A hordeum vulgare nsspecific genome-specific chromosome painting probe, reagent, kit and application thereof

Abstract

The application belongs to the technical field of molecular cytogenetics, and particularly relates to a specific chromosome painting probe of the Lophiumporphyrogaster Ns genome, a reagent, a kit and application thereof.The nucleotide sequence of the specific chromosome painting probe of the Lophiumporphyrogaster Ns genome is shown as SEQ ID NO.1, and the reagent and the kit are developed based on the specific chromosome painting probe of the Lophiumporphyrogaster Ns genome.After fluorescence in situ hybridization is performed on chromosome preparation, the Lophiumporphyrogaster Ns genome chromosomes present clear red fluorescence signals, and the wheat chromosome background is clean and free of cross signals.The specific chromosome painting probe of the Lophiumporphyrogaster Ns genome, the reagent and the kit can be applied to detecting the Lophiumporphyrogaster Ns chromosomes in the chromosome set of the wheat-Lophiumporphyrogaster distant hybrid offspring, can greatly improve the screening efficiency of the wheat-Lophiumporphyrogaster derivative offspring, reduce experimental cost, and lay a foundation for improving wheat breeding by using the Lophiumporphyrogaster Ns genome and widening the genetic diversity of wheat.

Description

Technical Field

[0001] This invention belongs to the field of molecular cytogenetics technology, specifically relating to a Bimbisara Ns genome-specific chromosome staining probe, reagent, kit, and their applications. Background Technology

[0002] Binmai ( Leymus mollis (Trin.) Pilger, NsNsXmXm, 2 n =28) is a perennial cross-pollinated plant belonging to the genus Leymus ( Leymus Wheat (Triticum aestivum), primarily found in sandy riverbanks along the northern coast of China, is characterized by its large ears, numerous small florets, and extended root system, serving both as a soil stabilizer and natural forage. As an allotetraploid species, its Xm genome chromosome has been extensively studied due to its unknown donor source; therefore, the letter X (unknown) is used as a substitute. Its Ns genome chromosome has been confirmed to carry numerous superior genes, making it a primary focus of current research. Due to its resistance to fungal diseases such as stripe rust and powdery mildew, as well as its tolerance to cold, drought, salinity, and poor soil conditions, Wheat is considered an important germplasm resource for wheat genetic improvement. Scientists have introduced superior genes from the Wheat Ns chromosome into common wheat through distant hybridization, creating wheat-Wheat derivative lines carrying the Wheat chromosome. These lines represent excellent germplasm resources for broadening the genetic base of wheat and breeding new stress-resistant varieties. my country possesses abundant wheat-Wheat derivative line materials. Systematic identification and utilization of superior genes in the Wheat Ns genome chromosome are of significant strategic importance for promoting wheat chromosome engineering breeding.

[0003] However, the accurate identification and tracking of *Lysimachia christinae* Ns genome chromosomes within the wheat genetic background remains a key bottleneck hindering the improvement of wheat breeding using *Lysimachia christinae*. On the one hand, the morphological characteristics of *Lysimachia christinae* Ns genome chromosomes are difficult to distinguish from those of wheat itself and closely related species (such as *Leymus chinensis* and rye) in conventional karyotype analysis. On the other hand, the Ns genome exhibits complex evolutionary relationships with various wheat genomes and other genomes of the *Lysimachia* genus, with high homology of repetitive sequences, making it difficult for traditional cytological identification methods to accurately identify the state and genetic behavior of Ns chromosomes within the wheat genetic background. Currently, the Giemsa C banding technique and basic karyotype analysis, primarily based on biochemical methods, are not only cumbersome to operate, have low resolution and reproducibility, but also cannot efficiently distinguish between Ns genome chromosomes and wheat genome chromosomes. Due to the lack of effective and specific chromosome physical markers, key scientific questions regarding the transmission patterns, structural variations, and introduction status of exogenous fragments of *Lysimachia christinae* Ns genome chromosomes in heterologous genetic backgrounds have long remained unresolved, severely restricting the effective utilization of *Lysimachia christinae*'s superior genetic resources.

