Primers, kit and method for identifying whether sugarcane material contains saccharum arundinaceum chromosome 9 or not

Abstract

The invention discloses primers, a kit and a method for identifying whether a sugarcane material contains a saccharum arundinaceum chromosome 9 or not. According to the method, whether the sugarcane material contains the saccharum arundinaceum chromosome 9 or not can be stably detected by utilizing the saccharum arundinaceum specific sequence PCR primer TaChr09-F / R (the sequence is shown as SEQ ID NO.1-2) through a conventional common PCR reaction program, and the method has the advantages of high efficiency, good stability, low cost, simplicity and convenience in operation and good generalization performance, and can be applied to improvement of sugarcane germplasm resources or sugarcane breeding.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and in particular relates to a primer, a kit and a method for identifying whether a sugarcane material contains chromosome 9 of Ampelopsis fasciata. Background Art

[0002] Sugarcane's closely related genera are rich in resources, with advantages such as high biomass, strong tillering ability, good perennial rooting, and strong resistance to drought, waterlogging, barrenness, and pests. In sugarcane breeding, hybridization of wild sugarcane germplasm materials with sugarcane has the potential to obtain new sugarcane varieties with high yield and high quality. Imperata sphaerocephala is one of the closely related wild species that has attracted much attention in sugarcane genetic improvement and breeding. After more than ten years of hybridization, several high-quality hybrid parents and registered cultivated varieties with Imperata sphaerocephala bloodline have been obtained. Chromosomes are the carriers of genes. In order to further utilize the excellent characteristics of Imperata sphaerocephala, it is necessary to understand the chromosome composition of its offspring. At present, the main methods for identifying sugarcane chromosomes are genomic in situ hybridization (GISH) and molecular markers. GISH can accurately mark chromosomes and intuitively reflect the distribution and changes of heterologous chromosomes and chromatin. GISH can be used to study the chromosome inheritance of sugarcane mitosis and meiosis and analyze the chromosome composition of sugarcane from different sources. Wang et al. used GISH analysis to reveal the genomic relationship between Chinese cherry and diploid closely related species. In addition to GISH, the Oligo-FISH technology that has emerged in recent years is also an important cellular means to identify and accurately recognize chromosomes. It uses oligonucleotides to synthesize probes, and the fluorescent probes bind to the chromosomes of cells to mark the chromosomes. Compared with GISH, Oligo probes are more specific. Through the constructed chromosome physical map, a specific chromosome segment and chromosome translocation can be identified, and the chromosome inheritance law can be analyzed. Jiang designed and synthesized Oligo probes using Chorus2 software, and conducted cytological studies on multiple plants such as corn, potatoes, and wheat, and found that different degrees of chromosome rearrangement occurred in the genetic process. Specific repetitive sequences can be prepared into probes to identify chromosomes. Specific repetitive sequences can be prepared into probes to identify chromosomes. Huang et al. discovered the centromere repetitive sequence specific to the cut-handed mitochondria and prepared it into a probe to mark and identify the chromosomes of the cut-handed mitochondria. However, GISH, Oligo-FISH and repetitive sequence in situ hybridization techniques require rich cytogenetics experience to obtain good experimental results. In addition, GISH uses the metaphase chromosomes of the root tip meristem cells of the sugarcane stem for detection, and it usually takes half a year to a year for sugarcane to mature. Therefore, the use of cytological methods cannot achieve the purpose of rapid detection.

