Variation site combination related to salt tolerance trait of wheat and detection product and application thereof

Abstract

A variation site combination related to salt tolerance of wheat, and detection products and applications thereof, belong to the field of molecular genetic breeding technology, the variation site combination is composed of 94 SNP sites and 1 Indel site, the physical position of each variation site is determined based on sequence alignment of wheat reference genome IWGSC RefSeq v.2.1, the Indel is located in the region of 540783807 to 540783852 of 5D chromosome, and is marked as 5D-Indel. The application also provides a nucleotide probe combination and a liquid gene chip developed based on the variation site combination. By using the above variation site combination and detection products, the salt tolerance related genotype of wheat germplasm resources and hybrid offspring can be rapidly screened in early breeding, the accurate identification and directional polymerization of salt tolerance excellent allelic variation can be realized, the dependence on field saline phenotype identification is reduced, the breeding cycle is shortened, and the detection cost and selection efficiency are considered.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetic breeding technology, specifically relating to the field of crop salt tolerance molecular breeding, and particularly to a combination of variant sites related to wheat salt tolerance traits, its detection products, and applications. Background Technology

[0002] Wheat is a vital global food crop, and its production security continues to be severely threatened by soil salinization. Due to the large and highly complex genome of wheat, the genetic analysis and molecular breeding progress of its salt tolerance traits have long lagged behind other major crops such as rice and maize. Currently, in wheat salt tolerance breeding practices, the selection of superior salt-tolerant individual plants mainly relies on traditional phenotypic identification methods, which involve planting breeding materials in saline-alkali environments and evaluating individual salt tolerance through visual inspection or measurement of indicators such as plant height and yield components. This method is not only time-consuming and inefficient, but also highly dependent on the stability of environmental conditions. Saline-alkali land is characterized by uneven salt distribution; even within the same field, soil salinity exhibits significant spatial variability. This leads to poor repeatability and reduced reliability of phenotypic identification results, severely limiting the accuracy of breeding selection.

[0003] Salt tolerance in wheat is a complex quantitative trait controlled by multiple genes and regulated by numerous minor genes and gene-gene interaction networks. Assessing a single or a few indicators is insufficient to reflect the overall salt tolerance level of a material. In recent years, with the rapid development of single nucleotide polymorphism (SNP) marker technology, it has become possible to genetically improve complex traits such as salt tolerance using marker-assisted selection. By statistically associating genomic-level variant sites with salt tolerance phenotypes, the salt tolerance potential of materials can be predicted early in the breeding process based on genotype information, thus bypassing the interference of environmental factors on phenotypic identification and significantly improving selection efficiency. However, the reported molecular markers related to wheat salt tolerance are numerous and scattered, often facing problems such as insufficient detection throughput, high costs, or unstable marker effects in practical breeding applications. There is a lack of a systematically validated, concise, and multi-dimensional combination of dedicated variant sites.

[0004] Based on the above situation, there is an urgent need to provide a technical solution that balances detection cost and selection efficiency. This solution should be based on a standardized combination of wheat salt tolerance-related variant sites, combined with corresponding detection products, to achieve rapid screening of salt-tolerant germplasm resources and accurate evaluation of breeding parent materials. This will promote the transformation of wheat salt tolerance molecular breeding from empirical selection based on phenotype to genotype-based targeted selection. Summary of the Invention

[0005] Technical Problem Solved: To address the problems of long cycles, low efficiency, and susceptibility to uneven salinity in saline-alkali soils in traditional phenotypic identification methods for wheat salt tolerance breeding, as well as the redundancy, high detection costs, and lack of systematic validation of existing molecular markers, which hinder their practical application in breeding, this invention provides a combination of variant sites related to wheat salt tolerance traits, along with its detection products and applications. This enables early and rapid screening of salt-tolerant germplasm resources and accurate genotypic evaluation of breeding materials through a small number of systematically validated variant site combinations, thereby reducing detection costs while improving the efficiency and accuracy of molecular marker-assisted selection.

