Liquid gene chip for wheat low cadmium and comprehensive agronomic trait identification and breeding, kit and application thereof

Abstract

The application belongs to the technical field of plant molecular breeding, biochip and molecular detection, and particularly relates to a liquid gene chip for wheat low-cadmium and comprehensive agronomic trait identification and breeding, a kit thereof and application thereof.The gene chip comprises detection reagents for specifically detecting a SNP site set, and the SNP site set is selected from the following: a cadmium-related SNP site set, a homologous function gene cadmium-related SNP site set, other metal ion-related SNP site set, a SNP site set related to Nramp, HMA, ABC, ZIP, PCR, MTP, YSL and CAX gene families, and a whole genome background filled SNP site set.The application realizes systematic detection of wheat cadmium absorption, transport, accumulation and detoxification related genetic variation, realizes multi-metal coordinated monitoring and heavy metal accumulation panoramic scanning, can be used for comprehensive risk assessment of wheat heavy metal accumulation traits, improves complex variation detection capability, has excellent detection performance, is high in information acquisition efficiency, and is suitable for large-scale application.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular breeding, biochips and molecular detection technology, specifically relating to a liquid-phase gene chip for identifying and breeding wheat with low cadmium and comprehensive agronomic traits, as well as its kit and applications. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of the world's most important food crops, and the safety of its grains is directly related to food security and human health. Cadmium (Cd) is a heavy metal element with strong biotoxicity. It can enter wheat grains through root absorption, xylem transport, and phloem redistribution, and then enter the human body through the food chain, causing damage to the kidneys, bones, and immune system. With the acceleration of industrialization, improper discharge of cadmium-containing industrial wastewater and solid waste, as well as the long-term application of cadmium-containing phosphate fertilizers in agricultural production, have led to an increasingly serious problem of cadmium pollution in global arable land soils.

[0003] To address the problem of excessive cadmium levels, current efforts mainly focus on "soil remediation" and "agronomic regulation," but both have significant limitations: soil remediation technologies require high costs and have long remediation cycles, making them highly unsuitable for large-scale arable land and difficult to meet the needs of large-scale wheat production in major wheat-producing areas; while agronomic regulation technologies can reduce cadmium absorption in the short term, their effectiveness is greatly affected by soil type and climate conditions, exhibiting poor stability and failing to fundamentally block the cadmium transport pathway to grains, thus making it difficult to ensure that wheat cadmium content consistently meets standards. Therefore, cultivating low-cadmium-accumulation wheat varieties is an important way to reduce the risk of cadmium exposure in grains and ensure food security.

[0004] Cadmium accumulation in wheat grains is a complex quantitative trait regulated by multiple genes and significantly influenced by environmental factors such as soil pH, organic matter content, cultivation management practices, and climatic conditions. Current screening methods for low-cadmium-accumulating materials mainly rely on field phenotypic identification and grain cadmium content determination. These methods suffer from problems such as long detection cycles, significant environmental impact, low throughput, and difficulty in early generation selection, failing to meet the need for rapid breeding of low-cadmium wheat varieties.

[0005] Furthermore, the selection of a single low-cadmium trait often fails to take into account comprehensive agronomic traits such as yield, quality, disease resistance, and stress resistance, which restricts the promotion and application of low-cadmium wheat varieties.

[0006] Molecular marker technology, especially single nucleotide polymorphism (SNP) markers, has become an important tool in crop molecular breeding due to its abundant quantity, genetic stability, and suitability for high-throughput detection. Liquid-phase gene chip technology, based on the principle of targeted capture sequencing, achieves simultaneous detection of a large number of target sites by designing oligonucleotide probes complementary to the target sites, offering advantages such as high flexibility, low cost, and scalability. Compared with whole-genome resequencing, liquid-phase gene chips can significantly reduce the cost per sample while ensuring coverage of key sites; compared with solid-phase chips, liquid-phase chips offer greater flexibility in site selection and can be customized according to breeding objectives.

[0007] Currently, existing wheat breeding chips (such as 660K and 55K) mainly focus on whole-genome genetic diversity analysis, variety identification, or conventional agronomic trait improvement, with insufficient systematic coverage of gene loci related to cadmium absorption, translocation, accumulation, and detoxification. Existing chips rarely integrate cadmium-related loci, polymetallic ion-related loci, and pan-genome structural variation loci into a single detection system, making it difficult to support the synergistic improvement of low-cadmium traits with yield, quality, and resistance, and also unable to achieve early prediction of low-cadmium traits based on genotype data.

