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Method for building celery EST-SSR (expressed sequence tags-simple sequence repeat) fingerprint and application thereof

A technology of fingerprints and construction methods, which is applied in the field of vegetable molecular assisted breeding and application in agriculture, and can solve problems such as fingerprints that have not been reported in literature

Inactive Publication Date: 2012-10-10
TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no literature report on the construction of the EST-SSR fingerprint of marked celery

Method used

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  • Method for building celery EST-SSR (expressed sequence tags-simple sequence repeat) fingerprint and application thereof
  • Method for building celery EST-SSR (expressed sequence tags-simple sequence repeat) fingerprint and application thereof
  • Method for building celery EST-SSR (expressed sequence tags-simple sequence repeat) fingerprint and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Searching for Candidate EST-SSR Sites in Celery

[0056] (1) Database: GenBank EST database

[0057] (2) Analysis software: BatchPrimer3 v1.0 online software

[0058] (3) Query celery EST sequences published by GenBank (http: / / www.ncbi.nlm.nih.gov / nucest / term=celery) and other databases with the keyword celery, and get 1122 EST sequences;

[0059] Using the SSR screening and primers function of BatchPrimer3 v1.0 online software (http: / / probes.pw.usda.gov / cgi-bin / batchprimer3 / batchprimer3.cgiPRIMER_TYPE=2&CLEAR_FORM=no&LOAD_EXAMPLE=no), 1122 celery EST sequences were analyzed According to the analysis, 194 candidate EST-SSR sites were obtained, and a pair of EST-SSR primers were designed based on each site.

Embodiment 2

[0061] Screening and Validation of Candidate EST-SSR Loci in Celery

[0062] (1) Reagents: GoTaq® Master Mix solution produced by Promega; EST-SSR primers synthesized by Shanghai Sangon;

[0063] (2) EST-SSR primer sequences are shown in Table 1;

[0064] Table 1 Eight celery EST-SSR loci and their PCR amplification primers

[0065]

[0066] (3) Experimental materials: 11 celery genome DNA templates;

[0067] (4) Taking 11 celery genomic DNA templates (50±1 ng / μL) stored in the laboratory as the research object, 47 pairs of primers were selected for PCR amplification and polyacrylamide gel electrophoresis, and the polymorphism was screened out. 8 EST-SSR sites (Table 1, figure 1 );

[0068] The total volume of the PCR amplification reaction was 10 μL, and the amplification reaction system was: 2 × GoTaq? Master Mixes 5 μL, 10 μmol / L upstream and downstream primers 0.25 μL each; Make up 10 μL with double distilled water; PCR reaction program is 95°C pre-denaturation for...

Embodiment 3

[0071] Analysis of EST-SSR loci and construction of fingerprints in celery

[0072] (1) Gel image analysis software: GeneTools (Sygene) software;

[0073] (2) Fingerprint construction software: EXCEL software;

[0074] After polyacrylamide gel electrophoresis staining, put the gel into the gel imaging system to take pictures, import the pictures into GeneTools (Sygene) software, define the molecular weight marker, and perform band molecular weight analysis on the target band according to the molecular weight marker;

[0075] Construct the EST-SSR fingerprint (Table 2) based on the EXCEL data format, record the molecular weight of the target band, and mark the place where there is a band as × or 1, and the place where there is no band is not recorded or recorded as 0, see Table 2 for details:

[0076] Table 2 Fingerprints of 8 EST-SSR loci of 11 celery breeding materials:

[0077]

[0078]

[0079]

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Abstract

The invention discloses a method for building a celery EST-SSR (expressed sequence tags-simple sequence repeat) fingerprint and application of the fingerprint. The method comprises the steps of: by taking a genome DNA (deoxyribonucleic acid) template of celery breeding material as a research object, designing an EST-SSR primer by utilizing BatchPrimer3v1.0 online primer design software by enquiring a celery EST (simple sequence repeat) sequence issued on a GenBank database; screening out an EST-SSR site with strong polymorphism by PCR (polymerase chain reaction) amplification and polyacrylamide gel electrophoresis, carrying out band molecular weight analysis by Genetools (Sygene) software, and building the EST-SSR fingerprint. The EST-SSR fingerprint can be applied to analysis such as identification of genetic relationship of celery varieties and varieties and the like.

Description

technical field [0001] The invention belongs to the technical field of agricultural vegetable molecular assisted breeding and application, in particular to a method for constructing celery EST-SSR fingerprints and its application, and is an analysis based on EST-SSR molecular marker technology. Background technique [0002] Celery belongs to Umbelliferae. There are two kinds of cress and celery, which have similar functions, and celery is the best medicinal one. Celery has a strong aroma, also known as "parsley", also known as "medicine celery". Celery is rich in protein, carotene, carbohydrates, fat, B vitamins, vitamin C, sugars, amino acids, minerals and dietary fiber, among which phosphorus and calcium are high in content. Celery is a high-fiber food. It produces a lignin or intestinal fat substance through intestinal digestion. This kind of substance is an antioxidant. Often eating celery, especially celery leaves, is beneficial to the prevention of high blood pressur...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 兰青阔王永赵新陈锐朱珠李欧静徐石勇郭永泽
Owner TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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