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SSR markers for plants and uses thereof

A technology of markers and plants, applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of undetected polymorphisms, etc.

Inactive Publication Date: 2014-04-30
ACGT INTELLECTUAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, according to the study by Sun et al., 2008, 17 SSR primers developed by the FIASCO (Fast Isolation of Repeat-Containing AFLP) protocol were not detected in 56 listed Chinese Jatropha curcas (J.curcas) polymorphism

Method used

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  • SSR markers for plants and uses thereof
  • SSR markers for plants and uses thereof
  • SSR markers for plants and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] [Example 1: PCR amplification of SSR markers using oligonucleotide primers in Table 1 and detection by capillary electrophoresis]

[0079] 10 μl of reaction mixture for SSR marker amplification consisted of: 2 mM MgCl 2 , 1×PCR buffer, 0.2 mM of dNTP mix, 250 nM of each primer of the primer pair, 1 U of Taq polymerase and 20 ng of DNA. Cycle conditions are denaturation at 94°C, 5min, 5 cycles: 94°C, 30s; 62°C, 30s (decrease 2°C to 52°C); 25 cycles: 94°C, 30s; 52°C, 30s, 72°C, 30s; Then at 72°C for 7min, and kept at 10°C. in 96-well Amplification is performed in the PCR system 9700. Other reaction mixtures, cycling conditions and thermal cyclers with heated lids can also be used.

[0080] For capillary electrophoresis, fluorescently labeled primers are used to amplify the products. Amplified products were analyzed by capillary electrophoresis. Dimensions are determined using standards. Samples and size standards (GeneScan600LIZ) were heated and loaded into an App...

Embodiment 2

[0082] [Example 2: Polyacrylamide gel electrophoresis (PAGE)]

[0083] PCR was performed using the same conditions as in Example 1. Amplification products were separated using polyacrylamide gel electrophoresis.

[0084] The amplified PCR products were heated with conventional formamide loading dye and 10 μL of each sample was separated in a 7% polyacrylamide gel (1 mm thick and 25 cm long) at 450 V for 6 h. Table 3 shows an example of polyacrylamide gel formulation.

[0085] Table 3: Polyacrylamide Formulations

[0086] 17.5ml

Concentrated stock acrylamide solution (19g acrylamide, 1g bisacrylamide in 100mL water)

10ml

5×TBE (1×TBE=0.09M Tris-boric acid, 0.002M EDTA)

22.5ml

water

220μl

10% ammonium persulfate (10%APS)

20 μL

TEMED

[0087] Add 10% APS and TEMED to aggregate.

[0088] After electrophoresis, the separated amplification products were visualized by silver staining or ethidium bromide staining. D...

Embodiment 3

[0089] [Example 3: Cluster Analysis]

[0090] Genotyping of 927 samples was performed using the 10 SSR markers of the present invention. For each marker, PCR was performed using the PCR mix shown in Table 4 in a volume of 10 μl.

[0091] Table 4: PCR mix

[0092] PCR mix

10 μl PCR

h 2 o

1.69

10×PCR buffer

1.00

50mM MgCl 2

0.40

10mM dNTPs

0.20

Primer mix F:R (25μM:50μM)

0.05

M13-labeled fluorescent forward primer (2.5 μM)*

0.50

Taq5u / μl

0.16

DNA (3ng / μl)

6.00

total capacity

10.00

[0093] * Indicates M13-labeled fluorescent forward primers (labeled fluorescent forward primers are also shown in Table 1 with *).

[0094] Samples and size standards (GeneScan600LIZ) were heated and loaded into an Applied Biosystems ABI3730xl Genome Analyzer (a fluorescence-based sequencer) according to the manufacturer's instructions. Scoring was performed with Applied B...

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Abstract

Simple sequence repeat (SSR) markers identified in Jatropha curcas and useful for the molecular genotyping of plants. are described. These markers may be used for identifying allele polymorphisms, identifying identical or related plants, differentiating plants and studying genetic diversity in a population. The markers may also be used in genetic and phenotype studies using statistical methods, for example, linkage analysis, association mapping, linkage disequilibrium and the like. The information may be used for breeding and / or selection of plants.

Description

【Technical field】 [0001] The present invention relates to the field of molecular genotyping. In particular, the invention relates to the identification and isolation of simple sequence repeat (SSR) markers and their application to genotyping. 【Background technique】 [0002] Jatropha curcas (Euphorbiaceae), also known as coral tree, is a non-food crop oleaginous-seeded tree (or large shrub) which can grow up to 5m. Jatropha (J. curcas) appears to be native to Central or South America. It now grows across the tropics and subtropics, such as in Africa and Asia. Naturally, it is cross-pollinated by insects, but can also be grown from cuttings. Jatropha (J. curcas) has never been widely cultivated for yield. [0003] The degree of genetic diversity is a prerequisite for crop improvement programs. In Jatropha curcas (J. curcas), the morphological characterization of genetic diversity can be biased by the strong influence of the environment even on highly heritable seed traits...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q2600/156
Inventor 谢钻珠杨淑华李荣华叶孙裕傅映鸣李盈桦
Owner ACGT INTELLECTUAL
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