SNP (Single Nucleotide Polymorphism) molecular marker combination related to content of neutral detergent fibers in oat, chip and application of SNP molecular marker combination
By constructing a combination of SNP molecular markers and a prediction system for the neutral detergent fiber content of oats, the problems of long cycle and low efficiency in traditional breeding have been solved, and early accurate prediction and improved overall benefits have been achieved.
Patent Information
- Application Number
- CN202610516630.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-17
- Publication Date
- 2026-05-19
AI Technical Summary
Traditional oat breeding relies on chemical testing of mature plants to screen for neutral detergent fiber content, resulting in long breeding cycles, cumbersome and destructive testing, and a limited number of existing molecular markers, making it impossible to predict target traits in the early stages and leading to low breeding efficiency.
We constructed a combination of SNP molecular markers that were significantly associated with the content of neutral detergent fiber in oats. Combined with a genome-wide selection model, we used a liquid-phase prediction chip and prediction system to achieve accurate prediction during the seedling stage, shorten the breeding cycle and improve screening efficiency.
It enables the completion of genotype potential analysis and phenotypic prediction within 3-4 months after sowing, shortening the breeding cycle by 60%, reducing costs by 45%-55%, and simultaneously improving agronomic traits such as stress resistance and yield.
Smart Images
Figure CN122060913A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology, specifically relating to a combination of SNP molecular markers, a chip, and their applications related to the content of neutral detergent fiber in oats. Background Technology
[0002] oat( Avena sativa L. Oat is an important dual-purpose crop for both food and feed. The neutral detergent fiber (NDF) content of its grains is a key quality trait that determines its palatability, nutritional value for feed, and processing adaptability. An appropriate NDF content helps maintain the functional characteristics of oat products, while an excessively high content will significantly reduce their digestibility and palatability.
[0003] In traditional breeding, the screening of neutral detergent fiber content in oats relies on chemical detection methods on mature plants, requiring sample processing and component analysis after the crop growth cycle is completed. This approach has significant limitations: the breeding cycle is long, typically requiring 3-4 years to complete one round of screening; the detection process is cumbersome and destructive, consuming substantial manpower, resources, and time; furthermore, traditional methods cannot predict target traits in early generations, leading to a large retention of ineffective breeding materials and low breeding efficiency.
[0004] Molecular marker-assisted breeding technology has been widely used in crop breeding, but there are still shortcomings in the research on the neutral detergent fiber content of oats: the number of existing molecular markers is limited, the genome coverage is narrow, the correlation with the target trait is not strong, and the prediction accuracy is low; there is a lack of stable marker combinations across environments and varieties, making it difficult to adapt to the screening needs of different oat germplasm resources; and no efficient prediction model and detection system have been established, making it impossible to achieve early and rapid screening and failing to fundamentally shorten the breeding cycle. Summary of the Invention
[0005] This invention aims to provide a combination of SNP molecular markers, a chip, and their applications related to the neutral detergent fiber content of oats. By screening SNP sites that are significantly associated with the neutral detergent fiber content of oats, an efficient detection probe set and prediction system are constructed to achieve accurate prediction of the neutral detergent fiber content of oat seedlings. This significantly shortens the oat breeding cycle, improves screening efficiency, and reduces breeding screening costs, providing technical support for targeted oat breeding.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A combination of SNP molecular markers associated with the neutral detergent fiber content of oats includes 109 SNP molecular markers that are significantly associated with the high neutral detergent fiber content of oats. The physical locations of the 109 SNP molecular markers were determined by sequence alignment based on the oat reference genome GWHGDJH00000000.1, and the specific site information is shown in Table 1.
[0007] Table 1 SNP molecular marker site information
[0008] Preferably, the SNP site contains a dominant allele that is positively or negatively associated with the neutral detergent fiber content of oats.
[0009] Preferably, the allele frequency of the SNP site is greater than 0.01; the deletion rate is less than 0.2; there are no InDel markers within 50 bp upstream and downstream; the upstream and downstream 100 bp sequences are single copies in the whole genome; the GC content is 40%-60%; and there are no short fragment duplications or N bases in the sequence.
[0010] The present invention also provides a liquid phase prediction chip for oat neutral detergent fiber content, the liquid phase chip comprising a primer set and / or probes for detecting the SNP molecular marker combinations related to oat neutral detergent fiber content.
[0011] The present invention also provides a kit comprising a primer set and / or probes for detecting the SNP molecular marker combination associated with the neutral detergent fiber content of oats.
