A probe for identifying the short arm of wheat chromosome 2A and its use and identification method

Abstract

The present invention discloses a probe for identifying the short arm of wheat chromosome 2A, its use and identification method, and belongs to the technical field of molecular cytogenetics. The nucleic acid sequence of the probe is shown in SEQ ID NO.1. The present application discloses a new 2A chromosome short arm near centromere region cytological detection probe Oligo‑pTa166‑1, which can quickly identify wheat chromosome 2A and 2A chromosome short arm in fluorescence in situ hybridization analysis.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular cytogenetics, and in particular relates to a probe for identifying the short arm of wheat 2A chromosome, and a use and an identification method thereof. Background Art

[0002] Wheat is one of the most important cereal crops in the world. Fluorescence in situ hybridization technology is widely used in its research on distant hybridization breeding, allopolyploidy and chromosome evolution. After the release of the wheat chromosome-level genome assembly, predecessors have systematically developed a series of oligonucleotide probes based on it. However, due to the limitations of the discovery methods at the time, the discovery of high-abundance repetitive sequences in the wheat reference genome was not sufficient, and there have been reports of the development of practical probes based on new types of repetitive sequences. At present, the hybridization signals of existing probes are still less distributed on some wheat chromosomes, which is not conducive to the development of genetic analysis and breeding research.

[0003] The probe combination developed based on pSc119.2 and pTa535 (Tang et al., 2014) can identify 21 pairs of chromosomes of wheat, but often has hybridization signals on multiple chromosomes at the same time, some signals are in similar positions, and the segments that can generate signals are limited. When the chromosome undergoes structural variations such as breakage, deletion, inversion or translocation, the recognition ability is limited.

[0004] In order to accurately identify specific chromosomes and their fragments in cytological analysis, it is necessary to develop more probes that can produce strong fluorescent signals in specific chromosome segments. Currently, few probes have been reported to produce hybridization signals in the near-centromere region of the short arm of wheat chromosome 2A (2AS): (1) Oligo-pTa535-1 (Tang et al., 2014), (AAG)5 (Cuadrado et al., 2000) and their similar probes have signals in the near-centromere region of 2AS, but they have too many signal points in the wheat genome, and they all have signals of similar intensity in the near-centromere region of 3AS, which lacks specificity; (2) Oligo-428 (Tang et al., 2018) produces weak hybridization signals in the near-centromere region of 2AS; (3) Using Oligo-2A or Oligo-2A plus (Zhang Jie et al., 2022) Wheat chromosome 2A can be specifically identified in fluorescence in situ hybridization analysis, but its hybridization signal is in the pericentromeric region of the 2A long arm (2AL), not the pericentromeric region of 2AS.

[0005] Therefore, it is necessary to develop a probe that can produce specific hybridization signals in the pericentromeric region of wheat 2AS. This will facilitate the direct identification of chromosome 2A with a single probe in cytological analysis and help identify structural variations resulting from the breakage of the 2AS centromeric region.

[0006] The conventional idea for finding cytological probes related to wheat chromosome 2A is to screen for repetitive sequences that are specifically enriched in chromosome 2A based on the reference genome. However, since the wheat genome is extremely large (about 17G), the repetitive sequence-enriched regions often cannot be assembled correctly. In the currently released reference genome, the repetitive sequences may be mounted in the wrong position and the copy number is also incorrect. Therefore, how to obtain a cytological probe that can quickly identify chromosome 2A is the key. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a probe for identifying the short arm of wheat chromosome 2A, and its use and identification method, which can quickly and accurately identify wheat chromosome 2A and the short arm of chromosome 2A.

[0008] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0009] A probe for identifying the short arm of wheat chromosome 2A, the nucleic acid sequence of the probe is shown in SEQ ID NO.1, and the specific sequence is as follows:

[0010] 5'-CAAAAATTATAAACTTTTTGTTAGTGCCATTAGTTTTCAAATTTG-3'(SEQ ID NO.1)

[0011] Furthermore, the 5' end of the probe is connected with a fluorescent labeling group.

[0012] Furthermore, the fluorescent labeling group is 6-carboxyfluorescein.

[0013] Further, the probe corresponds to the pericentromeric region of the short arm of wheat chromosome 2A.

[0014] A kit for identifying the short arm of wheat 2A chromosome, comprising the above-mentioned probe.