[0006] Technical solution: A combination of variant sites associated with wheat salt tolerance, consisting of 94 SNP sites and 1 Indel site. The physical locations of the 95 variant sites were determined based on wheat reference genome IWGSC RefSeq v.2.1 sequence alignment. The chromosomal location and allelic variant type of each variant site are as follows:

[0007] .

[0008] The aforementioned 95 variation sites are associated with at least two of the physiological, yield, and developmental traits of wheat salt tolerance.

[0009] The physiological traits mentioned above are selected from at least two of the aboveground sodium ion content, aboveground potassium ion content, sodium-potassium ratio, salt tolerance index, and total chlorophyll content; the yield traits are selected from at least two of the spike length, thousand-grain weight, number of grains per spike, and grain size; the developmental traits are selected from at least two of the plant height, root length, stem length, root-shoot ratio, and biomass.

[0010] A combination of nucleotide probes for detecting the above-mentioned combinations of variant sites.

[0011] A liquid-phase gene chip comprising the above-mentioned combination of nucleotide probes.

[0012] A method for detecting variant sites related to salt tolerance traits in wheat includes the following steps: Step 1, preparing a DNA library of the wheat sample to be tested; Step 2, using the nucleotide probe combination described in claim 4 or the liquid-phase gene chip described in claim 5 to perform targeted hybridization capture of the DNA library; Step 3, amplifying and sequencing the captured products; Step 4, comparing the sequencing data with the wheat reference genome IWGSC RefSeq v.2.1 to identify the allelic variant types of the 95 variant sites described in claim 1.

[0013] The application of the above-mentioned combination of variant sites, the nucleotide probe combination, or the liquid-phase gene chip in wheat salt-tolerant breeding.

[0014] The above applications include using the combination of mutation sites, the combination of nucleotide probes, or the liquid-phase gene chip to identify salt-tolerant allelic variations in breeding parent materials, and selecting parent materials carrying the target dominant allelic variations to formulate hybridization combinations based on the identification results.

[0015] The above applications include using the combination of the mutation sites, the combination of the nucleotide probes, or the liquid-phase gene chip to perform early genotyping of hybrid offspring populations, and screening individual plants with salt-tolerant target allelic variations based on the detection results, which are then retained for subsequent field identification.

[0016] Beneficial Effects: The combination of variant loci provided in this invention is based on GWAS analysis of the field phenotypes of 498 wheat varieties in saline-alkali land by our team, combined with macro-QTL analysis of reported wheat salt tolerance QTLs. This identified 95 variant loci that are environmentally stable, control multiple phenotypes, and have high genetic effects. These loci cover 94 SNP loci and 1 Indel locus, a concise number of loci, and each locus is significantly associated with at least two salt tolerance-related traits. These loci are distributed across multiple wheat chromosomes, enabling multi-dimensional evaluation of salt tolerance from physiological levels such as aboveground sodium and potassium ion content, sodium-potassium ratio, salt tolerance index, and chlorophyll content, as well as developmental and yield levels such as plant height, root length, thousand-grain weight, number of grains per spike, and grain size. Different variant loci are associated with different physiological mechanisms, developmental stages, and organ sites related to salt tolerance, and their effects are often weak when present individually. The combination of 95 variant loci makes the prediction accuracy of the comprehensive genotype far superior to that of single or a few loci, thus avoiding the omission of superior germplasm selection due to the assessment bias of a single minor locus.

[0017] In terms of detection products, the nucleotide probe combinatorial and liquid-phase gene chip developed based on this variant site combination utilizes GBTS targeted sequencing genotyping technology. The platform boasts broad adaptability, outstanding labeling flexibility, and high detection efficiency. The small and concentrated number of sites reduces the cost of probe synthesis and hybridization capture, resulting in significantly lower single-sample detection costs compared to high-density chips, making it suitable for gene screening in large-scale breeding populations. Furthermore, the sequence information is additive, allowing for flexible addition or removal of detection sites as needed for breeding without requiring a completely new chip, providing convenient support.

