Primers, kits and methods for identifying whether a sugar cane material contains chromosome 6 of brome 6

Abstract

This invention discloses primers, kits, and methods for identifying whether sugarcane materials contain chromosome 6 of *Imperata cylindrica*. The method utilizes *Imperata cylindrica*-specific sequence PCR primers TaChr06-F / R (sequence shown in SEQ ID NO. 1-2) to reliably detect the presence of chromosome 6 in sugarcane materials using a standard PCR procedure. This method offers advantages such as high efficiency, good stability, low cost, ease of operation, and broad applicability, and can be applied to sugarcane germplasm resource improvement or sugarcane breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to primers, reagent kits, and methods for identifying whether sugarcane material contains chromosome 6 of Imperata cylindrica. Background Technology

[0002] Sugarcane is rich in closely related genera, possessing advantages such as high biomass, strong tillering ability, good ratooning ability, and strong resistance to drought, waterlogging, poor soil, and pests. In sugarcane breeding, hybridization between wild sugarcane germplasm and sugarcane has the potential to obtain high-yielding and high-quality new sugarcane varieties. *Imperata cylindrica* is one of the closely related wild species that has received much attention in sugarcane genetic improvement breeding. After more than a decade of hybridization, several high-quality hybrid parents and cultivated varieties containing *Imperata cylindrica* lineage have been obtained. Chromosomes are the carriers of genes; to further utilize the excellent characteristics of *Imperata cylindrica*, it is necessary to understand the chromosome composition of its offspring. Currently, the main methods for identifying sugarcane chromosomes are genomic in situ hybridization (GISH) and molecular markers. GISH can accurately mark chromosomes, visually demonstrating the distribution and changes of heterologous chromosomes and chromatin. GISH can be used to study the chromosome inheritance during mitosis and meiosis in sugarcane and to analyze the chromosome composition from different sources in sugarcane. Wang et al. used GISH analysis to reveal the genomic relationship between Chinese cherry and diploid closely related species. Besides GISH, the Oligo-FISH technique, which has emerged in recent years, is also an important cellular method for identifying and accurately recognizing chromosomes. It utilizes oligonucleotides to synthesize probes, which carry fluorescence and bind to the chromosomes of the cell, thereby labeling them. Compared to GISH, Oligo probes are more specific. By constructing a chromosome physical map, specific segments of chromosomes and chromosomal translocations can be identified, allowing for the analysis of chromosomal inheritance patterns. Jiang designed and synthesized Oligo probes using Chorus2 software to conduct cellular studies on multiple plants, including corn, potatoes, and wheat, discovering varying degrees of chromosomal rearrangements during the genetic process. Specific repetitive sequences can be prepared into probes for chromosome recognition. Huang et al. discovered specific centromere repetitive sequences in *Dendrobium nobile* and prepared them into probes for chromosome labeling and identification in *Dendrobium nobile*. However, GISH, Oligo-FISH, and repetitive sequence in situ hybridization techniques require extensive experience in cytogenetics to achieve good experimental results. Furthermore, GISH uses metaphase cells from the root tip meristem of sugarcane stalks for detection, and sugarcane typically takes six months to a year to mature. Therefore, cytological methods cannot achieve rapid detection.

[0003] To rapidly identify and analyze sugarcane chromosome composition, Yang et al. used ITS sequence analysis to discover multiple SNP sites among different species and developed specific primers for identifying tropical species and *Imperata cylindrica*. Zhuang Nansheng et al. isolated genome-specific sequences of *Imperata cylindrica* by recovering AFLP amplification products and converted them into SCAR molecular markers, which can be used to track *Imperata cylindrica* chromosomes or fragments in the offspring of sugarcane-*Imperata cylindrica* hybrids. The use of molecular markers, such as SSRs and gene microsatellites, in sugarcane research has proven crucial for disease resistance identification, genetic diversity analysis, and marker-assisted selection in breeding projects. Currently, no molecular markers have been developed for direct identification of *Imperata cylindrica* chromosome 6, making the development of molecular markers for *Imperata cylindrica* chromosome 6 of significant importance. Summary of the Invention

[0004] This invention addresses the current lack of a molecular identification method for directly identifying chromosome 6 of *Imperata cylindrica*, providing primers, a kit, and a method for identifying whether sugarcane materials contain chromosome 6. The *Imperata cylindrica*-specific sequence PCR primers TaChr06-F / R enable rapid identification of chromosome 6 in sugarcane materials. The PCR results directly reflect the presence or absence of chromosome 6 in the tested sugarcane materials, promoting the widespread application of *Imperata cylindrica* germplasm resources in sugarcane genetic improvement.

