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Preparation method and application of hawthorn EST-SSR (Expression Sequence Tag-Simple Sequence Repeats) marker primer

A technology for labeling primers and hawthorn, which is applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of backwardness and less research on hawthorn genome background, and achieves the promotion of research level and improvement of research level. Effect

Inactive Publication Date: 2018-07-27
SHENYANG AGRI UNIV
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Problems solved by technology

At present, the research on the genetic basis of hawthorn resources is relatively backward, and morphological analysis is still the main method for resource evaluation and identification, and there are few reports on molecular biology research on hawthorn, and there are few studies on the background of hawthorn genome. However, there is no report on the development of EST-SSR primers using the hawthorn transcriptome sequence.

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  • Preparation method and application of hawthorn EST-SSR (Expression Sequence Tag-Simple Sequence Repeats) marker primer
  • Preparation method and application of hawthorn EST-SSR (Expression Sequence Tag-Simple Sequence Repeats) marker primer
  • Preparation method and application of hawthorn EST-SSR (Expression Sequence Tag-Simple Sequence Repeats) marker primer

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preparation example Construction

[0024] See Figure 1-6 , A preparation method of hawthorn EST-SSR marker primer, including the following steps:

[0025] 1) Screen the fragment sequences with assembly length greater than 5000bp from the hawthorn transcriptome data for the subsequent development of hawthorn EST-SSR markers;

[0026] 2) Use SSRHunter software to perform SSR discovery on each fragment, and pick out EST sequences that are rich in two, three, four or pentanucleotide repeats and length ≥ 10bp from a single sequence;

[0027] 3) Use primer3 software to screen the EST-SSR primers for the 150bp upstream and downstream base sequences of the SSR to obtain the hawthorn EST-SSR marker primers. The length of the primer is 18-23bp, the annealing temperature is 55-63°C, the GC content is 40-60%, and the length of the PCR amplified product is 150-300bp. Preferably, the optimal primer length is 20 bp, the annealing temperature is 58° C., the GC content is 50%, and the PCR amplification product length is 200 bp.

[00...

Embodiment 1

[0031] 1. Test materials: use the hawthorn variety'Da Mianqiu' as the female parent and'Autumn Jinxing' as the male parent to create a hybrid population, select 92 hybrid F1 generations as the mapping population, and construct a hawthorn molecular genetic map using SSR molecular marker technology;

[0032] 2. Select hawthorn varieties and the tender green leaves of hybrid plants to extract total DNA. Genomic DNA extraction is based on the improved CTAB method proposed by Ma Ming et al. (2007);

[0033] 3. Using the hawthorn EST-SSR marker primers of the present invention for analysis, the specific steps are as follows:

[0034] 3.1. EST-SSR screening and amplification;

[0035] 3.1.1. PCR reaction system: The SSR-PCR reaction system in this experiment is 16μL, and the components are as follows: 10×Buffer (containing Mg 2+ ) 1.6μL, dNTP 0.8μL (concentration 2.5mM / μL), primer 2*0.8μL (concentration 5nmol / μL), Taq polymerase 1μL (1U / μL), template DNA 1μL (20ng / μL), ddH2O 10μL;

[0036] 3.1...

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Abstract

The invention discloses a preparation method of a hawthorn EST-SSR (Expression Sequence Tag-Simple Sequence Repeats) marker primer. The preparation method comprises the following steps: 1) screening and assembling segment sequences with the length greater than 5000bp from hawthorn transcriptome data; 2) carrying out SSR digging on each segment by utilizing SSR Hunter software; picking out EST sequences which contain rich dinucleotide, trinucleotide, tetranucleotide or pentanucleotide repeats and has the length greater than or equal to 10bp from a single sequence; 3) screening an EST-SSR primeron 150bp base sequences at upstream and downstream of SSR by utilizing primer 3 software, so as to obtain the hawthorn EST-SSR marker primer. The invention provides the preparation method and application of the hawthorn EST-SSR marker primer, reference is provided for related researches of hawthorns in the future and a basic research level of the hawthorns is promoted and improved.

Description

Technical field [0001] The invention relates to the technical field of molecular marker preparation and application, in particular to a preparation method and application of a hawthorn EST-SSR marker primer. Background technique [0002] Crataegus is a plant of the genus Crataegus L. of Rosa-ceae. There are 18 species and 6 varieties of Crataegus native to China. After long-term cultivation and selection, it has formed a rich variety of types. Accurate identification and classification of hawthorn resources is the basis for further effective use of hawthorn resources. At present, the research on the genetic basis of hawthorn resources is relatively backward. So far, the morphological analysis method is still the main method for resource evaluation and identification. There are also fewer reports on molecular biology research on hawthorn. The related information of related species is referred to, and there is no report on the use of hawthorn transcriptome sequence to develop EST-...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6811C12N15/11
CPCC12Q1/6811C12Q1/6895C12Q2600/156
Inventor 赵玉辉刘诗佳郭印山刘丽婷
Owner SHENYANG AGRI UNIV
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