Probes and methods for constructing single chromosome maps of wild oat species with high repetitive size and large genome

Abstract

The present invention discloses a probe group preparation method, a probe group sequence and a map construction method for constructing a single chromosome map of a species with a high repetitive sequence and a large genome, and belongs to the field of genetic engineering technology. The probe sequence is all sequences or their reverse complementary sequences contained in the OMIX database https: / / ngdc.cncb.ac.cn / omix / with the accession number OMIX006067. The probe group and method disclosed in the present invention have high density and long interval, and achieve the effect of clear signal visualization, which can be applied to the analysis of plant chromosome structural variation and auxiliary mining of planting resources.

Description

Technical Field

[0001] The invention belongs to the technical field of dye nucleic acid probe preparation and detection, and specifically relates to a probe set and method for constructing a single chromosome map of a species with a high repetitive sequence and a large genome. Background Art

[0002] Fluorescence in situ hybridization (FISH) plays an important role in the study of karyotype evolution. The large size of the oat genome (11.0 Gb) and the high repetition ratio (>80%) limit the development of basic oat biology and molecular breeding. Oligonucleotide (Oligo) probe libraries include single-stranded probe libraries and double-stranded probe libraries. The single-stranded probe library is obtained by transcribing the target library into RNA and then reversely transcribing it into cDNA, and then labeling it with a fluorescent group. For example, it has been used in chromosome rearrangements of rice (Oryza sativa) and potato (Solanum tuberosum) (doi:10.1534 / genetics.117.300344; doi:10.1186 / s12870-019-1769-z). The disadvantage is that the probe library preparation process is cumbersome and the reagents used are expensive. Double-stranded probe libraries can be quickly obtained through PCR amplification and are suitable for large genomes (>3.5Gb). Their disadvantages are low universality between different species, more thorough shielding of repetitive sequences by probe library design, and high quality requirements for white film production.

[0003] Breakage and recombination are the main causes of chromosome structural variation. For example, comparative genomic studies have speculated that spring oat chromosome 7D may have an inversion event (doi:10.1038 / s42003-022-03256-5). Since most inversions are concentrated in repetitive sequence-rich regions, it is difficult to determine the breakpoint position of the inversion based solely on genomic sequence information. With the help of single chromosome research, the colinear genes flanking the breakpoints can be visually identified, which opens up new ideas for clarifying the breakpoints and rejoining sites. Inversions affect the positional effect of certain genes, such as genes related to the lipid synthesis pathway and beta-glucan synthase (doi.org / 10.2135 / cropsci2015.11.0678), indicating that the detection technology of single chromosomes has great scientific research value and economic benefits in the exploration of nutrition-related germplasm resources of wild oats. At present, the construction of visualized single chromosome fluorescent marker maps of species with high repetitive sequences and large genomes is still challenging, and there has been no report on the visualization of alternative methods for the construction of single chromosome maps of wild species of the genus Avena. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a probe set and a construction method for constructing a single chromosome map of a species with a high repetitive sequence and a large genome.

[0005] In one aspect, the present invention provides a method for designing a chromosome fluorescent labeling probe set, characterized in that the method comprises the following steps:

[0006] 1) Remove repetitive sequences based on the reference genome mask;

[0007] 2) Obtaining a 45 bp single copy oligonucleotide sequence library;

[0008] 3) Remove short fragments containing ≥6 consecutive identical base repeats and retain short sequences with a GC content of 25-80%;

[0009] 4) retain short sequence libraries with dTm values ​​(Tm – hairpin Tm) greater than 10°C;

[0010] 5) Align the short sequence library back to the target region and remove the probe fragments in the non-target region to obtain the probe set;

[0011] Furthermore, the fluorescent labeling probe set is suitable for constructing a visualized single chromosome fluorescent labeling map of species with high repetitive sequences and large genomes.

[0012] In a preferred embodiment, step 1) can use RepeatMasker2 (http: / / www.repeatmasker.org) or any other software that can achieve the same effect, step 2) can use Chorus2 software or any other software that can achieve the same effect, step 4) can use Primer3 or any other software that can achieve the same effect, and step 5) can use Geneious Primer v.2022.0.2 (https: / / www.geneious.com / ) or any other software that can achieve the same effect.

[0013] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 60% and a genome greater than or equal to 1G.

[0014] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 70% and a genome greater than or equal to 2G.

[0015] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 80% and a genome greater than or equal to 3G.

[0016] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 80% and a genome greater than or equal to 3.5G.

[0017] In a preferred embodiment, the highly repetitive sequence large genome species is a plant.

[0018] In a preferred embodiment, the high repetitive sequence large genome species is a Poaceae plant.

[0019] In a preferred embodiment, the species with a large genome and high repetitive sequences is a plant of the genus Avena.

[0020] In a preferred embodiment, the species with high repetitive sequence and large genome is Oat.

[0021] In a preferred embodiment, the reference genome is a reference genome of a wild species of oat, and its GenBank number is GCA_030063025.1.

[0022] In a preferred embodiment, the base sequences of the chromosome intervals shown in Table 3 are selected as reference sequences for designing probes.

[0023] In a preferred embodiment, the base sequence of the chromosome interval shown in Table 3 or its completely reverse complementary sequence is finally selected as the probe.

[0024] In a preferred embodiment, at least one of the four probe libraries is finally selected as the probe group, and the four probe libraries are G12405, G16009, R41087 and R416126, respectively, wherein the probe contained in G12405 corresponds to the nucleotide sequence contained in the file ID OMIX006067-01 of OMIX006067 or its complete reverse complementary sequence, the probe contained in G16009 corresponds to the nucleotide sequence contained in the file ID OMIX006067-02 of OMIX006067 or its complete reverse complementary sequence, the probe contained in R41087 corresponds to the nucleotide sequence contained in the file ID OMIX006067-03 of OMIX006067 or its complete reverse complementary sequence, and the probe contained in R41626 corresponds to the nucleotide sequence contained in the file ID OMIX006067-04 of OMIX006067 or its complete reverse complementary sequence.

