A method for constructing a Pore-C library suitable for complex polyploid plants

Abstract

The present invention discloses a method for constructing a Pore-C library suitable for complex polyploid plants, which relates to the technical field of plant genetic engineering. In the present invention, plant tissues are ground into fine powder and then added to a nuclear extraction solution, while DNA and proteins are crosslinked. Nuclear precipitates are obtained through filtration and sucrose density gradient centrifugation; after washing the nuclei with 1×PBS and resuspending the nuclei with the nuclear extraction solution, Percoll density gradient centrifugation is carried out and an equal volume of 0.25 M sucrose is added to separate and obtain intact and pure nuclei; then in situ nuclear enzyme digestion, ligation, and de-crosslinking are carried out; then it is extracted twice with a DNA extraction solution, and sodium chloride, sodium acetate, and absolute ethanol are added to the supernatant to obtain DNA. The extracted DNA is 5-9 μg, which can be used for 3-7 cells on the machine, obtaining 300 G-1 T of data, greatly reducing the cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a method for constructing a Pore-C library suitable for complex polyploid plants. Background Art

[0002] A high-quality reference genome is the basis for the exploration and utilization of crop germplasm resources and breeding improvement. However, the assembly of high-ploidy complex genomes is extremely challenging, facing problems such as the lack of sufficient and accurate read information for connecting contigs and a high chimeric error rate of contigs during chromosome scaffolding. It is difficult to complete the assembly of high-quality complex polyploid plant genomes under traditional techniques. Pore-C is a new three-dimensional genomics research technology that combines chromatin conformation capture (3C) and the third-generation long-read sequencing platform of Oxford Nanopore Technologies (ONT). With its advantages of long reads, capturing multiple interactors, and distinguishing allelic methylation, it can assist genome assembly and chromosome scaffolding, increase the discrimination of homologous chromosomes, and solve the chromosomal typing assembly of complex genomes. At the same time, it can analyze the three-dimensional structure of chromosomes and obtain a genome-wide chromosome interaction map.

[0003] Currently, existing Pore-C methods are all developed for humans or model plants (such as Arabidopsis thaliana). Non-model plants, especially those with complex genomes, are rich in secondary metabolites such as polysaccharides and polyphenols, and it is impossible to obtain high-quality and high-yield data. Currently, the output of Pore-C applied to polyploid model plants (such as allopolyploid upland cotton TM-1) is about 52-61G (PromethION platform), and the average proportion of multiple interactors is about 43.56%. Its high data production cost and low proportion of interactors are the fundamental reasons restricting its application in complex genome assembly and three-dimensional structure research. Summary of the Invention

[0004] Aiming at the above problems existing in the prior art, the technical problem to be solved by the present invention is a method for constructing a Pore-C library suitable for complex polyploid plants, which is used to construct a Pore-C library suitable for complex polyploid plants.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for constructing a Pore-C library suitable for complex polyploid plants: Grind the plant tissue into fine powder and add it to the nuclear extraction solution, while cross-linking DNA and proteins. Obtain nuclear precipitate through filtration and sucrose density gradient centrifugation; After washing the nuclei with 1×PBS and resuspending the nuclei with the nuclear extraction solution, perform Percoll density gradient centrifugation and then add an equal volume of 0.25 M sucrose to separate and obtain intact and pure nuclei; Then perform in situ nuclear digestion, ligation, and de-crosslinking; Then extract with DNA extraction solution twice, take the supernatant and add sodium chloride, sodium acetate, and absolute ethanol to obtain DNA.

[0007] The plant tissue is fresh or frozen materials at various ages.

[0008] The composition of the nuclear extraction solution is: 0.4 M sucrose, 10 mM Tris-HCl, pH 8, 10 mM KCl, 0.5% Triton X-100, 10 mM EDTA, pH 8, 1% PVP40, 0.25% β-Mercaptoethanol, 0.1 mM PMSF.

[0009] The composition of the sucrose gradient centrifugation solution is: 1.7 M sucrose, 20 mM Tris-HCl, pH 8, 2 mM KCl, 0.2% Triton X-100, 2 mM EDTA, pH 8, 0.25% β-Mercaptoethanol, 0.1 mM PMSF.

