CN113528699B - Marker combination and application thereof in mitotic recombination hotspot detection - Google Patents

Abstract

The invention relates to the technical field of synthetic biology, in particular to a marker combination and application thereof in mitotic recombination hotspot detection. The PCRTag tag combination provided by the invention can be used for rapidly detecting mitotic recombination hot spots of eukaryotic genome. By utilizing the tag combination, mitotic recombination hotspots of homologous chromosomes can be rapidly identified through a specific watermark tag, and a convenient method is provided for researching mitotic recombination events of eukaryotes.

Description

Marker combination and application thereof in mitotic recombination hotspot detection

Technical Field

The invention relates to the technical field of synthetic biology, in particular to a marker combination and application thereof in mitotic recombination hotspot detection.

Background

Homologous recombination between DNA molecules refers to a biological process in which DNA sequences undergo double strand breaks, recombinational repair, and chromosome separation processes to achieve recombination between sequences, forming novel DNA molecules. Homologous recombination can not only repair double-stranded break (DSB) of DNA, maintain genome stability, but also realize cross exchange of genetic information, generate daughter cell genetic diversity and accelerate the biological evolution process.

Homologous recombination comprises both meiotic and mitotic recombination, and naturally, genetic material in a cell is more predisposed to meiotic recombination, and mitotic recombination occurs at a rate 10 times less than meiotic recombination events ⁴ ～10 ⁵ And thus, meiotic recombination events have been more extensively studied. However, mitotic recombination also has important roles in eukaryotes, including repair of spontaneously occurring DSBs, restarting arrested replication forks, providing an alternative pathway for telomere replication in telomerase-deficient cells, and facilitating genome evolution by generating new chromosomal rearrangements. Chromosomal rearrangements during cell mitosis lead to loss of heterozygosity (LOH) at tumor suppressor sites, which in turn induces malignant growth of cells and induces cancer cells, although there are many causes of LOH, about half of the LOH events are associated with mitotic recombination in a study on retinoblastoma, and thus, the study on mitotic recombination mechanism can also provide guidance for the mechanism of cancer induction.

In the early period, only a single recombination hotspot region in yeast is researched, and a great deal of research is carried out to characterize the chromosome rearrangement rate by constructing a meiosis specific integration plasmid, the integration plasmid can be integrated into a genome during meiosis to cause ectopic recombination of homologous regions, and the research finds that the meiosis recombination rate of a DNA fragment close to THR4 at the position of 2-2.5cM/kb of chromosome III is 6 times higher than the average recombination rate of chromosomes, and the site is a strong DSB site. Using a similar approach, the following recombination hotspots for saccharomyces cerevisiae were also located: HIS2, HIS4, ARG4, CYS3, DED81, ARE1/IMG1, CDC19, LEU2-CEN3. By performing sequence analysis on the hot spots, most recombination hot spots are DSB sites, and are usually tandem repeat sequences sensitive to DNase I, but there are exceptions, for example, the recombination hot spot of ade6-M26 is not located at the DSB site, but is located in the sequence 500bp upstream of the DSB site, the site can be combined with the transcription factor Atf1/Pcr1, and the hot spot of White and colleagues also need to be combined with the transcription factors Bas1, bas2 and Rap1 through an experiment HIS4, the hot spot needing to be combined with the transcription factor is called an 'alpha' hot spot, and the hot spot needing not to be combined with the transcription factor is called a 'beta' hot spot. The above experiments indicate that the DSB site is the first site for recombination fragmentation, but the DSB site is not the only factor affecting the recombination rate, and the recombination rate is also closely related to the chromatin structure and the environment of chromosomes.

