A molecular marker affecting oat crude protein content and application thereof

Abstract

The present application relates to the technical field of molecular genetics, and particularly relates to a kind of molecular marker affecting oat crude protein content and application thereof, the base sequence of the SNP molecular marker is as shown in SEQ ID No.1, and the 301th site in the sequence of SEQ ID No.1 is T or C.The present application overcomes the defects of time and energy consumption, low accuracy and lack of effective related molecular markers in the prior art oat high crude protein variety breeding relying on traditional phenotype selection, realizes the rapid and accurate prediction and screening of oat crude protein content, improves the breeding efficiency of oat high crude protein variety, and shortens the breeding cycle.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics, specifically to a molecular marker that affects the crude protein content of oats and its application. Background Technology

[0002] As an important high-quality feed crop, the crude protein content of oats is one of the key indicators for measuring its feed value. Oats with high crude protein content can provide herbivores with richer nutrition and promote their growth and development. Therefore, breeding oat varieties with high crude protein content has important economic significance and application value.

[0003] Traditional breeding of high-crude-protein oat varieties for feed mainly involves selecting individual plants based on the crude protein content of the offspring. This method requires waiting for the plants to reach a certain stage of growth before the crude protein content can be measured, which is not only time-consuming and labor-intensive, but also susceptible to environmental factors (such as soil, climate, water and fertilizer management), resulting in low accuracy and severely restricting the efficiency and progress of forage oat breeding.

[0004] Currently, molecular marker technology is widely used in crop breeding. By finding molecular markers associated with target traits, early, rapid, and accurate selection of target traits can be achieved. However, in the research of molecular markers related to crude protein content in forage oats, no efficient, stable molecular markers that are significantly associated with crude protein content have been discovered and applied. Therefore, there is a lack of effective molecular marker-assisted breeding methods to improve the breeding efficiency of high-crude-protein forage oat varieties.

[0005] Some studies have attempted to use general molecular markers (such as SSR markers) to perform association analyses on certain traits of oats. However, these markers show weak associations with crude protein content, low phenotypic contribution rates, and are difficult to accurately predict and screen oat materials with high crude protein content. Furthermore, the detection methods for these markers are relatively complex and costly, making them unsuitable for large-scale breeding screening. Additionally, while some molecular markers associated with crude protein content have been found in other crops (such as wheat, maize, and alfalfa), the significant differences in the genetic background of different crops mean that these markers cannot be directly applied to oat breeding.

[0006] Therefore, there is an urgent need to develop a molecular marker related to the crude protein content of oats in order to effectively solve the problem of low breeding efficiency of high crude protein oat varieties. Summary of the Invention

[0007] To address the above problems, this invention provides a molecular marker that affects the crude protein content of oats and its application.

[0008] This invention is achieved through the following technical solution:

[0009] A molecular marker that affects the crude protein content of oats, wherein the base sequence of the SNP molecular marker is shown in SEQ ID NO.1, and the 301st position in the SEQ ID NO.1 sequence is T or C.

[0010] A KASP primer combo for amplifying the molecular marker, the KASP primer combo consisting of specific primer 1, specific primer 2 and universal primer.

[0011] The base sequence of the specific primer 1 is shown in SEQ ID No. 3; the base sequence of the specific primer 2 is shown in SEQ ID No. 4; and the base sequence of the universal primer is shown in SEQ ID No. 5.

[0012] Preferably, the 5' end of the specific primer 1 is connected to a FAM fluorescent adapter to bind the T allele; the 5' end of the specific primer 2 is connected to a HEX fluorescent adapter to bind the C allele.

[0013] A method for identifying oat varieties with high crude protein content, the method comprising the following steps:

[0014] Genomic DNA was extracted from the oat material to be tested.

[0015] KASP-PCR amplification was performed using the described KASP primer combination to obtain the amplification product.

[0016] When the fluorescence signal of the amplified product is a co-occurrence of HEX fluorescence signal and FAM fluorescence signal, the oat material to be tested is an oat material with high crude protein content; when the fluorescence signal is only FAM fluorescence signal, the oat material to be tested is an oat material with low crude protein content.

[0017] The high crude protein content is ≥12.6%; the low crude protein content is ≤9.3%.

[0018] Preferably, the oat material is the young leaves of oats.

[0019] Preferably, the KASP-PCR amplification program is as follows: pre-denaturation stage: 94℃ for 15 minutes; landing stage: 95℃, denaturation for 20 seconds, 10 cycles; annealing extension at 64℃~55℃ for 60 seconds; amplification stage: 95℃, denaturation for 20 seconds, 30 cycles; annealing extension at 56℃ for 60 seconds; and incubation stage: storage at 4℃.

