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Primer pair, castanopsis hystrix SSR8 (Simple Sequence Repeat 8) marker and preparation method and application thereof

A primer pair, red cone technology, applied in biochemical equipment and methods, DNA preparation, microbial assay/inspection, etc., can solve problems such as inability to directly apply molecular marker-assisted breeding and QTL positioning, poor stability, etc.

Inactive Publication Date: 2014-04-23
GUANGXI FORESTRY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although scholars at home and abroad have used multiple markers such as ISSR and RAPD to study the genetic diversity of red cone populations, the genetic structure of red cone populations, and the genetic relationship and distance between red cones of different provenances, the two markers of ISSR and RAPD All are dominant markers with poor stability and cannot be directly applied to molecular marker-assisted breeding and QTL mapping

Method used

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  • Primer pair, castanopsis hystrix SSR8 (Simple Sequence Repeat 8) marker and preparation method and application thereof
  • Primer pair, castanopsis hystrix SSR8 (Simple Sequence Repeat 8) marker and preparation method and application thereof
  • Primer pair, castanopsis hystrix SSR8 (Simple Sequence Repeat 8) marker and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Extraction and enzyme digestion of total DNA of red cone

[0029] Select the young leaves of Red Cone, and use CTAB method to extract the nuclear genome; digest with EcoRV at 37°C for 3 hours, and inactivate the endonuclease at 65°C for 8 minutes; the reaction system is 20 μL, including 2.0 μL of 10×Buffer, 1.0 μL of EcoRV endonuclease, 2 μL of DNA, 15.0 μL of sterilized double distilled water;

[0030] Connection of connector

[0031] Connect the red cone genomic DNA digested by step EcoRV to the upper and lower adapters (the upper adapter sequence GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT, the sequence table SEQ.ID.No.4; the lower adapter sequence ACCAGCCC-NH 2 , sequence table SEQ.ID.No.5); the 20 μL ligation system contains 4 μL of enzyme-digested red cone genomic DNA, and 100 pmol of the upper and lower adapters; the ligation reaction is performed under T4 ligase buffer conditions (T4 ligase 1.0 μL, ligase buffer2. 0 μL), ligated at 16°C overnight; react...

Embodiment 2

[0044] Extraction and enzyme digestion of total DNA of red cone

[0045] Select the young leaves of Red Cone, and extract the nuclear genome by CTAB method; use EcoRV to digest at 37°C for 8 hours, and inactivate the endonuclease at 65°C for 14 minutes; the reaction system is 20 μL, including 2.0 μL of 10×Buffer, 1.0 μL of EcoRV endonuclease, 2 μL of DNA, 15.0 μL of sterilized double distilled water;

[0046] Connection of connector

[0047] Connect the red cone genomic DNA digested by step EcoRV to the upper and lower adapters (the upper adapter sequence GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT, the sequence table SEQ.ID.No.4; the lower adapter sequence ACCAGCCC-NH 2 , sequence table SEQ.ID.No.5); the 20 μL ligation system contains 4 μL of enzyme-digested red cone genomic DNA, and 100 pmol of the upper and lower adapters; the ligation reaction is performed under T4 ligase buffer conditions (T4 ligase 1.0 μL, ligase buffer2. 0 μL), ligate overnight at 16°C; react t...

Embodiment 3

[0060] Extraction and enzyme digestion of total DNA of red cone

[0061] Select the young leaves of Red Cone, and extract the nuclear genome by CTAB method; use EcoRV to digest at 37°C overnight, and inactivate the endonuclease at 65°C for 20 minutes; the reaction system is 20 μL, including 2.0 μL of 10×Buffer, 1.0 μL EcoRV endonuclease, 2 μL DNA, 15.0 μL sterilized double distilled water;

[0062] Connection of connector

[0063] Connect the red cone genomic DNA digested by step EcoRV to the upper and lower adapters (the upper adapter sequence GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT, the sequence table SEQ.ID.No.4; the lower adapter sequence ACCAGCCC-NH 2 , sequence table SEQ.ID.No.5); the 60 μL ligation system contains 9 μL of enzyme-digested red cone genomic DNA, and 100 pmol of the upper and lower joints; the ligation reaction is performed under T4 ligase buffer conditions (T4 ligase 2.0 μL, ligase buffer2. 0 μL), ligated overnight at 16°C; reacted the ligated...

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Abstract

The invention discloses a primer pair, a castanopsis hystrix SSR8 (Simple Sequence Repeat 8) marker and a preparation method and application thereof. According to the preparation method, the total DNA (Deoxyribonucleic Acid) of castanopsis hystrix is subjected to PCR (Polymerase Chain Reaction) amplification by using the specific primer pair HZ8, thereby obtaining the SSR8 marker. The method is simple and feasible and is simple and convenient in operation; shown by tests which are carried out in members of two natural castanopsis hystrix populations and among the members, the obtained SSR has relatively high polymorphism. The castanopsis hystrix SSR8 marker is a co-dominant marker; compared with other molecular markers, the SSR marker is good in repeatability and high in reliability, can be applied to the genetic diversity evaluation of castanopsis hystrix, the genetic map construction of castanopsis hystrix, the research on the spatial distribution pattern of the genetic variation of castanopsis hystrix population and the origination and evolution of castanopsis hystrix and the standardization of target genes, and can also be applied to the molecular marker assisted breeding and QTL (Quantitative Trait Locus) research of castanopsis hystrix.

Description

technical field [0001] The invention belongs to the technical field of molecular markers in biological sciences, and in particular relates to a red cone SSR8 marker, a pair of primers and a preparation method and application thereof. Background technique [0002] Castanopsis hystrix is ​​an evergreen tree belonging to the genus Castanopsis of the Fagaceae family. It is an important native broad-leaved, high-quality and precious timber tree species in South China. It grows fast, adapts widely, and has high benefits. It can be used for construction, shipbuilding, and high-end furniture. It has strong germination power, dense branches and leaves, is more resistant to shade, and has good mixed growth performance. It can be planted in pure forests or mixed forests. It is a mixed forest of coniferous species. One of the most ideal companion tree species; the red cone has strong adaptability to the habitat, and is located in the arbor layer in the community. Because red cone wood ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 刘海龙蒋燚朱积余何应会覃玉凤韦颖文覃子海曾永明
Owner GUANGXI FORESTRY RES INST
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