阅读说明：本技术 一种利用长穗偃麦草特异序列开发的寡聚核苷酸探针 (Oligonucleotide probe developed by utilizing elytrigia elongata specific sequence ) 是由 戴毅 石俊涛 陈建民 于 2021-08-28 设计创作，主要内容包括：本发明涉及一种用长穗偃麦草特异序列开发的寡聚核苷酸探针,主要是通过基因组测序的手段,获取长穗偃麦草中高度重复的序列,设计成寡核酸探针Oligo-pTh1,实验中可与其他探针组合使用,可以丰富染色体FISH核型图,用于识别远缘杂交种质中染色体的结构变异。小麦远缘杂交材料常常作为中间种质在小麦育种中得以应用,Oligo-pTh1探针可以辅助鉴定出小麦背景中的染色体的结构变异,能够显著提外源染色体的鉴定准确性,便于培育出优质的小麦新种质。(The invention relates to an oligonucleotide probe developed by using a long spike elytrigia repens specific sequence, which is mainly used for obtaining a highly repetitive sequence in the long spike elytrigia repens by means of genome sequencing, is designed into an oligonucleotide probe Oligo-pTh1, can be combined with other probes in an experiment for use, can enrich a chromosome FISH karyotype chart and is used for identifying structural variation of chromosomes in distant hybridization germplasm. The wheat distant hybrid material is often used as an intermediate germplasm in wheat breeding, the Oligo-pTh1 probe can assist in identifying the structural variation of chromosomes in a wheat background, the identification accuracy of exogenous chromosomes can be obviously improved, and high-quality new wheat germplasm can be conveniently cultured.)

1. An oligonucleotide probe developed by utilizing a long spike elytrigia repens specific sequence is characterized by comprising the following steps during preparation:

(1) extracting the DNA of the elytrigia elongata to obtain a DNA sample, and completing the preparation of the whole library by the DNA sample, wherein the steps of completing the preparation of the whole library comprise enzyme digestion, sequencing joint addition, purification and PCR amplification; sequencing the constructed library by an Illumina sequencer, and performing quality control analysis on the obtained original sequence to obtain a high-quality sequence;

(2) randomly selecting the obtained high-quality sequence by using a Seqtk program, wherein the selected sequence is 0.25 times of the sequencing depth;

(3) integrating the selected sequence by utilizing a prepressing of FASTQ Pair ordered-End reads program under a repeatExplorer utility tool in a repeatExplorer website, wherein the specific parameters are Read sampling No, Quality cut: 10, Percent above cut: 95, Trim reads: Yes, Start position:1, End position:101 and Rename reads: Yes;

(4) analyzing the integrated data by utilizing a RepeatExplorer2 clustering program under a RepeatExplorer2 tool in a RepeatExplorer website;

(5) downloading the analyzed data, opening a tarean report in a file, and selecting a sequence listed in the positive assets (high confidence) in the report: AGCCTAGTTCAAATAATTTTACACTAGAGTTGAACTAGCTCTAT, synthesizing the sequence, and carrying out TAMRA modification at the 5' end of the sequence to obtain Oligo-pTh1 probe.

2. The oligonucleotide probe developed by using the specific sequence of elytrigia elongata according to claim 1, further comprising the step (6):

and (3) verifying the synthesized Oligo-pTh1 probe by using a fluorescence in situ hybridization technology, observing the signal position of the Oligo-pTh1 probe in the Chinese spring elytrigia elongata additional line, and drawing a chromosome FISH karyotype chart according to a fluorescence signal.

Technical Field

The invention relates to an oligonucleotide probe developed by using a long spike elytrigia repens specific sequence, belonging to the field of cell biology.

Technical Field

Wheat is one of three major food crops, and provides more than 20% of the world population with required energy and protein. However, with the annual increase of the total population of the world, the arable area is reduced, the global climate change is added, the wheat planting area is reduced year by year, and the parent utilization in breeding is single, so that a breakthrough variety is lacked. In the face of the current situation of wheat breeding, enriching and expanding the genetic basis of wheat is very important for the wheat genetic breeding work. Practice proves that the wheat kindred wild species carry a plurality of excellent agronomic traits, and the excellent genes are introduced into common wheat, so that the genetic basis of the wheat can be widened, and the method is one of the common approaches in the research of wheat genetic breeding.