With the development of DNA sequencing technology, people are beginning to focus on the exploration of recombination hot spots and cold spots in the whole genome. Gerton et al performed microarray analysis on 6200 ORFs by using a DNA probe method, ranked the region with the recombination rate of 12.5% as the recombination hotspot region, and finally detected 177 hotspots in total. As a result, all chromosomes have at least one hot spot, the significant correlation exists between the size of the chromosomes and the number of the hot spots, the comparative analysis of all recombination hot spots shows that no single significant characteristic exists in all hot spot regions, and the factor with the strongest correlation among the hot spots is high GC content. Mancera et al genotyped the parental strain S288c/YJM789 heterozygote and 204 spores thereof produced by 51 meioses using ssGengenotyping to map the high resolution map of meiotic recombination of the yeast genome. Studies have shown that on average each meiosis produces 90.5 CO and 46.2 NCO, and that the amount of CO is linear with chromosome length. By counting the number of strains appearing at each marker, the recombination rate of each locus of the genome is characterized, and 84% of recombination hotspot regions are found to be overlapped with the promoter. Most of the genomes have differences in the distribution ratio of CO/NCO, and the differences can reach the maximum CO/NCO event ratio of 14:0 and 0:7. by analyzing the GC content of the recombination hotspot region, it was found that chromosomes tended to undergo fragmentation recombination at high GC content. Charles explores the mitotic recombination rule of chromosome IV of yeast by using a SNP microarray method, and finds that the region where an inverted repeat sequence is positioned is very easy to be a breaking hot spot region, because the inverted repeat sequence is frequently subjected to intrachain pairing during recombination, a hairpin structure is formed to prevent the movement of a replication fork, a DSB gap is formed, a cross-shaped structure similar to a holliday knot is formed sometimes, and the cross-shaped structure is mistakenly recognized and cut by endonuclease, and the research shows that the recombination rate of hot spot HS4 is obviously related to the inverted Ty element pairs (YDRWTy 2-3 and RCYDTy 1-3) positioned between 981 and 992 kb. Chromosomal genomic recombination at palindromic, tandem repeats, G4 DNA sequences, tRNA's, intron-containing gene sequences, ARS elements, ty elements and their long terminal repeats, highly transcribed genes, replication termination regions, high gamma-H2 AX regions, high GC content regions, rrm3p termination sites, and the like, has been found to occur more readily by characterizing chromosomal fragile sites in yeast. Yang et al investigated the chromosomal rearrangement hot spots in the spontaneous mitosis process of diploid yeast and found that large deletions/duplications and translocations are often mediated by homologous recombination between repeated sequences such as Ty element or delta element, that the break point of I-LOH (Interstial LOH) in the yeast genome is significantly related to the replication termination (Ter) sequence and the region with abnormal GC content, while T-LOH (Terminal LOH) is mostly located at the G4 quadruplex sequence, the region with high gamma-H2 AX content, the region with high GC content and the non-coding RNA gene.

Although a great deal of research has been conducted on the genome variation law of mitotic recombination of yeast, research on the hot and cold spot regions of yeast recombination is insufficient because of limitation in the construction of mitotic libraries and screening of recombinant strains. The SCRAMBLE system in the synthetic yeast can quickly construct a genome recombinant library, and secondly, the introduction of the PCRtag watermark provides a simple and efficient method for positioning the recombination hotspot region of the genome recombinant strain. The process of random disorganization and recombination of the synthetic chromosomes is very similar to the process of forming cancers, so that a theoretical model is hopefully provided for determining target genes related to human chromosome diseases by researching the recombination rule of the yeast synthetic chromosomes in the artificial induced rearrangement process, and a reference is provided for target screening and designing of antitumor drugs. The rules of the synthetic diploid yeast and the artificially constructed tetraploid yeast with doubled whole genome in artificial induction rearrangement are analyzed by comparison, so that the method is favorable for analyzing the influence of genome doubling on genome recombination, and has guiding significance on polyploidization and chromosome fragmentation research in cancer. However, no suitable PCRtag has been found for the study of mitotic recombination hotspots.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a marker combination and its application in mitotic recombination hotspot detection.

The method adopted by the invention can be suitable for the exploration of all chromosome rearrangement hot spots containing specific watermark labels. However, due to the specificity of the chromosome tags of different species, when exploring different chromosomes, attention needs to be paid to selecting the corresponding watermark tag on the chromosome. The invention aims at the research of saccharomyces cerevisiae, and in the research, a yeast strain containing artificially synthesized chromosomes is adopted. Each synthetic chromosome is introduced with different numbers of specific watermark tags at the beginning of design, such as 532, 186, 339, 164, 490 and 681 pairs of PCRTag tags introduced into synII, synIII, synV, synVI, synX and synXII respectively, and the tags can be used for screening genome recombinant strains. However, in the process of detecting mitotic recombination hot spots, the selection of the watermark label should be performed according to the length of the chromosome, and the aim is to finally obtain a good detection effect.

