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EST-SSR (expressed sequence tag-simple sequence repeats) molecular markers for heat-resistant radish, primers of molecular markers and application thereof

A molecular marker, radish technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems that cannot meet the requirements of breeding or other fields of research, narrow genetic background, and phenotypic similarity of cultivars higher question

Inactive Publication Date: 2017-05-10
HUANGGANG NORMAL UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0004] Radish is an important vegetable crop in my country, but basic research such as molecular biology is relatively backward, which is far from meeting the needs of breeding or other fields of research. At the same time, radish germplasm resources are abundant, but the phenotype similarity of cultivated varieties is high, and the genetic background is narrow For example, Cui Na et al reported that the average number of alleles, the average number of effective alleles and the value of the gene diversity index of 75 radish materials are not high, specifically from Korean white radish varieties Chenxiao and Xuefenghuang, etc. The similarity coefficient between 15 pairs of radish varieties is 1.00, indicating that they may have the same genetic background
In view of this, it is difficult to distinguish between different varieties of radish. At present, there is no effective SSR molecular marker that can distinguish the purity of heat-tolerant radish varieties.

Method used

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  • EST-SSR (expressed sequence tag-simple sequence repeats) molecular markers for heat-resistant radish, primers of molecular markers and application thereof
  • EST-SSR (expressed sequence tag-simple sequence repeats) molecular markers for heat-resistant radish, primers of molecular markers and application thereof
  • EST-SSR (expressed sequence tag-simple sequence repeats) molecular markers for heat-resistant radish, primers of molecular markers and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] According to the EST sequence of radish published in NCBI, Primer3.0 primer design software was used to design SSR primers, and a pair of SSR primers from SEQ ID No.3 to SEQ ID No.62 was synthesized by Nanjing GenScript Company.

[0061] Extract heat-resistant radish-Xia Kang 40 days and the DNA of its parents, specifically including the following steps:

[0062] 1. Put the young radish leaves into a 2ml flat-bottomed EP tube and put them in liquid nitrogen.

[0063] 2. Grind quickly until it becomes powder.

[0064] 3. Take out the EP tube and quickly add 0.6ml of CTAB solution preheated to 65°C (2% CTAB, 20gm CTAB, 20mM EDTA, 100mM Tris-Cl pH 8.0, 1.4M NaCl dissolved in one liter of water

[0065] Adjust the pH to 7.5-8.0, add 0.2% mercaptoethanol after sterilization), mix quickly and place in a dry bath at 65°C for 20 minutes, and slowly invert once every 5 minutes.

[0066] 4. Use a snipped pipette tip to suck out the supernatant into a new EP tube, add 300ul of c...

Embodiment 2

[0084] According to the method of Example 1, PCR amplification of the SSR17 locus was performed on the DNA of the parents of Xiakang 40 days old.

[0085] The amplified SSR17 amplification product of Xia Kang 40 days and the amplification product of SSR17 site of the DNA of Xia Kang 40 days parents were subjected to PAGE gel electrophoresis.

[0086] The detection of the amplified product by PAGE gel is to amplify the carrot genomic DNA with the primers initially screened out by agarose gel electrophoresis, and to amplify the carrot genomic DNA in the PCR amplification product (Xia Kang 40-day male parent, Xia Kang 40-day female parent , Xia Kang 40 days) amplification products, were added 5ul sample buffer, with 8% non-denaturing polyacrylamide gel. The setting parameters of the electrophoresis instrument are generally a voltage of 1200-1500V and a current of 60mA-80mA for 1.5 hours of electrophoresis. The buffer in the electrophoresis tank is generally 0.5×TBE or 1×TBE. Af...

Embodiment 3

[0098] According to the method of Example 1, PCR amplification of the SSR75 site was performed on the DNA of the Xiakang 40-day parents.

[0099] PAGE gel electrophoresis was performed on the SSR75 amplification product of Xia Kang 40 days amplified in Example 1 and the DNA SSR75 amplification products of Xia Kang 40 days parents. The steps of PAGE gel electrophoresis are the same as those in Example 2, and will not be repeated here.

[0100] The PAGE gel electrophoresis picture of the amplification product of SSR75 is shown in image 3 . from image 3It can be seen from the figure that the band sizes of Xia Kang’s 40-day male parent and Xia Kang’s 40-day female parent are different, indicating that there are alleles. The bands were the same as those of the male parent and the female parent respectively, indicating that the SSR75 locus of the 40-day-resistant summer radish variety was co-dominant.

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Abstract

The invention provides EST-SSR (expressed sequence tag-simple sequence repeats) molecular markers for heat-resistant radish. The EST-SSR molecular markers comprise a type of or two types of SSR 17 and SSR 75. Nucleotide sequences of the SSR 17 are shown as SEQ ID No.1 in sequence tables; nucleotide sequences of the SSR 75 are shown as SEQ ID No.2 in the sequence tables. The EST-SSR molecular markers have the advantages that heat-resistant radish varieties, namely Summer Resistance 40-day, are screened to obtain the two EST-SSR molecular markers, the SSR 17 and the SSR 75 have codominant expression characteristics, the population purity of the commodity heat-resistant radish varieties, namely the Summer Resistance 40-day, is detected by the aid of the two EST-SSR molecular markers in codominant relationships, the purity can reach 92% as shown by results and is consistent with the purity of field phenotype, and accordingly the two EST-SSR molecular markers can be used for detecting the purity of the heat-resistant radish varieties.

Description

technical field [0001] The invention belongs to the technical field of molecular markers, and in particular relates to heat-resistant radish EST-SSR molecular markers, primers for molecular markers and applications thereof. Background technique [0002] Molecular markers are genetic markers based on nucleotide sequence variation in genetic material between individuals, and are a direct reflection of genetic polymorphism at the DNA level. Compared with several other genetic markers - morphological markers, biochemical markers, and cytological markers, DNA molecular markers have an unlimited number of markers, can analyze species at different stages of biological development, and do not affect the expression of target traits, so , DNA molecular markers have advantages over other methods. [0003] The commonly used marking methods of DNA molecular markers include RFLP molecular markers, RAPD molecular markers and SSR (Simple Sequence Repeats) molecular markers. SSR molecular ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 李世升向福李竟才方元平项俊陈娟张娜娜张与晨
Owner HUANGGANG NORMAL UNIV
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