Populus cold-resistant whole genome SNP site combination, probe, chip and application thereof
By developing a combination of cold-resistant whole-genome SNP sites and probe chips for poplar, the problem of low efficiency in artificial selection in cold-resistant poplar breeding has been solved, achieving efficient and accurate prediction of cold resistance and significantly accelerating the breeding process.
Patent Information
- Application Number
- CN202511850403.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-09
- Publication Date
- 2026-01-23
Smart Images
Figure CN121380422A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of molecular breeding, in particular to a poplar cold tolerance whole genome SNP site combination, a probe, a chip and application thereof. BACKGROUND
[0002] Poplar (Populus) is a common tree, which is widely used as protective vegetation, soil compaction, soil and water conservation and afforestation plant. However, the frost resistance of poplar as an important biological trait is closely related to temperature change. Many poplar varieties are classified as mild zone plants, which are prone to frost damage symptoms in low temperature environment. Therefore, it is of great significance to study the frost resistance characteristics of poplar and the influence of temperature on the occurrence of frost damage.
[0003] Poplar cold tolerance breeding is mainly based on artificial selection, which is labor-intensive. Molecular marker assisted breeding is to use molecular markers closely linked to cold tolerance traits to detect the presence of target genes, and further breed cold-tolerant poplar hybrid offspring. This technology is fast, accurate and not affected by environmental conditions. For poplar cold tolerance breeding, no major genes have been located, and molecular marker assisted breeding is difficult to promote in poplar breeding. Whole genome selection is to use molecular markers distributed throughout the genome to predict the genomic estimated breeding value of individuals as a screening basis, which is a further application of molecular marker assisted breeding. In whole genome selection, first, a modeling population is constructed, and its genotype and phenotype are determined. A prediction model is established according to the phenotype and genotype data. Second, the genotype of the predicted population is determined. Finally, the breeding value is calculated using the established prediction model and the genotype data of the predicted population, and selection is carried out. After the whole genome selection model is established, the DNA of poplar seeds or seedlings can be detected to complete the cold tolerance breeding of poplar hybrid offspring, greatly reducing the number of plantings and accelerating the breeding process. SUMMARY
[0004] In view of the above problems in the prior art, the present application provides a poplar cold tolerance whole genome SNP site combination, a probe, a chip and application thereof, which effectively solves the problems of traditional poplar cold tolerance breeding relying on artificial selection, large workload and low efficiency.
[0005] To achieve the above purpose, the technical scheme adopted by the present application to solve its technical problems is to provide a poplar cold tolerance whole genome SNP site combination. The SNP site combination comprises 40141 SNP sites, and the physical position and typing information of the 40141 SNP sites are shown in Table 2.
[0006] Further, the poplar is Populus simonii.
[0007] A probe combination for detecting the poplar cold tolerance whole genome SNP site combination.
[0008] The probe combination is applied in preparation of a poplar cold tolerance whole genome chip.
[0009] A poplar cold tolerance whole genome chip comprises the probe combination.
[0010] Further, the probe combination is used for genotyping of each SNP site in the poplar cold tolerance whole genome SNP site combination.
[0011] The poplar cold tolerance whole genome SNP site combination, the probe combination or the poplar cold tolerance whole genome chip is applied in screening of a cold tolerance poplar variety.
[0012] A method for poplar cold tolerance whole genome genotyping detection comprises the following steps:
[0013] S1, obtaining genomic DNA of a poplar sample to be detected;
[0014] S2, detecting the genomic DNA obtained in step S1 by using the poplar cold tolerance whole genome chip to obtain raw data;
[0015] S3, analyzing the raw data obtained in step S3 to obtain a genotyping result.
[0016] In summary, the present application has the following beneficial effects:
[0017] 1. The present application realizes efficient and accurate cold tolerance prediction of a poplar hybrid offspring by developing a whole genome SNP site combination highly associated with cold tolerance and constructing a high-accuracy whole genome selection model based on the SNP site combination, thereby significantly reducing field planting scale and greatly accelerating the breeding process.