The invention researches on the chromosome V of yeast, and the PCRTag tag combination provided by the invention comprises: at least two of YEL038W _ amp1, YEL013W _ amp1, YER026C _ amp1, YER056C _ amp1, YER086W _ amp1, YER113C _ amp2, YER144C _ amp2, YER170W _ amp1, YEL032W _ amp2, YEL029C _ amp1, YEL023C _ amp1, YEL019C _ amp1. In some embodiments, the PCRTag tag combination of the present invention includes: YEL038W _ amp1, YEL013W _ amp1, YER026C _ amp1, YER056C _ amp1, YER086W _ amp1, YER113C _ amp2, YER144C _ amp2, and YER170W _ amp1.

In the PCRTag tag combination provided by the invention, YEL038W _ amp1, YEL013W _ amp1, YER026C _ amp1, YER056C _ amp1, YER086W _ amp1, YER113C _ amp2, YER144C _ amp2 and YER170W _ amp1 are uniformly distributed on the synV chromosome, wherein 2 PCRTags are uniformly distributed on the left arm of the V chromosome, and the remaining 6 PCRTags are uniformly distributed on the right arm of the V chromosome. More mitotic recombination events of chromosomes can be observed relative to other combinations of PCRTag tags.

In addition, for strains with chromosome breakage between YEL038W _ amp1 and YEL013W _ amp1, 10kb refinement analysis was also performed, and one PCRTag was reselected every 10kb, verifying genome loss at YEL032W _ amp2, YEL029C _ amp1, YEL023C _ amp1, YEL019C _ amp1 for the above samples, respectively.

The invention also provides application of the label combination in mitotic recombination hotspot detection.

The invention realizes mitotic recombination of homologous chromosomes through manual intervention, and quickly constructs a recombinant strain library through a specific watermark label, thereby providing a convenient method for researching mitotic recombination events. In the present invention, the mitosis is a mitosis of a eukaryote. The eukaryote is a yeast, and in some embodiments, the eukaryote is saccharomyces cerevisiae.

The strains involved in the research of the invention comprise diploid saccharomyces cerevisiae, triploid saccharomyces cerevisiae and tetraploid saccharomyces cerevisiae.

The diploid s.cerevisiae contains one wild type chromosome V (wtV) and one synthetic type chromosome V (synV). Triploid Saccharomyces cerevisiae contains one wild type chromosome V (wtV) and two synthetic chromosomes V (synV). Tetraploid Saccharomyces cerevisiae contains two wild type V chromosomes (wtV) and two synthetic V chromosomes (synV).

The invention also provides a reagent for detecting the mitotic recombination hot spots of the yeast, which comprises a primer for detecting the label combination. The primer of the invention comprises:

the nucleic acid sequence of the amplification primer of YEL038W _ amp1 is shown as SEQ ID NO. 1-2;

the nucleic acid sequence of the amplification primer of YEL013W _ amp1 is shown in SEQ ID NO. 3-4;

the nucleic acid sequence of the amplification primer of YER026C _ amp1 is shown in SEQ ID NO. 5-6;

the nucleic acid sequence of the amplification primer of YER056C _ amp1 is shown as SEQ ID NO. 7-8;

an amplification primer of YER086W _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 9-10;

the nucleic acid sequence of the amplification primer of YER113C _ amp2 is shown in SEQ ID NO. 11-12;

the nucleic acid sequence of the amplification primer of YER144C _ amp2 is shown in SEQ ID NO. 13-14;

the nucleic acid sequence of the amplification primer of YER170W _ amp1 is shown in SEQ ID NO. 15-16.

In the present invention, the reagent further includes a PCR reaction reagent. The PCR reagent comprises Taq enzyme.

The invention also provides a recombinant hot spot method for detecting the mitosis of the yeast, which is used for detecting the yeast by using the reagent.

The invention takes chromosome V as a research object to research the mitosis recombination hotspot of the yeast.

The yeast comprises diploid saccharomyces cerevisiae, triploid saccharomyces cerevisiae and tetraploid saccharomyces cerevisiae. Wherein the diploid s.cerevisiae contains one wild type V chromosome (wtV) and one synthetic type V chromosome (synV). Triploid s.cerevisiae contains one wild-type V chromosome (wtV) and two synthetic V chromosomes (synV). Tetraploid Saccharomyces cerevisiae contains two wild type V chromosomes (wtV) and two synthetic V chromosomes (synV).

Before amplification detection, the yeast is subjected to induced rearrangement to construct a rearrangement library. The induction was with estradiol. The concentration of estradiol in the induced medium was 1. Mu. Mol/L.