[0010] The composition of the DNA extraction solution is: phenol:chloroform:isoamyl alcohol = 25:24:1.

[0011] The composition of the digestion reaction solution is: 30 μL of 20 U / μL Hind III, 50 μL of NEB buffer 2.1, 20 μL of enzyme-free water; or, 40 μL of 10 U / μL Dpn II, 50 μL of NEB buffer 3.1, 10 μL of enzyme-free water.

[0012] The composition of the ligation reaction solution is: 260 μL of sterile enzyme-free water, 100 μL of 10% Triton X-100, 100 μL of 10×ligation buffer, 5 μL of 20 μg / μL Recombinant albumin, 30 μL of 400 U / μL T4 DNA ligase; or, 415 μL of sterile enzyme-free water, 100 μL of 10×ligation buffer, 5 μL of 20 μg / μL Recombinant albumin, 30 μL of 400 U / μL T4 DNA ligase.

[0013] The sucrose density gradient centrifugation method determines the centrifugal force according to the genome size.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] 1) 1% PVP is added to the nuclear extraction solution of the present invention to separate polyphenols and polysaccharide secondary metabolites from DNA, facilitating subsequent filtration and removal.

[0016] 2) The present invention uses a vacuum dryer for crosslinking, performs 4 vacuum / degas exchanges, and the crosslinking is sufficient, increasing the proportion of high-order interaction fragments obtained.

[0017] 3) The present invention uses the sucrose density gradient centrifugation method to determine the centrifugal force according to the genome size, obtaining more cell nuclei.

[0018] 4) The present invention uses 50% Percoll density gradient centrifugation for nuclear purification, effectively removing secondary metabolites, obtaining complete and pure cell nuclei, and improving the efficiency of enzymatic digestion and ligation.

[0019] 5) The present invention uses phenol:chloroform:isoamyl alcohol = 25:24:1 as the DNA extraction solution, extracts DNA twice, effectively removes impurities such as proteins, and improves data output.

[0020] 6) For the DNA extracted by the method of the present invention, the library length can be increased without cutting the gel to remove small fragments, and the N50 can exceed 9 kb (Hind III endonuclease) at most.

[0021] 7) The DNA extraction rate of the method of the present invention is high, 5-9 μg of DNA can be obtained, which can be used for 3-7 cells on the machine, and 300 G-1 T of data can be obtained, greatly reducing the cost.

[0022] 8) The method of the present invention is not limited to young and fresh materials, and is suitable for plant materials of various ages and frozen in the refrigerator. Description of the Drawings

[0023] Figure 1 It is the agarose gel detection diagram of the alfalfa Pore-C library prepared by the method of the present invention;

[0024] Figure 2 It is the distribution diagram of the number of multiple interaction fragments of the alfalfa Pore-C library prepared by the method of the present invention;

[0025] Figure 3 It is the agarose gel detection diagram of the sugarcane Pore-C library prepared by the method of the present invention;

[0026] Figure 4 It is the distribution diagram of the number of multiple interaction fragments of the sugarcane Pore-C library prepared by the method of the present invention. Detailed implementation manners

[0027] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well-known to those skilled in the art.

[0028] Example 1

[0029] A method for constructing a Pore-C library suitable for complex polyploid plants. The plant material selected in this example is autotetraploid alfalfa, which is planted in the experimental base in Yongning County, Yinchuan City, Ningxia Hui Autonomous Region. After the materials are collected, they are stored in a -80 °C refrigerator for later use. The steps are as follows:

[0030] 1) Take 1-2 g of young and tender leaves of autotetraploid alfalfa, quickly grind them into fine powder in liquid nitrogen, transfer them to a 50 mL centrifuge tube containing 15 mL of pre-cooled nuclear extraction buffer (0.4 M sucrose, 10 mM Tris-HCl, pH 8, 10 mM KCl, 0.5% TritonX-100, 10 mM EDTA, pH 8, 1% PVP40, 0.25%

