Pennisetum subgenomic specific retrotransposon and use thereof

Abstract

The application discloses a sub-genome-specific retrotransposon of elephant grass and application thereof, and belongs to the field of bioinformatics and molecular cytogenetics. The application mainly comprises the following steps: 2 sub-genome-specific retrotransposon sequences are screened out through bioinformatics analysis of short fragment sequences of the sub-genome of elephant grass; primers are designed according to the sequences, and the genomic DNA of elephant grass is used as a template to amplify the primers through PCR, and the target fragments are recovered through purification of PCR products; and it is verified that the 2 retrotransposon sequences produce clear and bright signals on the respective 14 chromosomes of elephant grass through probe labeling, chromosome sheet preparation and fluorescence in situ hybridization. The retrotransposon sequences can be directly used for specific identification of the sub-genome of elephant grass, more accurate information is provided for identification of the sub-genome of elephant grass, and a foundation is laid for chromosome engineering breeding of elephant grass.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioinformatics and molecular cytogenetics, and particularly relates to elephant grass sub-genome specific retrotransposon and application thereof. BACKGROUND

[0002] In the field of genomics research, repetitive sequences are a key component of the genome, and retrotransposons, as an important type of repetitive sequence, account for a considerable proportion of the genome. Retrotransposons replicate and transpose through a reverse transcription mechanism, generating a large number of copies in the genome, and have a profound impact on the structure, function and evolution of the genome. They not only change the size and stability of the genome, but also may affect gene expression and function by inserting into the interior or adjacent region of the gene, thereby regulating biological trait performance.

[0003] Elephant grass (Pennisetum purpureum) is a perennial grass of the family Poaceae with important economic and ecological value in tropical and subtropical regions. It grows rapidly, has a large biomass, and is highly adaptable, and is widely used in many fields such as livestock feed, ecological restoration, and biomass energy production. The elephant grass genome is relatively complex and contains a large number of repetitive sequences dominated by retrotransposons. However, current research on retrotransposons in the elephant grass genome is relatively limited, especially the study of sub-genome specific retrotransposon sequences is still in its infancy.

[0004] Existing methods for identifying retrotransposons mainly rely on sequence alignment and conserved domain analysis. These methods can identify some retrotransposon sequences, but for sub-genome specific retrotransposons in complex genomes, the identification effect is not good. On the one hand, it is difficult to distinguish similar retrotransposon sequences from different sub-genomes; on the other hand, the detection sensitivity is low for low copy number or retrotransposons with large sequence variations. In addition, the two diploid ancestral species of elephant grass have become extinct, and traditional in situ hybridization of diploid ancestral species genomic DNA is difficult to distinguish between sub-genomes.

[0005] In summary, existing research lacks a systematic and efficient identification process and standard, and takes into account the characteristics of the elephant grass genome and the differences between sub-genomes, leading to slow progress in the study of elephant grass sub-genome specific retrotransposons, which seriously hinders the in-depth development of elephant grass genomics research and genetic improvement in practical applications. Therefore, the present application aims to provide a method for identifying elephant grass sub-genome specific retrotransposon sequences to improve the accuracy and efficiency of identification, and to provide a powerful tool for elephant grass genomics research and genetic improvement. SUMMARY

[0006] The present application aims at the problem that the prior art cannot distinguish the A' and B subgenomes of Pennisetum, provides two subgenome-specific retrotransposon sequences of Pennisetum, develops a chromosome identification method based on fluorescence in situ hybridization (FISH), and accurately distinguishes the A' and B subgenome chromosomes by the subgenome-specific retrotransposon markers, thereby providing a cytological identification tool for chromosome engineering breeding and genome evolution research of Pennisetum polyploidy plants.

[0007] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a subgenome-specific retrotransposon of Pennisetum, comprising an A' subgenome retrotransposon SubA'-Athila and a B subgenome retrotransposon SubB-Retand, wherein the sequence of SubA'-Athila is shown as SEQ ID NO: 1, and the sequence of SubB-Retand is shown as SEQ ID NO: 2.

