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Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing

A transcriptome sequencing and transcriptome technology, applied in the fields of molecular biology and bioinformatics, can solve the problem of a small number of mung bean SSR primers and other problems

Inactive Publication Date: 2014-03-19
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no complete genome sequence information of mung bean, and the number of mung bean SSR primers is relatively small

Method used

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  • Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing
  • Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing
  • Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 RNA-seq analysis and design of SSR primers

[0043] 1. Acquisition of transcriptome data

[0044] The total RNA of mung bean seedlings was extracted by Trizol reagent, and the mRNA was enriched by Oligo (dT) magnetic beads. Add fragmentation buffer to break mRNA into short fragments, use mRNA as a template, use six base random primers to synthesize the first cDNA strand, then add buffer, dNTPs, RNase H and DNA polymerase I to synthesize the second cDNA strand, in After purification with QiaQuick PCR kit and elution with EB buffer, end repair was performed, A was added and sequencing adapters were connected, and then agarose gel electrophoresis was used for fragment size selection, and finally PCR amplification was performed. The constructed sequencing library was used with IlluminaHiseq2000 Perform sequencing.

[0045] Reverse transcribe and synthesize double-stranded cDNA, purify cDNA, perform end repair, add A and connect sequencing adapters, then use agar...

Embodiment 2

[0069] Example 2 Discovery of mung bean high-throughput SSR sites

[0070]Using the above method, mung bean leaves were used as materials for high-throughput sequencing, and high-throughput SSR sites were excavated from mung bean transcriptome sequences using Perl language, and 83,542 transcriptome sequences and 48,693 unigenes were obtained (Table 1). The SSR density distribution has the highest frequency of single-base microsatellites, and the highest proportion is A / T, followed by tetranucleotides (Table 2, Figure 5 , Figure 6 ).

[0071] Table 1 Frequency distribution of splicing length

[0072]

[0073] Table 2 Repeated primitives

[0074]

[0075] A total of 13134 pairs of SSR primers were designed using primer3.0 batch design software. Randomly select 50 pairs of primers (see the nucleotide sequence list) from it, and use mung bean leaf DNA to carry out primer design success rate detection. The results show that a total of 46 pairs of SSR primers detect clea...

Embodiment 3

[0076] Example 3 Using SSR primers to identify polymorphisms in 8 mung bean DNAs from different countries

[0077] The DNA of 8 parts of mung bean was extracted, 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. The DNA of the materials used for primer development was used to identify the success rate of primer design by PCR. The PCR reaction system uses a 10 μL reaction system, which includes 40ng of genomic DNA, 1 × Taq enzyme buffer (10mmolL -1 Tris-HCl, pH8.8; 10mmol L -1 KCl; 10mmol L -1 (NH 4 ) 2 SO 4 ;1.5mmol L -1 MgCl 2 ; 0.1%Triton X-100), 1mmol L -1 dNTPs, upstream and downstream primers 0.25 μmol L -1 and 1U Taq DNA polymerase. The SSR reaction program was: pre-denaturation at 95°C for 5 min, deformation at 95°C for 30 s, annealing at 51-60°C for 45 s, extension at 72°C for 45 s, 32-35 cycles, and finally extension at 72°C for 5 min. After the reaction, add 2 ...

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Abstract

The invention provides a method for developing a mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing. The method comprises the following steps: obtaining a set of mung bean genome-wide transcription, and forming a sequence database; splicing sequencing sequences into a transcriptome by Trinity; taking the longest transcript in each gene as Unigene; carrying out bioinformatics analysis of a Unigene sequence; carrying out SSR detection on the Unigene by adopting MISA1.0; carrying out SSR primer design by using a Primer 3, and carrying out SSR primer polymorphism identification. 13134 pairs of SSR primers are successfully designed by application of the method; 50 pairs of primers are randomly selected to verify 8 parts of mung bean deoxyribonucleic acids (DNAs) from different countries, wherein 32 pairs of polymorphic primers are formed in all; the mung bean materials with different geographical origins can be distinguished by using the 32 pairs of SSR primers. The method disclosed by the invention is convenient, fast and accurate, and low in cost, and a new thought is provided for development of the mung bean SSR primer.

Description

technical field [0001] The invention relates to molecular biology and bioinformatics, in particular to a method for developing mung bean SSR primers based on transcriptome sequencing. Background technique [0002] Mung bean (Vigna radiata) is a plant of the genus Vigna of the Fabaceae and Papilionaceae, native to India and Myanmar. Now it is widely planted in East Asian countries, and a small amount is also planted in Africa, Europe, and the United States. China is one of the birthplaces of mung bean [Vigna radiata (L.) Wilczek], and has a wide variety of mung bean varieties. China, Myanmar and other countries are the main mung bean exporters. Due to its short growth period, wide adaptability, and good nitrogen fixation capacity, it is an indispensable food crop for rational allocation of planting resources, crop rotation, intercropping, disaster reduction and relief, and an important economic crop for farmers in poor areas to get rich; at the same time Mung bean is rich i...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6811C12Q2525/191
Inventor 陈红霖程须珍王素华王丽侠
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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