[0018] 2. The whole genome selection model constructed by using the SNP marker combination can efficiently predict the cold tolerance of a poplar hybrid offspring, and has high accuracy (the prediction accuracy of indicators such as chlorophyll content is >0.7), thereby greatly reducing the number of plants that need to be planted in the field and significantly accelerating the breeding process. The present application provides a reliable tool for cold tolerance breeding: the probes and chips developed based on the SNP combination can efficiently and accurately detect target sites, and provide a stable and reliable technical means for molecular marker assisted selection and whole genome selection breeding of poplar cold tolerance traits. The present application has wide application value: the technical scheme is not only suitable for small leaf poplar, but also has good detection efficiency (capture efficiency is generally >90%) for other poplar factions (such as green poplar faction and black poplar faction), and has wide applicability and popularization value. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 It is a number distribution diagram of 30 half-sib families.
[0020] It is a number distribution diagram of 30 half-sib families.Figure 2 Distribution and correlation analysis chart of 6 indexes at T1 time point;
[0021] Figure 3 Distribution and correlation analysis chart of 6 indexes at T2 time point;
[0022] Figure 4 Distribution and correlation analysis chart of 6 indexes at T3 time point;
[0023] Figure 5 Distribution and correlation analysis chart of 6 indexes at T4 time point;
[0024] Figure 6 Distribution chart of EC-associated SNP markers;
[0025] Figure 7 Distribution chart of Chl-associated SNP markers;
[0026] Figure 8 Distribution chart of AOC-associated SNP markers;
[0027] Figure 9 Distribution chart of SOD-associated SNP markers;
[0028] Figure 10 Distribution chart of sugar-associated SNP markers;
[0029] Figure 11 Distribution chart of GSH-associated SNP markers;
[0030] Figure 12 Prediction accuracy result chart of whole genome selection model. DETAILED DESCRIPTION
[0031] The principles and features of the present application are described below, and the examples are only used to explain the present application, and are not used to limit the scope of the present application. If no specific conditions are indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If no manufacturer of the reagent or instrument is indicated, it is a conventional product that can be obtained by market purchase.
[0032] Example 1
[0033] I. Introduction of materials
[0034] A total of 639 Populus simonii materials from 30 half-sib families were collected. They were from Inner Mongolia, Liaoning, Shanxi, Jilin, Shaanxi and other main distribution areas of P. simonii, which were excellent materials for screening cold-tolerant poplar. The quantity distribution of 30 half-sib families is shown in Table 1. Figure 1
[0035] II. Analysis of resequencing data
[0036] 639 Populus whole genome resequencing data were analyzed, including the following processes:
[0037] (1) DNA extraction:
[0038] Whole genome DNA was extracted from 639 Populus leaves. 1.5% agarose electrophoresis showed clear and complete electrophoretic bands, no degradation, qubit quantification of total DNA concentration was more than 600 ng, and resequencing experiment was performed.
[0039] (2) Whole genome resequencing library construction, using ABclone library construction standard kit (item number RK20261), steps as follows:
[0040] a. The genomic DNA was broken into 200-300 bp fragments, and the DNA fragment ends were repaired.
[0041] b. Connect Huada DNBSEQ-T7 sequencing adapter and index.
[0042] c. Library amplification and purification, qubit quantification of library, DNA total amount is more than 200 ng.
[0043] d. Sequencing by Huada DNBSEQ-T7.
[0044] (3) Data filtering:
[0045] Using Trimmomatic-0.36 to filter the fq.gz data, removing reads with adapter, N content more than 10% and quality value less than 10 more than 50%.
[0046] (4) Variation detection:
[0047] a. Filtered fq.gz data, using bwa software to align Populus GCA_015852605.2 genome (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_015852605.2 / ) to generate bam file. Using GATK software to analyze SNP variation data.