[0008] Therefore, there is an urgent need to develop a liquid-phase gene chip for low-cadmium wheat breeding to achieve precise detection of low-cadmium-related genetic loci, synergistic monitoring of multiple metal accumulation traits, and comprehensive agronomic trait assessment, thereby promoting the transformation of low-cadmium wheat breeding from traditional methods to precision and intelligence. Summary of the Invention

[0009] The first aspect of the present invention provides a gene chip, including a detection reagent for specifically detecting a set of cadmium-related SNP sites, the set of cadmium-related SNP sites including the SNP site set shown in Table 2 of the specification, and the detection reagent being a combination of point-type capture probes for specifically detecting SNP sites.

[0010] Optionally, the cadmium-related SNP sites also include a set of cadmium-related SNP sites of homologous functional genes as shown in Table 3 of the specification; the detection reagent is a combination of point-capture probes for specific detection of SNP sites.

[0011] Optionally, the gene chip may also include detection reagents for specifically detecting at least one set of the following SNP sites: (Group 1) A set of 514 SNP sites associated with other metal ions, as shown in Table 4 of the instruction manual; (Group 2) SNP sites associated with the Nramp, HMA, ABC, ZIP, PCR, MTP, YSL, and CAX gene families, as shown in Table 5 of the instruction manual; (Group 3) SNP sites used for whole-genome background filling, as shown in Table 6 of the instruction manual; The detection reagent is a combination of spot capture probes for specific detection of SNP sites.

[0012] A second aspect of the present invention provides a gene chip, including a detection reagent for specifically detecting a set of wheat deletion-type structural variant sites; the set of wheat deletion-type structural variant sites is shown in Table 7 of the specification; the detection reagent is a cross-breakpoint capture probe combination, with one forward and one reverse probe designed for each deletion-type structural variant site.

[0013] A third aspect of the present invention also provides a liquid-phase gene chip for identifying and breeding wheat with low cadmium levels and comprehensive agronomic traits, comprising the above-mentioned gene chip.

[0014] The fourth aspect of the present invention also provides a kit for identifying and breeding wheat with low cadmium content and comprehensive agronomic traits, comprising the above-mentioned gene chip.

[0015] The fifth aspect of this invention also provides the application of SNP site combinations related to wheat breeding in the identification and breeding of wheat with low cadmium levels and comprehensive agronomic traits. The SNP site combinations include one or more sets of the following SNP site sets: cadmium-related SNP site sets as shown in Table 2 of the specification; cadmium-related SNP site sets of homologous functional genes as shown in Table 3 of the specification; SNP site sets related to other metal ions as shown in Table 4 of the specification; SNP site sets related to the Nramp, HMA, ABC, ZIP, PCR, MTP, YSL, and CAX gene families as shown in Table 5 of the specification; SNP site sets for whole-genome background filling as shown in Table 6 of the specification; and wheat deletion-type structural variation site sets as shown in Table 7 of the specification.

[0016] A sixth aspect of the present invention also provides a method for obtaining wheat breeding information, comprising the following steps: S1. Extract genomic DNA from the wheat sample to be tested; S2. Construct a library from genomic DNA to obtain a sequencing library; S3. Using the gene chip of the present invention, the sequencing library is used to perform liquid-phase hybridization to capture the target site, and the captured product is obtained. S4. Perform high-throughput sequencing on the captured products to obtain sequencing data; S5. Perform data analysis on the sequencing data to obtain breeding information for the wheat samples to be tested.

[0017] The seventh aspect of the present invention also provides applications of the above-mentioned gene chip, or liquid-phase gene chip or reagent kit, including at least one of the following uses: Screening of low-cadmium wheat germplasm; targeted breeding of low-cadmium wheat varieties; molecular marker-assisted selection of low-cadmium wheat; risk assessment of heavy metal accumulation in wheat.

[0018] The technical solution provided by this invention has at least the following beneficial effects: (1) Precise coverage of low-cadmium sites and reliable screening sources; the technical solution of this invention includes 2277 cadmium ion-related SNP sites (Tables 2-3), a set of SNP sites related to 5797 Nramp, HMA, ABC, ZIP, PCR, MTP, YSL, and CAX gene families (Table 5); and a set of SNP sites for filling 3968 whole-genome backgrounds (Table 6). The coverage area is at least 3.43 million bp, enabling systematic detection of genetic variations related to cadmium absorption, transport, accumulation, and detoxification in wheat.

[0019] (2) Multi-metal synergistic monitoring to achieve panoramic scanning of heavy metal accumulation; The technical solution of the present invention includes 514 SNP sites related to more than ten metal ions such as As, Hg, Pb, Cu, Ni (Table 4), which can be used for comprehensive risk assessment of heavy metal accumulation traits in wheat, and is not limited to the detection of a single cadmium element.

