CN107619855B - Method for rapidly identifying common wheat A, B, D genome chromosome - Google Patents

Abstract

The invention provides a method for rapidly identifying a common wheat A, B, D genome chromosome. The invention firstly provides 3 oligonucleotide probes, the nucleotide sequences of which are respectively shown as SEQ ID No.1-3, the probes SEQ ID No.1 and SEQ ID No.2 can respectively generate completely-covered clear signals on wheat B and D genome chromosomes, and the probe SEQ ID No.3 generates clear signals on the A genome chromosome. The combination of 3 probes for ND-FISH can completely distinguish common wheat A, B, D genome chromosomes, can identify translocation chromosomes among chromosome groups in wheat, and has the advantages of simple operation, short time and low cost. Has good application prospect in the field of wheat breeding.

Description

Method for rapidly identifying common wheat A, B, D genome chromosome

Technical Field

The invention relates to the field of molecular cytogenetics, in particular to a method for detecting a wheat A, B, D genome chromosome and a translocation chromosome between wheat chromosome groups by using an oligonucleotide probe and an ND-FISH method.

Background

The chromosome sequences of higher organisms are complex and various, and a large number of repeated sequences exist in different intervals, so that a new thought and a new method are provided for marking and identifying chromosomes. In the field of molecular cytogenetics, a chromosome can be marked by constructing a repetitive sequence probe, and the distribution state of a probe signal on the chromosome is analyzed, so that a genome and a chromosome set are distinguished. Furthermore, it is also common practice to analyze chromosome behavior by comparing changes in probe signals on chromosomes.

Common wheat has been obtained by the fusion evolution of three different diploid ancestral species, contains A, B and D three genomes, and is a typical allohexaploid. Thus, common wheat is a useful material for studying species evolution (Marcussen et al 2014). Meanwhile, common wheat is also an important grain crop. The three genomes of common wheat are accurately identified and recognized, which is beneficial to researching the evolution of the wheat genome and finding out the chromosome translocation among the wheat genomes, thereby researching the function of the translocation chromosome. Therefore, the rapid and accurate identification of the chromosomes of the three genomes of the wheat A, B, D is very important, and particularly, the accurate identification of the chromosomal translocation among the three genomes is greatly helpful for the application of the translocation chromosome in wheat breeding. Although in situ hybridization techniques are available to achieve this goal, the existing in situ hybridization techniques for identifying the three genomic chromosomes of wheat A, B, D have respective defects, and the technical means need to be updated. On the one hand, specific probes are found which can generate full-coverage signals on chromosomes of A, B, D three genomes or two genomes, namely, the probes can generate full-coverage signals on A genome chromosome only, B genome chromosome only or D genome chromosome only; on the other hand, the found specific probe is convenient to prepare, and the hybridization process of the probe and the chromosome is simplified.

GISH (Genome in situ hybridization): the marked total genome DNA of animal and plant is used as probe to hybridize with chromosome of animal and plant to find out existence and mode of exogenous chromosome. In the prior art, the genome DNA of common wheat ancestral materials Triticum urartu, Aegiros spheres and Aegiros tauschii are taken as probes, the three genome DNAs are marked into different colors, the three genome chromosomes of common wheat A, B, D are distinguished by using a multicolor genome in situ hybridization (multicolor GISH) technology, and the distribution mode of the three genome DNA probes on the chromosome is a full coverage mode (Han et al 2004). The multicolor GISH technique requires extraction of genomic DNA from the three wheat progenitor materials Triticum urartu, aegiops spheres and aegiops tauschii, followed by labeling of the three genomic DNAs separately. After the probe is labeled, the probe and chromosome are denatured and hybridized overnight during in situ hybridization. The process is very tedious, time and labor consuming, and the cost of probe labeling is high.

