Prunus mume weeping trait SNP molecular marker and application thereof

A molecular marker, plum blossom technology, applied in recombinant DNA technology, DNA/RNA fragment, microbial determination/inspection, etc., can solve the problems of low efficiency, long screening cycle of plum blossom weeping traits, etc., achieve good repeatability, improve breeding Efficiency, the effect of shortening the breeding cycle

Active Publication Date: 2018-10-30
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems such as long screening period and low efficiency of weeping traits of plum blossoms existing in the

Method used

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  • Prunus mume weeping trait SNP molecular marker and application thereof
  • Prunus mume weeping trait SNP molecular marker and application thereof
  • Prunus mume weeping trait SNP molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Based on the SLAF-seq technology, the development of SNP molecular markers for plum blossom weeping traits 1. Experimental materials

[0067] The test materials included 'Liupetal' plum as the female parent, 'Fentai Weeping Branch' as ​​the male parent and F 1 Separate groups, straight and weeping plum varieties. The parents had significant differences in phenotypic traits such as weeping branch and flower color, and all the materials were planted in He Village, Moganshan Town, Huzhou City, Zhejiang Province (30.566389°N, 119.879582°E).

[0068] 2. Genomic DNA extraction

[0069] choose F 1 There are 20 individual plants of straight branch and weeping branch plant type in the group. DNA extraction was carried out according to the instructions of the high-efficiency plant genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), and 1.0% agarose gel was prepared, and 3 μL DNA was mixed with about 1 μL loading-buffer, and the voltage was 150 V, a...

Embodiment 2

[0072] The amplification of embodiment 2 SNP molecular marker

[0073] Primers were designed according to the differential markers of plum blossom weeping characters, as follows:

[0074] According to the Sanger sequencing results, two Markers (Marker301243, Marker311414) were selected, and primers were designed using Primer Premier 5.0. The primer sequences are shown in Table 1.

[0075] Table 1 Information of 2 pairs of primers used for Sanger sequencing

[0076]

[0077] Several straight and weeping branches were randomly selected for PCR amplification. PCR amplification system 20μL, containing 100ng·μL -1 Template DNA 2 μL, 2×Taq PCR Master Mix (BIOMIGA) 10 μL, 10 mol / L forward and reverse primers (Beijing Synthesis Department of Sangon Bioengineering Co., Ltd.) 1 μL each, ddH 2 O 10.3 μL. The PCR program was: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 20 s, annealing at 56°C for 30 s, extension at 72°C for 20 s, and 30 cycles; final extension at 7...

Embodiment 3

[0079] Example 3 Establishing an AS-PCR method for genotyping based on SNP markers

[0080] Two markers (Marker301243 and Marker311414, whose nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively) were obtained by Sanger sequencing, and AS-PCR primers were designed for corresponding SNP sites. When designing allele-specificity, the SNP sites of Marker301243(A / T) and Marker311414(C / T) were designed respectively. In order to improve the specificity of primers, at the same time, introduce 1 mismatched base, another primer (common primer) was designed according to conventional methods, and the specificity of the set primer was detected by NCBI (Table 2). During the AS-PCR operation, two AS-PCR primers should be paired with common primers, and the same DNA template should be amplified by PCR in two PCR tubes. If the 3' end of the AS-PCR primer matches the allelic site in the sample, effective amplification can be performed; otherwise, effective amplification can...

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Abstract

The invention discloses a prunus mume weeping trait SNP molecular marker and the application thereof. The prunus mume weeping trait SNP molecular marker specifically comprises molecular Marker 301243and/or Marker 311414; the nucleotide sequences of the molecular markers are respectively shown as SEQ ID NO.1 and SEQ ID NO.2. The prunus mume weeping trait SNP molecular marker is characterized in that two prunus mume weeping trait SNP molecular markers of Marker 301243 and Marker 311414 are obtained by using a SLAF-seq technology to develop, and sequence amplification primers and allele-specificPCR primers of the molecular markers are provided. The molecular markers Marker 301243 and/or Marker 311414 and the primers thereof have higher accuracy, stability and repeatability of identifying prunus mume varieties. When the molecular Marker 301243 and/or Marker 311414 is/are used for molecular assisted selection breeding, early selection of seedling stage can be realized and the prunus mumeweeping trait is rapidly identified, so that the workload is reduced and the breeding time of prunus mume is greatly shortened.

Description

technical field [0001] The invention relates to the fields of molecular biology and plant molecular breeding, in particular to SNP molecular markers for weeping branch traits of plum blossoms and applications thereof. Background technique [0002] Plum blossom (Prunus mume Sieb.et Zucc.) belongs to Rosaceae (Rosaceae) Prunus (Prunus). Many years of introduction and cultivation history (Chen Junyu. Illustration of Chinese Plum Blossom Varieties [M]. China Forestry Press, 2010). There are various types of plum blossoms, and rich flower patterns and plant types. Weeping plum blossoms are one of the nine varieties of plum blossoms with unique plant patterns. Plays an important role in landscaping. [0003] Zhang Jie (Zhang Jie. Construction of high-density genetic map of plum blossoms and QTL analysis of some ornamental traits [D]. Beijing Forestry University, 2016.) used SLAF-seq, Specific-Locus Amplified Fragment Sequencing (SLAF-seq, Specific-Locus Amplified Fragment Sequen...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 张启翔李素珍卓孝康郑唐春李璐璐邱丽珂孙丽丹程堂仁王佳
Owner BEIJING FORESTRY UNIVERSITY
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