Construction method of peanut a genome single chromosome specific probe library and application thereof

Abstract

This invention provides a method for constructing a single-chromosome-specific probe library for peanut A-genome species and its application. The method for constructing this single-chromosome-specific probe library for peanut A-genome species includes the following steps: designing the single-chromosome-specific probe library for peanut A-genome; and labeling the oligonucleotides in the oligonucleotide library with probes to obtain an Oligos LS-8A probe library labeled with fluorescent groups. This invention utilizes the chromosome sequence of the wild peanut species *A. duranensis* A08 to develop a single-copy oligonucleotide probe library that can specifically stain the A chromosome in A-genome species, solving the problems of accurately identifying peanut A-genome species at the chromosome level and effectively distinguishing between A-genome and non-A-genome species.

Description

Technical Field

[0001] This invention relates to a method for identifying wild peanut species, and more particularly to the construction of a single chromosome-specific probe library for peanut A genome species and a method for distinguishing peanut A genome species. Background Technology

[0002] Peanuts are an important source of edible oil and plant protein worldwide, and my country is the world's largest producer and consumer of peanuts. In recent years, peanut diseases and pests have become increasingly serious, posing a significant bottleneck to high peanut yields. Wild peanut species possess abundant genes for disease and pest resistance, making them an important gene resource for improving cultivated peanut varieties.

[0003] Peanut species are divided into nine groups based on geographical distribution, morphological characteristics, and hybridization compatibility: Peanut Group, Large-root Group, Erect Group, Perivecular Group, Heteromorphic Flower Group, Creeping Group, Rhizome Group, Trifoliate Group, and Trifoliate Group. To date, 15 genomes have been reported (A, B, C, D, K, F, G, E, Ex, H, Pr, R1, R2, Te, and T). Species with genome A contain a pair of "small chromosomes," while species with other genomes do not. Therefore, peanut species can be divided into A-genome species and non-A-genome species. Initially, researchers identified "small chromosomes" (also known as A chromosomes) primarily based on chromosome size. Later, using the fluorescent dye DAPI to stain chromosomes, it was discovered that the centromeres of small chromosomes have bright centromere bands, while the chromosome arms are lightly stained. This was thus used as another characteristic for identifying "small chromosomes." Nevertheless, due to issues with slide preparation and DAPI staining quality, errors in identifying "small chromosomes" are common.

[0004] Peanut distant hybridization is the most important method for utilizing wild peanut genetic resources. Different degrees of hybridization incompatibility exist between cultivated and wild peanut species. Wild species that are closely related to cultivated species are generally easier to hybridize with, primarily those within the *Arinum* section, especially those with the A genome. Currently, the diploid wild species *A. diogoi*, *A. correntina*, and *A. cardenasii*, all frequently used in distant hybridization research, are all A genome species. Therefore, accurately identifying the characteristic "small chromosome" of the A genome is crucial for distinguishing A genome species from non-A genome species at the chromosome level, identifying "small chromosome" introgression lines formed through interspecific hybridization, and facilitating peanut distant hybridization. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for constructing a single-chromosome-specific probe library for peanut A-genome species and its application in distinguishing peanut A-genome species. Utilizing the chromosome sequence of the wild peanut species *A. duranensis* A08, a single-copy oligonucleotide probe library is developed that can specifically stain the "A chromosome" in A-genome species, solving the problems of accurately identifying peanut A-genome species at the chromosome level and effectively distinguishing between A-genome and non-A-genome species.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for constructing a single-chromosome-specific probe library for peanut A genome species includes the following steps:

[0008] (1) Design of a single-chromosome-specific probe library for the peanut A genome:

[0009] The shortest chromosome sequence, A08, was extracted from the genome sequence map of wild peanut species A. duranensis published by PeanutBase. This chromosome is chromosome A of the genus Peanut. A single-copy oligonucleotide library of this chromosome was designed using software and named Oligos LS-8A.

[0010] (2) The oligonucleotides in the oligonucleotide library Oligos LS-8A were labeled with probes to obtain the Oligos LS-8A probe library labeled with fluorescent groups.

[0011] The design method of the single-copy oligonucleotide library is as follows: the A. duranensis chromosome A08 chromosome sequence file is input using Chorus2 software, and a 0.48Mb region is selected every 1.90-1.92Mb for oligonucleotide design. The analysis parameters are: (a) oligonucleotide length 42-48nt; (b) oligonucleotide density 0.54oligos / kb; (c) oligonucleotide dTm > 10℃.