[0025] In a preferred embodiment, fluorescent labels are added to both ends of the probe.

[0026] In a preferred embodiment, universal adapters F and R for amplification are further designed at both ends of the probe, and the probe group is amplified and enriched using fluorescently labeled primers.

[0027] In a preferred embodiment, the fluorescently labeled primer is a primer labeled with Biotin and / or Digoxygenin at the 5' end.

[0028] On the other hand, the present invention provides a probe set for constructing a visualized single chromosome fluorescent labeling map of a species with a high repetitive sequence and a large genome, wherein the probe set is designed according to any of the aforementioned methods.

[0029] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 60% and a genome greater than or equal to 1G.

[0030] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 70% and a genome greater than or equal to 2G.

[0031] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 80% and a genome greater than or equal to 3G.

[0032] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 80% and a genome greater than or equal to 3.5G.

[0033] In a preferred embodiment, the highly repetitive sequence large genome species is a plant.

[0034] In a preferred embodiment, the high repetitive sequence large genome species is a Poaceae plant.

[0035] In a preferred embodiment, the species with a large genome and high repetitive sequences is a plant of the genus Avena.

[0036] In a preferred embodiment, the species with high repetitive sequence and large genome is Oat.

[0037] In a preferred embodiment, the reference genome is a reference genome of a wild species of oat, and its GenBank number is GCA_030063025.1.

[0038] In a preferred embodiment, the probe set is a base sequence designed from the chromosome interval shown in Table 3 by the aforementioned method or its completely reverse complementary sequence.

[0039] In a preferred embodiment, the probe group is at least one of four probe libraries, and the four probe libraries are G12405, G16009, R41087 and R416126, respectively, wherein the probe contained in G12405 corresponds to the nucleotide sequence contained in the file ID OMIX006067-01 of OMIX006067 or its complete reverse complementary sequence, the probe contained in G16009 corresponds to the nucleotide sequence contained in the file ID OMIX006067-02 of OMIX006067 or its complete reverse complementary sequence, the probe contained in R41087 corresponds to the nucleotide sequence contained in the file ID OMIX006067-03 of OMIX006067 or its complete reverse complementary sequence, and the probe contained in R41626 corresponds to the nucleotide sequence contained in the file ID OMIX006067-04 of OMIX006067 or its complete reverse complementary sequence.

[0040] In a preferred embodiment, the probe group comprises four probe libraries: G12405, G16009, R41087 and R416126.

[0041] In a preferred embodiment, both ends of the probe are linked to fluorescent labels.

[0042] In a preferred embodiment, the two ends of the probe are linked with universal adapters F and R for amplification, and the probe group can be amplified and enriched using fluorescent labeled primers.

[0043] In a preferred embodiment, the probe is obtained by amplification using a fluorescently labeled primer, and the fluorescently labeled primer is a primer labeled with Biotin and / or Digoxygenin at the 5' end.

[0044] In a preferred embodiment, the two ends of the probes in the four probe libraries G12405, G16009, R41087 and R416126 are linked to universal adapters F and R, and the probe group can be amplified and enriched using fluorescent labeled primers, and the fluorescent labeled primers are shown in Table 1.

[0045] On the other hand, the present invention provides the use of the aforementioned probe set in constructing a visualized single chromosome fluorescent labeling map of a species with a high repetitive sequence and a large genome.

[0046] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 60% and a genome greater than or equal to 1G.

[0047] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 70% and a genome greater than or equal to 2G.

[0048] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 80% and a genome greater than or equal to 3G.

[0049] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 80% and a genome greater than or equal to 3.5G.

[0050] In a preferred embodiment, the highly repetitive sequence large genome species is a plant.

[0051] In a preferred embodiment, the high repetitive sequence large genome species is a Poaceae plant.

[0052] In a preferred embodiment, the species with a large genome and high repetitive sequences is a plant of the genus Avena.

[0053] In a preferred embodiment, the species with high repetitive sequence and large genome is Oat.

[0054] On the other hand, the present invention provides a method for constructing a visualized single chromosome fluorescent marker map of a species with a high repetitive sequence and a large genome, characterized in that the method comprises the following steps:

[0055] 1) Design and synthesis of probe sets;

[0056] 2) PCR fluorescent labeling of probe sets;

[0057] 3) In situ hybridization of the probe set with chromosomes of the target species.

[0058] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 60% and a genome greater than or equal to 1G.

[0059] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 70% and a genome greater than or equal to 2G.

[0060] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 80% and a genome greater than or equal to 3G.

[0061] In a preferred embodiment, the high-repetitive sequence large genome species is a species with a genome repeat sequence ratio higher than 80% and a genome greater than or equal to 3.5G.

[0062] In a preferred embodiment, the highly repetitive sequence large genome species is a plant.

[0063] In a preferred embodiment, the high repetitive sequence large genome species is a Poaceae plant.

[0064] In a preferred embodiment, the species with a large genome and high repetitive sequences is a plant of the genus Avena.

[0065] In a preferred embodiment, the species with high repetitive sequence and large genome is Oat.

[0066] In a preferred embodiment, the design in step 1) is performed using the aforementioned design method for chromosome fluorescent labeling probe sets.

[0067] In a preferred embodiment, the probe set is a base sequence from the chromosome interval shown in Table 3 or its complete reverse complementary sequence.

