Method for identifying sugarcane germplasm resources based on SSR (Simple Sequence Repeats) and CE (capillary electrophoresis) technique
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An identification method and sugarcane technology, applied in the field of high-throughput detection, can solve problems such as difficulty in identification of sugarcane germplasm resources, and achieve the effects of reducing workload, accurate experimental results, and reducing human errors
Inactive Publication Date: 2014-09-03
广西泰格瑞科技有限公司
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[0006] The purpose of the present invention is to solve the problem of difficult identification of sugarcane germplasm resources, and provide a high-throughput, simple operation, accurate and reliable identification method for sugarcane seed resources
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Embodiment 1
[0015] Example 1: 12 pairs of SSR fluorescent primers were used to establish plant maps of 52 sugarcane cultivars, and the selected 52 sugarcane cultivars are shown in the table below.
[0016]
[0017] 1. Genomic DNA extraction
[0018] (1) Take young sugarcane leaves in a mortar, add liquid nitrogen to grind them into powder, and put them into a 2 ml centrifuge tube;
[0019] (2) Add 1.2 ml 65°C preheated SDS extraction solution (Tris-HCl 200 mmol / L, pH8.0; EDTA 50 mmol / L pH8.0; NaCl 500 mmol / L; 3% SDS) into the centrifuge tube , Shake the sample powder and the extract quickly to prevent them from forming agglomerates, put in a water bath at 65°C for 45 minutes, and shake gently 4-6 times during the period;
[0020] (3) Add 200 μl KAc (5 mol / L, pH 4.8), mix well, and place in ice bath for 15 min;
[0021] (4) Centrifuge at 10,000 rpm for 15 min in a refrigerated centrifuge at 4°C;
[0022] (5) Take 800 μl supernatant, add 800 μl chloroform to mix, and centrifuge at 120...
Embodiment 2
[0040] Embodiment 2: the identification of sugarcane variety to be tested
[0041] 1. Select two kinds of unknown sugarcane varieties as materials to be tested, and carry out DNA extraction, SSR-PCR amplification, capillary gel electrophoresis detection and result data analysis according to the method in Example 1;
[0042] 2. According to the read band size, according to the 141 specific SSR marker fragments in Example 1, if it contains these 141 specific SSR marker fragments, it will be marked as "A", if there is no extension, it will be marked as "C". , to construct the SSR fingerprint of the sugarcane variety to be tested. And comparing the results with the established standard sugarcane fingerprints in Example 1, the sugarcane variety was identified as Guitang No. 19.
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Abstract
The invention discloses a method for identifying sugarcane germplasm resources. According to the method, SSR (Simple Sequence Repeats) molecular marker and CE (capillary electrophoresis) technique are combined, and fingerprint spectrums of 52 sugarcane varieties are established by utilizing 12 pairs of primers and are applied to identification of sugarcane germplasm, variety identification and protection. The specific method comprises the following steps: extracting genomic DNAs of different varieties of sugarcane, carrying out PCR (polymerase chain reaction) amplification, detecting amplification results by utilizing CE, establishing fingerprint spectrums of different sugarcane germplasms according different sizes of DNA strips and utilizing the fingerprint spectrums in the identification of germplasm resources. Compared with the conventional germplasm resource identification and biochemical detection, the method for identifying the sugarcane germplasm resources are not influenced by growth environment of plants and seasons and has the advantages that the resolution is high, the result is exact and reliable, the operation is simple, quick and direct, and the method is especially suitable for mass multiple-batch experimental data integration.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to a high-throughput detection method for identifying sugarcane germplasm resources by using SSR and capillary electrophoresis techniques. Background technique [0002] my country is one of the ancient sugarcane-growing countries in the world, and there are many types of sugarcane cultivars in the past dynasties. In recent years, China has introduced and bred more than 120 sugarcane varieties suitable for promotion in various ecological types of sugarcane areas. However, the genetic basis of current sugarcane cultivars is narrow, and the biological characteristics such as morphology and agronomic traits of the varieties are not much different. How to identify sugarcane cultivars and their hybrid offspring has become one of the most difficult points in current sugarcane scientific research. [0003] Before DNA molecular techniques were widely used, people mainly used experimental methods ...
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