Wild wheat grain and cultivated wheat grain 4K liquid-phase gene chip and application thereof in breeding

Abstract

The invention discloses a 4K liquid-phase gene chip for wild wheat and cultivated wheat and application of the 4K liquid-phase gene chip in breeding, and belongs to the technical field of liquid-phase chips. The chip is developed based on a targeted capture sequencing technology, 4252 specific target gene sequences are screened out by comparing a wild wheat TA299 genome, a cultivated wheat PI306540 genome and a TA10622 genome with a common hexaploid wheat genome, and the specific target gene sequences comprise 70 wild specific gene sequences, 55 specific cultivation gene sequences and co-holding sites of the wild specific gene sequences and the cultivated specific gene sequences. A biotin-modified probe is designed according to the principles of 110bp length, 30-80% GC content and the like, and the chip is prepared after the probe is coupled with fluorescent microspheres. The method can accurately distinguish different wheat genomes, is uniform in coverage, can be used for distant hybridization derivative material identification, blood relationship analysis, QTL positioning, molecular assisted breeding and other scenes, realizes localization of detection, and accelerates excellent gene mining and variety improvement processes.

Description

Technical Field

[0001] This invention relates to the field of liquid phase chip technology, specifically to a 4K liquid phase gene chip for wild and cultivated wheat and its application in breeding. Background Technology

[0002] wild einkorn wheat ( T. monococcum L . ssp boeoticum , 2n=2x=14, A b A b ) is the ancestral species of cultivated wheat, cultivated wheat ( T. monococcum L . ssp monococcum , 2n=2x=14, A m A m This variety is closely related to the common wheat A genome donor, *Wheat urartu*, and both possess excellent stress resistance, capable of surviving in harsh environments such as drought and high temperatures, making them important components of wheat germplasm resources. Cultivated *Wheat urartu* also contains abundant protein and minerals, as well as high-quality subunits that improve wheat flour quality. Furthermore, both exhibit excellent disease resistance, exhibiting resistance to powdery mildew, stripe rust, and leaf rust. Currently, a powdery mildew resistance gene has been identified in wild *Wheat urartu*. Pm25 , PmTb7A.1 and PmTb7A.2 ; Stem rust resistance gene Sr22 Stripe rust resistance gene QYrtb.pau-5A and YrZ15-1370 Among the disease resistance genes already located in cultivated wheat, there is a gene for resistance to leaf rust. Lr63 Powdery mildew resistance gene Pm1b , Pm2026 and Pm4d ; Stem rust resistance gene Sr21 and Sr35 Stripe rust resistance gene Yr34, Yr48 (as later proved) Yr34 and Yr48 (It is the same disease-resistant gene) YrZ15-1949 and QYrtm.pau-2A Wild and cultivated wheat are both important foundational species and genetic resources of the wheat genus, and are of great value for improving modern wheat varieties.

[0003] Traditional breeding techniques rely on morphological screening methods, selecting superior individual plants by observing their plant type, ear type, and grain characteristics in the field. However, this method is highly susceptible to environmental influences; plants with the same genotype may exhibit different morphologies under different growing conditions, leading to low screening accuracy. Especially for breeding, it is difficult to identify the genetic characteristics of plants in early generations, resulting in long breeding cycles and low efficiency. While cytological identification methods, such as chromosome karyotype analysis and chromosome banding techniques, can obtain some genetic information at the cellular level, they are complex to operate, have low throughput, and are insufficient to meet the needs of large-scale breeding material screening.

[0004] With the development of molecular biology techniques, marker-assisted breeding (MAS) has been increasingly applied to wheat breeding. However, commonly used molecular markers, such as simple sequence repeat markers (SSR), amplified fragment length polymorphism (AFLP) markers, and restriction fragment length polymorphism (RFLP) markers, suffer from problems such as cumbersome detection procedures, high technical requirements, high costs, and low detection efficiency. Liquid-phase microarray technology, a novel high-throughput detection technology, is a tool developed based on targeted sequencing and liquid-phase capture technology, primarily used for wheat genetic research and molecular breeding. Its principle involves designing specific probes to capture target DNA fragments, combined with high-throughput sequencing to achieve simultaneous detection of multiple gene loci. It offers advantages such as flexibility, economy, and high efficiency, and has important applications in the field of marker-assisted breeding.