[0020] (3) Integrating pan-genome structural variations to enhance the ability to detect complex variations; The gene chip of this invention integrates 142 deletion-type structural variation sites obtained based on wheat pan-genome screening (Table 7), which can make up for the problem of insufficient ability of conventional SNP chips to detect structural variations and improve the accuracy of detection of complex genetic variations.

[0021] (4) Excellent detection performance and high information acquisition efficiency, suitable for large-scale application; verified by 10 wheat varieties, the gene chip detection rate reached 98.6%, and the individual detection rate reached 97.89%; the sequencing alignment rate was 99.95%~99.97%, and the target region coverage rate was 96.21%~98.46%; the sequencing data Q20≥99.25%, Q30≥97.29%, and the SNP transition / transversion ratio (ts / tv) was 2.612, indicating reliable data quality that can meet the needs of large-scale detection of a large number of samples. In addition, this invention uses liquid-phase capture sequencing technology, which can simultaneously obtain variation information of flanking sequences in the target region while achieving accurate detection of the target site, and the information acquisition efficiency is better than that of solid-phase chips.

[0022] (5) Supports early prediction and breeding decision-making, significantly improving breeding efficiency; Based on 448 resequencing wheat core germplasm populations and phenotypic data, this invention constructs a "phenotype-effect value" association model, which can predict low cadmium traits and comprehensive breeding value at the seed or seedling stage, improve breeding selection efficiency, and promote the transformation of low cadmium wheat breeding towards precision and intelligence. Attached Figure Description

[0023] Figure 1 This is a distribution map of the genetic marker sites of the liquid-phase gene chip of the present invention on the 21 chromosomes of wheat; Figure 2Density distribution of InDel variants in the wheat genome for 10 validation samples; Figure 3 Density distribution of SNP variants in the wheat genome for 10 validation samples; Figure 4 This is a scatter plot of the linear fit between the model's predicted values ​​and the actual values; where the horizontal axis represents the actual values ​​and the vertical axis represents the predicted values; RMSE is the root mean square error, PCC is the Pearson correlation coefficient, y = ax + b represents the fitted linear equation, and R² is the coefficient of determination. Figure 4 (A) in the figure is a scatter plot of linear fit predicted by the GBLUP method for the validation population in the TG environment; Figure 4 (B) in the figure is a scatter plot of the linear fit predicted by the GBLUP method in the JY environment for the validation population. Figure 4 (C) in the figure is a scatter plot of the linear fit predicted by the GBLUP method for the validation population in the BLUE environment; Figure 4 (D) in the figure is a scatter plot of linear fit predicted by the GBLUP+219 SNP matrix method for the validation population in the TG environment; Figure 4 (E) in the figure is a scatter plot of linear fit predicted by the validation population using the GBLUP+219 SNP matrix method in the JY environment; Figure 4 In the diagram, (F) is a scatter plot of linear fit predicted by the validation population using the GBLUP+219 SNP matrix method in the BLUE environment. Detailed Implementation

[0024] The abbreviations used in this application are as follows: SNP (Single Nucleotide Polymorphism); InDel (Insertion / Deletion); GWAS (Genome-Wide Association Study); Nramp (Natural Resistance-Associated Macrophage Protein); HMA (Heavy Metal ATPase); ABC (ATP-Binding Cassette Transporter); ZIP (Zinc-regulated transporter, Iron-regulated transporter-like Protein); MTP (Metal Tolerance Protein); YSL (Yellow Stripe 1-Like); CAX (Cation Exchanger); PCR (Plant Cadmium Resistance); DNA (Deoxyribonucleic Acid); GBLUP (Genomic Best Linear Unbiased) Prediction: Optimal linear unbiased prediction model; GEBV (Genomic Estimated Breeding Value); MLM (Mixed Linear Model); As: Arsenic; Hg: Mercury; Pb: Lead; Cu: Copper; Ni: Nickel; PCC (Pearson correlation coefficient); R² (coefficient of determination); RMSE (Root Mean Square Error).

[0025] The first aspect of this invention proposes a gene chip, including a detection reagent for specifically detecting a set of cadmium-related SNP loci, which includes the SNP loci set shown in Table 2. This invention uses grain cadmium content as the target trait and employs a mixed linear model (MLM) to conduct genome-wide association analysis. False positive associations are controlled through population structure and kinship matrices, and cadmium-related SNP loci reaching a significant threshold are screened. In the significant association results, the representative loci have a p-value of 10.-5 The explanatory power of a single site phenotype is approximately 5%–6%. After significant screening, LD block organization, and redundant site removal, the aforementioned set of SNP sites is finally obtained. In one implementation, the detection reagent is a combination of point-capture probes for detecting SNP sites.