[0068] In a preferred embodiment, the probe group is at least one of four probe libraries, and the four probe libraries are G12405, G16009, R41087 and R416126, respectively, wherein the probe contained in G12405 corresponds to the nucleotide sequence contained in the file ID OMIX006067-01 of OMIX006067 or its complete reverse complementary sequence, the probe contained in G16009 corresponds to the nucleotide sequence contained in the file ID OMIX006067-02 of OMIX006067 or its complete reverse complementary sequence, the probe contained in R41087 corresponds to the nucleotide sequence contained in the file ID OMIX006067-03 of OMIX006067 or its complete reverse complementary sequence, and the probe contained in R41626 corresponds to the nucleotide sequence contained in the file ID OMIX006067-04 of OMIX006067 or its complete reverse complementary sequence.

[0069] In a preferred embodiment, the probe group comprises four probe libraries: G12405, G16009, R41087 and R416126.

[0070] In a preferred embodiment, both ends of the probe are linked to fluorescent labels.

[0071] In a preferred embodiment, the two ends of the probe are linked with universal adapters F and R for amplification, and the probe group can be amplified and enriched using fluorescent labeled primers.

[0072] In a preferred embodiment, the probe is obtained by amplification using a fluorescently labeled primer, and the fluorescently labeled primer is a primer labeled with Biotin and / or Digoxygenin at the 5' end.

[0073] In a preferred embodiment, the two ends of the probes in the four probe libraries G12405, G16009, R41087 and R416126 are linked to universal adapters F and R, and the probe group can be amplified and enriched using fluorescent labeled primers, and the fluorescent labeled primers are shown in Table 1.

[0074] In a preferred embodiment, step 3) includes 3.1) metaphase chromosome preparation, 3.2) fluorescence in situ hybridization, and 3.3) photography and image processing.

[0075] In a preferred embodiment, step 3.1) comprises:

[0076] (1) Collecting root tips: Place the seeds in a culture dish covered with moist filter paper and germinate them in a 25°C incubator for 48 h, keeping the filter paper moist (sampling time is 8:45–9:00 am).

[0077] (2) Pretreatment: When the root tip grows to 1–2 cm, place the root tip in an ice-water mixture for 24 h.

[0078] (3) Fixation: 1–2 cm of the root apex was cut and placed in freshly prepared Carnoy’s fixative (anhydrous ethanol: glacial acetic acid = 3:1, v / v). The root was fixed at 37°C for 30 min, placed at room temperature for 2 h, and then stored at -20°C. The shelf life is half a year.

[0079] (4) Enzymatic hydrolysis: The fixed root tips were removed and washed three times with distilled water for 10 min each time; soaked in 1× enzyme buffer (40 ml 10 mM citric acid + 60 ml 10 mM trisodium citrate diluted 10 times) for 10 min; placed in 37°C enzyme mixture [2% (w / v) Cellulase (20 U / ml, Sigma C1184) + 0.2% (w / v) Cellulase (10 U / ml, Onozuka RS) + 3% Pectinase (20 U / ml, Sigma P4716)] for enzymatic hydrolysis for about 90 min; the root tips were aspirated and soaked in 1× enzyme buffer for 10 min.

[0080] (5) Preparation of white slides: Use a pipette to carefully pipette the root apex onto a clean adhesive slide (Citglas, #C / N188105), use filter paper to absorb excess enzyme buffer, add one drop (20ul) of 60% acetic acid, use a dissecting needle to crush the root apex and remove all tissues except the meristem, smear the slide, cover with a coverslip (18×18mm), squeeze gently, and examine under a phase contrast microscope. Select white slides that are well dispersed and have ≥5 metaphases, mark with a pencil, remove with liquid nitrogen, and dry for later use.

[0081] In a preferred embodiment, step 3.2) comprises:

[0082] (1) Pretreatment: The slides were treated with freshly prepared fixative (anhydrous ethanol: glacial acetic acid = 3:1, v / v) for 15 min, then treated twice with 100% ethanol, 5 min each time, and dried at room temperature.

[0083] (2) RNase treatment: Prepare RNAse solution [20 μl RNaseA (10 mg / μl) + 1980 μl 2×SSC], add 250 μl RNAse solution to each slide, cover with sealing film, place in a wet box and incubate at 37°C for 1 h, then wash twice with 2×SSC for 2 min and 10 min respectively.

[0084] (3) Pepsin treatment: 0.01 M HCl treatment for 2 min, add 250 μl 7.5 μg / ml pepsin solution [15 μl Pepsin (1 mg / ml) + 1985 μl 0.01 M HCl] to each slide, incubate in a wet box at 37°C for 10 min, wash with distilled water for 1 min, and wash twice with 2× SSC, 5 min each time.

[0085] (4) Paraformaldehyde fixation: Place the slides in a 4% paraformaldehyde solution in a ventilated place, incubate at room temperature for 10 min, and wash twice with 2×SSC, for 2 min and 10 min respectively.

[0086] (5) Dehydration: Place the slides in 50%, 70%, 85%, and 100% ethanol solutions in sequence, dehydrate for 2 min each, and air-dry.

[0087] (6) Hybridization: Prepare hybridization mixture, dispense 34 μl into each tube (Table 4), add appropriate amount of probe (Table 5), denature at 85°C for 10 min, keep on ice for 10 min, drop the hybridization mixture with probes on the chromosome region of the slide, cover with high temperature resistant sealing film, denature at 72°C for 7 min in the first cycle in the hybridizer, and keep at 37°C overnight for 16–20 h in the second cycle. The components of the hybridization mixture system are shown in Table 4.

