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Method for SSR (simple sequence repeat) analysis of tetraploid alfalfa by multiplex PCR (polymerase chain reaction) fluorescence labeling technique

A fluorescent labeling and alfalfa technology, which is applied in biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of low efficiency and high cost, and achieve the effect of high efficiency, low cost and easy identification

Inactive Publication Date: 2018-10-09
TIANJIN AGRICULTURE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] However, the above method still has the problems of low efficiency and high cost, and there is no method for SSR analysis of tetraploid alfalfa using multiple PCR fluorescent labeling technology.

Method used

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  • Method for SSR (simple sequence repeat) analysis of tetraploid alfalfa by multiplex PCR (polymerase chain reaction) fluorescence labeling technique
  • Method for SSR (simple sequence repeat) analysis of tetraploid alfalfa by multiplex PCR (polymerase chain reaction) fluorescence labeling technique
  • Method for SSR (simple sequence repeat) analysis of tetraploid alfalfa by multiplex PCR (polymerase chain reaction) fluorescence labeling technique

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Genomic DNA extraction and primer synthesis

[0049] Using the alfalfa cultivar Supersonic as the test material, 12 individual plants were randomly selected, and enough young leaves were cut out, frozen and ground in liquid nitrogen, and the genomic DNA was extracted by the CTAB method of Doyle et al. The quality and quantity of genomic DNA were detected by electrophoresis on % agarose gel.

[0050] Synthesize group A primers BBI131, GBG230 and E776153 according to the primer sequences in Table 1, add the M13(-21) universal primer sequence to the 5' end of the primer leading strand, and then covalently bind the fluorescent label Cy5 before M13(-21) .

[0051] 2. Multiplex PCR

[0052] (1) The reaction system comprises the following components:

[0053] Template DNA 1.5ng / μL, Taq DNA polymerase 0.035U / μL, dNTPs 0.25mM, 1×PCR Buffer (containing 1.5mM MgCl 2 ), the forward and reverse primer concentrations of primers BBI131F, GBG230 and E776153 are shown in Table 2,...

Embodiment 2

[0061] 1. Genomic DNA extraction and primer synthesis

[0062] 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.

[0063] Synthesize group B primers DMt1H10, CBF156 and DBI28 according to the primer sequences in Table 1, add M13(-21) universal primer sequence to the 5' end of the primer leading strand, and then covalently bind fluorescent marker 6 before M13(-21) ' - FAM.

[0064] 2. Multiplex PCR

[0065] (1) The reaction system comprises the following components:

[0066] Template DNA 1.5ng / μL, Taq DNA polymerase 0.030U / μL, dNTPs 0.25mM, 1×PCR Buffer (containing 1.5mM MgCl 2 ), the forward and reverse primer concentrations of primers DMt1H10, C...

Embodiment 3

[0074] 1. Genomic DNA extraction and primer synthesis

[0075] Using the alfalfa variety Juneng as the test material, 12 individual plants were randomly selected, a sufficient amount of young leaves were cut, and after freezing and grinding with liquid nitrogen, genomic DNA was extracted by the CTAB method of Doyle et al. The quality and quantity of genomic DNA were detected by electrophoresis on % agarose gel.

[0076] Synthesize group C primers ABE93, CIC338 and AMTIC95 according to the primer sequences in Table 1, add the M13(-21) universal primer sequence to the 5' end of the primer leading strand, and then covalently bind the fluorescent label Cy5 before M13(-21) .

[0077] 2. Multiplex PCR

[0078] (1) The reaction system comprises the following components:

[0079] Template DNA 1.50ng / μL, Taq DNA polymerase 0.030U / μL, dNTPs 0.25mM, 1×PCR Buffer (containing 1.5mM MgCl 2), the forward and reverse primer concentrations of primers ABE93, CIC338 and AMTIC95 are shown in ...

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Abstract

The invention relates to a method for SSR (simple sequence repeat) analysis of tetraploid alfalfa by a multiplex PCR (polymerase chain reaction) fluorescence labeling technique. The method involves nine multiplex PCR groups (including 23 pairs of SSR primers and a universal primer) and corresponding PCR reaction systems and reaction programs. Amplification product separation is realized by conventional denatured polyacrylamide gel electrophoresis, a gradient multifunctional laser scanning imager (Amersham Biosciences, USA) is adopted for scanning and recording images under different wave lengths. 23 pairs of SSR labels are uniformly distributed on chromosomes and a label interval is not smaller than 10Mbp, labeled genotypes are easily recognizable, and the average genotype accuracy rate ishigh than 95%. By adoption of the scheme for genetic diversity analysis, group structure analysis and variety identification of tetraploid alfalfa, short time and high accuracy are realized, and testefficiency is remarkably improved.

Description

technical field [0001] The invention relates to a method for SSR analysis of tetraploid alfalfa by using multiple PCR fluorescent markers, 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 there are many varieties under study, the number of analysis units (genotypes) will be very large. Due to the limitations of actual ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6895
CPCC12Q1/6858C12Q1/6895C12Q2525/151C12Q2537/143C12Q2563/107C12Q2531/113
Inventor 桂枝高建明谢晓东
Owner TIANJIN AGRICULTURE COLLEGE
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