[0026] In one embodiment, the cadmium-related SNP sites also include a set of cadmium-related SNP sites of homologous functional genes as shown in Table 3. In one embodiment, the detection reagent is a set of point-capture probes for detecting SNP sites. Based on GWAS screening of significantly associated sites, this invention further identifies homologous genes in the wheat reference genome based on reported genes related to cadmium absorption, transport, accumulation, isolation, and detoxification, extracting SNP variant sites in gene coding regions and upstream and downstream regulatory regions to obtain homologous functional gene SNP sites.

[0027] In one implementation, the gene chip further includes a detection reagent that specifically detects at least one set of the following SNP sites: (Group 1) The set of SNP sites related to other metal ions, as shown in Table 4 of the specification; (Group 2) The set of SNP sites associated with the Nramp, HMA, ABC, ZIP, PCR, MTP, YSL, and CAX gene families, as shown in Table 5 of the instruction manual; (Group 3) The set of SNP sites for whole-genome background filling, as shown in Table 6 of the instruction manual.

[0028] In this embodiment of the invention, 514 SNP sites related to other metal ions were screened. These other metal ions include at least As ions, Hg ions, Pb ions, Cu ions, and Ni ions, as detailed in Table 4.

[0029] In this embodiment of the invention, members of gene families related to cadmium and heavy metal transport, such as Nramp, HMA, ABC, ZIP, PCR, MTP, YSL, and CAX, in the wheat genome were systematically identified. SNP variant sites in their coding and regulatory regions were extracted, and a total of 5797 key gene family SNP sites were obtained, as shown in Table 5.

[0030] In this embodiment of the invention, 3968 SNP sites were screened to fill the background of the whole genome, as shown in Table 6, thereby ensuring that the gene chip uniformly covers the whole genome of wheat subgenomes A, B, and D.

[0031] In one implementation, the detection reagent is a set of point-capture probes for detecting SNP sites. In actual detection and analysis, those skilled in the art can select detection reagents for different SNP site sets according to the purpose of the detection.

[0032] To ensure probe stability, probes were designed using existing technology principles. For each SNP site, flanking sequences (e.g., 100-200 bp) were extracted from the upstream and downstream sides of the SNP site on the reference genome. Probes containing the target variant site were designed, with one probe for each SNP site and one probe for each strand, ensuring precise capture of the target variant site. The reference genome used was the wheat reference genome IWGSC RefSeq v2.1.

[0033] In one specific implementation, the above-mentioned set of cadmium-related SNP sites (Table 2), the set of cadmium-related SNP sites of homologous functional genes (Table 3), the set of 514 SNP sites related to other metal ions (Table 4), the set of SNP sites related to the Nramp, HMA, ABC, ZIP, and PCR gene families (Table 5), and the set of SNP sites used for whole-genome background filling (Table 6) are incorporated into the chip.

[0034] A second aspect of this invention provides a gene chip including detection reagents for wheat deletion-type structural variant sites, the set of which is shown in Table 7. Based on wheat pan-genome data, this invention identifies structural variations among different wheat materials, focusing on screening deletion-type structural variant sites associated with cadmium accumulation and important agronomic traits. After rigorous quality control, 142 key deletion-type structural variant sites were obtained. In one embodiment, the detection reagent is a cross-breakpoint capture probe combination. To ensure probe stability, the probes are designed using existing technology principles, and the detection reagent is a cross-breakpoint capture probe combination, with one forward and one reverse strand probe designed for each deletion-type structural variant site.

[0035] The third aspect of this invention proposes a liquid-phase gene chip for identifying and breeding wheat with low cadmium levels and comprehensive agronomic traits. The above-mentioned cadmium-related SNP loci set (Table 2), the homologous functional gene cadmium-related SNP loci set (Table 3), the 514 SNP loci set related to other metal ions (Table 4), the SNP loci set related to the Nramp, HMA, ABC, ZIP, and PCR gene families (Table 5), the SNP loci set used for whole-genome background filling (Table 6), and the wheat deletion-type structural variation loci set (Table 7) are all included in the gene chip. Furthermore, a liquid-phase gene chip is preferred to obtain a liquid-phase gene chip for identifying and breeding wheat with low cadmium levels and comprehensive agronomic traits.

[0036] All probes were designed based on the wheat reference genome IWGSC RefSeq v2.1 and followed the following quality control standards: probe sequence length 110bp~150bp, GC content 40%~69%, theoretical melting temperature not higher than 64℃, no nitrogen bases in the sequence, sequence complexity not less than 0.9, maximum homopolymer length not exceeding 6, and removal of non-specific probes and repetitive sequence probes from multiple alignments on the reference genome. All probes were biotin-tagged at the 5' end for liquid-phase hybridization capture.