[0009] In a second aspect, the present application provides primers for amplifying the aforementioned retrotransposon, wherein,

[0010] The primers for amplifying SubA'-Athila are as follows:

[0011] The upstream primer is 5'-TGATGTTACCACTCGGCAAGTCCCT-3', shown as SEQ ID NO: 3;

[0012] The downstream primer is 5'-ACCTTGGAGCCAGCGTCAGTGTCAT-3', shown as SEQ ID NO: 4;

[0013] The primers for amplifying SubB-Retand are as follows:

[0014] The upstream primer is 5'-CCCCGTAACGATAGAG-3', shown as SEQ ID NO: 5;

[0015] The downstream primer is 5'-ATCCATCCTTAGCGTC-3', shown as SEQ ID NO: 6.

[0016] In a third aspect, the present application provides the aforementioned retrotransposon of Pennisetum and the aforementioned primers for use in (A1) or (A2) as follows:

[0017] (A1) for distinguishing the A' and B subgenomes of Pennisetum;

[0018] (A2) for use in the preparation of a product for distinguishing the A' and B subgenomes of Pennisetum.

[0019] In a specific embodiment, the application comprises the following specific steps.

[0020] In a fourth aspect, the present application protects a method for distinguishing A' and B subgenomes of E. arizonicum, which comprises the following specific steps.

[0021] In a specific embodiment, the application comprises the following specific steps.

[0022] (1) PCR amplification: PCR amplification of the aforementioned A' subgenomic retrotransposon SubA'-Athila and B subgenomic retrotransposon SubB-Retand, and purification of the PCR product;

[0023] (2) Probe preparation: preparation of double-color FISH probes by Nick Translation using digoxin-labeled A' subgenomic retrotransposon sequence SubA'-Athila probe and biotin-labeled B subgenomic retrotransposon sequence SubB-Retand probe;

[0024] (3) Mitotic metaphase chromosome preparation: incubation of young roots with 8-hydroxyquinoline at 25°C for 3 h, treatment with Carnoy's fixative (absolute ethanol (v): glacial acetic acid (v) = 3:1) for 24 h; cutting of the root tip meristematic zone and placing it in a mixture of 2% cellulase + 1% pectinase for enzymatic digestion at 37°C for 3 h; drop method for root tip mitotic metaphase chromosome preparation;

[0025] (4) Fluorescence in situ hybridization (FISH) identification:

[0026] (B1) First, prepare the probe mixture: prepare 10 μL of hybridization buffer, including 50% formamide, 10% dextran sulfate, 2x SSC, and then add 1 μL of digoxin probe and 1 μL of biotin probe;

[0027] (B2) Incubate the denatured probe with the denatured chromosomal DNA in the hybridization buffer (50% formamide, 10% dextran sulfate, 2x SSC) in a wet box at 37°C for 16 h;

[0028] (B3) Color development by anti-digoxigenin rhodamine and AF488 antibody, and then observation under microscope.

[0029] In a specific embodiment, in step (1), the primers used in PCR amplification are shown as SEQ ID NO: 3-6.

[0030] In a more specific embodiment, the primers for amplifying SubA'-Athila are shown as SEQ ID NO: 3-4, and the primers for amplifying SubB-Retand are shown as SEQ ID NO: 5-6.

[0031] In a specific embodiment, in step (1), the PCR amplification reaction system is: 5 μL 2× ExTaq buffer, 4 μL 2.5 mM dNTP Mixture, 0.5 μL 5 U / μL ExTaq, 1.25 μL 10 μM upstream primer, 1.25 μL 10 μM downstream primer, 2 μL 100 ng / μl Erianthus arundinaceus genomic DNA, 36.75 μL ddH2O.

[0032] In a specific embodiment, in step (1), the PCR amplification conditions are: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 10 min.

[0033] In a specific embodiment, in step (B2), the probe is denatured at 95℃, and the chromosomal DNA is denatured at 75℃.

