CN101974516B - Method for discriminating Chinese milk vetch variety by using SSR (Simple Sequence Repeat)fingerprint spectrum - Google Patents
Method for discriminating Chinese milk vetch variety by using SSR (Simple Sequence Repeat)fingerprint spectrum Download PDFInfo
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Abstract
The invention provides a method for discriminating Chinese milk vetch varieties by using an SSR (Simple Sequence Repeat)fingerprint spectrum. A specific SSR site and bilateral primer sequences thereof (SEQ.NO.1-18) in a Chinese milk vetch genome, and a fingerprint spectrum which is obtained by carrying out PCR (Polymerase Chain Reaction) amplification on screened primers can be used for discriminating Chinese milk vetch varieties. The discriminating method mainly comprises the following steps of: extracting Chinese milk vetch total DNA (Deoxyribonucleic Acid); screening the specific SSR site and designing primers from genome DNA by using magnetic-bead enrichment method; and finally obtaining 6 SSR site primer pairs which can form obvious banded difference by carrying out the PCR amplification on different varieties of different Chinese milk vetch. The constructed fingerprint spectrum can be effectively used for discriminating Chinese milk vetch varieties.
Description
Technical field
The invention belongs to biology field, more specifically relate to the method that a kind of SSR of utilization finger printing is differentiated green manure crop Herba Astragali Melilotoidis (Herba Astragali Sinici) kind.
Background technology
Herba Astragali Melilotoidis (Herba Astragali Sinici) (formal name used at school:
Astragalus sinicus) be the plant of pulse family Astragalus.Belong to annual or more year sward this.Claim clover again, Astragalus sinicus.Be distributed widely in north latitude 25～33 degree areas, be grown in small stream limit, hillside and moist place more.China is as far back as the establishing in large scale in area, the middle and lower reach of Yangtze River just of bright, clear epoch.The Herba Astragali Melilotoidis (Herba Astragali Sinici) main root is loose, generally buries 40～50 centimetres, and lateral root buries more shallow.On the radicula on Herba Astragali Melilotoidis (Herba Astragali Sinici) main root, lateral root and the face of land can both the knurl of taking root, with number in the majority on the lateral root.Stem is cylindrical, hollow, and tender succulence has thin fine hair.General 80～120 centimetres of Cultivar plant height, wild has only 10～30 centimetres.The leaf majority is an imparipinnate leaf, 7～13 pieces of leaflets of tool.Leaflet full edge, obovoid or ellipse.Flower is umbel, and general armpit is given birth to, and give birth on few top, and Xiao Hua more than 8～10 is often arranged, and family gives birth on bennet, is arranged in colyliform.Pod two row are unified into trilateral, and are curved slightly, do not have hair, and there is beak on the top.Every pod has 4～10 in seed, the seed kidney shape, and the kind skin is smooth, general yellow-green colour.Thousand seed weight 3～3.5 grams.How southern china is as the plantation of rice field green manure in winter now, and generally cover is sowed in the late rice in the fall, makes the base manure of early rice.Herba Astragali Melilotoidis (Herba Astragali Sinici) is except that as the green manure, can also be directly or the ensiling Herba Astragali Melilotoidis (Herba Astragali Sinici) make feed, nutritive value is quite high.
At present, the kind of Herba Astragali Melilotoidis (Herba Astragali Sinici) differentiates that with fertility characteristic and form outward appearance be main: can divide early, middle and late 3 types by breeding time and ripening stage.The main improved seeds of Herba Astragali Melilotoidis (Herba Astragali Sinici), early seasonal strain have purple No. 1 of Xinyang kind, Leping kind, Fujian etc., and middle seasonal strain has purple No. 6 of the Da Ye of Yujiang County seed, peaceful No. 3 of duckweed, Changde kind, Fujian etc., and late variety has purple No. 5 of Ningbo bridge kind, Zhejiang etc.Because the Herba Astragali Melilotoidis (Herba Astragali Sinici) cross-pollination, hybrid rate is higher, depends merely on the discriminating that morphologic observation and growth characteristics are difficult to carry out exactly kind.In order to carry out kind identification better, reach the purpose of protection kind knowledge rights and interests, need and carry out the method that kind is differentiated more fast and accurately.
