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A kind of germplasm resource identification method of Sarsaparilla

A technology of smilax and smilax, which is applied in the field of germplasm resource identification of smilax, can solve the problems of long development cycle, low flux of primers, high cost, etc., achieve accurate and reliable results, simple and easy method, and applicable sex high effect

Inactive Publication Date: 2020-06-05
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Xu et al. (American Journal of Botany: e64–e66.2011) developed a small number of Smilax spica SSR primers through the dual suppression method, but this method only obtained SSR markers of the two-base repeat unit or two-base repeat combination unit type, The genetic diversity is not high, the locus coverage is insufficient, and the polymorphism richness is low, and it is only applicable to the Smilax sarsaparilla plant populations in Greece and Italy, and cannot effectively identify different provenances of Smilax sarsaparilla
In addition, the development cycle of this method is long, the throughput of primers obtained in one development is low, and the unit labeling cost is relatively high
If DNA gene fragments are used to identify Smilax fringae, from the perspective of identification efficiency, this method cannot effectively identify germplasm resources between populations in the region, usually the markers come from chloroplast haplotypes, and can only detect genetic information inherited from single parent , and if all gene fragments are sequenced, the cost is high, and the feasibility of large-scale samples is not good

Method used

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  • A kind of germplasm resource identification method of Sarsaparilla
  • A kind of germplasm resource identification method of Sarsaparilla
  • A kind of germplasm resource identification method of Sarsaparilla

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1. Construction of the transcriptome database of Smilax fruticosa

[0045] (1) Use the RNAprep Pure Plant Kit (Beijing Tiangen) kit to extract the total RNA from the fresh leaves of Smilax paniculata, and send it to the sequencing company for transcriptome sequencing.

[0046] (2) Use the De novo assembly function of GENEIOUS 9.0.2 to splice short-read sequences into transcriptome frame data, take the longest transcript in each gene as Unigene, and establish a transcriptome database capable of searching for microsatellite sites .

Embodiment 2

[0047] Example 2. Development of microsatellite SSR primers

[0048] (1) Use MISA (http: / / pgrc.ipk-gatersleben.de / misa / misa.html) software to scan different types of microsatellites for the above Unigene to identify and locate SSR sites, parameter settings (misa.ini configuration file) as follows: Identify mononucleotides, dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, hexanucleotides with a repeat count of at least 10, 6, 5, 4 , 3 times, 3 times, if the base distance between two adjacent SSRs is less than 100bp, it is considered as a composite SSR.

[0049] (2) Copy *.fasta, misa.pl and misa.ini to the same folder, run the command >misa.pl*.fasta in the Perl environment, and get *.fasta.misa and *.fasta after running Two files of .statistics, among which *.fasta.misa is used for subsequent primer design.

[0050] (3) Under the Perl environment, the Primer 3 module was used to design SSR primers in batches. The primer design parameters were primer length ...

Embodiment 3

[0053] Example 3. Genomic DNA Extraction

[0054] Using PlantZol (Hangzhou Laifeng) reagent, adopt the improved CTAB method, extract the genomic DNA of the above 72 parts of Smilax sarsaparilla materials, quantify with NanoDrop 2000 (Thermo Fisher Scientific, USA), dilute to 20ng / μl, 4 ℃ or - Store at 20°C until use.

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Abstract

The invention relates to a germplasm resource identification method of smilax aspera, which includes steps of (1), extracting gene group DNA of a sample to be detected; (2), using the DNA extracted in step (1) as a template, and performing PCR amplification by an SSR primer combination based on a smilax aspera transcription group sequence; (3), performing sequencing parting and gene segment length reading on the amplified product; (4), building up SSR genetic information characteristic base and a germplasm resource identification framework of the smilax aspera by means of genetics analysis software and carrying out validity verification. The method can effectively and rapidly classify and identify the smilax aspera from different producing areas such as Europe, Africa, Asia, and different natural group sources. The result is exact and reliable, the method is simple and easy to practice, and high in applicability; the method can be effectively applied to the germplasm resource identification of smilax aspera.

Description

technical field [0001] The present invention relates to the technical field of molecular markers, in particular to the development and application of primer sets in the identification of plant germplasm resources. Through the method of transcriptome sequencing, 47 pairs of SSR primers with strong specificity and good sensitivity have been developed efficiently. Smilax pachyrhiza plant resources from Africa are used as materials to cover its natural distribution area, so as to establish the SSR genetic information characteristic library and germplasm resource identification framework of Smilax pachyrhiza plants, and to conduct rapid germplasm resource identification methods. Background technique [0002] Smilax aspera Linnaeus (Smilax aspera Linnaeus, English name Italian sarsaparilla, roughbindweed) is a perennial climbing vine or small shrub of the genus Smilax of the family Smilax. It is widely distributed in the Mediterranean region of Europe, eastern Africa, and southern ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11G16B40/00
CPCC12Q1/6895C12Q2600/156G16B40/00
Inventor 祁哲晨王瑞红沈超李攀邱英雄赵云鹏傅承新梁宗锁
Owner ZHEJIANG SCI-TECH UNIV