The detection method comprises the following steps: the amplification primer of the invention is used for amplifying the microzyme. The system for amplification comprises: an upstream primer, a downstream primer, taq enzyme, template DNA and water.

Based on comparing the amplification results of the three strains, genomic mitotic diversity was obtained. The method mainly comprises the steps of counting the number of breakpoints between adjacent PCRTags after rearrangement to obtain the mutation condition of the sample. For example: the presence of all the PCRTag bands of the synthetic type V chromosome indicates that at least one copy of the DNA fragment of all the synthetic type V chromosomes is present in the test strain. The PCRTag bands of the synthetic type V chromosome were not detected at all, indicating that the synthetic type V chromosome was lost in the test strain at all. The presence of the PCRTag band portion of synthetic type V chromosome indicates that both copies of the partially synthetic type V chromosome DNA region were completely lost in the triploid test strain. If the number of amplification breakpoints is large, the segment is unstable and is a recombination hot spot region. And the small number of times of amplification breakpoints indicates that the part of the genome is not easy to lose during recombination and is a recombination cold spot region.

In addition, the rearranged libraries were sequenced and statistically analyzed. The sequencing method comprises the following steps: whole genome sequencing reads obtained by specific extraction paired-end sequencing. The statistical analysis comprises: analyzing the copy number variation of homologous chromosomes of cells in the genome rearrangement process by utilizing the difference of PCRTag sequences on the synthetic V chromosome and the wild V chromosome; reacting the number of synthetic type V chromosomes and wild type V chromosomes in the cells by sequencing; counting the mode and position of ectopic recombination, and analyzing unequal repetition/deletion caused by ectopic recombination among sister chromatids; and (4) counting the quantity of SNP sites of homologous chromosomes and analyzing the phenomenon of interstitial heterozygosity deletion.

The invention provides a PCRTag tag combination and provides application thereof in rapidly detecting mitotic recombination hot spots of eukaryotic genome. The mitotic recombination hotspot of homologous chromosomes can be quickly identified by utilizing the label combination through the specific watermark label, and a convenient method is provided for researching the mitotic recombination event of eukaryotes.

Drawings

FIG. 1 shows a process for constructing polyploid strains of different karyotypes;

FIG. 2 shows a schematic of the PCRTag detection of synthetic, wild-type chromosomes;

FIG. 3 shows the PCRTag detection of chromosome V presence pattern of the triploid strain;

FIG. 4 shows the existence pattern and proportions of the triploid SCRaMbLE mutant synV;

FIG. 5 shows statistics of the number of occurrences of breakpoint regions between adjacent PCRTags in a diploid;

FIG. 6 shows statistics of 10kb thinning breakpoint times between diploid synV 70.5kb-118.1 kb;

FIG. 7 shows a method for detecting the ORF to which a PCRTag-specific CDS belongs;

FIG. 8 shows a genome-wide sequencing depth map of a triploid synV completely deleted yeast strain;

FIG. 9 shows the phenomenon of unequal duplication/deletion caused by ectopic recombination between diploid yeast sister chromatids;

FIG. 10 shows I-LOH resulting from diploid mitotic recombination.

Detailed Description

The invention provides a marker combination and application thereof in mitotic recombination hotspot detection, and a person skilled in the art can appropriately modify process parameters for realization by referring to the content in the text. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. In the invention, the adopted strains are all from Tianjin university. The designation of the PCRtag is referred to in "Perfect" designer chromosome V and behavor of a ring derivative (Science, 2017,355 (1046): eaaf 4704.) and http:// synthetic layer. The invention is further illustrated by the following examples:

examples

1. Construction of rearranged Strain libraries

1.1 strains

(1) The diploid s.cerevisiae strain yLHM120, which is the starting strain, contains one wild-type chromosome V (wtV) and one synthetic chromosome V (synV), from the university of Tianjin.

(2) The triploid saccharomyces cerevisiae strain yXZX1892, which contains one wild type chromosome V (wtV) and two synthetic type chromosomes V (synV), was from the university of tianjin.

(3) The tetraploid s.cerevisiae strain yLHM086, which contains two wild type number V chromosomes (wtV) and two synthetic number V chromosomes (synV), was used as the starting strain from Tianjin university.

1.2 Induction of rearrangement:

from the SC-His plate, a single colony carrying pCRE4 was picked, inoculated into 3mL of SC-His medium, and cultured with shaking at 30 ℃ for 24 hours.