[0012] The present invention also provides a method for screening SNP molecular marker combinations related to the neutral detergent fiber content of oats, comprising the following steps: S1. Multi-environmental phenotypic identification was conducted on germplasm resource populations covering the main oat planting areas to obtain neutral detergent fiber content phenotypic data. S2. Perform whole-genome resequencing on the germplasm resource population described in S1, and obtain the whole-genome SNP marker set through bioinformatics analysis; S3. Perform genome-wide association analysis on the phenotypic data described in S1 and the SNP marker set described in S2 to screen out SNP sites that are significantly associated with the content of neutral detergent fiber; S4. The significantly associated SNP sites obtained in S3 are screened a second time according to the established probe design feasibility criteria to obtain the SNP molecular marker combination related to the neutral detergent fiber content of oats.
[0013] Preferably, in S4, the probe design feasibility criteria include: no InDel variants within 50bp upstream and downstream of the SNP site, single copies of the upstream and downstream 100bp sequences in the whole genome, GC content of 40%-60%, and no short fragment duplications or N bases in the sequence.
[0014] The present invention also provides a system for predicting the neutral detergent fiber content of oats, comprising: The SNP molecular marker combination was obtained, and the SNP sites were analyzed based on a genome-wide selection model. Phenotypic data of neutral detergent fiber content in oats under multiple environments were integrated to obtain a system for predicting the range and stability of neutral detergent fiber content in mature oats during the oat seedling stage.
[0015] Preferably, the model is selected from at least one of Bayesian-ALMT, GBLUP, LightGBM, and Random Forest.
[0016] This invention also provides a method for oat breeding, comprising the following steps: T1. Extract genomic DNA from oat seedlings; T2. Use the liquid phase prediction chip to detect SNP sites in the genomic DNA extracted in T1; T3. Input the test results into the prediction system to generate a neutral detergent fiber content prediction report; T4. Based on the T3 prediction report, select plants with the target neutral detergent fiber content phenotype for targeted breeding.
[0017] This invention also provides the application of one of the following oat breeding methods in oat variety selection or quality improvement: the SNP molecular marker combination, the liquid phase prediction chip, the reagent kit, the prediction system, and the breeding method.
[0018] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a combination of SNP molecular markers related to the neutral detergent fiber content of oats, a chip, and their applications. Based on the integration of 109 relevant SNP markers, combined with genome-wide selection model and time-series phenotypic integration technology, it can achieve quantitative prediction of the neutral detergent fiber content of oats under different environments and varieties. The prediction accuracy is greater than 0.70, which is significantly better than the prediction accuracy of traditional molecular markers.
[0019] By combining seedling-stage genotyping with a neutral detergent fiber (NDF) content prediction model, genotypic potential analysis and phenotypic prediction can be completed within 3-4 months after sowing, shortening the traditional 3-4 year cycle relying on post-maturity chemical testing to 12-18 months, improving efficiency by over 60%. Furthermore, by identifying materials with the target NDF content phenotype early on, the field planting and later testing of ineffective breeding materials are reduced, saving labor, resources, and planting space, resulting in a 45%-55% reduction in overall costs. In addition, while predicting NDF content, it is possible to correlate it with agronomical traits such as stress resistance and yield in oats, enabling simultaneous improvement of quality and agronomic traits and enhancing overall breeding benefits.
[0020] 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
[0021] Figure 1 This is a map showing the SNP density distribution based on whole-genome resequencing; Figure 2 For population genetic structure analysis based on genome-wide SNPs; Figure 3 Manhattan plot of genome-wide association analysis (GWAS) of neutral detergent fiber content; Figure 4 To evaluate the accuracy of neutral detergent fiber content prediction based on a genome-wide selection model. Detailed Implementation
[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0024] Source of experimental materials: In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.
[0025] Example 1 A method for screening SNP molecular marker combinations related to the neutral detergent fiber content of oats, comprising the following steps: S1. 1078 global oat germplasm resources (including local varieties, bred varieties, and core germplasm, covering major oat-growing areas) were introduced and collected from the National Germplasm Bank, local agricultural research institutes, and other institutions. These resources exhibited rich variation in crude protein content (range: 7.8%-16.1%). The germplasm resources were planted in different experimental fields (such as Guyuan, Ningxia and Zhangjiakou, Hebei). A randomized block design with three replicates was adopted. The aboveground parts of the plants were collected at the milk stage of oat growth, withered and yellow leaves were removed, and the plants were blanched at 105℃ for 30 minutes, dried at 80℃ to constant weight, pulverized, and passed through a 40-mesh sieve. The neutral detergent fiber content was determined by the Van der Waals washing fiber analysis method. Three replicates were set up for each germplasm resource. The average value and BLUP value were calculated as the phenotypic value of the material. The collected material phenotypic BLUP values were used for GWAS association analysis. S2. DNA Extraction and Sequencing: During the oat seedling stage, select 3 robust plants from each material, collect their tender leaves, mix them evenly, and use them as DNA extraction samples for that material.