[0012] The probe labeling method is as follows: Digoxigenin-11-dUTP upstream and downstream primers are added to both ends of the oligonucleotide as primer adapters. The oligonucleotide library is amplified, transcribed in vitro, and the probe is labeled by reverse transcription. RNA is removed by enzymatic digestion. Then, the downstream primer of Digoxigenin-11-dUTP is labeled onto the oligonucleotide sequence to obtain the Oligos LS-8A probe library.

[0013] The upstream primer is TAATACGACTCACTATA, and the downstream primer is CGTGGTCGCGTCTCA.

[0014] A method for distinguishing peanut A genome species using a single chromosome-specific probe library established by the aforementioned construction method is employed. Using the oligonucleotide library Oligos LS-8A as probes, the chromosomes in metaphase of mitosis of root tip cells of the peanut species to be identified are stained using a single chromosome staining method. Based on the distribution location of signal chromosomes and the number of chromosome pairs in the genome of the species to be identified, peanut A genome species and non-A genome species are distinguished.

[0015] The single-chromosome staining method includes the following steps:

[0016] (a) Preparation and denaturation of hybridization solution:

[0017] First, prepare the hybridization solution. Denature the hybridization solution at 105℃ for 13 min, and then place it in anhydrous ethanol at -20℃ for 10-15 min. The hybridization solution consists of: 7.5 μl of analytical grade deionized formamide per 13.5 μl, 1.5 μl of 20×SSC buffer, 2 μl of 50% (wt%) dextran sulfate solution, 0.5 μl of salmon sperm DNA at 10 mg / ml, and 2 μl of Oligos LS-8A oligonucleotide probe library.

[0018] (b) Degeneration of chromosome slides of the species to be identified:

[0019] Chromosome slides were prepared from root tip cells of the species to be identified during metaphase of mitosis. The slides were frozen at -80℃ and then dehydrated in anhydrous ethanol for 12 h. They were then denatured in 70% formamide solution at 75℃ for 70 s and then dehydrated in 70%, 95%, and 100% (v / v) ethanol at -20℃ for 5 min each. The slides were then dried.

[0020] (c) Single chromosome staining:

[0021] Place the hybridization solution obtained in step (a) onto the slide obtained in step (b), cover with a coverslip, and hybridize at 37°C for 36-48 hours. Discard the coverslip and rinse 4-5 times with 2×SSC buffer at 42°C. Then wash twice with 2×SSC buffer at 42°C for 5 minutes each time. Finally, wash with 1×TNT buffer at room temperature for 5 minutes. Drain the slides and add 50 μl of the solution to each slide. Incubate 1×TNB buffer and 1 μl of anti-digoxin rhodamine in a 37°C dark box for 50 min; then wash the slides three times with 1×TNT buffer at room temperature for 5 min each time; finally, dry the slides, add 500 μl of DAPI buffer (0.5 μg / ml) to each slide, stain for 3-4 min, rinse 4-5 times with DAPI buffer, add 6-7 μl of anti-fluorescence quenching mounting medium, cover with a 20 mm × 20 mm coverslip, and take pictures under a fluorescence microscope.

[0022] The method for distinguishing between species of the genus *Peanuta* with A genomes and those without A genomes, based on the distribution location of signal chromosomes and the number of chromosome pairs in the genome of the species to be identified, is as follows:

[0023] Using the signal distribution characteristics of chromosome A08 of the A genome species *A. duranensis* as a control, *A. duranensis* contains only one pair of chromosomes with a signal that covers the entire chromosome. If the staining results of the species to be identified are the same as the signal distribution characteristics of chromosome A08 of *A. duranensis*, that is, the genome of the species to be identified contains only one pair of chromosomes with a signal that covers the entire chromosome, then it is an A genome species; if the genome of the species to be identified contains two or more pairs of chromosomes with a signal, then it is a non-A genome species.

[0024] The 2×SSC buffer solution consists of 0.3M trisodium citrate dihydrate and 3M sodium chloride;

[0025] The 1×TNT buffer consists of 0.1M tris(hydroxymethyl)aminomethane hydrochloride, 0.15M sodium chloride, and 0.05% (v / v) Tween 20;

[0026] The 1×TNB buffer consists of 0.5M tris(hydroxymethyl)aminomethane hydrochloride, 0.15M sodium chloride, and 0.5% (wt%) blocking agent;

[0027] The DAPI buffer consists of 0.1M citric acid and 0.05M disodium hydrogen phosphate.

