Combined marker and method for rapidly detecting recombination positions and interference coefficients on different chromosomes of rice

Abstract

The invention discloses a combined marker and method for rapidly detecting recombination positions and interference coefficients on different chromosomes of rice, the combined marker comprises a recombination marker HEI10, a centromere marker CENH3 and a chromosome axis marker REC8, and the recombination marker HEI10 is a LOCOs02g13810 gene. According to the method, immunofluorescence is combined with an ultrahigh-resolution microscopic imaging technology, the recombination number and distribution of rice chromosomes are analyzed by utilizing a combined marker, and the interference coefficient is calculated through a cytological picture of microscopic imaging. Through the method, the distribution of the recombination sites on different chromosomes of the rice and the interference coefficient of the recombination sites are obtained, data support is provided for further analyzing a genetic mechanism of the recombination frequency, and a theoretical basis is provided for regulation and control of the recombination frequency in rice breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a combined marker and a method for rapidly detecting recombination positions and interference coefficients on different chromosomes of rice. Background Art

[0002] Rice( Oryza sativa ) As one of the world's major food crops, rice occupies an important position in genetic research and crop breeding. The complete sequencing of the rice genome and the in-depth study of gene function provide strong support for its genetic improvement. However, gene recombination is an important basis for rice genetic diversity and breeding improvement, and its research is of great significance for improving rice yield, stress resistance and other agronomic traits. In recent years, although technologies based on molecular markers and high-throughput sequencing have been widely used in rice recombination research, existing research methods rely on molecular markers or whole-genome sequencing, which often lack spatial resolution at the chromosome level and make it difficult to accurately capture the cytological mechanism of rice genome recombination. Therefore, cytological methods, especially direct observation and analysis of recombination events through microscopy and chromosome marker technology, are still an effective way to reveal the location and frequency of recombination. Through cytological methods, researchers can observe and analyze the process, location and frequency of rice genome recombination at a microscopic scale, and reveal the spatial distribution and genetic mechanism of recombination events.

[0003] At present, cytological research on rice genome recombination has made significant progress. Early studies focused on revealing recombination sites by observing the behavior of chromosomes during rice meiosis. With the advancement of fluorescent labeling, chromosome imprinting and immunofluorescence technology, the study of rice recombination events has gradually deepened. For example, researchers have successfully used immunofluorescence technology to mark rice chromosome axes and centromeres, and track the occurrence of recombination events through fluorescent signals, which has greatly facilitated the study of the recombination process of rice.

[0004] Cytological methods, especially the use of microscopes and chromosome labeling technology, are one of the most intuitive and accurate ways to analyze genetic recombination events by directly observing changes in chromosome behavior during meiosis. During rice meiosis, the pairing and crossing over of homologous chromosomes are key stages in the occurrence of recombination events. By using a high-resolution microscope to observe the pairing and crossing over of homologous chromosomes in rice meiotic cells, researchers can directly count the crossover points and then analyze the frequency and location of recombination. This method has a high spatial resolution and can capture detailed changes in chromosomes at the microscopic level.

[0005] With regard to the above existing technologies, it is difficult to simultaneously detect the co-localization of chromosome axes, centromeres, and recombination markers on chromosomes using ultra-high resolution microscopy technology. This greatly limits the feasibility of researchers using centromere positions to identify single chromosomes and analyze their recombination numbers and distribution. Summary of the invention

[0006] The present invention aims to solve the technical problem, overcomes the shortcomings of the prior art and provides a combined marker and method for quickly detecting the recombination positions and interference coefficients on different rice chromosomes. The method can directly observe and analyze rice recombination events at the chromosome level on the basis of ultra-high resolution imaging, and obtain the distribution of recombination sites on different rice chromosomes and their interference coefficients, which is of great significance for understanding rice genetic variation and evolution.

[0007] In order to achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a recombinant polyclonal antibody against HEI10, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] In a second aspect, the present invention provides a gene encoding the above-mentioned recombinant polyclonal antibody against HEI10, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0009] In a third aspect, the present invention provides use of the above-mentioned recombinant polyclonal antibody against HEI10 as a cytological marker indicating a class I exchange.

[0010] The present invention also provides a combined marker for rapidly detecting recombination positions and interference coefficients on different chromosomes of rice, including a recombination marker HEI10, a centromere marker CENH3 and a chromosome axis marker REC8. LOC_Os02g13810 Gene.

[0011] The centromere marker CENH3 is LOC_Os05g41080 gene, the chromosome axis marker REC8 is LOC_ Os05g50410 Gene.

