A chromosome-specific ND-FISH probe for *Leymus chinensis* from Huashan and its application and kit

Abstract

This invention relates to a chromosome-specific ND-FISH probe of *Leymus chinensis* and its applications and kits, belonging to the field of molecular genetics. The *Leymus chinensis* chromosome-specific ND-FISH probe of this invention has a nucleotide sequence of 5'-CAATGGGCCTCTACTATAGGGTGTTAGGCTCACACTAGAGAA-3', with a fluorescent label at the 5' end. This *Leymus chinensis* chromosome-specific ND-FISH probe can efficiently distinguish between wheat and *Leymus chinensis* chromosomes within four hours through non-denaturing fluorescent in situ hybridization, greatly improving the screening efficiency of wheat-*Leymus chinensis* derived progeny, reducing experimental costs, and laying the foundation for improving wheat breeding using the closely related species *Leymus chinensis*, thus broadening wheat genetic diversity.

Description

Technical Field

[0001] This invention relates to a chromosome-specific ND-FISH probe of *Leymus chinensis* from Huashan and its application and kit, belonging to the field of molecular genetics technology. Background Technology

[0002] *Psathyrostachys huashanica* Keng (2n=2x=14, NsNs) is a perennial diploid self-incompatible plant belonging to the tribe Triticeae, subtribe Hordeinae, and genus *Psathyrostachys* Nevski. It is mainly distributed in the Huashan region of Shaanxi Province (W34°25′~34°30′, E109°57′~110°05′), on rocky slopes at altitudes of 400-800 meters. *Psathyrostachys huashanica* plants grow in clusters with well-developed rhizomes. It possesses numerous excellent traits, including tolerance to abiotic stresses (salt-alkali soil, high altitude, cold, drought, and poor soil) and resistance to biotic stresses (rust, powdery mildew, take-all, Fusarium head blight, and yellow dwarf disease), making it an extremely valuable germplasm for wheat stress and disease resistance breeding. In the 1980s, the first distant hybridization between *Leymus chinensis* and common wheat was conducted by Chen Shuyang et al., resulting in hybrid offspring. The experiment involved first crossing the common wheat variety 7182 with *Leymus chinensis*, then obtaining the F1 generation hybrids using embryo rescue technology. Backcrossing the F1 generation with wheat parents yielded the heptaploid material H8911 (2n=7x=49, AABBDDNs), which is essentially a wheat heptaploid material with 7 *Leymus chinensis* chromosomes added to the 42 wheat chromosomes. Through self-pollination or hybridization with other wheat varieties, a batch of wheat-*Leymus chinensis*-derived offspring with excellent agronomic traits were harvested. These materials typically exhibit the multi-resistance and stress tolerance characteristics of the exogenous parent *Leymus chinensis* due to the introduction of *Leymus chinensis* chromosomes, especially showing good resistance to various common wheat diseases such as stripe rust and powdery mildew. Creating various types of new wheat-Huashan wheatgrass germplasm carrying exogenous chromosomes using chromosome engineering is of great significance for enriching wheat genetic diversity, discovering superior exogenous genes, and promoting innovation and breakthroughs in wheat breeding. Identifying whether the wheat-Huashan wheatgrass-derived progeny contain Huashan wheatgrass chromosomes is the first step in analyzing this material. Currently, the most commonly used identification methods are genomic in situ hybridization and EST-STS molecular marker analysis. Genomic in situ hybridization is a complex procedure, requiring two days for the complete process; when using EST-STS, hundreds of EST-STS primer pairs from seven homologous groups often require about a week of screening. Therefore, the rapid and accurate identification of Huashan wheatgrass chromosomes in distant hybrids of wheat-Huashan wheatgrass has become a bottleneck problem currently faced by researchers and breeders.

[0003] Definitions of abbreviations and key terms

[0004] EST-STS: Expressed Sequence Tags. EST refers to a sequence set obtained by randomly selecting clones from a species' cDNA library and sequencing them. STS refers to further screening of the sequences in the set for those that have been expressed multiple times and are of suitable length, indicating that the sequence has a high abundance in the set. Summary of the Invention

[0005] The purpose of this invention is to provide a chromosome-specific ND-FISH probe for *Leymus chinensis*, which can efficiently identify exogenous *Leymus chinensis* chromosomes in a wheat background through non-denaturing fluorescent in situ hybridization.

[0006] This invention also provides the application of the Huashan new wheatgrass chromosome-specific ND-FISH probe and its kit.

[0007] To achieve the above objectives, the technical solution adopted by the Huashan New Wheatgrass Chromosome-Specific FISH Probe of the present invention is as follows:

[0008] A chromosome-specific ND-FISH probe for *Leymus chinensis* has the nucleotide sequence 5'-CAATGGGCCTCTACTATAGGGTGTTAGGCTCACACTAGAGAA-3'; the 5' end is labeled with a fluorescent group.