[0028] Application of the single-chromosome-specific probe library of peanut A genome established using the above construction method in the differentiation and identification of peanut A genome species.

[0029] Beneficial effects of this invention:

[0030] 1. A peanut single-chromosome staining technique has been established, enabling the identification of individual peanut chromosomes, specific tracking of a particular chromosome or a class of chromosomes, and detection of homologous chromosome variations. This invention utilizes the A08 chromosome sequence of the wild peanut species *A. duranensis* to develop a single-copy oligonucleotide library of the peanut A08 chromosome, and for the first time establishes a peanut single-chromosome staining technique, providing a new method to improve the accuracy of chromosome identification in wild peanut species.

[0031] 2. This invention utilizes the Oligos LS-8A single-copy probe library to perform oligonucleotide staining on chromosomes of different genomic species of the genus Peanut. Using single-chromosome staining technology, chromosomes in metaphase of mitosis in root tip cells of wild peanut species to be identified were stained. It was found that the distribution location and number of signals on chromosomes are related to the species' genome. Based on the number of signal chromosomes and their distribution characteristics in the genome of the species to be identified, effective differentiation between peanut A-genome and non-A-genome species was achieved. Attached Figure Description

[0032] Figure 1 The signal distribution and electronic localization results of the oligonucleotide probe library Oligos LS-8A on the A. duranensis chromosome.

[0033] Among them, (a) shows the electronic localization results of the Oligos LS-8A probe library on A. duranensis, which shows that the Oligos LS-8A probe library is distributed in a spaced manner on the sequence map, which is consistent with the probe design and target region; (b) shows the DAPI staining map; (c) shows the overlay map of the Oligos LS-8A probe library signal and DAPI staining; (d) shows the FISH karyotype map of A. duranensis chromosome; (e) shows the distribution map of 45S and 5S rDNA signals; the white arrows in b to e indicate "small chromosomes".

[0034] It can be seen that A. duranensis contains only one pair of chromosomes with signals, and the signals cover the entire "small chromosome", and the karyotype shows that the chromosome is also the A08 chromosome.

[0035] Figure 2 Signal distribution of the oligonucleotide probe library Oligos LS-8A in A-genome and non-A-genome species.

[0036] In this diagram, (a) shows the signal distribution of Oligos LS-8A in A-genome species, including some species in the lineages A. duranensis (AA), A. cardenasii (AA), A. diogoi (AA), A. microsperma (AA), A. villosa (AA), A. stenosperma (AA), A. simpsonii (AA), and A. herzogii (AA); (b) shows the cut chromosomes with Oligos LS-8A signals in (a); (c) shows the signal distribution of Oligos LS-8A in non-A-genome species, including A. valida (BB), A. ipaensis (BB), A. batizocoi (KK), A. pintoi (CC), A. stenophylla (EE), and A. trinitensis (FF); and (d) shows the cut chromosomes with Oligos LS-8A signals in (c).

[0037] It can be seen that species with genome A contain only one pair of signaled chromosomes, and the signal covers the entire chromosome, while other species without genome A contain two or more pairs of signaled chromosomes. Detailed Implementation

[0038] The specific embodiments of the present invention will be further described in detail below with reference to examples. Unless otherwise specified, all percentages are weight percentages.

[0039] Some of the reagents used in this invention are as follows:

[0040] 2×SSC buffer: composed of 0.3M trisodium citrate dihydrate and 3M sodium chloride.

[0041] 1×TNT buffer: Composed of 0.1M tris(hydroxymethyl)aminomethane hydrochloride, 0.15M sodium chloride and 0.05% (v / v) Tween-20.

[0042] 1×TNB buffer: Composed of 0.5M tris(hydroxymethyl)aminomethane hydrochloride, 0.15M sodium chloride and 0.5% blocking agent.

[0043] DAPI buffer: composed of 0.1M citric acid and 0.05M disodium hydrogen phosphate.

[0044] Example 1: Method for constructing a single chromosome-specific probe library of peanut A genome species

[0045] (1) Extract the shortest chromosome sequence from the genome sequence map of the wild peanut species A. duranensis published by PeanutBase (https: / / peanutbase.org / gbrowse_aradu1.0), which is commonly referred to as the peanut "small chromosome" or "A chromosome" (the entire A08 chromosome with a length of 49.46 Mb), and design a single-copy oligonucleotide library of this chromosome.