Summary of the invention
The invention provides a kind of SSR of utilization finger printing and differentiate the method for Herba Astragali Melilotoidis (Herba Astragali Sinici) kind, can differentiate the Herba Astragali Melilotoidis (Herba Astragali Sinici) variety source from the molecular level of plant gene more accurately.
The present invention utilizes the SSR finger printing to be used to differentiate the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind; Wherein relate to SSR site specific in a kind of Herba Astragali Melilotoidis (Herba Astragali Sinici) genome; The sequence in said specific SSR site is made up of 6 sequences, and its sequence is arranged as from short to long: 1. ACACACACAACACACACAAACACA; 2. ACACACACACACACACAAACAAACACAAACAC; 3. CTCTCTCTGATCTCTCTCTCCCTCTCCC TCTCCCTCTCCCTCT; 4. CTCTTCCTCTTCTTCTCTTTCATATGCTCCACT CTTCCTCTCTTCTTCTCT; 5. CCC ACACCACCACTCACCAACATCACCACTCACACACCAGCACCTTCCACTCCCTTCAA CCACCCCCACCACTACTCAAACACCACCATCTCACCA; ⑥ACACACACACACACACACACACGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTACGTGCGCGTGTGTGTGTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGCGTGCGTGTGTGTGTGTGT。
To forming, primer is following to sequence by 6 pairs of primers for the both sides designed primer sequence in specific SSR site in the above-mentioned Herba Astragali Melilotoidis (Herba Astragali Sinici) genome: the 1. EG6-54 upper reaches: CGCCCATAAATCTCTTCCA downstream: CTCCCTTTGATCAATCTCGC; 2. the EG6-167 upper reaches: TTTCACTTCTAGAAATTTGTTGACA, downstream: ATGGATGGTCAACTCGGTAAC; 3. the Bm2-8 upper reaches: GCTCGGCCTTTCACATGG, downstream: GAGAAATAGGTACCAGTTCTAGGG; 4. the Lm2-44 upper reaches: GATTGACAAAGTCTGCCG, downstream: ATGCTCCCCTCTTCACACA; 5. the EG6-170 upper reaches: GGCCAACACTCACTACCCTCAT, downstream: CAACCAACGATCACTCCCACC; 6. the YAGT37 upper reaches: CGCCCATAAATCTCTTCCA, downstream: TGCGCACACACAACG.The screening method of primer comprises the following steps: the extraction of the total DNA of Herba Astragali Melilotoidis (Herba Astragali Sinici); With the SSR site in the affine enrichment with magnetic bead Herba Astragali Melilotoidis (Herba Astragali Sinici) genome, the screening of both sides, SSR site primer design; Pcr amplification; PCR product electrophoresis and dyeing, the primer that screening has polymorphum according to electrophoresis result.
Wherein said Herba Astragali Melilotoidis (Herba Astragali Sinici) total DNA extraction method; With blade of Herba Astragali Melilotoidis (Herba Astragali Sinici) kind seedling or ripe plant or in the stem section one or both is material; Extract genomic dna; Concrete operation steps is: 1. take by weighing 0.1 gram plant tissue, rinse well with deionized water, change over to rapidly in the 1.5ml centrifuge tube; 2. add liquid nitrogen, smash to pieces with electric drill rapidly, add 600mlCTAB; 3. 65 ℃ of water-bath 1h, mixing; 4. add isopyknic chloroform-primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24:1, mixing; 5. the centrifugal 6min of 12000rpm gets supernatant; 6. in supernatant, add the absolute ethyl alcohol of 2 times of volumes or the isopropanol precipitating DNA of 2/3 volume, place 20min for-20 ℃; 7. the centrifugal 6min of 12000rpm abandons supernatant; 8. add 75% ethanol cleaning DNA, the centrifugal 2min of 12000rpm abandons supernatant; 9. air-dry back adds the TE damping fluid dissolving DNA of 50ul, and is subsequent use.