[0034] Beneficial effects

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] The present application uses bioinformatics to cut the Erianthus arundinaceus subgenomic into short fragment sequences, and through sequence similarity-based clustering analysis, two Erianthus arundinaceus subgenomic specific retrotransposon sequences are screened out, which can be used for efficient mining of retrotransposon sequences for specific recognition of Erianthus arundinaceus subgenomic. Through FISH experiment verification, the two retrotransposon sequences only produce clear and bright signals on the corresponding subgenomic of Erianthus arundinaceus, indicating that the obtained Erianthus arundinaceus subgenomic retrotransposon can truly and reliably specifically recognize Erianthus arundinaceus subgenomic. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 : Hybridization results of Erianthus arundinaceus subgenomic specific retrotransposon sequences on Erianthus arundinaceus metaphase chromosomes; wherein, Figure 1 Metaphase chromosomes in mitosis A: Figure 1Middle B: Sequence 1-SubA'-Athila retrotransposon signal; Figure 1 Middle C: Sequence 2-SubB-Retand retrotransposon signal. DETAILED DESCRIPTION

[0038] The application will be further described below in conjunction with specific examples. The temperature in the examples not indicated is room temperature, and the reagents not indicated are conventional chemical reagents.

[0039] Example 1: Screening of elephant grass subgenomic specific retrotransposon

[0040] (1) The two subgenomic sequences of elephant grass were cut into fragments with a length of 100 bp.

[0041] (2) Sequence similarity-based clustering analysis was performed on all 100 bp fragments of the two subgenomes using RepeatExplorer2 software, and similar fragments were classified into the same repeat cluster (cluster) to obtain different repeat clusters.

[0042] (3) The number of occurrences of each cluster in the respective subgenome was counted to determine its abundance in the corresponding subgenome.

[0043] (4) The abundance of the same cluster in the two subgenomes was compared, and the cluster with the highest fold difference in copy number between the two subgenomes was selected as the candidate sequence.

[0044] (5) One subgenomic specific retrotransposon sequence was mined from each of the A' and B subgenomes of elephant grass, and the sequence information is shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0045] Example 2: Amplification and probe labeling of elephant grass subgenomic specific retrotransposon sequence

[0046] (1) Select elephant grass plants that grow well and have no pests and diseases, take their leaf tissues, extract genomic DNA using the CTAB method, and use Nanodrop 1000 to determine the quality and concentration of the extracted DNA.

[0047] (2) Primer Premier 5.0 was used to design primers for the retrotransposon sequence, and the primer sequence information is shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0048] (3) Using the extracted genome DNA of Pennisetum purpureum as template, the designed primers were respectively subjected to PCR reaction with the template DNA. The PCR reaction system (50 μL) was as follows: 5 μL 2 × ExTaq buffer, 4 μL 2.5 mM dNTP Mixture, 0.5 μL 5 U / μL ExTaq, 1.25 μL 10 μM upstream primer, 1.25 μL 10 μM downstream primer, 2 μL 100 ng / μl genome DNA of Pennisetum purpureum, and 36.75 μL ddH2O. The PCR reaction procedure was as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 30 s, for a total of 35 cycles; and 72 °C final extension for 10 min.

[0049] (4) The PCR amplification product was detected by agarose gel electrophoresis, and the appearance of specific bands was observed to confirm the amplification effect of the primers and the existence of the target sequence.

[0050] (5) The PCR product was purified using a GenElute™ PCR purification kit, the quality and concentration of the purified DNA were determined using a Nanodrop 1000, and the gap translation method was used to label SubA'-Athila with digoxin and SubB-Retand with biotin.

[0051] Example 3: Verification by fluorescence in situ hybridization (FISH)

[0052] (1) Chromosome specimen preparation

[0053] 1) Material treatment: 1-2 cm of tender root tips of Pennisetum purpureum plants were taken and placed in a 2 mmol / L 8-hydroxyquinoline solution for 3 h of pretreatment at 25 °C.

[0054] 2) Fixation and dissociation: fixation with Carnoy's fixative (absolute ethanol: glacial acetic acid = 3:1) for 24 h, and treatment with enzyme solution (2% cellulase + 1% pectinase) at 37 °C for 3 h.

[0055] 3) Drop piece: the cell suspension after dissociation was added dropwise to pre-cooled glass slides, and after air drying, it was stored at -20 °C for standby use.

[0056] (2) Fluorescence in situ hybridization

[0057] 1) Probe mixing: 10 μL hybridization buffer (50% formamide, 10% dextran sulfate, 2 × SSC) was taken, and 1 μL of digoxin probe and 1 μL of biotin probe (final concentration 2 ng / μL) were added.

