Development and application of tenfold elytrigia elongate blue-grained character special molecular marker and fluorescence in situ hybridization probe

A technology of molecular markers and in situ hybridization, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the lagging, time-consuming, and low efficiency of the development of molecular markers in E. elongatum. question

Active Publication Date: 2018-08-14
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

However, with the rapid development of molecular biology, the development of molecular markers of E. elongatum is still relatively lagging behind, and the sources of markers are mainly concentrated in diploid species.
Due to the large genome of the decaploid E. elongatum, the high homology between the genome and the wheat genome, and the lack of complete genome sequence information, the development and research of molecular markers of the decaploid E. elongatum are time-consuming, costly and difficult. The disadvantages such as low efficiency have seriously hindered the development and application of genetic resources of Echinopsis elongatum

Method used

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  • Development and application of tenfold elytrigia elongate blue-grained character special molecular marker and fluorescence in situ hybridization probe
  • Development and application of tenfold elytrigia elongate blue-grained character special molecular marker and fluorescence in situ hybridization probe
  • Development and application of tenfold elytrigia elongate blue-grained character special molecular marker and fluorescence in situ hybridization probe

Examples

Experimental program
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Effect test

Embodiment 1、 10

[0056] Example 1. Development of specialized molecular markers for the traits of blue-grained decaploid E. elongatum

[0057] 1. Genomic DNA extraction and purification

[0058] Genomic DNA of common wheat Chinese spring, decaploid Etyophony elongatum and Blue 58 were extracted by CTAB method, and the specific operation steps were as follows:

[0059] (1) Take an appropriate amount of tender tissue, freeze it in liquid nitrogen, quickly grind it into powder and put it into a 2mL centrifuge tube.

[0060] (2) Add 800 μL of CTAB extract preheated at 65°C, shake vigorously, place in a water bath at 65°C for 40-60 minutes, and shake gently occasionally.

[0061] (3) Take out the centrifuge tubes, add 800 μL of chloroform:isoamyl alcohol (24:1) to each tube, mix well by inverting up and down, centrifuge at room temperature, 12000 rpm / min for 15 min.

[0062] (4) Transfer about 600 μL of the supernatant to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix by in...

Embodiment 2

[0081] Example 2, Development of Specific Probes for Chromosomal Tandem Repeats of Decaploid Echinopsis elongatum 4Ag

[0082] Using the decaploid Thiopyrum elongatum (Th. ponticum, 2n=10x=70) as a template, the molecular marker 4Ag-19 obtained in Example 1 was used for PCR amplification to obtain a PCR product. The obtained PCR products were purified and labeled as FISH probes, and in situ hybridization was carried out on the metaphase of mitotic somatic cells of blue 58 and blue granule translocation lines L5, L9 and L13. At the same time, GISH analysis using the whole genome DNA of Decaploid Echinopsis elongatum as a probe was used as a control, and the steps of GISH analysis using the whole genome DNA of Decaploid Echinacea elongatum as a probe refer to the literature "Zheng Q., Luo Q .,Niu Z.,Li H.,Li B.,Xu S.S.,Han F.,Li Z.,Variation in Chromosome Constitution of the XiaoyanSeries Partial Amphiploids and Its Relationship to Stripe Rust and Stem RustResistance.J Genet Gen...

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Abstract

The invention discloses development and application of a tenfold elytrigia elongata blue-grained character special molecular marker and a fluorescence in situ hybridization probe. A specificity locusamplified fragment sequencing technique is utilized for sequencing common wheat, blue-grained wheat (blue 58) and elytrigia elongata; an elytrigia elongata 4Ag chromosome specific DNA sequence is obtained through sequence data comparative analysis; on the basis of the elytrigia elongate 4Ag chromosome specific DNA sequence, the elytrigia elongate 4Ag chromosome-derived blue-grained character special molecular marker and the special fluorescence probe are developed. The molecular marker and the probe provided by the invention can be used for identifying a blue-grained phenotype during a processof transferring a chromosome segment to the wheat from elytrigia elongata, so that not only a foundation is laid for positioning and cloning a blue aleurone gene, but also a basis is provided for marker-assisted selection of a blue-grained character.

Description

technical field [0001] The invention belongs to the field of crop genetics and breeding, and specifically relates to the development and application of a specialized molecular marker and a fluorescence in situ hybridization probe for the traits of the decaploid Etyophora elongatum blue grain. Background technique [0002] Thinopyrum ponticum (Thinopyrum ponticum, 2n=10x=70) is a perennial herb belonging to the genus Thinopyrum ponticum in the Poaceae family, and is an important wild relative of wheat. It has excellent characters such as long spikes and multiple flowers, wide adaptability, and strong stress tolerance. It contains many excellent genes that common wheat does not have (Shannon, 1978; Cox, 1991), and it is easy to hybridize with wheat. There is a valuable resource bank of wheat varieties. Through distant hybridization and chromosome engineering, Li Zhensheng et al. selected blue-grained wheat (Lan 58) from the hybrid progeny of common wheat and Echinopsis elonga...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6895C12Q1/686C12Q1/6841
CPCC12Q1/6841C12Q1/686C12Q1/6895C12Q2600/13C12Q2563/107C12Q2543/10
Inventor 郑琪刘利勤张静罗巧玲李宏伟李滨李振声
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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