CN103993098A - SRAP (Sequence-Related Amplified Polymorphism) molecular marking method related to color character of Ulmus pumila cv.jinye. and application thereof - Google Patents

Abstract

The invention discloses an SRAP (Sequence-Related Amplified Polymorphism) molecular marking method related to color character of Ulmus pumila cv.jinye. and an application thereof, and relates to an SRAP molecular marking method and an application thereof. The method comprises the following steps: I, collecting leaves of different colors of Ulmus pumila cv.jinye. and calibrating the colors; II, respectively extracting total RNAs (Ribonucleic Acid) of golden and forest green leaves on each Ulmus pumila cv.jinye.; III, detecting RNA concentration; IV, carrying out cDNA-SRAP (Complementary Deoxyribonucleic Acid) amplification on differential fragments; and V, recovering, sequencing and analyzing the differential fragments. The method provided by the invention has the characteristics of simplicity in operation, accuracy in result, visualized result, practicality and the like. The method provided by the invention is used for researching genes related to different colors of Ulmus pumila cv.jinye.

Description

Relevant SRAP molecule marking method and the application of China's gold leaf Siberian elm leaf look proterties

Technical field

The present invention relates to a kind of SRAP molecule marking method and application.

Background technology

In afforestation, that color leaf seeds have is bright in luster, viewing period long, be easy to form the feature of large color lump view, the garden landscape level that it is abundant, made up Urban Color dull, lack the shortcoming that aspect changes.Various countries for color leaf plant the application on gardens extraordinarily pay attention to.In the last hundred years, developed countries is being done a lot of work aspect the seed selection of color leaf plant variety and cultivation.In Germany, the Chang Seye seeds that are applied in city trees and shrubs have 64 kinds, belong to 17 kinds, and 35 subspecies or cross-fertilize seed, and colorful configure properly, build graceful garden landscape effect.Domestic in color leaf plant breeding the work aspect introducing a fine variety start more late, still in the starting stage.The most of color leaf seeds of China are all drawn from external, as Ligustrum vicaryi, photinia glabra etc. at present.The color leaf floristics that the northern area of China is introduced and applied with Beijing, Dalian is more.But the area to the north of Liaoning, applicable color-leaved woody plant kind reduces rapidly.The color leaf new variety of plant that China gold leaf Yu Shiyou Hebei Adademy of Forestry Sciences, Shijiazhuang City Lv Yuanda Garden Engineering company limited cultivate.On June 26th, 2004, passed through Hebei province's appraisal of scientific and technological achievements.Achievement aggregate level is international advanced, has passed through Hebei province's high quality tree species in November, 2004 and has assert.The features such as China gold leaf elm is the tall all suitable good color leaf seeds of filling with, and has blade golden yellow gorgeous, and tree crown is plentiful, and high resistance is cold, arid are the successful color leaf neies variety of plant of the autonomous cultivation of China.Rapidly, branch is intensive for China's gold leaf elm growth, and resistance to intensity is pruned, and moulding is abundant, both can cultivate as arbor, as Scenery of gardens tree, can cultivate into shrub again, is widely used in hedgerow, colour band.China's gold leaf elm well developed root system, impoverishment tolerant, soil conservation ability is strong, except for urban afforestation, also can be widely used in Landscape greening the landscape ecological forest and soil and water conservation forest.The mutation of China gold leaf Yu Shi China indigenous tree American elm, has extremely strong adaptability to cold, dry climate.At the vast northeast of China, the Northwest's well-grown, there is stronger saline-alkali tolerance simultaneously, can widespread use in coastland.Its growth district north to Heilungkiang, the Inner Mongol, to the east of the Yangze river and Huai river Plain of North of Yangtze River, to Gansu, Qinghai, Xinjiang, reach the provinces such as Jiangsu, Hubei in the south, and in China gardens, range of application is wider.

In gold leaf elm application process, we find that its tree crown is inner and bottom leaf colour cast is green, and shady face leaf look cadmium yellow degree is poor, and the whole tree crown leaf of the gold leaf elm look on driveway side is inhomogeneous, in some environment, its blade presents " atavism " in various degree, thereby has reduced its ornamental value.The mechanism that produces these phenomenons is not clear at present, should take the genetic stability of how measure, its kind and leaf look thereof to need further research to the response mechanism of environmental factors etc.