Said pcr amplification, its PCR reaction system is: comprise the DNA solution 1 μ L of extraction in the PCR reaction system of 25 μ L, 10nmol/L primer 2 μ L, 2.5 mmol/LdNTP, 2 μ L, 10 * PCRbuffer, 2.5 μ L,
TaqDNA polysaccharase 1 U; The PCR response procedures is: 94 ℃ of preparatory sex change 7min; 30 circulations subsequently, each 94 ℃ of sex change 30 s that circulate, 55 ℃ of annealing 30 s, 72 ℃ are extended 30s; Last 72 ℃ are extended 10 min.
Said PCR product electrophoresis and dyeing adopt 6% polyacrylamide gel electrophoresis and silver to dye colour developing.
A kind of method of utilizing the SSR finger printing to differentiate the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind of the present invention, it is characterized in that: said method comprises: the extraction of the total DNA of (1) Herba Astragali Melilotoidis (Herba Astragali Sinici); (2) according to said specific both sides, SSR site design primer; (3) primer is carried out pcr amplification; (4) the PCR product forms the SSR finger printing of different Herba Astragali Melilotoidis (Herba Astragali Sinici) kinds through electrophoresis and dyeing, carries out the kind of Herba Astragali Melilotoidis (Herba Astragali Sinici) then and differentiates.
Wherein said Herba Astragali Melilotoidis (Herba Astragali Sinici) total DNA extraction method; With blade of Herba Astragali Melilotoidis (Herba Astragali Sinici) kind seedling or ripe plant or in the stem section one or both is material; Extract genomic dna; Concrete operation steps is: 1. take by weighing 0.1 gram plant tissue, rinse well with deionized water, change over to rapidly in the 1.5ml centrifuge tube; 2. add liquid nitrogen, smash to pieces with electric drill rapidly, add 600mlCTAB; 3. 65 ℃ of water-bath 1h, mixing; 4. add isopyknic chloroform-primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24:1, mixing; 5. the centrifugal 6min of 12000rpm gets supernatant; 6. in supernatant, add the absolute ethyl alcohol of 2 times of volumes or the isopropanol precipitating DNA of 2/3 volume, place 20min for-20 ℃; 7. the centrifugal 6min of 12000rpm abandons supernatant; 8. add 75% ethanol cleaning DNA, the centrifugal 2min of 12000rpm abandons supernatant; 9. air-dry back adds the TE damping fluid dissolving DNA of 50ul, and is subsequent use.
Said pcr amplification, its PCR reaction system is: comprise the DNA solution 1 μ L of extraction in the PCR reaction system of 25 μ L, 10nmol/L primer 2 μ L, 2.5 mmol/LdNTP, 2 μ L, 10 * PCRbuffer, 2.5 μ L,
Taq DNA polysaccharase 1 U.The PCR response procedures is: 94 ℃ of preparatory sex change 7min; 30 circulations subsequently, each 94 ℃ of sex change 30 s that circulate, 55 ℃ of annealing 30 s, 72 ℃ are extended 30s; Last 72 ℃ are extended 10 min.
Remarkable advantage of the present invention:
1) be expert evidence with the total DNA of Herba Astragali Melilotoidis (Herba Astragali Sinici) that extracts, it can draw materials extraction and not influence reaction result in any growth period and the growth site of Herba Astragali Melilotoidis (Herba Astragali Sinici), thus interference and the influence of avoiding Herba Astragali Melilotoidis (Herba Astragali Sinici) growth conditions and growth period that kind is differentiated; 2) molecular marking technique that adopts is a microsatellite molecular marker, also claims the SSR mark.This mark extensively is randomly distributed in the eukaryotic gene group, has the repeatability and the codominant inheritance mode of the polymorphum of height, high information content, height.Therefore, the SSR marking method has characteristics such as allelic diversity height easy, quick, special, stable, that disclosed than other marking methods; 3) through a large amount of screening operations; Obtained in the Herba Astragali Melilotoidis (Herba Astragali Sinici) different varieties, to form the SSR site of polymorphum; With the primer of this SSR site both wings sequences Design, can be used to make up the finger printing of Herba Astragali Melilotoidis (Herba Astragali Sinici) different varieties, reach the purpose of differentiating the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind.