[0004] Traditionally, the identification of exogenous chromosomes in wheat backgrounds has been primarily achieved through in situ genomic hybridization. This involves competitive hybridization using total genomic DNA of wheat (Ns) as a probe and total genomic DNA of wheat as a blocking DNA. For example, a Chinese patent document with authorization announcement number CN103592169B discloses a method for identifying wheat-wheat multiple substitution lines, using cytological chromosome microscopy and GISH to identify and screen wheat-wheat multiple substitution lines. However, GISH technology has inherent limitations: it requires repeated optimization of blocking conditions, has a long experimental cycle (about two days), high cost, and poor reproducibility; moreover, because there are many conserved repetitive sequences between Ns and the wheat genome, it is easy to cause excessively deep hybridization background or weakened signal, resulting in false positive / false negative results, which is difficult to meet the needs of fine karyotype analysis and structural variation detection.

[0005] Existing publicly available probes for identifying closely related wheat species, especially staining probes, can only be developed based on species-specific repetitive sequences. Since genomes differ between species, these probes are strictly species-specific and cannot be used across species. The *Triticum aestivum* Ns genome is highly differentiated from traditional wheat relatives such as rye and *Triticum aestivum*, with significant differences in repetitive sequences. Reported probes for closely related species cannot identify *Triticum aestivum* chromosomes and cannot be used for the detection and identification of exogenous chromosomes in *Triticum aestivum*. To date, a chromosome staining probe identification system targeting all chromosomes of the *Triticum aestivum* Ns genome has not been established, and no oligonucleotide probes for specifically tracking Ns chromosomes against a wheat background have been reported. Summary of the Invention

[0006] The first objective of this invention is to provide a genome-specific chromosome staining probe for wheat Ns to address the problem in the prior art of lacking tools that can specifically and comprehensively identify all chromosomes of the wheat Ns group in a wheat background.

[0007] A second objective of this invention is to provide a reagent.

[0008] A third objective of this invention is to provide a reagent kit.

[0009] The fourth objective of this invention is to provide the application of the above-mentioned wheat Ns genome-specific chromosome staining probes, reagents, and kits in detecting wheat Ns chromosomes in the chromosome set of wheat-wheat distant hybrid offspring.

[0010] To achieve the above objectives, the technical solution adopted by the *Hymenopausal nucleus* Ns genome-specific chromosome staining probe in this invention is as follows: A *Prunus chinensis* Ns genome-specific chromosome staining probe, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0011] The specific chromosome staining probe of the Binmai Ns genome of the present invention is based on the principle of complementary base pairing. It can specifically anneal and bind to a portion of the chromosomes of the Binmai Ns group at a suitable temperature, and achieve precise tracking of the Binmai Ns genome chromosomes through incubation and elution.

[0012] Preferably, the 5' end of the Binmai Ns genome-specific chromosome staining probe is labeled with a TAMRA fluorescent group.

[0013] The TAMRA fluorescent group used in this invention has high brightness, strong stability, clear fluorescence signal, and good biocompatibility, making it suitable for fluorescence in situ hybridization experiments.

[0014] To achieve the above objectives, the technical solution adopted by the reagent in this invention is as follows: A reagent containing the above-mentioned Binmai Ns genome-specific chromosome staining probe.

[0015] The reagent of the present invention contains the above-mentioned wheat Ns genome-specific chromosome staining probe. After performing fluorescence in situ hybridization on chromosome samples using this reagent, the wheat Ns genome chromosomes can show a clear red fluorescent signal, while the wheat chromosome background is clean and there is no crossover signal, thereby achieving rapid and accurate identification of exogenous chromosomes.

[0016] To achieve the above objectives, the technical solution adopted in the reagent kit of this invention is as follows: A reagent kit comprising the reagents described above.

[0017] The reagents and kits developed based on the above-mentioned *Pteris vittata* Ns genome-specific chromosome staining probes can achieve accurate identification of *Pteris vittata* Ns chromosomes, which is of great significance for *Pteris vittata* chromosome karyotype analysis, differentiation of chromosomes from different species, and phylogenetic analysis.

[0018] Preferably, the kit further includes 2×SSC buffer.

[0019] Preferably, the kit further includes a mounting medium containing an anti-fluorescence quencher and a nucleic acid dye.

[0020] Preferably, the kit further includes a fixative, specifically Carnoy's fixative I.

