ND-FISH probe for oriental lily genome recognition and application of ND-FISH probe

Abstract

The invention discloses an oligonucleotide probe sequence capable of specifically recognizing genome chromosomes of lilium orientalis. The probe is suitable for an ND-FISH detection technology. The probe can be used for quickly, simply and economically identifying the chromosome segment of the oriental lily in the lilium hybrid, has the advantages of simplicity and convenience in operation, short detection period, low cost, strong signal specificity, stable and reliable result and the like, and is suitable for analyzing chromosome components and quickly identifying breeding materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular genetics, and particularly relates to an oligonucleotide probe sequence capable of specifically identifying genomic chromosomes of oriental lilies, and application of the oligonucleotide probe sequence in non-denaturing fluorescence in situ hybridization (ND-FISH) technology. Background Art

[0002] Lilium spp., as important ornamental and economic crops, possess an extremely complex genetic background, with diverse and mixed hybrid lineages. Currently, genomic in situ hybridization (GISH) remains the primary analytical method for lily germplasm research, hybrid identification, and chromosome analysis. Existing GISH technology has significant drawbacks for lily chromosome identification: the experimental process is cumbersome, requiring the extraction, purification, and labeling of whole-genomic DNA as probes. This involves numerous and complex steps, typically requiring a 3-5-day operation cycle. Probe preparation is laborious and requires the extraction of high-quality genomic DNA from different lilies and fluorescent labeling, a time-consuming process susceptible to material quality. Experimental costs are high, with fluorescent labeling reagent systems being expensive. Furthermore, the technology is demanding, requiring specialized personnel and comprehensive experimental facilities, making it difficult to widely apply in grassroots breeding units.

[0003] In contrast, non-denaturing fluorescence in situ hybridization (ND-FISH) uses short oligonucleotides as probes, enabling efficient and specific fluorescent labeling of chromosomes without the need for DNA extraction or denaturation. This technique offers advantages such as ease of use, short hybridization cycles, strong reproducibility, and a high signal-to-noise ratio. It has been widely used in crops such as wheat, demonstrating significant advantages in rapid chromosome identification and recombination monitoring.

[0004] However, the application of this technology in Lilium species remains largely unexplored, particularly with regard to probe design, which lacks systematic development of genome-specific sequences for the Oriental lily (Lilium oriental). As one of the most important cultivated groups in the genus, Oriental lily is widely used in hybrid breeding for OT, OA, and LO types due to its excellent ornamental value, genetic diversity, and strong affinity. Accurate identification of its chromosomal composition is crucial for germplasm innovation and hybrid authenticity verification.

[0005] Therefore, there is an urgent need to develop an oligonucleotide probe that can specifically identify the genomic region of Oriental lily and establish a chromosome detection method suitable for the ND-FISH system to achieve rapid, simple, economical and stable identification of Oriental lily chromosome fragments, thereby replacing the application of traditional GISH technology in chromosome analysis of Lilium. Summary of the Invention

[0006] In view of the problems that the existing technology of chromosome identification of Lilium plants relies on the GISH method, such as cumbersome operation, long cycle, high cost and complex probe preparation, the purpose of the present invention is to provide a simple, fast and economical chromosome identification solution that can replace the GISH technology.

[0007] The purpose of the present invention is achieved through the following technical solutions: An oligonucleotide probe Oligo-(AG) that can specifically identify chromosomes of the Oriental lily genome 10 After the probe is fluorescently labeled with TAMRA at the 5' end, it can specifically mark the Oriental lily (Oriental) genome part in the OT type lily, showing a clear red fluorescent signal.

[0008] Oligo-(AG) 10 The nucleotide sequence is: 5'-AGAGAGAGAGAGAGAGAGAG-3'.

[0009] A method for identifying chromosomes in the genome of Oriental lily using Oligo-(AG) 10 ND-FISH detection of lily was performed using the probe.

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention does not require the extraction and labeling of genomic DNA, avoiding the tedious DNA extraction, purification and labeling steps of traditional GISH technology. It does not require DNA denaturation, maintains the integrity of chromosome structure, and significantly shortens the hybridization time from the traditional overnight hybridization to 2 hours.

[0011] 2. The elution step of the method of the present invention is simplified, requiring only simple buffer washing, reducing the amount of reagents used and reducing costs by about 80%.

[0012] 3. The specific probes and methods described in this invention are suitable for identification, cultivar classification, and phylogenetic relationship studies of lily hybrids. By labeling specific genomic components, the genomic composition and chromosomal origin of hybrids can be intuitively determined. This method is particularly suitable for identification and chromosome analysis of hybrids containing the Oriental lily genome, such as the OT lily (Oriental × Trumpet), LO lily (Musk × Oriental), and OA lily (Oriental × Asiatic), accurately distinguishing chromosome segments derived from different parents.