Description of drawings
Fig. 1 is the fingerprint banding pattern of primer EG6-54;
Fig. 2 is the fingerprint banding pattern of primer EG6-167;
Fig. 3 is the fingerprint banding pattern of primer Bm2-8;
Fig. 4 is the fingerprint banding pattern of primer Lm2-44;
Fig. 5 is the fingerprint banding pattern of primer EG6-170;
Fig. 6 is the fingerprint banding pattern of primer YAGT37;
Fig. 7 is the fingerprint banding pattern that 9 tested varieties of Herba Astragali Melilotoidis (Herba Astragali Sinici) form in the 75bp-300bp scope.
Embodiment
1, the extraction of the total DNA of Herba Astragali Melilotoidis (Herba Astragali Sinici):
(1) the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind of participating in the experiment: 1: purple No. 1 of Fujian; 2: Xinyang kind; 3: Ningbo bridge kind; 4:8324411; 5: purple No. 6 of Fujian; 6:8410441; 7:8487711; 8: shoot a retrievable arrow the river seed; 9: the Da Ye of Yujiang County seed; Wherein purple No. 1,8324411 of Fujian, Fujian are provided by Fujian Academy of Agricultural Sciences for purple No. 6,8410441,8487711; Xinyang kind is provided by Henan Province Xinyang Agricultural Science Inst.; The Da Ye of Yujiang County seed is provided by Jiangxi Province Academy of Agricultural Sciences; Shooting a retrievable arrow the river seed is provided by the Academy of Agri-Science and Technology Anhui Province, and Ningbo bridge kind is provided by the Zhejiang Academy of Agricultural Science.
(2) be material with Herba Astragali Melilotoidis (Herba Astragali Sinici) kind seedling; Adopt the CTAB method to extract genomic dna, because the CTAB method is different for the extraction procedure of Different Crop, we are through improving; Simplified step to Herba Astragali Melilotoidis (Herba Astragali Sinici); Detailed process is: 1. take by weighing 0.1 gram plant tissue: plant tissue comprises the part of stem and the leaf of Herba Astragali Melilotoidis (Herba Astragali Sinici) seedling, rinses well with deionized water, changes over to rapidly in the 1.5ml centrifuge tube; 2. add liquid nitrogen, smash to pieces with electric drill rapidly, add 600mlCTAB (CTAB is available from Amresco company); 3. 65 ℃ of water-bath 1h (every 20min counter-rotating shakes up once); 4. add isopyknic chloroform-primary isoamyl alcohol (volume ratio 24:1), put upside down mixing; 5. the centrifugal 6min of 12000rpm gets supernatant in new pipe; 6. in supernatant, add absolute ethyl alcohol (or Virahol of 2/3 volume) the deposit D NA of 2 times of volumes, place 20min for-20 ℃; 7. the centrifugal 6min of 12000rpm removes supernatant; 8. add 75% ethanol cleaning DNA, the centrifugal 2min of 12000rpm removes supernatant; 9. air-dry back adds the TE damping fluid dissolving DNA of 50ul, and is subsequent use; The organic solvent of above-mentioned use is all available from the Beijing Chemical Plant.
2. with specific SSR sequence in the affine enrichment with magnetic bead Herba Astragali Melilotoidis (Herba Astragali Sinici) genome: the Dynal magnetic bead of this step application Invitrogen company and biotin labeled little satellite probe (CT)
15, (AG)
15, (GT)
15, (AC)
15With the hybridization of Herba Astragali Melilotoidis (Herba Astragali Sinici) genomic dna endonuclease bamhi, enzyme is cut the restriction endonuclease that uses and is EcoRI, MseI, and all available from NEB company, enzyme is cut step and is done; Get Herba Astragali Melilotoidis (Herba Astragali Sinici) DNA that 10 μ g extract and in 50 μ l reaction systems, cut and digested 3 hours, get 5 μ l enzymes and cut product electrophoresis detection enzyme and cut effect, confirm that enzyme cuts entirely with restriction enzyme EcoRI and MseI enzyme.Then contain the DNA fragment of microsatellite sequence by the operation instructions of Dynal magnetic bead in order to catch 300-1500 bp, be connected in the pMD18-T carrier (available from Takara company), the small segment that makes up the enrichment microsatellite sequence inserts the library.Utilize the sequence measuring joints primer (primer can be directly also can be synthetic) at pMD18-T carrier two ends and according to little satellite core sequence designed primer: (CT) by the sequence that its products catalogue provides available from Takara company
15, (AG)
15, (GT)
15, (AC)
15(it is synthetic to give birth to the worker by Shanghai) cooperates and forms primer to screening in order to directly to carry out library PCR; 236 positive colonies from 800 transformants, have been obtained; It is carried out sequencing analysis, obtained 133 microsatellite sequences, the bioaccumulation efficiency of microsatellite sequence reaches 16.7%.