[0005] Most existing microarrays are based on common wheat and lack specificity for distant germplasm resources such as wild and cultivated einkorn wheat. Therefore, they cannot accurately detect and analyze these genes, hindering the full exploration and utilization of their superior genetic resources. Thus, there is an urgent need to develop a highly efficient and accurate detection tool specifically for wild and cultivated einkorn wheat to advance the breeding process.

[0006] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a liquid-phase gene chip specific to wild and cultivated Eriny wheat, which is different from ordinary hexaploid wheat. It uniformly covers the chromosome set and can be better applied to the analysis of bloodline materials from wild and cultivated Eriny wheat sources. It has high accuracy and high detection efficiency and has application prospects in QTL gene mapping and mining, molecular-assisted breeding, and variety improvement.

[0008] To achieve the above objectives, the present invention provides a 4K liquid phase gene chip for wild emmer wheat and cultivated emmer wheat, containing a probe developed from 4252 specific gene sequences, which can be used to distinguish the phylogenetic relationship between wild emmer wheat and cultivated emmer wheat and common hexaploid wheat. The information on the 4252 specific gene sequences is shown in Table 1. In each sequence information, Chr1A~7A on the left indicates the chromosome where the sequence is located, and the following two sets of values ​​are the start and end positions of the 4252 specific gene sequences in wild wheat and cultivated wheat, respectively.

[0009] Furthermore, the aforementioned specific gene sequences are the distinguishing sequences obtained by screening the genomes of wild Eriny wheat TA299, cultivated Eriny wheat PI306540 ​​and TA10622 with common hexaploid wheat China Spring v2.1, including 70 unique to wild Eriny wheat (see Table 2), 55 unique to cultivated Eriny wheat (see Table 3), and sequences shared by both.

[0010] Preferably, the probe length in the liquid-phase gene chip provided by the present invention is 110 bp, the GC content is 30%~80%, and the number of homologous regions is ≤3.

[0011] Preferably, the probe in the liquid-phase gene chip provided by the present invention is modified with a biotin group at its 5' end and coupled to fluorescent microspheres through a C12 molecular arm and amino modification.

[0012] The liquid-phase gene chip provided by this invention can be used in the analysis of wild and cultivated einkorn wheat lineage materials, including the detection of A in wheat materials. b Or A m Introgression, substitution, or translocation of chromosome segments; including wheat materials such as addition lines, substitution lines, translocation lines, or introgression lines.

[0013] The liquid-phase gene chip provided by this invention can be used in wheat QTL gene localization and mining, wheat molecular-assisted breeding, and wheat variety improvement.

[0014] The present invention has the following advantages: This invention develops a 4K liquid-phase gene chip for wild and cultivated Erinum asiaticum based on targeted capture sequencing technology, which can accurately distinguish between common hexaploid wheat AABBDD and wild Erinum asiaticum A. b A b And cultivating a grain of wheat A m A m Different sources. This invention has completely independent intellectual property rights and is a product that achieves full-process domestic production of testing equipment and reagents. Applicable to common hexaploid wheat AABBDD and A b A b And Am A m Precise molecular identification of addition lines, substitution lines, translocation lines, and introgression lines from distant hybridization, A b A b and A m A m This chip has various applications, including pedigree analysis, authenticity verification, QTL gene mapping and mining, molecular-assisted breeding, and variety improvement. It lays the foundation for the analysis of germplasm resources from both wild and cultivated *Eriocheir sinensis*, facilitating the breeding of varieties derived from these two sources. Attached Figure Description

[0015] Figure 1 The 4K liquid-phase gene chip loci provided by this invention are distributed across the entire TA299 genome.

[0016] Figure 2 The 4K liquid-phase gene chip loci provided by this invention are distributed across the entire genome in PI306540.