[0046] The specific method is as follows: Using Chorus2 software, the target chromosome sequence file is input, and approximately 0.48 Mb regions are selected every 1.91 Mb for oligonucleotide design. The analytical parameters are: (a) oligonucleotide length 42-48 nt; (b) oligonucleotide density 0.54 oligos / kb; (c) oligonucleotide dTm (DNA melting temperature - hairpin DNA melting temperature) > 10℃. Based on the above procedures and parameters, we finally selected 27,392 sequences for the construction of a single-copy oligonucleotide library, named Oligos LS-8A. Figure 1 The electronic localization results of this probe library on chromosome A08 of *A. duranensis* show the distribution locations of the 27,392 oligonucleotides in the probe library on chromosome A08 and the number of oligonucleotides in each region. The Oligos LS-8A probe library is distributed in a spaced manner on the sequence map, which is consistent with the probe design and target regions.

[0047] (2) The obtained 27,392 oligonucleotide sequences covering the entire target chromosome were used to synthesize an oligonucleotide library. During synthesis, digoxigenin-11-dUTP-labeled upstream primer F (TAATACGACTCACTATA, SEQ1) and downstream primer R (CGTGGTCGCGTCTCA, SEQ2) were added to both ends of the oligonucleotides as primer adapters. Amplification products of a certain concentration (product concentration >40 ng / μl) were obtained through oligonucleotide library amplification. Then, after in vitro transcription, reverse transcription to label probes, and enzymatic digestion to remove RNA, the 5'-CGTGGTCGCGTCTCA-3' sequence of the downstream primer R containing digoxigenin-11-dUTP was labeled onto the 27,392 oligonucleotide sequences of the single-copy oligonucleotide library Oligos LS-8A to obtain the labeled Oligos LS-8A probe library. The specific labeling process is as follows:

[0048] (a) In a 0.2 mL centrifuge tube, add the following in sequence: 25 μl of KAPA enzyme, 0.25 μl of a mixture of equal volumes of 100 μM upstream primer F and downstream primer R, 24.75 μl of ddH2O, and 2.5 μl of the synthesized oligonucleotide library (0.07 ng / μl). Mix well and place in a PCR instrument. Set the program as follows: ① Denaturation at 95 °C for 3 min; ② Denaturation at 98 °C for 20 s; ③ Annealing at 54 °C for 15 s; ④ Extension at 72 °C for 30 s; ⑤ Repeat steps ② to ④ 4 times; ⑥ Denaturation at 98 °C for 20 s; ⑦ Annealing at 56 °C for 15 s; ⑧ Extension at 72 °C for 30 s; ⑨ Repeat steps ⑥ to ⑧ 14 times; ⑩ Maintain at 24 °C.

[0049] (b) Add the following to a centrifuge tube in sequence: 10 μl of KAPA enzyme, 1.2 μl of a mixture of equal volumes of 100 μM upstream primer F and downstream primer R, and 8.8 μl of ddH2O. Mix well and place in a PCR instrument. Set the program as follows: ① Denaturation at 95℃ for 3 min; ② Denaturation at 98℃ for 20 s; ③ Annealing at 54℃ for 15 s; ④ Extension at 72℃ for 30 s; ⑤ Repeat steps ② to ④ once; ⑥ Keep at 24℃ for later use.

[0050] (c) Add ddH2O to the centrifuge tube to bring the total volume to 100 μl, and purify the amplified DNA product using the Qiagen Qiaquick PCR Purification Kit (Qiagen Corporation).

[0051] (d) The amplified DNA product (product concentration > 40 ng / μl) was transcribed in vitro. In a 0.2 ml centrifuge tube, the following were added in sequence: X μl of nucleic acid-free water, Y μl of 480 ng amplified DNA product (calculated based on the amplified product concentration, the final X + Y = 16 μl), 4 μl of 10×T7 reaction buffer, 16 μl of a mixture of equal volumes of 10 mM ATP, 10 mM GTP, 10 mM CTP, and 10 mM UTP, and 4 μl of T7 enzyme. The reaction solution was mixed and incubated at 37 °C for 4 h.