[0020] The application of the molecular markers or the KASP primer combinations in oat genetic breeding.

[0021] Preferably, the crude protein content of offspring is increased by selecting individuals with the TC heterozygous genotype as parents.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a molecular marker that influences the crude protein content of oats, with its base sequence shown in SEQ ID No. 1, where the base is T or C at position 301. This molecular marker is significantly correlated with the crude protein content of oats, with a phenotypic contribution rate of 14.17%, and it has been stably and accurately used for the prediction and screening of crude protein content in two consecutive years of testing. The KASP primer combination designed based on this marker has high specificity and can directly distinguish different genotypes through fluorescence signals. The detection process is rapid and efficient, with a single reaction requiring only 2 hours, making it suitable for large-scale, high-throughput screening of breeding materials. Using the molecular marker and detection method provided by this invention, detection can be completed at the oat seedling stage without waiting for plant maturity, thereby significantly shortening the breeding cycle, improving breeding efficiency, and reducing breeding costs. The detection results of this molecular marker are not affected by environmental factors and have high accuracy, with a verified accuracy rate of up to 93.6%, effectively improving the accuracy of breeding high-crude-protein oat varieties. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0025] Figure 1 These are the results of a genome-wide association study (GWAS) in this embodiment of the invention;

[0026] Figure 2 This is a comparison of crude protein content among materials of different genotypes in the embodiments of the present invention. Detailed Implementation

[0027] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0029] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0030] Example 1

[0031] 1. Obtaining molecular markers

[0032] Experimental materials: Based on 1078 global oat germplasm resources (including local varieties, bred varieties and core germplasm, covering the main oat growing areas) introduced and organized by our laboratory from the National Germplasm Bank, local agricultural research institutes and other units, the materials have rich variation in crude protein content (range: 7.8%-16.1%).

[0033] Planting and Phenotypic Determination: The above-mentioned germplasm resources were planted in different experimental fields (such as Guyuan, Ningxia and Zhangjiakou, Hebei) using a randomized block design with three replicates. Aboveground parts of the oats were collected at the milk stage, with withered leaves removed, blanched at 105℃ for 30 minutes, dried at 80℃ to constant weight, pulverized, and passed through a 40-mesh sieve. Crude protein content was determined using the Kjeldahl method (GB / T 6432-2018). Each material was measured three times, and the average value was taken as the crude protein content phenotypic value for that material.

[0034] DNA Extraction and Sequencing: During the oat seedling stage, three robust plants were selected from each material, and their tender leaves were collected and mixed thoroughly as DNA extraction samples. A modified CTAB method was used to extract genomic DNA: The leaf samples were ground into powder in liquid nitrogen, and CTAB extraction buffer preheated to 65°C was added. The mixture was incubated at 65°C for 30 minutes, gently inverting to mix. An equal volume of chloroform-isoamyl alcohol (24:1) was added, and the mixture was gently inverted to mix for 10 minutes. The mixture was centrifuged at 12000 rpm for 15 minutes at 4°C. The supernatant was collected, and an equal volume of isopropanol was added. The mixture was incubated at -20°C for 30 minutes to precipitate the DNA. The mixture was centrifuged at 12000 rpm for 10 minutes at 4°C, the supernatant was discarded, and the precipitate was washed twice with 75% ethanol. After air-drying at room temperature, the precipitate was dissolved in TE buffer. The integrity of the extracted DNA was assessed by 1% agarose gel electrophoresis, and its concentration and purity were measured using a Nanodrop spectrophotometer to ensure OD... 260 / 280 Between 1.8 and 2.0, OD 260 / 230 ≥2.0, DNA concentration ≥50ng / μL. Qualified DNA samples were sent to a sequencing company for whole-genome resequencing using the Illumina NovaSeq platform with a PE150 sequencing strategy, yielding raw sequencing data with a total data volume of 106.57Tb and an average sequencing depth of approximately 9.06×.