[0009] The chromosome-specific ND-FISH probe of the present invention can specifically bind to the Ns chromosome of *Leymus chinensis* in the chromosome set of the offspring derived from distant hybridization of wheat and *Leymus chinensis*, emit a fluorescent signal, and identify the chromosome of *Leymus chinensis*.

[0010] The Huashan New Wheatgrass-specific ND-FISH probe of the present invention can efficiently distinguish the chromosomes of wheat and Huashan New Wheatgrass within four hours through non-denaturing fluorescent in situ hybridization. It can greatly improve the screening efficiency of wheat-Huashan New Wheatgrass-derived offspring, reduce experimental costs, and lay the foundation for improving wheat breeding by using the closely related species Huashan New Wheatgrass and broadening the genetic diversity of wheat.

[0011] All fluorescent groups used in existing oligonucleotide probes can be used as fluorescent groups for the ND-FISH probe of this invention. Further, the fluorescent group is a 6-FAM fluorescent group. When 6-FAM is used as the fluorescent group, after the ND-FISH probe specifically binds to the chromosome of *Leymus chinensis*, it emits a striking and bright green fluorescent signal under the corresponding excitation light, facilitating rapid microscopic examination.

[0012] The technical solution adopted in the application of the Huashan New Wheatgrass chromosome-specific ND-FISH probe of the present invention is as follows:

[0013] Application of a chromosome-specific ND-FISH probe for *Leymus chinensis* in detecting *Leymus chinensis* chromosomes in the chromosome set of wheat-*Leymus chinensis* distant hybrid offspring. The *Leymus chinensis* chromosome-specific ND-FISH probe of this invention distinguishes between wheat chromosomes and *Leymus chinensis* chromosomes in the chromosome set of the offspring derived from wheat-*Leymus chinensis* distant hybridization.

[0014] Furthermore, the application of the Huashan New Wheatgrass chromosome-specific ND-FISH probe in detecting Huashan New Wheatgrass chromosomes in wheat chromosome sets includes the following steps: preparing chromosome slides of the sample to be tested and performing non-denaturing fluorescent in situ hybridization with the Huashan New Wheatgrass chromosome-specific ND-FISH probe, followed by microscopic examination.

[0015] Further, the chromosomes of the sample to be tested are prepared by the following steps: a suspension of root tip cells from distant hybrids of wheat and wheatgrass is dropped onto the center of a glass slide and left to stand in a humidifier.

[0016] Furthermore, the fluorescence hybridization reaction uses a hybridization reaction solution prepared by mixing 2×SCC buffer solution and probe solution at a volume ratio of 7:3. The probe solution is obtained by diluting the Huashan wheatgrass-specific ND-FISH probe with 2×SCC buffer to a concentration of 20 ng / μL. Even further, the hybridization reaction is conducted at a temperature of 42℃ for 3 hours.

[0017] Furthermore, the 2×SCC buffer solution is obtained by dissolving 4.41g of trisodium citrate and 8.77g of NaCl in 480mL of deionized water, adjusting the pH to 7.0 with HCl, adding water to bring the volume to 500mL, and then sterilizing.

[0018] Furthermore, the system obtained after non-denaturing fluorescence in situ hybridization on the slide was immersed in a buffer solution, dried, covered with a coverslip, and placed in the dark before microscopic examination. Even further, the buffer solution used for immersion was 2×SSC buffer solution. The mounting medium was H-1200 (Vector Labs, USA), an anti-fluorescence quenching mounting medium. The placement time in the dark was 10 minutes.

[0019] Furthermore, the microscopic examination was performed using a fluorescence microscope equipped with DAPI and GFP modules.

[0020] The application of a chromosome-specific ND-FISH probe for *Leymus chinensis* in the preparation of a product for detecting the chromosome set of *Leymus chinensis* in the offspring of distant hybridizations of wheat and *Leymus chinensis*. The product for detecting the chromosome set of *Leymus chinensis* in the offspring of distant hybridizations of wheat and *Leymus chinensis* can be a kit.

[0021] The technical solution adopted by the reagent kit of the present invention is as follows:

[0022] The kit contains the aforementioned chromosome-specific ND-FISH probe from *Leymus chinensis*. This kit can be used to identify whether exogenous chromosomes are present in the offspring of distant hybridization of wheat and *Leymus chinensis*.