[0037] The fourth aspect of this invention provides a kit for identifying and breeding wheat with low cadmium levels and comprehensive agronomic traits, including the aforementioned gene chip. In addition to the core modules described above, the kit may also include an amplification / labeling pre-module, a blocking / anti-non-specific adsorption system, a magnetic bead / purification module, built-in quality control (QC) materials, consumables, and instructions.

[0038] The fifth aspect of this invention proposes an application of SNP locus combinations related to wheat breeding in the identification and breeding of wheat with low cadmium levels and comprehensive agronomic traits, including one or more sets of the following SNP locus sets: As shown in Table 2 of the instruction manual, the set of cadmium-related SNP sites; As shown in Table 3 of the instruction manual, the set of cadmium-related SNP sites of homologous functional genes; As shown in Table 4 of the instruction manual, the set of SNP sites related to other metal ions; The set of SNP sites associated with the Nramp, HMA, ABC, ZIP, PCR, MTP, YSL, and CAX gene families is shown in Table 5 of the instruction manual. The set of SNP sites for whole-genome background filling is shown in Table 6 of the instruction manual; The set of wheat deletion-type structural variation sites is shown in Table 7 of the instruction manual.

[0039] A sixth aspect of this invention provides a method for obtaining wheat breeding information using the aforementioned gene chip or reagent kit, comprising at least the following steps: S1. Extract genomic DNA from the wheat sample to be tested; S2. Construct a library from genomic DNA to obtain a sequencing library; S3. Use the above-mentioned gene chip to perform liquid-phase hybridization capture of the target site on the sequencing library to obtain the capture product; S4. Perform high-throughput sequencing on the captured products to obtain sequencing data; S5. Perform data analysis on the sequencing data to obtain breeding information for the wheat samples to be tested.

[0040] On the other hand, the present invention also provides a method for predicting the breeding value of the low cadmium trait in wheat by estimating its genomic value, which, based on the above detection method, further includes: S6. Construct a prediction model based on the genotype data and grain cadmium content data of the resequencing wheat core germplasm population. The prediction model includes at least one of the following: linear mixture model, genome best linear unbiased prediction model (GBLUP), Bayesian model, machine learning model, or multi-trait selection index model. S7. Based on breeding information, use a prediction model to predict the genomic estimated breeding value (GEBV) of the cadmium trait in the wheat material to be tested.

[0041] The seventh aspect of this invention provides an application of the gene chip or reagent kit as described above, including at least one of the following uses: Screening of low-cadmium wheat germplasm; directional breeding of low-cadmium wheat varieties; molecular marker-assisted selection of low-cadmium wheat; whole-genome selection breeding of wheat.

[0042] Example 1: Screening of microarray genetic marker sites

[0043] 1. Sample and Data Sources

[0044] Based on the 448 core wheat germplasm materials shown in Table 1, whole-genome resequencing was performed on all materials. The sequencing data were compared with the wheat reference genome IWGSC RefSeq v2.1, and after quality control and filtering, a high-quality SNP variation dataset was obtained. At the same time, grain cadmium content data and phenotypic data of key agronomic traits such as seedling traits, spike length, plant height, and thousand-grain weight were collected for the above materials.

[0045] Table 1: Source information of 448 core germplasm populations

[0046]

[0047]

[0048]

[0049] 2. SNP marker site screening

[0050] 2.1 Screening of cadmium ion-related SNP sites

[0051] GWAS-associated loci screening: Using grain cadmium content as the target trait, genome-wide association analysis was performed using a mixed linear model (MLM), and loci were screened based on a significant association threshold (p < 1 × 10⁻⁶). -5The SNP sites were identified, and 924 GWAS-significantly associated sites were obtained. The chromosome number, physical location, reference base and variant base information of the sites are detailed in Table 2.

[0052] Table 2: GWAS screening information on cadmium-related sites (924 sites in total)

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] 2.2 Screening of homologous functional gene loci

[0060] Based on the reported genes related to cadmium absorption, transport, accumulation, isolation, and detoxification, homologous genes were identified in the wheat reference genome IWGSCRefSeq v2.1. SNP sites were extracted from the gene coding regions and upstream and downstream regulatory regions, resulting in 1353 homologous functional gene SNP sites. Detailed information on the SNP sites is shown in Table 3.

[0061] Table 3: Information on cadmium-related SNP sites in homologous functional genes (total 1353)

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] After merging the above GWAS-associated sites with homologous functional gene sites to remove redundancy, a total of 2277 cadmium ion-related SNP sites were obtained.

[0072] 2.3 Screening of SNP sites related to other metal ions

[0073] Based on the reported genes related to the absorption, transport, and tolerance of metal ions such as arsenic (As), mercury (Hg), lead (Pb), copper (Cu), and nickel (Ni), homologous genes were identified in the wheat reference genome, and relevant SNP variant sites were extracted. A total of 514 other metal ion-related SNP sites were obtained. The specific information of the sites is detailed in Table 4.