[0012] The present invention uses a type of recombination marker HEI10, a centromere marker CENH3, and a chromosome axis marker REC8, combined with super-high resolution microscopic imaging technology (STED), to obtain cytological images that can distinguish the 12 complete rice chromosomes in a short time. The present invention provides an important cytological analysis method for the study of rice recombination frequency and its distribution law. HEI10 (Homologous Recombination Protein 10) is an E3 ubiquitin ligase involved in the homologous recombination process, which usually plays an important role in the recombination process of meiosis, especially in the formation of chiasma and maintaining the structural stability between homologous chromosomes. The localization and expression of HEI10 protein have temporal and spatial specificity during meiosis, and usually accumulate in the area where the recombination event occurs. Therefore, the use of HEI10 protein markers can accurately detect the location and distribution of recombination, becoming an effective tool for studying genome recombination.

[0013] The present invention provides a method for rapidly detecting recombination positions and interference coefficients on different chromosomes of rice, comprising the following steps: immunofluorescence combined with ultra-high resolution microscopic imaging technology, using combined markers to analyze the number and distribution of rice chromosome recombination, and calculating the interference coefficient through cytological pictures of microscopic imaging.

[0014] The present invention provides a method for combining rice immunofluorescence with ultra-high resolution microscopic imaging, and utilizes cytological images of microscopic imaging to analyze the recombination distribution and interference coefficient of different rice chromosomes.

[0015] Immunofluorescence combined with ultra-high resolution microscopic imaging technology first uses immunofluorescence technology to bind polyclonal antibodies to specific target molecules. The antibodies can specifically recognize and bind to the target molecules, providing labels for subsequent microscopic imaging. Then, ultra-high resolution microscopic imaging technology is used to image the samples combined with antibodies, which can break through the resolution limitations of traditional optical microscopes and provide nanometer-level resolution, so that the distribution and morphology of target molecules can be clearly observed.

[0016] The number and distribution of recombination events are analyzed using ultra-high resolution microscopic images. After obtaining ultra-high resolution microscopic images, the images are processed and analyzed to accurately locate the location of the recombination event by identifying and combining cytological markers. Then, by counting the number and distribution of these markers, the frequency and pattern of recombination events can be evaluated.

[0017] The method of calculating the interference coefficient from images. In ultra-high resolution microscopic imaging images, the interference coefficient refers to the "negative correlation" of crossover events within a chromosome interval. The larger the interference coefficient, the smaller the possibility of crossover in adjacent regions; conversely, it means that the interference of crossover is smaller and the distribution of crossover points is more random. By comparing the interference coefficients of recombination distribution on different chromosomes, the differences in recombination distribution on different chromosomes can be revealed, and the formation rules of recombination events can be further understood.

[0018] In order to improve the accuracy of statistics, it is necessary to collect at least a certain number of cell images. By counting the number of crossover points in each cell, the distribution pattern of crossover points can be obtained. Further statistical analysis of the number of crossover points can be performed to obtain the crossover frequency in different rice varieties and different genetic backgrounds. These statistical data will help evaluate the recombination hotspots of the rice genome and the genetic diversity between parents, and provide basic data for the analysis of the genetic mechanism of recombination frequency.

[0019] The method of the present invention can quickly detect the recombination position and interference coefficient on different chromosomes of rice, and provides a new technical means for rice genomics research and breeding. With the rapid development of precision agriculture and biotechnology, the demand for improved rice varieties is increasing, especially the breeding demand for improving yield, disease resistance and stress resistance is increasing. This method can efficiently detect recombination hotspots, optimize hybrid breeding strategies, and improve the accuracy and efficiency of rice breeding, and has broad market application prospects.

[0020] The invention adopts interference pattern analysis to analyze the crossover point distribution and interference intensity of the rice variety Yandao No. 8, and explores the distribution law of recombination exchange on the rice genome.

[0021] Through the above method, the present invention obtains the distribution of recombination sites on different rice chromosomes and their interference coefficients, provides data support for further analysis of the genetic mechanism of recombination frequency, and provides a theoretical basis for the regulation of recombination frequency in rice breeding. It can significantly shorten the breeding cycle, reduce variety development costs, improve breeding efficiency, and further accelerate variety improvement and the introduction of new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the immunofluorescence localization diagram of the HEI10 antibody in the present invention during the meiotic period.

[0023] Figure 2 This is a super-resolution signal diagram of the HEI10 antibody in the present invention at the pachytene stage.

[0024] Figure 3 This is a distribution diagram of the HEI10 large dots on the long arm of chromosome 4 of the present invention. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described in detail below in conjunction with the embodiments: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection authority of the present invention is not limited to the following embodiments. Sample selection and material preparation

[0026] 1. Preparation of HEI10, CENH3, and REC8 antibodies (1) Selection of antigen Rice HEI10 for LOC_Os02g13810 Gene, centromere marker CENH3 LOC_Os05g41080 Gene, chromosome axis marker REC8 LOC_Os05g50410 Genes. The full-length amino acid sequences of these genes were selected as prokaryotic expression proteins as antigens for immunizing mice. The CDS sequences of the antigens are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and the amino acid sequences of the antigens are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6. The target gene was connected to the pET30a expression vector.