CDNA-SRAP is New molecular marker SRAP (the sequence-related amplified polymorphism of a kind of PCR-based of calendar year 2001 Li and Quiros proposition, SRAP) technology, research also shows that SRAP also can be applied to cDNA Differential expression analysis.

The gordian technique of cDNA-SRAP is in the design of its primer.Study surperficial exon in the higher region of GC content, intron and other regulating and controlling sequence are in the higher region of AT content, according to this genomic gene characteristic distributions, Li and Quiros develop SRAP marking method, this marking method is used two based on the high exon region of GC content and the high intron of AT content and the random primer in regulation and control region, genome to be increased, thereby produce based on sequence variations and the dominant and codominant marker based on length of intron variation, it is worth mentioning that, the codominant marker's that SRAP produces efficiency for example, much larger than other labeling technique (AFLP), and codominant marker contributes to molecular mark.Another one distinguishing feature is that the differential fragment of cDNA-SRAP generation is mostly relevant with proterties, can regard potential functional gene as.CDNA-SRAP is a kind of new technology that can carry out differential expression research without any sequence information, one of this utilization is combined in the immobilized primer of exon 1 and one and is combined in intron, control region and transcribed spacer template cDNA is increased, research shows that general exon is in being rich in the region karyomit(e) of GC, goes out to comprise like this sequence of these components by CCGG sequence with regard to specific amplified likely.

Since cDNA-SRAP technological development, on the plants such as paddy rice, wild cabbage, muskmelon, be applied at present.Li etc. utilize SRAP technology to carry out increasing after reverse transcription to the mRNA of the hybridization double diploid of Cauliflower and asparagus broccoli, with 88 cDNA samples of 48 pairs of combination of primers marks, obtain 281 polymorphic bandses.Lu Yong congruence application SRAP technology is carried out difference to the salt-tolerance character of Value of Spartina Anglica and is shown research, and identifying one has the differential fragment of 30% similarity with the beta-1,3-glucanase gene of paddy rice.Ma Aifen etc. select the recombinant inbred lines (RILs) of cultivating 7 years, utilize cDNA-SRAP method to carry out difference and show research, 996 pairs of SRAP combination of primers amplify 2100 bands, 2 times amplification repetition rate is 65.2%, wherein between the black seed material of Huang, repeating the stable differential fragment occurring has 12, finds that 2 fragments have very high homology with NAD+ADP ribose transferring enzyme and the little peptide transfer protein 3 of Arabidopis thaliana respectively.But also do not have cDNA-SRAP technology to analyze in application aspect leaf look difference expression gene in screening.

Summary of the invention

The object of the present invention is to provide relevant SRAP molecule marking method and the application of a kind of Chinese gold leaf Siberian elm leaf look proterties.

The relevant SRAP molecule marking method of the present invention's China's gold leaf Siberian elm leaf look proterties, carries out according to the following steps:

One, the collection of the different leaf look of Chinese gold leaf elm blade and leaf colour code are fixed:

Select the identical and healthy Chinese gold leaf elm plant of the 10 strain age of trees, label is 1～10 respectively, leaf colour code adopts the comparison of international color Standard colour board surely, choose the healthy young leaflet tablet of identical colour, from every strain, gather the golden blade of #FFD700 and the green blade of #228B22 forest, seal immediately cryopreservation;

Two, extract respectively total RNA of the golden blade of #FFD700 and the green blade of #228B22 forest on every strain China gold leaf elm plant;

Three, RNA concentration detects:

Adopt trace dna Protein Detection instrument to detect RNA concentration, use the ddH without RNase ₂rNA between O balance sample makes its concentration identical, be 50ng/ μ l, the RNA of the golden blade of #FFD700 that gets 1～10 strain of equivalent builds golden blade RNA mixing pit, and the RNA of the green blade of #228B22 forest that gets 1～10 strain of equivalent builds green blade RNA mixing pit;

Four, cDNA-SRAP amplification differential fragment:

A, the first chain cDNA's is synthetic: adopt M-MLV reversed transcriptive enzyme, respectively golden blade RNA mixing pit is become to cDNA, the aseptic ddH of product with the RNA reverse transcription in green blade RNA mixing pit ₂5 times of O dilutions, save backup in-20 ℃;

SRAP amplification, PCR product electrophoresis and the detection of b, cDNA:

With 25 upstream primers and 25 downstream primers, carry out random combine, 2 cDNA mixing pits are carried out to pcr amplification;