The elytrigia elongata is a closely-related wild species of wheat, has the characteristic of high resistance to gibberellic disease, is an excellent gene resource for genetic improvement of wheat, and is widely applied to genetic and breeding work of wheat. Usually, the desired chromosome fragment is introduced into a cultivar by chromosome engineering. Therefore, how to accurately identify the elytrigia elongata chromosome is the basis for obtaining excellent germplasm. At present, the most common method for identifying the chromosome identity of elytrigia elongata is FISH molecular karyotype. In recent years, with the continuous progress of in situ hybridization technology, oligonucleotide probes have been widely used in the identification of foreign chromosomes. Compared with a plasmid probe, the oligonucleotide probe has the advantages of shorter hybridization time, high identification efficiency, good experimental result repeatability, low cost and the like. At present, oligonucleotide probes Oligo-pSc119.2, Oligo-pAs1 and the like are widely applied to wheat, have rich signals and can be used for FISH karyotype analysis. However, the probes are not from sequences specific to the elytrigia elongata, and the karyotype of the chromosome to be analyzed is relatively limited. Therefore, in order to enrich the FISH karyotype of the elytrigia elongata chromosome, the invention discloses an oligonucleotide probe developed by utilizing the specific sequence of the elytrigia elongata.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an oligonucleotide probe developed by using a long spike elytrigia repens specific sequence.

The purpose of the invention is realized as follows: an oligonucleotide probe developed by utilizing a specific sequence of elytrigia elongata is characterized by comprising the following steps:

(1) extracting the DNA of the elytrigia elongata to obtain a DNA sample, and completing the preparation of the whole library by the DNA sample, wherein the steps of completing the preparation of the whole library comprise enzyme digestion, sequencing joint addition, purification and PCR amplification; sequencing the constructed library by an Illumina sequencer, and performing quality control analysis on the obtained original sequence to obtain a high-quality sequence;

(2) randomly selecting the obtained high-quality sequence by using a Seqtk program, wherein the selected sequence is 0.25 times of the sequencing depth;

(3) integrating the selected sequence by utilizing a prepressing of FASTQ Pair ordered-End reads program under a repeatExplorer utility tool in a repeatExplorer website, wherein the specific parameters are Read sampling No, Quality cut: 10, Percent above cut: 95, Trim reads: Yes, Start position:1, End position:101 and Rename reads: Yes;

(4) analyzing the integrated data by utilizing a RepeatExplorer2 clustering program under a RepeatExplorer2 tool in a RepeatExplorer website;

(5) downloading the analyzed data, opening a tarean report in a file, and selecting a sequence listed in the positive assets (high confidence) in the report:

AGCCTAGTTCAAATAATTTTACACTAGAGTTGAACTAGCTCTAT, synthesizing the sequence, and carrying out TAMRA modification at the 5' end of the sequence to obtain Oligo-pTh1 probe.

Further comprising the step (6): and (3) verifying the synthesized Oligo-pTh1 probe by using a fluorescence in situ hybridization technology, observing the signal position of the Oligo-pTh1 probe in the Chinese spring elytrigia elongata additional line, and drawing a chromosome FISH karyotype chart according to a fluorescence signal.

The method for identifying the chromosome of the wheat kindred wild species is mainly FISH, namely, a repeat sequence is made into a probe, and the position condition of the repeat sequence appearing on different chromosomes is drawn into a FISH karyotype to identify the chromosome identity. However, the probe sequences commonly used at present are not derived from elytrigia elongata, and the chromosome karyotype analysis is relatively limited. Therefore, aiming at the existing technical defects, the invention provides an oligonucleotide probe developed by utilizing the long spike elytrigia repens specific sequence, and the probe can be used for enriching the FISH karyotype chart of the chromosome and is favorable for accurately identifying the chromosome with structural variation in distant hybridization germplasm.

The method is advanced and scientific, and by the oligonucleotide probe developed by using the specific sequence of the elytrigia elongata, the sequencing technology is used for obtaining the genomic sequence information of the elytrigia elongata and carrying out quality control analysis to obtain the high-quality elytrigia elongata genomic sequence; randomly selecting the obtained high-quality sequence by using a Seqtk program, wherein the selected sequence is 0.25 times of the sequencing depth; integrating the selected sequence by using a Preprocessing of FASTQ ordered-end reads program under a repeatExplorer utility tool in a repeatExplorer website; analyzing the integrated sequence by utilizing a RepeatExplorer2 clustering program under a RepeatExplorer2 tool in a RepeatExplorer website; downloading the analyzed data, opening a tarean report in a file, and selecting a sequence listed in the positive assets (high confidence) in the report: AGCCTAGTTCAAATAATTTTACACTAGAGTTGAACTAGCTCTAT, submitting the sequence to a company for synthesis, and carrying out TAMRA modification at the 5' end of the sequence to obtain an Oligo-pTh1 probe; the probe is used for verification in a Chinese spring-elytrigia elongata additional line, the position of a fluorescence signal is observed, and a chromosome FISH karyotype chart is drawn.

