KASP molecular marker for regulating oat flag leaf size and application thereof

Abstract

The application discloses a KASP (Kompetitive Allele Specific PCR) molecular marker for regulating oat flag leaf size and application thereof, and belongs to the technical field of molecular marker assisted breeding. AsFL1 The KASP molecular marker is KASP-2C, which is an SNP (Single Nucleotide Polymorphism) molecular marker; a DNA sequence is shown as SEQ ID NO. 1, a CDS (Coding Sequence) sequence is shown as SEQ ID NO. 2, and the 129th nucleotide in the CDS sequence is a C / A polymorphism; the application provides a specific KASP primer group, and rapid typing is realized through fluorescent quantitative PCR; FAM signal is detected to be a C homozygous small-flag-leaf genotype, and HEX signal is detected to be an A homozygous large-flag-leaf genotype. The application has high marker specificity, stable typing, and is not affected by the environment, and the coincidence rate of a phenotype and a genotype reaches 91.72%, so that the application can be used for oat germplasm identification and molecular marker assisted breeding, and significantly improves the directional screening efficiency of flag leaf size and shortens a breeding period.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, and in particular to a KASP molecular marker for regulating the size of oat flag leaves and its application. Background Technology

[0002] oat( Oatmeal Oat (Poaceae) belongs to the Poaceae family, Pooideae subfamily, and is the world's sixth largest dual-purpose crop for both food and forage. The flag leaf is the last leaf to unfold at the top of the oat plant. As the core functional leaf for photosynthesis in the later stages of oat growth, its size directly determines the accumulation efficiency of photosynthetic products during the grain-filling period and the final yield. Simultaneously, the size of the oat flag leaf is closely related to quality traits. For grain oats, an appropriately sized flag leaf can balance photosynthetic efficiency and nutrient distribution to the grain, improving the quality of grains such as protein and β-glucan. For forage oats, the flag leaf, as an important component of high-quality aboveground forage, affects the yield and palatability of forage per plant. Furthermore, the flag leaf's combination of high protein and palatability is one of the core phenotypic characteristics of high-quality forage oats. Furthermore, the size of the flag leaf in oats is closely related to stress resistance and plant type adaptability: in arid and semi-arid regions, a moderately smaller flag leaf can reduce transpiration and water loss, enhancing the drought resistance of oats; in areas with high water and fertilizer yields, a larger flag leaf can make full use of light resources and improve the photosynthetic efficiency of the population; at the same time, the size of the flag leaf also affects the field productivity of oats by influencing the canopy structure. Therefore, screening molecular markers closely related to the size of the flag leaf in oats is of irreplaceable importance for the precise regulation of the flag leaf size trait in oat genetic breeding, as well as for the rapid breeding of high-yielding grain, high-quality feed, and stress-resistant oat varieties.

[0003] Currently, there are no reports on the development of specific KASP molecular markers for the oat flag leaf size gene. Molecular-assisted breeding of oat flag leaf size remains in its infancy, lacking efficient and precise technical tools. Therefore, developing KASP molecular markers closely linked to the oat flag leaf size gene is of great practical significance and broad application prospects for overcoming the technical bottlenecks of traditional oat flag leaf size screening, shortening the breeding cycle, improving breeding efficiency, and promoting the industrial application of molecular-assisted breeding of oat flag leaf size traits. Summary of the Invention

[0004] The purpose of this invention is to provide a KASP molecular marker for regulating oat flag leaf size and its application, which solves the problems of long screening cycles, low efficiency and high cost of existing oat leaf size trait screening. At the same time, it provides a detection method and application of this molecular marker, providing technical support for early and accurate screening of oat leaf size traits and the breeding of superior varieties.