[0015] The use of the above probe or kit in identifying the short arm of wheat chromosome 2A.

[0016] The use of the above probe in wheat genetic analysis and breeding.

[0017] A method for identifying the short arm of wheat chromosome 2A comprises the following steps:

[0018] (1) synthesizing the above-mentioned probe sequence;

[0019] (2) Hybridizing the sample to be tested with the probe sequence obtained in step (1) and then examining under a microscope.

[0020] Furthermore, the probe in step (1) is prepared into a hybridization solution with a buffer solution, and then hybridization is performed; the probe concentration in the hybridization solution is 1 to 5 nmol / 100 μL.

[0021] Further, the buffer solution has a pH of 7.0, and includes 2×SSC and 1×TE in a volume ratio of 1:1.

[0022] Beneficial effects of the present invention:

[0023] By using the 2A chromosome short arm near centromere region cytological probe Oligo-pTa166-1 disclosed in the present invention, wheat 2A chromosome and 2A chromosome short arm can be quickly identified in fluorescence in situ hybridization analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 FISH images of hexaploid wheat; A is the FISH image using Oligo-pTa166-1 as a probe; B is the FISH image using Oligo-pSc119.2-1 and Oligo-pTa535-1 as probes;

[0025] Figure 2 FISH images of durum wheat-African rye amphidiploid; A is a FISH image using Oligo-pTa166-1 as a probe; B is a FISH image using Oligo-pSc119.2-1 and Oligo-pTa535-1 as probes;

[0026] Figure 3 These are the FISH images of some amphidiploids of wheat-Perennial Elaeagnus villosa; A is the FISH image using Oligo-pTa166-1 as a probe; B is the FISH image using Oligo-pSc119.2-1 and Oligo-pTa535-1 as probes;

[0027] Figure 4 FISH images of hexaploid triticale; A is the FISH image using Oligo-pTa166-1 as a probe; B is the FISH image using Oligo-pSc119.2-1 and Oligo-pTa535-1 as probes. DETAILED DESCRIPTION

[0028] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0029] Example 1 Synthesis of Oligo-pTa166-1 Probe

[0030] According to the reference genome sequence of wheat Chinese Spring IWGSC RefSeq v2.0 (https: / / www.wheatgenome.org), TR-toolkit (https: / / gitee.com / lhtk / TR-toolkit) and B2DSC (https: / / gitee.com / lhtk / b2dsc) were used to explore tandem repeat sequences and their fragments on single chromosomes in the genome. A 45bp sequence fragment was screened that was significantly enriched in the 687-688Mb region of chromosome 3A, with an enrichment peak greater than 200, and no 45bp sequence fragment with a distribution of more than 100 copies / Mb in other regions of the genome. Based on this fragment, a probe sequence was designed and obtained: 5'-CAAAAATTATAAACTTTTTGTTAGTGCCATTAGTTTTCAAATTTG-3' (SEQ ID NO.1).

[0031] The screened 45 bp sequence fragment was sent to a nucleic acid synthesis company to synthesize an oligonucleotide probe, and its 5' end was fluorescently labeled with green 6-carboxyfluorescein.

[0032] Example 2 Fluorescence in situ hybridization verification

[0033] 1. Take the root

[0034] (1) Hexaploid wheat, durum wheat-African rye amphidiploid, wheat-partial amphidiploid of perennial Eleutherodactylum and hexaploid triticale seeds were placed on moist filter paper and cultured at 23°C for 24 h.

[0035] (2) The seeds were cultured at 4°C for 24 h.

[0036] (3) The seeds were cultured at 23°C until they began to take root.

[0037] (4) When the root grows to 1-2 cm, take the root and place it in a moist 0.5 mL EP tube (the EP tube is placed on ice). A small hole with a diameter of about 3 mm should be poked on the lid of the 0.5 mL EP tube in advance to facilitate the nitrous oxide treatment of the root tip.

[0038] (5) Place the EP tube containing the roots into a nitrous oxide tank and allow the tank to be filled with nitrous oxide (0.9 Mpa) for 2 hours.

[0039] (6) Take out the EP tube and place it on ice. Add 90% acetic acid to it and fix the root tip for 8-10 minutes. Wash the roots twice with ddH2O.