[0027] (2) Transformation and screening The recombinant plasmid was transformed into the expression strain BL21, positive clones were screened using kanamycin-resistant agar plates, and the insertion of the target gene was verified by colony PCR.

[0028] (3) Induced protein expression Select a single colony of a positive clone and inoculate it into a liquid culture medium containing antibiotics and culture it until the OD600 value is 0.4-0.6. Add 0.1 Mm IPTG to induce gene expression at 37°C for 4 hours.

[0029] (4) Detection of protein expression SDS-PAGE was used to detect the protein expression in cell lysate.

[0030] (5) Cell collection and lysis The expressing cells were collected by centrifugation at 4000 g for 10 minutes and lysed using cell lysis buffer and ultrasonic disruption.

[0031] (6) Protein purification His tag affinity chromatography was used to wash away non-specifically bound impurities using a nickel column, and the target protein was finally eluted using 250 mM imidazole.

[0032] (7) Immunity The above protein was used to immunize mice and rabbits four times, with an interval of two weeks between each immunization. After the immunization, a small sample of serum was taken for testing. If it met the expected results, the sample was sacrificed and the whole serum was taken for subsequent experiments.

[0033] 2. Tissue Slice Preparation The rice cultivar Yandao No. 8 was selected, and the pollen mother cells of fresh rice panicles were selected for sample collection. At this time, homologous chromosomes began to pair and recombine, which is the period when HEI10 protein expression is active. Fresh pollen mother cells were selected for tissue samples, and chromosomes were extracted from pollen mother cells, fixed and stained using cytological techniques. Rice panicles were fixed with 4% Paraformaldehyde (PFA) for 30 minutes to maintain good cell structure and avoid cell rupture or chromatin loss for subsequent immunofluorescence experiments.

[0034] Example 2 Immunofluorescence experiment combined with ultra-high resolution microscopy 1. HEI10, CENH3, REC8 antibody incubation HEI10, CENH3, and REC8 antibodies are mixed with tissue sections for incubation, wherein CENH3 and REC8 are selected with rabbit antibodies, and HEI10 is selected with mouse antibodies (see the sera of mice and rabbits after immunization in step (7) of implementation 1), or CENH3 and REC8 are selected with mouse antibodies, and HEI10 is selected with rabbit antibodies. The incubation conditions of the antibodies and tissue sections are constant temperature at 37°C for 1-2 hours, or incubated at 4°C overnight. The antibody concentration is 1:5.

[0035] 2. Secondary Antibody Fluorescence Labeling Select a suitable commercial fluorescent conjugated secondary antibody, dilute it at 1:1000, and continue to incubate the sample with the diluted fluorescent conjugated secondary antibody at 37°C for 30 minutes. The commercial fluorescent conjugated secondary antibody uses abberior STAR ORANGE goat anti-mouse IgG (STORANGE-1001-500 UG) or abberior STAR RED goat anti-rabbit IgG (STRED-1002-500 UG). At this time, CENH3 and REC8 are labeled with the same colorimetric secondary antibody, while HEI10 is labeled with a separate secondary antibody for subsequent ultra-high resolution microscopic imaging observation.

[0036] 3. Ordinary microscopic imaging observation The localization of HEI10 protein in pollen mother cells was observed by ordinary fluorescence microscopy. HEI10 protein can indicate the location where a type of recombination occurs. From the leptotene stage to the pachytene stage, the HEI10 signal gradually changes from a dot to a line, and finally forms a large dot signal (see Figure 1 , Figure 1 The HEI10-marked recombination site is shown, scale bar: 5 μm). Figure 1 In the figure, from top to bottom are the zygotene stage, early pachytene stage and pachytene stage; the green signal (HEI10) indicates the dynamic changes of a type of recombination marker, and the red signal (REC8) indicates the dynamic changes of the chromosome axis. Using ordinary fluorescence microscopy imaging, we can only preliminarily analyze the number of recombination in the whole cell, and it is difficult to distinguish single rice chromosomes and observe the distribution of recombination on each chromosome.