FISH (fluorescence in situ hybridization): the marked DNA sequence is used as a probe to hybridize with the chromosome of the animal and plant, the distribution of the probe sequence on the chromosome is determined, and the specific landmark of the chromosome is established, so that the aims of identifying the chromosome, and knowing the structural function and the genome evolution of the chromosome are fulfilled. Three genomic chromosomes of Triticum aestivum A, B, D were identified using conventional FISH techniques (Zhang et al 2004; Komuro et al 2013). Cloning A, B, D group specific repeated sequences of common wheat, and identifying A, B, D chromosome of wheat by FISH technology by using the repeated sequences as probes. The method can also well distinguish three genome chromosomes of the common wheat A, B, D, and the effect is similar to that of a multicolor GISH technology. However, the conventional FISH technique requires a very complicated process for preparing the probe. Firstly, a large amount of specific repetitive sequences need to be obtained, which relates to complicated processes such as cloning, transformation of competent escherichia coli, plasmid extraction and the like. In addition, as with the GISH technique, the resulting repeat sequences also require labeling, and the purchase of a labeling kit is costly. Both the probe and chromosome also need to be denatured prior to hybridization of the probe to the chromosome. Therefore, the conventional FISH technology is complex in process and high in cost.

The C-banding technology (C-banding) is that plant chromosomes are treated under certain conditions and then dyed by Giemsa dye, different chromosomes display different dyeing shades, and different chromosome display different shade patterns, so that the aim of identifying the chromosomes is fulfilled. The C-banding technique can also be used to identify three genomic chromosomes of Triticum aestivum A, B, D (Friebe and Gill 1994). By the C-banding technology, different banding patterns of different chromosomes of wheat can be displayed, so that A, B, D three genome chromosomes are distinguished. However, the C-banding technology has long flow and complicated process, is not full-chromosome-covered, and particularly has not abundant banding on chromosomes of A and D genomes. Thus, the C-banding technique can accurately identify chromosomes only when the wheat chromosome or chromosome arm is intact, and cannot identify chromosomes if breaks and translocation of chromosomes occur in regions with poor or no banding.

ND-FISH (non-denaturing fluorescence in situ hybridization): generally, the synthesized single-stranded oligonucleotide is used as a probe to perform in situ hybridization with a non-denatured chromosome, so that the distribution of a probe sequence on the chromosome can be determined quickly and conveniently at low cost, and a specific landmark of the chromosome can be established, thereby achieving the purposes of identifying the chromosome, knowing the structural function of the chromosome and the genome evolution. By using oligonucleotide probes Oligo-pAs1, Oligo-pSc119.2 and Oligo-pTa535 and combining ND-FISH technology, chromosomes of three genomes of wheat A, B, D can be rapidly and accurately identified (Tang et al 2014; Fu et al 2015). The disadvantage of this technique is that the oligonucleotide probes used are designed based on the existing tandem repeat sequences pSc119.2, pAs1 and pTa-535, and the hybridization effect is the same as that of the original repeat sequences, mainly generating hybridization signals at the ends and sub-ends of A, B, D three genomic chromosomes and several signal points between the arms of a few chromosomes, all of which do not generate hybridization signals on chromosomes in a full coverage manner. Thus, these probes can only accurately identify chromosomes when the wheat chromosome or chromosome arm is intact, and cannot identify chromosomes if breaks and translocations occur in areas where these probes do not signal.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a method capable of distinguishing the chromosomes of the wheat A, B, D genome and identifying the translocation chromosomes among wheat chromosome groups.

In order to achieve the purpose, the technical scheme of the invention is as follows:

(1) the Wheat Genome reference sequence (IWGSC _ RefSeq _ V1) was downloaded from the public database International Wheat Genome Sequencing Consortium (IWGSC).

(2) Tandem Repeats in the wheat genome were annotated with Tandem Repeats Finder software (http:// Tandem. bu. edu/trf. html) to give about 390 ten thousand Tandem Repeats.

(3) And (3) carrying out primary screening on all the obtained tandem repeat sequences by using Python language programming under the condition that the repeat unit (Period Size) of the tandem repeat sequences is more than 6.