3. screening of SSR primer design and PCR amplification contain the dna fragmentation of microsatellite sequence: the SSR sequence is analyzed, and wherein all there is long base sequence (> 30bp in the two ends of 35 little satellite Tumor-necrosis factor glycoproteinss).Right according to its design special primer, 9 kind Herba Astragali Melilotoidis (Herba Astragali Sinici) complete genome DNAs are carried out 2 screenings.The PCR reaction system is: comprise the DNA solution 1 μ L of extraction in the PCR reaction system of 25 μ L, and 10nmol/L primer 2 μ L, 2.5 mmol/LdNTP, 2 μ L, 10 * PCRbuffer, 2.5 μ L,
TaqDNA polysaccharase 1 U.The PCR response procedures is: 94 ℃ of preparatory sex change 7min; 30 circulations subsequently, each 94 ℃ of sex change 30 s that circulate, 55 ℃ of annealing 30 s, 72 ℃ are extended 30s; Last 72 ℃ are extended 10 min.Because the SSR primer has very high specificity, these primers can be divided into general reaction result: do not react, do not have polymorphum and polymorphum is arranged.It is clear only to choose banding pattern according to the electrophoresis result of PCR, and the foundation differentiated as kind of the different fingerprint banding patterns that have the primer of polymorphum to form.Here the unnecessary result's statistics of carrying out makes up kind cluster crowd because the result of statistics is mainly used in, and is helpless to carry out interracial discriminating.It is clear that the result obtains in different varieties, can amplifying banding pattern altogether, and 6 pairs of primers of polymorphum are arranged.Prove these primers between the SSR site in the genome of Herba Astragali Melilotoidis (Herba Astragali Sinici) different varieties, exist tangible polymorphum, the electrophoresis result of PCR can be used for making up interracial finger printing.
4. polyacrylamide gel electrophoresis and silver dye colour developing, and specified operational procedure is: 1. prepare 6% polyacrylamide gel, draw the sample-loading buffer of PCR product 7 μ L and 1 μ L, fully appearance on the mixing.140 V voltage electrophoresis after last appearance finishes.When tetrabromophenol sulfonphthalein indicator during, finish electrophoresis apart from following 1 cm～2 cm.2. after electrophoresis finishes; Close electrophoresis apparatus, take out sheet glass and the medication the shank of the key is carefully pried open, remove upper glass plate; To face down with gel with the sheet glass of gel and immerse in the off-the-shelf clean porcelain dish that is placed with distilled water; Gel promptly falls, or the medication the shank of the key can fall into one jiao of glue strip off in the dish gently; 3. gel is fixing: outwell the water in the porcelain dish, add stationary liquid (100 mL/L ethanol), on shaking table, slowly shake 15 min; Outwell the stationary liquid in the porcelain dish, wash 2 times with distilled water; 4. the dyeing of gel: outwell the water in the porcelain dish, add staining fluid (20 g/L AgNO
3), on shaking table, slowly shake dyeing 20 min; 5. wash glue: outwell the staining fluid in the porcelain dish, wash 2 times, amount to 1 min with distilled water; 6. the development of gel: add a small amount of colour developing liquid (30 g/L Na
2CO
3) flushing 15 s, adding certain volume colour developing liquid again, the jog porcelain dish is generally 5 min～6 min up to seeing band clearly, and the time is not long, otherwise background can be dark excessively; 7. stop developing: outwell the developing solution in the porcelain dish, pour stop bath (100 mL/L HAC) rapidly into, slowly shake 2 min～3 min; 8. distinguish sequence: outwell the stop bath in the porcelain dish, wash 2 times with distilled water, each 2 min are placed on air drying with gel, the recognition sequence.Also available digital camera or the shooting of gel imaging appearance.