PCR reaction system: 10 * buffer1.2 μ L, Mg ²⁺25nmolL ^-11 μ L, upstream primer 5nmolL ^-11 μ L, downstream primer 5nmolL ^-11 μ L, dNTP2nmolL ^-12 μ L, Taq archaeal dna polymerase 0.5U0.5 μ L, the first chain cDNA cut back 1 μ L, adds water to 12 μ L;

Pcr amplification condition is: 94 ℃ of denaturation 5min, and 94 ℃ 1min-35 ℃ 1min-72 ℃ 1min5 circulation, 94 ℃ 1min-50 ℃ 1min-72 ℃ 1min35 circulation, 72 ℃ are extended 10min, 4 ℃ of termination reactions.

From the PCR reaction result selection differences band of 100 pairs of primers of random combine, clone;

Five, the recovery of differential fragment, order-checking and analysis.

Utilize above-mentioned SRAP molecule marking method to carry out Chinese gold leaf Siberian elm leaf aberration recessive allele research.

Beneficial effect of the present invention:

(1) the present invention utilizes international color Standard colour board comparison gold leaf Siberian elm leaf look, can effectively avoid error, realizes the stdn of color, for the leaf form and aspect correlation gene screening more convincing.

(2) adopt the present invention to adopt the different colours blade of a set of vegetable material to be built into the mixing pit of same colour system, this mixing pit is except leaf look color proterties difference, all the other proterties are identical, can effectively discharge other proterties differences and disturb, and guarantee the accuracy of result.

(3) the present invention have simple to operate, result accurately, the feature such as practical directly perceived, can promote cDNA-SRAP technology in leaf form and aspect, close on character analysis accurately, fast, application easily, apply this screening method simultaneously, to analyzing leaf form and aspect correlation gene, find that new gene is significant.

Accompanying drawing explanation

Fig. 1 is the amplification of two kinds of leaf looks of the Chinese gold leaf elm of embodiment 2

Embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.

Embodiment 1: Chinese gold leaf Siberian elm leaf look proterties related SR AP molecule marker primer screening

The present embodiment Chinese gold leaf elm used is that commercial sources purchase obtains.

One, the collection of the different leaf look of Chinese gold leaf elm blade and leaf colour code are fixed:

Select the identical and healthy Chinese gold leaf elm plant of the 10 strain age of trees, label is 1～10 respectively, leaf colour code adopts the comparison of international color Standard colour board surely, choose the healthy young leaflet tablet of identical colour, from every strain, gather the golden blade of #FFD700 and the green blade of #228B22 forest, seal immediately cryopreservation;

Two, the total RNA that extracts respectively the golden blade of #FFD700 and the green blade of #228B22 forest on every strain China gold leaf elm plant, the extracting method of total RNA adopts improved Trizol method, and concrete grammar is as follows:

A, get 0.2g China gold leaf Siberian elm leaf sheet, remove petiole and Zhong Mai, in liquid nitrogen, grind, add homogenate after 1ml Trizol;

The standing 5min of room temperature after B, homogenate, in 12000 turn/min, 4 ℃ of centrifugal 5min;

C, supernatant liquor is transferred in 1.5ml centrifuge tube, adds chloroform, the volume of chloroform is 1/5 of supernatant liquor volume, and concussion mixes, the standing 15min of room temperature;

D, in 12000 turn/min, 4 ℃ of centrifugal 15min, supernatant is transferred in another centrifuge tube, add isopyknic Virahol, the standing 10min of room temperature;

E, in 12000 turn/min, 4 ℃ of centrifugal 10min, in precipitation, add the ethanolic soln washing precipitation of the volumetric concentration 75% of 1ml, in 12000 turn/min, 4 ℃ of centrifugal 5min;

F, abandon supernatant, retain precipitation, dry, the DEPC that is dissolved in 30～50 μ l processes in water;

Three, RNA concentration detects:

Adopt trace dna Protein Detection instrument to detect RNA concentration, use the ddH without RNase ₂rNA between O balance sample makes its concentration identical, be 50ng/ μ l, the RNA of the golden blade of #FFD700 that gets 1～10 strain of equivalent builds golden blade RNA mixing pit, and the RNA of the green blade of #228B22 forest that gets 1～10 strain of equivalent builds green blade RNA mixing pit;

Four, cDNA-SRAP amplification differential fragment:

1, the first chain cDNA's is synthetic: adopt M-MLV reversed transcriptive enzyme (operating according to M-MLV reversed transcriptive enzyme specification sheets), respectively golden blade RNA mixing pit is become to cDNA, the aseptic ddH of product with the RNA reverse transcription in green blade RNA mixing pit ₂5 times of O dilutions, save backup in-20 ℃;

2, the SRAP of cDNA amplification, PCR product electrophoresis and detection:

With 25 upstream primers and 25 downstream primers in table 1, carry out random combine, 2 cDNA mixing pits are carried out to pcr amplification.