(4) And (3) classifying the series-connected repeated sequences obtained by screening in the step (3) into 4 classes (30-100, 100-200,200-300, >300) according to the Size of a repeated unit (Period Size) by using Python language programming.

(5) And (3) setting a similarity threshold value of 75% by using CD-HIT software (http:// weizhong-lab. ucsd. edu/CD-HIT /), clustering the 4 types of tandem repeat sequences obtained by the screening in the step (4) (namely, sequences with the similarity higher than 75% are clustered into one type), and counting the sum of copy numbers (copy number) of the tandem repeat sequences in each type.

(6) The Python language programming is used to extract the same kind of sequences with the copy number sum larger than 1000 and extract the consistency sequence (CONSENSUS).

(7) And (3) comparing the sequence obtained by screening in the step (6) with a known probe sequence by using a Blast localization tool, removing the probe sequence which is developed at present, and further filtering candidate tandem repeat sequences for probe development.

(8) And (3) comparing the tandem repeat sequence obtained by screening in the step (7) with the wheat genome sequence by using a Blast localization tool, analyzing the distribution characteristics of the tandem repeat sequence on the wheat chromosome, and obtaining the sequence finally used for probe design according to the characteristics of the chromosome position, copy number, sequence matching length and the like of the sequence.

(9) Oligonucleotide probes were designed based on the reported chromosome-specific interspersed repeat sequence 676D4(Zhang et al 2004) of the A genome. The design steps are as follows: the 676D4 sequence was divided into several segments in units of 60 bases, and then these segments were aligned by Python language programming, and probes were designed by taking sequences with a similarity of 45% or more.

(10) Synthesizing the designed probe sequence.

(11) And (3) after the probes are synthesized, performing ND-FISH test one by one to verify the probes. In the ND-FISH verification process, the probe is applied to the chromosome at room temperature controlled at 25-28 ℃, and then the slide glass is immediately placed in a plastic box with a length of 25 cm and a width of 15 cm, pre-incubated at 42 ℃ and placed in a 42 ℃ incubator for 1 hour.

(12) The effective oligonucleotide sequences obtained:

Chr2D242:5’-GGTTCAGGAATAGCCTCAGGAATTGGCTCAATT-3’(SEQ ID NO.1)

Chr6D978:5’-TACGG GTGCC AAACG AGTGT CTGAA AGACT CCTCG AGAGG AAAAT GCGAA-3’(SEQ ID NO.2)

Oligo-676D4:5'-AACTG ATTAT GTTTT TGCCA TGTTC ACATG CTTGC AATTG TATTT TCTGA TCCCT TTTG-3'(SEQ ID NO.3)

the invention firstly provides an oligonucleotide probe for identifying wheat B genome chromosome, and the nucleotide sequence is shown as SEQ ID NO. 1.

The invention provides an oligonucleotide probe for identifying wheat D genome chromosome, and the nucleotide sequence of the oligonucleotide probe is shown as SEQ ID NO. 2.

The invention provides an oligonucleotide probe for identifying wheat A genome chromosome, and the nucleotide sequence is shown in SEQ ID NO. 3.

Furthermore, the invention provides an oligonucleotide probe combination, which comprises 3 oligonucleotide probes, and the nucleotide sequences of the oligonucleotide probes are respectively shown in SEQ ID NO. 1-3.

The invention provides application of the oligonucleotide probe or the oligonucleotide probe combination in identifying the wheat A, B, D genome chromosome.

The invention provides application of the oligonucleotide probe combination in detecting translocation chromosomes between wheat chromosome groups.

The invention provides application of the oligonucleotide probe or the oligonucleotide probe combination in improvement or breeding of wheat germplasm resources.

