Pennisetum purpureum subgenome specific retrotransposon and application thereof

Abstract

The invention discloses a pennisetum purpureum subgenome specific retrotransposon and application thereof, and belongs to the field of bioinformatics and molecular cell genetics. The method mainly comprises the following steps: screening two subgenome specific reverse transcription transposon sequences through bioinformatics analysis of subgenome short fragment sequences of target grass; designing a primer according to the sequence, taking Pennisetum purpureum genome DNA as a template, carrying out PCR amplification, and purifying a PCR product to recover a target fragment; probe labeling, chromosome flaking and fluorescence in situ hybridization verify that the two reverse transcription transposon sequences generate clear and bright signals on respective 14 chromosomes of pennisetum purpureum. The reverse transcription transposon sequence disclosed by the invention can be directly used for specifically identifying the pennisetum purpureum subgenome, provides more accurate information for identification of the pennisetum purpureum subgenome, and lays a foundation for engineering breeding of the pennisetum purpureum chromosome.

Description

Technical Field

[0001] The invention belongs to the fields of bioinformatics and molecular cytogenetics, and in particular relates to a subgenome-specific retrotransposon of elephant grass and an application thereof. Background Art

[0002] In genomics, repetitive sequences are key components of the genome, and retrotransposons, a key type of repetitive sequence, account for a significant proportion of the genome. Retrotransposons replicate and transpose via reverse transcription, generating large numbers of copies within the genome and profoundly influencing its structure, function, and evolution. They can not only alter the size and stability of the genome but also potentially influence gene expression and function by inserting into or adjacent to genes, thereby regulating biological traits.

[0003] Elephant grass (Pennisetum purpureum) is a perennial grass with significant economic and ecological value in tropical and subtropical regions. Its rapid growth, high biomass, and strong adaptability have led to its widespread use in livestock feed, ecological restoration, and biomass energy production. The elephant grass genome is complex, containing numerous repetitive sequences, primarily retrotransposons. However, research on retrotransposons in the elephant grass genome is relatively limited, and the study of subgenome-specific retrotransposon sequences is still in its infancy.

[0004] Existing methods for identifying retrotransposons primarily rely on sequence alignment and conserved domain analysis. While these methods can identify some retrotransposon sequences, they are ineffective for identifying subgenome-specific retrotransposons within complex genomes. On the one hand, it is difficult to distinguish similar retrotransposon sequences from different subgenomes; on the other hand, detection sensitivity is low for retrotransposons with low copy numbers or those with large sequence variability. Furthermore, since the two diploid ancestral species of elephant grass are now extinct, traditional in situ hybridization methods using genomic DNA from these diploid ancestral species have difficulty distinguishing their subgenomes.

[0005] In summary, existing research lacks a systematic and efficient identification process and standards, taking into account the characteristics of the elephant grass genome and the differences between subgenomes. This has led to slow progress in the study of subgenome-specific retrotransposons in elephant grass, severely hindering the in-depth development of elephant grass genomics research and its practical genetic improvement. Therefore, the present invention aims to provide a method for identifying subgenome-specific retrotransposon sequences in elephant grass, thereby improving identification accuracy and efficiency and providing a powerful tool for elephant grass genomics research and genetic improvement. Summary of the Invention

[0006] To address the problem that existing technologies cannot distinguish between the A' and B subgenomes of Pennisetum, the present invention provides two subgenome-specific retrotransposon sequences of Pennisetum and develops a chromosome identification method based on fluorescence in situ hybridization (FISH). The A' and B subgenome chromosomes are accurately distinguished through subgenome-specific retrotransposon markers, providing a cytological identification tool for chromosome engineering breeding and genome evolution research of polyploid plants of the genus Pennisetum.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] In a first aspect, the present invention provides elephant grass subgenome-specific retrotransposons, including the A' subgenome retrotransposon SubA'-Athila and the B subgenome retrotransposon SubB-Retand, wherein the SubA'-Athila sequence is shown in SEQ ID NO: 1, and the SubB-Retand sequence is shown in SEQ ID NO: 2.

