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Method for differentiating indica japonica of weedy rice through two repeated DNA (Deoxyribonucleic Acid) sequences

A technology of DNA repetition, rice indica and japonica, applied in the field of identification of weedy rice indica and japonica classification, achieving high reliability, good repeatability, and simple rootlets

Active Publication Date: 2013-04-17
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using DNA-specific repeat sequences to identify the indica and japonica characters of weedy rice with molecular cytology methods has not been reported yet.

Method used

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  • Method for differentiating indica japonica of weedy rice through two repeated DNA (Deoxyribonucleic Acid) sequences

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Prepare Hybridization Solution Containing Probes

[0035] a) The 45S rDNA probe is obtained as follows: The pTA71 clone used contains the wheat 45S rDNA gene sequence, and its plasmid DNA is extracted by QIAGEN plasma miniprep kit. The nick translation method is used to label the probe. The 45S rDNA probe labeling method is as follows: put 2ug plasmid DNA in a 1.5ml Eppendorf tube, add 10×Buffer (0.5M Tris HCl pH7.5, 50mM MgCl 2) 10ul, 0.5mM dNTP 10ul, 0.5mM biotin-dUTP 10ul, DNA polymerase I 2.4ul (8unit / ul), DNase I 4ul (0.1unit / ul), make up to 100ul with sterile ultrapure water, and mix well; After placed in a water bath at 15°C for 2 hours, 10ul of 0.2M EDTA was added to terminate the reaction; 3ul of the labeled probe solution was taken and detected by 1% agarose gel electrophoresis. The size of the labeled probe fragment was 100bp-500bp.

[0036] The Os48 probe was obtained as follows: pOs48 clone contains 355bp rice AA chromosome subtelomeric tandem repeat seque...

Embodiment 2

[0040] Specimen preparation of weedy rice to be identified

[0041] a) Identified weedy rice

[0042] 19 parts of weedy rice materials, including: 1 part of Feidong Tang rice from Anhui, 1 part of Huaiyuan Tang rice, 1 part of Lu rice from Jiangsu, 5 parts of Yangzhong weedy rice, 1 part of Chuzhou weedy rice, 1 part of Gaoyou weedy rice, 1 part of Yandu weedy rice, 1 part of Guanyun weedy rice, 2 parts of Rugao weedy rice, 1 part of Funing weedy rice, 2 parts of Jiangyan weedy rice, from Liaoning Weed rice 2 parts.

[0043] b) Producing and denaturing the identified weedy rice prometaphase chromosomes

[0044] The seeds of different weedy rice varieties were placed in petri dishes lined with two layers of filter paper, and an appropriate amount of distilled water was added to accelerate germination in a 25°C incubator. When the root grows to 1-2cm, cut the young root with scissors, or directly cut the young root of the weedy rice transplanted to the field, soak it in 2mM 8...

Embodiment 3

[0046] a) Fluorescence in situ hybridization

[0047] Add 22ul of the denatured hybridization solution provided in Example 1 to each denatured chromosome preparation obtained in Example 2, cover with a 22×40mm cover glass, and place it in a humid box at 37°C in an incubator for 8-10 hours . After hybridization, the cover slip was removed and washed three times with 1×PBS, 5 min each time.

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Abstract

The invention relates to a method for differentiating indica japonica of weedy rice through two repeated DNA (Deoxyribonucleic Acid) sequences. In the method, plasmid DNAs of cloning pTA71 containing wheat 45SrDNA gene sequences and cloning pOs48 containing repeated serial sequences of subtelocentric rice AA group chromosome are extracted, and 45SrDNA and Os48 probes are formed by using pOs48 labeled by using digoxin-dUTP (deoxyuridine triphosphate) and pTA71 labeled by using biotin-dUTP through a nick translation method to differentiate the indica japonica of the weedy rice. The method comprises the following steps of: flaking and denaturing a metaphase chromosome of the differentiated weedy rice before karyokinesis; carrying out two-color fluorescence in-situ hybridization between the metaphase chromosome and a denatured hybridization solution containing the probes; detecting a signal of the metaphase chromosome in the dark; and classifying the indica japonica of the weedy rice of different sources according to the locus number and distribution characteristics of the 45SrDNA and the Os48 on the metaphase chromosome of the weedy rice before the karyokinesis.

Description

technical field [0001] The invention uses two DNA-specific repeat sequences as probes respectively, adopts two-color fluorescence in situ hybridization technology to study the distribution of repeat sequences on the prometaphase chromosomes of weedy rice, and analyzes the indica and japonica characteristics of weedy rice, and relates to a weedy rice The invention discloses an identification method for the classification of indica and japonica, which belongs to the technical field of plant molecular cytology. Background technique [0002] Weedy rice (weedy rice) is a type of wild rice and cultivated rice produced by natural selection and human intervention, also known as weedy rice or weedy rice (Wang Weixia et al., Weedy rice Science, 2005(2):1-5). In terms of plant classification, weedy rice belongs to the Poaceae (Poaceae) genus (Oryza), because it belongs to the AA genotype (2n=24) like cultivated rice, and is morphologically similar to Asian cultivated rice (O. sativa L...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 杨学明姚金保马鸿翔周淼平任丽娟杨杰陈志德
Owner JIANGSU ACAD OF AGRI SCI
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