[0037] 4. Ultra-high resolution microscopic imaging observation Using ultra-high resolution microscopic imaging technology (STEDYCON), the number and distribution of centromere markers (CENH3), chromosome axis markers (REC8) and a type of recombination marker (HEI10) on rice chromosomes can be simultaneously observed (see Figure 2 , Figure 2 The colocalization of HEI10, REC8 and CENH3 is shown, and the scale bar is 5 μm. The 12 pairs of rice chromosomes are distinguished by the position of the centromere and the length of the chromosome axis, and the interference coefficient between the recombination sites on each chromosome is calculated based on this. Figure 2 In the figure, the red arrow (CENH3) indicates the centromere region of rice chromosome 1, and the green arrow (HEI10) indicates the location where a type of exchange occurs on rice chromosome 1. According to the distribution of centromeres of rice chromosomes in the pachytene stage (see Table 1), different rice chromosomes were identified. Figure 2 The results showed that the centromere-to-arm ratio of rice chromosome 1 was about 1.6, and the recombination sites were located on the long and short arms of the chromosome. At the same time, this method can be used to monitor the number and distribution of recombination on the remaining 11 pairs of homologous chromosomes.

[0038] Table 1 Average length and arm ratio of chromosomes in pachytene stage of stalked rice

[0039] Example 3 Detection and analysis of recombination sites 1. Intersection distribution and quantity statistics Crossover statistics can be used to determine the number and location of recombination sites by analyzing the HEI10 information on chromosomes in the above-mentioned ultra-high resolution cell photos of rice. Taking the CO distribution on the long arm of chromosome 4 as an example, 10 ultra-high resolution imaging images of pollen mother cells in the pachytene stage were analyzed (see Figure 3 0 is relatively close to the telomere, and 1 is relatively close to the centromere. The results show that the positions of CO1 and CO2 on the long arm of chromosome 4 have a certain degree of clustering.

[0040] 2. Calculation of interference coefficient Interference refers to the effect of a recombination event in a chromosome region on the secondary recombination in the adjacent region. The interference intensity can be expressed by the "interference coefficient" (I).

[0041] The calculation of the interference coefficient can be performed by the following steps: Calculation of distance between crossover points: For each crossover point in the chromosome region, calculate the distance between two adjacent crossover points. If the distance between two crossover points is too close, it indicates that they may be subject to strong interference.

[0042] Calculation of interference coefficient: The classic "interference coefficient" (I) calculation method is used, I = 1 - (Observed Distance / Expected Distance), where Observed Distance is the actual observed intersection spacing, and Expected Distance is the expected intersection spacing when there is no interference. The interference coefficient is calculated by statistically analyzing the intersection spacing in different regions. The calculation method refers to Anderson LK, Lohmiller LD, Tang X, Hammond DB, Javernick L, Shearer L, Basu-Roy S, Martin OC, Falque M. Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers. Proceedings of the National Academy of Sciences 2014:111:13415-13420.

[0043] Compared with the traditional method for calculating the interference coefficient, this embodiment can distinguish the long arm and short arm of rice chromosomes and calculate the interference coefficient separately, thereby eliminating the influence of the centromere on the interference coefficient. Taking the distribution of HEI10 signals on the long arm of chromosome 4 of salt rice No. 8 as an example, the interference coefficients in 10 groups of samples were calculated (see Table 2).

[0044] Table 2 Distribution data of HEI10 large dots on chromosome 4 of salt rice variety 8

[0045] Combining the above methods, the distribution of recombination sites on different rice chromosomes and their interference coefficients can be obtained, providing data support for further analysis of the genetic mechanism of recombination frequency and a theoretical basis for the regulation of recombination frequency in rice breeding.

[0046] The present invention combines ultra-high resolution microscopic imaging with chromosome marker technology, and for the first time, accurately detects the recombination position on rice chromosomes at the cytological level, and can calculate the interference coefficient, thus providing a new technical means for studying the mechanism of rice genome recombination. Compared with traditional molecular marker methods, the present invention has significant advantages in spatial resolution, accuracy and ease of operation, and can provide important theoretical basis and data support for rice breeding and genome improvement.

[0047] The above description is only a specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or substitutions within the technical scope disclosed by the present invention, which should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A recombinant polyclonal antibody against HEI10, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the recombinant polyclonal antibody against HEI10 according to claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID NO.

1.

3. Use of the recombinant polyclonal antibody against HEI10 according to claim 1 as a cytological marker.

4. A combined marker for rapid detection of recombination positions and interference coefficients on different chromosomes of rice, characterized in that: It includes a recombination marker HEI10, a centromere marker CENH3 and a chromosome axis marker REC8, wherein the recombination marker HEI10 is LOC_ Os02g13810 Gene.

5. A combined marker for rapid detection of recombination positions and interference coefficients on different chromosomes of rice according to claim 4, characterized in that: The marker gene of the centromere marker CENH3 is LOC_Os05g41080 , the chromosome axis marker gene REC8 is LOC_ Os05g50410 .

6. A method for rapidly detecting recombination positions and interference coefficients on different chromosomes of rice, characterized in that: The method comprises the following steps: immunofluorescence is combined with super-high resolution microscopic imaging technology, combined markers are used to analyze the number and distribution of rice chromosome recombination, and interference coefficients are calculated through cytological images of microscopic imaging.

Images