CN117887879A - Primer combinations and methods for isolating the genomic sequence of an Opie transposon insertion site - Google Patents

Abstract

The invention belongs to the field of molecular biology, and provides a primer combination and a method for separating an Opie transposon insertion site genome sequence, in particular relates to an efficient detection primer and a method for an Opie whole genome site based on NGS sequencing, wherein the primer is a base sequence shown by an Opie1-inner, an Opie1-outer and an Opie 2-inner. By using the 3 primers and performing a method of establishing a library PCR and then performing NGS second-generation deep sequencing, about 10000-20000 Opie loci can be enriched from each corn material.

Description

Primer combinations and methods for isolating the genomic sequence of an Opie transposon insertion site

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a primer combination and a method for separating an Opie transposon insertion site genome sequence.

Background

Transposons in plants and animals are widespread and play an important role in the evolution, domestication and the formation of new varieties of species. Jump gene or transposon, a DNA sequence which can be copied or broken off separately from the original position, inserted into another site after cyclization and has the regulatory function on the subsequent gene. For example, transposons have found very wide application, the sleeping beauty trannoson (SleepingBeautytranposon, SB-Tn) system, a gene therapy technique capable of avoiding the defects of the viral transfer gene technique, capable of correcting the defects of genes that lead to sickle cell anemia (SCD) in the laboratory. Meanwhile, a large number of transposons are used for preparing mutant libraries, artificial variation is prepared, and new crop varieties are created. Also, in vitro and in vivo DNA cleavage tools are made using transposons for making fragmented DNA.

Retrotransposons with different sequence characteristics are currently found in different plants and play an important role in speciation, crop acclimatization and the like. The Opie is the biggest LTR transposon superfamily found in maize and varies in length according to the elements contained therein, about 8000-11000bp in length, and the types of the elements contained therein are Gag, integrase, protease, ribonuclease, a reverse transcriptase. The transposon is characterized by a substantially even distribution throughout the genome. In different corn varieties, the copy numbers of the Opies are basically consistent, the coverage is good, and the library generated by the method is a high-density molecular marker or a liquid chip, is a high-efficiency genotype detection tool, and has strong application potential.

In agricultural breeding, application scenes such as resource material background, breeding authenticity identification, whole genome locus scanning of breeding population, excellent haplotype screening and the like can be realized through the genome sequence markers obtained by the method. However, existing detection means, including such as SSR markers, indel markers, multiplex PCR, kasp markers, etc., have the disadvantage of low numbers of markers (typically less than 1000); while resequencing random sequences results in lower efficiency and excessive cost, the above methods limit the development of the field, and it is highly desirable to build a site number of up to 10 ⁴ And the capture site is clear, high-flux and low-cost detection means.

The genome size of maize is 4Gb, there are 4 ten thousand genes in total, and about 10-100kb on average distributes one gene, and when we have 10 ten thousand high density markers, there is a cognate marker near each gene. Theoretically 10 "ten thousand" level marks can be obtained. Calculated, 10 different types of 10 are shown ⁴ The accumulated superposition of copied transposons can reach 10 ⁵ Total copy number, achieving the ideal state of saturation of the genomic marker locus. Therefore, every "ten thousand" level transposon has valuable value.

The Opie is one of high-copy transposons, has a unique sequence and an insertion site in a corn genome, has higher frequency of occurrence in a coding region, and has good scientific research and industrial application prospects by utilizing the novel marker detection method and technology developed by the Opie.

Disclosure of Invention

It is an object of the present invention to provide a primer combination and method for isolating the genomic sequence of an Opie transposon insertion site.

To achieve the object of the present invention, in a first aspect, the present invention provides a specific primer for detecting an Opie transposon site in a maize genome, wherein the specific primer comprises an Opie1-inner, an Opie1-outer and an Opie2-inner, and the sequences of the specific primer are shown as SEQ ID No. 1-3 respectively.