[0088] In a preferred embodiment, step 3.3) includes: examining with a Nikon fluorescence microscope (Ni-U, Tokyo, Japan) in a dark room, capturing each metaphase phase via four fluorescence excitation blocks, UV-2A (EX355 / 50, DM400, BA410), FITC (EX465-495, DM505, BA512-558), TRITC (EX537-552, DM565, BA582-637), ET-Cy5 (ET620 / 60, T660lpxr, ET700 / 75), and using the software NIS-Elements v.2.3.4 (Nikon, Tokyo, Japan) to control the CCD camera pco.panda 4.2 (PCO, Dassow, Germany) to capture images, and the exposure times for the red, green, and blue channels are 5 sec, 250 millisec, and 90 millisec, respectively. Images were processed using Image J v.1.51j8 software (Wayne Rasband, NIH, USA) and Photoshop CS6 v.13.0 software (Adobe System, San Jose, USA).

[0089] In a preferred embodiment, the content of the probe combination used in step 3.2) is: 2.1ul of G12405_Dig+G16009_Dig (green), and 1.7ul of R41087_Bio+R416126_Bio (red).

[0090] Compared with the prior art, the present invention has the following advantages:

[0091] 1) The present invention provides a method for constructing a high-resolution, visualized in situ map of a single chromosome suitable for large genomes (greater than 3.5 Gb) with high repetitive sequences (greater than 80%).

[0092] 2) The present invention provides an oligonucleotide probe library for constructing a single chromosome identification of a large genome (greater than 3.5 Gb) with high repetitive sequences (greater than 80%), the probe library has high density (0.949-1.432 oligos / kb), long interval (4.49-9.93

[0093] Mb), the hybridization signal is discernible. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0095] Figure 1Shown is the marking pattern of oligonucleotide probe libraries G12405_Dig+G16009_Dig (green) and R41087_Bio+R416126_Bio (red) on the chromosome marker sequence of the long-leaved oat reference genome (see Table 3 for signal site names and chromosome locations).

[0096] Figure 2 Shown are the Oligo-FISH hybridization results of the control group of long-leaved oat probe library G12405_Dig (green) and R41087_Bio (red) (chromosomes are numbered from long to short): (A) Oligo-FISH results of probe library G12405Dig (green) plus 1.2ul + R41087Bio plus 1.7ul, the hybridization signal is not discernible. (B) Oligo-FISH results of probe library G12405Dig (green) plus 1.5ul + R41087Bio plus 1.2ul, the hybridization signal is not discernible. (C) Oligo-FISH results of probe library G12405Dig (green) plus 1.8ul + R41087Bio plus 1.5ul, the hybridization signal is not discernible. (D) Oligo-FISH results of probe library G12405Dig (green) plus 2.1ul + R41087Bio plus 1.7ul, the hybridization signal is not discernible.

[0097] Figure 3 The results of Oligo-FISH hybridization of the probe library of Oat longifolia are shown, and the volume gradient of the dual probe library is explored. (AC) Oligo-FISH results with unsatisfactory hybridization effects: (A) Oligo-FISH results with 1.2ul of probe library G12405Dig+G16009Dig (green) and 1.7ul of R41087Bio+R416126Bio (red), respectively, and the green signal is not discernible. (B) Oligo-FISH results with 1.5ul of probe library G12405Dig+G16009Dig (green) and 1.2ul of probe library R41087Bio+R416126Bio (red), respectively, and the green signal is not discernible. (C) Oligo-FISH results of adding 1.8ul of probe library G12405Dig+G16009Dig (green) and 1.5ul of probe library R41087Bio+R416126Bio (red). The green and red signals are identifiable but weak. (D) Oligo-FISH results of adding 2.1ul of probe library G12405Dig+G16009Dig (green) and 1.7ul of probe library R41087Bio+R416126Bio (red). Both green and red signals are bright. The cross represents trace impurities ( Figure 3 D hybridization results and Figure 1 model Figure 1 The results show that the hybridization effect of the double library with a span of 4.491–9.934 Mb and an oligonucleotide density of 0.949–1.432 oligos / kb is the best. The signal description is shown in Table 6. The chromosome number and Figure 1 Metaphase chromosomes were stained with DAPI, scale bar = 5 um.

[0098] Figure 4 The chromosome distribution pattern of the optimal combination probe. There are strong red patchy signals (AL01S1, AL01S2) on the short arms of chromosomes 1 to 2; there are a pair of strong green patchy signals (AL02L1) on the long arms of chromosomes 3 to 4, and a pair of strong red patchy signals (AL02L2) at the ends of the long arms; there are a pair of red patchy signals (AL03S1) at the ends of the short arms of chromosomes 5 to 6, weak red dot signals in the pericentromere region of the long arms (AL03L1), and a pair of strong green patchy signals at the ends of the long arms (AL03L 2); there is a pair of strong plaque-like green signals (AL04S1) at the ends of the short arms of chromosomes 7-8; there are strong plaque-like red signals (AL05S1) at the ends of the short arms of chromosomes 9-10, and weak red dot-like signals (AL05L1) at the ends of the long arms; there are strong plaque-like green signals (AL06S1) at the ends of the short arms of chromosomes 11-12, and red signals (AL06L1) at the ends of the long arms; there are dot-like to plaque-like red signals (AL07L1) in the inter-arm region of chromosomes 13-14. DETAILED DESCRIPTION

[0099] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0100] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs. The probe library and probe group mentioned in the present invention have the same meaning, both referring to a collection of multiple probes.

[0101] Example 1

[0102] Screening and preparation of oligonucleotide sequence library

[0103] The probe library was designed using the chromosome-level genome sequence of Avena longiglumis assembled in our laboratory (GenBank website https: / / identifiers.org / ncbi / insdc.gca:GCA_030063025.1). The genome size of Avena longiglumis is 3.97 Gb, the heterozygosity is 0.48%, and the repetitive sequences account for 82.08%.