[0035] SNP screening and association analysis: Quality control was performed on the raw sequencing data. FastQC software was used to evaluate the quality of raw reads, mainly including base quality distribution, GC content distribution, and adapter contamination. Trimmomatic software was used to filter out low-quality reads (reads with terminal base quality values ​​below 20 were truncated, and reads shorter than 50 bp were discarded) and adapter sequences. The filtered clean sequencing data was aligned to the oat reference genome (cultivation variety "Marvellous," genome data released to the National Genome Database, accession number GWHGDJH00000000.1) using the BWA-MEM algorithm, obtaining alignment results in SAM format. Samtools software was used to convert the SAM format files to BAM format, and then sorted and removed repetitive sequences. SNP calling was performed using GATK software: First, the HaplotypeCaller module was used to detect variants in each sample, generating a gVCF file; then, the GenotypeGVCFs module was used to merge the gVCF files of all samples to obtain the original VCF file. The original VCF files were rigorously filtered with the following parameters: Quality Depth (QD) ≥ 2.0, Fisher Strand FS ≤ 60.0, Strand Odds Ratio (SOR) ≤ 3.0, Mapping Quality (MQ) ≥ 40.0, Mapping Quality Rank Sum (MQRankSum) ≥ -12.5, and Read Pos Rank Sum (ReadPosRankSum) ≥ -8.0. Biasable SNPs were retained, while SNPs with a deletion rate greater than 20% and a minor allele frequency (MAF) less than 5% were filtered out, resulting in a high-quality SNP marker set of 31,644,360. Genome-wide association analysis (GWAS) was performed using TASSEL 5.0 software with a mixed linear model (MLM). Population structure (first three principal components) and kinship (kinship matrix) were included as covariates. Population structure was obtained using ADMIXTURE software, and the first three principal components were selected as covariates to correct for the influence of population structure on the association analysis results. The threshold for significant association was set to P≤1e-8, and a single SNP molecular marker significantly associated with oat crude protein content was finally obtained, such as... Figure 1 As shown.

[0036] Molecular marker information: This molecular marker is located on chromosome 2D of the oat reference genome, at physical location 526910324 bp, and the SNP genotype is T / C polymorphism. Statistical analysis showed that its correlation with oat crude protein content had a P-value of 9.12e-38, and the explainable phenotypic contribution rate was 14.17%. Specifically, the TC genotype indicates oat materials with high crude protein content, while the TT genotype indicates oat materials with low crude protein content. Figure 2 As shown.

[0037] 2. Base sequence of molecular markers

[0038] The base sequence containing the T allele is shown in SEQ ID No. 1, which is: (lowercase letters indicate SNP sites).

[0039] The base sequence containing the C allele is shown in SEQ ID No. 2, and is as follows:

[0040] (Lowercase letters indicate SNP sites).

[0041] 3. Primer combination design

[0042] For the aforementioned SNP molecular markers, a KASP primer combination was designed using Primer 5 software, consisting of two specific primers and one universal primer, with the specific sequences as follows:

[0043] The base sequence of specific primer 1 is shown in SEQ ID No. 3: GAAGGTGACCAAGTTCATGCTGCAAACACTAGCCCTGCGGTAGACACA. This primer binds to the T allele, with a FAM fluorescent adapter attached to its 5' end.

[0044] The base sequence of specific primer 2 is shown in SEQ ID No. 4: GAAGGTCGGAGTCAACGGATTGCAAACACTAGCCCTGCGGTAGACACG. This primer binds to the C allele and has a HEX fluorescent adapter at its 5' end.

[0045] The base sequence of the universal primer is shown in SEQ ID No. 5, which is GGAGGACCCGGGGATCAGAAAT.

[0046] The orientation of all the above primers is from the 5' to the 3' end.

[0047] 4. Detection Method

[0048] Five young leaves were extracted from the oat material to be tested, and genomic DNA was extracted using a modified CTAB method. Using the extracted DNA as a template (concentration 10 ng / μL), KASP-PCR amplification was performed using the primer combination described above. The PCR reaction system (10 μL) consisted of: 5 μL of 2×KASP Master Mix (containing Taq DNA polymerase, dNTPs, fluorescent probes, and ROX reference dye), 0.17 μL of 10 μM specific primer 1 (targeting the T allele, with a FAM fluorescent tag attached to the 5' end), 0.17 μL of 10 μM specific primer 2 (targeting the C allele, with a HEX fluorescent tag attached to the 5' end), 0.42 μL of 10 μM universal primer (without a fluorescent tag, binding to the shared sequences at two sites), 1 μL of genomic DNA template (20 ng / μL), and 3.24 μL of nuclease-free ultrapure water.

