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A Method for SSR Analysis of Tetraploid Alfalfa Using Multiplex PCR

An alfalfa and tetraploid technology, which is applied in the directions of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, can solve problems such as genetic diversity analysis of tetraploid alfalfa, and achieve easy identification. , high accuracy, uniform distribution effect

Active Publication Date: 2020-09-22
TIANJIN AGRICULTURE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no method for analyzing the genetic diversity of tetraploid alfalfa using multiplex PCR-SSR

Method used

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  • A Method for SSR Analysis of Tetraploid Alfalfa Using Multiplex PCR
  • A Method for SSR Analysis of Tetraploid Alfalfa Using Multiplex PCR
  • A Method for SSR Analysis of Tetraploid Alfalfa Using Multiplex PCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Genomic DNA extraction and primer synthesis

[0044]Using alfalfa variety WL323 as the test material, 12 individual plants were randomly selected, enough young leaves were cut, and after freezing and grinding in liquid nitrogen, genomic DNA was extracted by the CTAB method of Doyle et al. Agarose gel electrophoresis detection of the quality and quantity of genomic DNA.

[0045] Synthesize group A primers BBI131F, GBG230 and E776153 according to the primer sequences in Table 1.

[0046] 2. Multiplex PCR

[0047] (1) The reaction system comprises the following components:

[0048] Template DNA 1.5ng / μL, Taq DNA polymerase 0.035U / μL, dNTPs 0.25mM, 1×PCR Buffer (containing 1.5mM MgCl2), the concentrations of primers BBI131F, GBG230 and E776153 were 0.145μM, 0.170μM and 0.135μM ( Forward and reverse primers have the same concentration), and make up to 15 μL with deionized water.

[0049] (2) Carry out PCR amplification according to the following steps:

[0050] Pre-d...

Embodiment 2

[0056] 1. Genomic DNA extraction and primer synthesis

[0057] Using the alfalfa variety Algonquin as the test material, 12 individual plants were randomly selected, a sufficient amount of young leaves were cut off, frozen and ground in liquid nitrogen, and the genomic DNA was extracted by the CTAB method of Doyle et al., with λDNA as the reference standard. 0.8% agarose gel was used to detect the quality and quantity of genomic DNA by electrophoresis.

[0058] According to the primer sequences in Table 1, primers CBF96 and GAW212 of group H were synthesized.

[0059] 2. Multiplex PCR

[0060] (1) The reaction system comprises the following components:

[0061] Template DNA 1.5ng / μL, Taq DNA polymerase 0.035U / μL, dNTPs 0.25mM, 1×PCR Buffer (containing 1.5mM MgCl2), primers CBF96 and GAW212 were 0.160μM and 0.140μM (same concentration of forward and reverse primers) , make up to 15 μL with deionized water.

[0062] (2) Carry out PCR amplification according to the following ...

Embodiment 3

[0069] 1. Genomic DNA extraction and primer synthesis

[0070] Using alfalfa variety Zhonglu No. 1 as the test material, 12 individual plants were randomly selected, and enough young leaves were cut off, frozen and ground in liquid nitrogen, and the genomic DNA was extracted by the CTAB method of Doyle et al., with λDNA as the reference standard. The quality and quantity of genomic DNA were detected by electrophoresis using 0.8% agarose gel.

[0071] Synthesize group D primers GBF56, CAW306 and BBG28 according to the primer sequences in Table 1.

[0072] 2. Multiplex PCR

[0073] (1) The reaction system comprises the following components:

[0074] Template DNA 1.5ng / μL, Taq DNA polymerase 0.035U / μL, dNTPs 0.25mM, 1×PCR Buffer (containing 1.5mM MgCl2), primers GBF56, CAW306 and BBG28 were 0.285μM, 0.115μM and 0.125μM (positive and negative to the same concentration as the primers), make up to 15 μL with deionized water.

[0075] (2) Carry out PCR amplification according to ...

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Abstract

The invention relates to a method for performing SSR analysis on tetraploid alfalfa by utilizing multiple PCR. The method specifically comprises nine multiple PCR-SSR groups (24 pairs of SSR primers) and corresponding PCR systems and reaction programs. 24 SSR markers are uniformly distributed on chromosomes, the distance between the markers being not smaller than 10Mbp; marker genotypes are easy to identify, and the average genotype accuracy rate is higher than 95%. When the solution is adopted to perform genetic diversity analysis, group structure analysis and variety identification on the tetraploid alfalfa, the method has the advantages of short time, low cost and high accuracy, and the total efficiency can be improved by two to three times.

Description

technical field [0001] The invention relates to a method for SSR analysis of tetraploid alfalfa by multiplex PCR, and belongs to the field of biotechnology. Background technique [0002] Alfalfa (Medicago sativa) is the most important forage crop in temperate regions of the world. The two main planted subspecies (M.sativa subssp.sativa and M.sativa subssp.×varia) are all autotetraploid, natural outcrossing , severe inbreeding depression. Genetic diversity is the basis for plant breeding and genetic improvement. Since most alfalfa varieties are synthetic varieties, they are bred by continuous random mating of selected excellent individual plants and their progeny for several generations. Therefore, when analyzing genetic diversity, generally 20 to 40 individual plants should be selected from one alfalfa variety. When many species are studied, this often results in a very large number of analysis units (genotypes). Due to the limitations of actual workload, funds, time and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q2537/143C12Q2565/125
Inventor 桂枝高建明谢晓东
Owner TIANJIN AGRICULTURE COLLEGE
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