PCR reaction system: 10 * buffer1.2 μ L, Mg ²⁺25nmolL ^-11 μ L, upstream primer 5nmolL ^-11 μ L, downstream primer 5nmolL ^-11 μ L, dNTP2nmolL ^-12 μ L, Taq archaeal dna polymerase 0.5U0.5 μ L, the first chain cDNA cut back 1 μ L, adds water to 12 μ L;

Pcr amplification condition is: 94 ℃ of denaturation 5min, and 94 ℃ 1min-35 ℃ 1min-72 ℃ 1min5 circulation, 94 ℃ 1min-50 ℃ 1min-72 ℃ 1min35 circulation, 72 ℃ are extended 10min, 4 ℃ of termination reactions.

From the PCR reaction result selection differences band of 100 pairs of primers of random combine, clone.

Table 1

Primer title	Primer sequence	Primer title	Primer sequence
				Me1	TGAGTCCAAACCGGAAA	Em1	GACTGCGTACGAATTAAT
Me2	TGAGTCCAAACCGGAAT	Em2	GACTGCGTACGAATTAAC
				Me3	TGAGTCCAAACCGGAAC	Em3	GACTGCGTACGAATTATG
Me4	TGAGTCCAAACCGGAAG	Em4	GACTGCGTACGAATTACG
				Me5	TGAGTCCAAACCGGATA	Em5	GACTGCGTACGAATTAGC
Me6	TGAGTCCAAACCGGACA	Em6	GACTGCGTACGAATTTAG
				Me7	TGAGTCCAAACCGGACT	Em7	GACTGCGTACGAATTTGA
Me8	TGAGTCCAAACCGGACC	Em8	GACTGCGTACGAATTTGC
				Me9	TGAGTCCAAACCGGACG	Em9	GACTGCGTACGAATTTCA
Me10	TGAGTCCAAACCGGAGA	Em10	GACTGCGTACGAATTTCG
				Me11	TGAGTCCAAACCGGAGC	Em11	GACTGCGTACGAATTCAA
Me12	TGAGTCCAAACCGGAGG	Em12	GACTGCGTACGAATTCA?T
				Me13	TGAGTCCAAACCGGTAG	Em13	GACTGCGTACGAATTCAC
Me14	TGAGTCCAAACCGGTTG	Em14	GACTGCGTACGAATTCAG
				Me15	TGAGTCCAAACCGGTCA	Em15	GACTGCGTACGAATTCTA
Me16	TGAGTCCAAACCGGTGT	Em16	GACTGCGTACGAATTCTT
				Me17	TGAGTCCAAACCGGTGC	Em17	GACTGCGTACGAATTCTC
Me18	TGAGTCCAAACCGGCAG	Em18	GACTGCGTACGAATTCTG
				Me19	TGAGTCCAAACCGGCTA	Em19	GACTGCGTACGAATTCCA
Me20	TGAGTCCAAACCGG?GAC	Em20	GACTGCGTACGAATTCGA
				Me21	TGAGTCCAAACCGGGTA	Em21	GACTGCGTACGAATTCGG

Me22	TGAGTCCAAACCGGGGT	Em22	GACTGCGTACGAATTGAT
				Me23	TGAGTCCTTTCCGGTAA	Em23	GACTGCGTACGAATTGAC
Me24	TGAGTCCTTTCCGGTCC	Em24	GACTGCGTACGAATTGAG
				Me25	TGAGTCCTTTCCGGTGC	Em25	GACTGCGTACGAATTGTC

Five, the recovery of differential fragment, order-checking and analysis.

Selection repeat amplification protcol is stable, fragment same difference fragment long, performance, and totally 4 are checked order.Sequencing result is carried out to homology analysis (as table 2) on the net at NCBI, (sequence number is respectively AB625451.1 to found that wherein 2 Me15/Em19 and Me7/Em2, DQ469798.1) there is very high homology with the abc transport albumen of paddy rice and Arabidopis thaliana respectively, homology is respectively 99% and 81%, and 2 fragments are all positive in green leaf material.

