Long rachis elytrigia repens 7E chromosome specific molecular marker based on SLAF-seq development and application thereof

A technology of E. elongatum and SLAF27230, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems that E. elongatum has not been studied, and achieve short cycle time, good repeatability, Amplify the effect of stabilization

Inactive Publication Date: 2013-01-23
YANGZHOU UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

At present, ISSR markers have been widely used in the identification of various plant varieties, gene

Method used

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  • Long rachis elytrigia repens 7E chromosome specific molecular marker based on SLAF-seq development and application thereof
  • Long rachis elytrigia repens 7E chromosome specific molecular marker based on SLAF-seq development and application thereof
  • Long rachis elytrigia repens 7E chromosome specific molecular marker based on SLAF-seq development and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1 Experimental material

[0092] Common wheat Chinese spring (CS, 2n=6x=42), diploid Echinopsis elongatum ( Th. elongatum ,EE,2n=2x=14), Chinese spring-Thinopyrum elongata addition line (CS- Th. elongatum addition lines), Chinese spring-Elongated wheatgrass replacement line (CS- Th. elongatum substitutions lines), tetraploid Etynotrum elongatum ( Th. elongatum ,2n=4x=28), Decaploid Echinopsis elongatum ( Th.ponticum , PI179162, PI204383, 2n=10x=70), the above materials were donated by Dr. Fedak of the Canadian Department of Agriculture, and the original document of the materials is "Disomic and ditelosomic additions of diploid Agropyron elongatum chromosomes to Triticum aestivum "; Yangmai 14 (Y14, that is, Yang 0-139, a hybrid of Yangmai 158 and Yangmai 6 used by Jiangsu Lixiahe Agricultural Science Institute), Yangmai 16 (Y16, that is, Yang 0-126 is a Yang 91F138 and Yang 90-30 used by the Institute of Agricultural Sciences were bred by hybrid...

Embodiment 2

[0093] Example 2 Extraction of Genomic DNA

[0094] The test material grows to the stage of two leaves and one heart, and the genomic DNA is extracted by the SDS method. The steps are: (1) Take the young leaves (about 0.1g), cut them into pieces and put them in a 2ml centrifuge tube, place them in liquid nitrogen to cool, Grind to powder with a grinding rod;

[0095] (2) Place the centrifuge tube at room temperature to cool slightly, add 700 μl of buffer A (buffer A formula: 29.2g NaCl, 100ml of 1M Tris-HCl, 18.6g EDTA-Na, 15g SDS, ddH 2 O (dilute to 1L and sterilize), mix gently, then bathe in water at 65°C for 20 minutes, and mix by inverting up and down every 5 minutes during this period;

[0096] (3) Take it out and cool it down to room temperature, add 350 μl of phenol and chloroform each, turn it upside down, mix well, and extract for 5 minutes;

[0097] (4) 12000rpm, centrifuge for 10min, draw the supernatant into a new centrifuge tube;

[0098] (5) Add about 750 μl ...

Embodiment 3

[0103] Example 3 Obtaining a Specific Fragment of Chromosome 7E of Echinopsis elongatum Based on SLAF-seq Technology

[0104] (1) Simplified protocol design: use enzyme digestion prediction software to analyze the reference genome system, design a marker development protocol based on the GC content of the genome, repeat sequence conditions, and gene characteristics, and determine the enzyme digestion protocol, gel cutting range, and sequencing volume etc. to ensure the density and uniformity of its molecular marker development so as to ensure that the expected experimental purpose is achieved.

[0105] (2) Enzyme digestion of genomic DNA: Select the target fragment by enzyme digestion to meet the expected design index requirements for the number of tags. The enzyme digestion system is 500ng of genomic DNA (genomic DNA of Chinese Spring, Echinopsis elongatum, and Chinese Spring-Ethiopia elongata 7E addition line), 1 μl of NEB buffer4 (buffer 4 from New England Biolabs), Mse 1...

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Abstract

The invention belongs to the field of crop genetic breeding, and particularly relates to a long rachis elytrigia repens 7E chromosome specific molecular marker based on SLAF-seq development. 10 long rachis elytrigia repens 7E chromosome specific molecular markers are developed by using a specificity length amplification segment sequencing technology developed based on a new generation of sequencing technology and bioinformatics principle, sequencing the specific segment of ordinary wheat and ordinary wheat-long rachis elytrigia repens additional system to obtain magnanimity ordinary wheat and long rachis elytrigia repens sequence and carrying out sequence data comparison analysis by using a computer software to obtain a magnanimity long rachis elytrigia repens 7E chromosome specific segment sequence. The markers can not only be used for detecting the long rachis elytrigia repens 7E chromosome, but also can be used for auxiliary selection of molecular marking in wheat resistance breeding. Besides, the success in developing the long rachis elytrigia repens chromosome specific molecular marker based on the SLAF-seq technology provides reference for the molecular marker development of other species, and provides an important application basis for molecular breeding, system evolution and germplasm resource identification.

Description

technical field [0001] The present invention belongs to the field of crop genetics and breeding, specifically based on the specific length amplified fragment sequencing technology (Specific Length Amplified Fragment Sequencing, SLAF-seq) developed based on the principle of next-generation sequencing technology and bioinformatics, for common wheat and common wheat- Sequencing the specific fragments of the addition line of E. elongatum to obtain a large number of common wheat and E. elongatum sequences. Using computer software for sequence data comparison analysis, a large number of specific fragment sequences of E. elongatum 7E chromosomes were obtained, thereby developing a large number of long Chromosome-specific molecular markers for the 7E E. tripyrum. These markers can not only be used for the detection of Chromosome 7E in E. elongatum, but also provide an important theoretical and marker basis for molecular marker-assisted selection in wheat resistance breeding. In addit...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 陈建民陈士强高勇黄泽峰秦树文高营营
Owner YANGZHOU UNIV
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