[0013] 4. The method described in this invention can also accurately detect chromosomal recombination during hybridization, visually demonstrating the distribution and integration of different parental genomes in hybrid offspring. For example, in OT-type lily hybrids, chromosome segments from Oriental lily and Trumpet lily can be clearly distinguished, as well as potential chromosome structural variations, providing critical cytogenetic evidence for breeding.

[0014] 5. The present invention provides efficient and economical technical support for lily genome research and breeding, and provides a reliable molecular cytological tool for lily chromosome composition analysis, hybrid identification and variety classification. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 In Example 3, the OT type lily 'Robina' uses Oligo-(AG) 10 -ND-FISH detection results of the TAMRA probe (×600), showing a specific red signal in the genomic region of Oriental lily.

[0016] Figure 2 This is the result of testing the OT type lily 'Robina' in Example 3 using traditional GISH technology (×600).

[0017] Figure 3 The OT type lily 'Nymph' in Example 4 uses Oligo-(AG) 10 -ND-FISH detection results of the TAMRA probe (×600), showing a specific red signal in the genomic region of Oriental lily.

[0018] Figure 4 This is the result of testing the OT lily 'Nymph' in Example 4 using traditional GISH technology (×600).

[0019] Figure 5 The OT hybrid seedlings of XN×MJ-02 ('Nymph'×Minjiang lily) in Example 5 were grown using Oligo-(AG) 10 -ND-FISH test results using TAMRA probe (×600) showed that the hybrid seedling contained a total of 36 chromosomes, of which 12 were OO and 24 were TT, with no recombination.

[0020] Figure 6 This is the result of the traditional GISH technique for detecting XN×MJ-02 ('Nymph'×Lily of the Min River) in Example 5 (×600).

[0021] Figure 7 The hybrid seedlings of XN×MJ-044 ('Nymph'×Minjiang lily) in Example 6 were grown using Oligo-(AG) 10 -ND-FISH detection results of the TAMRA probe (×600) showed a total of 36 chromosomes, including 11 OO chromosomes, 24 TT chromosomes, and 1 recombinant chromosome.

[0022] Figure 8This is the result of the traditional GISH technique for detecting XN×MJ-044 ('Nymph'×Lily of the Min River) in Example 6 (×600). DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the examples, but the present invention is not limited to the examples.

[0024] Example 1 - Design and synthesis of specific probes In this example, the repetitive sequences of the lily genome were analyzed, and based on the analysis results, a specific oligonucleotide probe Oligo-(AG) was designed. 10 The sequence is 5'-AGAGAGAGAGAGAGAGAGAG-3'. The probe was synthesized by the inventors at Shanghai Sangon Biotechnology Co., Ltd. and directly labeled with a TAMRA fluorescent group at the 5' end to obtain a visible red fluorescent signal probe. Example 2

[0025] A method for identifying chromosomes in the genome of oriental lily comprises the following steps: (1) Material preparation: Select young root tips of lilies, water them the day before, cut off 2-4 cm long root tips between 8:00-10:00 the next day, and place them in a moist centrifuge tube.

[0026] (2) Root apex pretreatment: Treat with nitrous oxide (N2O) for 2-3 hours, then add 90% acetic acid and fix on ice for 10-15 minutes. After washing with ddH2O 3-5 times, add 70% ethanol and store at -20°C.

[0027] (3) Chromosome preparation: Wash the preserved root tips with ddH2O, cut the meristem tissue, and place it in a mixture of 2% cellulase and 1% pectinase for enzymatic hydrolysis at 37°C for 50-60 minutes. After enzymatic hydrolysis, wash the root tips with 70% ethanol, grind the tissue, and centrifuge (3500 rpm, 3 minutes) before pouring off the ethanol. Add acetic acid at a ratio of 28 μL glacial acetic acid per root tip and vortex to mix. Take 10 μL of the suspension and drop it on a glass slide to dry naturally. Mark the mitotic phase position under a microscope and store after UV crosslinking.

[0028] (4) ND-FISH detection: ① Probe dissolution: Dissolve the synthesized Oligo-(AG) 10 -TAMRA probe dry powder was centrifuged (12000 r / min, 3 minutes), and 100 μL of 1× TE solution was added at 1 OD to prepare a 100× concentration probe stock solution.

[0029] ② Probe dilution: Dilute the original solution to 1000× working solution using a 1:1 buffer consisting of 2×SSC (standard salt solution):1×TE.