[0018] In breeding applications, when using this locus combination and detection products for parental selection, the allelic variation composition of different germplasm resources at salt tolerance-related loci can be quickly identified, guiding the aggregation of dominant alleles. In the screening of hybrid offspring populations, materials with poor salt tolerance potential can be eliminated early on based on genotype, retaining only individual plants with high aggregation at target loci for subsequent field identification. This significantly reduces the population size for field phenotypic identification, decreases reliance on uniformity of saline-alkali soil environmental conditions, and overall shortens the breeding cycle and improves selection efficiency. Attached Figure Description

[0019] Figure 1 A step-by-step diagram was obtained for 95 variant sites.

[0020] Figure 2 This is a distribution map of the mutation sites of the present invention on chromosomes.

[0021] Figure 3 This is a distribution map of the mutation sites in the gene structure of the present invention.

[0022] Figure 4 This is a yield difference graph for the first embodiment in Example 3 of the present invention.

[0023] Figure 5 This is a diagram showing the relationship between breeding materials in the second embodiment of Example 3 of the present invention.

[0024] Figure 6 Figure a is a pedigree chart of the materials in the third embodiment of the present invention; Figure b shows the excellent salt tolerance sites contained in the offspring; Figure c is the verification of the plant height phenotype in saline-alkali land. Detailed Implementation

[0025] The technical method of the present invention will be clearly and completely described below with reference to specific embodiments. The described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1:

[0027] A combination of variant loci associated with wheat salt tolerance includes 94 SNP loci and 1 Indel, each locus containing two allelic variant types, used to detect allelic changes at that locus. The physical locations of the 95 variant loci were determined by sequence alignment using the wheat reference genome IWGSC RefSeq v.2.1, which can be obtained from https: / / wheatomics.sdau.edu.cn / . The variant information of the SNP loci is represented in the form of chromosome_physical location reference genotype / variant allelic genotype. The variant information of the 95 loci is shown below:

[0028]

[0029] Methods for obtaining combinations of 95 variant sites associated with wheat salt tolerance traits:

[0030] Step 1: Collection of information on variant sites:

[0031] Based on the GWAS analysis of the field phenotypes of 498 wheat varieties in saline-alkali land by our team, and combined with the reported wheat salt tolerance QTLs, we carried out macro-QTL analysis, summarized the variant sites and gene information related to wheat salt tolerance traits, formed a dataset, and collected a total of 1591 QTLs.

[0032] Step 2: Use a supercomputing platform to integrate and statistically analyze the variant sites:

[0033] The computation process was developed based on the supercomputing cluster. The whole genome segmentation computation task was submitted through the Slurm job management system. LD Blocks (linked disequilibrium blocks) were divided. With the help of R language, the collected and organized QTLs were anchored to the reference genome. After integration and statistics, a total of 783 block QTLs were obtained.

[0034] Step 3: Identify hotspot regions using the frequency and contribution rate of each variant site.

[0035] We analyzed and statistically analyzed multiple closely adjacent block QTL intervals. If an interval contains multiple salt-tolerant sites and has been located in different literature, it indicates that the interval has been co-located multiple times and has a high weight. It is considered to be a wheat salt-tolerant hotspot. A total of 67 hotspot intervals were identified.

[0036] Step 4: Determination of the mutation site:

[0037] We collected marker and haplotype information for all salt-tolerant index sites within hotspot regions, analyzed them individually, and selected salt-tolerant indexes with development potential. For different hotspot regions with multiple identical salt-tolerant indexes, we selected different salt-tolerant indexes for marker development.

[0038] Step 5: Result Verification

[0039] The collected variant sites and salt tolerance traits were analyzed, and variant sites with excellent trait performance were screened out to obtain the 95 variant site combinations related to wheat salt tolerance traits described in this invention.