[0031] β-Mercaptoethanol, 0.1 mM PMSF, PMSF and β-Mercaptoethanol are added before use), quickly mix well, add 417 μL of 37% formaldehyde, open the lid and place it in a desiccator, connect the pressure pump and adjust the pressure to 0.06 Mpa, incubate at room temperature for a complete 20 min, and evacuate once every 5 min; release the vacuum, open the desiccator, add 963 μL of 2 M glycine (glycine, final concentration 0.125 M) to the petri dish, adjust the pressure to 0.06 Mpa, incubate at room temperature for 5 min, and immediately place it on ice to terminate crosslinking for 10 min to obtain a tissue suspension.

[0032] 2) After filtering the tissue suspension through two layers of Magic Filter Cloth (Merck millipore, 475855-1r), add it to 30 mL of pre-cooled sucrose gradient centrifugation solution (1.7 M sucrose, 20 mM Tris-HCl, pH 8, 2 mM KCl, 0.2% Triton X-100, 2 mM EDTA, pH 8, 0.25% β-Mercaptoethanol, 0.1 mM PMSF, PMSF and β-Mercaptoethanol are added before use), centrifuge at 4 °C for 20 min, and adjust the centrifugal force according to the genome size using sucrose density gradient centrifugation method (Table 1); discard the supernatant, wash the nuclear precipitate with 10 mL of pre-cooled 1×PBS, and centrifuge for 10 min under the same conditions; discard the supernatant, resuspend the nuclei with nuclear extraction solution, and perform density gradient centrifugation to obtain a nuclear suspension.

[0033] Table 1 Genome size and centrifugal force (sucrose density gradient centrifugation)

[0034] Genome size (Mb) Centrifugal force (g) 25 4800 100 3970 500 3190 1000 2900 5000 2330 25000 1870 50000 1700

[0035] 3) Add 2 mL of 2.5 M sucrose to a new 15 mL centrifuge tube, then slowly add 5 mL of 50% Percoll along the tube wall, and slowly add all the nuclear suspension obtained in the previous step along the tube wall; perform gradient centrifugation at 4 °C and 1800 g for 30 min using a horizontal centrifuge; collect the intact nuclei in the middle Percoll layer into a new centrifuge tube, add an equal volume of 0.25 M sucrose, and centrifuge at 4 °C and 1800 g for 20 min; discard the supernatant, transfer the nuclear precipitate to a 2 mL centrifuge tube, wash the nuclear precipitate with 2 mL of 1×PBS, and centrifuge at 4 °C and 1200 g for 5 min; carefully remove the supernatant, add 100 μL of 0.5% SDS to the nuclear precipitate, gently pipette and mix with a wide-bore pipette tip, then transfer to a metal bath and incubate at 62 °C for 6 min, and cool to room temperature; then add 250 μL of nuclease-free water and 50 μL of 10% Triton X-100 (final concentration is 1.25%, and the volume is 400 μL at this time), gently pipette and mix with a wide-bore pipette tip, and incubate at 37 °C for 15 min; then add the digestion reaction solution (30 μL of 20 U / μL HindIII, 50 μL of NEB buffer 2.1 and 20 μL of nuclease-free water) to a total volume of 500 μL, gently invert and mix, then transfer to a metal bath and incubate at 37 °C for 18 h, with a rotation speed of 900 rpm, 10 s on, 30 min off.

[0036] 4) After the enzymatic digestion is completed, perform a brief centrifugation to remove the condensed water on the lid, then add 55 μL of 1% SDS (final concentration is 0.1%, and the total volume is 555 μL at this time), transfer it to a metal bath and incubate at 65 °C for 20 min (to inactivate the restriction enzyme); after cooling to room temperature, aspirate 50 μL and transfer it to a new 1.5 mL centrifuge tube, label it as the enzymatic digestion control sample, and temporarily store it at 4 °C; add the ligation reaction solution (100 μL of 10% Triton X-100, 100 μL of 10× ligation buffer, 5 μL of 20 μg / μL Recombinant albumin, 30 μL of 400 U / μL T4 DNA ligase, and 260 μL of sterile enzyme-free water) to the remaining nuclear suspension until the total volume is 1000 μL, gently pipette and mix the sample with a wide-bore pipette tip; transfer the nuclear suspension to a metal bath and incubate at 16 °C for 12 h, with a rotation speed of 900 rpm, 10 s on, 30 min off.

