est-ssr marker primers developed based on the transcriptome sequence of Sophora japonica and its application

A technology of transcriptome sequence and marker primers, which can be applied in recombinant DNA technology, biochemical equipment and methods, and microbial determination/inspection, etc., and can solve problems affecting the innovative research and utilization of Sophora japonica germplasm resources.

Active Publication Date: 2021-07-06
SHANDONG FOREST SCI RES INST
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are few studies on the molecular aspects of Sophora japonica, especially the analysis of genetic diversity of Sophora japonica is rarely reported, which seriously affects the innovative research and utilization of Sophora japonica germplasm resources
However, there is no report on the development of EST-SSR marker primers using the transcriptome sequence of Sophora japonica.

Method used

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  • est-ssr marker primers developed based on the transcriptome sequence of Sophora japonica and its application
  • est-ssr marker primers developed based on the transcriptome sequence of Sophora japonica and its application
  • est-ssr marker primers developed based on the transcriptome sequence of Sophora japonica and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1 Test materials and reagents

[0044] 1.1 Sophora japonica material

[0045] The experimental materials of this study are 233 germplasms of ancient Chinese pagoda tree (Table 1), mainly from Shandong Weifang, Tai'an, Dongying, Linyi, Heze, Yantai, Binzhou, Dezhou, Zibo, Jining, Rizhao, Liaocheng, Zaozhuang. old tree. All the germplasm of Chinese pagoda japonica ancient trees were collected from the original site in 2012 and then grafted and preserved in Yinmaquan Nursery and Zhangqiu Experimental Base of Shandong Academy of Forestry Sciences. The germplasm materials of Chinese pagoda tree ancient trees used in this study basically involve the ancient Chinese pagoda tree of various cities, counties and townships in Shandong Province.

[0046] Table 1 233 germplasms of Chinese pagoda japonica ancient trees tested

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] 1.2 Primer acquisition

[0053] The high-throughput sequencing platform Illumina HiSeq ...

Embodiment 2

[0057] 2.1 Extraction of Genomic DNA from Sophora japonica

[0058] The genomic DNA of Sophora japonica was extracted using the improved CTAB method, and the young leaves of Sophora japonica were extracted as materials. And improve according to the characteristics of Chinese pagoda tree containing more phenols and protein.

[0059] 1. Preheat DNA extraction buffer 2×CTAB (100mM Tris-HCl (PH8.0), 1.4mM NaCl, 50MmEDTA (PH8.0), 2% CTAB) 700μL at 65℃, 20μL of β-mercaptoethanol (add now Use), 10% PVP140μL, mix well.

[0060] 2. Weigh 0.5g of young Sophora japonica leaves, put them into a 2mL centrifuge tube, add two steel balls with a diameter of about 2mm, put them in a box filled with liquid nitrogen, freeze for about 15 seconds, and grind them with a retsch mixing grinder After about 1.5 minutes, until the leaves of Sophora japonica are in the form of fine powder, quickly add 700 μL of preheated CTAB to each centrifuge tube, and then put it in a 65°C water bath for about 30 mi...

Embodiment 3

[0098] 3.1 Detection of the concentration and purity of Genomic DNA of Sophora japonica

[0099] High-quality DNA is an important prerequisite to ensure the success of PCR amplification. The leaves of Sophora japonica contain secondary substances such as phenols and polysaccharides. These substances can inhibit the activity of Ex Taq enzyme and should be removed as much as possible during DNA extraction. In this study, PVP and β-mercaptoethanol were added to the DNA extraction liquid, and the modified CTAB method was used to extract the extracted DNA. After the UV spectrophotometer detected the extracted DNA, the results showed that the OD260 / OD280 value of the DNA was between 1.7 and 2.0 . The DNA was detected by 0.8% agarose gel electrophoresis, and the results were as follows: figure 1 As shown, it can be seen from the figure that the extracted DNA showed clear and complete bands after agarose gel electrophoresis without obvious tailing. This shows that the concentration ...

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Abstract

The invention belongs to the field of molecular marker technology development and application, and specifically relates to the EST‑SSR marker primer set of Sophora japonica. The primer set includes primer pairs No. 1 to No. 10. The primer set of the present invention has the advantages of stable amplification, clear electrophoretic bands, rich polymorphism, etc., and can be effectively used in the analysis of genetic diversity of Sophora japonica germplasm resources, the construction of high-density maps, the identification of variety purity and authenticity, and molecular markers. Assisted breeding and other research fields.

Description

technical field [0001] The invention belongs to the field of development and application of molecular marker technology, and in particular relates to EST (expressed sequence tags, ESTs) microsatellite marker primer set (EST-SSR). Background technique [0002] Sophora japonica L., also known as Chinese pagoda tree, Chinese pagoda tree, pagoda tree, etc., belongs to the leguminous family Leguminosaesp. Sophora japonica L. is a deciduous tree with a height of 25 meters. It is native to the north of my country and has a profound cultivation history in China. As early as the Qin and Han Dynasties, there were already records about planting locust trees along the passageways. Chinese pagoda tree is a positive tree species, slightly shade-tolerant, not strict on soil requirements, well-developed root system, fast growth, high transplanting survival rate, strong adaptability, and also has the characteristics of anti-pollution, smoke resistance, wind resistance, etc., can be extremely...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11C12Q1/6806
CPCC12Q1/6806C12Q1/6895C12Q2600/156
Inventor 庞彩红李双云夏阳张明静付茵茵臧真荣刘盛芳杨勇亓玉昆李自峰王守国
Owner SHANDONG FOREST SCI RES INST
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