[0074] Table 4: Information on SNP sites related to other metal ions (514 in total)

[0075]

[0076]

[0077]

[0078] 2.4 Screening of key gene family SNP sites

[0079] The gene families Nramp (natural resistance-associated macrophage protein), HMA (heavy metal ATPase), ABC (ATP-binding cassette transporter), ZIP (zinc / iron regulatory transporter), MTP, YSL, CAX, and PCR (plant cadmium resistance protein) were systematically identified. SNP variant sites were extracted from the coding and regulatory regions of these gene family members, resulting in a total of 5797 key gene family SNP sites, as shown in Table 5.

[0080] Table 5: SNP information for key gene families (5797 in total)

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] 2.5 Genome-wide background filling of SNP sites

[0101] To ensure uniform coverage of the wheat A, B, and D subgenomes by the gene chip, high-quality SNP loci were screened uniformly according to their chromosomal physical locations based on variation data from 448 core germplasm populations, resulting in the addition of 3968 genome-wide background-filling SNP loci. (See Table 6.)

[0102] Table 6: SNP loci information for genome-wide background filling (3968 sites)

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] After merging the above four types of SNP sites, a total of 12,556 SNP variant sites were included.

[0121] Example 2: Screening of pan-genome deletion-type structural variant sites

[0122] Based on wheat pangenome data, structural variations existing among different wheat materials were identified. Deletion-type structural variation sites related to cadmium accumulation and important agronomic traits were screened. After strict quality control, 142 key deletion-type structural variation sites were obtained. The specific information of the sites is shown in Table 7.

[0123] Table 7: Information on pan-genome deletion-type structural variant sites (142 in total)

[0124]

[0125] Example 3: Construction of Liquid-Phase Gene Chips

[0126] 1. Integration and Optimization

[0127] The SNP sites (12,556) obtained in Example 1 and the structural variation sites (142) obtained in Example 2 were integrated. After redundancy removal and uniformity optimization, the gene chip was ensured to uniformly cover the entire wheat genome (subgenomes A, B, and D). The distribution map of the genetic marker sites of the liquid-phase gene chip of this invention on the 21 chromosomes of wheat is shown below. Figure 1 As shown.

[0128] 2. Probe Design

[0129] All probes were designed based on the wheat reference genome IWGSC RefSeq v2.1 and followed the following unified quality control standards: Probe sequence length: 110bp~150bp; GC content: 40%~69%; Theoretical melting temperature (Tm): ≤64℃; No nitrogen bases in the probe sequence; Sequence complexity: ≥0.9; Maximum homopolymer length: ≤6; Non-specific probes with multiple alignments on the reference genome are removed; Probes containing repetitive sequences are removed; The following probe design strategies are adopted for different characteristics: SNP point capture probes: Centered on each SNP site, extract its upstream and downstream flanking sequences on the reference genome, and design point capture probes containing the target variant site. One forward and one reverse probe are designed for each SNP site to ensure accurate capture of the target variant site.

[0130] Cross-breakpoint capture probes: Targeting 142 deletion-type structural variant sites, cross-breakpoint capture probes are designed based on variant breakpoint sequences. The probe sequence spans both sides of the deletion breakpoint and can specifically capture DNA fragments containing deletion variants for the detection and typing of deletion-type structural variants.

[0131] All probes have a biotin-tagged 5' end for liquid-phase hybridization capture.

[0132] 3. Gene chip synthesis

[0133] The probe pool is synthesized according to the above design parameters, which is the liquid phase gene chip.

[0134] 4. Gene chip testing process

[0135] 4.1 Genomic DNA Extraction

[0136] Genomic DNA was extracted from wheat leaves or seeds using the CTAB method or a commercial plant genomic DNA extraction kit. After extraction, DNA concentration (≥20 ng / μL), purity (OD260 / 280 1.8–2.0, OD260 / 230 ≥1.5), and integrity (clear main band and no obvious degradation detected by agarose gel electrophoresis) were assessed.

[0137] 4.2 Library Construction

[0138] Take an appropriate amount of genomic DNA, fragment it, repair the ends, add an A tail, ligate the sequencing adapter, amplify it by PCR, and then perform library quality control to obtain a qualified sequencing library.

[0139] 4.3 Liquid-phase hybridization capture

[0140] The sequencing library was hybridized with a liquid-phase gene chip probe pool. Biotin-labeled probe-library hybridization complexes were captured by streptavidin magnetic beads, non-specific binding fragments were eluted, and the captured products were amplified by PCR to obtain an enriched library.