[0048] b. Using vcftools software to filter SNP sites with allele frequency greater than 0.05, deletion rate less than 0.01, 2 alleles, minimum sequencing depth greater than 3, and minimum average sequencing depth greater than 30, obtaining 55522 high-quality SNP sites. Software parameters: --max-missing 0.99 --min-meanDP 30 --maf 0.05 --minDP 3 --max-alleles 2 --remove-indels.
[0049] Example 2
[0050] Populus tomentosa cold tolerance genome-wide association analysis, including the following processes:
[0051] (1) Populus tomentosa cold tolerance trait collection
[0052] Based on the temperature change of Tongliao City as shown in Table 1, on September 19, 2024 to September 22 (T1), September 30 to October 3 (T2), October 14 to October 6 (T3), October 21 to October 22 (T4) respectively on half-sib family forest single plant phenotypic value observation, including chlorophyll content (Chl), relative conductivity (EC), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), soluble sugar content (suagr), reduced glutathione (GSH).
[0053] Table 1 Sampling time points and temperature conditions
[0054]
[0055] Determination of chlorophyll content: randomly select 3 single plants for each strain, and randomly select 3 mature leaves for each single plant. The chlorophyll content was determined by using a handheld chlorophyll meter (model: LD-YC, Shandong Laideng Intelligent Technology Co., Ltd.).
[0056] Determination of relative conductivity: randomly select 3 single plants for each strain, and randomly select 4 mature leaves for each single plant. Use a puncher to take 2 circular leaf tissues with a diameter of 0.5 cm at the same position away from the leaf vein of each leaf, and place them in deionized water. After shaking thoroughly, the conductivity R1 was measured by using a conductivity meter. Then the extraction solution was placed in a boiling water bath for 20 min, and the conductivity R2 was measured again. Finally, the relative conductivity was obtained by calculating the ratio of R1 to R2.
[0057] Determination of physiological and biochemical indicators: randomly select 3 single plants for each strain, and randomly select 4 mature leaves for each single plant. Place them in liquid nitrogen for quick freezing, and then grind thoroughly. The GSH, SOD, sugar and T-AOC were determined by using the biochemical reagent kit of Solabio Company. Each single plant was measured 3 times repeatedly.
[0058] T1-T4 time points 6 indicators are normally distributed. The phenotype distribution graphs of T1-T4 time points are shown in Figures 2-5 .
[0059] (2) Genome-wide association analysis
[0060] a. Use GAPIT software to perform genome-wide association based on logit regression model for binary traits.
[0061] b.4 models (Blink model, FarmCPU model, GLM model and MLM model) were respectively associated with 6 indexes of cold tolerance.
[0062] c. All SNP sites associated with each index of cold tolerance were combined.
[0063] The distribution of SNP markers associated with 6 indexes of cold tolerance (EC, Chl, AOC, SOD, sugar and GSH) is shown in FIG. 1. Figures 6-11 The physical position and mutation type of SNP sites are shown in Table 2.
[0064] Table 2 Physical position and mutation type of SNP sites
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[0231] Example 3
[0232] Genome-wide selection analysis
[0233] (1) Genome-wide selection model establishment and prediction
[0234] a. For the 6 indexes related to cold tolerance, BayesA, BayesB, BayesC and GBLUP were used to establish genome-wide selection models. The prediction accuracy results are shown in Table 1. Figure 12
[0235] b. Training set results: There was no significant difference in the prediction effect of 4 models of 6 phenotypic indicators. The prediction accuracy of chlorophyll content (Chl), relative conductivity (EC), superoxide dismutase (SOD), and soluble sugar content (suagr) was higher than 0.7. The prediction accuracy of reduced glutathione (GSH) and total antioxidant capacity (T-AOC) was between 0.3 and 0.4, which may be due to the instability of enzyme activity determination. Chlorophyll content (Chl) is simple and convenient to detect, and has good prediction accuracy. Subsequent use of chlorophyll content (Chl) as a cold tolerance screening indicator.
[0236] Example 4
[0237] The SNP screened in Example 3 was evaluated by probe. The screening principle is that there is no InDel marker within 50bp upstream and downstream of the SNP site; there is only one copy number of 100bp sequence upstream and downstream of the genome; the GC content is 40-60%; there is no short repeat sequence in the sequence; and there is no N base in the sequence.