Centrifugation was carried out at 5000rpm for 2min at room temperature, the supernatant was aspirated, the cells were collected and washed three times with sterile water.

The cells in step 2 were transferred to 3mL of complete Synthesis (SC) medium containing no histidine and glucose and 2% glycerol as a carbon source, and cultured overnight with shaking at 30 ℃.

Centrifugation was carried out at 5000rpm for 2min at room temperature, the supernatant was aspirated, the cells were collected and washed three times with sterile water.

Resuspend cells in 3mL complete Synthesis (SC) Medium without histidine and glucose, containing 2% glycerol and 2% galactose as carbon sources, to give initial OD ₆₀₀ = 0.6-1.0, estradiol concentration is added to 1 μ M, and shaking culture is performed at 30 ℃ for 8 hours. (Induction of pCRE4 plasmid in Saccharomyces cerevisiae cells to generate one CRE recombination during the cell cycleAn enzyme that causes DNA recombination between loxPsym sites of synthetic chromosome V in a cell to generate structural variation such as DNA deletion, insertion, and inversion, FIG. 1)

Cells were appropriately diluted with sterile water and plated on SC-His plates to ensure that similar numbers of colonies were observed on control and experimental (treated with galactose and estradiol) plates, and cultured at 30 ℃ for 72 hours, and the colony numbers and colony sizes were observed and counted.

2. Screening of genomic mitotic recombinant strains

The strain libraries were analyzed using a synthetic polymerase chain reaction tag (PCRTag). In 177 segments divided by loxPsym or loxP sites (a total of 174 loxPsym recombination sites and 2 loxP recombination sites on the synV chromosome, 176 recombination sites dividing the chromosome into 177 regions, numbering 1-177 from the start position of the left end of the chromosome). In 103 segments, there is at least one non-essential gene, and at least one pair of PCRtags is designed in the gene.

8 sets of non-redundant PCRtags in which the distribution is uniform were selected for analysis of chromosome V. The 8 genes to which the PCRTag sequences belong are YEL038W, YEL013W, YER026C, YER056C, YER085W, YER113C, YER144C and YER170W, respectively. Of these, 2 pcrtags are evenly distributed on the left arm of chromosome V, and the remaining 6 are evenly distributed on the right arm of chromosome V (fig. 2).

TABLE 1 PCRtag primer List

TABLE 2 Saccharomyces cerevisiae colony PCRtag assay reaction System (10. Mu.L)

Reaction reagent	Volume (mu L)
		Primer F(c＝10μΜ)	0.25
Primer R(c＝10μΜ)	0.25
		2×Rapid Taq Master Mix	5
Template(Boiled yeast)	1
		ddH 2 O	3.5

The results of the PCRTag detection of the triploid control strain yXZX1892 show that both the synthetic and wild-type PCRTag detection bands are present. Indicating that the synthetic V chromosome and the wild-type V chromosome are both present in the cell in their entirety. The PCRTag detection result shows that the mitotic variation of the genome rearrangement library presents diversity, and the existence form of the synthetic V chromosome is as follows:

1) The PCRTag bands of the synthetic type V chromosome were detected to be present in their entirety, as shown by the detection result of the sample yXZX2468 in FIG. 3. This indicates that at least one copy of the DNA fragment of all synthetic type V chromosomes was present in the test strain.

2) The PCRtag bands of the synthetic type V chromosome were not detected at all, as shown by the detection result of the sample yXZX1105 in FIG. 3. Indicating that the synthetic type V chromosome is totally lost in the test strain.

3) Detecting the existence of the PCRTag strip part of the synthetic type V chromosome. As shown by the results of the detection of the sample yXZX1937 in FIG. 3, only two synthetic PCRTags (PCRTag of the genes YEL038W and YER 026C) were present. Indicating that both copies of the partially synthesized type V chromosomal DNA region were lost in the triploid test strain.

Then, the genotype statistical analysis is carried out on the experimental group strains with different existence forms of the synthetic type V chromosome in the genome rearrangement library. In the research, 274 samples are screened from the triploid saccharomyces cerevisiae genome rearrangement library, and the number and the corresponding proportion of different genotype strains in the screened library are respectively displayed. The classification statistics shows that 68 samples of the synthetic type V chromosome exist completely, and the total samples account for 24.82% of the total samples of the triploid. The synthetic type V chromosome part has 10 samples, which accounts for 3.65% of the total triploid samples. The total loss of synthetic type V chromosome is 196 samples, which accounts for 71.53% of the total samples. This indicates that the triploid Saccharomyces cerevisiae control strain yXZX1892 has different probabilities of forming different genotypes after genomic rearrangement. Wherein the highest probability of occurrence of synthetic type V chromosome loss events promoted the formation of aneuploid s.cerevisiae cells (fig. 4).

