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Lagerstroemia indica microsatellite molecular markers and application in identifying interspecific hybrids of lagerstroemia indica

A technology of molecular markers and distant hybridization, applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc.

Inactive Publication Date: 2012-07-25
PALM ECO TOWN DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are only 15 published SSR loci of Lagerstroemia indica, and it is impossible to use these SSR loci to effectively carry out molecular marker-assisted breeding of Lagerstroemia urophylla and Lagerstroemia urophylla distant hybrids

Method used

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  • Lagerstroemia indica microsatellite molecular markers and application in identifying interspecific hybrids of lagerstroemia indica
  • Lagerstroemia indica microsatellite molecular markers and application in identifying interspecific hybrids of lagerstroemia indica
  • Lagerstroemia indica microsatellite molecular markers and application in identifying interspecific hybrids of lagerstroemia indica

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1 Obtaining of Lagerstroemia indica and Lagerstroemia urophylla hybrid offspring

[0091] Lagerstroemia indica cultivars 'Qiaojiaren', 'Baiyunyingxia', 'XTSC3', 'Pink Butterfly', 'Duohuafen' were selected for hybridization with Lagerstroemia indica. Before the anthers are scattered, the anthers of Lagerstroemia indica and Lagerstroemia urophylla with long and short stamens are mixed and dried in a color-changing silica gel for 6 hours. The pollen is collected with an 80-mesh molecular sieve and stored at 4°C. Before pollination, the pollen viability of each parent was measured by the hanging drop method. The culture medium was 150g / L sucrose + 20mg / L boric acid + 20mg / L calcium chloride + 100g / L PEG4000, and the culture condition was 25°C for 6h. The vigor of pollen of each parent can be used for pollination only when it is greater than 35%.

[0092] Select the inflorescence with a large amount of flowering that day, cut open the flowers that are about to op...

Embodiment 2

[0094]Example 2 Construction of Lagerstroemia microsatellite library

[0095] Including the following steps:

[0096] (1) Extract Lagerstroemia indica genomic DNA (DNA secure Plant Kit, purchased from Tiangen Biology), 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), digest at 37°C for 40 hours to obtain DNA with a size of 300-1000 bp DNA fragments;

[0097] (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;

[0098] (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 for mor...

Embodiment 3

[0108] Example 3 Screening and sequencing of positive clones containing crape myrtle microsatellite sequences and design of crape myrtle microsatellite primers

[0109] Including the following steps:

[0110] (1) Pick 136 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;

[0111] 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 TaqDNA polymerase, 1.5 μL bacterial broth, 12.8 μL dH 2 O; Reaction program: 95°C for 3min; 35 cycle reactions, 95°C for 20sec, 50°C for 20sec, 72°C for 1.5min, 15°C for heat preservation;

[0112] (2) Detect the reaction product with 1.5% agarose gel electrophoresis, select the bacteria solution with a single PCR band and a pr...

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Abstract

The invention provides lagerstroemia indica microsatellite DNA molecular markers, which comprise 12 lagerstroemia indica microsatellite loci. The invention also provides a method for screening the lagerstroemia indica microsatellite DNA molecular markers and application in identifying interspecific hybrids between the lagerstroemia indica and lagerstroemia caudata. In addition, the invention alsoprovides a primer sequence for amplifying the 12 lagerstroemia indica microsatellite loci and an amplification method thereof. Two DNA molecular markers which are suitable for identifying the interspecific hybrids of the lagerstroemia indica, such as Pretty Woman, White Cloud Reflecting Sun Glow, XTSC3, Pink Butterfly and Polyad and the lagerstroemia caudata are selected, and a technical system of the lagerstroemia indica microsatellite DNA molecular markers is built; and in the breeding practice of the lagerstroemia indica and the lagerstroemia caudata, by using the DNA molecular markers, the reality of the interspecific hybrids can be quickly evaluated, the germplasm innovation and the hybridization breeding efficiency of the lagerstroemia indica are improved, and the lagerstroemia indica microsatellite DNA molecular markers have high repeatability and are reliable and efficient DNA molecular markers.

Description

technical field [0001] The invention relates to DNA molecular marker technology, in particular to a crape myrtle microsatellite DNA molecular marker and a method for using the marker to identify distant hybrid offspring of crape myrtle and urophylla crape myrtle. Background technique [0002] Lagerstroemia indica, a deciduous shrub or small tree belonging to Lythraceae, with smooth bark, light red, purple or white flowers, 3-4 cm in diameter, panicles, 6 petals, shrunken, with long claws , the flowering period is June-September, and the fruiting period is September-December. Lagerstroemia indica has a long flowering period and is highly ornamental. It blooms in summer and has a cultivation history of more than 1,600 years. At present, there are about 260 varieties of Lagerstroemia in the world (DIX, 2005, Cultivars and names of Lagerstroemia, http: / / www.usna.usda.gov / ; ZHANG, 1991, Studies on cultivars of Crape-Myrtle (Lagerstroemia indica) and their uses in Urbangreening, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 张启翔蔡明潘会堂王学凤宋平高亦珂孙明程堂仁田苗王敏
Owner PALM ECO TOWN DEV CO LTD
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