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Method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome

A technology of transcriptome sequencing and bird's nest fern, applied in the field of molecular biology, can solve the problem of limited number of SSR primers, achieve the effect of real and reliable results, save time and money, and improve development efficiency

Inactive Publication Date: 2016-01-27
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no complete genome sequence information of bird's nest fern, and the number of SSR primers for it is very limited

Method used

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  • Method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome
  • Method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome
  • Method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 : Acquisition of transcriptome sequence data and design of EST-SSR primers

[0096] 1. Acquisition of transcriptome data:

[0097] The total RNA of bird's nest fern leaves was extracted by TRIzoL method, purified, and treated with DNase to obtain TotalRNA samples with a total amount ≥ 5 μg and an OD260 / 280 of 1.8-2.2, which were quickly frozen in liquid nitrogen and stored at -70°C for later use; RNA samples were detected by AgiLent2100 BioanaLyzer , extract RNA samples with a quality RIN value ≥ 7.0 for library construction;

[0098] Use magnetic beads with Oligo(dT) to enrich the mRNA with polyA, then use ultrasound to break the mRNA into short sequences, and recover 200-700bp fragments;

[0099] Using mRNA as a template, use six-base random primers to synthesize the first cDNA strand, then add buffer, dNTPs, RNaseH and DNApolymeraseI to synthesize the second cDNA strand; the cDNA is purified by the kit and eluted with EB buffer to make the end Repair, a...

Embodiment 2

[0112] Example 2 : Mining of EST-SSR loci in Bird's nest fern

[0113]The fern bird's nest fern was used as the experimental material, and the leaves of bird's nest fern were collected for transcriptome sequencing. A total of 42,907 EST sequences were obtained by sequencing. After removing redundancy, a total of 6,067 EST-SSR sites were detected, distributed among 5,178 EST sequences. Accounting for 12.07% of the total; the EST-SSR types of Bird’s nest fern are abundant, and dinucleotide, trinucleotide and mononucleotide are the dominant repeat types, accounting for 81.62% of the total EST-SSR, see Table 1; Bird’s nest fern EST- In SSR, a total of 106 motif types were observed, and the motif types of mononucleotide to hexanucleotide repeat types were 2, 4, 18, 21, 23, and 38, see figure 2 ;Among them, AG / CT has the highest frequency of 1371, accounting for 22.60%; followed by CA / GT, with a total of 1066, accounting for 17.57%; A / T, a total of 628, accounting for 10.35%; G / C...

Embodiment 3

[0120] Example 3 : Polymorphism Identification of 10 Fern Material DNA Using EST-SSR Primers

[0121] Genomic DNA was extracted from 10 fern materials, and its quality was detected by 0.8% agarose gel electrophoresis. The DNA concentration was diluted to 50 ng / μL and stored at -20°C for later use. Use the DNA of the materials used for primer development to carry out PCR identification of the success rate of primer design; the total volume of the PCR reaction system is 20 μL, which includes 2.0 μL of 10×PCR buffer and 25 mmol / L of Mg 2+ 1.6 μL, 2.5 mmol / L dNTPs 1.8 μL, 10 μmol / L SSR primer 1.0 μL, 30 ng / μL genomic DNA 2.0 μL, 2.5U / μL TaqDNA polymerase 0.2 μL and ddH 2 O11.4μL; PCR amplification program: 94°C pre-denaturation for 3min; 94°C denaturation for 30s, 50-60°C annealing for 45s, 72°C extension for 1min, 32 cycles; 72°C extension for 10min; 4°C storage; after the reaction, Add 2 μL of Loadingbuffer to the PCR product, and use 8% polyacrylamide gel for electrophoresis...

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Abstract

The invention belongs to the molecular biology technology field, and concretely relates to a method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome. The method comprises the following steps: firstly, a transcriptome library is constructed; secondly, transcriptome data is obtained, namely, sequencing data is subjected to splicing by utilization of a Trinity software and sequences are spliced into a complete transcriptome; thirdly, SSR site search is carried out, namely, combined with a Perl programming language method, a lot of sequence information is subjected to batch processing, and SSR site search is carried out; fourthly, batch design of EST-SSR primers is carried out. Different fiddlehead materials are selected to verify the designed SSR primers, if strips are detected, successful SSR primers are obtained. Through the method, 4063 pairs of SSR primers are designed, and a new method and thought are provided for development of Asplenium nidus L. EST-SSR primers and further for achieving molecule label auxiliary selection breeding.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for developing a bird's nest fern EST-SSR primer based on transcriptome sequencing. Background technique [0002] Ferns, also known as ferns, are a large transitional plant group between bryophytes and seed plants in terms of morphological structure and environmental adaptability, and occupy an important position in the plant evolution system. Bird's nest fern (Aspleniumnidus L.) belongs to Eufilicales (Eufilicales), Aspleniaceae (Aspleniaceae) and Asplenium (Asplenium), not only is an important part of indoor foliage plants and is used as a ground cover to build a variety of gardens Landscape, but also has high food value and medicinal value. Bird's nest fern also plays an important role in tropical forest ecosystems, which is reflected in the regulation of water and nutrient cycles in forest ecosystems, enrichment and maintenance of forest biodiversity. Alt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6895C12Q2600/156C12Q2600/166C12Q2535/122
Inventor 贾新平邓衍明孙晓波梁丽建
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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