[0023] Furthermore, the kit also contains nucleic acid hybridization solutions, such as 2×SSC buffer. Attached Figure Description

[0024] Figure 1 This is a diagram showing the results of non-denaturing fluorescent in situ hybridization of probe HS-TZ1 with chromosome 7182 of common wheat in an application example;

[0025] Figure 2 This is a diagram showing the results of non-denaturing fluorescent in situ hybridization of probe HS-TZ1 with the chromosome set of *Leymus chinensis* in an application example.

[0026] Figure 3 This is a diagram showing the results of non-denaturing fluorescent in situ hybridization of probe HS-TZ1 with the chromosome set of the wheat-Huashan New Wheatgrass addition line in the application example. The white arrows indicate the Huashan New Wheatgrass chromosomes in the wheat background. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0028] Non-denaturing fluorescence in situ hybridization (ND-FISH) is based on the principle of complementary base pairing. It uses a specific short sequence carrying a fluorescent group as a probe to hybridize with the mitotic chromosomes of root tip cells. After successful hybridization, a fluorescent signal can be observed at the target location on the chromosome. Because it does not involve chromosome denaturation, the hybridization signal can be washed away, facilitating rehysteresis. The ND-FISH probe sequence must be a repetitive sequence on the chromosome. After multiple bindings at multiple sites on the chromosome with its complementary sequence, signal aggregation occurs, resulting in fluorescence. This technique allows for the differentiation of target chromosomes by synthesizing a sequence with an added fluorescent group and using non-denaturing fluorescence in situ hybridization.

[0029] The Huashan wheatgrass-specific ND-FISH probe of this invention can specifically bind to the Huashan wheatgrass Ns chromosome in the chromosome set of the offspring derived from distant hybridization of wheat and Huashan wheatgrass, emitting a fluorescent signal. It can be used to rapidly and effectively identify Huashan wheatgrass chromosomes in marker-assisted selection breeding, which helps to utilize the many superior genes carried on the Huashan wheatgrass chromosomes to improve the disease resistance and high yield breeding process of wheat, accelerate the transfer of exogenous superior genes and excellent traits to recipient wheat, and improve the breeding efficiency of new varieties.

[0030] The fluorescence microscope used in Example 2 has DAPI and GFP modules.

[0031] Example 1

[0032] The nucleotide sequence of the Huashan wheatgrass-specific ND-FISH probe in this embodiment (as shown in SEQ ID NO.1) is 5'-CAATGGGCCTCTACTATAGGGTGTTAGGCTCACACTAGAGAA-3', with a 6-FAM fluorescent group labeled at the 5' end. The probe was directly synthesized by Shanghai Sangon Biotech (Shanghai) Co., Ltd., and stored in dry powder form or diluted in buffer and protected from light. The Huashan wheatgrass-specific ND-FISH probe in this embodiment is named probe HS-TZ1.

[0033] Example 2

[0034] The application of the Huashan new wheatgrass-specific ND-FISH probe in detecting Huashan new wheatgrass chromosomes in this embodiment includes the following steps:

[0035] 1. Chromosome preparation of experimental materials, the specific steps are as follows:

[0036] 1) Take the clean root tips of the experimental material that have been rinsed with water and put them into a 0.5 mL centrifuge tube. Add the mixed enzyme solution of cellulase and pectinase and let it stand in a 37℃ water bath for 50 min.

[0037] 2) Gently aspirate the enzyme solution (Note: Do not aspirate the root tip meristem), place the centrifuge tube on ice, gently add 1×TE solution into the tube and let it stand for 5 minutes, aspirate the TE solution and wash the root tip 3 times with 75% ethanol.

[0038] 3) Add glacial acetic acid at a ratio of 20 μL per root tip and grind the root tip.

[0039] 4) Use a pipette to draw 8 μL of cell suspension and drop it onto the center of a glass slide. Place the slide in a partially sealed, moisturizing black box and let it stand for 10 minutes. Then observe and select chromosomes with good mitotic phases to prepare slides.

[0040] 2. Non-denaturing fluorescent in situ hybridization

[0041] 1) Prepare 2×SSC buffer solution, total volume 500mL, containing the following components:

[0042] NaCl 8.77g

[0043] 4.41g of trisodium citrate

[0044] 480mL of deionized water;

[0045] To prepare the solution, dissolve the trisodium citrate and NaCl in deionized water, then add concentrated HCl to adjust the pH to 7.0, add water to bring the volume to 500 mL, autoclave, and store for later use.

[0046] 2) Prepare probe solution by diluting the synthesized probe HS-TZ1 dry powder to 20 ng / μL with 2×SSC buffer to obtain probe HS-TZ1 solution, and store it in the dark.

[0047] 3) Prepare the hybridization reaction solution. The total system is 10 μL, which is formed by mixing 7 μL of buffer solution 2×SSC and 3 μL of probe HS-TZ1 solution.