[0017] Figure 3 The 4K liquid phase gene chip loci provided by this invention are distributed across the entire genome at TA10622.

[0018] Figure 4 The 4K liquid-phase gene chip provided by this invention shows the coverage distribution of the infiltrated material Z15-1370 in chromosome substitution detection.

[0019] Figure 5 The image shows the FISH identification diagram of the translocation material CZZ15231-3.

[0020] Figure 6 The GISH identification diagram of the translocation system material CZZ15231-3.

[0021] Figure 7 The 4K liquid-phase gene chip provided by this invention shows the coverage distribution of the translocation material CZZ15231-3 in chromosome substitution detection. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0023] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1: Development of 4K liquid phase gene chips for wild and cultivated einkorn wheat 1. Preparation of 4K targeting and capture probes for wild and cultivated wheat grains, the process is as follows: Step 1: Screening for specific gene sequences of wild and cultivated einkorn wheat; Based on the publicly available genomes of one wild einkorn wheat (TA299) and two cultivated einkorn wheat (PI306540 ​​and TA10622) (see Ahmed HI, Heuberger M, Schoen A, et al. Einkorngenomics sheds light on history of the oldest domesticated wheat. Nature. 2023; 620:830–838; Wang X, Li H, Shen T, et al. A near-complete genome sequence of einkorn wheat provides insight into the evolution of wheat Asubgenomes. Plant Communications. 2024; 5:100768.), specific gene sequences that can distinguish between wild einkorn wheat, cultivated einkorn wheat, and common hexaploid wheat (Chinese Spring v2.1) were screened using sequence BLAST alignment. A total of 4252 target gene sequences were finally identified, and the locus information is shown in Table 1.

[0025] Table 1. Information on the selected specific sequence sites

[0026] Of the sites mentioned above, 70 have sequences unique to the wild wheat genome, and their site information is shown in Table 2 below.

[0027] Table 2. Information on unique sequence sites in the wild wheat genome.

[0028] Of the sites mentioned above, 55 have sequences unique to the cultivated wheat genome, as shown in Table 3.

[0029] Table 3. Information on unique sequence sites in the genome of cultivated wheat.

[0030] In addition to all loci listed in Tables 2 and 3 above, the sequences of all remaining loci are unique in both cultivated and wild Eriny mildew. The term "unique" refers to the presence of the sequence, either alone or simultaneously, in both wild and cultivated Eriny mildew compared to the hexaploid wheat *Wheat China Spring* v2.1 reference genome.

[0031] In this context, Chr1A~7A on the left side of the locus number indicates the chromosome where the sequence is located, and the following two sets of values ​​are the start and end positions of the 4K-specific sequence in wild and cultivated wheat, respectively.

[0032] Step 2: Design and prepare site probes, the process is as follows: ① Design principles: The probe length is designed to be 110bp, the GC content is between 30% and 80%, the number of homologous regions is ≤3, and the probes are evenly distributed on the chromosome.

[0033] ② Using the 110bp specific nucleotide sequence obtained in step 1, synthesize a single-stranded DNA nucleotide sequence with a Novochip biotin group modified at the 5' end to obtain wild wheat and cultivated wheat 4K probes.

[0034] 2. Preparation of liquid-phase gene chips: To achieve stable coupling, the 4K probes obtained above were coupled to Novochip fluorescent microspheres via C12 molecular arms and amino modification. Each fluorescent microsphere was coupled with one probe, and finally added to Novochip targeting capture reagent to obtain wild wheat and cultivated wheat 4K liquid phase gene chips.

[0035] The distribution of the corresponding loci in the liquid-phase gene chip obtained in this embodiment on the three reference genomes (TA299, PI306540, and TA10622) is shown in the figures below. Figure 1 , 2 As shown in Figure 3. Figure 1 The chip showed a 99.3% coverage of the 2Mb window in genome TA299; Figure 2 The chip showed a 98.1% coverage rate within the 1.2Mb window of genome PI306540; Figure 3 The results show that the chip achieved 97.5% coverage of the 2Mb window in the TA10622 genome, indicating good overall genome coverage with no large uncovered segments. This demonstrates that the invention designs different numbers of probes based on chromosome length, ensuring that capture sites cover the entire genome.