[0005] To achieve the above objectives, the present invention provides a KASP molecular marker for regulating the size of oat flag leaves, wherein the KASP molecular marker is KASP- AsFL1 -2C is a SNP molecular marker; The KASP- AsFL1 The DNA sequence of -2C is shown in SEQ ID NO.1; The KASP- AsFL1 The CDS sequence of -2C is shown in SEQ ID NO.2, and the nucleotide at position 129 in the CDS sequence is a C / A polymorphism; The KASP- AsFL1 The amino acid sequence of -2C is shown in SEQ ID NO.3; The KASP molecular marker is located on oat chromosome 2C and is associated with the oat flag leaf size gene. AsFL1 Co-separation.

[0006] Preferably, the KASP molecular marker exists only as a homozygous genotype in the natural oat population. When the 129th position of the KASP molecular marker CDS sequence is homozygous for A, it corresponds to the large flag leaf phenotype; when the 129th position of the KASP molecular marker CDS sequence is homozygous for C, it corresponds to the small flag leaf phenotype.

[0007] On the other hand, the present invention also provides a set of KASP primers for amplifying the above-mentioned KASP molecular markers, comprising: Upstream primer KASP- AsFL1 -2C-F1, nucleotide sequence as shown in SEQ ID NO.4; Upstream primer KASP- AsFL1 -2C-F2, the nucleotide sequence is shown in SEQ ID NO.5; Downstream primer KASP- AsFL1 -2C-R, the nucleotide sequence is shown in SEQ ID NO.6.

[0008] Preferably, the upstream primer KASP- AsFL1 -2C-F1 is labeled with a FAM fluorescent group at its 5′ end, and the upstream primer KASP- AsFL1 The 5′ end of -2C-F2 is labeled with the HEX fluorescent group.

[0009] On the other hand, the present invention also provides a kit for detecting the size trait of oat flag leaves, comprising the above-mentioned KASP primer set.

[0010] On the other hand, the present invention also provides an application of the above-mentioned KASP molecular marker in the screening of flag leaf size trait in oats. The application includes rapid identification of flag leaf size genotypes in oat natural germplasm resources, directional selection of target flag leaf size genotypes in oat hybrid offspring, and oat molecular marker-assisted breeding.

[0011] On the other hand, the present invention also provides an application of the above-mentioned KASP primer set in the screening of flag leaf size trait in oats. The application includes rapid identification of flag leaf size genotypes in oat natural germplasm resources, directional selection of target flag leaf size genotypes in oat hybrid offspring, and oat molecular marker-assisted breeding.

[0012] Preferably, the method for screening oat flag leaf size using the KASP primer set includes the following steps: Using the genomic DNA of the oat sample to be tested as a template, the KASP primer set was used for real-time PCR amplification, and the amplification results were genotyped. KASP- was not detected. AsFL1 KASP- was detected by detecting the base C corresponding to the -2C-F1 molecularly labeled primer. AsFL1 If the base A corresponding to the -2C-F2 molecular marker primer is A, then the natural oat population material to be tested is a homozygous flag leaf genotype; Or KASP- was detected AsFL1 The base C corresponding to the -2C-F1 molecularly labeled primer was not detected (KASP-). AsFL1 If the base A corresponding to the -2C-F2 molecular marker primer is A, then the natural oat population material to be tested is a homozygous flag leaf genotype.

[0013] On the other hand, the present invention also provides an application of the above-mentioned kit for detecting the flag leaf size trait of oats in the screening of oat flag leaf size trait, the application including rapid identification of flag leaf size genotypes in oat natural germplasm resources, directional selection of target flag leaf size genotypes in oat hybrid offspring, and oat molecular marker-assisted breeding.

[0014] Therefore, the KASP molecular marker for regulating oat flag leaf size and its application, as described in this invention, have the following beneficial effects: (1) The KASP molecular marker and oat flag leaf size gene provided by this invention AsFL1 Co-segregated on chromosome 2C, with a polymorphism of C / A, it is related to the oat flag leaf size gene. AsFL1 Close linkage can be used to locate the trait of flag leaf size in oats, thereby enabling targeted selection of hybrid offspring in oat variety breeding and achieving rapid screening of homozygous large-leaf plants among hybrid offspring of oat flag leaf size. (2) Using this molecular marker for screening has the advantages of being fast, accurate and unaffected by the environment. It can be used as an effective means to accelerate the improvement of flag leaf oat varieties and help improve the efficiency of breeding work.

