Optimized coix lacryma-jobi SRAP-PCR (Sequence-Related Amplified Polymorphism-Polymerase Chain Reaction) reaction system

A technology of SRAP-PCR and reaction system, applied in the field of optimized Coix Coix SRAP-PCR reaction system, can solve the problems of low efficiency, poor polymorphism, poor clarity of reaction product bands, etc., so as to improve efficiency and reduce costs. , good effect of polymorphism

Inactive Publication Date: 2014-01-22
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

The application of SRAP in the study of Coix is ​​still in the exploratory stage. At present, the reaction products of Coix SRAP-PCR have poor

Method used

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  • Optimized coix lacryma-jobi SRAP-PCR (Sequence-Related Amplified Polymorphism-Polymerase Chain Reaction) reaction system
  • Optimized coix lacryma-jobi SRAP-PCR (Sequence-Related Amplified Polymorphism-Polymerase Chain Reaction) reaction system
  • Optimized coix lacryma-jobi SRAP-PCR (Sequence-Related Amplified Polymorphism-Polymerase Chain Reaction) reaction system

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Embodiment 1

[0014] 1. Coix germplasm resources

[0015] Based on 90 germplasm resources collected from major producing areas from 2008 to 2010, 69 germplasm resources collected in Fujian Province and other domestic regions (Zhejiang, Liaoning, Shandong, Henan, Yunnan, Jiangsu, Hunan, Guangdong , Shanghai) and other 21 copies (including 6 copies from Taiwan) as materials. From 2011 to 2012, it was planted in Fuzhou, Fujian and Minqing respectively.

[0016] 2. Extraction of Coix Genomic DNA

[0017] Genomic DNA of Coix coix was extracted by improved CTAB method. Weigh 3g young leaves of Coix coix seedlings or tillers, remove the veins, cut them into pieces quickly with scissors, grind them quickly in liquid nitrogen, put them into 50ml centrifuge tubes, and add 15ml of CTAB extraction buffer preheated at 65℃ respectively and corresponding antioxidants, add different concentrations of CTAB extraction buffer and different concentrations of antioxidants to each tube, CTAB (m / M) 3%, β-merca...

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Abstract

The invention discloses an optimized coix lacryma-jobi SRAP-PCR (Sequence-Related Amplified Polymorphism-Polymerase Chain Reaction) reaction system which comprises 3mmol/L Mg<2+>, 2U of Taq enzyme, 0.15mmol/L dNTPs, a 0.30 mu mol/L primer and 40ng of template DNA (Deoxyribonucleic Acid). The PCR amplification reaction process comprises the following steps of: conducting initial denaturation for 5 minutes at 94 DEG C, conducting denaturation for 1 minute at 94 DEG C, annealing for 1 minute at 35 DEG C, extending for 1 minute at 72 DEG C, and repeating for five times in all; subsequently conducting denaturation for 1 minute at 94 DEG C, annealing for 1 minute at 50 DEG C, extending for 1 minute at 72 DEG C, repeating for 35 times; and finally extending for 10 minutes at 72 DEG C, and preserving an amplification product at 4 DEG C. Due to adoption of the system, the coix lacryma-jobi SRAP-PCR reaction efficiency can be improved, the clarity and the polymorphism of a coix lacryma-jobi SRAP-PCR reaction product stripe are remarkably improved, so that the system is applicable to study on relevant fields of identification of coix lacryma-jobi seed quality, analysis on genetic diversity, establishment of coix lacryma-jobi genetic linkage maps, and the like, and has the characteristics of high efficiency, clean strip, good polymorphism and the like.

Description

technical field [0001] The invention relates to an optimized Coix SRAP-PCR reaction system, which belongs to the field of agricultural technology. Background technique [0002] SRAP (Sequence-related amplified polymorphism) marker is a molecular marker based on PCR technology. It mainly designs a pair of unique primers according to the characteristics of gene exons and introns to amplify specific regions of ORFs (Open reading frames) of genes. The upstream primer (l7bp) and downstream primer (18bp) specifically amplify the exon region and intron region, respectively, because these regions are different in different species or different individuals, resulting in polymorphism. The marker has the advantages of high efficiency, stability, simplicity, rich polymorphism, not affected by environmental conditions, and convenient cloning of target fragments. At present, SRAP has been widely used in the genetic diversity analysis of rice, corn, cassava, wheat, highland barley and oth...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2535/139C12Q2531/113
Inventor 季彪俊夏法刚孙小芳程军徐良华毛大梅詹福杨邓邦柱
Owner FUJIAN AGRI & FORESTRY UNIV
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