[0005] The first objective of this invention is to provide a primer for identifying whether sugarcane material contains chromosome 6 of Imperata cylindrica, the sequence of which is:

[0006] TaChr06-F: 5'-CAAGATTATGCGGCCTAACTGATAAC-3' (SEQ ID NO. 1);

[0007] TaChr06-R: 5'-GATCTCTGGGTAAGTGCACCTCCAAG-3' (SEQ ID NO. 2).

[0008] A second objective of this invention is to provide the application of the above-described primers in the preparation of a kit for identifying whether sugarcane material contains chromosome 6 of Imperata cylindrica.

[0009] A third object of the present invention is to provide a kit comprising the primers described above.

[0010] A fourth objective of this invention is to provide the application of the aforementioned primers or kits in identifying whether sugarcane material contains chromosome 6 of the sedge.

[0011] A fifth objective of this invention is to provide the application of the aforementioned primers or kits in sugarcane germplasm resource improvement or sugarcane breeding.

[0012] The sixth objective of this invention is to provide a method for rapidly identifying whether sugarcane material contains chromosome 6 of Imperata cylindrica, comprising the following steps:

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

[0014] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the above-mentioned TaChr06-F / R primers;

[0015] (3) Perform gel electrophoresis on the amplification products and determine whether the sugarcane material contains chromosome 6 of Imperata cylindrica based on the electrophoresis results.

[0016] In step (2), the PCR reaction system includes: 12.5 μL of 2×PCR Mix, 2 μL of TaChr06-F, 2 μL of TaChr06-R, 2 μL of template DNA at 100 ng / μL, and 6.5 μL of ddH2O, for a total of 25 μL.

[0017] In step (2), the PCR reaction procedure is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, cycled 35 times; 72℃ final extension for 3 min, stored at 4℃.

[0018] In step (3), the determination of whether the sugarcane material contains chromosome 6 based on the electrophoresis results is as follows: if a band of 1601 bp is obtained, it is determined that the sugarcane material contains chromosome 6; if no band of 1601 bp is obtained, it is determined that the sugarcane material does not contain chromosome 6.

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

[0020] (1) High efficiency: The method of this invention can quickly detect whether sugarcane material contains chromosome 6 of Imperata cylindrica, and only requires DNA extraction from seedling leaves for detection.

[0021] (2) Good stability: With the use of Imperata cylindrica in sugarcane breeding, there are no stable molecular markers for the identification of the authenticity of sugarcane and Imperata cylindrica hybrid offspring in higher generations. However, the method of this invention has been tested in the early stage and can stably detect whether sugarcane material contains Imperata cylindrica chromosome 6, which has good stability.

[0022] (3) Low cost: The presence of chromosome 6 of Imperata cylindrica can be detected using ordinary PCR technology.

[0023] (4) Simple operation: In the early stage, it is only necessary to extract genomic DNA and then detect it by ordinary PCR amplification.

[0024] (5) Good scalability: This invention is highly practical and has good scalability. Attached Figure Description

[0025] Figure 1 Chromosome identification was performed on the 1891-07 progeny of *Imperata cylindrica* using GISH and Oligo-FISH. In the figure, A: red represents *Imperata cylindrica* chromosomes; 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 indicate the marked *Imperata cylindrica* chromosomes. The scale bar is 5 μm.

[0026] Figure 2 This is the result of specific detection using TaChr06-F / R primers. In the figure, M: 100bp Marker; 1: 1723-05; 2: 1849-09; 3: 1849-29; 4: 1891-27; 5: 1891-71; 6: Hainan 92-77; 7: Yunnan 82-114; 8: Badila; 9: ROC22; 10: ddH2O.

[0027] Figure 3 This is the result of the TaChr06-F / R primer stability test. In the figure, M: 2000bp Marker; 1: 1723-02; 2: 1723-13; 3: 1723-28; 4: 1723-39; 5: 1723-42; 6: 1849-09; 7: 1849-10; 8: 1849-14; 9: 1849-18; 10: 1849-54; 11: 1891-06; 12: 1891-12; 13: 1891-16; 14: 1891-20; 15: 1891-71; 16: Hainan 92-77; 17: Hainan 92-105; 18: Yunnan 82-114; 19: Yunnan 82-110; 20: Badila; 21:

[0028] Black car Liben; 22: ROC22; 23: Liucheng 05-136; 24: ddH2O. Detailed Implementation

[0029] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0030] Example 1

[0031] Chromosomal identification was performed on the 1891-07 progeny of *Imperata cylindrica* using GISH and Oligo-FISH. Different colored probes were used to mark the chromosomes of 1891-07 to determine whether they contained chromosome 6 of *Imperata cylindrica*, facilitating later verification. Results are shown below. Figure 1 .

