Primers, kit and method for identifying whether sugarcane materials contain chromosome 9 of Echinops sphaerocarpus

Abstract

The present invention discloses primers, a kit, and a method for identifying whether sugarcane materials contain Imperata gracilis chromosome 9. The method utilizes Imperata gracilis-specific PCR primers TaChr09-F / R (sequence shown in SEQ ID NOs. 1-2) to stably detect whether sugarcane materials contain Imperata gracilis chromosome 9 through a conventional PCR reaction procedure. The method has the advantages of high efficiency, good stability, low cost, simple operation, and good scalability, and can be applied to sugarcane germplasm resource improvement or sugarcane breeding.

Description

Technical Field

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

[0002] Sugarcane relatives are rich in resources, boasting high biomass, strong tillering ability, good perennial rooting, and strong resistance to drought, waterlogging, barrenness, and pests and diseases. Hybridization of wild sugarcane germplasm with sugarcane has the potential to yield new high-yield and high-quality sugarcane varieties. Imperata serrata is one of the wild relatives that has attracted considerable attention in sugarcane genetic improvement and breeding. After more than a decade of hybridization, several high-quality hybrid parents and registered cultivated varieties with Imperata serrata ancestry have been obtained. Chromosomes are the carriers of genes, and understanding the chromosomal composition of its offspring is essential to further exploit the excellent properties of Imperata serrata. Currently, the main methods for identifying sugarcane chromosomes include genomic in situ hybridization (GISH) and molecular markers. GISH allows for accurate chromosome labeling, visualizing the distribution and changes of heterologous chromosomes and chromatin. GISH can be used to study chromosomal inheritance during mitosis and meiosis in sugarcane and to analyze the chromosomal composition of different sugarcane sources. Using GISH, Wang et al. revealed the genomic relationship between Chinese cherry and its diploid relatives. In addition to GISH, the recently emerging Oligo-FISH technique is also an important cellular method for identifying and accurately recognizing chromosomes. It uses oligonucleotides to synthesize probes that carry fluorescence and bind to cell chromosomes, thereby marking the chromosomes. Compared to GISH, oligo probes are more specific. By constructing a physical chromosome map, it is possible to identify specific chromosome segments and chromosome translocations, and analyze the patterns of chromosome inheritance. Jiang designed and synthesized oligo probes using Chorus2 software and conducted cytological studies on several plants, including corn, potatoes, and wheat, finding that chromosome rearrangements occurred to varying degrees during the genetic process. Specific repetitive sequences can be prepared into probes to identify chromosomes. Huang et al. discovered a centromeric repetitive sequence specific to the scutellaria scutellaria , and used it as a probe to mark and identify chromosomes in the scutellaria scutellaria . However, GISH, Oligo-FISH, and repetitive sequence in situ hybridization techniques require extensive cytogenetic experience to achieve good results. Furthermore, GISH uses chromosomes from metaphase cells in the root apical meristem of sugarcane stems, and sugarcane typically takes six months to a year to mature. Therefore, rapid detection using cytological methods is not feasible.

[0003] To rapidly identify and analyze sugarcane chromosome composition, Yang et al. used ITS sequence analysis to discover multiple SNPs across species and developed specific primers for identifying tropical species and Psoralea corylifolia. Zhuang Nansheng et al. isolated genome-specific sequences from Imperata serrata by recovering AFLP amplification products and converted them into SCAR molecular markers, which can be used to track Imperata serrata chromosomes or fragments in sugarcane-Iserrata hybrids. The use of molecular markers, such as SSRs and microsatellites, in sugarcane research has proven crucial for disease resistance identification, genetic diversity analysis, and marker-assisted selection in breeding programs. Currently, molecular markers that can directly identify Imperata serrata chromosome 9 have not been developed, making the development of molecular markers that can do so of great significance. Summary of the Invention

[0004] In view of the current lack of molecular identification methods for directly identifying chromosome 9 in Imperata serrata, the present invention provides primers, a kit, and a method for identifying whether sugarcane materials contain chromosome 9 in Imperata serrata. Using the Imperata serrata-specific PCR primers TaChr09-F / R, the presence of chromosome 9 in sugarcane materials can be rapidly identified. The PCR results can intuitively indicate the presence of chromosome 9 in the tested sugarcane materials, promoting the widespread application of Imperata serrata germplasm resources in sugarcane genetic improvement.