[0037] 5) After the ligation is completed, perform a brief centrifugation to remove the condensed water on the lid, and sequentially add 280 μL of sterile enzyme-free water, 500 μL of 20% Tween-20, 100 μL of 10% SDS, 100 μL of 20 μg / μL Proteinase K, 20 μL of RNase A (the total volume is 2 mL at this time), gently invert and mix, incubate in a 56 °C metal bath for 18 h, with a rotation speed of 900 rpm, 10 s on, 30 min off; obtain the de-crosslinking reaction solution and cool it on ice.

[0038] 6) Divide the 2 mL sample (de-crosslinking reaction solution) equally into 2 tubes, 1 mL per tube, add an equal volume of pre-cooled DNA extraction solution (phenol:chloroform:isoamyl alcohol = 25:24:1) to the two samples respectively, then place the two lysed samples in a rotary mixer and mix by inversion at 10 rpm for 5 min, transfer to 15 °C, and centrifuge at 15000 g for 15 min; carefully aspirate 850 μL of the upper aqueous phase from each of the two tubes and transfer them to new 2 mL centrifuge tubes respectively, add an equal volume of pre-cooled DNA extraction solution (phenol:chloroform:isoamyl alcohol = 25:24:1), place the two lysed samples in a rotary mixer and mix by inversion at 10 rpm for 5 min, then transfer to 15 °C and centrifuge at 15000 g for 15 min; carefully aspirate 800 μL of the upper aqueous phase from each of the two tubes and transfer them to a new 15 mL centrifuge tube for mixing, add 0.02 times the volume of 5 M NaCl (final volume is 0.1 M), 0.1 times the volume of 3 M sodium acetate (pH 5.5), and 3 times the volume of absolute ethanol according to the volume of the recovered aqueous phase, gently invert and mix, and place it in a -80 °C refrigerator for precipitation for more than 1 h or overnight in a -20 °C refrigerator.

[0039] 7) Centrifuge at 4°C and 15,000 g for 30 min; discard the supernatant, add 4 mL of 80% ethanol to wash the pellet, centrifuge at 4°C and 15,000 g for 5 min; discard the supernatant, add 2 mL of 70% ethanol to wash the pellet, centrifuge at 4°C and 15,000 g for 5 min; discard the supernatant, centrifuge briefly, discard any remaining supernatant, and air-dry for 5 min.

[0040] 8) Resuspend the pellet in 100 μL of TE buffer (pH 8) and incubate at room temperature for 5 min; add 50 μL of Novizan magnetic beads (0.5×) to the resuspended DNA to screen for fragments larger than 1 kb, and operate according to the Novizan magnetic bead instructions; add 50 - 70 μL of TE buffer (pH 8) to elute the DNA, and quantify the DNA concentration using the Qubit 1× dsDNA hS Assay Kit; take 200 ng of DNA and detect it on a 0.7% agarose gel.

[0041] The results are as Figure 1 shown. Samples with complete Hind III digestion showed diffuse bands, mainly enriched at 2.5 - 7.5 kb, while samples with complete ligation showed an obvious upward shift and enrichment compared to the digestion bands, mainly enriched at 15 kb and above, indicating that the alfalfa Pore-C library obtained by this method had high digestion and ligation efficiency and good quality.

[0042] 9) Construct the library and perform on-machine sequencing

[0043] Construct the on-machine library according to the instructions of the Nanopore Ligation Sequencing Kit (SQK-LSK 114) and sequence using the PromethION platform. In this example, 6 μg of extracted DNA was loaded onto 3 cells (chips), and 382 G of data was obtained, greatly reducing the cost.