3. Identification result of mitotic hotspot of chromosome

The strain libraries were analyzed using a synthetic polymerase chain reaction tag (PCRTag). In 177 segments divided by loxPsym or loxP sites (a total of 174 loxPsym recombination sites and 2 loxP recombination sites on the synV chromosome, which 176 recombination sites divide the chromosome into 177 regions, numbered 1-177 from the start position at the left end of the chromosome). In 103 segments, there is at least one non-essential gene, and at least one pair of PCRtags is designed in the gene.

The number of breakpoints occurring between adjacent pcrtags after rearrangement in the yhlm 120 was counted and classified into 7 different types (fig. 5). Among them, 33 samples showed a breakpoint in a region between 70.5kb and 118.1kb, accounting for 33% of the total amount of partial deletion samples, type 1 was described by way of example of yFJ1779, and PCR results showed that this strain lost synV chromosomes in the region between 0 and 79.3kb and a chromosome break between YEL033W and YEL032W (B in fig. 5). 39% of the strains had a breakpoint region between 118.1kb and 193.1kb, in which the synthetic type V chromosome in yFJ1809 was broken near the centromere and the right chromosome of the centromere was lost. The two segments have a large proportion of chromosomal fragmentation and recombination, and in addition, 15% of the samples are fragmented between 193.1kb and 251.7kb, 12% between 251.7kb and 312.7kb, 8% between 312.7kb and 372.0kb, 6% between 372.0kb and 433.4kb, and 17% between 433.4kb and 494.0 kb.

The genome loss of these samples at YEL032W _ amp2, YEL029C _ amp1, YEL023C _ amp1, YEL019C _ amp1 was verified for 33 strains with breakpoints between 70.5kb and 118.1kb for yLHM120 (FIG. 6). The results showed that 17 strains showed breakpoints between 70.5kb and 79.3kb, 6 strains showed breakpoints between 79.3kb and 87.2kb, 9 strains showed breakpoints between 99.4kb and 109.2kb, 11 strains showed breakpoints between 109.2kb and 118.1kb, and none of the strains showed breakpoints between 87.2kb and 99.4kb, accounting for 52%, 18%, 27%, 33%, and 0% of the total sample size, respectively. The statistical result shows that 70.5 kb-79.3 kb are relative recombination hot spot regions of the segment, and the DNA sequence between 86.9kb and 99.4kb is relatively stable, is not easy to lose during genome recombination and is a recombination cold spot region.

4. Establishment of homologous chromosome copy number detection method

Whole genome sequencing refers to paired end sequencing of the genome of the strain using MGISEQ-2000. It is difficult to accurately identify sequencing results of synthetic V chromosomes from whole genome sequencing data of triploid saccharomyces cerevisiae carrying both synthetic V chromosomes and wild type V chromosomes during the course of the study. To overcome this difficulty, a method for high throughput analysis of genomic sequencing data was developed in the present study. I.e. whole genome sequencing reads (reads) obtained by specific extraction paired-end sequencing (figure 7). The depth of coverage of the homologous regions of the synthetic V chromosome and the wild type V chromosome is then defined as the number of reads of the PCRTag number in the same segment. The difference in the PCRTag sequences on the synthetic V chromosome and the wild-type V chromosome was used to analyze the copy number variation (CN) of homologous chromosomes that occurs in cells during genome rearrangement.

5. Method for detecting homologous chromosome loss and duplication

The PCRTag is used for detecting that 71.53 percent of triploid strains in the rearranged strains synthesize the complete loss of V chromosomes, so the occurrence of the chromosome variation event is never an accidental phenomenon. The present study showed that the number of V chromosomes changed after chromosomal rearrangement in polyploid saccharomyces cerevisiae based on the depth of coverage of the whole genome sequencing reads, as shown in fig. 8. In the figure, blue represents the sequencing depth of the synthetic type V chromosome, and orange represents the sequencing depth of the wild type V chromosome. The abscissa represents the position of the segment of the ORF to which the corresponding PCRtag read belongs on the V chromosome, and the number of synthetic V chromosomes and wild-type V chromosomes in the deeply responsive cells by sequencing.

