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Specific primers and screening method for EST-SSR (Express Sequence Tag-Simple Sequence Repeat) marker of torreya grandis transcriptome sequence

A technology of transcriptome sequence and specific primers, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc. It can solve the problems of complex steps, high development costs, and no EST-SSR marker research reports.

Active Publication Date: 2017-10-24
ZHEJIANG FORESTRY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional SSR primers are mainly obtained from genomic libraries, the steps are complicated, the workload is heavy, and the development cost is high
There is no research report on the different types of EST-SSR markers between Torreya species and within Torreya species

Method used

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  • Specific primers and screening method for EST-SSR (Express Sequence Tag-Simple Sequence Repeat) marker of torreya grandis transcriptome sequence
  • Specific primers and screening method for EST-SSR (Express Sequence Tag-Simple Sequence Repeat) marker of torreya grandis transcriptome sequence
  • Specific primers and screening method for EST-SSR (Express Sequence Tag-Simple Sequence Repeat) marker of torreya grandis transcriptome sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] 1. Source of SSR loci and screening of EST-SSR sequences

[0139] The inventors used Trinity software to assemble the transcriptome data of the small torreya seed kernel transcriptome data and the round torreya seed kernel transcriptome data in the laboratory, and obtained the transcript sequence. A total of 246862 Transcripts and 142213 Unigenes were assembled. Use the MISA program (MIcroSAtellite identification tool, http: / / pgrc.ipk-gatersleben.de / misa / misa.html) to search for the SSR site of the Unigene above 1 kb obtained through screening. For the microsatellite fragments whose repetition times are 9, 8, 5, 5, and 5 times in sequence for repeating units of 2-6 bases, ESTs sequences containing microsatellite repeats are obtained. Using the primer design software Primer3.0 to design specific primers on the flanking sequences at both ends of the microsatellites.

[0140] 2. Design of EST-SSR labeled primers

[0141] In the flanking sequence of the microsatellite re...

Embodiment 2

[0150] 1. Extract the DNA of the Torreya tree

[0151] Specific steps are as follows:

[0152]Put CTAB in a 65°C water bath for 30 minutes in advance, take a fresh sample with an ice box, cut it into pieces and put it in a 2ml centrifuge tube (the sample volume is about 1 / 4 of the tube). Add a steel ball with a diameter of about 4mm into each centrifuge tube, soak it in liquid nitrogen for 5-7 minutes, fully pre-cool it, and grind it with a homogenizer. Add 800 μl of CTAB extract that has been water-bathed in advance to the centrifuge tube (add 1% β-mercaptoethanol to the CTAB extract), place the centrifuge tube in a 65°C water bath for 40 minutes, and shake it upside down once during the period. Centrifuge at 12000rpm at 4°C for 10min, take the supernatant, add an equal volume of 24:1 chloroform-isoamyl alcohol (chloroform:isoamyl alcohol=24:1), shake well, and let stand for 5-8min. 12000rpm, 4℃, centrifuge for 10min, take the supernatant, add an equal volume of 24:1, shake...

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Abstract

The invention belongs to the field of molecular biology DNA (Deoxyribonucleic Acid) marker technology and application, and particularly relates to specific primers and a screening method for an EST-SSR (Express Sequence Tag-Simple Sequence Repeat) marker of a torreya grandis transcriptome sequence. The screening method comprises the following steps: firstly, assembling fine torreya grandis seed kernel and circular torreya grandis seed kernel transcriptome data by adopting Trinity software to obtain a transcript sequence; secondly, carrying out SSR site research on Unigene with 1kb or above obtained by screening by using an MISA (MIcroSAtellite identification tool) program, and carrying out screening and separating on 2 to 6 base repeat units and repeat times according to microsatellite fragments with repeat times of 9, 8, 5, 5 and 5 in sequence, thereby obtaining an ESTs sequence with microsatellite repeat; thirdly, designing the primers by using a Primer 3 program; finally, comprehensively evaluating repeatability, stability and polymorphism of the EST-SSR marker to obtain the EST-SSR marker. The specific primers disclosed by the invention can be used for conveniently and quickly analyzing germ plasm resources of different types and genetic diversity of interspecific and intraspecific torreya grandis.

Description

technical field [0001] The invention belongs to the field of molecular biology DNA labeling technology and application, in particular to EST (Express Sequence Tag-Simple Sequence Repeat, Express Sequence Tag)-SSR (Simple Sequence Repeat) marked specific primers and The screening method belongs to the technical field of molecular markers. Background technique [0002] Torreya grandis belongs to the genus Torreya Arn. There are 7 species and 2 varieties of Torreya plants in the world, including Torreya taxifolia, Torreya californica, Torreya nucifera, Torreya Torreya grandis, Torreya fargesii, Torreya parvifolia, Torreya fargesii var.yunnanensis and Torreya grandis Fort.ex Lind.var.jiulongshanensis. It is one of the tree species with the longest history of cultivation and utilization and the highest economic value. The intraspecific variation of Torreya species is complex, and there are many types of natural variation, including artificially cultivated Torreya species. Torr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 张敏张迟周彩红吴家胜宋丽丽戴文圣
Owner ZHEJIANG FORESTRY UNIVERSITY
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