Me11/Em14 and Cucumber Chloroplasts genome tRNA-Arg gene have higher homology, are 97%; A kind of putative protein of Me18/Em21 and Selaginella tamariscina has higher homology, is 96%, and these two fragments are the yellow leaf specific expressino of gold leaf elm.

Abc transport albumen is a class ATP driving pump, participates in the transhipment of energy in pigment synthesis, and differential expression may be abundant with its chlorophyll content in green leaf for this gene, and photosynthesis is relevant by force.

Connected biological chemistry event in vital two functions in the biosynthesizing of tRNA participation protein.The one, the amino acidylate by the tRNA of related Aminoacyl-tRNA Synthetases catalysis, to the single-minded identification of the sequence of tRNA and structural element and amino acidylate, makes tRNA be converted into aminoacyl tRNA by aaRS.Another kind is that aminoacyl tRNA provides amino acid on rrna, and it is matched and started by complementary base by the anticodon of tRNA and the codon of mRNA.These two events in order and collaborative carrying out, have determined the aminoacid sequence of each protein.Therefore, in the yellow blade of gold leaf elm, may morph due to several bases of this fragment, thereby tRNA is converted in the process of aminoacyl tRNA to morph, the aminoacid sequence of the protein that impact is synthetic relevant to chlorophyll, chlorophyllous building-up process is obstructed, causes leaf look generation yellow.

Table 2

Embodiment 2: according to the Chinese gold leaf Siberian elm leaf look proterties related SR AP molecule marker system of embodiment 1, the Chinese gold leaf elm of identifying different leaf looks.

Fig. 1 is the amplification of primer pair Me15/Em19 to yellow leaf and these two proterties of green leaf, and wherein Y1-Y5 is yellow leaf plant, the mixing cDNA pond of YMix for being combined by Y1-Y5, and G1-G4 is green leaf plant, GMix is the mixing cDNA pond that G1-G4 forms.By SRAP molecule marking method of the present invention, can well distinguish two kinds of leaf looks.Method of the present invention is reproducible, and polymorphism is good, and band is clear, and background is low.

Claims (3)

1. the relevant SRAP molecule marking method of Chinese gold leaf Siberian elm leaf look proterties, is characterized in that the method carries out according to the following steps:

One, the collection of the different leaf look of Chinese gold leaf elm blade and leaf colour code are fixed:

Select the identical and healthy Chinese gold leaf elm plant of the 10 strain age of trees, label is 1～10 respectively, leaf colour code adopts the comparison of international color Standard colour board surely, choose the healthy young leaflet tablet of identical colour, from every strain, gather the golden blade of #FFD700 and the green blade of #228B22 forest, seal immediately cryopreservation;

Two, extract respectively total RNA of the golden blade of #FFD700 and the green blade of #228B22 forest on every strain China gold leaf elm plant;

Three, RNA concentration detects:

Adopt trace dna Protein Detection instrument to detect RNA concentration, use the ddH without RNase ₂rNA between O balance sample makes its concentration identical, be 50ng/ μ l, the RNA of the golden blade of #FFD700 that gets 1～10 strain of equivalent builds golden blade RNA mixing pit, and the RNA of the green blade of #228B22 forest that gets 1～10 strain of equivalent builds green blade RNA mixing pit;

Four, cDNA-SRAP amplification differential fragment:

A, the first chain cDNA's is synthetic: adopt M-MLV reversed transcriptive enzyme, respectively golden blade RNA mixing pit is become to cDNA, the aseptic ddH of product with the RNA reverse transcription in green blade RNA mixing pit ₂5 times of O dilutions, save backup in-20 ℃;

SRAP amplification, PCR product electrophoresis and the detection of b, cDNA:

With 25 upstream primers and 25 downstream primers, carry out random combine, 2 cDNA mixing pits are carried out to pcr amplification;

PCR reaction system: 10 * buffer1.2 μ L, Mg ²⁺25nmolL ^-11 μ L, upstream primer 5nmolL ^-11 μ L, downstream primer 5nmolL ^-11 μ L, dNTP2nmolL ^-12 μ L, Taq archaeal dna polymerase 0.5U0.5 μ L, the first chain cDNA cut back 1 μ L, adds water to 12 μ L;

Pcr amplification condition is: 94 ℃ of denaturation 5min, and 94 ℃ 1min-35 ℃ 1min-72 ℃ 1min5 circulation, 94 ℃ 1min-50 ℃ 1min-72 ℃ 1min35 circulation, 72 ℃ are extended 10min, 4 ℃ of termination reactions.