[0049] Amplification program: Pre-denaturation stage: 94℃ for 15 minutes (activates Taq enzyme, ensuring sufficient denaturation); Touchdown stage (10 cycles): 95℃ denaturation for 20 seconds; 64℃→55℃ annealing and extension for 60 seconds (decreases by 0.9℃ per cycle to improve amplification specificity); Amplification stage (30 cycles): 95℃ denaturation for 20 seconds; 56℃ annealing and extension for 60 seconds (efficient amplification at stable temperature); Incubation stage: 4℃ storage (short-term preservation of amplification products).

[0050] After amplification, fluorescence signals were detected using a quantitative real-time PCR instrument (e.g., Bio-Rad CFX96). The FAM (492nm excitation / 518nm emission) and HEX (535nm excitation / 556nm emission) fluorescence channels were selected for signal detection, with ROX used as a reference fluorescence to correct for inter-well differences. Scatter plots were generated using the instrument's built-in analysis software (e.g., Bio-Rad CFX Manager), and genotypes were determined based on fluorescence signal intensity. Samples showing co-occurrence of HEX and FAM fluorescence signals were classified as TC genotype; samples showing only FAM fluorescence signals were classified as TT genotype.

[0051] Genotyping criteria: If the genotyping result is TC, the oat material is a high crude protein material (average crude protein content ≥12.6%); if it is TT, it is a low crude protein material (average crude protein content ≤9.3%).

[0052] 174 oat materials with unknown crude protein content were randomly selected as samples. The above detection method was used to genotype these 174 samples, and the crude protein content of each sample was detected by Kjeldahl method. The genotyping results and crude protein content results of the 174 samples are shown in Table 1.

[0053] Table 1. Classification results and crude protein content

[0054]

[0055] The typing results and crude protein content detection results of the above 174 samples show that the detection results of this molecular marker are not affected by environmental factors and have high accuracy. The verified accuracy rate can reach 93.6%, indicating that the molecular marker provided by this invention can be used for rapid identification, accurate prediction and screening of high crude protein oat varieties, improve the breeding efficiency of high crude protein oat varieties and shorten the breeding cycle.

[0056] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0057] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. The application of a KASP primer set containing molecular markers affecting crude protein content in oats in the identification of crude protein content in oats, characterized in that, The base sequence of the molecular marker is shown in SEQ ID No. 1, where position 301 is either T or C. The KASP primer combination consists of specific primer 1, specific primer 2, and universal primers; The base sequence of the specific primer 1 is shown in SEQ ID No. 3; The base sequence of the specific primer 2 is shown in SEQ ID No. 4; the base sequence of the universal primer is shown in SEQ ID No.

5.

2. The application as described in claim 1, characterized in that, The 5' end of specific primer 1 is connected to a FAM fluorescent adapter to bind the T allele; the 5' end of specific primer 2 is connected to a HEX fluorescent adapter to bind the C allele.

3. The application as described in claim 1, characterized in that, The determination of crude protein content in oats includes the following steps: Genomic DNA was extracted from the oat material to be tested; KASP-PCR amplification was performed using the KASP primer combination as described in claim 1 to obtain the amplification product; When the fluorescence signal of the amplification product is a co-occurrence of HEX fluorescence signal and FAM fluorescence signal, the oat material to be tested is an oat material with high crude protein content; when the fluorescence signal is only FAM fluorescence signal, the oat material to be tested is an oat material with low crude protein content. The high crude protein content is ≥12.6%; the low crude protein content is ≤9.3%.

4. The application as described in claim 3, characterized in that, The oat material mentioned is the young leaves of oats.

5. The application as described in claim 3, characterized in that, The KASP-PCR amplification program is as follows: pre-denaturation stage: 94℃ for 15 minutes; landing stage: 95℃, denaturation for 20 seconds, 10 cycles; annealing extension at 64℃~55℃ for 60 seconds; amplification stage: 95℃, denaturation for 20 seconds, 30 cycles; annealing extension at 56℃ for 60 seconds. Insulation stage: Store at 4℃.

6. The application of a KASP primer combination containing molecular markers affecting crude protein content in oat crude protein content genetic breeding, characterized in that, The base sequence of the molecular marker is shown in SEQ ID No. 1, where position 301 is either T or C. The KASP primer combination consists of specific primer 1, specific primer 2, and universal primers; The base sequence of the specific primer 1 is shown in SEQ ID No. 3; The base sequence of the specific primer 2 is shown in SEQ ID No. 4; the base sequence of the universal primer is shown in SEQ ID No.

5.

7. The application as described in claim 6, characterized in that, By selecting individuals with the TC heterozygous genotype as parents, the crude protein content of offspring can be increased.

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