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Waxberry EST-SSR molecular markers and application thereof

A technology of labeling and bayberry, applied in the direction of DNA/RNA fragments, microbial determination/inspection, recombinant DNA technology, etc.

Inactive Publication Date: 2016-03-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of EST-SSR molecular markers in myrica rubra is not enough to meet the needs of genetic diversity system analysis and genetic map construction of germplasm resources, and more EST-SSR markers with high polymorphism need to be developed

Method used

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  • Waxberry EST-SSR molecular markers and application thereof
  • Waxberry EST-SSR molecular markers and application thereof
  • Waxberry EST-SSR molecular markers and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Genomic DNA was extracted by CTAB method.

[0019] (1) The formula for preparing CTAB buffer solution (hexadecyltriethylammonium bromide, Hexadecyltrimethylammonium bromide) is: 2% CTAB, 0.1MTris, 20mMEDTA, 1.4MNaCl, pH value 8.0.TE buffer solution (10mMTris, 1mMEDTA, pH8 .0).

[0020] (2) Harvesting the young and tender leaves of bayberry, the variety information of bayberry is as follows figure 1 shown.

[0021] (3) Put it into liquid nitrogen and grind it to powder form, weigh about 0.7g and transfer it into a 10ml centrifuge tube filled with 4ml CTAB solution and 80μl β-mercaptoethanol (preheated at 65°C), bathe in 65°C water for 1h; add 4ml chloroform / isoamyl alcohol (24:1, v / v), mix well, centrifuge at 12000rpm for 10min, take the supernatant and add 4ml chloroform / isoamylalcohol (24:1, v / v), mix well, and centrifuge at 12000rpm for 10min . Aspirate the supernatant, add 2ml of 5M NaCl, 4ml of isopropanol (pre-cooled at -20°C), mix well, and place a...

Embodiment 2

[0022] Embodiment 2: Design EST-SSR primers

[0023] The invention obtains the EST sequence of Myrica rubra through RNA-Seq and develops EST-SSR primers. The primer design software is: NCBIPrimer-BLAST (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHomeAd). The specific conditions for primer design are: PCR product length 100-350bp; primer length 18-25bp; avoid primer dimer. In primer synthesis, 18 bp of M13-tail (sequence TGTAAAACGACGGCCAGT) was added to the 5' end of the forward primer. A total of 17 pairs of EST-SSR primers were developed, and the sequences are shown in Table 2.

[0024] Table 2 Marker characteristics of bayberry EST-SSR

[0025]

[0026]

Embodiment 3

[0027] Example 3: Polymorphic Primer Screening

[0028] Use the bayberry leaf DNA extracted in Example 1 as a template, and use the 17 pairs of primers designed in Example 2 to amplify.

[0029] 1. PCR reaction system: 10×ExTaqBuffer (containing Mg 2+ ), 1.6 μl of 2.5mMdNTP, 5units / μl ExTaqDNApolymerase0.1μl, forward primer 2pmol, reverse primer 8pmol, M13 universal fluorescent primer 6pmol, 10ng / μl DNA template 1μl, ddH 2 O Supplement the system to 20 μl.

[0030] 2. Use Eppendorf Mastercycler (Eppendorf Scientific, Inc.) PCR instrument to amplify. The reaction program is: 94°C pre-denaturation for 5min, then 94°C (30s) / 60°C (30s) / 72°C (30s) cycle 20 times, then 94°C (30s) / 55°C (30s) / 72°C (30s) ) cycled 12 times, and finally extended at 72°C for 15min.

[0031] 3. Electrophoresis detection: Take 5 μl of PCR products and add 1 μl of 6×loading buffer, detect on agarose gel with a concentration of 1%, and take pictures for records.

[0032] 4. STR fragment length analysis: ...

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Abstract

The invention provides waxberry EST-SSR molecular markers. Sequences of four EST-SSR molecular markers with polymorphism are as shown in SEQ ID NO. 1-8. According to sequence information of a waxberry transcriptome, SSR sites taking three basic groups as repeated elements are selected, and Primer5.0 is adopted to design an EST-SSR primer. After PCR amplification, a fragment size of a product is measured to obtain polymorphism information of the primer. The invention screens four EST-SSR molecular markers with high polymorphism, which can effectively differentiate 24 waxberry varieties, and the molecular markers can be used for fields such as variety identification, genetic diversity analysis and molecular auxiliary breeding of waxberry.

Description

technical field [0001] The invention relates to molecular marker technology, in particular to four EST-SSR molecular marker technologies of bayberry varieties and application thereof. Background technique [0002] Red bayberry (Morellarubra) is a characteristic fruit in southern my country. Its fruit is bright in color, unique in flavor, and rich in nutritional value, and is deeply loved by consumers. [0003] Molecular markers are widely used in germplasm diversity research and molecular assisted breeding. Among them, the simple sequence repeat (SSR) widely exists in the genome of eukaryotes, with a large number and high repeatability, and is one of the most commonly used molecular markers. SSR can be mined from expressed sequence tag (Express sequence tag, EST) library or transcriptome sequencing data, called EST-SSR. Compared with genomic SSR, EST-SSR is relatively easy to develop, but its polymorphism is relatively low. Screening highly polymorphic EST-SSR molecular m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
CPCC12Q1/6895C12Q2600/156
Inventor 徐昌杰王文婷冯超丁毛毛陈昆松
Owner ZHEJIANG UNIV
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