[0104] The steps for probe design are as follows:

[0105] In the first step, RepeatMasker2 (http: / / www.repeatmasker.org) software was used to mask and remove repeated sequences with default parameters.

[0106] In the second step, Chorus2 software ( https: / / github.com / zhangtaolab / Chorus2) to obtain the long Oat 45bp single copy oligonucleotide sequence library .

[0107] In the third step, short fragments containing ≥6 consecutive identical base repeats were removed, and short sequences with a GC content of 25-80% were retained.

[0108] The fourth step is to call Primer3 software to retain the short sequence library with a dTm value (Tm-hairpin Tm) greater than 10°C.

[0109] In the fifth step, Geneious Primer v.2022.0.2 software (https: / / www.geneious.com / ) was used with the parameters of Custom Sensitivity, maximum mismatches per oligo 60%, maximum gap size 3, and minimum overlap identity 50%. The short sequence library was aligned back to the target region and the probe fragments in the non-target region were removed ( Figure 1 , Figure 2 ).

[0110] Step 6: Design F and R primers for the probe.

[0111] The software in the above steps can be replaced by any other software or script that has the function of implementing the step.

[0112] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize oligonucleotide probe libraries (probe sets) G12405, G16009, R41087 and R416126 using the semiconductor chip method.

[0113] Probe libraries G12405, G16009, R41087 and R416126 consist of all probe sequences designed in the chromosome intervals described in Table 3 in steps 1 to 5 above. The sequences of probe libraries G12405, G16009, R41087 and R416126 corresponding to the genome of oat is all the sequences corresponding to the accession number OMIX006067 in the OMIX database (https: / / ngdc.cncb.ac.cn / omix / ).

[0114] The probe contained in G12405 corresponds to the nucleotide sequence contained in the file ID OMIX006067-01 of OMIX006067, the probe contained in G16009 corresponds to the nucleotide sequence contained in the file ID OMIX006067-02 of OMIX006067, the probe contained in R41087 corresponds to the nucleotide sequence contained in the file ID OMIX006067-03 of OMIX006067, and the probe contained in R41626 corresponds to the nucleotide sequence contained in the file ID OMIX006067-04 of OMIX006067.

[0115] Furthermore, the probes contained in the above probe library are completely consistent with the sequences contained in the corresponding OMIX database accession numbers, or are completely complementary pairs.

[0116] Optimize the labeling of the oligonucleotide probe library. Preliminary experiments have shown that Biotin and Digoxygenin are the 5' primer modifications with the best labeling effect. Guangzhou Qingke Biotechnology Co., Ltd. was commissioned to synthesize the modified primers (Table 1).

[0117] Table 1 List of modified primers

[0118]

[0119] PCR amplification labeled double-stranded probe library. Using oligonucleotide probe library as template, PCR amplification method is used to label the probe library. The purpose of PCR is to enrich the probe sequence, and different fluorescent labeled primers are used to amplify the probe library fragments. The fluorescent substance is coupled to the 5' position of the F primer and the 5' position of the R primer (Table 1). The PCR reaction system is shown in Table 2. The reaction procedure is: pre-denaturation 98℃ / 30sec, followed by 24 cycles (denaturation 98℃ / 10sec, annealing 64℃ / 10sec, extension 72℃ / 20sec), and final extension 72℃ / 3min. After the reaction is completed, 2.5% agarose gel electrophoresis detection (60v, 60min) is performed to detect the product length. Nanodrop ultra-micro spectrophotometer (Thermo Scientific, Delaware, USA) was used to detect DNA purity, and agarose (NuSieve 3:1 Agarose 3:1 agarose, catalog number 10220ES25) gel electrophoresis was used to obtain a single bright band to determine the specificity and target length of the PCR product.

[0120] Table 2 Probe library PCR reaction system

[0121]

[0122] Oligo-FISH signal pattern diagram of long-flowered oat chromosome. Designed long-flowered oat chromosome marker pattern diagram, green dots represent the positions of probe libraries G12405 and G16009, and red dots represent the positions of probe libraries R41087 and R41626 ( Figure 1 , Table 3).

[0123] Table 3 Chromosomal location of signal sites in oligonucleotide sequence library

[0124]

[0125]

[0126] The chromosome number of the Oat genome corresponds to the Chromosome number of GenBank assembly: GCA_030063025.1 (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_030063025.1 / ).

[0127] Example 2

[0128] Fluorescence in situ hybridization assay

[0129] The Oligo-FISH process of the control group and the experimental group was the same, except that only one pair of probes G12405_Dig+R41087_Bio( Figure 2). The optimization probe library design method includes: adding two pairs of probes, adding newly designed probe libraries G16009 and R416126, and expanding the coverage range of the probe library (Table 3, Table 7). The purpose is to obtain a clear hybridization signal with position specificity and signal resolvable intensity ( Figure 3 D).

[0130] Metaphase chromosome preparation. Refer to Schwarzacher (2016; doi:10.1007 / 978-1-4939-3142-2_7). The experimental steps are as follows:

[0131] (1) Collecting root tips: Place the seeds in a culture dish covered with moist filter paper and germinate them in a 25°C incubator for 48 h, keeping the filter paper moist (sampling time is 8:45–9:00 am).

[0132] (2) Pretreatment: When the root tip grows to 1–2 cm, place the root tip in an ice-water mixture for 24 h.

[0133] (3) Fixation: 1–2 cm of the root apex was cut and placed in freshly prepared Carnoy’s fixative (anhydrous ethanol: glacial acetic acid = 3:1, v / v). The root was fixed at 37°C for 30 min, placed at room temperature for 2 h, and then stored at -20°C. The shelf life is half a year.