The numbers of the Opie transposon sequences published in the attachments of the scientific paper "De novo assembly, annostation, and comparative analys is of, diverse maize genomes" (https #// www.science.org/doi/full/10.1126/science. A bg 5289) are the OPie_AC185480_1137#LTR, the OPie_AC187149_1780#LTR, the OPie_AC187207_1792#LTR, the OPie_AC188002_2029#LTR, the OPie_AC196469_5133#LTR, the OPie_AC197084_5382#LTR, the OPie_AC197201_5474#LTR, the OPie_AC197691_5727 LTR, the OPie_AC198173_5898 LTR, the ie_AC198924_6206 LTR, the OPie_AC201793 #LTR, the OPie_AC7053#LT83_LTR, the OPie_L7272_LTR, the OPie_AC7472#LT32_LTR, the OPie_C7272_LTR, the OPie_C7235_SL7235_LTR, the OPie_C7235_Lvj7272#LTR, the OPie_C7235_Lvjvjvjvjvjvjvjvjvjb.

In a second aspect, the invention provides a primer combination for separating the genomic sequence of the Opie transposon insertion site, comprising a specific primer with the sequence shown in SEQ ID No. 1-3 and a linker primer with the sequence shown in SEQ ID No. 6.

In a third aspect, the invention provides a kit for separating the genomic sequence of the Opie transposon insertion site, which comprises the primer combination and a connector 1 and a connector 2 with sequences shown as SEQ ID No. 4 and SEQ ID No. 5 respectively.

In a fourth aspect, the invention provides a specific primer with a sequence shown as SEQ ID NO. 1-3, and application of the primer combination or the kit in high-throughput detection of the whole genome locus of the Opie transposon based on NGS sequencing.

In a fifth aspect, the present invention provides a method for isolating the genomic sequence of an Opie transposon insertion site, comprising the steps of:

a. extracting corn genome DNA, breaking the DNA by using breaking enzyme, and obtaining a DNA fragment of 250bp-5 kb;

b. c, connecting the DNA fragment obtained in the step a with the linker 1 and the linker 2 by using DNA ligase to obtain a DNA fragment with the linker, and recovering the DNA fragment by using a kit (such as an Axygen gel recovery kit AP-GX-250);

c. performing PCR amplification on the recovered product in the step b by using an Opie2-inner, an Opie1-outer, an Opie1-inner and a joint primer, respectively performing two rounds of PCR, firstly amplifying by using the Opie1-outer and the joint primer for 3-5 cycles, and then adding the Opie1-inner, the Opie2-inner and the joint primer for 10-20 cycles;

d. and c, recovering the amplified product in the step c, quantifying, sequencing by using an NGS sequencer, and comparing the sequencing result with the corn genome sequence to obtain the corresponding locus.

Wherein, opie2-inner, opie1-outer, opie1-inner, adaptor primer, adaptor1 and adaptor2 are from the above kit.

Further, the disruption enzyme in the step a is a DNase with a recognition sequence of 4-6 base cohesive ends, preferably NlaIII and NspI homotail enzyme restriction endonucleases, so that the disruption efficiency is improved; the disruption conditions were 37℃for 1-5 hours.

Further, the DNA ligase in the step b is T4 DNA ligase, and the ligation condition is that the incubation is carried out at 25 ℃ for 1-3 hours.

Further, the ratio of linker 1, linker 2 to DNA fragment in step b was 10pM:10pM:50ng.

Further, the amplification conditions in step c are: amplifying with Opie1-outer and adaptor primer for 5 cycles at 98℃for 2 min, at 98℃for 10 sec, 62℃for 20 sec, and 72℃for 1 min; adding the Opie1-inner, the Opie2-inner and the adaptor primer for amplification for 20 cycles at 98 ℃ for 10 seconds, 62 ℃ for 20 seconds and 72 ℃ for 1 minute; and finally, the temperature is 72 ℃ for 8 minutes.

Further, the amplification product is recovered in step d using magnetic beads (e.g., beckman's AMPure XP beads).

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

current high copy number transposon sites are generally obtained by resequencing. The existing OPIE transposon site identification method can only obtain whole genome information through re-sequencing without specificity and randomly, then carries out sequence filtering, screens out 99% of useless sequences, and has high cost and low efficiency in both library construction and sequencing. In contrast, the method provided by the invention can be enriched to almost all loci on a genome, and has extremely high efficiency; in addition, the method is suitable for high-throughput detection because the label sequences on the primers help separate the sites of the single sample from large-scale data after mixed sequencing; and the cost of library construction and sequencing is greatly reduced by mixing PCR and sequencing. The specific design of the primer not only ensures that the length of the tail end of OPIE is 150-350bp specific sequence in the subsequent analysis, but also ensures that the flanking genome sequence of 150-350bp is obtained in the subsequent second-generation NGS sequencing process, thus being a ingenious and efficient novel design.