[0021] Preferably, the kit further includes an enzymatic hydrolysate containing 1% cellulase and 0.5% pectinase by weight-to-volume ratio.

[0022] Preferably, the kit further includes glacial acetic acid.

[0023] To achieve the above objectives, the technical solution adopted in this invention for the application of the above-mentioned wheat Ns genome-specific chromosome staining probe, reagent, and kit in detecting the wheat Ns chromosome in the chromosome set of wheat-wheat distant hybrid offspring is as follows: The above-mentioned *Strombyx mori* Ns genome-specific chromosome staining probes, reagents, and kits were used to detect *Strombyx mori* Ns chromosomes in the chromosome set of wheat-*Strombyx mori* distant hybrid offspring.

[0024] The application method of this invention is simple, efficient and economical. It can quickly and specifically track wheat Ns genome chromosomes or fragments in a wheat background within five hours, accurately identify their quantity, morphology and structural variations, and provide reliable technical support for the identification of exogenous chromosomes in wheat distant hybridization breeding. Attached Figure Description

[0025] Figure 1 This is a diagram showing the results of fluorescence in situ hybridization of wheat as the experimental material in an application example of this invention; Figure 2 This is a diagram showing the fluorescence in situ hybridization results of common wheat 7182 as the experimental material in an application example of this invention; Figure 3 This is a diagram showing the results of fluorescence in situ hybridization of wheat-Bingmai disomic substitution lines in an application example of this invention. Figure 4 This is a diagram showing the results of fluorescence in situ hybridization of wheat-Binma tetrasomal substitution lines in an application example of the present invention. Detailed Implementation

[0026] Chromosomal painting technology is based on the principle of complementary nucleic acid pairing. It uses fluorescently labeled oligonucleotide sequences as probes to hybridize in situ with target chromosome sequences, forming dense clusters of fluorescent signals in the target chromosome region, enabling genome-wide visualization and identification. Unlike traditional FISH, which relies on localized spot fluorescence signals on chromosomes, the oligonucleotide sequences of painting probes cover the entire target genome and all its segments, producing a bright, specific signal covering the entire chromosome set. This significantly improves the efficiency of foreign chromosome identification while avoiding false positives.

[0027] Oligo nucleotide probes combined with FISH-painting technology (Oligo-Painting) provide a novel solution for the precise identification and tracking of wheat Ns genome chromosomes within the wheat genetic background. Oligo-Painting enables precise "painting" of target chromosomes or chromosome sets, allowing for intuitive identification of target chromosomes among numerous chromosomes. Oligo-Painting eliminates the need for genomic DNA and blocking DNA preparation, exhibits high specificity, stable hybridization signals, clean background, and can be flexibly designed as probes covering the entire genome or targeting single chromosomes as needed.

[0028] This invention is the first to develop a painting probe for the Brussels wheat Ns chromosome, which can specifically bind to the Brussels wheat Ns chromosomes to produce a fluorescent signal. Based on this probe, a fluorescent staining kit for Brussels wheat Ns chromosomes was developed. After performing fluorescence in situ hybridization on chromosome slides using this kit, the Brussels wheat Ns genome chromosomes showed a clear red fluorescent signal, while the wheat chromosomes showed a clean background with no crossover signal.

[0029] This invention does not simply apply existing FISH identification methods for wheat-related species to *Triticum aestivum*-wheat materials. Instead, it specifically screens for repetitive sequences unique to the *Triticum aestivum* Ns genome to develop a *Triticum aestivum*-specific probe. This probe can specifically identify *Triticum aestivum* Ns chromosomes, showing no hybridization signal on wheat chromosomes, thus achieving accurate identification of exogenous chromosomes in *Triticum aestivum*. Compared to existing identification techniques for closely related species, this invention differs clearly in probe source, target sequence, and applicable detection system. It is not a direct transfer and application of conventional techniques in this field, and possesses corresponding innovation and technological uniqueness.

[0030] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0031] Example 1: Binmai Ns genome-specific chromosome painting probe This embodiment provides a genome-specific chromosome staining probe for Binmai Ns, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0032] A fluorescent group is labeled at the 5' end of the probe. Any fluorescent group used for oligonucleotide probes in the prior art can be used as the fluorescent group of the painting probe of the present invention.