[0040] (7) Add 70% ethanol to the EP tube and place it in a -20℃ refrigerator to preserve the roots for a long time.

[0041] 2. Production

[0042] (1) Wash the roots twice with ddH2O to remove ethanol from the roots.

[0043] (2) Cut off the root tip and place it in enzyme solution for enzymatic hydrolysis at 37°C for 1 h.

[0044] (3) Rinse the root tip twice with 70% ethanol to wash away the enzyme solution.

[0045] (4) Leave a small amount of ethanol and crush the root apex with a needle tip. Centrifuge (4000 rpm, 2 min) and then invert the EP tube to drain the ethanol.

[0046] (5) Add 15-50 μL of acetic acid to the EP tube and vortex to obtain a root tip cell suspension.

[0047] (6) Place the tablets in the moistened paper box.

[0048] 3. Hybridization:

[0049] (1) Preparation of hybridization solution: The synthesized probe powder is diluted with hybridization buffer to form a working solution for use. The final concentration of the working solution is 1 nmol / 100 μL. When in use, take 2 μL / slice of working solution, then add 8 μL / slice of buffer and mix well. Finally, drop 10 μL of hybridization solution on each slide. Hybridization buffer formula: 2×SSC:1×TE=1:1(V:V), pH=7.0.

[0050] (2) Hybridize for 3 h at 37°C in the dark.

[0051] (3) DAPI staining, cover with coverslip, and examine under Leica fluorescence microscope DM2500. Use black and white cooled CCD to take pictures, and add false color to the fluorescence signal. The results are as follows Figure 1 , 2 As shown in , 3, and 4, the white arrow points to chromosome 2A, and the white triangle points to chromosome 3A.

[0052] Depend on Figure 1 B. Figure 2 B. Figure 3 B and Figure 4B shows that the probes Oligo-pSc119.2-1 and Oligo-pTa535-1 can identify intact 2A and 3A chromosomes, but this pair of probes has signals on all 21 pairs of wheat chromosomes. Careful observation is required to distinguish between 2A and 3A chromosomes. In addition, there are Oligo-pTa535-1 hybridization signals in the near-centromeric regions of 2AS and 3AS with comparable intensities, which is not conducive to the identification of 2AS in the case of structural variations such as deletion of the short arm end.

[0053] And by Figure 1 A. Figure 2 A. Figure 3 A and Figure 4 A shows that the probe Oligo-pTa166-1 has the brightest, most stable and high-intensity hybridization signal in the near-centromere region of the short arm of chromosome 2A, while on other wheat, African rye, rye and tricholoma villosa chromosomes, there are signals only at the ends and near the ends, indicating that it has good specificity in the near-centromere region of the short arm of chromosome 2A and can be used to identify and distinguish the short arm of chromosome 2A.

[0054] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A probe for identifying the short arm of wheat chromosome 2A, characterized in that: The nucleic acid sequence of the probe is shown as SEQ ID NO.

1.

2. The probe according to claim 1, characterized in that The 5' end of the probe is connected with a fluorescent labeling group.

3. The probe according to claim 2, characterized in that: The fluorescent labeling group is 6-carboxyfluorescein.

4. The probe according to any one of claims 1 to 3, characterized in that The probe corresponds to the pericentromeric region of the short arm of wheat chromosome 2A.

5. A kit for identifying the short arm of wheat chromosome 2A, characterized in that: The invention comprises the probe according to any one of claims 1 to 4.

6. Use of the probe according to any one of claims 1 to 4, or the kit according to claim 5, in identifying the short arm of wheat chromosome 2A.

7. Use of the probe according to any one of claims 1 to 4 in wheat genetic analysis and breeding.

8. A method for identifying the short arm of wheat chromosome 2A, characterized in that: The following steps are involved: (1) synthesizing the probe sequence according to any one of claims 1 to 4; (2) Hybridize the sample to be tested with the probe sequence obtained in step (1) and examine under a microscope.

9. The method according to claim 8, characterized in that The probe in step (1) is prepared into a hybridization solution with a buffer solution, and then hybridization is performed; the probe concentration in the hybridization solution is 1-5 nmol / 100 μL.

10. The method according to claim 9, characterized in that The buffer solution has a pH of 7.0 and includes 2×SSC and 1×TE in a volume ratio of 1:1.

Images