5. interpretation of result of SSR finger printing and judgement: the fingerprint banding pattern that the SSR site of 6 pairs of primer correspondences forms in different Herba Astragali Melilotoidis (Herba Astragali Sinici) kinds is seen Fig. 1-Fig. 6.Wherein Fig. 1 can form 4 kinds of fingerprint banding patterns in the 150bp-300bp scope to the Herba Astragali Melilotoidis (Herba Astragali Sinici) tested variety;
Fig. 2 can form 5 kinds of fingerprint banding patterns in the 80bp-150bp scope to the Herba Astragali Melilotoidis (Herba Astragali Sinici) tested variety; Fig. 3 can form 5 kinds of fingerprint banding patterns in the 75bp-100bp scope to the Herba Astragali Melilotoidis (Herba Astragali Sinici) tested variety; Fig. 4 can form 4 kinds of fingerprint banding patterns in the 70bp-150bp scope to the Herba Astragali Melilotoidis (Herba Astragali Sinici) tested variety; Fig. 5 can form 3 kinds of fingerprint banding patterns in the 240bp-300bp scope to the Herba Astragali Melilotoidis (Herba Astragali Sinici) tested variety; Fig. 6 can form 3 kinds of fingerprint banding patterns in the 250bp-650bp scope to the Herba Astragali Melilotoidis (Herba Astragali Sinici) tested variety;
Below we are how example explanation is distinguished 9 kinds of participating in the experiment according to its electrophoretic finger printing banding pattern with 2 sites in 6 SSR sites.
One, the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind of participating in the experiment: 1: purple No. 1 of Fujian; 2: Xinyang kind; 3: Ningbo bridge kind; 4:8324411; 5: purple No. 6 of Fujian; 6:8410441; 7:8487711; 8: shoot a retrievable arrow the river seed; 9: the Da Ye of Yujiang County seed; Wherein purple No. 1,8324411 of Fujian, Fujian are provided by Fujian Academy of Agricultural Sciences for purple No. 6,8410441,8487711; Xinyang kind is provided by Henan Province Xinyang Agricultural Science Inst.; The Da Ye of Yujiang County seed is provided by Jiangxi Province Academy of Agricultural Sciences; Shooting a retrievable arrow the river seed is provided by the Academy of Agri-Science and Technology Anhui Province, and Ningbo bridge kind is provided by the Zhejiang Academy of Agricultural Science.
Two, the identification result of SSR mark:
(M is a molecular weight standard to the fingerprint banding pattern that the banding pattern collection of illustrative plates of the Herba Astragali Melilotoidis (Herba Astragali Sinici) sample of participating in the experiment that 1, is formed by different SSR site design primer amplification: Fig. 7 forms in the 75bp-300bp scope 9 tested varieties of Herba Astragali Melilotoidis (Herba Astragali Sinici) for primer EG6-54 (right side among Fig. 7) and primer EG6-167 (on the left of among Fig. 7); 0 is blank, and 1-9 is the sample number into spectrum of participating in the experiment).
2, the fingerprinting of Herba Astragali Melilotoidis (Herba Astragali Sinici) kind banding pattern: the tangible master tape of amplification among Fig. 7 is defined as different banding patterns, capitalization English letter A, the B of using respectively as shown in Figure 7; C, D, E; F, G represent, can the finger printing of the banding pattern of Herba Astragali Melilotoidis (Herba Astragali Sinici) tested variety be summed up through table 1.
The fingerprint map analyzing of the banding pattern of table 1 Herba Astragali Melilotoidis (Herba Astragali Sinici) tested variety
The banding pattern that "+" expression 1-9 tested variety is had.
Visible by table 1:, promptly between No. 3 and No. 9, can not distinguishing, all different between all the other samples as long as just can 9 samples of participating in the experiment be divided into 8 types according to the polymorphum in 2 used SSR sites.Thereby can differentiate the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind from molecular level.