[0015] 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

[0016] 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.

[0017] Figure 1 Oat leaf size gene AsFL1 Location map on chromosome 2C.

[0018] Figure 2 KASP- molecular marker in oat materials with different leaf sizes AsFL1 The fluorescence readings for -2C detection are shown in the figure; where A represents the genotyping results of natural population group 1, B represents the genotyping results of natural population group 2, C represents the genotyping results of natural population group 3, and D represents the genotyping results of natural population group 4. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] 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.

[0021] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0022] Example (1) 866 oat germplasm resources from around the world were collected, covering hulled oats and naked oats, including high-yielding varieties and stress-resistant varieties, ensuring the genetic diversity of the population.

[0023] (2) Phenotypic identification of natural populations: Oats were planted in Wenjiang District of Chengdu City, Chongzhou City of Chengdu City, and Baicheng City of Jilin Province from 2023 to 2025. The row length was 2m and the row spacing was 0.3m. There were 15 seeds per row. Conventional field management was carried out. No serious diseases, pests, or lodging occurred during the growth period. During the grain-filling stage of oats, 5 plants from each material were randomly selected to measure the flag leaf length and width. The flag leaf area was calculated according to the formula: Flag leaf area = Flag leaf length × Flag leaf width × 0.73. Based on the phenotypic data over many years, the BLUE method was used to correct the phenotypic values.

[0024] (3) Gene localization: a. DNA extraction: Genomic DNA was extracted from 866 natural population materials using the CTAB method.

[0025] b. Resequencing was performed using the Illumina NovaSeq 6000 platform at a sequencing depth of 3X to obtain raw sequencing data. Sequencing reads were aligned to the oat reference genome using BWA software, and SNP calling was performed using GATK software to filter out SNPs with a minor allele frequency (MAF) <0.05 and a deletion rate >10%, ultimately yielding 129,542 high-quality SNPs. Based on normalized phenotypic values ​​and high-quality SNP data, genome-wide association analysis (GWAS) was performed using the GCTA-MLMA model.

[0026] like Figure 1 As shown, the results indicated that a gene significantly associated with flag leaf size exists within the 536.5-540.1 Mb region of oat chromosome 2C. A C / A polymorphic site was found in the coding region of this gene; materials with large flag leaves all showed A at this site, while materials with small flag leaves were all homozygous for C, with no heterozygous genotypes. This demonstrates that this SNP site is associated with... AsFL1 Gene co-segregation can serve as a target site for developing KASP molecular markers.

