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Method for converting monomorphic SSR (Simple Sequence Repeat) marker into polymorphic marker

A polymorphic marker and monomorphism technology, which is applied in DNA preparation, recombinant DNA technology, etc., can solve the problem of converting polymorphic markers from monomorphic SSR markers, and achieves a wide range of applications, low cost, and easy operation. simple effect

Inactive Publication Date: 2012-01-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] So far, there is no report on the conversion of monomorphic SSR markers into polymorphic markers by SSCP electrophoresis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The present invention comprises the following steps:

[0026] (1) Preparation of 8% non-denaturing polyacrylamide gel, the ratio of acrylamide to methylene bisacrylamide is 25:1, 10x TBE 100ml, and then dilute to 1L with distilled water;

[0027](2) PCR amplification of the two parents of the mapping population with monomorphic SSR primers under conventional electrophoresis conditions, the PCR reaction system (10ul) is: DNA template 25ng, 1x Buffer, 2.0mmol L-1 MgCl2, 0.25mmolL-1 dNTPs , 0.2μmol L-1 primer, 0.8U Taq DNA polymerase, the insufficient part was filled with sterile double distilled water; the PCR amplification program was: 95°C pre-denaturation 5min; 94°C denaturation 50sec, 56°C renaturation 45sec, Extend at 72°C for 60sec, 34 cycles; finally extend at 72°C for 5min;

[0028] (3) Electrophoresis of the amplified product on 8% non-denaturing polyacrylamide gel, under normal temperature conditions, 15W constant power electrophoresis for 3h;

[0029] (4) Gen...

Embodiment 2

[0031] The present invention comprises the following steps:

[0032] (1) Preparation of 8% non-denaturing polyacrylamide gel, the ratio of acrylamide to methylenebisacrylamide is 30:1, 10x TBE 100ml, and then dilute to 1L with distilled water;

[0033] (2) PCR amplification of the two parents of the mapping population with monomorphic SSR primers under conventional electrophoresis conditions, the PCR reaction system (10ul) is: DNA template 25ng, 1x Buffer, 2.0mmol L-1 MgCl2, 0.25mmolL-1 dNTPs , 0.2μmol L-1 primer, 0.8U Taq DNA polymerase, the insufficient part was filled with sterile double distilled water; the PCR amplification program was: 95°C pre-denaturation 5min; 94°C denaturation 50sec, 56°C renaturation 45sec, Extend at 72°C for 60sec, 34 cycles; finally extend at 72°C for 5min;

[0034] (3) Electrophoresis of the amplified product on 8% non-denaturing polyacrylamide gel, under normal temperature conditions, 15W constant power electrophoresis for 5h;

[0035] (4) Gen...

Embodiment 3

[0037] The present invention comprises the following steps:

[0038] (1) Preparation of 8% non-denaturing polyacrylamide gel, the ratio of acrylamide to methylenebisacrylamide is 29:1, 10x TBE 100ml, and then dilute to 1L with distilled water;

[0039] (2) PCR amplification of the two parents of the mapping population with monomorphic SSR primers under conventional electrophoresis conditions, the PCR reaction system (10ul) is: DNA template 25ng, 1x Buffer, 2.0mmol L-1 MgCl2, 0.25mmolL-1 dNTPs , 0.2μmol L-1 primer, 0.8U Taq DNA polymerase, the insufficient part was filled with sterile double distilled water; the PCR amplification program was: 95°C pre-denaturation 5min; 94°C denaturation 50sec, 56°C renaturation 45sec, Extend at 72°C for 60sec, 34 cycles; finally extend at 72°C for 5min;

[0040] (3) Electrophoresis of the amplified product on 8% non-denaturing polyacrylamide gel, 15W constant power electrophoresis at room temperature for 4 hours;

[0041] (4) Generate polymo...

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Abstract

The invention belongs to the breeding technology field of cotton molecules and particularly relates to a method for converting a monomorphic SSR (Simple Sequence Repeat) marker into a polymorphic marker. The method comprises the following steps: firstly, preparing 8 percent of non-denaturing polyacrylamide gel, ensuring that the proportion of acrylamide and methylene diacrylamide is 25-30:1, adopting 100 ml of 10x TBE (Tetrabromoethane) and then ensuring that the constant volume is 1 L with distilled water; secondly, amplifying the PCR (Polymerase Chain Reaction) of two parents of a mapping population with monomorphic SSR (Simple Sequence Repeat) primers under the conventional electrophoresis conditions, wherein a PCR (Polymerase Chain Reaction) reaction system (10 ul) is a 25 ng of DNA (Deoxyribose Nucleic Acid) template, a 1x Buffer, 2.0 mmol L of 1 MgCI2, 0.25 mmolL of 1 dNTPs (Deoxyribonucleotide Triphosphates), 0.2 umol L of 1 primers and 0.8 U of Taq DNA polymerases, and filling insufficient parts with aseptic double distilled water; thirdly, conducting electrophoresis to an amplified product on the 8 percent of non-denaturing polyacrylamide gradient gel; and fourthly, generating polymorphic SSR primers. The method has low cost and wide application scope, is simple to operate and can effectively convert the monomorphic SSR marker into the polymorphic marker, thereby greatly improving the utilization efficiency of the SSR marker.

Description

technical field [0001] The invention belongs to the technical field of cotton molecular breeding, in particular to a method for converting monomorphic SSR markers into polymorphic markers. Background technique [0002] SSR markers have rich polymorphisms, good repeatability, co-dominant markers, scattered distribution in the genome and are based on PCR, and have been proven to exist in most eukaryotic genomes, so they have been widely used in genetics Map construction, genetic diversity analysis, variety fingerprint drawing, variety purity detection and target trait molecular marker screening and other fields. Some researchers obtained 58 haploids and their diploids produced by the hybrid offspring of TM-1×Hai7124 by using hemigamy, and finally constructed the first genetic map based on PCR-based molecular markers. It was the first time that SSR markers were used in the construction of genetic maps in the map; some people also used the (E22×3-79)×E22 BC1 population containi...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 林忠旭张献龙李夕梅
Owner HUAZHONG AGRI UNIV
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