From the PCR reaction result selection differences band of 100 pairs of primers of random combine, clone;

Five, the recovery of differential fragment, order-checking and analysis;

Described in step 4,25 upstream primers and 25 downstream primer sequences are as follows:

Primer title Primer sequence Primer title Primer sequence Me1 TGAGTCCAAACCGGAAA Em1 GACTGCGTACGAATTAAT

Me2 TGAGTCCAAACCGGAAT Em2 GACTGCGTACGAATTAAC Me3 TGAGTCCAAACCGGAAC Em3 GACTGCGTACGAATTATG Me4 TGAGTCCAAACCGGAAG Em4 GACTGCGTACGAATTACG Me5 TGAGTCCAAACCGGATA Em5 GACTGCGTACGAATTAGC Me6 TGAGTCCAAACCGGACA Em6 GACTGCGTACGAATTTAG Me7 TGAGTCCAAACCGGACT Em7 GACTGCGTACGAATTTGA Me8 TGAGTCCAAACCGGACC Em8 GACTGCGTACGAATTTGC Me9 TGAGTCCAAACCGGACG Em9 GACTGCGTACGAATTTCA Me10 TGAGTCCAAACCGGAGA Em10 GACTGCGTACGAATTTCG Me11 TGAGTCCAAACCGGAGC Em11 GACTGCGTACGAATTCAA Me12 TGAGTCCAAACCGGAGG Em12 GACTGCGTACGAATTCA?T Me13 TGAGTCCAAACCGGTAG Em13 GACTGCGTACGAATTCAC Me14 TGAGTCCAAACCGGTTG Em14 GACTGCGTACGAATTCAG Me15 TGAGTCCAAACCGGTCA Em15 GACTGCGTACGAATTCTA Me16 TGAGTCCAAACCGGTGT Em16 GACTGCGTACGAATTCTT Me17 TGAGTCCAAACCGGTGC Em17 GACTGCGTACGAATTCTC Me18 TGAGTCCAAACCGGCAG Em18 GACTGCGTACGAATTCTG Me19 TGAGTCCAAACCGGCTA Em19 GACTGCGTACGAATTCCA Me20 TGAGTCCAAACCGG?GAC Em20 GACTGCGTACGAATTCGA Me21 TGAGTCCAAACCGGGTA Em21 GACTGCGTACGAATTCGG Me22 TGAGTCCAAACCGGGGT Em22 GACTGCGTACGAATTGAT Me23 TGAGTCCTTTCCGGTAA Em23 GACTGCGTACGAATTGAC Me24 TGAGTCCTTTCCGGTCC Em24 GACTGCGTACGAATTGAG Me25 TGAGTCCTTTCCGGTGC Em25 GACTGCGTACGAATTGTC

。

2. the relevant SRAP molecule marking method of Chinese gold leaf Siberian elm leaf look proterties according to claim 1, is characterized in that the extracting method of total RNA in step 2 adopts improved Trizol method, and concrete grammar is as follows:

A, get 0.2g China gold leaf Siberian elm leaf sheet, remove petiole and Zhong Mai, in liquid nitrogen, grind, add homogenate after 1ml Trizol;

The standing 5min of room temperature after B, homogenate, in 12000 turn/min, 4 ℃ of centrifugal 5min;

C, supernatant liquor is transferred in 1.5ml centrifuge tube, adds chloroform, the volume of chloroform is 1/5 of supernatant liquor volume, and concussion mixes, the standing 15min of room temperature;

D, in 12000 turn/min, 4 ℃ of centrifugal 15min, supernatant is transferred in another centrifuge tube, add isopyknic Virahol, the standing 10min of room temperature;

E, in 12000 turn/min, 4 ℃ of centrifugal 10min, in precipitation, add the ethanolic soln washing precipitation of the volumetric concentration 75% of 1ml, in 12000 turn/min, 4 ℃ of centrifugal 5min;

F, abandon supernatant, retain precipitation, dry, the DEPC that is dissolved in 30～50 μ l processes in water.

3. utilize SRAP molecule marking method described in claim 1 to carry out Chinese gold leaf Siberian elm leaf aberration recessive allele research.