The invention provides a method for detecting a wheat A, B, D genome chromosome, which utilizes the oligonucleotide probe combination to carry out ND-FISH method detection on a wheat material to be detected. And the following effects can be achieved by detection: Oligo-676D4(SEQ ID NO.3) produced a clearly defined hybridization signal on the A genome chromosome, but no obvious hybridization signal was observed on the B and D genome chromosomes, and the signals on 14A genome chromosomes were clearly contrasted with those on B, D genome chromosomes, so that 14A genome chromosomes could be distinguished from other 28 chromosomes (B, D genome chromosome) by naked eyes; chr2D242(SEQ ID NO.1) generates obvious and clear hybridization signals of a full coverage type on the B genome chromosome, but no obvious hybridization signals can be seen on the A and D genome chromosomes, and the signals on 14B genome chromosomes are obviously contrasted with those on A, D genome chromosomes, so that 14B genome chromosomes can be distinguished from other 28 chromosomes (A, D genome chromosomes) by naked eyes; chr6D978(SEQ ID NO.2) produced a clear hybridization signal on the D genome chromosome with full coverage, but no hybridization signal was seen on the A and B genome chromosomes, and the signals on 14D genome chromosomes were clearly contrasted with those on A, B genome chromosomes, so that 14D genome chromosomes could be distinguished from the other 28 chromosomes (A, B genome chromosome) by naked eyes.

On the other hand, the invention provides a method for detecting translocation chromosomes between wheat chromosome groups, which utilizes the oligonucleotide probe combination to carry out ND-FISH method detection on wheat materials to be detected. To illustrate the effect it should achieve: for example, wheat B genome chromosome and D genome chromosome are translocated, one segment of the translocated chromosome is provided with obvious signal of Chr2D242(SEQ ID NO.1), and the other segment is provided with obvious signal of Chr6D978(SEQ ID NO. 2). If the wheat B genome chromosome and the wheat A genome chromosome are translocated, one segment of the translocated chromosome is provided with an obvious signal of Chr2D242(SEQ ID NO.1), and the other segment is provided with an obvious signal of Oligo-676D4(SEQ ID NO. 3).

In the above method, the 3' -end base of the probe described in SEQ ID NO.1 is fluorescently labeled, the 5' -end base of the probe described in SEQ ID NO.2 is fluorescently labeled, and the 5' -end base of the probe described in SEQ ID NO.3 is fluorescently labeled, and diluted with 1 XTE of pH7.0, respectively;

the concentration of the working solution of the probe described in SEQ ID NO.1 is 35.69ng/ul, the concentration of the working solution of the probe described in SEQ ID NO.2 is 21.85ng/ul, the concentration of the working solution of the probe described in SEQ ID NO.3 is 24.53ng/ul, 0.4ul of each of the three working solutions is taken and mixed into 10ul of hybridization buffer solution to prepare hybridization solution.

The detection kit containing the oligonucleotide probes shown in SEQ ID NO.1, SEQ ID NO.2 and/or SEQ ID NO.3 and the oligonucleotide probes belonging to the same repetitive sequence family with the 3 oligonucleotide probes also belong to the protection scope of the invention.

The method of the invention has the following characteristics: the oligonucleotide probe Chr2D242 developed by the invention can be used for ND-FISH, generates an obvious and clear hybridization signal only with the B genome chromosome of the common wheat, can distinguish the B genome chromosome of the common wheat from the A and D genome chromosomes, and generates a signal on the B genome chromosome in a full-coverage manner. The oligonucleotide probe Chr6D978 can be used for ND-FISH, generates a clear hybridization signal only with the ordinary wheat D genome chromosome, can distinguish the ordinary wheat D genome chromosome from the A and B genome chromosomes, and generates a signal on the D genome chromosome in a full coverage mode. The oligonucleotide probe Oligo-676D4 can be used for ND-FISH, generates a clear hybridization signal with the ordinary wheat A genome chromosome only, and can distinguish the ordinary wheat A genome chromosome from the B and D genome chromosomes. Therefore, the combination of Chr2D242, Chr6D978 and Oligo-676D4 for ND-FISH can simultaneously distinguish three genomic chromosomes of Triticum aestivum A, B, D (FIG. 1). These three oligonucleotide probes, in combination, also enable the identification of chromosomes that have translocated between groups of chromosomes in wheat (FIG. 2). This overcomes the disadvantages of long flow, time and labor consumption and high cost of the C-band, GISH and conventional FISH techniques, and the disadvantage of the prior art that the oligonucleotide probe signal does not completely cover the entire chromosome.