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

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

[0011] Upstream primer: 5'-TGATGTTACCACTCGGCAAGTCCCT-3', as shown in SEQ ID NO: 3;

[0012] Downstream primer: 5'-ACCTTGGAGCCAGCGTCAGTGTCAT-3', as shown in SEQ ID NO: 4;

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

[0014] Upstream primer: 5'-CCCCGTAACGATAGAG-3', as shown in SEQ ID NO: 5;

[0015] Downstream primer: 5′-ATCCATCCTTAGCGTC-3′, as shown in SEQ ID NO:6.

[0016] In a third aspect, the present invention provides the use of the aforementioned elephant grass genomic retrotransposon and the aforementioned primers in the following (A1) or (A2):

[0017] (A1) Application in distinguishing A' and B subgenomes of elephant grass;

[0018] (A2) Use in preparing products for distinguishing the A' and B subgenomes of elephant grass.

[0019] In a specific embodiment, the application is to prepare the A' subgenomic retrotransposon SubA'-Athila and the B subgenomic retrotransposon SubB-Retand described above through PCR amplification and purification, and the purified PCR products are prepared into fluorescently labeled probes, and fluorescent in situ hybridization is performed on the metaphase chromosomes of elephant grass to distinguish the A' and B subgenomes of elephant grass.

[0020] In a fourth aspect, the present invention protects a method for distinguishing the A' and B subgenomes of elephant grass. The method comprises preparing the A' subgenome retrotransposon SubA'-Athila and the B subgenome retrotransposon SubB-Retand described above through PCR amplification and purification, preparing the purified PCR products into fluorescein-labeled probes, and performing fluorescence in situ hybridization on the metaphase chromosomes of elephant grass to distinguish the A' and B subgenomes of elephant grass.

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

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

[0023] (2) Probe preparation: Digoxigenin-labeled A' subgenomic retrotransposon sequence SubA'-Athila probe and biotin-labeled B subgenomic retrotransposon sequence SubB-Retand probe were used to prepare dual-color FISH probes by nick translation.

[0024] Preparation of mitotic metaphase chromosomes: Incubate young roots with 8-hydroxyquinoline at 25°C for 3 h and treat with Carnoy's fixative (anhydrous ethanol (v): glacial acetic acid (v) = 3:1) for 24 h. Root tip meristems were excised and enzymatically hydrolyzed in a mixture of 2% cellulase and 1% pectinase at 37°C for 3 h. Prepare sections of mitotic metaphase chromosomes from the root tip using the drop slide method.

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

[0026] (B1) Prepare the probe mixture: Prepare 10 μL of hybridization buffer containing 50% formamide, 10% dextran sulfate, and 2× SSC, and add 1 μL each of digoxigenin probe and biotin probe.

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

[0028] (B3) The cells were visualized using anti-digoxin rhodamine and AF488 antibodies and observed under a microscope.

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

[0030] In a more specific embodiment, the primers for amplifying SubA'-Athila are shown as SEQ ID NOs: 3-4, and the primers for amplifying SubB-Retand are shown as SEQ ID NOs: 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 elephant grass genomic DNA, and 36.75 μL ddH2O.

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

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

[0034] Beneficial effects

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

[0036] This study uses bioinformatics to slice the elephant grass subgenome into short fragment sequences. By performing sequence similarity cluster analysis on these fragments, two retrotransposon sequences specific to the elephant grass subgenome were screened, effectively mining retrotransposon sequences that can be used to specifically identify the elephant grass subgenome. FISH experiments confirmed that the two retrotransposon sequences produced clear, bright signals only on the corresponding elephant grass subgenomes, demonstrating that the obtained elephant grass subgenomic retrotransposons can truly and reliably identify the elephant grass subgenome. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : The hybridization results of the subgenome-specific retrotransposon sequences of elephant grass on the metaphase chromosomes of elephant grass; Among them, Figure 1 Middle A: chromosomes in metaphase of mitosis; Figure 1Middle B: Sequence 1-SubA'-Athila retrotransposon signal; Figure 1 Middle C: Sequence 2-SubB-Retand retrotransposon signal. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to specific examples. In the examples, temperatures not specified are all room temperature, and reagents not specified are all conventional chemical reagents.

[0039] Example 1: Screening of specific retrotransposons in subgenomes of elephant grass

[0040] (1) Cut the two subgenomic sequences of Elephant Grass into fragments of 100 bp in length.