[0026] Genomic DNA was extracted using a modified CTAB method. The specific experimental protocol was as follows: 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 12,000 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 12,000 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 OD500. 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×. S3. SNP Screening and Association Analysis: Quality control was performed on the raw sequencing data. FastQC software was used to evaluate the quality of the raw reads, mainly including the distribution of base quality values, 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.
[0027] The filtered clean sequencing data was aligned to the oat reference genome (cultivar "Marvellous", genome data released to the National Genome Database, accession number GWHGDJH00000000.1) using the BWA-MEM algorithm, yielding alignment results in SAM format. Samtools software was used to convert the SAM format files to BAM format, followed by sorting and removal of 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 file was rigorously filtered with the following parameters: Quality Depth (QD) ≥ 2.0, FisherStrand 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. Bisellelic SNPs were retained, while SNPs with a deletion rate greater than 20% and a minor allele frequency (MAF) less than 5% were filtered out. This resulted in a high-quality SNP marker set of 31,644,360. S4. Using emmax, gemma, rmvp (FARMCPU model, GLM model, MLM model), and tassel (GLM model), six models were used to associate the oat neutral detergent fiber content trait. The p-value threshold was set to 1E-7. Probe evaluation was performed on the screened SNPs. The screening criteria were: no InDel marker within 50bp upstream and downstream of the SNP site, only one copy number of the upstream and downstream 100bp sequences in the whole genome, GC content of 40%-60%, no short repetitive sequences and N bases in the sequence. Finally, 109 SNP sites remained. The selected SNP loci were evaluated using eight genome-wide selection models: LightGBM, Random Forest, SVR, BL, GBLUP, Kernel, XGBoost, and Gradient Boosting, combined with genotypic and neutral detergent fiber content phenotypic data from 420 materials. The results showed that the overall prediction accuracy was greater than 0.79, indicating good prediction performance.
[0028] The probe set for detecting SNP sites is shown in Table 2.
[0029] Table 2 Probe Sequences
[0030] The probe set includes probe sequences targeting 109 SNP sites related to the content of neutral detergent fibers. The probe sequences cover a 100bp region upstream and downstream of each SNP site, ensuring efficient and specific capture of the target SNP sites.
[0031] A probe is designed for each SNP site, with the nucleotide sequence of the probe being inversely complementary to the nucleotide sequence of the target site, thus obtaining a probe set.
[0032] The probe set consists of 109 single-stranded nucleotide probes that specifically capture 109 SNP sites. Each probe captures one SNP site, achieving specific binding to 109 target sites.
[0033] Fabrication of liquid phase prediction chips: The liquid-phase prediction chip contains the above-mentioned probe set and captures target SNP sites through liquid-phase hybridization technology, achieving efficient detection of SNP sites related to neutral detergent fiber content in the oat genome. The chip fabrication process conforms to the standard for conventional liquid-phase chip fabrication and was synthesized by Beijing Novogene Technology Co., Ltd.
[0034] Thirty different oat varieties were selected, and the capture efficiency was evaluated using the aforementioned liquid-phase prediction chip. The results showed that the SNPs related to high and medium neutral detergent fiber content had a detection rate range of 97.83%-99.02%, with a coverage depth of 402.15-548.63X. The overall capture efficiency was good, indicating that the SNP chip disclosed in this invention has a high detection rate of target sites and accurate and reliable genotyping results. It can be used for subsequent SNP detection and genome-wide selection evaluation of other samples.
[0035] Among them, the SNP density distribution map based on whole genome resequencing is as follows: Figure 1 As shown.
[0036] Depend on Figure 1 It can be seen that SNPs are relatively evenly distributed throughout the genome, with higher density in chromosome arm regions and lower density in centromere regions, providing ample marker resources for subsequent molecular marker development and high-density association analysis.
[0037] The results of population genetic structure analysis based on genome-wide SNPs are as follows: Figure 2 As shown.
[0038] Depend on Figure 2 It can be seen that oat germplasm can be divided into 6 main subgroups, which are derived from different geographical regions (such as East Asia, Europe, and North America), indicating that this germplasm collection has rich genetic diversity and provides a good material basis for screening superior germplasm. Manhattan plot of genome-wide association analysis (GWAS) of neutral detergent fiber content Figure 3 As shown.
