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A molecular marker, primer pair and application thereof for identifying male and female bayberry

A molecular marker and primer pair technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as research gaps, and achieve the effect of high guiding value

Active Publication Date: 2020-04-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sex determination mechanism related to other fruit trees has been reported a lot at home and abroad, such as papaya, kiwi, etc. However, the relevant research on bayberry is blank

Method used

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  • A molecular marker, primer pair and application thereof for identifying male and female bayberry
  • A molecular marker, primer pair and application thereof for identifying male and female bayberry
  • A molecular marker, primer pair and application thereof for identifying male and female bayberry

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Extraction of genomic DNA.

[0035] The genomic DNA of 197 parts (100 parts of male plants and 97 parts of female plants) of bayberry varieties were extracted respectively with the improved CTAB method, and the specific methods were as follows:

[0036] 1. Prepare buffer

[0037] CTAB buffer: 2% CTAB, 0.1M Tris, 20mM EDTA, 1.4M NaCl, pH 8.0;

[0038] DNA lysis buffer TE: 10 mM Tris; 1 mM EDTA; pH 8.0.

[0039] 2. Extraction of Waxberry Genomic DNA

[0040] ① Use a balance to quickly weigh about 1.0 g of young bayberry leaf tissue stored in a -80°C refrigerator, add a little PVP to prevent oxidation, grind the powder fully in a mortar with liquid nitrogen, and quickly transfer the powder into a container pre-prepared at 65°C. Put hot 4mL CTAB solution and 80μL β-mercaptoethanol in a 10mL centrifuge tube, bathe in water at 65°C for 1h;

[0041] ② Add 4 mL of chloroform / isoamyl alcohol (24:1) to the mixture in step ①, vortex to mix, centrifuge at 12000 rpm for 10 min, ...

Embodiment 2

[0046] Genome sequencing analysis, primer design.

[0047] Through the sequencing of bayberry genome, 100 female and male mixed pool sequencing, and 5 male and female re-sequencing, it was found that a female-specific genome segment, chromosome 2 (chr2) has a flowering-related functional gene FT1 (SEQ IDNo .3), the sequencing analysis results are as follows figure 1shown. Gene sequence FT2 (SEQ ID No. 4), which is 80% homologous to this sequence, exists in both males and females. Based on this, 2 pairs of PCR primers Mr-F1 and Mr-F2 for identifying the sex of red bayberry were designed and commissioned to synthesize by Hangzhou Jinbaiao Biotechnology Co., Ltd. The primer information is shown in Table 1.

[0048] Table 1

[0049]

Embodiment 3

[0051] PCR amplification.

[0052] ①Reaction system:

[0053]

[0054] ②Reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 10 s, a total of 35 cycles; extension at 72°C for 10 min.

[0055] ③ The product was detected by electrophoresis on 2.0% agarose gel, observed under ultraviolet light and recorded by photography.

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Abstract

The invention discloses a molecular marker for identifying male and female Myrica rubra, a primer pair and an application thereof. Genome of male and female Myrica rubra plants are sequenced, a femaleplant-specific gene FT1 and a FT2 gene which has high sequence homology with the gene FT1 and exists in both male and female plants are discovered, the sequences are compared by analyzing, and the primer pair is designed. The primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequences as follows: an upstream primer is 5'-GTATCAAATCGATCTTTTC-3'; a downstream primer is 5'-ACAGCTACCTATTTTGGATT-3'. The primer pair is used for identifying plant sex of Myrica rubra, and prediction accuracy of female plants reaches 99%. The primer pair is used for identifying sex of Myrica rubra with PCR, and has important meanings for seedling identification, cross breeding and the like of Myrica rubra and has high guidance values for practice and production of Myrica rubra.

Description

technical field [0001] The invention relates to the technical field of molecular identification, in particular to a molecular marker for identifying male and female bayberry, a pair of primers and an application thereof. Background technique [0002] Myrica rubra Sieb.&Zucc. belongs to the genus Myrica rubra. It originates in the southeastern provinces of my country and the Yunnan-Guizhou Plateau. It has a long history of cultivation and is a characteristic fruit in southern my country. At present, the main cultivars of Myrica rubra are distributed in Zhejiang, Jiangsu and Fujian. , Jiangxi, Hunan, Guangdong, Guizhou and Yunnan and other provinces, among which Zhejiang Province has the best cultivation area, yield and texture. The ripening period of bayberry fruit (June) coincides with the off-season of fruit in a year, which can effectively fill the fruit vacancy period in the market. In addition, bayberry fruit is bright in color, unique in flavor, rich in anthocyanins, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/13C12Q2600/156C12Q2565/125
Inventor 高中山王妍贾慧敏赵海波汪国云
Owner ZHEJIANG UNIV
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