[0134] (4) Enzymatic hydrolysis: The fixed root tips were removed and washed three times with distilled water for 10 min each time; soaked in 1× enzyme buffer (40 ml 10 mM citric acid + 60 ml 10 mM trisodium citrate diluted 10 times) for 10 min; placed in 37°C enzyme mixture [2% (w / v) Cellulase (20 U / ml, Sigma C1184) + 0.2% (w / v) Cellulase (10 U / ml, Onozuka RS) + 3% Pectinase (20 U / ml, Sigma P4716)] for enzymatic hydrolysis for about 90 min; the root tips were aspirated and soaked in 1× enzyme buffer for 10 min.

[0135] (5) Preparation of white slides: Use a pipette to carefully pipette the root apex onto a clean adhesive slide (Citglas, #C / N188105), use filter paper to absorb excess enzyme buffer, add one drop (20ul) of 60% acetic acid, use a dissecting needle to crush the root apex and remove all tissues except the meristem, smear the slide, cover with a coverslip (18×18mm), squeeze gently, and examine under a phase contrast microscope. Select white slides that are well dispersed and have ≥5 metaphases, mark with a pencil, remove with liquid nitrogen, and dry for later use.

[0136] Fluorescence in situ hybridization. The specific steps are as follows:

[0137] (1) Pretreatment: The slides were treated with freshly prepared fixative (anhydrous ethanol: glacial acetic acid = 3:1, v / v) for 15 min, then treated twice with 100% ethanol, 5 min each time, and dried at room temperature.

[0138] (2) RNase treatment: Prepare RNAse solution [20 μl RNaseA (10 mg / μl) + 1980 μl 2×SSC], add 250 μl RNAse solution to each slide, cover with sealing film, place in a wet box and incubate at 37°C for 1 h, then wash twice with 2×SSC for 2 min and 10 min respectively.

[0139] (3) Pepsin treatment: 0.01 M HCl treatment for 2 min, add 250 μl 7.5 μg / ml pepsin solution [15 μl Pepsin (1 mg / ml) + 1985 μl 0.01 M HCl] to each slide, incubate in a wet box at 37°C for 10 min, wash with distilled water for 1 min, and wash twice with 2× SSC, 5 min each time.

[0140] (4) Paraformaldehyde fixation: Place the slides in a 4% paraformaldehyde solution in a ventilated place, incubate at room temperature for 10 min, and wash twice with 2×SSC, for 2 min and 10 min respectively.

[0141] (5) Dehydration: Place the slides in 50%, 70%, 85%, and 100% ethanol solutions in sequence, dehydrate for 2 min each, and air-dry.

[0142] (6) Hybridization: Prepare hybridization mixture, dispense 34 μl into each tube (Table 4), add appropriate amount of probes (Table 5), denature at 85°C for 10 min, keep on ice for 10 min, drop the hybridization mixture with probes on the chromosome region of the slide, cover with high temperature sealing film, denature at 72°C for 7 min in the first cycle in the hybridizer, and keep at 37°C overnight for 16–20 h in the second cycle. The components of the hybridization mixture system are shown in Table 4. Due to the high cost of ordering and labeling the probe library, the appropriate volume of the dual probe library (3A-C) was first explored. The optimal combination of G12405_Dig+G16009_Dig (green) was added with 2.1 ul, and R41087_Bio+R416126_Bio (red) was added with 1.7 ul, and the probe concentration was 687.6–783.5 ng / ul ( Figure 3 D, Table 5).

[0143] Table 4 Hybridization mixture composition

[0144]

[0145]

[0146] Table 5 Exploring the appropriate volume of the dual probe library

[0147]

[0148] For elution, detection, DAPI staining and sealing, add 21 μl of a mixture of anti-fade agent and DAPI (100 μg / μl) (50 μl anti-fade + 50 μl sigma water + 1 μl DAPI) to each slide, cover with a large cover glass (No. 0, 24×24 mm, Agar Scientific, UK), seal with transparent nail polish, and store in a dark box at 4°C overnight.

[0149] Photography and image processing: The cells were examined under a Nikon fluorescence microscope (Ni-U, Tokyo, Japan) in a dark room. Each metaphase was captured by four fluorescence excitation blocks, UV-2A (EX355 / 50, DM400, BA410), FITC (EX465-495, DM505, BA512-558), TRITC (EX537-552, DM565, BA582-637), and ET-Cy5 (ET620 / 60, T660lpxr, ET700 / 75). The images were captured using a CCD camera pco.panda4.2 (PCO, Dassow, Germany) controlled by the software NIS-Elements v.2.3.4 (Nikon, Tokyo, Japan). The exposure times for the red, green, and blue channels were 5 sec, 250 millisec, and 90 millisec, respectively. Images were processed using Image J v.1.51j8 software (Wayne Rasband, NIH, USA) and Photoshop CS6 v.13.0 software (Adobe System, San Jose, USA).

[0150] Oligo-FISH results of metaphase chromosomes of oat.

[0151] The hybridization signal of the control group was not discernible ( Figure 2 , Table 8), indicating that the hybridization effect of the library with a span of 1.927–4.990 Mb and an oligonucleotide density of 0.993–1.611 oligos / kb was not ideal.