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

[0042] (3) Count the number of occurrences of each cluster in each subgenome and determine its abundance in the corresponding subgenome.

[0043] (4) Compare the abundance of the same cluster in the two subgenomes and select the cluster with the highest copy number difference between the two subgenomes as the candidate sequence.

[0044] (5) One subgenome-specific retrotransposon sequence was extracted from each of the A' and B subgenomes of Elephant Grass, and the sequence information is shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0045] Example 2: Amplification of subgenome-specific retrotransposon sequences and probe labeling of elephant grass

[0046] (1) Select elephant grass plants with good growth and no pests and diseases, take their leaf tissues, extract genomic DNA using the CTAB method, and use Nanodrop1000 to measure the quality and concentration of the extracted DNA.

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

[0048] (3) Using the extracted genomic DNA of the elephant grass as a template, the designed primers were reacted with the template DNA for PCR reaction. PCR reaction system (50 μL): 5 μL 2× ExTaq buffer, 4 μL 2.5 mM dNTP Mixture, 0.5 μL 5U / μL ExTaq, 1.25 μL 10 μM upstream primer, 1.25 μL 10 μM downstream primer, 2 μL 100 ng / μl elephant grass genomic DNA, 36.75 μL ddH2O. PCR reaction procedure: 95℃ initial denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 35 cycles; final extension at 72℃ for 10 min.

[0049] (4) The PCR amplification products were detected by agarose gel electrophoresis to observe the appearance of specific bands and confirm the amplification effect of the primers and the presence of the target sequence.

[0050] (5) The PCR products were purified using the GenElute™ PCR purification kit. The quality and concentration of the purified DNA were determined using Nanodrop1000. The gap translation method was used to label SubA'-Athila with digoxigenin and SubB-Retand with biotin.

[0051] Example 3: Fluorescence in situ hybridization (FISH) verification

[0052] (1) Chromosome specimen preparation

[0053] 1) Material treatment: 1-2 cm young root tips of Elephant Grass plants were placed in 2 mmol / L 8-hydroxyquinoline solution at 25°C for 3 h.

[0054] 2) Fixation and dissociation: Fix the cells in Carnoy's fixative (anhydrous ethanol: glacial acetic acid = 3:1) for 24 h, and then treat with enzymatic solution (2% cellulase + 1% pectinase) at 37°C for 3 h.

[0055] 3) Dropping: Add the dissociated cell suspension to the pre-cooled slide, air dry and store at -20℃ until use.

[0056] (2) Fluorescence in situ hybridization

[0057] 1) Probe mixing: Take 10 μL of hybridization buffer (50% formamide, 10% dextran sulfate, 2× SSC) and add 1 μL each of digoxigenin probe and biotin probe (final concentration 2 ng / μL).

[0058] 2) Denaturation and hybridization: Add the probe mixture denatured at 95°C to the chromosome specimen heat-denatured at 75°C, transfer to a humidified chamber at 37°C and hybridize for 16 h.

[0059] 3) Elution and Detection: Elute three times with 2× SSC and once with 1× PBS at 25°C, each elution time being 5 min. Then, add anti-digoxin rhodamine and AF488 antibodies dropwise, incubate at 37°C in the dark for 1 h, and then seal and observe.

[0060] 4) After incubation, the antibody was eluted three times with 1× PBS at room temperature, each elution time was 5 minutes;

[0061] 5) After drying the slides, apply anti-fluorescence quencher on the slides, cover with coverslips, and observe and analyze the hybridization results using a fluorescence microscope.

[0062] Using fluorescence microscopy for image acquisition, it was observed that on the A' and B subgenome chromosomes, the retrotransposon sequence probes from these two subgenomes produced clear and bright signals on each of the 14 different chromosomes, indicating that these 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 generated by the sequences of the present invention only generate clear and bright signals on 14 different chromosomes, verifying that these retrotransposon sequences can be used to specifically identify elephant grass subgenomes.

[0064] Since there are a large number of homologous sequences in the elephant grass subgenome, only common conserved repeat sequence probes are used, and the two diploid ancestral species of elephant grass are both extinct, it is impossible to use the genomic DNA of the diploid ancestral species to identify the elephant grass subgenome. The present invention uses the elephant grass subgenome-specific retrotransposon sequences obtained by cluster analysis of the elephant grass subgenome data, and uses FISH to identify two retrotransposons that specifically identify the elephant grass subgenome, proving that the method of the present invention is effective.