Drawings

FIG. 1 shows the 3' end alignment consensus sequences of different Opie family members of the maize genome of the present invention.

FIG. 2 shows the alignment of the 5' ends of different Opie family members of the maize genome of the present invention to consensus sequences.

FIG. 3 shows a sequence structure of a sequencing sample based on an Illumina sequencing platform, wherein the sequences marked with Opie1-inner and Opie2-inner are Opie transposons 5 'and 3', respectively.

FIG. 4 shows the results of the Opie transposon NGS library construction quality control of the present invention.

FIG. 5 shows the distribution of the Opie transposon of the present invention found in the maize DH-1 genome.

FIG. 6 shows the distribution of the Opie transposon of the invention found in the maize K17 genome.

FIG. 7 shows the distribution of the Opie transposon of the present invention found in the maize Zheng 58 genome.

Detailed Description

The present invention aims at providing a whole genome level capture site greater than 10 ⁵ The capture site immobilization, high throughput, high efficiency, low cost primers and methods.

The invention also provides a method for detecting the insertion site of an Opie transposon.

The invention adopts the following technical scheme:

the invention provides a specific primer for separating genomic sequences of an Opie transposon insertion site, wherein the specific primer comprises an Opie1-inner, an Opie1-outer and an Opie2-inner, and the Opie1-inner, the Opie1-outer and the Opie2-inner meet the following conditions a, b, c or d:

a. a conserved consensus sequence flanking the transposon on the genomic sequence;

the inner distance from the last base of the transposon end is about 70-300bp, and the outer distance from the last base of the transposon end is about 300-600bp;

c. carrying a 4-8bp tag sequence for distinguishing samples;

d. comprising an anchor sequence for NGS depth sequencing.

The genome is corn genome, the Opie1-inner and the Opie1-outer are sequences shown in SEQ ID NO. 1-2, and the Opie2-inner is a sequence shown in SEQ ID NO. 3.

Further, the specific primer combinations are Opte 1-inner, opte 1-outer, opte 2-inner and adapter primer (adapter primer, SEQ ID NO: 6).

The invention also provides a method for separating the genomic sequence of the Opie transposon insertion site, which comprises the following specific steps:

a. taking corn genome DNA, breaking by using DNA breaking enzyme to obtain breaking product, wherein the breaking DNA fragment is between 250bp and 5 kb;

b. c, connecting the disruption product obtained in the step a by using a synthesized linker 1 and a synthesized linker 2 and DNA ligase to obtain a DNA fragment with the linker;

c. c, recycling the product obtained in the step b by using a kit;

d. performing PCR amplification on the recovered product in the step c by using the Opie2-inner, the Opie1-outer, the Opie1-inner and the adaptor PCR primer adaptor primer, designing two rounds of PCR for ensuring the sequence specificity, performing 3-5 cycles of amplification by using the outer and the adaptor, and performing 10-20 cycles of amplification by adding the inner and the adaptor primer;

e. and d, recovering the amplified product in the step, quantifying, sequencing by using an NGS sequencer, and comparing the sequencing result with the genome sequence to obtain the corresponding locus.

Wherein the disruption enzyme in the step a is NlaIII or other DNase with recognition sequence of 4-6 base cohesive ends, and the disruption condition is 37 ℃ for 1-5 hours.

The linker 1in the step b is an adapter 1, the sequence of which is a sequence 3 in a sequence table, the linker 2 is an adapter 2, and the sequence of which is a sequence 4 in the sequence table.

The ligase in the step b is T4 DNA ligase, and the ligation condition is 25 ℃ for 1-3 hours; the kit in the step c is an Axygen gel recovery kit AP-GX-250; the PCR amplification conditions of step d are: the primer was pre-denatured at 98℃for 2 minutes, the outer primer and the adaptor primer were added in the first 3-5 cycles, the inner primer and the adaptor primer were added in the last 10-20 cycles (98℃for 10 seconds, 62℃for 20 seconds, 72℃for 1 minute), and the primer was extended at 72℃for 8 minutes.

