Method for identifying erianthus arundinaceus consanguinity in sugarcane by sub telomere sequence
A subtelomere and sequence table technology, which is applied in the field of plant detection and identification, can solve the problems such as the inability to use the authenticity of high-generation hybrid offspring, and achieve an efficient and stable identification method with high efficiency and good sensitivity
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Embodiment 1
[0015] Embodiment 1, a kind of method that utilizes subtelomeric sequence identification to comprise the blood lineage of sagegrass in sugarcane, it is characterized in that identification steps are as follows:
[0016] 1. Preparation of root tip cell slides: select healthy sugarcane stems and culture them at 28°C under constant temperature and humidity. A 2 cm root tip was excised and pretreated with 1,4-dichlorobenzene for 2 h. Then transfer to Carnoy's fixative solution and fix at 4°C for 24h. The fixed apical ddH 2 O was hypotonic for 0.5 h, and at 37°C, the fixed root tips were hydrolyzed in a mixed enzyme (4% Onozuka R10cellulose, 0.5% pectolyase Y-23 and 0.5% pectinase) for 3 h. Put the root tip into Carnoy's fixative solution for 0.5h and fix it on a clean glass slide to dry to obtain the root tip slide.
[0017] 2. Preparation of labeled probes: The reaction system for Digoxigenin labeled subtelomere sequences is shown in Table 1:
[0018] Table 1 Reaction system ...
Embodiment 2
[0027] Example 2. Mutual verification of subtelomeric sequences using PCR method to detect the blood relationship of Banmao
[0028] In order to further facilitate the detection process, we designed a pair of amplified P. chinensis specific primers 257F and 257R by using the subtelomeric sequence. Nucleotide sequence; 70 parts of plant material genomic DNA were extracted by CTAB method, including 5 parts of the original species of Banmao, tropical species of Saccharum, wild species with large stems, wild species with thin stems, Chinese species, Indian species and cultivated sugarcane species (such as Shown in Table 3), the hybrid progeny of Banmao and sugarcane F 1 、BC 1 、BC 2 、BC 3 A total of 35 materials (as shown in Table 4), OD 260 / OD 280 The quality of the DNA whose ratio is between 1.6 and 1.8 meets the requirements of the experiment, and the concentration is determined and diluted to 50ng / uL for aliquots as template DNA.
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