[0003] In order to quickly identify and analyze the chromosomal composition of sugarcane, Yang et al. used ITS sequence analysis to find that there are multiple SNP sites between different species, and developed specific primers that can identify tropical species and cut-handed dense. Zhuang Nansheng et al. isolated the specific sequence of the genomic sequence of Ampelopsis fasciata by recovering the AFLP amplification product and converted it into SCAR molecular markers, which can be used to track the chromosomes or fragments of Ampelopsis fasciata in the hybrid offspring of sugarcane and Ampelopsis fasciata. The use of molecular markers such as SSR and gene microsatellites in sugarcane research has been proven to be essential for disease resistance identification, genetic diversity analysis and marker-assisted selection in breeding programs. At present, molecular markers that can directly identify chromosome 9 of Ampelopsis fasciata have not been developed, so it is of great significance to develop molecular markers that can identify chromosome 9 of Ampelopsis fasciata. Summary of the invention

[0004] The present invention provides a primer, a kit and a method for identifying whether a sugarcane material contains chromosome 9 of Imperata sphaerocephala, as there is no molecular identification method that can directly identify chromosome 9 of Imperata sphaerocephala at present. The Imperata sphaerocephala specific sequence PCR primer TaChr09-F / R is used to quickly identify whether a sugarcane material contains chromosome 9 of Imperata sphaerocephala, and the PCR result can intuitively reflect whether the detected sugarcane material contains chromosome 9 of Imperata sphaerocephala, thereby promoting the wide application of Imperata sphaerocephala germplasm resources in sugarcane genetic improvement.

[0005] The first object of the present invention is to provide a primer for identifying whether a sugarcane material contains chromosome 9 of Echinochloa sphaerocephala, wherein the sequence of the primer is:

[0006] TaChr09-F: 5'-CATATCAGTATTCGTGTTCTCAAATTTC-3' (SEQ ID NO. 1);

[0007] TaChr09-R: 5'-GTAAAACATAGGCTAGGACATAAAC-3' (SEQ ID NO. 2).

[0008] The second object of the present invention is to provide the use of the above primers in preparing a kit for identifying whether sugarcane materials contain chromosome 9 of Echinochloa sphaerocephala.

[0009] The third object of the present invention is to provide a kit comprising the above-mentioned primers.

[0010] The fourth object of the present invention is to provide the use of the above primers or kit in identifying whether sugarcane materials contain chromosome 9 of Echinochloa sphaerocephala.

[0011] The fourth object of the present invention is to provide the use of the above primers or kit in sugarcane germplasm resource improvement or sugarcane breeding.

[0012] The fifth object of the present invention is to provide a method for rapidly identifying whether a sugarcane material contains chromosome 9 of Echinochloa crusgalli, comprising the following steps:

[0013] (1) Extracting genomic DNA from sugarcane materials;

[0014] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the above-mentioned TaChr09-F / R primers;

[0015] (3) The amplified product was subjected to gel electrophoresis, and the results of electrophoresis were used to determine whether the sugarcane material contained chromosome 9 of Echinochloa sphaerocephala.

[0016] In step (2), the PCR reaction system includes: 12.5 μL of 2×PCR Mix, 2 μL of TaChr09-F, 2 μL of TaChr09-R, 2 μL of 100 ng / μL template DNA and 6.5 μL of ddH2O, a total of 25 μL.

[0017] In step (2), the PCR reaction procedure is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, 35 cycles; final extension at 72°C for 3 min, and storage at 4°C.

[0018] In step (3), judging whether the sugarcane material contains chromosome 9 of Echinops sphaerocephala based on the electrophoresis result is as follows: if a band of 1006 bp is obtained, it is judged that the sugarcane material contains chromosome 9 of Echinops sphaerocephala; if no band of 1006 bp is obtained, it is judged that the sugarcane material does not contain chromosome 9 of Echinops sphaerocephala.

[0019] The present invention has the following beneficial effects:

[0020] (1) High efficiency: The method of the present invention can be used to quickly detect whether sugarcane materials contain chromosome 9 of Echinochloa sphaerocephala, and only DNA from seedling leaves needs to be extracted for detection.

[0021] (2) Good stability: With the use of Imperata sphaerocephala in sugarcane breeding, there is no stable molecular marker for the authenticity of the hybrid offspring of sugarcane and Imperata sphaerocephala of higher generations. However, the method of the present invention has been tested in the early stage and can stably detect whether the sugarcane material contains Imperata sphaerocephala chromosome 9, with good stability.