[0152] After exploring the appropriate probe amount by gradient method, it was found that the signals of 1.2ul, 1.5ul, and 1.8ul of the oligonucleotide probe library G12405 (green) and G16009 (green) labeled with digoxigenin were diffuse green, weak green double dots, and green double dots swelled into plaques ( Figure 3A, 3B, 3C), the signals of the digoxigenin-labeled oligonucleotide probe library R41087 (red), R41626 (red) 1.7ul, 1.2ul, 1.5ul were red double dots, weak red diffuse or double dots, red double dots with weak brightness ( Figure 3 A, 3B, 3C). The optimal combination was 2.1ul each of the digoxigenin-labeled oligonucleotide probe library G12405 (green) and G16009 (green), and 1.7ul each of the biotin-labeled oligonucleotide probe library R41087 (red) and R41626 (red). High-definition hybridization signals were obtained, and 13 signal sites on the single chromosome of Oat had specific and bright hybridization signals ( Figure 3 D).

[0153] Oligo-FISH signal pattern after optimal probe quantity combination ( Figure 4, Table 6) are: the oligonucleotide sequence AL01S1 (length of 9,628 oligos) labeled with Biotin has a patchy hybridization signal on chromosomes 1 to 2; the oligonucleotide sequence AL01S2 (length of 10,527 oligos) labeled with Biotin has a patchy hybridization signal on chromosomes 1 to 2; the oligonucleotide sequence AL02L2 (length of 12,130 oligos) labeled with Biotin has a patchy hybridization signal on chromosomes 3 to 4 ... The oligonucleotide sequence AL03S1 (length of 9,683 oligos) had patchy hybridization signals on chromosomes 5 and 6; the oligonucleotide sequence AL03L1 (length of 4,312 oligos) labeled with Biotin had dot-like hybridization signals on chromosomes 5 and 6; the oligonucleotide sequence AL05S1 (length of 9,352 oligos) labeled with Biotin had patchy hybridization signals on chromosome 9 and scattered dot-like hybridization signals on chromosome 10; the oligonucleotide sequence AL05L1 ( The length of the oligonucleotide sequence AL06L1 (7,580 oligos) had scattered dot-like hybridization signals on chromosome 9 and weaker scattered dot-like hybridization signals on chromosome 10. The oligonucleotide sequence AL06L1 labeled with Biotin (8,084 oligos) had patchy hybridization signals on chromosome 11, and the hybridization signals on chromosome 12 were covered by the red double electric signals due to the curling of the chromosome ends. The oligonucleotide sequence AL07L1 labeled with Biotin (11,417 oligos) had patchy hybridization signals on chromosomes 13-14. Blocky hybridization signals. When chromosomes are prepared, some chromosomes are stained in three-dimensional space at the places where they fold or overlap with other chromosomes. The three-dimensional spaces at these folded and overlapped positions are prone to residual probes that are not bound to the chromosomes, and are not easy to clean. In addition, since the amount of probe added is 2.1ul for green and 1.7ul for red, these places where non-bound probes are likely to remain basically show green fluorescence (green fluorescence masks red fluorescence), which is false positive fluorescence. This means that in order to reduce the occurrence of false positive fluorescence, high-quality white films need to be prepared, and the chromosomes need to be located on the same horizontal plane and dispersed.

[0154] The oligonucleotide sequence AL02L1 (7,023 oligos in length) labeled with Digoxigenin has plaque-like hybridization signals on chromosomes 3 and 4; the oligonucleotide sequence AL03L2 (7,258 oligos in length) labeled with Digoxigenin has plaque-like hybridization signals on chromosomes 5 and 6; the oligonucleotide sequence AL04S1 (7,558 oligos in length) labeled with Digoxygenin has plaque-like hybridization signals on chromosomes 7 and 8; the oligonucleotide sequence AL06S1 (6,575 oligos in length) labeled with Digoxygenin has plaque-like hybridization signals on chromosomes 11 and 12. Therefore, the probe libraries G12405_Dig+G16009_Dig (green) and R41087_Bio+R416126_Bio (red) spanned 4.491–9.934 Mb and had a density of 0.949–1.432 oligos / kb, achieving the purpose of identifying single chromosomes of Avena longimmensis ( Table 6 ).

[0155] Table 6 Oligonucleotide fluorescence in situ hybridization signal site names and hybridization signal distribution patterns

[0156]

[0157] In Table 6, the chromosome-level genome number AL01 corresponds to AL1 / 2, i.e. sister chromosomes 1 to 2; AL02 corresponds to AL3 / 4, i.e. sister chromosomes 3 to 4; AL03 corresponds to AL5 / 6, i.e. sister chromosomes 5 to 6; AL04 corresponds to AL7 / 8, i.e. sister chromosomes 7 to 8; AL05 corresponds to AL9 / 10, i.e. sister chromosomes 9 to 10; AL06 corresponds to AL11 / 12, i.e. sister chromosomes 11 to 12; AL07 corresponds to AL13 / 14, i.e. sister chromosomes 13 to 14. The same expressions in other tables or text have the same meanings as in Table 6. Since the oat genome is a diploid genome (7×2) with 14 chromosomes, and genome sequencing only assembles one representative sequence for sister chromosome monomers, the chromosome numbers or chromosome positions in Tables 6 and 7 (i.e., the chromosomes corresponding to the probe hybridization experiments) refer to the numbers of the 14 diploid chromosomes, rather than the representative chromosome sequence numbers in the genome database. When the genome number or chromosome genome number (i.e., the reference genome used for primer development) is referred to in this article, it refers to the Chromosome number from the long-leaved oat genome database GenBank assembly: GCA_030063025.1 or the corresponding digital number (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_030063025.1 / ).