Therefore, the technical scheme of the invention is mainly distinguished from the prior art by:

(1) although the prior art utilizes oligonucleotide probes and ND-FISH technology to identify the chromosomes of the three genomes of wheat A, B, D, the signal points generated by these probes are chromosome-local and not full-chromosome-covering. Thus, these oligonucleotide probes readily identify chromosomes only when they are in an intact state, and cannot identify chromosomes if breaks and translocations occur in regions where these probes do not signal. The technical scheme of the invention develops a novel oligonucleotide probe which can also be hybridized with the wheat chromosome through an ND-FISH technology, the hybridization mode is a chromosome full-coverage type, and chromosome breakage or translocation occurs in any region, so that the chromosome of which genome can be identified.

(2) GISH uses ancestral material genome DNA of common wheat as a probe, and needs to extract genome DNA of three ancestral species, then carries out labeling, denatures the probe and chromosome, and finally hybridizes. The present invention does not require these processes, and only a short probe sequence is synthesized, and neither chromosome nor probe is denatured at the time of hybridization.

(3) The C-band technology requires chromosome pretreatment before chromosome staining, the process is complicated, and the chromosome bands of wheat A group and D group are not abundant and can be accurately identified by very experienced people. The invention does not need complex chromosome pretreatment, and can clearly distinguish the chromosomes of three genomes of the common wheat.

(4) Conventional FISH techniques require acquisition of large numbers of sequences for probe labeling, followed by probe labeling, and denaturation of the probe and chromosome prior to hybridization. The present invention does not require these processes, only short probe sequences are synthesized, and neither chromosomes nor probes need to be denatured during hybridization.

Drawings

FIG. 1 shows that the oligonucleotide probes Oligo-676D4, Chr2D242 and Chr6D978 and ND-FISH technology are used to analyze chromosomes of metaphase cells of root tips of common wheat material Chinese spring, and A, B and C are chromosomes of the same cell. In Panel A of FIG. 1, the arrow indicates the A genome chromosome, and the hybridization signal with the probe Oligo-676D4 is clearly distinct, and the signal is clearly contrasted with the other 28 chromosomes; in panel B of FIG. 1, the arrow indicates the B genome chromosome, and the clear hybridization signal with Chr2D242 is generated, and the signal is compared with the rest 28 chromosomes; in FIG. 1C, the arrow indicates the D genome chromosome, and the clear hybridization signal with Chr6D978 is generated, and the signal is clearly contrasted with the rest 28 chromosomes.

FIG. 2 shows the analysis of metaphase cell chromosomes of common wheat materials 12FT2115.12 and 12FT2115.23 by using oligonucleotide probes Chr2D242, Chr6D978, Oligo-676D4 and ND-FISH technology. Panel A and B show chromosomes from the same cell as 12FT 2115.12. The probe used in panel A was Chr2D242 and the probe used in panel B was Chr6D 978. Arrows in panels A and B indicate translocation of wheat chromosomes B and D. Panel C and D are chromosomes from the same cell as 12FT 2115.23. The probe used in Panel C was Oligo-676D4, and the probe used in Panel D was Chr2D 242. Arrows in panels C and D indicate translocation of wheat chromosome A and B.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The wheat material of the verification probe used in the invention is a material which can be obtained by the public in China. Wheat materials 12FT2115.12 and 12FT2115.23 are materials which the laboratory was self-created by irradiation methods and have been published (Shulan Fu, Zhenglong Ren, Xiaoming Chen, Benju Yan, Feiquan Tan, Tihua Fu, Zongxing Tang. New where-r.t.5 DS-4RS.4RL and 4RS-5DS.5DL transition lines with a pore volume resistance. journal of Plant Research,2014, 127(6 743): 753). The biochemical reagents used in the present invention are all commercially available.