[0027] AsFL1 The DNA sequence, SEQ ID NO.1: AsFL1 The CDS sequence of, SEQ ID NO.2, where the underlined positions are the SNP (C / A) site positions: ATGAGGGTTCACCATTTGCATGTGGCCTACTTGGAGAAAACGGCCGTGGCCTCTCCATCTCCAGCCGTAGCTGGACCAGCACCATCAACCTCCCCCCTCTTCCCCTCCTCCTCCTCTTCGGCTGTGTT C CCTTTCGCCTTCCGATGCCTCCGCCCGCTCGCGCCCAAGCTCTCCCTCCCGGAGCAGCCGAAGAAGATGGTCGCGCCGGCCGACGTCTTGGGTCGGGTCAGAAACGCCACCAAGCTGCTCAGCTGCACCGTCAGAAACCACACCGTGCAGGTGCCGGTGGGCGGGACGACGCGGTGGAACCCCTCGGCGGAGCAGATCAAGGTGCTGGAGGCACTGTACCGCGGCGGGATGCGCACCCCGAACGCGGCGCAGATCGAGCGCATCACGGAGGAGCTGGGGAAGCACGGCCGGATCGAGGGGAAGAACGTCTTCTACTGGTTCCAGAACCACAAGGCCCGCGAGCGCCAGAAGCAGAAGCGCGCCGCACTCCTCACCCTCAGCACCCTCGACTCACCCGCCCTGCCCGCAACGACGACGACCAAGCAGGAGGCGTCTGACGACGTGCCGAGCAGCAAGCGACGGTGCACGGCTTGGGGTGATAATGGGCATGGCGATGCGGCGACGACGACGGAGGTGCCCGTCGACTGCACGGGCGGCAATGCCACGCTGGAGCTCTTCCCGTTGCGTCCGCAAGGGAAAGCTTAA AsFL1 The protein sequence of, SEQ ID NO.3: MRVHHLHVAYLEKTAVASPSPAVAGPAPSTSPLFPSSSSSSAVFPFAFRCLRPLAPKLSLPEQPKKMVAPADVLGRVRNATKLLSCTVRNHTVQVPVGGTTRWNPSAEQIKVLEALYRG GMRTPNAAQIERITEELGKHGRIEGKNVFYWFQNHKARERQKQKRAALLTLSTLDSPALPATTTTKQEASDDVPSSKRRCTAWGDNGHGDAATTTEVPVDCTGGNATLELFPLRPQGKA c. Select chromosome 2C based on sequencing data AsFL1 SNP sites with shared differences in the size of oat flag leaves within the candidate gene region were identified. Based on the KASP molecular marker design principle and using the oat bioinformatics database "OatBioDB, www.waooat.cn" for primer specificity detection, a specific sequence tag that can be recognized by "FAM" and "HEX" fluorescence was added to the front end of the marker, and the markers were sent to Sangon Biotech (Shanghai) Co., Ltd. for primer synthesis.

[0028] KASP molecular marker sequence: Upstream primer F1, SEQ ID NO.4: 5′-GAAGGTGACCAAGTTCATGCTCTCCTCCTCTTCGGCTGTGTTC-3′.

[0029] Upstream primer F2, SEQ ID NO.5: 5′-GAAGGTCGGAGTCAACGGATTCTCCTCCTCTTCGGCTGTGTTA-3′.

[0030] Downstream primer R, SEQ ID NO.6: 5′-TTTCTGACCCGACCCAAGACGT-3′.

[0031] In this study, upstream primers F1 and F2 had fluorescent modification groups FAM and HEX added to their 5′ ends, respectively.

[0032] d. Genotyping was performed using a BIO-RAD real-time quantitative PCR instrument. The KASP molecular marker primers obtained in step c were used to amplify the DNA of the natural population to identify the genotype and obtain molecular marker data. The specific process is as follows: Using genomic DNA from 866 oat samples from a natural population as templates, the KASP molecular marker primers (denoted as KASP-) in step (3) were used. AsFL1 -2C) DNA template was subjected to PCR amplification. The PCR amplification system is shown in Table 1: Table 1 PCR amplification system

[0033] The primer mixture was prepared by adding 60 μL, 60 μL, and 150 μL of upstream primer F1, upstream primer F2, and downstream primer R at a concentration of 10 ng / μL, respectively, and then adding 230 μL of ddH2O.

[0034] The PCR reaction procedure is shown in Table 2: Table 2 PCR reaction procedure

[0035] PCR products were obtained and detected using a Bio-Rad iQ5 real-time quantitative PCR instrument at 37℃. The PCR products were randomly divided into four groups, with one blank control group in each group (without oat DNA template, the volume was made up with sterile, enzyme-free water, and all other components were identical to the sample wells). The PCR products were genotyped using the bioinformatics software BioRad CFXManager. Plants with the same genotype as *Oatella macrophylla* were designated as "A", and those with the same genotype as *Oatella simonii* were designated as "B".

[0036] Genotyping results as follows Figure 2 As shown in Table 3. Figure 2 The blue square represents the large flag leaf type, the orange circle represents the small flag leaf type, and the black diamond is a blank control.