< 110>Fujian Province Agriculture InstituteSoil and Fertilizer Institute
< 120>a kind of method of utilizing the SSR finger printing to differentiate the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind
<160>?18
<210>?1
<211>?24
<212>?DNA
<213>Herba Astragali Melilotoidis (Herba Astragali Sinici) (
Astragalus sinicus)
<400>?1
acacacacaa?cacacacaaa?caca?24
<210>?2
<211>?32
<212>?DNA
<213>Herba Astragali Melilotoidis (Herba Astragali Sinici) (
Astragalus sinicus)
<400>?2
acacacacac?acacacaaac?aaacacaaac?ac?32
<210>?3
<211>?43
<212>?DNA
<213>Herba Astragali Melilotoidis (Herba Astragali Sinici) (
Astragalus sinicus)
<400>?3
ctctctctga?tctctctctc?cctctccctc?tccctctccc?tct?43
<210>?4
<211>?51
<212>?DNA
<213>Herba Astragali Melilotoidis (Herba Astragali Sinici) (
Astragalus sinicus)
<400>?4
ctcttcctct?tcttctcttt?catatgctcc?actcttcctc?tcttcttctc?t?51
<210>?5
<211>?96
<212>?DNA
<213>Herba Astragali Melilotoidis (Herba Astragali Sinici) (
Astragalus sinicus)
<400>?5
cccacaccac?cactcaccaa?catcaccact?cacacaccag?caccttccac?tcccttcaac?60
cacccccacc?actactcaaa?caccaccatc?tcacca?96
<210>?6
<211>?348
<212>?DNA
<213>Herba Astragali Melilotoidis (Herba Astragali Sinici) (
Astragalus sinicus)
<400>?6
acacacacac?acacacacac?acgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?60
gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgcgtgt?gtgtgtgtgt?120
gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?180
gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgcgagt?240
gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtgt?gtgtgtgtac?gtgcgcgtgt?gtgtgtgcgt?300
gtgtgtgtgt?gtgtgtgtgt?gtgtgcgtgc?gtgcgtgtgt?gtgtgtgt?348
<210>?7
<211>?19
<212>?DNA
< 213>artificial sequence
<400>?7
cgcccataaa?tctcttcca?19
<210>?8
<211>?20
<212>?DNA
< 213>artificial sequence
<400>?8
ctccctttga?tcaatctcgc?20
<210>?9
<211>?25
<212>?DNA
< 213>artificial sequence
<400>?9
tttcacttct?agaaatttgt?tgaca?25
<210>?10
<211>?21
<212>?DNA
< 213>artificial sequence
<400>?10
atggatggtc?aactcggtaa?c?21
<210>?11
<211>?18
<212>?DNA
< 213>artificial sequence
<400>?11
gctcggcctt?tcacatgg?18
<210>?12
<211>?24
<212>?DNA
< 213>artificial sequence
<400>?12
gagaaatagg?taccagttct?aggg?24
<210>?13
<211>?18
<212>?DNA
< 213>artificial sequence
<400>?13
gattgacaaa?gtctgccg?18
<210>?14
<211>?19
<212>?DNA
< 213>artificial sequence
<400>?14
atgctcccct?cttcacaca?19
<210>?15
<211>?22
<212>?DNA
< 213>artificial sequence
<400>?15
ggccaacact?cactaccctc?at?22
<210>?16
<211>?21
<212>?DNA
< 213>artificial sequence
<400>?16
caaccaacga?tcactcccac?c?21
<210>?17
<211>?19
<212>?DNA
< 213>artificial sequence
<400>?17
cgcccataaa?tctcttcca?19
<210>?18
<211>?15
<212>?DNA
< 213>artificial sequence
<400>?18
tgcgcacaca?caacg?15
Claims (5)
1. the specific SSR site of enrichment in the Herba Astragali Melilotoidis (Herba Astragali Sinici) is characterized in that said specific SSR site is made up of 6 different SSR sites, and its sequence is arranged as from short to long: 1. ACACACACAACACACACAAACACA; 2. ACACACACACACACACAAACAAACACAAACAC; 3. CTCTCTCTGATCTCTCTCTCCCTCTCCC TCTCCCTCTCCCTCT; 4. CTCTTCCTCTTCTTCTCTTTCATATGCTCCACT CTTCCTCTCTTCTTCTCT; 5. CCC ACACCACCACTCACCAACATCACCACTCACACACCAGCACCTTCCACTCCCTTCAA CCACCCCCACCACTACTCAAACACCACCATCTCACCA; ⑥ACACACACACACACACACACACGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTACGTGCGCGTGTGTGTGTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGCGTGCGTGTGTGTGTGTGT。