[0052] (e) Add 260 μl of ultrapure water to the reaction solution and divide it into three equal 100 μl samples. Perform subsequent RNA purification using the RNeasy Mini Kit (Qiagen) to obtain the amplified RNA product;

[0053] (f) Reverse transcribe the purified RNA (RNA concentration > 10¹⁰ ng / μl). Add X μl of nucleic acid-free water, 42 μg of amplified RNA product Y μl (calculated based on the amplified product concentration, final X + Y = 21.6 μl), 2.4 μl of 1 mM digoxigenin-labeled primer sequence 5'-CGTGGTCGCGTCTCA-3' solution, 15 μl of 10 mM dNTPs, and 1 μl of 20 U / μl SUPERase-In RNase inhibitor (Invitrogen) to a centrifuge tube. Mix well and incubate at 65°C for 5 min. Then add 4 μl of enzyme-free water, 20 μl of 5× First-strand buffer, 10 μl of 0.1 M dithiothreitol, and 1 μl of 20 U / μl SUPERase-In. Mix well and incubate at 42°C for 5 min. Add 200 U / μl of SuperScript. Add 2.5 μl of reverse transcriptase II, mix well, and incubate at 42 °C for 2 h. Add 11 μl of exonuclease I buffer and 2 μl of exonuclease I, mix well, and incubate at 37 °C for 15 min. Add 12 μl of 0.5 M EDTA (pH = 8.0), mix well, and immediately place the tube in 80 °C for 20 min. Then immediately place the tube on ice and terminate the reaction using the Zymo Quick-RNA MiniPrep kit (Zymo Corporation) to obtain the reverse transcription product.

[0054] (g) Add 6 μl of nucleic acid-free water, 10 μl of 10×RNase H enzyme buffer, 4 μl of 5 U / μl RNase H enzyme, and 80 μl of reverse transcription product to a centrifuge tube in sequence, mix well, incubate at 37℃ for 2 h, add 4 μl of RNase A, mix well, and perform the PCR program: ① digestion at 37℃ for 60 min; ② incubation at 70℃ for 20 min; ③ incubation at 50℃ for 60 min; ④ denaturation at 95℃ for 5 min; ⑤ cooling from 95℃ to 50℃; ⑥ annealing at 50℃ for 60 min; ⑦ storage at 4℃.

[0055] (h) The purification reaction was performed using the Zymo Quick-RNA MiniPrep kit, and approximately 100 μL (concentration >100 ng / μL) of purified probe labeled with a fluorescent group was finally obtained.

[0056] (3) Single chromosome staining of the root tip metaphase chromosome of A. duranensis was performed using the probe library Oligos LS-8A to evaluate the specificity of the oligonucleotide probe library.

[0057] (a) Preparation and denaturation of hybridization solution: Prepare hybridization solution (hybridization solution system includes: 7.5 μl of analytical grade deionized formamide, 1.5 μl of 20×SSC buffer, 2 μl of 50% dextran sulfate solution, 0.5 μl of 10 mg / ml salmon sperm DNA, and 2 μl of 200 ng / μl oligonucleotide probe library Oligos LS-8A), denature the hybridization solution at 105 °C for 13 min, and then place it in anhydrous ethanol at -20 °C for 10-15 min;

[0058] (b) Chromosome specimen denaturation: During metaphase of mitosis, slides were prepared, frozen overnight at -80°C, and then dehydrated in anhydrous ethanol for 12 h. They were then placed in 70% formamide solution and denatured at 75°C for 70 s. Finally, they were dehydrated in gradients of 70%, 95%, and 100% (v / v) ethanol at -20°C for 5 min each, and then dried.

[0059] (c) Single chromosome staining: Drop the hybridization solution from step (a) onto the denatured slide, cover with a coverslip, and hybridize at 37°C for 36-48 h; discard the coverslip, and rinse 4-5 times with 2×SSC buffer at 42°C; then wash twice with 2×SSC buffer at 42°C for 5 min each time; then wash for 5 min with 1×TNT buffer at room temperature; drain the slides, and add 50 μl of the solution to each slide. 1×TNB and 1 μl of anti-digoxin rhodamine were incubated in a dark box at 37°C for 50 min. The slides were then washed three times at room temperature with 1×TNT buffer for 5 min each time. Finally, the slides were dried, and 500 μl of DAPI buffer (0.5 μg / mL) was added to each slide. The slides were stained for 3-4 min, rinsed 4-5 times with DAPI buffer, and 6-7 μl of anti-fluorescence quenching mounting medium was added. The slides were then covered with a 20 mm × 20 mm coverslip and photographed under a fluorescence microscope.