[0058] 2) denaturation and hybridization: the 95℃ denatured probe mixture was added to the 75℃ heat-denatured chromosome sample, transferred to a wet box and hybridized at 37℃ for 16 h.

[0059] 3) elution and detection: eluted with 2x SSC for 3 times and 1x PBS for 1 time at 25℃, each for 5 min; then added anti-digoxin rhodamine and AF488 antibody, observed after incubation at 37℃ for 1 h in the dark.

[0060] 4) after incubation, eluted with 1x PBS for 3 times at room temperature, each for 5 min;

[0061] 5) after blowing dry the slide, added anti-fluorescence quencher on the slide, covered with a cover glass, observed and analyzed the hybridization results by fluorescence microscopy.

[0062] Image acquisition was performed by fluorescence microscopy, and it was observed that the probes from the two subgenomic retrotransposon sequences produced clear and bright signals on 14 different chromosomes respectively, indicating that the two retrotransposon sequences can be effectively used to distinguish the A' and B subgenomes of elephant grass.

[0063] From Figure 1 It can be seen that the signals produced by the sequences of the present application are only clear and bright signals on 14 different chromosomes respectively, verifying that these retrotransposon sequences can be used for specific recognition of elephant grass subgenomes.

[0064] Due to the presence of a large number of homologous sequences in the elephant grass subgenome, only common conservative repeat sequence probes can be used, and both of the two diploid ancestral species of elephant grass have been extinct, so it is impossible to use diploid ancestral species genomic DNA to identify elephant grass subgenome, the present application uses the elephant grass subgenome specific retrotransposon sequences obtained by clustering analysis of elephant grass subgenome data, and identifies 2 retrotransposons specific for elephant grass subgenome by FISH, proving that the method of the present application is effective.

[0065] The above only describes the preferred embodiments of the present application, and any changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.

Claims

1. A PCR amplification product for distinguishing between the A' and B subgenomes of ryegrass, characterized in that, The genomic DNA of elephant grass is used as a template, and primers for amplifying A' subgenomic specific retrotransposon sequences and primers for amplifying B subgenomic specific retrotransposon sequences are used respectively to obtain the following sequences: The upstream primer is 5'-TGATGTTACCACTCGGCAAGTCCCT-3', as shown in SEQ ID NO:

3. The downstream primer is 5'-ACCTTGGAGCCAGCGTCAGTGTCAT-3', as shown in SEQ ID NO:

4. The primers for amplifying B subgenomic specific retrotransposon sequences have the following sequences: The upstream primer is 5'-CCCCGTAACGATAGAG-3', as shown in SEQ ID NO:

5. The downstream primer is 5'-ATCCATCCTTAGCGTC-3', as shown in SEQ ID NO:

6.

2. The PCR amplification product of claim 1 is used in (A1) or (A2) as follows: (A1) for distinguishing between A' and B subgenomes of elephant grass; (A2) for preparing a product for distinguishing between A' and B subgenomes of elephant grass.

3. Use according to claim 2, characterized in that, The PCR amplification product of claim 1 is prepared by PCR amplification using the primers for amplifying A' subgenomic specific retrotransposon sequences and the primers for amplifying B subgenomic specific retrotransposon sequences, respectively, with the genomic DNA of elephant grass as a template, and the purified PCR product is prepared into a fluorescein-labeled probe, which is used for fluorescence in situ hybridization on metaphase chromosomes of elephant grass to distinguish between A' and B subgenomes of elephant grass.

4. A method for distinguishing between the A' and B subgenomes of elephant grass, characterized by, The method uses the primers for amplifying A' subgenomic specific retrotransposon sequences and the primers for amplifying B subgenomic specific retrotransposon sequences, respectively, to prepare the PCR amplification product of claim 1 by PCR amplification with the genomic DNA of elephant grass as a template, and the purified PCR product is prepared into a fluorescein-labeled probe, which is used for fluorescence in situ hybridization on metaphase chromosomes of elephant grass to distinguish between A' and B subgenomes of elephant grass.

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