Primers, kits and methods for identifying whether a sugar cane material contains chromosome 10 of brome

Abstract

The application discloses primers, kits and methods for identifying whether sugarcane material contains Chromosome 10 of Eremochloa ophiuroides. The method of the application can stably detect whether the sugarcane material contains Chromosome 10 of Eremochloa ophiuroides by using the Eremochloa ophiuroides specific sequence PCR primers TaChr10-F / R (sequences shown in SEQ ID NO. 1-2) through a conventional general PCR reaction procedure. The method has the advantages of high efficiency, good stability, low cost, simple operation and good popularization, and can be applied to sugarcane germplasm resource improvement or sugarcane breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology technology, and particularly relates to a primer, a kit and a method for identifying whether a sugarcane material contains Chromosome 10 of Erianthus arundinaceum. BACKGROUND

[0002] Sugarcane wild relatives are rich in resources, and have the advantages of high biomass, strong tillering ability, good ratooning ability, drought tolerance, waterlogging tolerance, drought tolerance, strong disease and pest resistance, etc. In sugarcane breeding, through hybridization of sugarcane wild germplasm materials with sugarcane, there is potential to obtain high-yielding sugarcane varieties. Erianthus arundinaceum is one of the wild relatives of sugarcane that are concerned in genetic improvement and breeding. After more than ten years of hybridization and utilization, a number of high-quality hybrid parents and registered varieties containing Erianthus arundinaceum blood have been obtained. Chromosomes are the carriers of genes. In order to further utilize the excellent characteristics of Erianthus arundinaceum, it is necessary to understand the composition of its offspring chromosomes. At present, the methods for identifying sugarcane chromosomes mainly include genomic in situ hybridization (GISH) and molecular markers. GISH can accurately mark chromosomes and intuitively reflect the distribution and changes of alien chromosomes and chromatin. Using GISH, the genetic characteristics of mitosis and meiosis of sugarcane chromosomes and the composition of chromosomes from different sources can be studied. Wang et al. used GISH to reveal the genomic relationship between Chinese cherry and diploid relatives. In addition to GISH, Oligo-FISH technology has also emerged in recent years and is an important cell method for identifying and accurately identifying chromosomes. It is a technology that uses oligonucleotide probes to synthesize probes, which are combined with the chromosomes of cells, and then the chromosomes are marked. Compared with GISH, Oligo probes are more specific. Through the construction of a physical map of the chromosome, a specific segment of the chromosome and the translocation of the chromosome can be identified, and the genetic rules of the chromosome can be analyzed. Jiang designed and synthesized Oligo probes using Chorus2 software to conduct cytological studies on corn, potatoes, and wheat, and found that different degrees of chromosome rearrangement occurred during the genetic process. Specific repetitive sequences can be used to prepare probes to identify chromosomes. Huang et al. mined specific centromere repetitive sequences of Erianthus arundinaceum and prepared probes to mark and identify the chromosomes of Erianthus arundinaceum. However, GISH, Oligo-FISH, and repetitive sequence in situ hybridization technology require rich experience in cytogenetics to obtain good experimental results. In addition, GISH uses metaphase chromosome cells in the root tip meristem zone of sugarcane stems for detection, and sugarcane maturity usually takes six months to a year. Therefore, cytological methods cannot achieve rapid detection.

[0003] In order to quickly identify and analyze the composition of sugarcane chromosome, Yang et al. used ITS sequence analysis to find multiple SNP sites between different species, and developed specific primers that can identify tropical species and Erianthus. Zhuang et al. isolated Erianthus genomic specific sequences by recovering AFLP amplification products and converted them into SCAR molecular markers, which can be used to track Erianthus chromosomes or fragments in the offspring of sugarcane and Erianthus hybridization. The use of molecular markers such as SSR and gene microsatellite in sugarcane research has been shown to be crucial for disease resistance identification, genetic diversity analysis, and marker-assisted selection in breeding programs. Currently, no molecular markers that can directly identify Erianthus chromosome 10 have been developed, so it is of great significance to develop molecular markers that can identify Erianthus chromosome 10. SUMMARY

[0004] The present application aims to provide a primer, kit and method for identifying whether sugarcane material contains Erianthus chromosome 10, as there is currently no molecular identification method that can directly identify Erianthus chromosome 10. The use of Erianthus-specific sequence PCR primers TaChr10-F / R can quickly identify whether sugarcane material contains Erianthus chromosome 10, and the PCR results can directly reflect whether the detected sugarcane material contains Erianthus chromosome 10, promoting the widespread application of Erianthus germplasm resources in sugarcane genetic improvement.

[0005] The first object of the present application is to provide a primer for identifying whether sugarcane material contains Erianthus chromosome 10, the sequence of which is:

[0006] TaChr10-F: 5'-CCAGTTTTCTAATTAGATTAAAGTC-3'(SEQ ID NO.1);

[0007] TaChr10-R: 5'-CTAGAAGGCTCACTATCACAATCC-3'(SEQ ID NO.2).