The invention also provides application of the primer or the method in separating the genomic sequence of the Opie transposon insertion site.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

Example 1Opie specific primer design and test

According to 2021 scientific paper "De novo assembly, analysis, and comparative analysis of, diverse maize genomes" (https:// www.science.org/doi/full/10.1126/science. Abg 5289) the published Opie sequence was used as a reference (but not limited to this sequence), a scanning alignment was performed in the maize B73 genome. Based on the characteristics of the common sequences of the Opies, sequences conserved at the 3 'end and the 5' end are cut out, and the comparison results are shown in figures 1 and 2. Finally, all the Opie sites at the whole genome level will be obtained using the NGS sequencing technology. The read length of the NGS is generally 150bp at a single end, the sum of the two ends is 300-700bp, and the read length is short. In order to ensure that adjacent genome sequences larger than 100bp can be obtained within the reading length range, the invention designs that 5 'or 3' end primers on the Opie are about 70bp away from the tail ends of the directions, and the sequences on the estimated obtained genome are not equal to 70-700 bp. We designed specific primers, opie1-inner, opie1-outer and Opie2-inner, at the 5 'and 3' ends of Opie, as shown in Table 1, the anchor sequence, where the transposon end specific sequence is located. In addition, the corresponding Adaptor sequences Adaptor1 and Adaptor2, adaptor PCR primer Adaptor primer, are designed. The adapter and the primer are matched with an anchor sequence and a sequencing primer sequence adopted in NGS second-generation high-throughput sequencing, and the primer can anneal with the same sequence of the adapter to finish PCR amplification, so that a product is obtained for sequencing.

According to the invention, a genome fragment interrupted by NlaIII (NEB, cat. No. R0125L) is matched to load a specific joint, nlaIII is a bright point of the invention, and a large number of cutting sites can be obtained from a chromosome by adopting the enzyme cutting sites (4 bp), so that the capturing of an Opie sequence is facilitated. Meanwhile, the adaptor sequences adaptor1 and adaptor2 with high specificity are designed. The adaptors 1 and 2 were melted at high temperature and then annealed, the procedure was 95℃for 2 minutes, and the adaptors 1 and 2 were mixed and then slowly cooled to room temperature.

The sequences of specific primers, adaptors, adaptor primers used to detect the whole genome Opie sites are shown in table 1:

TABLE 1 specific primers, adaptors, adaptor primer sequences for detection of whole genome Opie sites

The invention is based on an Illumina sequencing platform, and sequences marked with Opie1-inner and Opie2-inner are respectively 5 'and 3' of an Opie transposon (figure 3).

Example 2NGS sequencing method to enrich the corn genome for the Opie site

Selecting K17, zheng 58 and DH-1 corn samples, and performing the following steps:

(1) 50ng of maize genomic DNA was taken and disrupted with (NEB, cat. No. R0125L) at 37℃for 5 hours to give a disrupted product, the resulting DNA fragment was between 250bp-5 kb.

(2) Adding 10pM linker and 1 mu l T DNA ligase of each of the adapter 1 and the adapter 2 into the disruption product, and reacting for 3 hours at 25 ℃ to carry out ligation to obtain a DNA fragment with the linker; in this step, the ratio of linker to cleavage product was 10pM:50ng.

(3) Recovered with a PCR product purification kit.

(4) 10pM of Optie 1-outer,10pM adaptor primer, and 1. Mu.g of the recovered product of step (3) were added to the system, PCR was performed under conditions of pre-denaturation at 98℃for 2 minutes, 5 cycles (98℃for 10 seconds, 62℃for 20 seconds, 72℃for 1 minute), 5pM of Optie 1inner, optie 2inner were further added, PCR was continued, and amplification conditions were 20 cycles (98℃for 10 seconds, 62℃for 20 seconds, 72℃for 1 minute), and extension at 72℃for 8 minutes.

(5) The product was recovered by equal volume using Beckman's AMPure XP beads.

(6) NGS novaseq sequencing, library quality inspection results are shown in figure 4.

(7) Based on the sequencing analysis result, the number of effective sequences refers to the genome sequence comprising the Opie sequence and the flanking wings (the number of effective sequences on each chromosome and the ratio thereof are specifically shown in Table 2).