Example 1 determination of common wheat chromosome-specific oligonucleotide probes for A, B and D genomes

According to the published wheat genome sequence, various bioinformatics methods are comprehensively utilized, specific parameters are set, and a proper probe sequence is developed.

1. Determination of oligonucleotide probe nucleotide sequence and probe design

(1) Download the newly published wheat genome sequence: the Wheat Genome reference sequence (IWGSC _ RefSeq _ V1) was downloaded from the public database International Wheat Genome Sequencing Consortium (IWGSC).

(2) Performing primary screening on the downloaded sequences: tandem Repeats in the wheat genome were first annotated with Tandem Repeats Finder software (http:// Tandem. bu. edu/trf. html) to give about 390 million Tandem Repeats.

(3) Self-programming by using a bioinformatics method, setting specific parameters, and reducing the target range: carrying out primary screening on all the obtained tandem repeat sequences by using Python language programming, wherein the screening condition is that the repeat unit (Period Size) of the tandem repeat sequences is more than 6; then, the serial repetitive sequences obtained by the screening step are divided into 4 types (30-100, 100-200,200-300, >300) according to the repetitive unit Size (Period Size) by using Python language programming; setting a similarity threshold value of 75% by using CD-HIT software (http:// weizhong-lab. ucsd. edu/CD-HIT /), clustering the 4 types of tandem repeat sequences (namely, sequences with similarity higher than 75% are clustered into one type), and counting the sum of copy numbers (copy number) of the tandem repeat sequences in each type; using Python language programming to extract the same kind of sequences with the copy number sum larger than 1000 and extracting a consistency sequence (CONSENSUS); and finally, comparing the sequence obtained by screening in the step (6) with a known probe sequence by using a Blast localization tool, removing the probe sequence which is developed at present, and further filtering candidate tandem repeat sequences for probe development.

(4) Determination of the probe sequence for synthesis: and comparing the screened tandem repeat sequences with the wheat genome sequence by using a Blast localization tool, analyzing the distribution characteristics of the tandem repeat sequences on the wheat chromosome, and obtaining the sequence finally used for probe design according to the characteristics of the chromosome position, copy number, sequence matching length and the like of the sequence. Furthermore, oligonucleotide sequences were designed based on the published wheat A genome chromosome-specific interspersed repeat sequence 676D4(Zhang et al 2004). The specific operation is as follows: the 676D4 sequence was divided into several segments in units of 60 bases, and then these segments were aligned by Python language programming, and probes were designed by taking sequences with a similarity of 45% or more. These sequences were then sent to the company to synthesize oligonucleotide probes, which were verified by ND-FISH method to find sequences that specifically hybridized to wheat A, B or the D genome, thereby obtaining functional oligonucleotide probes.

Chr2D242:5’-GGTTCAGGAATAGCCTCAGGAATTGGCTCAATT-3’ (SEQ ID NO.1)

Chr6D978:5’-TACGG GTGCC AAACG AGTGT CTGAA AGACT CCTCG AGAGG AAAAT GCGAA-3’(SEQ ID NO.2)

Oligo-676D4:5'-AACTG ATTAT GTTTT TGCCA TGTTC ACATG CTTGC AATTG TATTT TCTGA TCCCT TTTG-3'(SEQ ID NO.3)

Example 2 establishment of a method for discriminating wheat A, B, D genome chromosomes

The three oligonucleotide probes (Chr2D242, Chr6D978 and Oligo-676D4) determined in example 1 were mixed and used in ND-FISH method to identify the A, B, D genomic chromosome of Triticum aestivum and the translocation chromosome between wheat genomes in Triticum aestivum materials 12FT2115.12 and 12FT 2115.23. Mid-root chromosome preparation in chinese spring, 12FT2115.12 and 12FT2115.23 the method described with reference to Han et al (2006), and ND-FISH analysis procedure the method described with reference to Fu et al (2015). The method comprises the following specific steps:

(1) root tip metaphase chromosome preparation

Treating root tips with laughing gas for 2 hours, fixing with 95% acetic acid for 10 minutes, washing off the acetic acid with double distilled water, cutting off a root tip meristem area, placing the root tip meristem area in a mixed solution of 2% cellulase and 1% pectinase, treating in a water bath at 37 ℃ for 50 minutes, cleaning the enzyme with 70% alcohol, mashing the root tips in 70% alcohol, centrifuging to remove alcohol, adding acetic acid to prepare a root tip cell suspension, dripping the suspension on a clean glass slide (8-10 ul suspension per glass slide), performing microscopic examination on the slide after the acetic acid volatilizes, and selecting the well-split slide to perform an ND-FISH experiment.

(2) ND-FISH experiment

For probe synthesis, the 3' -end base of the Chr2D242 sequence was fluorescently labeled, the 5' -end base of the Chr6D978 sequence was fluorescently labeled, and the 5' -end base of the Oligo-676D4 sequence was fluorescently labeled. The synthetic oligonucleotide probes Oligo-676D4, Chr2D242 and Chr6D978 were diluted to working solution concentration with 1 XTE pH7.0, working solution concentration of Chr2D242 was 35.69ng/ul, working solution concentration of Chr6D978 was 21.85ng/ul, and working solution concentration of Oligo-676D4 was 24.53 ng/ul. Then 0.4ul of each of Oligo-676D4, Chr2D242 and Chr6D978 working solutions was mixed with 10ul of hybridization buffer (hybridization buffer was composed of 1mM/L Tris-HCl,0.1mM/L EDTA,17.5g/L NaCl,8.8 g/L trisodium citrate) to prepare a hybridization solution. The prepared hybridization solution and the metaphase cell chromosome are not subjected to any denaturation treatment, 10ul of the prepared hybridization solution is directly taken at the room temperature of 25-28 ℃, dripped on a glass slide with the metaphase cell chromosome, covered with a cover glass, immediately placed in a wet common lunch box preheated at 42 ℃, placed for 1 hour at 42 ℃, eluted by 2 XSSC buffer preheated at 42 ℃, the elution time is about 15 seconds, when the slide is slightly dry, 10ul of anti-fading agent containing DAPI is added to each slide, covered with the cover glass, observed and photographed under an OLYMPUS BX51 fluorescence microscope after 2 minutes, the hybridization effect of the probe is detected, and the result shows that Chr2D242 only generates obvious and clear hybridization signals with the wheat B genome chromosome and does not generate obvious hybridization signals with the wheat A and D genomes. The Chr6D978 only generates obvious and clear hybridization signals with wheat D genome chromosomes, but does not generate obvious hybridization signals with wheat A and B genome chromosomes. Oligo-676D4 only hybridized with wheat A genome chromosome and did not generate obvious signal with wheat B and D genome chromosome.