[0040] The salt tolerance traits include physiological traits, yield traits, and developmental traits. Physiological traits include aboveground sodium ion content, aboveground potassium ion content, sodium-potassium ratio, salt tolerance index, and total chlorophyll content. Yield traits include ear length, thousand-grain weight, number of grains per ear, and grain size. Developmental traits include plant height, root length, stem length, root-to-shoot ratio, and biomass.

[0041] Step 6: Analyze the distribution of the 95 variant sites on the chromosome. The results are shown in […]. Figure 2 The distribution of 95 loci in gene structure was analyzed, and the results are shown in [the table below]. Figure 3 .

[0042] Implementation steps are as follows Figure 1 As shown.

[0043] Example 2

[0044] Based on the obtained 95 marker sites, probes for detecting these 95 marker sites were designed to obtain probe combinations. The method for functional gene detection using the designed liquid-phase gene chip includes the following steps:

[0045] Step 1: Preparation of the sample library;

[0046] Genomic DNA was extracted from wheat leaf samples using the Qiagen DNeasy Plant Mini Kit. The concentration and purity of the extracted DNA were determined using the Qubit dsDNA HS Assay and Nanodrop 2000, with a purity A260 / A280 of approximately 1.8–2.0. The extracted DNA was sonicated to 200–400 bp, and sequencing libraries were constructed using the Agilent SureSelect XT Target Enrichment Kit. The library adapters were paired-end adapters and included index sequences to support multi-sample pooled sequencing. Library purification was performed using magnetic beads (AMPure XP), and quantification was performed using qPCR or Qubit.

[0047] Step 2, probe hybridization;

[0048] Step 2.1, Probe preparation;

[0049] Step 2.2: DNA library hybridization capture;

[0050] The library and liquid-phase chip probes were mixed according to the recommended ratio in the kit, and hybridization buffer (containing SSC, SDS and blocking agent) was added. Hybridization was carried out at 65°C for 16–24 h.

[0051] Step 2.3: The hybridization fragment is combined with the hybridization magnetic beads;

[0052] After hybridization, the DNA bound to the probe was captured using magnetic beads and washed: 3 times at 65°C with low-salt buffer (0.1×–0.5× SSC + 0.1% SDS), 5 min each time.

[0053] Step 2.4: Elute to remove unbound DNA;

[0054] Step 3: PCR enrichment;

[0055] The captured DNA was enriched by PCR using a high-fidelity DNA polymerase (such as Q5 or KAPA HiFi) under the following cycling conditions: initial denaturation at 95℃ for 3 min; 10–14 cycles: 95℃ for 30 s → 60℃ for 30 s → 72℃ for 30 s; final extension at 72℃ for 5 min; the PCR products were purified by magnetic beads and then used for sequencing.

[0056] Step 4: Sequencing;

[0057] Sequencing was performed using the Illumina NovaSeq 6000 platform.

[0058] Step 5: Compare;

[0059] Sequencing data were first quality controlled (low-quality and adapter sequences were removed), and then aligned to the wheat reference genome IWGSC RefSeq v2.1 using BWA-MEM (v0.7.17). The alignment parameters were set to -t 8 -M to retain high-quality alignments with a mapping quality ≥ 30.

[0060] Example 3

[0061] A method for molecular breeding of salt-tolerant wheat includes the following steps:

[0062] Step a: Use the liquid-phase gene chip described in Example 2 to detect germplasm resources and breeding parent materials to determine whether they contain the dominant genotypes among the 95 mutation sites.

[0063] Step b: Based on the measurement results of step a, select germplasm resources with certain dominant genotypes among the 95 variation sites as parental materials, construct hybrid combinations, and carry out wheat breeding to obtain offspring wheat samples.

[0064] Step c: Use the liquid phase gene chip described in Example 2 to perform genotyping on the offspring wheat samples obtained in step b. Based on the association between genotype and salt tolerance, determine the salt tolerance trait of the offspring samples and identify the parental material with better overall traits of the offspring samples, thereby achieving marker-assisted selection.