[0022] (3) Low cost: Ordinary PCR technology can be used to detect whether the sugarcane material contains chromosome 9 of E. altissima.

[0023] (4) Easy operation: In the early stage, it is only necessary to extract genomic DNA and then perform conventional PCR amplification detection.

[0024] (5) Good promotional properties: The present invention has strong practicability and good promotional properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 GISH and Oligo-FISH were used to identify chromosomes of the offspring of Echinochloa sphaerocephala 1891-07. In the figure, A: red is the chromosome of Echinochloa sphaerocephala; B: red Chr1 and green Chr2; C: red Chr3 and green Chr4; D: red Chr5 and green Chr6; E: red Chr7 and green Chr8; F: red Chr9 and green Chr10. The arrows point to the marked chromosomes of Echinochloa sphaerocephala. The scale bar is 5μm.

[0026] Figure 2 This is the specificity test result of TaChr09-F / R primers. In the figure, M: 2000bp Marker; 1: 1723-05; 2: 1723-13; 3: 1849-29; 4: 1891-07; 5: 1891-15; 6: Hainan 92-77; 7: Yunnan 82-114; 8: Badila; 9: ROC22; 10: ddH2O.

[0027] Figure 3 This is the result of the stability test of TaChr09-F / R primers. In the figure, M: 2000bp Marker; 1: 1723-05; 2: 1723-13; 3: 1723-28; 4: 1723-39; 5: 1723-42; 6: 1849-09; 7: 1849-10; 8: 1849-14; 9: 1849-32; 10: 1849-54; 11: 1891-06; 12: 1891-12; 13: 1891-15; 14: 1891-16; 15: 1891-67; 16: Hainan 92-77; 17: Hainan 92-105; 18: Yunnan 82-114; 19: Yunnan 82-110; 20: Badila; 21:

[0028] HeiCheLiBen;22:ROC22;23:LiuCheng05-136;24:ddH2O. DETAILED DESCRIPTION

[0029] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0030] Example 1

[0031] GISH and Oligo-FISH were used to identify chromosomes of the offspring of Imperata sphaerocephala 1891-07. Different colored probes were used to mark the chromosomes of 1891-07 to determine whether it contained Imperata sphaerocephala chromosome 9, which was convenient for later verification. The results are shown in Figure 1 .

[0032] 1. Test materials

[0033] The materials used to detect the specificity of primer TaChr09-F / R in Imperata sphaerocephala and sugarcane materials include Imperata sphaerocephala original species, tropical species, large-stemmed wild species, thin-stemmed wild species and 9 sugarcane materials with sugarcane ancestry, as shown in Table 1.

[0034] The materials used to detect the stability of primer TaChr09-F / R in Imperata sphaerocephala and sugarcane materials include Imperata sphaerocephala original species, tropical species, large-stem wild species, thin-stem wild species and 23 sugarcane materials with sugarcane ancestry, as shown in Table 2.

[0035] Table 1 Specific detection materials

[0036]

[0037]

[0038] Table 2 Stability test materials

[0039]

[0040] 2. Experimental Procedure

[0041] (1) DNA extraction: The genomic DNA of the leaves of the sugarcane materials in Table 1 and Table 2 was extracted by the CTAB method. The extracted genomic DNA was detected by an ELISA instrument, and its OD260 / OD280 ratio should be between 1.8 and 2.0, and was stored in a -20°C refrigerator for use.

[0042] (2) Prepare PCR reaction solution: In 25 μL reaction solution, on ice, add 12.5 μL 2×PCR Mix, 2 μL TaChr09-F, 2 μL TaChr09-R, 2 μL 100 ng / μL gDNA, and 6.5 μL ddH2O.

