Preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment

A technology of kidney beans and magnetic beads, applied in the field of bioengineering

Inactive Publication Date: 2012-07-04
WUHAN VEGETABLE RES INST
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Problems solved by technology

At present, only microsatellites with known coding sequences have been screened and identified, and a large number of microsatellites with non-coding regions have yet to be developed and utilized

Method used

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  • Preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment
  • Preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment
  • Preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment

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Embodiment 1

[0060] A kind of magnetic bead enrichment method develops the preparation method of kidney bean SSR primer, and its steps are:

[0061] A. Extract the genome DNA of kidney bean, carry out enzyme digestion to the genome, and establish the AFLP-PCR library:

[0062] The bean variety Qiao Yu (an excellent bean variety provided by Wuhan Institute of Vegetable Science, creeping, green leaves, light yellow flowers). This variety is a premium string bean variety with the general characteristics of string bean varieties. Plant according to conventional cultivation management and fertilizer and water measures, wait for the seedlings to grow 5-6 leaves, take 0.4 g of young leaves and use the CTAB method to extract genomic DNA.

[0063] (1) Add 4ml of 3×CTAB extract (containing 0.2% β-mercaptoethanol (W / V)) to a 10ml Eppeddorf tube and preheat in a 65°C water bath.

[0064] (2) Take 0.4 g of young leaves of kidney bean and put them in a mortar, add appropriate amount of liquid nitrogen...

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Abstract

The invention discloses a preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment. The method comprises the following steps of: (1) digesting Phaseolus vulgaris genome DNA with Msel, and establishing a library by AFLP; (2) hybridizing the genome PCR library with a biotin-labeled repetitive sequence probe; (3) adding the hybridization mixed liquid into streptavidin-coated magnetic beads; (4) amplifying micro-satellite DNA fragments by PCR using eluent-purified micro-satellite DNA as DNA template; (5) linking the purified PCR amplification product on a T vector for cloning to obtain a DNA sequence with inserted fragments; and (6) repeating on the two sides of the core area to design primers, and screening primers to obtain effective microsatellite loci. The method is simple and feasible, increases efficiency of the primers, screens 20 pairs of effective primers from 48 pairs of primers, and provides guarantee for large-batch development of SSR primers.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a preparation method for developing SSR primers of kidney bean by a magnetic bead enrichment method, and the method is applicable to all varieties, strains or germplasm resource materials of kidney beans. Background technique [0002] Although SSR markers have good effect and high reliability, the key point lies in the development of SSR primers. Analysis of SSR markers for a species is only possible after a certain number of SSR primers are available. Especially for biological species that have not been sequenced, species that know little or nothing about their DNA base sequence can only use some special methods to obtain this repeat sequence and its flanking sequences on both sides to design SSR primers. At present, the sources of SSR primers can be summed up in the following four ways: 1. related literature; 2. primers of related species (for example, belonging to different specie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 汪爱华周国林林处发丁毅胡侦华黄兴学邓耀华王斌才姚芳
Owner WUHAN VEGETABLE RES INST
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