[0039] Depend on Figure 3 A total of 109 significantly associated SNP sites were detected (P < 1.0 × 10⁻⁶). -6 These sites are mainly distributed on chromosomes 3A, 4C, 7A, and 7D, forming a combination of SNP molecular markers that are significantly associated with the neutral detergent fiber content of oats, and can effectively distinguish between high and low neutral detergent fiber materials.
[0040] The accuracy assessment results of neutral detergent fiber content prediction based on the whole-genome selection model are as follows: Figure 4 As shown.
[0041] Depend on Figure 4 It is known that the Bayesian Lasso (BL) model has the highest prediction accuracy, which can provide a reliable basis for breeding decisions, increase the probability of breeding success, and ensure the cultivation of germplasm with high and medium neutral detergent fiber content.
[0042] Example 2 An oat breeding method includes the following steps: T1. Collect oat seedling leaves, preserve them with ice packs and transport them back to the laboratory in a timely manner, and extract total DNA using the magnetic bead method. T2. Use the above liquid-phase prediction chip to detect SNP molecular marker combinations: Fragment the genomic DNA (200-300bp), repair the ends and connect the adapters, then perform Pre-PCR amplification to construct a library. Hybridize the prepared SNP probes with the Pre-PCR library, enrich the target DNA library, and then perform high-throughput sequencing by Beijing Novogene Technology Co., Ltd. T3. Input the genotyping information of the detected SNP sites into the prediction model to generate a neutral detergent fiber content prediction report. Based on a dataset of 420 oat germplasm resources, the prediction accuracy can reach 0.79, and the breeding cycle is shortened to 12-18 months. T4. Based on the T3 prediction report, select plants with the target neutral detergent fiber content phenotype for targeted breeding.
[0043] 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 combination of SNP molecular markers related to the neutral detergent fiber content of oats, characterized in that, This includes 109 SNP molecular markers that are significantly associated with the high neutral detergent fiber content of oats. The physical locations of these 109 SNP molecular markers were determined by sequence alignment based on the oat reference genome GWHGDJH00000000.
1. The specific site information is shown in the table below: 。 2. A liquid phase prediction chip for the content of neutral detergent fiber in oats, characterized in that, The liquid-phase chip comprises a primer set and / or probes for detecting the SNP molecular marker combinations related to the neutral detergent fiber content of oats as described in claim 1.
3. A reagent kit, characterized in that, The kit contains a primer set and / or probes for detecting the SNP molecular marker combination associated with the neutral detergent fiber content of oats as described in claim 1.
4. A method for screening SNP molecular marker combinations related to the neutral detergent fiber content of oats as described in claim 1, characterized in that, Includes the following steps: S1. Multi-environmental phenotypic identification was conducted on germplasm resource populations covering the main oat planting areas to obtain neutral detergent fiber content phenotypic data. S2. Perform whole-genome resequencing on the germplasm resource population described in S1, and obtain the whole-genome SNP marker set through bioinformatics analysis; S3. Perform genome-wide association analysis on the phenotypic data described in S1 and the SNP marker set described in S2 to screen out SNP sites that are significantly associated with the content of neutral detergent fiber; S4. The significantly associated SNP sites obtained in S3 are screened a second time according to the established probe design feasibility criteria to obtain the SNP molecular marker combination related to the neutral detergent fiber content of oats.
5. The screening method according to claim 4, characterized in that, In S4, the feasibility criteria for probe design include: no InDel variants within 50bp upstream and downstream of the SNP site, single copies of the upstream and downstream 100bp sequences in the whole genome, GC content of 40%-60%, and no short fragment duplications or N bases in the sequence.
6. A system for predicting the fiber content of oat neutral detergent, characterized in that, include: The SNP molecular marker combination related to the neutral detergent fiber content of oats as described in claim 1 was obtained, the SNP sites were analyzed based on a genome-wide selection model, and phenotypic data of neutral detergent fiber content of oats under multiple environments were integrated to obtain a system for predicting the range and stability of neutral detergent fiber content of mature oats during the oat seedling stage.
7. A method for oat breeding, characterized in that, Includes the following steps: T1. Extract genomic DNA from oat seedlings; T2. Use the liquid phase prediction chip described in claim 2 to detect SNP sites in the genomic DNA extracted in T1; T3. Input the test results into the prediction system described in claim 6 to generate a neutral detergent fiber content prediction report; T4. Based on the T3 prediction report, select plants with the target neutral detergent fiber content phenotype for targeted breeding.
8. The application of any of the following oat breeding methods in oat variety selection or quality improvement: the SNP molecular marker combination related to the neutral detergent fiber content of oats as described in claim 1, the liquid phase prediction chip as described in claim 2, the kit as described in claim 3, the prediction system as described in claim 6, and the oat breeding method as described in claim 7.