[0008] The second object of the present application is to provide the use of the above-mentioned primer in the preparation of a kit for identifying whether sugarcane material contains Erianthus chromosome 10.

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

[0010] The fourth object of the present application is to provide the use of the above-mentioned primer or kit in identifying whether sugarcane material contains Erianthus chromosome 10.

[0011] The fourth object of the present application is to provide the use of the above-mentioned primer or kit in the improvement of sugarcane germplasm resources or sugarcane breeding.

[0012] A fifth object of the present application is to provide a method for rapidly identifying whether a sugarcane material contains Chromosome 10 of Erianthus arundinaceus, comprising the following steps:

[0013] (1) extracting genomic DNA of the sugarcane material;

[0014] (2) using the extracted genomic DNA as a template, PCR amplification is performed using the above-mentioned TaChr10-F / R primers;

[0015] (3) subjecting the amplification product to gel electrophoresis, and determining whether the sugarcane material contains Chromosome 10 of Erianthus arundinaceus according to the electrophoresis result.

[0016] In step (2), the reaction system of the PCR comprises 2x PCR Mix 12.5 μL, TaChr10-F 2 μL, TaChr10-R 2 μL, 100 ng / μL of template DNA 2 μL, and ddH2O 6.5 μL, with a total of 25 μL.

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

[0018] In step (3), the determination of whether the sugarcane material contains Chromosome 10 of Erianthus arundinaceus according to the electrophoresis result is specifically as follows: if a band with a size of 1350 bp is obtained, it is determined that the sugarcane material contains Chromosome 10 of Erianthus arundinaceus, and if no band with a size of 1350 bp is obtained, it is determined that the sugarcane material does not contain Chromosome 10 of Erianthus arundinaceus.

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

[0020] (1) high efficiency: the present application can rapidly detect whether a sugarcane material contains Chromosome 10 of Erianthus arundinaceus, and only needs to extract leaf DNA of a seedling for detection.

[0021] (2) good stability: as Erianthus arundinaceus is used in sugarcane breeding, no stable molecular marker has been used for detecting the authenticity of a high-generation sugarcane and a hybrid offspring of Erianthus arundinaceus, but the present application can stably detect whether a sugarcane material contains Chromosome 10 of Erianthus arundinaceus, and has good stability.

[0022] (3) low cost: ordinary PCR technology can be used to detect whether a sugarcane material contains Chromosome 10 of Erianthus arundinaceus.

[0023] (4) simple operation: only genomic DNA needs to be extracted, and then ordinary PCR amplification detection can be performed.

[0024] (5) Good popularization: the application has strong practicability and good popularization. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is the chromosome identification of 1891-07 by GISH and Oligo-FISH. In the figure, A: red is the chromosome of Deyeuxia; 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 arrow points to the labeled Deyeuxia chromosome. The scale size is 5 μm.

[0026] Figure 2 is the specific detection result of TaChr10-F / R primer. In the figure, M: 2000bp Marker; 1: 1849-09; 2: 1849-29; 3: 1891-27; 4: 1891-71; 5: 1723-05; 6: Hainan92-77; 7: Yunnan82-114; 8: Badila; 9: ROC22; 10: ddH2O.

[0027] Figure 3 is the stability detection result of TaChr10-F / R primer. In the figure, M: 2000bp Marker; 1: 1723-02; 2: 1723-05; 3: 1723-13; 4: 1723-28; 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: 1891-16; 14: 1891-20; 15: 1891-67; 16: Hainan92-77; 17: Hainan92-105; 18: Yunnan82-114; 19: Yunnan82-110; 20: Badila; 21:

[0028] Hecheliuben; 22: ROC22; 23: Liucheng05-136; 24: ddH2O. DETAILED DESCRIPTION

[0029] The following examples are further illustrations of the application and are not intended to limit the same.

[0030] Example 1

[0031] GISH and Oligo-FISH are used to identify the chromosome of 1891-07, different color probes are used to label the chromosome of 1891-07, and it is determined whether the chromosome contains Deyeuxia No. 10 chromosome, which is convenient for verification in the later stage, and the results are shown in Figure 1.

[0032] 1. Test materials

[0033] Materials for detecting the specificity of primers TaChr10-F / R in Erianthus arundinaceus and sugarcane materials, including 9 Erianthus arundinaceus materials, 1 tropical species, 1 large stem wild species, 1 slender stem wild species and 9 sugarcane materials containing sugarcane bloodlines, see Table 1.

[0034] Materials for detecting the stability of primers TaChr10-F / R in Erianthus arundinaceus and sugarcane materials, including 23 Erianthus arundinaceus materials, 1 tropical species, 1 large stem wild species, 1 slender stem wild species and 23 sugarcane materials containing sugarcane bloodlines, see Table 2.

