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Microsatellite DNA molecular markers of lagerstroemia caudate and application

A DNA molecule and microsatellite technology, applied in the field of crape myrtle microsatellite DNA molecular markers

Inactive Publication Date: 2012-08-15
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are only studies on the single population structure of Lagerstroemia urophylla (Huang Chenhui et al., 2008, A Preliminary Study on the Population Structure and Dynamics of Lagerstroemia urophylla, Tropical Forestry, 36(4), 24-26), while the distribution of Lagerstroemia urophylla resources, Studies on population genetic diversity and germplasm resources protection have not been reported

Method used

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  • Microsatellite DNA molecular markers of lagerstroemia caudate and application
  • Microsatellite DNA molecular markers of lagerstroemia caudate and application
  • Microsatellite DNA molecular markers of lagerstroemia caudate and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Construction of Lagerstroemia urophylla microsatellite library

[0079] Including the following steps:

[0080] (1) Extract Genomic DNA of Lagerstroemia urophylla (DNA secure Plant Kit, purchased from Tiangen Biotechnology), and digest with endonucleases Rsa I and Xmn I. The enzyme digestion system is: 2.5 μL ligation buffer (NEB), 0.25 μL 100×BSA (NEB), 0.25 μL NaCl (5M), 1 μL Rsa I (NEB), 1 μL Xmn I (NEB), 20 μL DNA (100ng / L), at 37 ° C for 40 hours to obtain a size of 300 ~1000bp DNA fragment;

[0081] (2) Mix 10 μM oligonucleotide SuperSNX24F (5'-GTTTAAGGCCTAGCTAGCAGAATC-3') and terminal phosphorylated SuperSNX24R (5'-pGATTCTGCTAGCTAGGCCTTAAACAAAA-3') in equal volumes, denature at 95°C for 10 minutes, cool slowly to room temperature, and form connector;

[0082] (3) Mix 7 μL of the linker in step (2), 2 μL of T4 DNA ligase (NEB) and 1 μL of 10× ligation buffer, add to the digested product of (1), connect at 22°C for 2 hours, and place in 4 ℃ connected ...

Embodiment 2

[0092] Example 2 Screening and sequencing of positive clones containing Lagerstroemia urophylla microsatellite sequences and design of microsatellite primers for Lagerstroemia urophylla

[0093] Including the following steps:

[0094] (1) Pick 72 white colonies with a toothpick and inoculate them into LB medium containing 50 μg / mL ampicillin, and culture at 37° C. at 150 rpm for 40 hours. Use the bacterial solution as a template to carry out PCR reaction;

[0095] The PCR reaction system is: 2.5 μL 250 μg / mL BSA, 2.5 μL 10×PCR buffer, 1 μL 10 mM primer M13F: 5’-GTAAAACGACGGCCAG-3’, 1 μL 10 mM primer M13R: 5’-CAGGAAACAGCTATGAC-3’, 2 μL 25 mM MgCl 2 , 1.5 μL 2.5mM dNTP, 0.2 μL Taq DNA polymerase, 1.5 μL bacterial solution, 12.8 μL dH 2 O; Reaction program: 95°C for 3min; 95°C for 20sec, 50°C for 20sec, 72°C for 1.5min, 35 cycle reactions; 15°C for heat preservation;

[0096] (2) Detect the reaction product with 1.5% agarose gel electrophoresis, select the bacteria solution with...

Embodiment 3

[0098] Example 3 Using microsatellite DNA molecular markers of Lagerstroemia urophylla to analyze genetic differences among different individuals of Lagerstroemia urophylla

[0099] The specific analysis method is:

[0100] (1) Carry out the 6' end of the forward primer of the specific primer of I1G, I2B, I2G, I2H, I3G, I9H, I10H, I12H, II1C, II2C, II3A, II3H, II6A, II8A, II12A sites respectively - FAM or JOE mark;

[0101] (2) Genomic DNA of different individual plants of Lagerstroemia urophylla was extracted (DNA secure Plant Kit, purchased from Tiangen Biology);

[0102] (3) Use the specific primers of I1G, I2B, I2G, I2H, I3G, I9H, I10H, I12H, II1C, II2C, II3A, II3H, II6A, II8A, II12A sites to amplify 20 individuals of Lagerstroemia urophylla Genomic DNA. The 10 μL reaction system included 10ng genomic DNA, 1×PCR buffer (10mM Tris-HCl, 50mM KCl, pH 8.3), dNTPs (250μM each), MgCl 2 (1.5mM), forward and reverse primers (0.5μM each), Taq enzyme (0.5U), the reaction program...

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Abstract

The invention provides microsatellite DNA molecular markers of lagerstroemia caudate. The molecular markers comprise15 microsatellite sites of lagerstroemia caudate. The invention also provides a screening method of the microsatellite DNA molecular markers of lagerstroemia caudate and an application of the microsatellite DNA molecular markers to studies on genetic diversity of lagerstroemia caudate. Besides, the invention also provides primer sequences and a method for amplifying the 15 microsatellite sites of lagerstroemia caudate. The molecular markers have the following advantages that themicrosatellite DNA molecular marker technology system of lagerstroemia caudate is established; and the markers are utilized for analysis of genetic diversity of lagerstroemia caudate, fingerprint construction and molecular marker-assisted selection, have good repeatability and are reliable and effective.

Description

[0001] The present invention is the application number 200910244439.8, the application date is December 31, 2009, and the title of the invention is a divisional application of the mother case of a Lagerstroemia urophylla microsatellite DNA molecular marker and its application. technical field [0002] The invention relates to DNA molecular marker technology, in particular to a Lagerstroemia urophylla microsatellite DNA molecular marker and application thereof. Background technique [0003] Lagerstroemia caudata is a large deciduous tree belonging to the genus Lagerstroemia in the family Lythraceae, with a height of 18-30 meters and a diameter at breast height of about 40 cm; the bark is smooth, brown, and peels off in flakes; the whole plant is glabrous; flowering April-May, fruit period July-October. Lagerstroemia urophylla has dense flowers and strong fragrance, and is an excellent parent for cultivating aromatic crape myrtle varieties. It grows well on the slightly shade...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 张启翔蔡明潘会堂王学凤孙明高亦珂程堂仁宋平田苗
Owner BEIJING FORESTRY UNIVERSITY
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