A combination of SNP molecular markers related to yield traits of forage oats and application thereof

Abstract

The application discloses a SNP molecular marker combination related to yield traits of forage oats and application thereof, and includes 160 SNP loci, information of the SNP loci is shown in table 1, and the base position on the chromosome in table 1 is referred to the published oat genome sequence Marvellous. By using the molecular marker, precise screening of high-yield potential materials can be realized in early breeding, interference of environmental factors on phenotypes is avoided, and breeding efficiency and selection accuracy are significantly improved. Meanwhile, the marker combination provides a reliable basis for molecular marker assisted selection, helps to speed up the breeding process of high-yield varieties, and has high application value.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics, specifically to a combination of SNP molecular markers related to the yield trait of feed oats and their application. Background Technology

[0002] Forage oats are an important annual grass forage crop, possessing advantages such as balanced nutrition, good palatability, and easy digestibility. They can meet the growth needs of various livestock and are of significant practical importance in alleviating the current shortage of high-quality forage. Furthermore, forage oats are cold-resistant, drought-resistant, and tolerant of poor soil conditions, with flexible soil requirements, enabling efficient use of land resources. This plays a crucial supporting role in promoting sustainable agricultural development and safeguarding national food security.

[0003] Traditional oat breeding for forage relies on phenotypic selection, which is easily affected by environmental factors and suffers from limitations such as long breeding cycles and low selection efficiency, making it difficult to meet the current demand for high-yield and high-quality new forage oat varieties. With the rapid development of molecular biology and genomics technologies, molecular marker-assisted selection and genome-wide selection have become new technical pathways to overcome the bottlenecks of traditional breeding, providing key technical support for improving the efficiency of forage oat breeding.

[0004] Yield-related traits (including core agronomic traits such as plant height, tiller number, spike length, and spikelet number) are key phenotypic indicators determining the production potential of forage oats, and their synergistic optimized expression is a core prerequisite for achieving the integrated goal of high yield and high quality in varieties. Locating yield-related SNP molecular markers through genome-wide association analysis can provide a theoretical basis for systematically elucidating the genetic basis of yield traits in forage oats and screening superior genetic materials, thereby providing key technical support for marker-assisted selection breeding and genome-wide selection breeding of yield traits. Summary of the Invention

[0005] Based on the above needs, a combination of SNP molecular markers for identifying yield traits in feed oats has been invented. This combination can provide an efficient and practical technical tool for the field of molecular breeding of feed oats, and has important theoretical value and practical significance for promoting molecular genetics research and breeding technology innovation in feed oats.

[0006] To achieve the above objectives, this invention provides a combination of SNP molecular markers associated with yield traits in feed oats, comprising 160 SNP loci. The information of the SNP loci and their associated yield traits are shown in Table 1.

[0007] Table 1

[0008]

[0009] The base positions on the chromosomes in Table 1 are referenced to the publicly available oat genome sequence Marvellous.

[0010] The present invention also provides probes for detecting the above-described SNP molecular marker combinations, the nucleotide sequences of which are shown in SEQ ID NO.1-SEQ ID NO.160.

[0011] The present invention also provides a gene chip for detecting SNP molecular marker combinations in Table 1, wherein the chip is loaded with probes having nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.160.

[0012] The above-mentioned SNP molecular markers, probes, and gene chips are used in any of the following applications:

[0013] (1) Identify the yield-related traits of feed oats;

[0014] (2) Early prediction of traits related to feed oat yield;

[0015] (3) Molecular marker-assisted selection breeding for yield traits of feed oats;

[0016] (4) Used for trait evaluation and superior gene discovery of feed oat germplasm resources;

[0017] (5) Genotyping of yield traits in feed oat varieties.

[0018] The present invention also provides a method for detecting the yield trait of feed oats, which uses the above-mentioned probe or gene chip to detect the feed oats to be tested, and determines the yield of the feed oats to be tested based on the detection results.

[0019] The detection of feed oats using the aforementioned probes or gene chips includes: extracting genomic DNA from the feed oats; constructing a library from the genomic DNA; hybridizing and capturing the library using probes with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.160 to obtain enriched target DNA fragments; sequencing the target DNA fragments to obtain sequencing data for SNP detection; and performing quality control, sequence alignment, and SNP molecular marker identification on the sequencing data to obtain genotype detection results.

[0020] The determination of the yield of the feed oats to be tested based on the test results is based on the genotype, which is used to evaluate or predict the yield-related traits of the feed oats.