[0048] 4) Add the hybridization reaction solution to the target position of the chromosome slide, cover with a coverslip, and incubate in a 42°C incubator for 3 hours.

[0049] 5) After the incubation is complete, remove the slide, discard the coverslip, immerse it in 2×SSC buffer solution for 3 minutes, and then remove and air dry.

[0050] 6) Add 8 μL of H-1200 mounting medium to the target location, cover with a coverslip, and let stand in the dark for 10 minutes.

[0051] 3. Microscopic examination under a fluorescence microscope

[0052] The chromosomes were located under the DAPI light channel of the microscope, and the fine adjustment knob was adjusted to the clearest image before taking a picture. Then, the image was taken under the same field of view using the GFP light channel. The images from the two channels were combined into one image as the result of non-denaturing fluorescent in situ hybridization of this material.

[0053] Experimental Example 3

[0054] The kit in this embodiment includes the probe HS-TZ1 from Example 1 and the buffer 2×SSC from Example 2.

[0055] Application examples

[0056] Common wheat variety 7182, *Leymus chinensis*, and wheat-*Leymus chinensis* disomal addition line DH18 (this material has two additional chromosomes from *Leymus chinensis* added to the wheat chromosome set) were used as experimental materials and cultured until the taproot length reached 2-3 cm. Root tips were then cut and preserved in Carnoy's fixative. The *Leymus chinensis* chromosomes were detected using the method and steps applied in Example 2. The non-denaturing fluorescence in situ hybridization results for each experimental material are shown in the figures below. Figures 1 to 3 .

[0057] Image of non-denaturing fluorescent in situ hybridization results of probe HS-TZ1 with common wheat chromosome 7182 ( Figure 1The absence of hybridization signal indicates that the probe does not pair with wheat chromosomes.

[0058] Figure 1 shows the results of non-denaturing fluorescent in situ hybridization of probe HS-TZ1 with chromosomes of *Leymus chinensis*. Figure 2 The presence of green fluorescent signals on each chromosome indicates that the probe specifically paired and bound to each chromosome of *Hypericum huashanense*.

[0059] Figure 1 shows the results of non-denaturing fluorescent in situ hybridization of probe HS-TZ1 with the wheat-Huashan wheatgrass chromosome disomy addition line. Figure 3 The presence of green fluorescent signals on only two chromosomes indicates that the probe specifically paired and bound to the chromosomes of *Leymus chinensis* in the wheat background.

[0060] The results show that the above-mentioned Huashan new wheatgrass chromosome-specific ND-FISH probe can quickly and accurately identify whether wheat-Huashan new wheatgrass distant hybrid offspring contain exogenous chromosomes, laying the foundation for improving wheat breeding by utilizing the many excellent genes carried on the chromosomes of the closely related species Huashan new wheatgrass. <110> Henan University of Science and Technology <120> A chromosome-specific ND-FISH probe for *Leymus chinensis* from Huashan and its application and kit <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 42 <212> DNA <213> Artificial Sequence <400> 1 caatgggcct ctactatagg gtgttaggct cacactagag aa 42

Claims

1. A chromosome-specific ND-FISH probe for *Leymus chinensis*, characterized in that: The nucleotide sequence is as follows: 5'-CAATGGGCCCTACTATAGGGTGTTAGGCTCACACTAGAGAA-3'; The 5' end is labeled with a fluorescent group.

2. The Huashan New Wheatgrass Chromosome-Specific ND-FISH Probe according to claim 1, characterized in that: The fluorescent group is a 6-FAM fluorescent group.

3. The application of the Huashan New Wheatgrass chromosome-specific ND-FISH probe as described in claim 1 or 2 in detecting the Huashan New Wheatgrass chromosome in the chromosome set of wheat-Huashan New Wheatgrass distant hybrid offspring.

4. The application of the Huashan New Wheatgrass chromosome-specific ND-FISH probe according to claim 3, characterized in that: Includes the following steps: After preparing chromosome slides of the sample to be tested, non-denaturing fluorescent in situ hybridization was performed with the Huashan New Wheatgrass chromosome-specific ND-FISH probe. The slides were then examined under a microscope or soaked in a buffer solution and dried, then covered with a mounting medium and examined under a microscope.

5. The use of the Huashan New Wheatgrass chromosome-specific ND-FISH probe as described in claim 1 or 2 in the preparation of a product for detecting the chromosome set of Huashan New Wheatgrass in the offspring of distant hybridization of wheat and Huashan New Wheatgrass.

6. A reagent kit, characterized in that: It contains the chromosome-specific ND-FISH probe of *Leymus chinensis* as described in claim 1 or 2.

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