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Creation method of sweet pepper SSR finger-print and application thereof

A fingerprint and sweet pepper technology, which is applied in the field of establishment of sweet pepper SSR fingerprints, can solve problems such as the lack of and no establishment of methods for constructing sweet pepper DNA fingerprints, and achieve the effect of protecting intellectual property rights

Inactive Publication Date: 2010-06-23
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is a lack of research on the DNA fingerprint of sweet pepper SSR, and there is no establishment of a perfect method for constructing the DNA fingerprint of sweet pepper.

Method used

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  • Creation method of sweet pepper SSR finger-print and application thereof
  • Creation method of sweet pepper SSR finger-print and application thereof
  • Creation method of sweet pepper SSR finger-print and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the extraction of sweet pepper total DNA

[0033] (1) Test method:

[0034] 1. Collect fresh seedlings (stored at -80°C), grind them into powder with liquid nitrogen in a pre-cooled mortar at -20°C.

[0035] 2. Transfer the powder obtained in step 1 to a 1.5ml centrifuge tube pre-cooled at -20°C, and store in a -20°C refrigerator until use (overnight).

[0036] 3. Add 600ul SDS extract solution to the centrifuge tube containing the powder in step 2, shake well, and incubate at 65°C for 30min. Oscillate 3-4 times occasionally.

[0037] 4. Then add 1 / 4 volume (about 100 μl) of KAC, shake well, and ice-bath for 30 minutes.

[0038] 5. Then add 1 / 2 volume (about 300-400 μl) of chloroform:isoamyl alcohol (24:1, volume ratio), and shake well on a shaker (120 rpm, 30 min).

[0039] 6. Centrifuge in a desktop centrifuge: 6000-8000rpm, 15min, take the supernatant.

[0040] 7. Then add an equal volume (about 400ul) of chloroform:isoamyl alcohol (24:1, volume ra...

Embodiment 2

[0052] Example 2: PCR amplification of SSR molecular markers

[0053] (1) Test method:

[0054] Reagents used

[0055] (1) SSR primer: synthesized by Takara Company, the concentration is 2pmol / μl

[0056] (2) TEMED: Tetramethylethylenediamine

[0057] (3) DNA Ladder (50bp): Nanjing Sangon Company

[0058] (4) Indicator:

[0059] 0.25% (w / v) Bromophenol Blue (stored at 4°C)

[0060] 0.25% (w / v) xylene cyanol FF (Xglue Cyanoll FF)

[0061] 40% (w / v) sucrose in water

[0062] (5) 40% (w / v) Acri / bis-Acri gel stock solution

[0063] Acrylamide (Acri) 38 grams

[0064] N, N-methylenebisacrylamide (bis-Acri) 2 g

[0065] ddH 2 O to 100ml, store at 4°C

[0066] (6) 10% (w / v) of AP (ammonium persulfate)

[0067] (7) 10% (w / v) sodium thiosulfate (mother liquor)

[0068] 1.2 PCR reaction

[0069] PCR reaction system (10μl)

[0070] DNA (10ng / μl) 1μl

[0071] Primer (2pmol / μl) 0.7μl

[0072] 10×Buffer (freeMgCl 2 ) 1μl

[0073] dNTP (2.5mM) 0.2μl

[0074] MgCl 2 (25mM...

Embodiment 3

[0099] Embodiment 3: data statistics and analysis

[0100] (1) Test method:

[0101] According to the amplified band type, select the primers that only amplify the main band in most of the materials, and have polymorphism and good repeatability to distinguish all the test materials, and are used to construct 10 primers for this experiment. Fingerprints of sweet pepper varieties and inbred lines.

[0102] (2) Test results:

[0103] Among the SSR primers, there are 12 pairs of primer combinations, CM2, CM26, CM28, CM46, CM53, CM62, CM63, CM76, CM77, CM86, and CM96, which can separate the tested materials. Primer CM63 has a characteristic band for material 4; primer CM28 has a characteristic band for material 5 (see figure 1 ); Primer CM76 has characteristic bands to materials 4 and 6; Primer CM86 has characteristic bands to materials 4 and 5; Primer CM96 has characteristic bands to material 6 (see figure 2 ); primers CM46 and CM62 have characteristic bands for material 10 a...

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Abstract

The invention discloses a creation method of sweet pepper SSR finger-print and an application thereof. The method is as follows: screening SSR primer for different sweet pepper varieties and inbred line total DNA to obtain 12 pairs of SSR polymorphism molecular markers, and performing electrophoretic separation to obtain corresponding SSR molecular marker polymorphism gram through the 12 pairs of SSR polymorphism molecular markers. The technology can make definite conclusion on the sweet pepper genetype, performs genetic classification to sweet pepper breeding material which is not recorded on an exact pedigree, makes up the deficiency of the pedigree record, also can be used for identifing the hereditary feature of the inbred line of sweet pepper, and protects the variety of the inbred line against invasion.

Description

technical field [0001] The invention relates to a germplasm identification technology, in particular to a method for establishing a sweet pepper SSR fingerprint and its application. Background technique [0002] DNA molecular marker technology essentially refers to a specific DNA fragment that can reflect a certain difference in the genome of an individual or population. Simple sequence repeat (SSR), also known as microsatellite DNA (microsatellite DNA), refers to a tandem repeat of 1 to 5 nucleotides in a DNA molecule. With its advantages of random distribution, high information content and polymorphism, co-dominance and Mendelian inheritance in animal and plant genomes, SSR is widely used in the construction of genetic maps, genetic diversity analysis, kinship identification, DNA fingerprinting, and functional genes. Marking and other aspects have recognized advantages and application prospects. DNA molecular markers mostly represent the DNA differences between biologica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 薛永姚泉洪彭日荷熊爱生李贤龚繁荣查丁石朱为民
Owner SHANGHAI ACAD OF AGRI SCI
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