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DNA fingerprint of peony, acquisition method and special primer thereof
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A DNA fingerprint and peony technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, biochemical equipment and methods, etc., to achieve high polymorphism, high co-dominance, and good repeatability
Inactive Publication Date: 2011-08-10
INST OF BOTANY CHINESE ACAD OF SCI
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TRAP is a new type of molecular marker, which has the advantages of simplicity, high efficiency, high co-dominance, repeatability, and easy sequencing, and overcomes the shortcomings of other markers such as poor repeatability, few detection sites, and complicated analysis procedures.
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[0025] Operation process: Genomic DNA extraction→PCR amplification→electrophoresis→EB color development→obtain clear fingerprints→statistical analysis results. The specific method and process are as follows:
[0027] The young leaves were harvested during the vigorous growth period of the plants, rinsed with tap water, dried and used for extraction of total DNA. The total DNA in the young leaves of peony was extracted according to the method in Xiaoyan Han et al. (Biochem. Genet. 2008, 46: 162-179).
[0028] 2. Primer design
[0029] A peony cDNA library was established, and 2187 EST sequences were obtained through large-scale sequencing. According to the EST sequence information, primer pairs were designed as follows:
[0030] Fixed primer (sequence 1 of the sequence listing): 5-GAGCAACAATGGCGTCTA-3;
[0031] Random primer (SEQ ID NO: 2 of the Seque...
Embodiment 2
[0048] Embodiment 2, the optimization of parameter
[0049] Taking 'Guanshi Moyu' as the test material, the following parameters were optimized.
[0050] 1. Optimization of primer concentration
[0051] The young leaves were harvested during the vigorous growth period of the plants, rinsed with tap water, dried and used for extraction of total DNA. The total DNA in the young leaves of peony was extracted according to the method in Xiaoyan Han et al. (Biochem. Genet. 2008, 46: 162-179).
[0052] PCR was performed using the total DNA as a template. Set five primer concentrations: 0.2 μM, 0.33 μM, 0.4 μM, 0.53 μM, 0.6 μM. See Table 2 for the five PCR systems (15 μL).
[0053] Table 2 PCR system with optimized primer concentration
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Abstract
The invention discloses a DNAfingerprint of peony, an acquisition method and a special primer thereof. The invention provides a special primer which is used for acquiring the DNAfingerprint of peony and comprises a DNA sequence shown in sequence 1 in a sequence table and a DNA sequence shown in sequence 2 in the sequence table. The method comprises the following steps: 1) extracting genomeDNA of peony; 2) carrying out PCR by the special primer with the genomeDNA as a template; 3) carrying out polyacrylamidegel electrophoresis on PCR products, separating DNA markers and showing the DNA fingerprint of peony by EB staining. The invention acquires the TRAP fingerprint with good expansion effect and high resolution and lays technical foundation for improving accuracy and efficiency of selective breeding of peony, shortening breeding cycle and carrying out molecular marker assisted breeding of peony by modern biotechnology.
Description
technical field [0001] The invention relates to a DNA fingerprint of tree peony, its acquisition method and special primers. Background technique [0002] Peony belongs to the woody group of the genus Paeoniae. It is a unique traditional flower in China and is known as the "King of Flowers". The traditional cross-breeding method has a long cycle and cannot screen the target traits early, which severely limits the speed of breeding new varieties. Molecular marker technology can link markers with plant traits, providing a new means for peony breeding. The use of DNA marker-assisted selection breeding will bring revolutionary changes to traditional cross-breeding. The traditional breeding selection is to select the variation of phenotypic traits, but some important traits (such as flower color, flower shape and other traits closely related to ornamental quality) cannot be detected in the early stage, molecular marker technology can be used for DNA analysis of seeds and seedli...
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