[0065] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A subgenome-specific retrotransposon of elephant grass, characterized in that: The retrotransposon consists of an A' subgenomic retrotransposon SubA'-Athila and a B subgenomic retrotransposon SubB-Retand, wherein the sequence of SubA'-Athila is shown in SEQ ID NO: 1, and the sequence of SubB-Retand is shown in SEQ ID NO:

2.

2. Primers for amplifying the subgenome-specific retrotransposon of the elephant grass according to claim 1.

3. The primer according to claim 2, characterized in that The primer sequences for amplifying SubA'-Athila are as follows: Upstream primer: 5'-TGATGTTACCACTCGGCAAGTCCCT-3', as shown in SEQ ID NO: 3; Downstream primer: 5'-ACCTTGGAGCCAGCGTCAGTGTCAT-3', as shown in SEQ ID NO: 4; The primer sequences for amplifying SubB-Retand are as follows: Upstream primer: 5'-CCCCGTAACGATAGAG-3', as shown in SEQ ID NO: 5; Downstream primer: 5′-ATCCATCCTTAGCGTC-3′, as shown in SEQ ID NO:

6.

4. Use of the elephant grass subgenome-specific retrotransposon according to claim 1 or the primer according to any one of claims 2-3 in the following (A1) or (A2): (A1) Application in distinguishing A' and B subgenomes of elephant grass; (A2) Use in preparing products for distinguishing the A' and B subgenomes of elephant grass.

5. The use according to claim 4, characterized in that The application is to prepare the A' subgenomic retrotransposon SubA'-Athila and the B subgenomic retrotransposon SubB-Retand described in claim 1 through PCR amplification and purification, and the purified PCR products are prepared into fluorescently labeled probes, and fluorescent in situ hybridization is performed on the metaphase chromosomes of elephant grass to distinguish the A' and B subgenomes of elephant grass.

6. A method for distinguishing A' and B subgenomes of elephant grass, characterized in that: The method prepares the A' subgenomic retrotransposon SubA'-Athila and the B subgenomic retrotransposon SubB-Retand described in claim 1 through PCR amplification and purification, prepares the purified PCR products into fluorescently labeled probes, and performs fluorescence in situ hybridization on elephant grass metaphase chromosomes to distinguish the elephant grass A' and B subgenomes.

7. The use according to claim 5 or the method according to claim 6, characterized in that The specific steps include: (1) PCR amplification: PCR amplify the A' subgenomic retrotransposon SubA'-Athila and the B subgenomic retrotransposon SubB-Retand described in claim 1, and purify the PCR products; (2) Probe preparation: Digoxigenin was used to label the A' subgenomic retrotransposon sequence SubA'-Athila probe, and biotin was used to label the B subgenomic retrotransposon sequence SubB-Retand probe; (3) Preparation of mitotic metaphase chromosomes: Incubate young roots with 8-hydroxyquinoline at 25°C for 3 h and treat with Carnoy's fixative for 24 h. Cut the root tip meristem and place it in a mixture of 2% cellulase and 1% pectinase at 37°C for 3 h. Prepare the root tip metaphase chromosomes using the drop slide method. (4) Fluorescence in situ hybridization identification: (B1) First, prepare a probe mixture: prepare 10 μL of hybridization buffer containing 50% formamide, 10% dextran sulfate, and 2× SSC, and then add 1 μL each of the digoxigenin probe and biotin probe prepared in step (2); (B2) Incubate the denatured probe and denatured chromosomal DNA in hybridization buffer at 37°C for 16 h; (B3) The cells were visualized using anti-digoxin rhodamine and AF488 antibodies and observed under a microscope.

8. The use or method according to claim 7, characterized in that 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 elephant grass genomic DNA, and 36.75 μL ddH2O.

9. The use or method according to claim 7, characterized in that In step (1), the conditions for PCR amplification are: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; and final extension at 72°C for 10 min.

10. The use or method according to claim 7, characterized in that In the step (3), the Carnoy's fixative is a mixture of anhydrous ethanol and glacial acetic acid in a volume ratio of 3:1.

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