[0033] After screening, this invention selected TAMRA as the fluorescent group for the Binmai Ns genome-specific chromosome staining probe.

[0034] Example 2 Reagents and Kits This embodiment provides a reagent or kit for detecting the Ns chromosome of wheat in the chromosome set of wheat-wheat distant hybrid offspring, which includes the following components: ① A *Binma* Ns genome-specific chromosome staining probe, the nucleotide sequence of which is shown in SEQ ID NO.1, with a TAMRA fluorescent group labeled at the 5' end. The probe was synthesized by Shanghai Sangon Biotech (Shanghai) Co., Ltd., and a dry powder form of the *Binma* Ns genome-specific chromosome staining probe was obtained.

[0035] ②2×SSC buffer, the preparation method is as follows: Weigh 0.88 g of trisodium citrate and 1.75 g of sodium chloride, add 90 mL of deionized water to dissolve them completely, adjust the pH to 7.0 and bring the volume to 100 mL, autoclave and obtain 2×SSC buffer.

[0036] The dry powder of the *Strombocys edulis* Ns genome-specific chromosome staining probe was diluted with 2×SSC buffer to a concentration of 20 ng / μL to obtain a reagent for detecting *Strombocys edulis* Ns chromosomes in the chromosome set of wheat-*Strombocys edulis* distant hybrid offspring. The reagent was named LMPT1 solution and stored in the dark at -20°C.

[0037] ③ Mounting tablets, containing nucleic acid dyes and anti-fluorescence quenchers, purchased from Vector Laboratories, catalog number H1200.

[0038] ④ Carnoy's fixative I, composed of anhydrous ethanol and glacial acetic acid, with anhydrous ethanol: glacial acetic acid = 3:1 (v / v).

[0039] ⑤ Enzymatic hydrolysate, containing 1% cellulase and 0.5% pectinase by weight-volume ratio.

[0040] ⑥Glacial acetic acid.

[0041] A kit for detecting the Ns chromosome of wheat in the chromosome set of wheat-wheat distant hybrid offspring was prepared by combining LMPT1 solution, mounting medium, Carnoy's fixative I, enzyme digest, and glacial acetic acid.

[0042] Application examples This application example provides an example of the application of the wheat Ns genome-specific chromosome staining probe from Example 1, and the reagents and kits from Example 2, in detecting the wheat Ns chromosome in the chromosome set of wheat-wheat distant hybrid offspring, including the following steps: 1. Preparation of mitotic slides from *Bambusa textilis* root tip cells, the specific steps are as follows: 1) Soak the seeds of *Ilex chinensis* in water overnight. Take out the germinating seeds and place them on moist filter paper. After the main root is exposed, transfer them to a 4°C refrigerator and treat them at low temperature for 24 h. Then take them out and place them in a 25°C incubator. When the root tip grows to 1.5-2 cm, cut off the root tip and place it in a centrifuge tube with ice water added. After bathing in ice water for 24 h, take it out and preserve it in Carnoy's fixative I. 2) Take out a root tip of *Ilex chinensis*, rinse it with clean water, put it into a 0.5 mL centrifuge tube, add 20 μL of enzyme hydrolysate, and incubate in a 37 ℃ water bath for 50 min; 3) Gently aspirate the enzyme solution, add 75% ethanol and slowly wash the root tip three times, then add 20 μL of glacial acetic acid and grind the root tip with a grinding rod; 4) Use a pipette to draw 8 μL of cell suspension and drop it vertically into the center of a glass slide. Place the slide in a sealed, humidified black box and let it stand for 10 min. Examine the slide under a microscope and select chromosomes with good mitotic phases.

[0043] 2. Prepare the hybridization reaction solution for the Ns genome chromosome markers of *Strombocys edulis*. The entire process must be conducted in the dark. The specific steps are as follows: 1) Measure 7 μL of 2×SSC buffer and 3 μL of LMPT1 solution, mix thoroughly, and prepare the hybridization reaction solution.