[0035] Table 1 Specificity detection materials

[0036]

[0037]

[0038] Table 2 Stability detection materials

[0039]

[0040] 2. Experimental steps

[0041] (1) DNA extraction: The genomic DNA of the leaf of the sugarcane materials in Table 1 and Table 2 was extracted by the CTAB method. The extracted genomic DNA was detected by a microplate reader, and the OD260 / OD280 ratio should be between 1.8-2.0, and was stored in a -20℃ refrigerator for use.

[0042] (2) Preparation of PCR reaction solution: In a 25μL reaction solution, operate on ice, add 12.5μL 2×PCR Mix, 2μL TaChr10-F, 2μL TaChr10-R, 2μL 100ng / μL gDNA and 6.5μL ddH2O.

[0043] TaChr10-F: 5'-CCAGTTTTCTAATTAGATTAAAGTC-3'(SEQ ID NO. 1);

[0044] TaChr10-R: 5'-CTAGAAGGCTCACTATCACAATCC-3'(SEQ ID NO. 2).

[0045] (3) PCR amplification and detection: 95°C pre-denaturation 5 min; 95°C denaturation 30 s, 55°C annealing 30 s, 72°C extension 2 min, cycle 35 times; 72°C final extension 3 min, 4°C storage. 1.5% agarose gel electrophoresis was used for detection, and gel imaging instrument was used for photograph analysis.

[0046] PCR amplification results of 9 sugarcane materials in Table 1 are shown in Figure 2 . Figure 2 It is shown that specific fragments of 1350 bp (TaChr10, sequence as shown in SEQ ID NO. 3) are amplified in the materials of E. arundinaceus, while no fragments are amplified in the materials of Saccharum without E. arundinaceus bloodline, which indicates that the specificity of primers TaChr10-F / R is good, and the primers can be used as specific molecular markers of E. arundinaceus to identify whether the sugarcane materials contain the 10th chromosome of E. arundinaceus.

[0047] PCR amplification results of 23 sugarcane materials in Table 2 are shown in Figure 3 . Figure 3 It is shown that through the conventional PCR reaction, the primers TaChr10-F / R can accurately detect the materials containing E. arundinaceus bloodline in 23 materials, and stably detect whether the sugarcane materials contain the 10th chromosome of E. arundinaceus.

[0048] > TaChr10 (SEQ ID NO. 3)

[0049]

[0050] In summary, the primer TaChr10-F / R has good specificity and stability, and using the primer can quickly identify whether the sugarcane material contains the 10th chromosome of Eremochloa.

[0051] The above merely describes the preferred embodiments of the present application, and it should be noted that the above preferred embodiments should not be regarded as a limitation to the present application, and the protection scope of the present application should be defined by the scope of the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A primer for identifying whether a sugar cane material contains Chromosome 10 of Eremochloa 10, characterized in that, The sequence of the primer is: TaChr10-F: 5'-CCAGTTTTCTAATTAGATTAAAGTC-3'; TaChr10-R: 5'-CTAGAAGGCTCACTATCACAATCC-3'.

2. Use of the primer of claim 1 in the preparation of a kit for identifying whether a sugarcane material contains the 10th chromosome of Erianthus arundinaceus.

3. A kit characterized in that, The kit comprises the primer of claim 1.

4. Use of the primer of claim 1 or the kit of claim 3 in identifying whether a sugarcane material contains the 10th chromosome of Erianthus arundinaceus.

5. A method for rapid identification of sugarcane material containing Chromosome 10 of Eremochloa ophiuroides, characterized by, The method comprises the following steps: (1) extracting genomic DNA of the sugarcane material; (2) using the extracted genomic DNA as a template, performing PCR amplification using the TaChr10-F / R primer of claim 1; (3) performing gel electrophoresis on the amplification product, and determining whether the sugarcane material contains the 10th chromosome of Erianthus arundinaceus according to the electrophoresis result.

6. The method of claim 5, wherein, The reaction system of the PCR comprises 2xPCR Mix 12.5 μL, TaChr10-F 2 μL, TaChr10-R 2 μL, 100 ng / μL template DNA 2 μL, and ddH2O 6.5 μL, with a total volume of 25 μL.

7. The method of claim 5, wherein, The reaction procedure of the PCR is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 2 min, for 35 cycles; final extension at 72℃ for 3 min, and storage at 4℃.

8. The method of claim 5, wherein, The determination of whether the sugarcane material contains the 10th chromosome of Erianthus arundinaceus according to the electrophoresis result is as follows: if a band with a size of 1350 bp is obtained, it is determined that the sugarcane material contains the 10th chromosome of Erianthus arundinaceus, and if no band with a size of 1350 bp is obtained, it is determined that the sugarcane material does not contain the 10th chromosome of Erianthus arundinaceus.

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