[0030] ③ Hybridization reaction: Prepare hybridization solution according to the hybridization system (1 μL probe + 9 μL buffer), and add 10 μL of hybridization solution to the split phase area.

[0031] ④ Hybridization conditions: Hybridization at 37°C for 2 hours, without DNA denaturation step.

[0032] ⑤ Elution: Elute with 2×SSC solution preheated to 42°C and blow dry quickly.

[0033] ⑥ Staining: add 8 μL DAPI anti-fading agent for counterstaining and observe and photograph under a fluorescence microscope.

[0034] ⑦Test results: Through fluorescence microscopy, Oligo-(AG) 10 -TAMRA produces a specific red signal in the genomic region of Oriental lily, which can clearly identify the part of the Oriental lily genome in the OT type lily. Example 3

[0035] OT type lily 'Robina' was tested by ND-FISH and traditional GISH technology using the method described in Example 2. The results are as follows: Figure 1-Figure 2 shown.

[0036] The results of ND-FISH detection using the method described in Example 2 are as follows: Figure 1 As shown in the figure, 24 chromosomes of the OT lily 'Robina' show significant red fluorescent signals, indicating that this part is derived from the genomic region of Oriental lily (OO); the remaining 12 chromosomes that do not show red signals are derived from trumpet lily (TT) and appear blue after DAPI staining. This result shows that Oligo-(AG) 10 The probe can efficiently and specifically mark the repetitive sequence region unique to the Oriental lily genome, thereby enabling rapid identification of the chromosome composition of OT-type lilies.

[0037] To verify the accuracy of the ND-FISH method, this example also used traditional GISH technology to detect the same material, and the results were as follows: Figure 2 Although GISH can also identify 24 chromosomes from Oriental lilies, the procedure is cumbersome and requires extraction and labeling of genomic DNA, followed by high-temperature denaturation. The hybridization process is time-consuming and results in high fluorescence background, making it unsuitable for high-throughput applications. Example 4

[0038] To further verify the universality and applicability of the method of the present invention in the rapid identification of chromosome composition of OT lily, this example selected OT lily 'Nymph' as ​​the experimental material, and used the ND-FISH method described in Example 2 and the traditional GISH technology for detection. The results are as follows Figure 3-Figure 4 shown.

[0039] Figure 3 This is a chromosome hybridization image obtained using the method described in Example 2. Twelve chromosomes clearly display a distinct red fluorescent signal, indicating that these chromosome segments originate from the Oriental lily (OO) genome. The remaining 12 chromosomes, not marked by the red signal, are displayed with a blue DAPI signal and correspond to the Trumpet Lilium (TT) genome. The clear distribution of the red fluorescent signal and low background interference demonstrate the accurate identification of genomic components in the Oriental lily 'Nymph', confirming the excellent versatility of this probe across different OT-type lily accessions. Figure 4 This figure shows the detection results of the same sample using traditional GISH technology. The red fluorescent signal on the chromosomes of Oriental lily can also be observed in the figure. However, the signal distribution is relatively diffuse, and there is local nonspecific background fluorescence. Example 5

[0040] Hybrid seedlings numbered XM-02 ('Nymph'×Minjiang lily) were subjected to hybrid identification by ND-FISH detection and traditional GISH detection using the method described in Example 2. The results are as follows: Figure 5-Figure 6 shown.

[0041] Figure 5 This image shows an XN×MJ-02 hybrid seedling detected using the method described in Example 2. A total of 36 metaphase chromosomes were observed, 12 of which showed a distinct red fluorescent signal, indicating their origin from the Oriental Lilium (OO) genome. The remaining 24 chromosomes showed no red fluorescent signal but were stained blue with DAPI, indicating their origin from the Trumpet Lilium (TT) genome. The concentrated distribution of the red signal, with strong specificity and minimal background interference, clearly distinguished the two sets of chromosome components, validating the ability of this method to accurately determine genomic composition ratios in actual hybrids.

[0042] Figure 6 These results are from the same sample tested using traditional GISH technology. Red fluorescent signals from chromosomes of OO origin are also observed in the image, but the signal distribution is uneven, with diffuse or blurred signals appearing on some chromosomes. Furthermore, this method requires the preparation and fluorescent labeling of genomic DNA from Oriental lilies. The hybridization process is complex, requiring high-temperature denaturation and prolonged incubation, resulting in a long experimental cycle and unsuitable for high-throughput screening of batches of samples. Example 6

[0043] Hybrid seedlings numbered XM-044 ('Nymph'×Minjiang lily) were subjected to hybrid identification by ND-FISH detection and traditional GISH detection using the method described in Example 2. The results are as follows: Figure 7-Figure 8 shown.