[0036] Application Example 1 The application method of the 4K liquid phase gene chip provided in this embodiment includes the following steps: (1) DNA extraction: Wheat DNA was extracted using the CTAB method.

[0037] (2) Construction and capture of DNA libraries: DNA samples were subjected to sonication or enzyme digestion to break down DNA, end repair and 3' end addition of "A", adapter ligation and purification, and Pre-PCR library amplification to obtain the libraries required for hybridization capture. The capture method was performed according to the Novochip® hybridization capture system guidelines of Beijing Baiyu Biotechnology Co., Ltd. After library and probe hybridization, probe binding to magnetic beads, rinsing of non-specifically bound libraries, post-capture PCR amplification, library quantification and quality control, sequencing was performed.

[0038] (3) Library quality control and quantification: Library concentration was detected using a Qubit 4.0 Fluorometer. A library with a pre-capture concentration >25 ng / L was considered a qualified library. The ABI Qsep 100 was used for detection. The main peak of the library should be around 200-500 bp, with no extraneous peaks before or after the main peak.

[0039] (4) Sequencing: The MGI T7 next-generation sequencing platform was used for 2*150bp paired-end sequencing.

[0040] (5) Data analysis: mainly includes the following steps 5.1) Raw Sequencing Data: The raw image data obtained from high-throughput sequencing (MGI platform, Nova platform, etc.) is identified and converted into raw sequencing data in FASTQ format (called raw data) by a specific program. This data is relatively large and is generally difficult to open under Windows after decompression. It needs to be opened under Linux using the less command or with other editors.

[0041] 5.2) Data Filtering and Quality Control: By setting certain filtering criteria, connector information, low-quality bases, and undetected bases (represented by N) that may interfere with subsequent information analysis are removed. The final data obtained is called cleandata or clean reads. The data filtering criteria are as follows:

[0042] ① Filter out reads containing adapter sequences; ② When the content of N in a single-end sequencing read exceeds 3% of the length of that read, remove this pair of paired reads; ③ When the number of low-quality (less than 3) bases in a single-end sequencing read exceeds 50% of the length of that read, remove the paired reads.

[0043] 5.3) Data analysis workflow: The samtools / bcftools workflow was used for variant analysis, and the filtered sequencing sequences were compared with the reference genome.

[0044] 5.4) Analysis Methods and Software Versions: Raw sequencing reads were processed using Fastap (version 0.12.4) to remove low-quality reads and adapters. BWA (version 0.7.16) mem was applied to align high-quality reads with the wild and cultivated *Erinella* genomes using default parameters. Samtools (version 1.17) was used for BAM sorting, read PCR duplicate removal, and sample alignment statistics. Genomic variants were then retrieved from the BAM using bcftools. The minimum mapping quality parameter was set to 30, used only for retrieving high-quality aligned reads of variants. bcftools (version 1.17) was used for variant quality filtering, with "QUAL>30" and "DP>5". For variant annotation, a custom database containing wild and cultivated *Erinella* HC / LC genes was created, and custom annotated genes were created using snpEff.

[0045] Application Example 2 Resource material Z15-1370 was selected and identified as a wild *Eriocheir sinensis* introgression line, as referenced in (Zhang M, Liu X, Peng T, et al. Identification of a recessive gene). YrZ15- 1370 conferring adult plant resistance to stripe rust in wheat- Triticum boeoticum introgression line[J]. Theoretical and Applied Genetics, 2021,134(9): 2891-2900.).

[0046] Following the above-described 4K liquid phase chip application method, the A in Z15-1370 was detected and tracked. b A b Regarding the introgression exchange fragments and sizes in hexaploid wheat AABBDD, it was found that the designed probes could accurately capture their respective characteristic fragments, while hybridization signals could not be detected for the corresponding hexaploid wheat AABBDD. See results below. Figure 4 As shown.

