CN102622634B

CN102622634B - Method for preparing bar code identities of apple germplasm resources

Info

Publication number: CN102622634B
Application number: CN201210094690.2A
Authority: CN
Inventors: 高源�; 王昆; 龚欣; 刘立军; 王大江; 刘凤之
Original assignee: Fruit Tree Institute of CAAS
Current assignee: Fruit Tree Institute of CAAS
Priority date: 2012-03-31
Filing date: 2012-03-31
Publication date: 2014-04-23
Anticipated expiration: 2032-03-31
Also published as: CN102622634A

Abstract

The invention provides a method for preparing bar code identities of apple germplasm resources. The method comprises the following steps of: extracting genomes DNA (Deoxyribonucleic Acid) of the apple germplasm resources and choosing SSR (Simple Sequence Repeat) molecular marker primers with good polymorphism; carrying out PCR (Polymerase Chain Reaction) amplification on the DNA of the apple germplasm resources; detecting PCR products by using polyacrylamide gel electrophoresis to obtain an identification map; performing data analysis to acquire data 0 or 1, and sequentially ordering the data 0 or 1 acquired at all SSR sites; and generating the special bar code identity of each apple germplasm resource by using bar code generation software. The method provided by the invention can be used for generating simple and available bar code identities for the authentication data of the apple germplasm resources by using the SSR molecular marks, so that the identities are conveniently distinguished and have the advantages of good stability, reliable results, strong practicability and suitableness for application to production, scientific research or breeding of apple species.

Description

A kind of preparation method of apple germplasm resources bar code identification

Technical field

The present invention relates to Fruit Tree Germplasm Resources recognition technology, specifically a kind of preparation method of apple germplasm resources bar code identification.

Background technology

Malus ( malusmill.) germ plasm resource is abundant, and variety type is various, comprises a large amount of wild species, semi-wild kind and cultivar.China is one of important centre of origin of apple germplasm resources in the world, and germ plasm resource is extremely abundant, and originating in Chinese apple has 24 kinds, and its kind of lower mutation, modification are various especially.Except wild species, apple resource individual difference is very unobvious, and various places exchange frequently in addition, cause synonym, homonym phenomenon general.The precise Identification of apple germplasm resources is the prerequisite that germ plasm resource is preserved and utilized, and the authentication method of apple germplasm resources was mainly the methods such as morphological observation, isodynamic enzyme in the past at present.Along with the development of molecular marking technique, molecular marking technique is more and more applied in the characterization and evaluation of apple germplasm resources.

From Litt and Luty first by SSR technology (simple sequence repeat, be that simple sequence repeats, claim again micro-satellite) for since carrying out human genetics research, SSR technology because polymorphism is high, it is wide, reproducible to distribute, codominance and cost be widely used compared with the advantage such as low, be also therefore applied in the evaluation of apple germplasm resources.But the appraising datum that utilizes SSR molecular labeling to obtain apple genome DNA cloning is more abstract, disperse, and be unfavorable for directly application in production or scientific research.

Summary of the invention

In order to utilize SSR molecular marker data to obtain apple germplasm resources bar code identification, the present invention proposes a kind of preparation method of apple germplasm resources bar code identification.The method utilizes the genomic DNA of SSR primer pair apple germplasm resources to increase, utilize polyacrylamide gel electrophoresis to detect, by data analysis, obtain 0 or 1 data, 0 or the 1 data arranged in sequence that all SSR site is obtained, utilize bar code to generate software, generate bar code identification every part of apple germplasm resources uniqueness and that can directly utilize in production or scientific research, solve the technical matters of apple germplasm resources identification.

The scheme that technical solution problem of the present invention adopts is:

Extract the genomic DNA of apple germplasm resources, select the SSR molecular labeling primer that polymorphism is good, the DNA of apple germplasm resources is carried out to PCR, and PCR, increases, utilize polyacrylamide gel electrophoresis to detect PCR product, obtain and identify collection of illustrative plates, by data analysis, obtain 0 or 1 data, the 0 or 1 data arranged in sequence that all SSR site is obtained, utilize bar code to generate software, generate the bar code identification of every part of apple germplasm resources uniqueness.