[0060] like Figure 1 b shows the DAPI staining results for the A. duranensis chromosome. Figure 1 c represents the result of staining the A. duranensis chromosome with the probe library Oligos LS-8A.

[0061] Then, sequential FISH karyotype analysis was performed using repeating sequence oligonucleotide probes in the form of Multiplex#3 (FAM-TIF-439, FAM-TIF-185-1, FAM-TIF-134-3, FAM-TIF-165-3), Multiplex#4 (TAMER-DP-1, TAMER-DP-5, TAMER-Ipa-1162, TAMER-Ipa-1137), and FAM-DP-1, TAMER-Ipa-1162, TAMER-Ipa-1137, respectively. Figure 1 d) and FISH analysis of 45S and 5S rDNA probes (e.g. Figure 1 e) The results showed that the chromosome stained by the oligonucleotide probe library was chromosome A08, which is specific.

[0062] Example 2: A method for distinguishing peanut A-genome species from non-A-genome species using a single-chromosome-specific probe library for peanut A genome species.

[0063] (1) Eight A-genome species with well-defined genomes were selected: A. duranensis (A genome), A. cardenasii (A genome), A. diogoi (A genome), A. microsperma (A genome), A. villosa (A genome), A. stenosperma (A genome), A. simpsonii (A genome), and A. herzogii (A genome), and six non-A-genome species were selected: A. valida (B genome), A. ipaensis (B genome), A. batizocoi (K genome), A. pintoi (C genome), A. stenophylla (E genome), and A. trinitensis (F genome).

[0064] Referring to step (3) of Example 1, chromosome slides of root tip cells from wild-type species undergoing metaphase mitosis were prepared. After freezing overnight at -80°C, the slides were dehydrated with anhydrous ethanol and air-dried. The prepared cytology slides were then stained with the Oligos LS-8A probe library, photographed, and the chromosomes obtained were... Figure 2 a and Figure 2 c.

[0065] (2) Integrate the staining results images of all species, as shown in the figure. Figure 2 As shown, using the signal distribution characteristics of chromosome 8A of *A. duranensis* as a control, species with the same signal distribution characteristics as those of chromosome 8A of *A. duranensis*, i.e., species whose genomes contain only one pair of chromosomes with signals, and whose signals cover the entire chromosome, are classified as A genome species (e.g., *A. duranensis*). Figure 2a) Species with signals on two or more pairs of chromosomes are non-A genome species (e.g., Figure 2 c).

[0066] Cut out Figure 2 Chromosome diagrams with Oligos LS-8A signals in a and 2c, such as Figure 2 As shown in b and 2d, it can be seen that species with genome A contain only one pair of chromosomes with signals that cover the entire chromosome, while other species without genome A contain two or more pairs of chromosomes with signals.

[0067] The classification of these 14 species (15 wild materials) using this method was consistent with the actual results, indicating that this method can effectively distinguish between A-genome species and non-A-genome species.

[0068] The above are merely preferred embodiments of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are within the scope of protection of the present invention.

Claims

1. A method of distinguishing between peanut A genome species, characterized by, It comprises the following steps: (1) Designing of the single chromosome specific probe library of peanut A genome: Extraction of peanut PeanutBase published wild species of peanut A. duranensis The smallest length of A08 chromosome sequence in the genomic sequence map, namely Arachis A chromosome, a single copy oligonucleotide library of the chromosome was designed by software and named Oligos LS-8A; the design method of the single copy oligonucleotide library is: inputting the sequence of the chromosome into Chorus2 software A. A. duranensis The chromosome A08 chromosome sequence file, a region of 0.48 Mb is selected every 1.90-1.92 Mb for oligonucleotide design, wherein the analysis parameters are: (a) oligonucleotide length 42-48 nt; (b) oligonucleotide density 0.54 oligos / kb; (c) oligonucleotide d Tm>10℃; (2) Probe labeling of the oligonucleotides in the oligonucleotide library Oligos LS-8A to obtain the Oligos LS-8A probe library labeled with a fluorescent group; (3) Using the Oligos LS-8A probe library to perform single chromosome staining on the chromosomes in the mitotic metaphase of the root tip cells of the peanut to be identified, and distinguishing the A genome of the peanut from the non-A genome species according to the distribution position of the chromosomes with signals and the logarithm of the chromosomes of the genome of the species to be identified; Specific differentiation method: based on species with genome A A. duranensis The A08 chromosome signal distribution characteristics are used as a control, and the A genome species A. duranensis If only one pair of chromosomes has a signal and the signal covers the entire chromosome, and the staining result of the species to be identified is different from that of the chromosome, then the staining result of the chromosome is different from that of the chromosome. A. duranensis The A08 chromosome signal distribution characteristics are the same, that is, if the genome of the species to be identified contains only one pair of chromosomes with signals and the signals cover the entire chromosome, then it is an A genome species; if the genome of the species to be identified contains two or more pairs of chromosomes with signals, then it is a non-A genome species.

