CN103525813A

CN103525813A - SSR primer group and method for identification of hot pepper species thereby

Info

Publication number: CN103525813A
Application number: CN201310516095.8A
Authority: CN
Inventors: 何建文; 苏丹; 姜虹; 韩世玉; 刘永翔; 刘作易
Original assignee: GUIZHOU PEPPER RESEARCH INSTITUTE
Current assignee: GUIZHOU PEPPER RESEARCH INSTITUTE
Priority date: 2013-10-28
Filing date: 2013-10-28
Publication date: 2014-01-22
Anticipated expiration: 2033-10-28
Also published as: CN103525813B

Abstract

The invention relates to the molecular biological technology field, and concretely relates to an SSR primer group and a method for identification of hot pepper species thereby. The SSR primer group is composed of 20 primers. The method for identification comprises steps: first, DNA extraction of hot pepper leaves; second, PCR amplification; third, electrophoresis detection; fourth, data recording; and fifth, result determination. The primers have high specificity and high amplification efficiency, and can identify different species of hot peppers rapidly. The method is advantaged by short period, low cost, reliable results, manpower saving, material resource saving and land resource saving.

Description

SSR primer sets and utilize this primer sets to identify the method for capsicum variety

Technical field

The present invention relates to field of molecular biotechnology, is a kind of primer and the method for utilizing this primer evaluation capsicum variety thereof specifically.

Background technology

Cultivar identification is the important method in agriculture production, crop breeding and Seed Inspection, is also cover crop kind, prevents the important means that fake and forged kind comes into the market.Traditional seed testing method has field macroscopical identification and Grain Morphology evaluation etc., and still, the required cycle of field macroscopical identification is long, expends a large amount of human and material resources and financial resources.Effect of Pepper Seed Quality monitoring is the important step that capsicum produces.In capsicum produces, it is serious that local variety mix phenomenon, and conventional discriminating is consuming time longer, field differentiates that difficulty is larger, along with the develop rapidly of Protocols in Molecular Biology, molecular marking technique is constantly ripe, has opened up approach to the evaluation of crop kind genomic level.

Summary of the invention

For realizing the problem occurring in above-mentioned capsicum variety evaluation, the object of the invention is to, propose the molecular detecting method that capsicum variety is identified, the method is utilized molecular marking technique, realizes capsicum variety identify in seedling stage, there is the cycle short, cost is low, and reliable results is saved manpower, material resources and land resources, improved the accuracy of cultivar identification greatly.

To achieve these goals, the technical solution used in the present invention is: a kind of SSR primer sets, and this SSR primer sets sequence is as follows:

Another object of the present invention is to provide a kind of method that the SSR of utilization primer sets is identified capsicum variety, comprises the steps:

(1) Pepper Leaves DNA extraction;

(2) pcr amplification;

(3) electrophoresis detection;

(4) data logging;

(5) result is judged.

Further, in described step (2), pcr amplification program is 94 ℃ of denaturation 5min; 94 ℃ of sex change 45s, 60 ℃ of renaturation 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.

Further, in described step (2), pcr amplification system comprises 20ng template DNA, 2.5mmol/L Mg2+, 0.2mmol/L dNTPs, 1U TaqDNA polysaccharase, every primer 400nmol/L, 10 * Buffer.

Further, when described step (5) result is judged, if two kinds (strain or self-mating system) have different equipotentials at two or more SSR marker sites, be judged to be different varieties.

Useful technique effect of the present invention is: primer specificity of the present invention is high, and amplification efficiency is good, and the different varieties of energy Rapid identification capsicum, has the cycle short, and cost is low, and reliable results is saved human and material resources and land resource characteristics.

Accompanying drawing explanation

Fig. 1 the inventive method detects H224, H137, H089, H204, H042023A, H009, H015, H004, H102, H042025D totally 10 self-mating system electrophoresis result figure;

Fig. 2 utilizes the present invention to detect peppery No. four of perfume, first peppery Hua Shuai, first peppery Hong Feng and graceful enlightening gold bar electrophoresis result figure.

Embodiment

Below in conjunction with concrete embodiment, the present invention will be further described

Embodiment 1

(1) capsicum extracting genome DNA: select H224, H137, H089, H204, H042023A, H009, H015, H004, H102, H042025D Gong10Ge Ben institute self-fertile self-mating system material, treat to gather seedling stage the fresh young leaflet tablet 2～3g of plant, adopt improved CTAB method to extract DNA sample.Concrete steps are: 1. with 1.5ml centrifuge tube lid, get seedling leaves (approximately 12～15mg), be placed on ice; 2. add extract 100 μ l, grind pulping; 3. 65 ℃ of water-bath 20-40min, put upside down centrifuge tube therebetween twice; 4. take out micro-centrifuge tube, place 5-10min, be chilled to room temperature; 5. the phenol that adds 100 μ l: chloroform: primary isoamyl alcohol (25:24:1), turns upside down and mixes 5min, the centrifugal 10min of 3200-12000rpm; 6. shift supernatant to another 1.5ml centrifuge tube, add 7.5 μ l5mol/L NaCl, with 100 μ l dehydrated alcohol (freezing or room temperature) precipitation DNA; 7. of short duration centrifugal, remove ethanol, use 75% washing with alcohol, the centrifugal 5min of 12000rpm, abandons ethanol, and seasoning is to there is no ethanol smell; 8. add 200 μ l sterilizing ddH2O or TE (pH=8.0), 4 ℃ of preservations are stand-by.