[0005] The first object of the present invention is to provide a primer for identifying whether sugarcane materials contain chromosome 9 of Echinops sphaerocarpa, 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 Echinops sphaerocarpa.

[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 Echinops sphaerocarpa chromosome 9.

[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] A fifth object of the present invention is to provide a method for rapidly identifying whether a sugarcane material contains chromosome 9 of Echinops sphaerocarpa, 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 Echinops sphaerocarpa.

[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), the determination of whether the sugarcane material contains Echinops sphaerocarpa chromosome 9 is performed based on the electrophoresis results. Specifically, if a band of 1006 bp is obtained, it is determined that the sugarcane material contains Echinops sphaerocarpa chromosome 9; if no band of 1006 bp is obtained, it is determined that the sugarcane material does not contain Echinops sphaerocarpa chromosome 9.

[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 Echinops sphenanthera. It only requires DNA extraction from seedling leaves for testing.

[0021] (2) Good stability: With the use of Imperata spp. in sugarcane breeding, there is no stable molecular marker for the authenticity of the hybrid offspring of sugarcane and Imperata spp. in 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 spp. 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 Echinops sphaerocarpa.

[0023] (4) Easy to operate: 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 Chromosome identification of the offspring of Imperata spp. 1891-07 was performed using GISH and Oligo-FISH. In the figure, A: Red Imperata spp. 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. Arrows point to the labeled Imperata spp. chromosomes. Scale bar, 5 μm.

[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 The results of the stability test of TaChr09-F / R primers are shown in the figure. 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] Heiche Liben; 22: ROC22; 23: Liucheng 05-136; 24: ddH2O. DETAILED DESCRIPTION

[0029] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0030] Example 1

[0031] Chromosome identification of the offspring of Imperata sphaeroides 1891-07 was performed using GISH and Oligo-FISH. Different colored probes were used to mark the chromosomes of 1891-07 to determine whether it contained Imperata sphaeroides 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 sphaerocarpa and sugarcane materials included Imperata sphaerocarpa original species, tropical species, large-stemmed wild species, thin-stemmed wild species and 9 sugarcane materials with sugarcane lineage, as shown in Table 1.

[0034] The materials used to test the stability of primer TaChr09-F / R in Imperata sphaerocarpa and sugarcane materials included Imperata sphaerocarpa original species, tropical species, large-stemmed wild species, thin-stemmed wild species, and 23 sugarcane materials with sugarcane lineage, 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: 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 detected by a microplate reader, and its OD260 / OD280 ratio should be between 1.8 and 2.0. The DNA was then stored in a -20°C refrigerator until use.

[0042] (2) Prepare PCR reaction solution: In a 25 μL reaction solution, operate 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; 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min; final extension at 72°C for 3 min, and storage at 4°C. Detection was performed using 1.5% agarose gel electrophoresis and photographed 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 shown in SEQ ID NO. 3) was amplified in all the E. truncatula materials, while the fragment was not amplified in the Saccharum materials that were not related to E. truncatula. This result indicates that the primer TaChr09-F / R has good specificity and can be used as an E. truncatula-specific molecular marker to identify whether the sugarcane materials contain E. truncatula 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 the materials with Echinops spp. bloodline among 23 materials through conventional PCR reaction, and stably detect whether the sugarcane materials contained Echinops spp. 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 Echinops sphenanthera chromosome 9.

[0051] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer for identifying whether sugarcane material contains chromosome 9 of Echinops sphaerocarpa, 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 sugarcane materials contain chromosome 9 of Echinops sphaerocarpa.

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 sugarcane materials contain chromosome 9 of Echinops sphaerocarpa.

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

6. A method for rapidly identifying whether sugarcane material contains chromosome 9 of Echinops sphaerocarpa, 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 Echinops sphaerocarpa.

7. The method according to claim 6, characterized in that The PCR reaction system includes: 2×PCR Mix 12.5 μL, TaChr09-F 2 μL, TaChr09-R 2 μL, 100 ng / μL template DNA 2 μL and ddH 2 O 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 as follows: 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 2 min were repeated 35 times; final extension at 72°C for 3 min was performed, and the tube was stored at 4°C.

9. The method according to claim 6, characterized in that The method of judging whether the sugarcane material contains Echinops elegans chromosome 9 based on the electrophoresis results is as follows: if a band of 1006 bp is obtained, it is judged that the sugarcane material contains Echinops elegans chromosome 9; if no band of 1006 bp is obtained, it is judged that the sugarcane material does not contain Echinops elegans chromosome 9.

Images