[0238] Example 5
[0239] Using the SNP marker data set described in Example 5 to detect poplar
[0240] (1) Genomic DNA extraction
[0241] Poplar leaves were collected and stored with ice packs and transported back to the laboratory in time. The total DNA of poplar was extracted by magnetic bead method.
[0242] (2) Genomic DNA library construction
[0243] The genomic DNA was fragmented (200-300bp), the DNA fragment ends were repaired and connected to the adapter, and the library was amplified by PCR.
[0244] (3) Probe hybridization with target region
[0245] The prepared SNP probe was hybridized with the Pre-PCR library, the streptomycin-modified probe was complementary captured with the complementary DNA library, and after enrichment of the target DNA library, other library DNA was eluted, i.e. the Pre-PCR library after capture.
[0246] (4) DNA purification
[0247] After magnetic bead purification, the DNA concentration required for high-throughput sequencing was amplified and enriched.
[0248] (5) High-throughput sequencing
[0249] The library obtained after capturing and amplifying step (4) is subjected to high-throughput sequencing by using DNBSEQ-T7 sequencing platform of Huada Intelligent Manufacturing to obtain the sequencing results of genomic DNA, and the obtained data is subjected to basic cleaning treatment.
[0250] (6) Evaluation of liquid phase probe capture rate
[0251] The cleaned sequencing data is aligned to the poplar GCA_015852605.2 reference sequence by using bwa software, and the capture efficiency is evaluated.
[0252] Example 6
[0253] Evaluation of liquid phase probe capture efficiency for 38 poplar varieties
[0254] (1) Experimental materials
[0255] 38 poplar leaves were selected, stored in the refrigerator and transported to the laboratory in time, and the total DNA of each single plant was extracted by using CTAB method. The sample numbers are shown in Table 3.
[0256] Table 3 Capture efficiency of different samples
[0257]
[0258] (2) Evaluation of capture efficiency of 38 poplar varieties
[0259] The capture efficiency of black poplar is 93.98% to 94.26%, the capture efficiency of white poplar is 84.66% to 92.73%, the capture efficiency of green poplar is 93.85% to 98.92%, the capture efficiency of large leaf poplar is 91.12% to 96.73%, and the capture efficiency of poplar is 94.16% to 96.17%. The present application screens SNP marker combination for 639 materials of small leaf poplar of green poplar, and the capture efficiency of small leaf poplar is the highest, and the capture efficiency of other poplars is also good.
[0260] The above only describes the preferred embodiments of the present application, and does not limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims
1. A genome-wide SNP locus combination for cold-resistant poplar trees, characterized in that, The SNP locus combination contains 40,141 SNP loci, and the physical location and genotyping information of the 40,141 SNP loci are shown in Table 2.
2. A probe array for detecting the combination of cold-resistant whole-genome SNP sites of poplar as described in claim 1.
3. The application of the probe combination according to claim 2 in the preparation of a poplar cold-resistant whole genome chip.
4. A poplar cold-resistance whole genome chip, characterized in that, Includes the probe assembly described in claim 2.
5. The poplar cold-resistance whole genome chip as described in claim 4, characterized in that, The probe combination is used to identify the genotype of each SNP site in the poplar cold-resistant whole genome SNP site combination described in claim 1.
6. The application of the poplar cold-resistant whole genome SNP site combination as described in claim 1, the probe combination as described in claim 2, or the poplar cold-resistant whole genome chip as described in any one of claims 4-5 in screening cold-resistant poplar varieties.
7. A method for detecting cold-resistant whole-genome genotyping of poplar, characterized in that, Includes the following steps: S1. Obtain the genomic DNA of the poplar sample to be tested; S2. Detect the genomic DNA obtained in step S1 using the poplar cold-resistant whole genome chip described in claim 4 or 5 to obtain raw data; S3. Analyze the raw data obtained in step S3 to obtain the genotyping results.