[0044] The Pore-C data statistics of alfalfa are shown in Table 2. It can be seen that the DNA extracted by the method of the present invention can increase the library length without cutting the gel to remove small fragments, and the N50 is up to more than 9 kb (Hind III endonuclease).

[0045] Table 2 Pore-C data of alfalfa

[0046] Output Average length Maximum length N50 Q20 Q30 124G 6835bp 105926bp 9087bp 82.58％ 69.66％

[0047] Figure 2It is a distribution map of the number of multiple interaction fragments in the alfalfa Pore-C library. This high-order interaction map is obtained by aligning the Pore-C reads after quality control with the third-generation alignment software minimap 2 to the corresponding reference genome, filtering through cphasing-rs, filtering out fragments with an alignment quality lower than 1, a length less than 30, a sequence identity less than 0.75, and single fragments, calculating the number of aligned fragments for each Pore-C read, which is the high-order number (order), and finally counting the number of Pore-C reads for each order. Figure 2 It shows that the proportion of high-order chromatin interaction fragments (the number of aligned fragments per read ≥ 3) is approximately 62%, indicating that the library prepared by the library construction method of the present invention can obtain a high proportion of high-order chromosome interaction fragments and detect more high-order chromatin interactions.

[0048] Example 2

[0049] A method for constructing a Pore-C library suitable for complex polyploid plants. The plant material selected in this example is the cultivated sugarcane, which is planted in the experimental base of the Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences. The steps are as follows:

[0050] 1) Take 1 - 2 g of sugarcane leaves at the mature stage, quickly grind them into fine powder in liquid nitrogen, transfer them to a 50 mL centrifuge tube containing 15 mL of pre-cooled nuclear extraction buffer (0.4 M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM KCl, 0.5% Triton X-100, 10 mM EDTA, pH 8.0, 1% PVP 40, 0.25% β-Mercaptoethanol, 0.1 mM PMSF, PMSF and β-Mercaptoethanol are added before use), quickly mix well, add 417 μL of 37% formaldehyde, open the lid and place it in a desiccator, connect the pressure pump and adjust the pressure to 0.06 Mpa, incubate at room temperature for a complete 20 min, and evacuate once every 5 min; release the vacuum, open the desiccator, add 963 μL of 2 M glycine (glycine, final concentration 0.125 M) to the petri dish, adjust the pressure to 0.06 Mpa, incubate at room temperature for 5 min, and immediately place it on ice to terminate the crosslinking for 10 min to obtain a tissue suspension.

[0051] 2) After filtering the tissue suspension through two layers of Miracloth (Merck millipore, 475855-1r), add it to 30 mL of pre-cooled sucrose gradient centrifugation solution (1.7 M sucrose, 20 mM Tris-HCl, pH 8.0, 2 mM KCl, 0.2% Triton X-100, 2 mM EDTA, pH 8.0, 0.25% β-Mercaptoethanol, 0.1 mM PMSF, PMSF and β-Mercaptoethanol are added before use). Centrifuge at 4°C for 20 min, and adjust the centrifugal force according to the genome size using sucrose density gradient centrifugation (Table 1); discard the supernatant, wash the nuclear pellet with 10 mL of pre-cooled 1×PBS, and centrifuge at the same conditions for 10 min; discard the supernatant, resuspend the nuclei with nuclear extraction solution, and perform density gradient centrifugation to obtain a nuclear suspension.