The three maps A, B, C in FIG. 1 are chromosomes of the same cell in China spring of triticum aestivum, and compared with the three maps, the three probes respectively generate obvious and clear hybridization signals with three different chromosome sets (one set is composed of 14 chromosomes). As can be seen from the A diagram in FIG. 1, the oligonucleotide probe Oligo-676D4 only produces clearly clear signals with 14 chromosomes of 42 chromosomes of China spring wheat, wherein the 14 chromosomes are the A genome chromosome, and no clearly clear hybridization signals exist on the other 28 chromosomes (B and D genome chromosomes), indicating that Oligo-676D4 can distinguish the A genome chromosome from the B and D genome chromosomes. As can be seen from the B diagram in FIG. 1, the oligonucleotide probe Chr2D242 only generates clear hybridization signals with 14 chromosomes in 42 chromosomes of China spring wheat, the signal pattern of the Chr2D242 on the 14 chromosomes is full coverage, the 14 chromosomes are B genome chromosomes, and the other 28 chromosomes (A and D genome chromosomes) have no clear Chr2D242 signals, which shows that the Chr2D242 can distinguish the B genome chromosomes from the A and D genome chromosomes. As can be seen from the C diagram in FIG. 1, the oligonucleotide probe Chr6D978 also only produces clearly clear hybridization signals with 14 chromosomes of 42 chromosomes of China spring wheat, and the signal pattern of Chr6D978 on the 14 chromosomes is also full-coverage, and the 14 chromosomes are D genome chromosomes. While the other 28 chromosomes (A and B genomic chromosomes) had no clear signal for Chr6D978, indicating that Chr6D978 could distinguish the D genomic chromosome from the A and B genomic chromosomes. Therefore, the oligonucleotide probes Oligo-676D4, Chr2D242 and Chr6D978 combined can simultaneously distinguish the wheat A, B, D genome chromosomes under simple hybridization conditions.

As can be seen from the A and B plots in FIG. 2, the oligonucleotide probes Chr2D242 and Chr6D978 bound and a translocated chromosome between the genomes occurring in wheat material 12FT2115.12 was identified. In the A diagram of FIG. 2, each of the 3 chromosomes indicated by the arrows has a segment carrying a signal of Chr2D242, and in the B diagram of FIG. 2, the other segment of the 3 chromosomes carries a signal of Chr6D978, indicating that the 3 chromosomes are translocations between the B genome and the D genome chromosomes.

As can be seen from the C and D plots in FIG. 2, the oligonucleotide probes Oligo-676D4 in combination with Chr2D242 identified a translocating chromosome that occurred between the genomes in wheat material 12FT 2115.23. In the C diagram of FIG. 2, each of the 3 chromosomes indicated by the arrows has a signal carrying Oligo-676D4, and the other segment of the 3 chromosomes has a signal carrying Chr2D242 corresponding to the D diagram of FIG. 2, indicating that the 3 chromosomes are translocated between the A genome and the B genome.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain modifications and improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> Sichuan university of agriculture

<120> method for rapidly identifying genome chromosome of common wheat A, B, D

<130> KHP171114779.8

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<170> PatentIn version 3.5

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Claims (7)

1. An oligonucleotide probe combination is characterized by comprising 3 oligonucleotide probes, and the nucleotide sequences of the oligonucleotide probes are respectively shown as SEQ ID NO. 1-3.

2. Use of the oligonucleotide probe combination of claim 1 for identifying a wheat A, B, D genomic chromosome or in wheat germplasm resource improvement or breeding.

3. Use of the oligonucleotide probe combination of claim 1 for detecting a translocation chromosome between wheat chromosome sets.

4. A method for detecting the genome chromosome of wheat A, B, D, which is characterized in that the oligonucleotide probe combination of claim 1 is used for carrying out ND-FISH method detection on a wheat material to be detected.

5. A method for detecting translocation chromosomes between wheat chromosome groups, which is characterized in that the oligonucleotide probe combination of claim 1 is used for carrying out ND-FISH method detection on wheat materials to be detected.

6. The method according to claim 4 or 5, wherein the base at the 3' end of the probe of SEQ ID No.1 is fluorescently labeled, the base at the 5' end of the probe of SEQ ID No.2 is fluorescently labeled, and the base at the 5' end of the probe of SEQ ID No.3 is fluorescently labeled, each diluted with 1 XTE at pH 7.0;

the concentration of the working solution of the probe described in SEQ ID NO.1 is 35.69ng/ul, the concentration of the working solution of the probe described in SEQ ID NO.2 is 21.85ng/ul, the concentration of the working solution of the probe described in SEQ ID NO.3 is 24.53ng/ul, 0.4ul of each of the three working solutions is taken and mixed into 10ul of hybridization buffer solution to prepare hybridization solution.

7. A test kit comprising the oligonucleotide probe set of claim 1.

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