[0047] Previously, our research group identified a stripe rust resistance gene derived from wild Erinum melitensis G52 on the long arm of chromosome 6A of Z15-1370, ranging from 601.5 Mb to 603.3 Mb (see reference above). This indicates that the introgression line Z15-1370 definitely contains an introgression fragment from wild Erinum melitensis at the long arm of chromosome 6A. Liquid chromatography-mass spectrometry (LC-MS) analysis confirmed the presence of a hybridization signal at the long arm of chromosome 6A, demonstrating the accuracy and reliability of the LC-MS detection results constructed in this technical approach. (See reference above.) Figure 4 As shown.

[0048] Application Example 3 This liquid phase chip can be used to identify the chromosome composition of wild-ear wheat-derived materials, and the identification process is as follows. Specifically, the sample preparation, detection scheme, and analysis procedure for genetic composition analysis of wild-ear wheat-derived materials using this liquid phase chip are the same as in Example 3.

[0049] The resource material selected for this test is CZZ15231-3, with a pedigree of Shumai 114 / Z18-1244 / / Chuanyu 43 / 3 / SM830F5. Among them, Z18-1244 has been confirmed to be common wheat-wild wheat 4A. b / 4B substitution line. References (Liu X, Feng Z, Liang D. et al. Development, identification, and characterization of blue-grained wheat- Triticum boeoticum Substitution lines[J]. Journal of Applied Genetics, 2020; 61:167–177.). Cellular FISH was performed on the materials (results see...). Figure 5 ) and GISH (see results) Figure 6 The analysis results showed that the material was 4A. b S+4A b L·4BS translocation line. The translocation line was analyzed using the liquid-phase gene chip constructed above; the results are shown below. Figure 7 As shown, there is indeed a hybridization signal at the 4A long arm of the translocation material, proving that this liquid phase chip can effectively achieve rapid identification of the genome composition of wild wheat-derived materials.

[0050] In summary, this invention has developed a 4K liquid phase gene chip for wild and cultivated wheat based on targeted capture sequencing technology. This chip can be effectively applied to various scenarios such as genetic diversity analysis of wild and cultivated wheat, variety authenticity identification, QTL gene mapping and mining, molecular marker-assisted selection breeding, and variety improvement.

[0051] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A Triticum boeoticum and Triticum aestivum 4K liquid gene chip, characterized by, The liquid phase gene chip comprises probes developed from 4252 specific gene sequences, which can distinguish the genetic relationship between wild einkorn and cultivated einkorn and common hexaploid wheat; The 4252 specific gene sequence information is as follows: ； In the sequence information, Chr1A~7A on the left side represents the chromosome where the sequence is located, and the subsequent two groups of values are the start and end positions of the 4252 specific gene sequences in wild einkorn and cultivated einkorn, respectively.

2. The liquid phase gene chip according to claim 1, wherein The specific gene sequences are distinguishing sequences obtained by screening wild einkorn TA299, cultivated einkorn PI306540 and TA10622 and common hexaploid wheat Chinese Spring v2.1 genomes, and include 70 sequences unique to wild einkorn, 55 sequences unique to cultivated einkorn, and sequences common to both.

3. The liquid gene chip according to claim 1, wherein The probe length is 110 bp, the GC content is 30%~80%, and the number of homologous regions is ≤3.

4. The liquid phase gene chip according to claim 3, wherein The probe is modified at the 5' end with a biotin group, coupled to a fluorescent microsphere through a C12 molecular arm and an amino modification.

5. The liquid phase gene chip according to any one of claims 1~4 is applied in the analysis of wild einkorn and cultivated einkorn blood materials.

6. Use according to claim 5, characterized in that, A b or A m introgression, substitution, or translocation of a chromosome segment.

7. Use according to claim 6, characterized in that, The wheat materials include addition lines, substitution lines, translocation lines or introgression lines.

8. The liquid phase gene chip according to any one of claims 1~4 is applied in the positioning and mining of wheat QTL genes.

9. The liquid phase gene chip according to any one of claims 1~4 is applied in wheat molecular assisted breeding.

10. The liquid phase gene chip according to any one of claims 1~4 is applied in wheat variety improvement.

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