[0052] 3) Add 2 mL of 2.5 M sucrose to a new 15 mL centrifuge tube, and then slowly add 5 mL of 50% Percoll along the tube wall. Slowly add all the nuclear suspension obtained in the previous step along the tube wall; centrifuge at 4°C and 1800 g for 30 min using a horizontal centrifuge; collect the intact nuclei in the middle Percoll layer into a new centrifuge tube, add an equal volume of 0.25 M sucrose, and centrifuge at 4°C and 1800 g for 20 min; discard the supernatant, transfer the nuclear pellet to a 2 mL centrifuge tube, wash the nuclear pellet with 2 mL of 1×PBS, and centrifuge at 4°C and 1200 g for 5 min; carefully remove the supernatant, add 100 μL of 0.5% SDS to the nuclear pellet, gently pipette and mix with a wide-bore pipette tip, and transfer to a metal bath. Incubate at 62°C for 6 min, and cool to room temperature; then add 250 μL of nuclease-free water and 50 μL of 10% Triton X-100 (final concentration is 1.25%, and the volume is 400 μL at this time), gently pipette and mix with a wide-bore pipette tip, and incubate at 37°C for 15 min; then add the restriction enzyme reaction solution (40 μL of 10 U / μL DpnII, 50 μL of NEB buffer 3.1, and 10 μL of nuclease-free water) to a total volume of 500 μL, gently invert and mix, and transfer to a metal bath. Incubate at 37°C for 18 h, with a rotation speed of 900 rpm, 10 s on, 30 min off.

[0053] 4) After the digestion is completed, centrifuge briefly to remove the condensed water on the lid, and then transfer the sample to a metal bath at 65°C and incubate at 300 rpm for 20 min to inactivate the restriction endonuclease. After cooling to room temperature, aspirate 50 μL and transfer it to a new 1.5 mL centrifuge tube, labeled as the digestion control sample, and temporarily store it at 4°C. Add the ligation reaction solution (100 μL of 10× ligation buffer, 5 μL of 20 μg / μL Recombinant albumin, 30 μL of 400 U / μL T4 DNA ligase, and 415 μL of sterile enzyme-free water) to the remaining nuclear suspension to a total volume of 1000 μL. Gently pipette the sample to mix well using a wide-bore pipette tip. Transfer the nuclear suspension to a metal bath and incubate at 16°C for 12 h at a rotation speed of 900 rpm, with 10 s on and 30 min off.

[0054] 5) After the ligation is completed, centrifuge briefly to remove the condensed water on the lid. Then, add 280 μL of sterile enzyme-free water, 500 μL of 20% Tween-20, 100 μL of 10% SDS, 100 μL of 20 μg / μL Proteinase K, and 20 μL of RNase A (the total volume is 2 mL at this time). Gently invert to mix well and incubate in a metal bath at 56°C for 18 h at a rotation speed of 900 rpm, with 10 s on and 30 min off. Obtain the de-crosslinking reaction solution and cool it on ice.

[0055] 6) Divide the 2 mL sample (de-crosslinking reaction solution) equally into 2 tubes, 1 mL per tube. Add an equal volume of pre-cooled DNA extraction solution (phenol:chloroform:isoamyl alcohol = 25:24:1) to each of the two samples. Then, place the two lysed samples in a rotary mixer and mix by inversion at 10 rpm for 5 min. Transfer to 15°C and centrifuge at 15,000 g for 15 min. Carefully aspirate 850 μL of the upper aqueous phase from each of the two tubes and transfer them to new 2 mL centrifuge tubes respectively. Add an equal volume of pre-cooled DNA extraction solution (phenol:chloroform:isoamyl alcohol = 25:24:1). Place the two lysed samples in a rotary mixer and mix by inversion at 10 rpm for 5 min, then transfer to 15°C and centrifuge at 15,000 g for 15 min. Carefully aspirate 800 μL of the upper aqueous phase from each of the two tubes and transfer them to a new 15 mL centrifuge tube for mixing. Add 0.02 times the volume of 5 M NaCl (final volume is 0.1 M), 0.1 times the volume of 3 M sodium acetate (pH 5.5), and 3 times the volume of absolute ethanol according to the volume of the recovered aqueous phase. Gently invert to mix well and place in a -80°C refrigerator to precipitate for more than one hour or overnight in a -20°C refrigerator.

[0056] 7) Centrifuge at 4°C, 15,000 g for 30 min; discard the supernatant, add 4 mL of 80% ethanol to wash the precipitate, and centrifuge at 4°C, 15,000 g for 5 min; discard the supernatant, add 2 mL of 70% ethanol to wash the precipitate, and centrifuge at 4°C, 15,000 g for 5 min; discard the supernatant, centrifuge briefly, discard any residual supernatant, and air dry for 5 min.

