A method for identification of bloodlines containing Pseudomonas in sugarcane using subtelomeric

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 good sensitivity and stability.

Inactive Publication Date: 2021-03-26
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, the specific sequences of the P. chinensis genome that they isolated did not cover all P. chinensis chromosomes. These molecular markers can only be used to identify the authenticity of the early hybrid offspring of sugarcane and P. spp. Authenticity of high-generation hybrids

Method used

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  • A method for identification of bloodlines containing Pseudomonas in sugarcane using subtelomeric
  • A method for identification of bloodlines containing Pseudomonas in sugarcane using subtelomeric
  • A method for identification of bloodlines containing Pseudomonas in sugarcane using subtelomeric

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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 syste...

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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Abstract

The invention relates to a method for identifying erianthus arundinaceus consanguinity in sugarcane by a sub telomere sequence. The method comprises the following steps: preparing a root-tip cell slide, preparing a label probe, pre-treating the root-tip slide, preparing a denatured hybrid mixture, and hybridizing the denatured hybrid mixture and the pretreated root-tip slide. If a green fluorescence signal is generated on the hybrid slide, judging that a to-be-detected sample comprises erianthus arundinaceus consanguinity which is a real filial generation of sugarcane and erianthus arundinaceus. A nucleotide sequence of the generated green fluorescence signal probe is a nucleotide sequence as shown in SEQ ID NO: 1 in a sequence table. The method integrates a fluorescent in-situ hybridization technique, utilizes a special sub telomere sequence probe, relative to the sugarcane, of erianthus arundinaceus to identify the real filial generation of sugarcane and erianthus arundinaceus, has the characteristics of being high in efficiency, good in sensitivity, good in stability and high in reliability, can detect reality of relatively high filial generation by only needing to obtain root-tips of sugarcane, and provides a stable and effective identifying method for hybrid utilization of erianthus arundinaceus in a sugarcane genetic breeding improvement process.

Description

technical field [0001] The invention relates to a method for identifying bloodlines of sagegrass in sugarcane, in particular to a method for identifying bloodlines of sagegrass in sugarcane by using subtelomeric sequences, and belongs to the technical field of plant detection and identification. Background technique [0002] Wild sugarcane species have rich genetic diversity and contain a large number of excellent traits that can be used in sugarcane genetics and breeding. Banmao is one of the closely related wild species of sugarcane genetic improvement and breeding. Sugarcane breeders hope to introduce the wild species of Banmao and other closely related genera into the sugarcane background through the method of distant hybridization to broaden the genetic basis of sugarcane . Through the unremitting efforts of sugarcane breeders, a large number of hybrid offspring of sugarcane and Banmao have been obtained. At present, the authenticity identification methods of the hybr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6841C12Q1/6804C12N15/11
CPCC12Q1/6804C12Q1/6841C12Q1/6895C12Q2563/107C12Q2563/131
Inventor 邓祖湖黄永吉徐良年余凡杨善李雪停
Owner FUJIAN AGRI & FORESTRY UNIV
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