2. both sides designed primer of utilizing the specific SSR site of enrichment in the said Herba Astragali Melilotoidis (Herba Astragali Sinici) of claim 1; It is characterized in that; To forming, primer is following to sequence by 6 pairs of primers for said primer sequence: the 1. EG6-54 upper reaches: CGCCCATAAATCTCTTCCA downstream: CTCCCTTTGATCAATCTCGC; 2. the EG6-167 upper reaches: TTTCACTTCTAGAAATTTGTTGACA, downstream: ATGGATGGTCAACTCGGTAAC; 3. the Bm2-8 upper reaches: GCTCGGCCTTTCACATGG, downstream: GAGAAATAGGTACCAGTTCTAGGG; 4. the Lm2-44 upper reaches: GATTGACAAAGTCTGCCG, downstream: ATGCTCCCCTCTTCACACA; 5. the EG6-170 upper reaches: GGCCAACACTCACTACCCTCAT, downstream: CAACCAACGATCACTCCCACC; 6. the YAGT37 upper reaches: CGCCCATAAATCTCTTCCA, downstream: TGCGCACACACAACG.
3. method of utilizing the SSR finger printing to differentiate the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind, it is characterized in that: said method comprises: the extraction of the total DNA of (1) Herba Astragali Melilotoidis (Herba Astragali Sinici); (2) both sides, SSR site according to claim 1 design primer; (3) carry out pcr amplification; (4) the PCR product forms the SSR finger printing of different Herba Astragali Melilotoidis (Herba Astragali Sinici) kinds through electrophoresis and dyeing, carries out the kind of Herba Astragali Melilotoidis (Herba Astragali Sinici) then and differentiates.
4. the method for utilizing the SSR finger printing to differentiate the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind according to claim 3; It is characterized in that: said Herba Astragali Melilotoidis (Herba Astragali Sinici) total DNA extraction method; With blade of Herba Astragali Melilotoidis (Herba Astragali Sinici) kind seedling or ripe plant or in the stem section one or both is material, extracts genomic dna, and concrete operation steps is: 1. take by weighing 0.1 gram plant tissue; Rinse well with deionized water, change over to rapidly in the 1.5ml centrifuge tube; 2. add liquid nitrogen, smash to pieces with electric drill rapidly, add 600mlCTAB; 3. 65 ℃ of water-bath 1h, mixing; 4. add isopyknic chloroform-primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24:1, mixing; 5. the centrifugal 6min of 12000rpm gets supernatant; 6. in supernatant, add the absolute ethyl alcohol of 2 times of volumes or the isopropanol precipitating DNA of 2/3 volume, place 20min for-20 ℃; 7. the centrifugal 6min of 12000rpm abandons supernatant; 8. add 75% ethanol cleaning DNA, the centrifugal 2min of 12000rpm abandons supernatant; 9. air-dry back adds the TE damping fluid dissolving DNA of 50ul, and is subsequent use.
5. the method for utilizing the SSR finger printing to differentiate the Herba Astragali Melilotoidis (Herba Astragali Sinici) kind according to claim 3; It is characterized in that: said pcr amplification; Its PCR reaction system is: comprise the DNA solution 1 μ L of extraction in the PCR reaction system of 25 μ L, 10nmol/L primer 2 μ L, 2.5 mmol/LdNTP, 2 μ L; 10 * PCRbuffer, 2.5 μ L
TaqDNA polysaccharase 1 U; The PCR response procedures is: 94 ℃ of preparatory sex change 7min; 30 circulations subsequently, each 94 ℃ of sex change 30 s that circulate, 55 ℃ of annealing 30 s, 72 ℃ are extended 30s; Last 72 ℃ are extended 10 min.
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| Lin-kai Huang等.A Comparative Analysis of Molecular Diversity of Erect Milkvetch (Astragalus adsurgens) Germplasm from North China Using RAPD and ISSR Markers.《Biochemical Genetics》.2009,第47卷92-99页. * |
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