[0021] The SNP molecular marker combination provided by this invention is significantly associated with yield-related traits in forage oats, and can be used for rapid identification and early prediction of forage oat yield traits. By employing the molecular markers of this invention, precise screening of high-yield potential materials can be achieved in the early stages of breeding, avoiding interference from environmental factors on phenotypes and significantly improving breeding efficiency and selection accuracy. Simultaneously, this marker combination provides a reliable basis for marker-assisted selection, helping to accelerate the breeding process of high-yield varieties and possessing high application value. Attached Figure Description

[0022] Figure 1 This is a normal distribution diagram of the correlation traits of feed oat yield in the examples. Detailed Implementation

[0023] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Example 1: Obtaining SNP molecular markers for yield-related traits in feed oats

[0027] (1) Construction of natural populations of feed oats

[0028] The natural population germplasm of feed oats was provided by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences, and included 797 feed oat cultivars. These feed oat germplasms were sown in Daqing, Heilongjiang Province in 2022 using single-seed planting, with a plant spacing of 30 cm and a row length of 3 m. Each germplasm was planted in 4 rows, and conventional field management was implemented.

[0029] (2) Resequencing of feed oat germplasm resources

[0030] Fresh young leaves from 797 oat germplasm resources were collected, and total genomic DNA was obtained using the CTAB method. High-depth whole-genome resequencing was performed using the BGI sequencing platform. After the data was processed, low-quality paired-end reads were filtered out to obtain high-quality sequencing data (clean data), with an average genome sequencing depth of 8×.

[0031] After filtering the obtained resequencing data, clean reads were aligned to the oat T2T reference genome (Marvellous) using BWA software. The results were then processed using Samtools, Picard-tools, and Reseqtools (sorting, deduplication, adding IDs, etc.). Individual SNP detection for the samples was then performed using GATK4's HaplotypeCalle. CombineGVCFs and GenotypeGVCFs were then used to obtain the raw.VCFs of the population's original latent variants. VariantFiltration and VCFtools were used to filter and remove SNPs with allele frequencies below 0.05, deletion rates greater than 20%, and heterozygosity greater than 20%, resulting in a high-confidence SNP dataset for subsequent analysis.

[0032] (3) Investigation and analysis of yield phenotypes of oat germplasm resources for feed

[0033] Phenotypic traits of the tested oat germplasm resources for forage, including plant height, total number of tillers, number of effective tillers, stem diameter, length and width of flag leaf and second leaf from the top, spike length, number of spikelets, number of leaves, number of whorls, and biomass, were measured at the milk stage. Following the "Oat Germplasm Resource Description Specification and Data Standards," 10 individual plants were randomly selected for phenotypic investigation. The specific measurement methods are as follows:

[0034] Plant height: Measure the height from the ground to the top of the panicle and calculate the average. Total tillers: Count the total number of tillers per plant and calculate the average. Effective tillers: Count the effective tillers per plant and calculate the average. Stem diameter: Measure the diameter of the second internode at the base using calipers and calculate the average. Flag leaf and penultimate leaf length and width: Measure the length and width at the widest point of the flag leaf and penultimate leaf respectively and calculate the average. Panicle length: Measure the straight-line distance from the base of the panicle to the top and calculate the average. Number of spikelets: Count the total number of spikelets on the main stem and calculate the average. Number of leaves: Count the total number of leaves on the main stem and calculate the average. Number of panicle whorls: Count the number of whorls on the main stem and calculate the average. Biomass: After harvesting and drying all above-ground parts of a single plant, weigh them and calculate the average.

[0035] Phenotypic data from 797 oat germplasm resources for feed were obtained. Statistical analysis showed that all phenotypic data exhibited a significant normal distribution (e.g., ...). Figure 1 As shown in the figure, it meets the requirements for subsequent genome-wide association analysis (GWAS).

[0036] (4) SNP screening

[0037] Genome-wide association analysis (GWAS) was performed on 797 forage oat phenotypic data (plant height, tillering, spike length, spikelet number) and 31,644,360 high-quality SNPs (MAF > 0.05, deletion rate ≤ 0.2) genotype data to screen for SNP loci significantly associated with traits across the entire genome. The results are shown in Table 2, revealing 160 SNP loci associated with different forage oat yield-related traits. Further analysis revealed that among the 160 SNP loci, 19 were related to spikelet number, 12 to total tillering, 14 to effective tillering, 20 to spike length, 23 to plant height, 11 to leaf number, 8 to second-to-last leaf width, 11 to second-to-last leaf length, 8 to biomass, 13 to stem diameter, 9 to flag leaf length, 8 to flag leaf width, and 4 to spike whorl number.