The invention has the beneficial effects that: the obtained Oligo-pTh1 probe sequence is from elytrigia elongata, is different from the current commonly used probes, has stable hybridization signals and rich positions, can enrich the chromosome FISH karyotype chart when being combined with other probes, not only can accurately identify the chromosome identity, but also can provide an identification mode for the evolutionary relationship among species.

In conclusion, the invention discloses an oligonucleotide probe developed by utilizing a long spike elytrigia repens specific sequence, which is mainly used for obtaining a highly repetitive sequence in the long spike elytrigia repens by means of genome sequencing and is designed into an oligonucleotide probe Oligo-pTh1, and the oligonucleotide probe can be combined with other probes in an experiment to be used, so that a chromosome FISH karyotype diagram can be enriched and is used for identifying the structural variation of chromosomes in distant hybridization germplasm.

The wheat distant hybrid material is often used as an intermediate germplasm in wheat breeding, the Oligo-pTh1 probe can assist in identifying the structural variation of chromosomes in a wheat background, the identification accuracy of exogenous chromosomes can be obviously improved, and high-quality new wheat germplasm can be conveniently cultured.

Drawings

FIG. 1 shows the result of fluorescence in situ hybridization of an addition line of Chinese spring-thinopyrum elongatum;

in the figure:

a to G: adding DA1E-DA7E to Chinese spring-elytrigia elongata;

A1-G1: chromosomes following the DAPI chromosome;

A2-G2: fluorescence signal of Oligo-probe Oligo-pTh 1;

A3-G3: fluorescence signal of Oligo-probe Oligo-pTh 2;

A4-G4: fluorescence signals of the combination of Oligo-probes Oligo-pTh1 and Oligo-pTh 2.

Detailed Description

The invention is further illustrated by the following examples.

Example 1

In order to verify the feasibility of the oligonucleotide probe Oligo-pTh1 and determine the fluorescence signal intensity, the combination of Oligo-pTh1 and Oligo-pTh2 is used for carrying out a fluorescence in situ hybridization experiment on the Chinese spring thinopyrum elongatum additional line, and the specific operation is as follows:

(1) sending the sequences of Oligo-pTh1 and Oligo-pTh2 to companies for synthesis, and performing TAMRA and FAM modification at the 5' end of the sequences respectively;

(2) diluting the synthesized probe according to the concentration requirement, preparing a hybridization solution according to the system in the table 1, placing the prepared hybridization solution in a metal bath at 103 ℃ for denaturation for 13min, immediately placing the hybridization solution in alcohol precooled at-20 ℃ after the denaturation is finished, and placing the hybridization solution in a refrigerator at-20 ℃;

(3) putting the prepared chromosome sheet of the Chinese spring-elytrigia elongata additional line in 70% alcohol solution of 0.15mol/L NaOH for denaturation for 5 min; dehydrating in 70%, 90% and 100% ethanol for 5min each time after tabletting and denaturation, and blow-drying;

(4) adding 40 μ L hybridization solution to each denatured slide, covering with a cover slip, and placing in a 37 deg.C incubator for more than 6h (or hybridizing overnight);

(5) eluting hybridized slices in 1 × PBS for 3 times at room temperature for 5min each time, and drying after elution;

(6) mu.L of DAPI (VECTOR, H-1200) was added to the blow-dried plate, covered with a cover slip and counterstained for 10min, observed under a Nikon (Ni-U) fluorescence microscope, and photographed with a Nikon DS-Qi1 Mc.

(7) Photographs taken were processed with Photoshop and synthesized with NIS-Elements D4.30.00 for analysis of the signal of Oligo-pTh1 probe on chromosome. As is clear from FIG. 1, the Oligo-pTh1 probe has a distinct fluorescence signal on a chromosome, and combined with the fluorescence signals of other probes, the FISH karyotype of each chromosome can be mapped, and structural variations of the chromosome can also be identified.

The above experimental results show that the oligonucleotide probe Oligo-pTh1 can generate fluorescent signals on chromosomes of distant hybridization germplasm, and can be used for drawing a FISH karyotype chart of the chromosomes and analyzing the variation of the chromosome structure.

TABLE 1 fluorescent in situ hybridization solution System

The sequences used

Sequence listing

<110> Yangzhou university

<120> oligonucleotide probe developed by utilizing long spike elytrigia repens specific sequence

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agcctagttc aaataatttt acactagagt tgaactagct ctat 44

<210> 2

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caggactgca gctaagcccc cgagtgagag ggttgctcat cactcggtag gat 53