[0158] Table 7 Statistical information of single chromosome probe libraries G12405+G16009 and R41087+R416126 of Oat

[0159]

[0160]

[0161] Table 8 Statistical information of the control group G12405 and R41087 of the single chromosome probe library of Oat

[0162]

[0163] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for designing a chromosome fluorescent labeling probe set for a species with a high repetitive sequence and a large genome, characterized in that: The species is a plant of the genus Avena, the proportion of genome repeat sequences is higher than 80%, and the genome is greater than or equal to 3G. The method comprises the following steps: 1) Remove repetitive sequences based on the Avena reference genome; 2) Obtaining a 45 bp single copy oligonucleotide sequence library; 3) Remove short fragments containing ≥6 consecutive identical base repeats and retain short sequences with a GC content of 25-80%; 4) retain the short sequence library with dTm value greater than 10°C; 5) Align the short sequence library back to the target region and remove the probe fragments in the non-target region to obtain the probe set; 6) Add fluorescent labels to both ends of the probe.

2. A probe set for chromosome fluorescence labeling of species with high repetitive sequences and large genomes, characterized in that: The probe group is designed according to the method according to claim 1.

3. The probe set according to claim 2, characterized in that: The probe group includes 4 probe libraries, which are G12405, G16009, R41087 and R41626 respectively. The reference genome intervals corresponding to the four probe libraries are shown in the following table, and each probe library is composed of all probes corresponding to the chromosome intervals in the following table: 。 4. Use of the probe set according to any one of claims 2 to 3 in constructing a visualized chromosome fluorescent labeling map of Avena plants.

5. A method for constructing a single chromosome fluorescent labeling map of a species with a high repetitive sequence and a large genome, characterized in that: The high-repetitive sequence large genome species is a species with a repeat sequence ratio higher than 80% and a genome larger than 3G. The method comprises the following steps: using the method of claim 1 to design and synthesize probes, and in situ hybridization of the probes with chromosomes of the target species.

6. The method according to claim 5, characterized in that The probes are all the probes of the four probe libraries described in claim 3. In the hybridization reaction system, 2.1 μL of digoxigenin-labeled oligonucleotide probe libraries G12405 and G16009 are used, and 1.7 μL of biotin-labeled oligonucleotide probe libraries R41087 and R41626 are used, and the probe concentration is 687.6-783.5 ng / μL.

7. The method according to claim 6, characterized in that Probes for in situ hybridization with chromosomes of target species include: Metaphase chromosome preparation (1) Collecting root tips: Place the seeds in a petri dish covered with moist filter paper and germinate them in a 25°C incubator for 48 h, keeping the filter paper moist; (2) Pretreatment: When the root tip grows to 1-2 cm, place the root tip in an ice-water mixture for 24 hours; (3) Fixation: Cut 1-2 cm root tips and place them in freshly prepared Carnoy’s fixative at 37°C for 30 min. Place them at room temperature for 2 h and store them at -20°C. (4) Enzymatic hydrolysis: (4.1) Remove the fixed root apex and wash it with distilled water three times, 10 minutes each time; (4.2) Soak in 1× enzyme buffer for 10 min. The enzyme buffer is 40 ml 10 mM citric acid + 60 ml 10 mM trisodium citrate diluted 10 times; (4.3) Place at 37°C for enzymatic hydrolysis for 90 min, wherein the enzyme mixture is 2% (w / v) 20U / ml Cellulase + 0.2% (w / v) 10U / ml Cellulase + 3% (w / v) 20U / ml Pectinase; (4.4) Aspirate the root tip and soak it in 1× enzyme buffer for 10 min; (5) Preparation of white slides: Use a pipette to carefully draw the root apex onto a clean adhesive slide, use filter paper to absorb the excess enzyme buffer, add a drop of 60% acetic acid, use a dissecting needle to crush the root apex and remove all tissues except the meristem, smear on the slide, cover with a cover slip, squeeze gently, and examine under a phase contrast microscope. Select white slides with good dispersion and ≥5 metaphases, mark with a pencil, remove with liquid nitrogen, and dry for later use; Fluorescence in situ hybridization (1) Pretreatment: Treat the slides with freshly prepared Carnoy's fixative for 15 min, treat twice with 100% ethanol, 5 min each time, and dry at room temperature; (2) RNase treatment: Prepare an RNAse solution, which is 20 μL of 10 mg / μL RNase A + 1980 μL of 2×SSC. Add 250 μL of RNAse solution to each slide, cover with sealing film, and place in a wet box for incubation at 37°C for 1 h. Then wash twice with 2×SSC for 2 min and 10 min respectively. (3) Pepsin treatment: 0.01 M HCL treatment for 2 min, add 250 μL of 7.5 μg / ml pepsin solution to each slide, the pepsin solution is 15 μL of 1 mg / ml Pepsin + 1985 μL of 0.01 M HCl, incubate in a wet box at 37°C for 10 min, wash with distilled water for 1 min, and wash twice with 2×SSC, 5 min each time; (4) Paraformaldehyde fixation: Place the slides in a 4% paraformaldehyde solution in a ventilated place, incubate at room temperature for 10 min, and wash twice with 2×SSC, for 2 min and 10 min respectively; (5) Dehydration: Place the slides in 50%, 70%, 85%, and 100% ethanol solutions in sequence for 2 min each, and air dry. (6) Hybridization: Prepare a hybridization mixture, the composition of which is as follows ， Aliquot 34 μL into each tube, add 2.1 μL of digoxigenin-labeled oligonucleotide probe library G12405 and G16009, 1.7 μL of biotin-labeled oligonucleotide probe library R41087 and R41626, respectively, with a probe concentration of 687.6 ~ 783.5 ng / μL, denature at 85℃ for 10 min, keep on ice for 10 min, add the hybridization mixture with probes to the chromosome region of the slide, cover with high-temperature resistant sealing film, denature at 72℃ for 7 min in the first cycle in the hybridizer, and keep at 37℃ overnight for 16~20 h in the second cycle.

Images