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SLAF-seq-based developed elytrigia elongata 1E chromosome specific molecular markers and application thereof

A long-spiked wheatgrass, specific marker technology, applied in application, plant genetic improvement, recombinant DNA technology and other directions, can solve problems such as no research on long-spiked wheatgrass, and achieve short cycle, good repeatability, and stable amplification. Effect

Inactive Publication Date: 2013-02-13
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, ISSR markers have been widely used in the identification of various plant varieties, genetic mapping, gene mapping, genetic diversity, etc., but have not been studied in Echinopsis elongatum.

Method used

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  • SLAF-seq-based developed elytrigia elongata 1E chromosome specific molecular markers and application thereof
  • SLAF-seq-based developed elytrigia elongata 1E chromosome specific molecular markers and application thereof
  • SLAF-seq-based developed elytrigia elongata 1E chromosome specific molecular markers and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 Experimental material

[0046] Common wheat China spring (CS, 2n=6x=42), diploid Th. elongatum (Th. elongatum, EE, 2n=2x=14), China spring-Thn. elongatum addition line (DA lines), China Spring-Elongatum replacement lines (DS lines), Langdon (LD), Langdon-Elongatum diploid (8801), tetraploid Elongatum (Th.elongatum, 2n=4x=28) , Decaploid Elongated wheatgrass (Th.ponticum, PI179162, PI204383, 2n=10x=70), the above materials were donated by Dr.George Fedak of the Canadian Department of Agriculture. The original document of the materials is "Disomic and ditelosomic additions of diploidAgropyron elongatum chromosomes to Triticum aestivum", etc.; Yangmai 10 (Y10, formerly known as 93-111, bred by the Institute of Agricultural Sciences in Lixiahe, Jiangsu and Nanjing Agricultural University), Yangmai 14 (Y14, namely Yang 0-139, Jiangsu Li Yangmai 158 and Yangmai 6 used by Xiahe Agricultural Science Institute), Yangmai 16 (Y16, that is, Yang 0-126 is a hybrid of Y...

Embodiment 2

[0047] The extraction of embodiment 2 genome DNA

[0048] 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;

[0049] (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;

[0050] (3) Take it out and cool it down to room temperature, add 350l each of phenol and chloroform, turn it upside down, mix well, and extract for 5 minutes;

[0051] (4) 12000rpm, centrifuge for 10min, draw the supernatant into a new centrifuge tube;

[0052] (5) Add about 750...

Embodiment 3

[0057] Example 3 Obtaining a Specific Fragment of Chromosome 1E of Echinopsis elongatum Based on SLAF-seq Technology

[0058] (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.

[0059] (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, Ethiopyrum elongatum, and Chinese spring-Ethiopyrum elongatum 1E addition line), 1 μl of NEB buffer4 (buffer 4 from New England Biolabs), 0.12...

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Abstract

The invention belongs to the field of crop genetic improvement, comprising the following steps of: sequencing the specific segments of common wheat and a common wheat-elytrigia elongata addition line to obtain a mass of common wheat and elytrigia elongata sequences, carrying out comparative analysis on the sequence data by computer software to obtain a mass of elytrigia elongata 1E chromosome specific segmental sequences, randomly selecting 8 sequences, and successfully developing 5 elytrigia elongata 1E chromosome specific molecular markers such as one of SED ID NOs.1, 4, 7, 10 and 13. The markers not only can be used for detecting the elytrigia elongata 1E chromosome, but also can be used for the assistant selection of the molecular markers in the wheat resistance breeding. Furthermore, the SLAF-seq technology-based successfully developed elytrigia elongata chromosome specific molecular markers can provide an important reference for the molecular marker development technologies of the other species, and provide an important application basis for the molecular breeding, the system evolution and the 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, and using computer software for sequence data comparison and analysis, a large number of specific fragment sequences of E. elongatum 1E chromosome were obtained, thereby developing a large number of long Chromosome 1E specific molecular markers in Etienia tachyphylla. These markers can not only be used to detect the 1E chromosome of E. elongatum, but also provide 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68A01H1/04
Inventor 陈建民陈士强高勇黄泽峰秦树文戴毅高营营
Owner YANGZHOU UNIV
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