[0065] like Figure 4 As shown in this embodiment, genotype association analysis using 660K sequencing data from 500 natural populations revealed that the materials containing germplasm resources with a higher number of dominant genotypes exhibited significantly better yield traits than materials with fewer dominant genotypes.

[0066] like Figure 5As shown, the parental materials selected in this embodiment are Zhoumai 16 and Aikang 58. Among them, according to genotype association analysis, Zhoumai 16 has root length-related loci, and Aikang 58 has potassium ion content, plant height and thousand-grain weight-related loci. The offspring sample obtained by hybridization is Zhoumai 27. According to genotype association analysis, it has salt tolerance loci of both parents. The salt tolerance loci are as follows: 3B_745957827, 3D_550513764, 3B_762917691, 4B_24954785, 5A_443899374, 6A_76366111, 7A_57958549.

[0067] like Figure 6 As shown, the maternal parent material used in this embodiment is Ji037042 (intermediate breeding material), and the paternal parent material is Jimai 20. Genotype association analysis showed that Jimai 60 possesses salt tolerance loci related to germination rate, plant height, grain size, and thousand-grain weight that the paternal parent does not have. The loci are as follows: 3D_550513764, 2A_696085982, 4B_49803112, 4D_420378324, 5A_415498300, 5A_403875699, and 6A_54905423. In field trials on saline-alkali land, the plant height of Jimai 60 was significantly higher than that of Jimai 20, consistent with the genotype analysis results.

[0068] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A combination of variants associated with a salt tolerance trait in wheat, characterized in that, Composed of 94 SNP sites and 1 Indel site, the physical locations of the 95 variant sites were determined based on wheat reference genome IWGSC RefSeq v.2.1 sequence alignment. The chromosomal location and allelic variant type of each variant site are as follows: 。 2. The variant site combination of claim 1, wherein, The 95 variant sites are associated with at least two of the physiological, yield, and developmental traits of wheat salt tolerance.

3. The variant site combination of claim 2, wherein, The physiological traits are selected from at least two of the aboveground sodium ion content, aboveground potassium ion content, sodium-potassium ratio, salt tolerance index, and total chlorophyll content; the yield traits are selected from at least two of the spike length, thousand-grain weight, number of grains per spike, and grain size; the developmental traits are selected from at least two of the plant height, root length, stem length, root-shoot ratio, and biomass.

4. A combination of nucleotide probes for detecting the combination of variant sites as described in claim 1.

5. A liquid phase gene chip, characterized by, It comprises the nucleotide probe combination of claim 4.

6. A method of detecting a variant site associated with salt tolerance trait in wheat, characterized in that, Includes the following steps: Step 1: Prepare a DNA library of the wheat sample to be tested; Step 2: Use the nucleotide probe combination described in claim 4 or the liquid phase gene chip described in claim 5 to perform targeted hybridization capture of the DNA library; Step 3: Amplify and sequence the captured products; Step 4: Compare the sequencing data with the wheat reference genome IWGSC RefSeq v.2.1 to identify the allelic variant types of the 95 variant sites described in claim 1.

7. The application of the combination of variant sites according to claim 1, the combination of nucleotide probes according to claim 4, or the liquid phase gene chip according to claim 5 in wheat salt tolerance breeding.

8. Use according to claim 7, characterized in that, The application includes using the combination of mutation sites, the combination of nucleotide probes, or the liquid-phase gene chip to identify salt-tolerant allelic variations in breeding parent materials, and selecting parent materials carrying the target dominant allelic variations to formulate hybridization combinations based on the identification results.

9. The application according to claim 7, characterized in that, The application includes using the combination of the mutation sites, the combination of the nucleotide probes, or the liquid-phase gene chip to perform early genotyping of the hybrid offspring population, and screening individual plants with salt-tolerant target allelic variants based on the detection results, retaining them for subsequent field identification.

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