(2) take the capsicum genomic dna extracting is template, utilizes 20 pairs of SSR primers that filter out to carry out pcr amplification.

Above-mentioned PCR reaction is totally 25 μ L: comprising 20ng template DNA, and 2.5mmol/L Mg ²⁺, 0.2mmol/L dNTPs, 1U TaqDNA polysaccharase, every primer 400nmol/L, 10 * Buffer(damping fluid).Pcr amplification program is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 45s, 60 ℃ of renaturation 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.

(3) electrophoresis detection is inserted electrophoresis chamber by the gel slab making, and establishes 45 ℃, rated output 70W, first prerunning 30min, then detection sample is added in groove, establish 50 ℃, under rated output 75-80W, electrophoresis 1h, when indicator dimethylbenzene green grass or young crops is during apart from about 1cm left and right, sheet glass bottom, finishes electrophoresis.

(4) data logging, is placed on running gel under lamp box and observes, and different varieties is designated as " 1 " at the band of the clear appearance in same site, does not have band to be designated as " 0 ", generates thus " 1 ", " 0 " matrix.

(5) result is judged

Specificity criterion with molecule marker difference value >=2 as kind (or strain, self-mating system), that is if two kinds (or strain, self-mating system) have different equipotentials at two or more SSR marker sites, can be judged to be different varieties (or strain, self-mating system) (Fig. 1).

Fig. 1 mark CA514272 can differentiate H224, H089, H042023A and H042025D to come respectively at other kind.

Embodiment 2

(1) capsicum extracting genome DNA: select fragrant peppery No. four, first peppery Hua Shuai, first peppery Hong Feng and 4 capsicum varieties of graceful enlightening gold bar, treat to gather seedling stage the fresh young leaflet tablet 2-3g of plant, adopt improved CTAB method to extract DNA sample.Concrete steps are: 1. with 1.5ml centrifuge tube lid, get seedling leaves (about 12-15mg), be placed on ice; 2. add extract 100 μ l, grind pulping; 3. 65 ℃ of water-bath 20-40min, put upside down centrifuge tube therebetween twice; 4. take out micro-centrifuge tube, place 5-10min, be chilled to room temperature; 5. the phenol that adds 100 μ l: chloroform: primary isoamyl alcohol (25:24:1), turns upside down and mixes 5min, the centrifugal 10min of 3200-12000rpm; 6. shift supernatant to another 1.5ml centrifuge tube, add 7.5 μ l5mol/L NaCl, with 100 μ l dehydrated alcohol (freezing or room temperature) precipitation DNA; 7. of short duration centrifugal, remove ethanol, use 75% washing with alcohol, the centrifugal 5min of 12000rpm, abandons ethanol, and seasoning is to there is no ethanol smell; 8. add 200 μ l sterilizing ddH2O or TE (pH=8.0), 4 ℃ of preservations are stand-by.

Above-mentioned PCR reaction is totally 25 μ L: comprising 20ng template DNA, and 2.5mmol/L Mg2+, 0.2mmol/L dNTPs, 1U TaqDNA polysaccharase, every primer 400nmol/L, 10 * Buffer(damping fluid).Pcr amplification program is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 45s, 60 ℃ of renaturation 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.

(3) electrophoresis detection is inserted electrophoresis chamber by the gel slab making, if 45 ℃, rated output 70-75W, first prerunning 30min, then adds detection sample in groove, establishes 50 ℃, under rated output 75-80W, electrophoresis 1-2 hour, when indicator dimethylbenzene green grass or young crops is during apart from about 1cm left and right, sheet glass bottom, finish electrophoresis.

Result is judged

(5) the specificity criterion as kind (or strain, self-mating system) with molecule marker difference value >=2, that is if two kinds (or strain, self-mating system) have different equipotentials at two or more SSR marker sites, can be judged to be different varieties (or strain, self-mating system) (Fig. 2).

Fig. 2 mark Hpms1-43 can come graceful enlightening gold bar, perfume respectively for peppery No. four with the peppery red rich discriminating of the peppery Hua Shuai of unit and unit.

Claims

1.SSR primer sets, is characterized in that, this SSR primer sets sequence is as follows:

2. utilize SSR primer sets to identify a method for capsicum variety, it is characterized in that, comprise the steps:

(1) Pepper Leaves DNA extraction;

(2) pcr amplification;

(3) electrophoresis detection;

(4) data logging;

(5) result is judged.

3. utilize according to claim 2 SSR primer sets to identify the method for capsicum variety, it is characterized in that, in described step (2), pcr amplification program is 94 ℃ of denaturation 5min; 94 ℃ of sex change 45s, 60 ℃ of renaturation 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of preservations.

4. utilize according to claim 3 SSR primer sets to identify the method for capsicum variety, it is characterized in that, in described step (2), pcr amplification system comprises 20ng template DNA, 2.5mmol/L Mg2+, 0.2mmol/L dNTPs, 1U TaqDNA polysaccharase, every primer 400nmol/L, 10 * Buffer.

5. utilize according to claim 2 SSR primer sets to identify the method for capsicum variety, it is characterized in that, when described step (5) result is judged, if two kinds have different equipotentials at two or more SSR marker sites, be judged to be different varieties.