[0044] Figure 7 This is a chromosome image of an XN×MJ-044 hybrid seedling obtained using the method described in Example 2. There are 36 chromosomes in total, 11 of which show a clear red fluorescent signal, belonging to the Oriental lily (OO) genome. Twenty-four unlabeled chromosomes show a blue signal stained with DAPI, corresponding to the Trumpet Lilium (TT) genome. Furthermore, a chromosome with a partial red fluorescent signal is clearly visible in the image, appearing to be stained at one end and unlabeled at the other, presumably a chromosome recombinant between Oriental lily and Trumpet Lilium. These results demonstrate that ND-FISH can not only accurately identify the origin of purebred chromosomes but also detect fragmented recombinant chromosomes that occur during hybridization, demonstrating excellent resolution and specificity.

[0045] Figure 8 These are the results obtained using traditional GISH technology for the same material. The figure also shows red signals on some chromosomes, identifying chromosomes of OO origin. A recombinant chromosome with a red signal at only one end is also clearly visible. However, the chromosome processing may have been prolonged, resulting in chromosome swelling. Furthermore, traditional GISH relies on genomic DNA extraction, fluorescent labeling, and denaturing hybridization, which is a complex and time-consuming process, hindering the efficient analysis of large numbers of samples in hybrid breeding.

[0046] The results of Example 5 and Example 6 show that the method of the present invention achieves equally clear results in chromosome identification of lily hybrids compared with the traditional GISH technology, and is simpler to operate, takes less time, and has lower costs.

[0047] According to the test results of Examples 3 to 6, the method of the present invention has multiple advantages over the traditional GISH technology, as shown in Table 1.

[0048]

[0049] While the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection of the present invention.

Claims

1. An oligonucleotide probe Oligo-(AG) that can specifically identify chromosomes in the Oriental lily genome 10 , characterized in that, Its nucleotide sequence is: 5'-AGAGAGAGAGAGAGAGAGAG-3'.

2. A method for identifying chromosomes in the genome of Oriental lily, characterized in that: Utilize the oligonucleotide probe Oligo-(AG) that can specifically identify the chromosome of the oriental lily genome according to claim 1 10 ND-FISH detection was performed on lily.

3. The method for identifying chromosomes in the oriental lily genome according to claim 2, wherein: The specific steps include: (1) Material preparation: Select young lily root tips, water them the day before, cut 2-4 cm long root tips between 8:00-10:00 the next day, and place them in a moist centrifuge tube; (2) Root apex pretreatment: Treat with N2O for 2-3 hours, then add 90% acetic acid and fix on ice for 10-15 minutes, wash with ddH2O 3-5 times, and then add 70% ethanol and store at -20°C; (3) Chromosome preparation: The preserved root tips were washed with ddH2O, and the meristem tissue was cut and placed in a mixture of 2% cellulase and 1% pectinase for enzymatic hydrolysis at 37°C for 50-60 minutes; the root tips after enzymatic hydrolysis were washed with 70% ethanol, and the tissue was ground and centrifuged at 3500 r / min for 3 minutes, and then the ethanol was poured out; acetic acid was added at a ratio of 28 μL glacial acetic acid per root tip, and vortexed to mix; 10 μL of the suspension was dropped on a glass slide and naturally dried, and the mitotic phase position was marked under a microscope, and preserved after UV crosslinking; (4) ND-FISH detection: ① Probe dissolution: Dissolve the synthesized Oligo-(AG) 10 -TAMRA probe powder was centrifuged at 12000 r / min for 3 minutes, and 100 μL of 1× TE solution was added to 1 OD to prepare a 100× concentration probe stock solution; ② Probe dilution: dilute the original solution to 1000× working solution with 2×SSC:1×TE=1:1 buffer; ③ Hybridization reaction: Mix 1 μL probe and 9 μL buffer to prepare hybridization solution, and add 10 μL hybridization solution to the split phase area; ④ Hybridization conditions: Hybridization at 37°C for 2 hours; ⑤ Elution: Elute with 2×SSC solution preheated to 42°C and blow dry quickly; ⑥ Staining: add 8 μL DAPI anti-fading agent for counterstaining and observe and photograph under a fluorescence microscope.

4. The oligonucleotide probe Oligo-(AG) capable of specifically identifying the chromosome of the Oriental lily genome according to claim 1 10 Application in identifying chromosomes in lily genome.

5. Containing the oligonucleotide probe Oligo-(AG) capable of specifically identifying the chromosome of the Oriental lily genome according to claim 1 10 detection kit.

Images