2. The differentiation method of claim 1, wherein, The method of probe labeling in step (2) is as follows: adding the upstream primer and the downstream primer of Digoxigenin-11-dUTP as primer adaptors to both ends of the oligonucleotide, marking the probe through oligonucleotide library amplification, in vitro transcription, reverse transcription, removing RNA by enzymatic method, and then labeling the downstream primer of Digoxigenin-11-dUTP to the oligonucleotide sequence to obtain the Oligos LS-8A probe library.

3. The method of distinguishing of claim 2, wherein, The upstream primer is TAATACGACTCACTATA, and the downstream primer is CGTGGTCGCGTCTCA.

4. The method of distinguishing of claim 1, wherein, The single chromosome staining in step (3) comprises the following steps: (I) Preparation and denaturation of hybridization solution: First, prepare the hybridization solution, denature the hybridization solution at 105℃ for 13 min, and then place it in anhydrous ethanol at -20℃ for 10-15 min; wherein the composition of the hybridization solution is: 7.5 μl of deionized formamide analytical pure, 1.5 μl of 20×SSC buffer, 2 μl of 50% (wt%) dextran sulfate solution, 0.5 μl of 10 mg / ml salmon sperm DNA, and 2 μl of oligonucleotide Oligos LS-8A probe library; (II) Denaturation of the chromosome preparation of the species to be identified: Prepare the chromosome preparation of the root tip cells in the mitotic metaphase of the species to be identified, freeze the slide at -80℃, then dehydrate the slide in anhydrous ethanol for 12 h, then denature it in 70% formamide solution at 75℃ for 70 s, and then gradient dehydrate it in 70%, 95%, and 100% (v / v) alcohol at -20℃ for 5 min each time, and blow dry the slide; (III) Single chromosome staining: The hybridization solution obtained in step (I) is dropped onto the slide obtained in step (II), a cover glass is covered, and hybridization is performed at 37°C for 36-48 h; the cover glass is removed, and the slide is washed with 2×SSC buffer at 42°C for 4-5 times; then the slide is washed with 2×SSC buffer at 42°C for 2 times, each for 5 min; then the slide is washed with 1×TNT buffer at room temperature for 5 min; the slide is drained, 50 µl of 1×TNB buffer and 1 µl of anti-digoxigenin rhodamine are added to each slide, and the slide is incubated in a dark box at 37°C for 50 min; then the slide is washed with 1×TNT buffer at room temperature for 3 times, each for 5 min; finally, the slide is dried, 500 µl of DAPI buffer with a DAPI concentration of 0.5 µg / ml is added to each slide, and the slide is stained for 3-4 min; the slide is washed with DAPI buffer for 4-5 times, 6-7 µl of anti-fluorescence quenching mounting medium is dropped, a 20 mm×20 mm cover glass is covered, and a picture is obtained by fluorescence microscopy.

5. The distinguishing method of claim 4, wherein, the 2×SSC buffer is composed of 0.3 M trisodium citrate dihydrate and 3 M sodium chloride; the 1×TNT buffer is composed of 0.1 M tris-hydroxymethyl aminomethane hydrochloride, 0.15 M sodium chloride, and 0.05 % (v / v) Tween 20; the 1×TNB buffer is composed of 0.5 M tris-hydroxymethyl aminomethane hydrochloride, 0.15 M sodium chloride, and 0.5 % (wt%) blocking agent; the DAPI buffer is composed of 0.1 M citric acid and 0.05 M disodium hydrogen phosphate.

6. Application of the distinguishing method of claim 1 in distinguishing and identifying peanut A genome species.

Images