A method for identifying the blood relationship of zebra in sugarcane by using dispersal sequence

A technology of in-sequence list and sequence, applied in the field of plant detection and identification, can solve the problems such as inability to know and accurately detect the chromosomes or fragments of P. High, high efficiency and good stability

Inactive Publication Date: 2021-03-02
FUJIAN AGRI & FORESTRY UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Zhuang Nansheng et al. and Wang Ying et al. successively used AFLP marker technology to screen out 3 specific DNA sequences of P. chinensis, and developed them into 3 specific molecular markers of P. chinensis. However, none of them were tested for stability, and it is impossible to know that these markers can be used for Accurate detection of sarcophagus chromosomes or fragments in high-generation clones
In addition, D'hont et al. designed a pair of primers on the 5S rDNA conserved sequence of sugarcane and Banmao by taking advantage of the different lengths of the 5S rDNA non-transcribed spacer NTS sequences between sugarcane and Banmao. According to the electrophoresis detection strip The difference in band size accurately identifies the F 1 However, the 5S rDNA sequence only exists on a certain chromosome of Banmao, which cannot stably and effectively detect the hybrid offspring that lost this chromosome

Method used

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  • A method for identifying the blood relationship of zebra in sugarcane by using dispersal sequence
  • A method for identifying the blood relationship of zebra in sugarcane by using dispersal sequence
  • A method for identifying the blood relationship of zebra in sugarcane by using dispersal sequence

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Experimental program
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Embodiment 1

[0015] Embodiment 1, a kind of method that utilizes interspersed sequence identification to comprise the blood lineage of sagegrass in sugarcane, it is characterized in that identification step is 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 interspersed sequences is shown in Table 1:

[0018] Table 1 Reaction system...

Embodiment 2

[0027] Example 2, using the PCR method to mutually confirm the interspersed sequence to detect the ancestry of Banmao

[0028] In order to further simplify the detection process, we designed and screened a pair of amplification primers 128F and 128R with reference to the specific scattered sequence of P. chinensis, the nucleotide sequences of which are shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence list 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 varieties of sugarcane ( 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 concen...

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Abstract

The invention relates to a method for identifying consanguinity of saccharum arundinaceum in sugarcane via a spread sequence. The method comprises steps of preparing a root-tip cell slide, preparing alabeled probe, pre-processing the root-tip slide, preparing a denaturing hybrid mixed solution and hybridizing the denaturing hybrid mixed solution and the pre-processed root-tip slide. A to-be-detected sample is judged that the sample contains the consanguinity of saccharum arundinaceum and is a real hybrid later generation of sugarcane and saccharum arundinaceum when a green fluorescence signalis generated on the hybrid slide. The nucleotide sequence of a probe which generates the green fluorescence signal is shown as SEQ ID NO:1 in a sequence list. The method provided by the invention, through the combination with fluorescence in-situ hybridization technology and by identifying the real hybrid later generation of sugarcane and saccharum arundinaceum through the spread sequence probe that saccharum arundinaceum is specific to sugarcane, has the characteristics of being high in efficiency, good in sensitivity, good in stability and high in reliability; and the reality of any hybridgenerations can be detected just by acquiring root tips of sugarcane seedlings, so that an efficient and stable identifying method is provided for hybridization utilization of the saccharum arundinaceum in a sugarcane genetic breeding improving process.

Description

technical field [0001] The invention relates to a method for identifying the lineage of sagegrass in sugarcane, in particular to a method for identifying the lineage of sagegrass in sugarcane by using scattered sequences, and belongs to the technical field of plant detection and identification. Background technique [0002] Sugarcane belongs to annual or perennial tropical and subtropical C 4 Crops, primarily used to make sugar and as a clean energy substitute. Modern sugarcane cultivars are mainly derived from hybrids of a few tropical species and wild species with thin stems, which limits the improvement of sugarcane quality. In order to broaden the genetic basis of sugarcane, sugarcane breeders have introduced wild species of closely related genera such as Pseudomonas spp. into the background of sugarcane by means of distant hybridization, and have obtained a large number of hybrid offspring of sugarcane and Pseudomonas spp. At present, the authenticity identification m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6841
CPCC12Q1/6841C12Q2563/107C12Q2565/518
Inventor 邓祖湖余凡徐良年黄永吉杨善罗玲
Owner FUJIAN AGRI & FORESTRY UNIV
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