[0032] 1. Test materials

[0033] The materials used to detect the specificity of primer TaChr06-F / R in Imperata cylindrica and sugarcane materials included nine sugarcane materials: the original Imperata cylindrica species, tropical species, large-stemmed wild species, small-stemmed wild species, and sugarcane-related materials, as shown in Table 1.

[0034] The materials used to test the stability of primer TaChr06-F / R in Imperata cylindrica and sugarcane materials included 23 sugarcane materials, including the original Imperata cylindrica species, tropical species, large-stemmed wild species, small-stemmed wild species, and sugarcane-related materials, as shown in Table 2.

[0035] Table 1 Specific Detection Materials

[0036]

[0037]

[0038] Table 2. Materials for Stability Testing

[0039]

[0040] 2. Experimental Procedure

[0041] (1) DNA extraction: Genomic DNA was extracted from the leaves of the sugarcane materials in Tables 1 and 2 using the CTAB method. The extracted genomic DNA was tested by an enzyme-linked immunosorbent assay (ELISA) reader, and the OD260 / OD280 ratio should be between 1.8 and 2.0. The extracted DNA was then stored at -20°C for later use.

[0042] (2) Preparation of PCR reaction solution: In 25 μL of reaction solution, on ice, add 12.5 μL of 2×PCR Mix, 2 μL of TaChr06-F, 2 μL of TaChr06-R, 2 μL of 100 ng / μL of gDNA and 6.5 μL of ddH2O.

[0043] TaChr06-F: 5'-CAAGATTATGCGGCCTAACTGATAAC-3' (SEQ ID NO. 1);

[0044] TaChr06-R: 5'-GATCTCTGGGTAAGTGCACCTCCAAG-3' (SEQ ID NO. 2).

[0045] (3) PCR amplification and detection: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, cycled 35 times; 72℃ final extension for 3 min, stored at 4℃. Detection was performed using 1.5% agarose gel electrophoresis, and images were taken and analyzed using a gel imaging system.

[0046] The results of PCR amplification of the nine sugarcane materials in Table 1 are shown below. Figure 2 . Figure 2 The results showed that a specific fragment of 1601 bp (TaChr06, sequence as shown in SEQ ID NO.3) was amplified in *Imperata cylindrica*, while the fragment was not amplified in *Suaeda salsa* materials without *Imperata cylindrica* lineage. This result indicates that the primer TaChr06-F / R has good specificity and can be used as a *Imperata cylindrica*-specific molecular marker to identify whether sugarcane materials contain *Imperata cylindrica* chromosome 6.

[0047] The results of PCR amplification of the 23 sugarcane materials in Table 2 are shown below. Figure 3 . Figure 3 The results showed that, using conventional PCR, primers TaChr06-F / R were able to accurately detect materials with *Imperata cylindrica* lineage in 23 samples, and stably detect whether sugarcane materials contained *Imperata cylindrica* chromosome 6.

[0048] TaChr06 (SEQ ID NO.3)

[0049]

[0050] In summary, primer TaChr06-F / R exhibits good specificity and stability, and its use enables rapid identification of whether sugarcane materials contain chromosome 6 of Imperata cylindrica.

[0051] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, 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 considered within the scope of protection of the present invention.

Claims

1. A primer for identifying whether a sugar cane material contains Chromosome 6 of Eremochloa baulana, characterized in that, The sequence of the primer is: TaChr06-F: 5'-CAAGATTATGCGGCCTAACTGATAAC-3'; TaChr06-R: 5'-GATCTCTGGGTAAGTGCACCTCCAAG-3'.

2. Use of the primer of claim 1 in the preparation of a kit for identifying whether a sugarcane material contains the 6th chromosome of Erianthus arundinaceum.

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 6th chromosome of Erianthus arundinaceum.

5. A method for rapidly identifying whether a sugarcane material contains Chromosome 6 of Erianthus arundinaceus, 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 TaChr06-F / R primer of claim 1; (3) performing gel electrophoresis on the amplification product, and determining whether the sugarcane material contains the 6th chromosome of Erianthus arundinaceum according to the electrophoresis result.

6. The method of claim 5, wherein, The reaction system of the PCR comprises 2x PCR Mix 12.5 μL, TaChr06-F 2 μL, TaChr06-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: 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, 4℃ storage.

8. The method of claim 5, wherein, The determination of whether the sugarcane material contains the 6th chromosome of Erianthus arundinaceum according to the electrophoresis result is as follows: if a band with a size of 1601 bp is obtained, it is determined that the sugarcane material contains the 6th chromosome of Erianthus arundinaceum; if no band with a size of 1601 bp is obtained, it is determined that the sugarcane material does not contain the 6th chromosome of Erianthus arundinaceum.

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