[0141] 4.4 High-throughput sequencing

[0142] High-throughput sequencing was performed on the enriched library to obtain raw sequencing data (Raw reads).

[0143] 4.5 Data Quality Control

[0144] The raw data was filtered using FASP software to remove low-quality reads, connector sequences, and reads with excessively high N content, resulting in high-quality clean reads. Quality control standards: Q20 ≥ 95%, Q30 ≥ 85%.

[0145] 4.6 Reference Genome Alignment and Variation Detection

[0146] Clean reads were aligned to the wheat reference genome IWGSC RefSeq v2.1 using BWA software, SNP / InDel detection was performed using GATK software, deletion structural variant detection was performed using the corresponding software, and a high-quality variant set was obtained after quality control filtering. The genotype data matrix of the sample to be tested was then output.

[0147] It should be noted that liquid-phase gene chips are based on the principle of targeted capture sequencing. The probe captures the DNA fragment (usually 200bp~500bp) containing the target site. After sequencing, the entire captured fragment is analyzed for mutations. Therefore, the actual detected mutation sites include the chip's target site and the mutations in the adjacent regions covered by the captured fragment. The total number of detected mutations is usually greater than the number of sites designed for the chip. Gene chip performance is evaluated based on the detection rate of the target site. The additional mutation information detected in the adjacent regions can be used as supplementary genetic information.

[0148] Example 4: Performance Verification of Gene Chips

[0149] 1. Validation Sample

[0150] Ten wheat varieties (numbered D_27, D_34, D_41, D_50, D_161, D_174, D_259, D_273, D_281, and D_JM22) were selected as validation samples, and the gene chip of Example 3 was analyzed according to the method of Example 3. The comparative statistical chart is shown below. Figure 2 As shown.

[0151] 2. Sequencing data quality

[0152] The statistical results of the sequencing data quality of the 10 validation samples are shown in Table 8.

[0153] Table 8: Statistical Analysis of Sequencing Data Quality for Validation Samples

[0154] 3. Comparative statistics

[0155] The statistical results of the reference genome alignment for the 10 validation samples are shown in Table 9.

[0156] Table 9: Statistical Analysis of Verification Samples

[0157] The alignment rate of all samples was 99.95%–99.97%, the coverage rate of the target region was 96.21%–98.46%, and the average sequencing depth was 30×–96×.

[0158] 4. Gene chip detection rate

[0159] According to statistics, the gene chip in Example 3 had a chip detection rate of 98.6% and an individual detection rate of 97.89% for 10 verification samples, indicating that the gene chip has excellent detection performance and can meet the needs of large-scale application.

[0160] 5. Variation detection quality

[0161] Because liquid-phase gene chips are based on the principle of targeted capture sequencing, the actual detected variant sites include variants in the flanking regions covered by the chip's target sites and its captured fragments. The total number of detected variants is greater than the number of sites designed for the chip. Chip performance is evaluated based on the target site detection rate (Example 3). A total of 5,476,149 SNP variants were detected in 10 validation samples, with an SNP transition / transversion ratio (ts / tv) of 2.612, within the normal range (2.0~3.0), indicating that the SNP detection results are reliable. A total of 422,018 InDel variants were detected, including 236,394 deletions and 185,624 insertions. The density distribution of SNPs and InDel variants on the wheat genome is shown in the figure below. Figure 3 As shown.

[0162] Example 5: Application of gene chips in low-cadmium molecular breeding of wheat

[0163] 1. Screening of low-cadmium germplasm

[0164] The liquid-phase gene chip of Example 3 was used to detect the wheat breeding materials to be tested. After obtaining the genotype data, the genotype information of 2277 cadmium ion-related SNP loci was extracted. Based on the known low cadmium alleles, the low cadmium potential of the materials to be tested was preliminarily assessed, and excellent germplasm materials carrying low cadmium alleles were screened.

[0165] 2. GEBV Prediction

[0166] A phenotypic-effect value association model was constructed based on 448 core germplasm populations. Genotypic data obtained from gene chip detection were used to calculate the GEBV value of the tested materials, predicting the breeding value of low cadmium traits and comprehensive agronomic traits. A scatter plot of the linear fit between the model's predicted values ​​and the actual values ​​is shown below. Figure 4 As shown in Table 10.

[0167] Table 10: Predictive power of the Genomic Best Linear Unbiased Prediction (GBLUP) model on two datasets

[0168] 3. Synergistic selection of integrated agronomic traits

[0169] Based on the genotypic analysis of low-cadmium loci, genotypic information of loci related to yield, development, disease resistance, stress resistance, and quality was extracted simultaneously. A comprehensive selection index was constructed to screen wheat materials that have both low cadmium accumulation potential and excellent comprehensive agronomic traits, thereby achieving synergistic improvement of low cadmium and comprehensive agronomic traits.