[0057] 8) Add 100 μL TE buffer (pH 8) to resuspend the precipitate and incubate at room temperature for 5 min; add 50 μL Novezan magnetic beads (0.5×) to the resuspended DNA to screen fragments larger than 1 kb, and operate according to the Novezan magnetic beads instructions; add 50-70 μL TE buffer (pH 8) to elute the DNA, and use Qubit 1×dsDNA hS Assay Kit to quantify the DNA concentration; take 200 ng DNA for detection on 0.7% agarose gel.

[0058] The results are as follows Figure 3 As shown, the samples with complete Dpn II digestion showed diffuse bands, which were mainly enriched below 2 kb, while the samples with complete ligation showed obvious upward shift and enrichment compared with the digestion bands, which were mainly enriched at 1 kb and above, indicating that the digestion and ligation efficiency of cultivated sugarcane DNA samples obtained by this method was high, and high-quality libraries could also be obtained from mature leaves.

[0059] 9) Build the library on the machine

[0060] The library was constructed according to the instructions of the Nanopore Ligation Sequencing Kit (SQK-LSK 114) and sequenced using the PromethION platform. In this example, 7 μg of DNA was extracted and sequenced on 5 cells (chips) to obtain 655G data, which greatly reduced the cost.

[0061] Figure 4 This is a distribution diagram of the number of multiple interaction fragments of the sugarcane Pore-C library in this example. The proportion of the number of high-order chromatin interaction fragments (the number of aligned fragments for each read segment ≥3) is about 70%, indicating that the library prepared by the library construction method of the present invention can obtain a high proportion of high-order chromosome interaction fragments, and the use of Dpn II restriction endonuclease can capture more high-order chromatin interactions than Hind III, and has a higher proportion of ≥5 high-order chromosome interaction fragments.

[0062] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A method for constructing a Pore-C library suitable for complex polyploid plants, characterized in that: The plant tissue was ground into fine powder and added to the nuclear extract solution, and DNA and protein were cross-linked at the same time. The nuclear precipitate was obtained by filtration and sucrose density gradient centrifugation. The nuclei were washed with 1×PBS and resuspended in nuclear extract solution. After Percoll density gradient centrifugation, the same volume of 0.25 M sucrose was added to separate the pure nuclei. Then, the nuclei were enzymatically digested, connected, and cross-linked in situ. The nuclei were extracted twice with DNA extract solution, and the supernatant was added with sodium chloride, sodium acetate, and anhydrous ethanol to obtain DNA. The plant tissues are fresh or frozen materials of various ages; The cell nuclear extract solution consists of: 0.4 M sucrose, 10 mM Tris-HCl, pH 8, 10 mM KCl, 0.5% Triton X-100, 10 mM EDTA, pH 8, 1% PVP40, 0.25% β-Mercaptoethanol, 0.1 mM PMSF; The composition of the sucrose gradient centrifugation solution was: 1.7 M sucrose, 20 mM Tris-HCl, pH 8, 2 mM KCl, 0.2% Triton X-100, 2 mM EDTA, pH 8, 0.25% β-Mercaptoethanol, 0.1 mM PMSF; The composition of the DNA extraction solution is: phenol: chloroform: isoamyl alcohol = 25:24:1; The composition of the enzyme digestion reaction solution is: 30 µL 20 U / µL Hind III, 50 µL NEB buffer 2.1, 20 µL enzyme-free water; or, 40 µL 10 U / µL Dpn II, 50 µL NEB buffer 3.1, 10 µL enzyme-free water; The ligation reaction solution consists of: 260 µL sterile enzyme-free water, 100 µL 10% Triton X-100, 100 µL 10×ligation buffer, 5 µL 20 µg / µL Recombinant albumin, 30 µL 400 U / µL T4 DNA ligase; or, 415 µL sterile enzyme-free water, 100 µL 10×ligation buffer, 5 µL 20 µg / µL Recombinant albumin, 30 µL 400 U / µL T4 DNA ligase; The sucrose density gradient centrifugation method determines the centrifugal force according to the genome size.

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