[0044] 3. Fluorescence in situ hybridization requires complete darkness. The specific steps are as follows: 1) Drop the hybridization reaction solution onto the appropriate area of ​​the chromosome slide, cover with a coverslip, and incubate at 42 ℃ for 4.5 h; 2) Remove the slide, knock off the coverslip, immerse it in pure water to wash away the hybridization solution, and then place it on the table to air dry; 3) Add 10 μL of mounting medium to the original hybridization solution drop position, cover with a coverslip and place in the dark for natural staining for 20 min.

[0045] 4. Microscopic examination under a fluorescence microscope, the specific steps are as follows: 1) Place the prepared slide on the stage of a fluorescence microscope, locate the metaphase phase with well-dispersed chromosomes and complete morphology under the DAPI channel, and take a bright field image of the chromosomes after fine focusing. 2) Without moving the field of view, switch to the Rhoda channel and simultaneously acquire hybridization signal images within that field of view; 3) Use image processing software to overlay and integrate the DAPI and Rhoda two-channel images to obtain the fluorescence in situ hybridization result image of this material.

[0046] Common wheat 7182, *Binmai*, wheat-Binmai disomic substitution line (this material has two *Binmai* Ns chromosomes introduced into the wheat chromosome set), and wheat-Binmai tetrasomic substitution line (this material has four *Binmai* Ns chromosomes introduced into the wheat chromosome set) were used as experimental materials. The corresponding fluorescence in situ hybridization results were obtained after the above steps, as shown in the figures below. Figures 1-4 .

[0047] Figure 1 The image shows the results of fluorescence in situ hybridization of wheat as the experimental material. The Ns genome chromosome shows a red fluorescent signal, while the Xm genome chromosome shows no hybridization signal, indicating that the probe does not pair and bind with chromosomes other than the Ns genome.

[0048] Figure 2 The image shows the results of fluorescence in situ hybridization of common wheat 7182 as the experimental material. There is no hybridization signal, indicating that the probe does not pair and bind with the wheat chromosome.

[0049] Figure 3 The image shows the results of fluorescence in situ hybridization of wheat-Binmaie disomic substitution lines. Only two chromosomes showed red fluorescence signals, indicating that the probe specifically paired and bound to the Binmaie Ns genome chromosomes in the wheat background.

[0050] Figure 4 The image shows the results of fluorescence in situ hybridization of wheat-Binma tetrasomal substitution lines. Only four chromosomes showed red fluorescence signals, indicating that the probe specifically paired and bound to Binma Ns genome chromosomes in the wheat background.

[0051] The results show that the Ns chromosome painting probe developed in this invention can efficiently and accurately identify the Ns chromosome in wheat and the Ns chromosome in wheat-wheat distant hybrid offspring, laying the foundation for improving wheat breeding by utilizing the superior genes carried on the Ns chromosome of the closely related wheat species.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A genome-specific chromosome staining probe for *Bambusa Ns*, characterized in that, The nucleotide sequence of the Binmai Ns genome-specific chromosome painting probe is shown in SEQ ID NO.

1.

2. The *Binma* Ns genome-specific chromosome painting probe as described in claim 1, characterized in that, The 5' end of the Binmai Ns genome-specific chromosome staining probe is labeled with the TAMRA fluorescent group.

3. A reagent, characterized in that, The reagent contains the Binmai Ns genome-specific chromosome staining probe as described in claim 1 or 2.

4. A reagent kit, characterized in that, The kit includes the reagent as described in claim 3.

5. The kit according to claim 4, characterized in that, The kit also includes 2×SSC buffer.

6. The kit as described in claim 4 or 5, characterized in that, The kit also includes a mounting medium containing an anti-fluorescence quencher and a nucleic acid dye.

7. The kit according to any one of claims 4 to 6, characterized in that, The kit also includes a fixative, specifically Carnoy's fixative I.

8. The kit according to any one of claims 4 to 7, characterized in that, The kit also includes an enzymatic hydrolysate containing 1% cellulase and 0.5% pectinase by weight-volume ratio.

9. The kit according to any one of claims 4 to 8, characterized in that, The kit also includes glacial acetic acid.

10. The application of the wheat Ns genome-specific chromosome staining probe as described in claim 1 or 2, the reagent as described in claim 3, and the kit as described in any one of claims 4 to 9 in detecting the wheat Ns chromosome in the chromosome set of wheat-wheat distant hybrid offspring.

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