[0038] Table 2 SNP location information and trait relationships

[0039]

[0040] Example 2 Probes for Yield-Related Traits in Feed Oats

[0041] (1) Design and fabrication of probes

[0042] A probe design window was formed by extending 30 bp on each side of each SNP locus associated with a yield trait. Multiple candidate probe center locations were generated using a 5 bp step-size sliding window method. Starting from each candidate probe center, a 110 bp probe sequence was extracted and evaluated according to physicochemical parameters such as GC content (0.3–0.65), absence of nitrogen bases, annealing temperature Tm ≥ 68℃, sequence complexity ≥ 0.9, single-base tandem repeat count ≤ 6, and arbitrary-base tandem repeat count ≤ 3. Probes meeting the criteria were screened. The probe sequences were then aligned to the entire genome using BLASTN, and only high-specificity probes with ≤ 3 matching sites within the entire genome were retained. Their sequences are shown in SEQ ID NO.1-SEQ ID NO.160, as detailed in Table 3.

[0043] Table 3

[0044]

[0045] (2) Verification of probe synthesis and typing accuracy

[0046] The 160 high-quality probe sequences obtained from screening were synthesized in batches, quantified, and mixed in equimolar amounts to prepare a mixed probe pool. Twelve oat accessions were randomly selected from 797 oat feed crops for genomic DNA extraction and library construction. The libraries were hybridized with the mixed probe pool for capture, enriched with magnetic beads, and eluted multiple times. After quality control of the captured target DNA fragments, paired-end sequencing was performed to obtain high-quality SNP detection data.

[0047] The raw sequencing data underwent FastQC quality control to remove low-quality reads and adapter sequences. High-quality data were compared with the reference genome using BWA software, and SNP variants were detected and genotyped using GATK or SAMtools. After further quality control, a high-quality SNP genotyping matrix was generated.

[0048] The genotyping matrix was compared with the genotypes of 12 oat germplasms obtained by resequencing. The results showed that the accuracy rate was 100%, indicating that the SNPs and probes provided by this invention have high accuracy and reliability.

[0049] Analysis of feed oat yield in application examples

[0050] The probe provided in Example 2 can be used to accurately genotype 160 target sites in oats. Then, the frequency value of the genotyping result for each site is calculated. The breeding value of the sample is calculated based on the frequency value of each genotyping result. The breeding value is compared with the population threshold. If it is higher than the population threshold, it is judged as a high-yield oat variety. If it is lower than the population threshold, it is judged as a low-yield oat variety.

[0051] Specifically, the population threshold in this application is obtained by analyzing populations with different yields. Those skilled in the art can also adjust it according to breeding objectives, and this invention does not impose any limitations.

[0052] The results of the analysis of high-yield and low-yield feed oat populations in this application were subjected to a significance test (independent samples Man Whitney U test), and the difference was P<0.01, indicating that the results obtained by using the method of this invention are accurate and effective.

[0053] This result fully confirms that the 160 selected molecular markers can effectively distinguish the differences in yield-related traits among different germplasm resources, further verifying the scientific validity and practicality of using them as liquid phase chips for the precise identification of yield-related traits in feed oat germplasm resources, and providing a reliable molecular basis for subsequent germplasm screening and genome selection breeding applications.

[0054] Of course, those skilled in the art can also use the SNP sites provided by this invention in breeding models to analyze and predict breeding values, and this invention does not impose any limitations.

[0055] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of 160 SNP loci in analyzing the yield size of feed oats, characterized in that, The location information of the 160 SNP sites is shown in the table below: ； The base positions on the chromosomes in the table are referenced to the publicly available oat genome sequence Marvellous.

2. A probe combination for detecting the 160 SNP sites described in claim 1, characterized in that, The nucleotide sequences of the probe are shown in SEQ ID NO.1-SEQ ID NO.

160.

3. A gene chip for detecting the 160 SNP sites of claim 1, wherein the probe of claim 2 is loaded thereon.

4. The application of the 160 SNP sites described in claim 1 in identifying the yield size of feed oats.

5. The application of the probe according to claim 2 in identifying the yield size of feed oats.

6. The application of the gene chip according to claim 3 in identifying the yield size of feed oats.

7. The application of the 160 SNP loci described in claim 1 for early prediction of feed oat yield.

8. The application of the probe of claim 2 for early prediction of the yield of feed oats.

9. The application of the gene chip of claim 3 for early prediction of the yield of feed oats.

10. A method for detecting the yield of feed oats, characterized in that, include: (1) Extract genomic DNA from the feed oats to be tested; (2) Construct a library from the genomic DNA; (3) The library is hybridized and captured using a probe pool containing nucleic acid probes targeting the SNP molecular markers of claim 1 to obtain enriched target DNA fragments; (4) Sequencing the target DNA fragment to obtain sequencing data for SNP site detection; (5) Perform quality control, sequence alignment and SNP molecular marker identification on the sequencing data to obtain the genotypes of the 160 SNP sites as described in claim 1; (6) Evaluate or predict the yield of feed oats based on the genotype.

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