TABLE 2 number of Opie loci on each chromosome of the genome of 3 maize Material detected by the method of the present invention

The distribution of the Opie transposon in the maize DH-1 genome is shown in FIG. 5, the distribution of the Opie transposon in the maize K17 genome is shown in FIG. 6, and the distribution of the Opie transposon in the maize Zheng 58 genome is shown in FIG. 7.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The specific primer is used for detecting the Opie transposon site in the corn genome and is characterized by comprising an Opie1-inner, an Opie1-outer and an Opie2-inner, and the sequences of the specific primer are shown as SEQ ID NO. 1-3 respectively;

the sequence numbers of the Opie transposons are the OPie_AC185480_1137#LTR, the OPie_AC187149_1780#LTR, the OPie_AC187207_1792#LTR, the OPie_AC188002_2029#LTR, the OPie_AC196469_5133#LTR, the OPie_AC197084_5382#LTR, the OPie_AC197201_5474#LTR, the OPie_AC197691_5727#LTR, the OPie_AC198173_5898#LTR, the OPie_AC198924_6206#LTR, the OPie_AC201793_7083#LTR, the OPie_AC202020_7258#LTR, the OPie_AC202033_7274#LTR, the OPie_AC210610 _72#LTR, the OPie_AC197691 _LTR, the OPie_AC3727#LTR, the OPie_AC3740#LTR, and the OPie_pc3929#LTR.

2. A primer combination for separating the genomic sequence of the Opie transposon insertion site, which is characterized by comprising the specific primer as claimed in claim 1 and a linker primer with a sequence shown in SEQ ID No. 6.

3. A kit for separating the genomic sequence of the Opie transposon insertion site, which is characterized by comprising the primer combination as claimed in claim 2 and a linker 1 and a linker 2 with sequences shown in SEQ ID No. 4 and SEQ ID No. 5 respectively.

4. Use of the specific primer of claim 1, the primer combination of claim 2 or the kit of claim 3 in high throughput detection of the whole genome locus of the Opie transposon based on NGS sequencing.

5. A method for isolating the genomic sequence of an Opie transposon insertion site, comprising the steps of:

a. extracting corn genome DNA, breaking the DNA by using breaking enzyme, and obtaining a DNA fragment of 250bp-5 kb;

b. c, connecting the DNA fragment obtained in the step a with the linker 1 and the linker 2 by using DNA ligase to obtain a DNA fragment with the linker, and recovering the DNA fragment by using a kit;

c. performing PCR amplification on the recovered product in the step b by using an Opie2-inner, an Opie1-outer, an Opie1-inner and a joint primer, respectively performing two rounds of PCR, firstly amplifying by using the Opie1-outer and the joint primer for 3-5 cycles, and then adding the Opie1-inner, the Opie2-inner and the joint primer for 10-20 cycles;

d. c, recovering the amplified product of the step c, quantifying, sequencing by using an NGS sequencer, and comparing the sequencing result with a corn genome sequence to obtain a corresponding locus;

wherein, opie2-inner, opie1-outer, opie1-inner, adaptor primer, adaptor1 and adaptor2 are from the kit of claim 3.

6. The method according to claim 5, wherein the disruption enzyme in step a is a dnase recognizing a cohesive end of 4-6 bases in sequence, preferably a NlaIII, nspI isotail restriction endonuclease; the disruption conditions were 37℃for 1-5 hours.

7. The method according to claim 5, wherein in step b the DNA ligase is T4 DNA ligase and the ligation conditions are 25℃for 1-3 hours.

8. The method according to claim 5, wherein the ratio of linker 1, linker 2 to DNA fragment in step b is 10pM:10pM:50ng.

9. The method according to claim 5, wherein the amplification conditions in step c are: amplifying with Opie1-outer and adaptor primer for 5 cycles at 98℃for 2 min, at 98℃for 10 sec, 62℃for 20 sec, and 72℃for 1 min; adding the Opie1-inner, the Opie2-inner and the adaptor primer for amplifying for 20 cycles, wherein the amplification condition is 98 ℃ for 10 seconds, 62 ℃ for 20 seconds and 72 ℃ for 1 minute; and finally, the temperature is 72 ℃ for 8 minutes.

10. The method according to any one of claims 5 to 9, wherein the amplification product is recovered in step d using magnetic beads.