[0170] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A gene chip, characterized in that, The invention includes a detection reagent for specifically detecting a set of cadmium-related SNP sites, the set of cadmium-related SNP sites including the SNP site set shown in Table 2 of the specification; the detection reagent is a combination of point-capture probes for specifically detecting SNP sites.

2. The gene chip according to claim 1, characterized in that, The cadmium-related SNP sites also include a set of cadmium-related SNP sites of homologous functional genes as shown in Table 3 of the specification; the detection reagent is a combination of point-capture probes for specific detection of SNP sites.

3. The gene chip according to claim 1, characterized in that, The gene chip also includes a detection reagent that specifically detects at least one of the following sets of SNP sites: Group 1: The set of SNP sites associated with other metal ions, as shown in Table 4 of the instruction manual; Group 2: The set of SNP sites associated with the Nramp, HMA, ABC, ZIP, PCR, MTP, YSL, and CAX gene families, as shown in Table 5 of the instruction manual. Group 3: The set of SNP sites used for genome-wide background filling, as shown in Table 6 of the instruction manual; The detection reagent is a combination of point-capture probes used for the specific detection of SNP sites.

4. A gene chip, characterized in that, The invention includes a detection reagent specifically designed for detecting a set of wheat deletion-type structural variant sites; the set of wheat deletion-type structural variant sites is shown in Table 7 of the specification. The detection reagent is a cross-breakpoint capture probe combination, with one forward and one reverse probe designed for each deletion-type structural variation site.

5. A liquid-phase gene chip for identifying and breeding wheat with low cadmium levels and comprehensive agronomic traits, comprising the gene chip as described in any one of claims 1 to 4.

6. A kit for identifying and breeding wheat with low cadmium levels and comprehensive agronomic traits, characterized in that, The kit contains the gene chip as described in any one of claims 1 to 5.

7. The application of a combination of SNP loci related to wheat breeding in the identification and breeding of wheat with low cadmium levels and comprehensive agronomic traits, characterized in that, The SNP locus combination includes one or more sets of the following SNP locus sets: As shown in Table 2 of the instruction manual, the set of cadmium-related SNP sites; As shown in Table 3 of the instruction manual, the set of cadmium-related SNP sites of homologous functional genes; As shown in Table 4 of the specification, the set of SNP sites related to other metal ions; The set of SNP sites associated with the Nramp, HMA, ABC, ZIP, PCR, MTP, YSL, and CAX gene families is shown in Table 5 of the instruction manual. The set of SNP sites for whole-genome background filling is shown in Table 6 of the instruction manual; The set of wheat deletion-type structural variation sites is shown in Table 7 of the instruction manual.

8. A method for obtaining wheat breeding information using a gene chip as described in any one of claims 1 to 5 or a reagent kit as described in claim 6, characterized in that, Includes the following steps: S1. Extract genomic DNA from the wheat sample to be tested; S2. Construct a library from the genomic DNA to obtain a sequencing library; S3. Using the chip, the sequencing library is used to capture the target site by liquid-phase hybridization to obtain the capture product; S4. Perform high-throughput sequencing on the captured product to obtain sequencing data; S5. Perform data analysis on the sequencing data to obtain the breeding information of the wheat sample to be tested.

9. A method for predicting the genomic estimation breeding value of the low cadmium trait in wheat using the gene chip as described in any one of claims 1 to 5 or the kit as described in claim 6, characterized in that, Includes the following steps: S1. Extract genomic DNA from the wheat sample to be tested; S2. Construct a library from the genomic DNA to obtain a sequencing library; S3. Using the gene chip, the sequencing library is used to capture the target site by liquid-phase hybridization to obtain the capture product; S4. Perform high-throughput sequencing on the captured product to obtain sequencing data; S5. Perform data analysis on the sequencing data to obtain the breeding information of the wheat sample to be tested; S6. Construct a prediction model based on the genotype data and grain cadmium content data of the resequencing wheat core germplasm population. The prediction model includes at least one of the following: linear mixture model, genome-optimal linear unbiased prediction model, Bayesian model, machine learning model, or multi-trait selection index model. S7. Based on the breeding information, use the prediction model to predict the genomic estimated breeding value of the cadmium trait of the wheat material to be tested.

10. The application of a gene chip as described in any one of claims 1 to 4, or a liquid-phase gene chip as described in claim 5, or a kit as described in claim 6, characterized in that, Including at least one of the following uses: Screening of low-cadmium wheat germplasm; Targeted breeding of low-cadmium wheat varieties; Low-cadmium molecular marker-assisted selection in wheat; Risk assessment of heavy metal accumulation in wheat.

Images