[0037] Table 3 Statistical analysis of different oat sample typing results

[0038] Depend on Figure 2 As shown in Table 3, the genotyping results indicate that using this KASP- AsFL1 The -2C molecular marker primers were used as templates for genotyping of different oat samples using genomic DNA. The results showed that the phenotype and genotype consistency rate was as high as 91.72%. The KASP molecular marker provided by this invention has excellent effect on genotyping of oat flag leaf size.

[0039] 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. A KASP molecular marker for regulating the size of oat flag leaves, characterized in that: The KASP molecular marker is KASP- AsFL1 -2C is a SNP molecular marker; The KASP- AsFL1 The DNA sequence of -2C is shown in SEQ ID NO.1; The KASP- AsFL1 The CDS sequence of -2C is shown in SEQ ID NO.2, and the nucleotide at position 129 in the CDS sequence is a C / A polymorphism; The KASP- AsFL1 The amino acid sequence of -2C is shown in SEQ ID NO.3; The KASP molecular marker is located on oat chromosome 2C and is associated with the oat flag leaf size gene. AsFL1 Co-separation.

2. The KASP molecular marker for regulating oat flag leaf size according to claim 1, characterized in that: The KASP molecular marker exists only as a homozygous genotype in the natural oat population. When the 129th position of the KASP molecular marker CDS sequence is homozygous (A), it corresponds to the large flag leaf phenotype; when the 129th position of the KASP molecular marker CDS sequence is homozygous (C), it corresponds to the small flag leaf phenotype.

3. A set of KASP primers for amplifying the KASP molecular marker as described in claim 1 or 2, characterized in that, include: Upstream primer KASP- AsFL1 -2C-F1, nucleotide sequence as shown in SEQ ID NO.4; Upstream primer KASP- AsFL1 -2C-F2, the nucleotide sequence is shown in SEQ ID NO.5; Downstream primer KASP- AsFL1 -2C-R, the nucleotide sequence is shown in SEQ ID NO.

6.

4. The KASP primer set according to claim 3, characterized in that: The upstream primer KASP- AsFL1 -2C-F1 is labeled with a FAM fluorescent group at its 5′ end, and the upstream primer KASP- AsFL1 The 5′ end of -2C-F2 is labeled with the HEX fluorescent group.

5. An application of the KASP molecular marker as described in claim 1 or 2 in the screening of oat flag leaf size trait, characterized in that: The applications include rapid identification of flag leaf size genotypes in oat natural germplasm resources, targeted selection of target flag leaf size genotypes in oat hybrid offspring, and oat molecular marker-assisted breeding.

6. A reagent kit for detecting the size trait of oat flag leaves, characterized in that: It includes the KASP primer set as described in claim 3 or 4.

7. An application of the KASP primer set as described in claim 3 or 4 in the screening of oat flag leaf size trait, characterized in that: The applications include rapid identification of flag leaf size genotypes in oat natural germplasm resources, targeted selection of target flag leaf size genotypes in oat hybrid offspring, and oat molecular marker-assisted breeding.

8. The application according to claim 7, characterized in that: The method for screening oat flag leaf size using the aforementioned KASP primer set includes the following steps: Using the genomic DNA of the oat sample to be tested as a template, the KASP primer set was used for real-time PCR amplification, and the amplification results were genotyped. KASP- was not detected. AsFL1 KASP- was detected by detecting the base C corresponding to the -2C-F1 molecularly labeled primer. AsFL1 If the base A corresponding to the -2C-F2 molecular marker primer is A, then the natural oat population material to be tested is a homozygous flag leaf genotype; Or KASP- was detected AsFL1 The base C corresponding to the -2C-F1 molecularly labeled primer was not detected (KASP-). AsFL1 If the base A corresponding to the -2C-F2 molecular marker primer is A, then the natural oat population material to be tested is a homozygous flag leaf genotype.

9. The application of the kit for detecting the size trait of oat flag leaves as described in claim 6 in the screening of oat flag leaf size traits, characterized in that: The applications include rapid identification of flag leaf size genotypes in oat natural germplasm resources, targeted selection of target flag leaf size genotypes in oat hybrid offspring, and oat molecular marker-assisted breeding.

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