[0043] TaChr09-F: 5'-CATATCAGTATTCGTGTTCTCAAATTTC-3' (SEQ ID NO. 1);

[0044] TaChr09-R: 5'-GTAAAACATAGGCTAGGACATAAAC-3' (SEQ ID NO. 2).

[0045] (3) PCR amplification and detection: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, 35 cycles; final extension at 72°C for 3 min, storage at 4°C. Detection was performed using 1.5% agarose gel electrophoresis, and photographic analysis was performed using a gel imager.

[0046] The results of PCR amplification of the 9 sugarcane materials in Table 1 are shown in Figure 2 . Figure 2 The results showed that a specific fragment with a size of 1006 bp (TaChr09, sequence as shown in SEQ ID NO.3) was amplified in all the materials of Imperata sphaerocephala, while the fragment was not amplified in the Saccharum materials without Imperata sphaerocephala kinship. The results showed that the primer TaChr09-F / R had good specificity and could be used as an Imperata sphaerocephala-specific molecular marker to identify whether the sugarcane materials contained Imperata sphaerocephala chromosome 9.

[0047] The results of PCR amplification of the 23 sugarcane materials in Table 2 are shown in Figure 3 . Figure 3 The results showed that primer TaChr09-F / R could accurately detect materials with Echinops sphaerocephala bloodline among 23 materials through conventional PCR reaction, and stably detect whether sugarcane materials contained Echinops sphaerocephala chromosome 9.

[0048] >TaChr09 (SEQ ID NO.3)

[0049]

[0050] In summary, primer TaChr09-F / R has good specificity and stability, and can be used to quickly identify whether sugarcane materials contain chromosome 9 of E. altissima.

[0051] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A primer for identifying whether a sugarcane material contains chromosome 9 of Echinochloa sphaerocephala, characterized in that: The sequences of the primers are: TaChr09-F: 5'-CATATCAGTATTCGTGTTCTCAAATTTC-3'; TaChr09-R: 5'-GTAAAACATAGGCTAGGACATAAAC-3'.

2. Use of the primers according to claim 1 in preparing a kit for identifying whether a sugarcane material contains chromosome 9 of Echinochloa sphaerocephala.

3. A kit, characterized in that: Comprising the primer according to claim 1.

4. Use of the primers according to claim 1 or the kit according to claim 3 in identifying whether a sugarcane material contains chromosome 9 of Echinochloa sphaerocephala.

5. Use of the primer according to claim 1 or the kit according to claim 3 in sugarcane germplasm resource improvement or sugarcane breeding.

6. A method for rapidly identifying whether a sugarcane material contains chromosome 9 of Ampelopsis fasciatus, characterized in that: The following steps are involved: (1) Extracting genomic DNA from sugarcane materials; (2) using the extracted genomic DNA as a template and performing PCR amplification using the TaChr09-F / R primers described in claim 1; (3) The amplified product was subjected to gel electrophoresis, and the results of electrophoresis were used to determine whether the sugarcane material contained chromosome 9 of Echinochloa sphaerocephala.

7. The method according to claim 6, characterized in that The PCR reaction system includes: 2×PCR Mix12.5 μL, TaChr09-F 2 μL, TaChr09-R 2 μL, 100 ng / μL template DNA 2 μL and ddH2O 6.5 μL, a total of 25 μL.

8. The method according to claim 6, characterized in that The reaction procedure of the PCR is: pre-denaturation at 95°C for 5 min; Denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, cycle 35 times; final extension at 72°C for 3 min, and storage at 4°C.

9. The method according to claim 6, characterized in that The method of judging whether the sugarcane material contains Chromosome 9 of Psoralea corylifolia according to the electrophoresis result is as follows: if a band of 1006 bp is obtained, it is judged that the sugarcane material contains Chromosome 9 of Psoralea corylifolia; if no band of 1006 bp is obtained, it is judged that the sugarcane material does not contain Chromosome 9 of Psoralea corylifolia.

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