Flax SSR molecular marker and application thereof

A molecular marker and molecular marker-assisted technology, which is applied in the direction of DNA/RNA fragments, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems of SSR molecular marker development that have not been reported, and achieve stable amplification and band clear effect

Inactive Publication Date: 2019-03-26
INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are no relevant reports on the search for SSR loci ...

Method used

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  • Flax SSR molecular marker and application thereof
  • Flax SSR molecular marker and application thereof
  • Flax SSR molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 The acquisition of flax genome-wide SSR loci

[0114] First, NCBI downloaded the whole genome sequence information of flax

[0115] ( https: / / www.ncbi.nlm.nih.gov / assembly / GCA_000224295.2 ), further use MISA software (MIcroSAtellite identification tool)

[0116] (http: / / pgrc.ipk-gatersleben.de / misa / ) Detection of microsatellite and composite microsatellite loci in the flax genome sequence. The detection parameters adopted are 2-6, 3-5, 4-5, 5-5, 6-5 (the number of repetitions of the 2-base repeating unit motif>=6 times; the number of repetitions of the 3-base repeating unit>=5 ; and so on.); when the distance between two microsatellites was less than 100bp, a composite microsatellite was formed, and 28571 SSR loci were detected in the whole flax genome.

Embodiment 2

[0117] Embodiment 2 flax SSR primer design and synthesis

[0118] According to the SSR detection results of MISA, using Primer3

[0119] (http: / / pgrc.ipk-gatersleben.de / misa / primer3.html) software default parameters (length 100bp-300bp; Tm value 55°C-60°C, primer length 20-25bp) for primer design, try to avoid primer two Polymer (dimer), hairpin structure (hairp-in), mismatch (false primer), etc. In the design results, the 3 pairs of primers with the highest scores were selected for subsequent experiments, and a total of 71184 pairs of SSR primers were obtained. Then 96 pairs of primers were randomly selected and synthesized by Hunan Qingke Biological Company.

Embodiment 3

[0120] The amplification of embodiment 3 SSR primers and the genetic diversity analysis of 26 flax germplasm resources

[0121] The TPS method was used to extract 26 flax germplasms (see Table 1), and the specific method can be referred to (Zhang Youchang et al., 2016). Then use DNA to measure the DNA concentration of each germplasm, and finally dilute to the working solution 30ng / μL. The SSR reaction system was 10 μL, which included 50 μM dNTPs, 0.2 μM primers, 0.5U Taq polymerase (TaKaRa, Dalian) and 30ng of DNA template. The PCR reaction was carried out in a LongGeneA200 gene thermal cycler. The overall reaction system includes: PCR reaction is carried out with rTaq DNA polymerase from Takara Company, and the reaction system is as follows (10 μL): DNA template (10ng / μL) 2.0 μL, primer (2 pmol / μL) 1 μL, 10×Buffer 1.0 μL, dNTP ( 1 mM) 0.3 μL, rTaq (5U / μL) 0.1 μL, ddH2O 5.4 μL. The PCR amplification program includes: specific procedures are as follows: pre-denaturation at 9...

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Abstract

The invention relates to the technical field of plant variety identification and breeding, in particular to a building method of flax SSR fingerprint chromatography. According to the method, through MISA software, a flax genome is searched for to obtain 28751 SSR sites, Primer 5.0 is used for designing 71184 pairs of primers, and 26 flax genetic resources are used for screening out the 11 pairs ofSSR markers rich in polymorphism: RM4-5, RM4-7, RM6-2, RM6-6, RM6-7, RM7-1, RM7-3, RM8-1, RM9-4, RM13-5 and RM15-1. The SSR primers which are developed based on the flax genome have the advantages ofbeing stable in amplification, clear in electrophoretic band, rich in polymorphism and the like, and the building method of the flax SSR fingerprint chromatography can be effectively used for the study fields of flax genetic resource genetic diversity analysis, high-density chromatography building, variety purity and authenticity identification, molecular marker-assisted breeding and the like.

Description

technical field [0001] The invention relates to the technical field of plant variety identification and breeding, in particular to flax SSR molecular markers and applications thereof. Background technique [0002] Flax (Linum usitatissimum L.) is an important economic crop, which is widely planted in Northwest and Northeast China. The fiber produced from flax stems is an important raw material for the textile industry. The oil content of flax seeds is 30%-45%. The further development of the flax industry was limited by the lack of high-quality fiber and flaxseed. Previous studies have shown that molecular breeding is one of the important means to improve crop quality, and the development of molecular markers is one of the important steps in molecular breeding. Although some molecular markers based on PCR fragments such as RAPD, AFLP, and ISSR have been widely used in flax research, these markers are generally time-consuming and low in polymorphism (Yurenkova et al., 2005; ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 潘根张义李德芳赵立宁陈安国李建军黄思齐唐慧娟常丽
Owner INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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