Good effect: the inventive method can generate simple available bar code identification to the appraising datum of apple germplasm resources by SSR molecular labeling, makes it to be convenient to identification, has good stability, reliable results, practical advantage.Suitablely in production, scientific research or apple variety seed selection, apply.

Accompanying drawing explanation

Fig. 1 is bar code data Pareto diagram of the present invention

Bar code has contour not wide markings, and bottom is the coding of genomic data 0 or 1.

Embodiment

The pcr amplification of SSR primer: the genomic DNA that extracts high-quality apple germplasm resources, select the SSR molecular labeling primer that polymorphism is good, utilize 20 μ l reaction systems (containing 20～40ng genomic DNA, Tris – HCl10 mmol/L (25 ℃, pH9.0), KCl 50mmol/L, MgCL21.5mmol/L, dNTP0.2 mmol/L, primer 0.5 μ mol/L tagpolymerase 1U), on PCR instrument, the genomic DNA to apple germplasm resources increases.

Electrophoresis detection and data analysis: utilize polyacrylamide gel electrophoresis to detect the amplified production of every pair of SSR primer pair apple germplasm resources genomic DNA, obtain gel electrophoresis spectrum, by data statistic analysis, obtain 0 or 1 data.

Bar code generates: the 0 or 1 data arranged in sequence that all SSR site is obtained, and utilize bar code to generate software, generate the bar code identification of every part of apple germplasm resources uniqueness.

Embodiment

Shown in Fig. 1, using 41 parts of Apple Materials as examination material, utilize SSR primer, under 58 ℃ of conditions, pcr amplification is carried out in annealing, utilizes polyacrylamide gel electrophoresis to carry out electrophoretic process to PCR product, obtains evaluation collection of illustrative plates, according to collection of illustrative plates, determines allele data.Utilize 5 pairs of SSR primers to identify and distinguish all for the apple germplasm resources material trying, the allele data in 5 SSR sites that obtain are converted into 0 or 1 data, utilize bar codes technique, the specific data by 41 parts of apple germplasm resources materials in 5 SSR sites changes into the bar code identification of every part of material uniqueness.The Fuji apple of take is wherein the generative process of example explanation bar code:

Fuji apple in the allele data in 5 SSR sites in Table 1.

Table 1 Fuji apple is in the allele data in 5 SSR sites

Primer numbering	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16
																	XX0004	0	0	0	0	0	0	0	0	1	0	0	0	1	?	?	?
XX0005	0	0	0	0	0	0	0	0	1	0	1	0	0	0	1	1
																	XX182	0	0	0	0	0	0	0	0	0	0	0	0	?	?	?	?
XX201	0	0	0	0	0	1	0	0	0	0	0	?	?	?	?	?
																	XX203	1	0	0	1	0	0	0	0	0	0	0	?	?	?	?	?

The allele data arranged in sequence in 5 sites that Fuji apple is obtained is: 00,000,000,100,010,000,000,010,100,011,000,000,000,000,000,001,000,001,001 0000000.

With primer XX004, XX005, XX182, XX201, XX203, obtain respectively allele data.According to allele data, on bar code, carry out 0 or 1 mark, form genomic data coding, with strip line arranged side by side, represent.

This coding can be understood and identify.

The bar code identification that utilizes bar code software to generate Fuji apple uniqueness is:

Formed bar code can scan every part of apple germplasm resources material in conjunction with bar code scanner, is convenient to the evaluation of apple germplasm resources in production and scientific research, has solved to a certain extent the problem of homonym and synonym.

Claims

1. the preparation method of an apple germplasm resources bar code identification, it is characterized in that: the pcr amplification of SSR primer: the genomic DNA that extracts high-quality apple germplasm resources, select the SSR molecular labeling primer that polymorphism is good, utilize 20 μ l reaction systems, containing 20～40ng genomic DNA, temperature is the Tris – HCl10 mmol/L that 25 ℃, pH are 9.0, KCl 50mmol/L, MgCL21.5mmol/L, dNTP0.2 mmol/L, primer 0.5 μ mol/L tagpolymerase 1U, the genomic DNA to apple germplasm resources on PCR instrument increases;

Electrophoresis detection and data analysis: utilize polyacrylamide gel electrophoresis to detect the amplified production of every pair of SSR primer pair apple germplasm resources genomic DNA, obtain gel electrophoresis spectrum, by data statistic analysis, obtain 0 or 1 data;