performed using Taqman Genotyper Software. The fluorescence values ​​corresponding to HEX and FAM for each PCR reaction well were obtained and divided by the value of the reference dye (ROX) for that well. The fluorescence values ​​were standardized to obtain the relative fluorescence values ​​of HEX and FAM for each PCR reaction well (FAM fluorescent tag sequences were observed at excitation wavelength of 485nm and emission wavelength of 520nm, and HEX fluorescent tag sequences were observed at excitation wavelength of 528nm and emission wavelength of 560nm). Based on the relative fluorescence values, the samples were clustered. The detection results are as follows: Figure 4 As shown.

[0055] Depend on Figure 4 It can be seen that there are two genotypes at this locus: blue dots represent genotypes A / A, and red dots represent genotypes G / G.

[0056] The correlation between the number of tassel branches in maize materials tested in Example 4 and the typological results is analyzed. The correspondence between the number of tassel branches in maize materials and the typological results, as well as the degree of correlation, are shown in Tables 3-5. Figure 5 As shown.

[0057] Table 3. KASP molecular marker scanning results from 195 maize tassel branching studies

[0058]

[0059] Table 4 (Continued)

[0060]

[0061] Table 5. Statistical results of KASP genotyping and phenotype.

[0062]

[0063] From Table 3, Table 5 and Figures 4-5 It can be seen that when the fluorescence signal of the amplification product is orange-red, the trait of the number of male tassel branches in maize is identified as having a high number of male tassel branches, and the corresponding genotype is GG; when the fluorescence signal of the amplification product is blue, the trait of the number of male tassel branches in maize is identified as having a low number of male tassel branches homozygous, i.e., AA; in general, there is a significant difference in the number of male tassel branches between the two haplotype varieties. In 195 samples to be tested, the KASP detection results and the trait are 100% consistent, indicating that the KASP experiment using this molecular marker can effectively detect the genotype of the test material, thereby completing the identification of germplasm.

[0064] Therefore, this invention provides SNP and KASP markers closely linked to the number of branches on maize tassels; the SNP is located at 24904636 bp on chromosome 3 of the B73 genome V4 version; the forward primers for the KASP marker are 1TBN23-KASP-106F1 and 1TBN23-KASP-106F2, and the reverse primer is 1TBN23-KASP-106R; the provided SNP and KASP markers can effectively genotype the number of branches on maize tassels, and can be used for the identification of the trait of the number of branches on maize tassels and for molecular marker-assisted breeding, which has important theoretical and practical guiding significance for accelerating the genetic selection and improvement of maize varieties.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of a detection reagent for SNP loci closely linked to the number of branches in maize tassels in the identification of the number of branches in maize tassels, characterized in that: The SNP locus is located at 24904636 bp on chromosome 3 of the B73 genome V4 version; the SNP locus is AA for maize plants with fewer tassel branches and GG for maize plants with more tassel branches; fewer tassel branches are defined as <6 branches and more tassel branches are defined as ≥6 branches.

2. The application of a detection reagent for SNP loci closely linked to the number of branches in maize tassels in maize tassel branch number breeding, characterized in that: The SNP locus is located at 24904636 bp on chromosome 3 of the B73 genome V4 version; the SNP locus for maize plants with fewer tassel branches is AA, and the SNP locus for maize plants with more tassel branches is GG; fewer tassel branches are defined as <6 branches, and more tassel branches are defined as ≥6 branches.

3. The application of the KASP primer set for identifying the number of branches on maize tassels in the identification of the number of branches on maize tassels, characterized by: The KASP primer set is a set of KASP primers for identifying SNP sites closely linked to the number of branches of maize tassels. The KASP primer set consists of two forward primers, 1TBN23-KASP-106F1 and 1TBN23-KASP-106F2, and one reverse primer, 1TBN23-KASP-106R. The sequence of 1TBN23-KASP-106F1 is shown in SEQ ID NO.1, the sequence of 1TBN23-KASP-106F2 is shown in SEQ ID NO.2, and the sequence of 1TBN23-KASP-106R is shown in SEQ ID NO.

3. The SNP locus is located at 24904636 bp on chromosome 3 of the B73 genome V4 version; the SNP locus for maize plants with fewer tassel branches is AA, and the SNP locus for maize plants with more tassel branches is GG; fewer tassel branches are defined as <6 branches, and more tassel branches are defined as ≥6 branches.

4. The application of the KASP primer set for identifying the number of branches on maize tassels in maize tassel branch number breeding, characterized by: The KASP primer set is a set of KASP primers for identifying SNP sites closely linked to the number of branches of maize tassels. The KASP primer set consists of two forward primers, 1TBN23-KASP-106F1 and 1TBN23-KASP-106F2, and one reverse primer, 1TBN23-KASP-106R. The sequence of 1TBN23-KASP-106F1 is shown in SEQ ID NO.1, the sequence of 1TBN23-KASP-106F2 is shown in SEQ ID NO.2, and the sequence of 1TBN23-KASP-106R is shown in SEQ ID NO.

3. The SNP locus is located at 24904636 bp on chromosome 3 of the B73 genome V4 version; the SNP locus for maize plants with fewer tassel branches is AA, and the SNP locus for maize plants with more tassel branches is GG; fewer tassel branches are defined as <6 branches, and more tassel branches are defined as ≥6 branches.

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