From the sequencing depth map, it can be seen that the sequencing depth of the synthetic V chromosome in the triploid control strain yXZX1892 was about 140, and the sequencing depth of the wild type V chromosome was about 70. The sequencing depth of the synthetic V chromosome was 2 times that of the wild type V chromosome. Therefore, the copy number of the synthetic V chromosome in the cell is2, the copy number of the wild V chromosome is 1, and no chromosome variation event occurs in the cell. The sequencing depth of the synthetic V-shaped chromosome in the experimental group strain yXZX2471 is 0, so that the copy number of the synthetic V-shaped chromosome in the cell is 0, namely the synthetic V-shaped chromosome is completely lost, and the correctness of the primary detection result of the PCRTag is verified. Further analysis shows that the sequencing depth of the corresponding wild type V chromosomes in the cells of the experimental group is changed to about 140, namely the wild type V chromosomes are amplified by 1 time, namely the triploid rearrangement strain contains two wild type V chromosomes to form 3n-1 type aneuploid yeast cells. This indicates that complete loss of synthetic type V chromosome does occur after genomic rearrangement in triploid yeast cells, and that such chromosomal variation events are high frequency and stably exist.

6. Unequal repeat/deletion phenomena caused by ectopic recombination between sister chromatids

Translocation across sister chromatids results in unequal chromosomal crossovers, with partial deletion or replication of one homologous chromosome and the other homologous chromosome remaining unchanged (a in figure 9). We further divided ectopic recombination between sister chromatids into four types (B in fig. 9) based on the manner and location of ectopic recombination, the first being interstitial deletion, i.e. deletion of part of the middle segment of synV, while wtV remains unchanged (yFJ 1803). 20 samples in the diploid sequencing sample are subjected to interstitial deletion, the positions and frequency of breakpoints of the interstitial deletion of the samples are counted, a hot spot distribution diagram shown as C in figure 9 is drawn, breakpoints with the occurrence frequency of more than 4 are defined as deletion fracture hot spot regions, and 4 hot spot regions are detected in total and are respectively positioned at 105.4 kb-106.8 kb, 118.6 kb-122.3 kb, 446.4 kb-446.7 kb and 518.0 kb-519.1 kb. As the literature reports that the GC content influences gene conversion and further influences the distribution of genome recombination hot spots, the GC content of the fracture sites is determined, and the GC content of the areas from 105.4kb to 106.8kb and 446.4kb to 446.7kb is larger than the average value (38%) of synV, and is respectively 1 percent and 4 percent higher than the GC content of the areas from 118.6kb to 122.3kb and 518.0kb to 519.1kb which are respectively 1 percent and 2 percent lower than the average value of the synV.

If the deletion occurs at the end of the chromosome, we define it as an end deletion. As shown in FIG. 9, yFJ1865 underwent chromosome fragmentation at both the left and right ends of 31.9kb to 33.2kb and 233.5kb to 235.8kb, and had two terminal deletion fragmentation sites. The end deletion of synV of 20 strains in total of the diploid occurs, and the positions of the breakpoints in the sample are counted, so that 23 percent of the strains generate chromosome rearrangement at 75.1-77.5 kb and 135.9-140.0 kb, and therefore the two regions are regarded as high-frequency fracture sites of the diploid strains in the end deletion, wherein the GC content of 75.1-77.5 kb is 3 percent higher than the average value, and the GC content of 135.9-140.0 kb is 3 percent lower than the average value. 135.9kb to 140.0kb flank the centromere, and this region contains a large number of repeated sequences and promotes ectopic recombination between sister chromatids.

The third is interstitial duplication, the synV middle segment is replicated in one copy, wtV is unchanged. Only 1 strain of the diploid test strain developed a phenomenon of interstitial repetition (yFJ 1798), which occurred in multiple segments, mainly including 4 segments, of synV interstitial repetition: 123.5 kb-124.0 kb, 130.4 kb-131.0 kb, 131.8 kb-133.8 kb, 191.8 kb-192.6 kb, except that the GC content of the 131.8 kb-133.8 kb segment is less than 3% of the average value, the GC content of the 130.4 kb-131.0 kb segment is equal to that of the control, and the remaining two segments are 4% and 2% higher than the average value, respectively.

If the repeats occur on both sides of the telomere, they are called terminal repeats, and 1 sample of the diploid has terminal repeats (yFJ 1843), synV is fragmented at 105.4kb to 106.8kb, the sequence between 0kb to 106.8kb on the left is duplicated, and terminal deletions occur between 145.7kb to 536.0kb on the right.

6. Interstitial heterozygous deletion (I-LOH)

If recombination occurs between homologous chromosomes, a mitotic gene switch/cross-over event occurs that mediates a SNP to 0 in one homologous chromosome portion and a doubling of the original level in the homologous portion in the other chromosome, resulting in LOH. I-LOH is usually produced when DSB in G2 stage is repaired, and is the product of gene conversion in a small region (FIG. 10). We observed the I-LOH phenomenon in diploid strains yFJ1805, yFJ1815 and yFJ1836, wherein the SNP of synV is reduced to 0,wtV at 321.6 kb-339.9 kb and 344.1 kb-357.7 kb of yFJ1805 and yFJ1836 respectively, and gene transformation occurs at two sites of 438.8 kb-456.1 kb and 481.9 kb-507.8 kb of yFJ1815, and the length is not more than 30kb.

We found that in I-LOH, 3 breakpoints were located inside the gene, which were not detected when the sister chromatids were ectopically recombined, and all the samples that underwent ectopic recombination underwent chromosomal breaks between the two ORF frames. It has been reported that most of the ectopic recombination is mediated by homologous recombination between repeated sequences. LoxPsym is a reversed palindrome sequence which is 34bp outside the ORF frame, cre acts on the loxPsym to generate a DSB gap during induction of rearrangement, and the gap can be repaired between any two loxPsym through homologous recombination between repeated sequences, so that ectopic recombination tends to occur between the loxPsym outside the ORF frame, and chromosome breakage does not easily occur inside the gene. Compared to the mitotic gene conversion process, ectopic repeats between sister chromatids place higher demands on the repeat sequence.

The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Tianjin university

<120> marker combination and application thereof in mitotic recombination hotspot detection

<130> MP21013006

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tccagctcac gacagcttag acttaaac 28

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aactggagcg ttgcctggtc tactg 25

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tagcttgtta agcagtactg acccagac 28

<210> 4

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aactaagtgt ggtaaaccgc cggctcta 28

<210> 5

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgatttgctg atcataccgc aaccgtga 28

<210> 6

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cggcaagcca cactatgttc agagagct 28

<210> 7

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aaaagccaag ccggcgacca aactaaag 28

<210> 8

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgcttatgag aagtggagct gggttcca 28

<210> 9

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tagccaaggc gttggcttaa gcagtaga 28

<210> 10

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tctctcttcg gctaacttag cgcattcg 28

<210> 11

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

accaacgctg ttagccaatg acatccag 28

<210> 12

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccactgccca ggtgctagca aaaattat 28

<210> 13

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

agtgcttggg gtgttcaact tgctcaag 28

<210> 14

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cagcgttatc aagccattat caggtacc 28

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tcgttacgtc catgtcccat caggtcgt 28

<210> 16

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggtttcaccg ctaacagtgc cgaagata 28

Claims (2)

1. The application of the amplification primer group combined by the PCRTag tag in preparing a reagent for detecting the mitotic recombination hot spots of the yeast;

   the PCRTag tag combination consists of YEL038W _ amp1, YEL013W _ amp1, YER026C _ amp1, YER056C _ amp1, YER086W _ amp1, YER113C _ amp2, YER144C _ amp2, and YER170W _ amp 1;

   the primers of the amplification primer group are as follows:

   the nucleic acid sequence of the amplification primer of YEL038W _ amp1 is shown as SEQ ID NO. 1 to 2;

   an amplification primer of YEL013W _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO 3 to 4;

   an amplification primer of YER026C _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 5 to 6;

   an amplification primer of YER056C _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO. 7-8;

   an amplification primer of YER086W _ amp1, the nucleic acid sequence of which is shown in SEQ ID NO 9-10;

   the nucleic acid sequence of the amplification primer of YER113C _ amp2 is shown in SEQ ID NO. 11 to 12;

   an amplification primer of YER144C _ amp2, the nucleic acid sequence of which is shown in SEQ ID NO 13-14;

   the nucleic acid sequence of the amplification primer of YER170W _ amp1 is shown in SEQ ID NO. 15-16.
2. 2. Use according to claim 1, wherein the yeast is Saccharomyces cerevisiae.

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