CN102533745B - Saline-alkaline-tolerance Fraxinus velutina sequence-characterized amplified region (SCAR) marker and applications in assistant selection breeding - Google Patents
Saline-alkaline-tolerance Fraxinus velutina sequence-characterized amplified region (SCAR) marker and applications in assistant selection breeding Download PDFInfo
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Abstract
The invention discloses a saline-alkaline-tolerance Fraxinus velutina sequence-characterized amplified region (SCAR) marker. The SCAR marker is a specific fragment of which the overall length is 592bp, and the nucleic acid sequence of the SCAR marker is shown by SEQ ID NO.1. The SCAR marker disclosed by the invention can be widely used for saline-alkaline-tolerance Fraxinus velutina molecule-marked assistant selection breeding, and simultaneously, is capable of laying a solid foundation for cloning the saline-alkaline-tolerance gene of Fraxinus velutina and analyzing gene sequences. The saline-alkaline-tolerance Fraxinus velutina SCAR marker can be used for performing seedling stage identification on individuals, thereby, the breeding efficiency is greatly increased, and the breeding process is shortened.
Description
Technical field
The present invention relates to a kind of SCAR mark and application thereof, relate in particular to a kind of Salt And Alkali Tolerance Fraxinus velutina SCAR mark and the application in assisted selection thereof, belong to biological technical field.
Background technology
Approximately more than 70 plant in the Fraxinus whole world, and great majority are distributed in warm temperate zone, the Northern Hemisphere, and existing 34 kinds of China wherein originates in 26 kinds, introduces 8 kinds abroad.Fraxinus velutina (Fraxinus VelutinaTorr.) is Oleaceae (Oleaceae) Fraxinus (Fraxinus Linn.) deciduous tree, originate in and be the U.S., growth is fast, trunk is perfectly straight, wooden structures is even, beautiful texture, easily breeding, degeneration-resistant is good saline-alkali soil forestation, afforestation and precious commerical tree species.Introduced a fine variety to Jinan (Meng Zhao and etc., 2001) as far back as 1911, nineteen fifty-three is quoted to Tianjin (Yang Ruixing etc., 1996), extensively cultivates in the East China, North China after the eighties.China North China, main coastal cities, East China and varieties in saline-alkali areas afforestation and afforestation fine tree species (Shi Mingzhi, 1996 have been become at present; Wang Qin etc., 2000; Ni Guoxiang etc., 1995; Wang You equality, 2007).
Domestic research about Chinese wax ecosystem characterization, habit aspect is more, mainly lays particular emphasis on the aspect such as salt tolerant alkalescence, seedling-wood breeding, afforestation technology of Chinese wax.Fan Baomin (1992) report, Fraxinus velutina is comparatively responsive to the marine solonchak of react acid; The seeds seedling that Meng Kangmin (1999) introduces a fine variety 5 beach saline lands carries out potted plant salt tolerance test, comprehensively pass judgment on according to 5 Salt-tolerance Physiologicals such as photosynthetic rate, chlorophyll content, cell leakage, mda content, free proline contents, the result shows that the Fraxinus velutina Salt-endurance is the strongest; Liu Dexi etc. (2008) 1-3 under the salt marsh habitat gives birth to red Ash as material, and salty ions distribution rule in each vegetative organ of the red Ash of the different age of trees is studied; Wang You equality (2009) has been studied under the salt marsh habitat 1-3 and has been given birth to salty ions distribution rule in the Fraxinus velutina Different Nutrition organ.The Abroad in Recent Years report focuses mostly on and separates at the little satellite of the nuclear DNA of European Chinese wax (F.excelsior), Using Chloroplast Simple mark, EST-SST label screening, and microsatellite marker is in population genetic analysis and phylogeography research.Heurtz etc. (2004) are by thinking to chloroplast DNA and the Using Chloroplast Simple of the wild F.excelsior of all Europe colony, and lower monoploid is polymorphic to be because the gene flow impact of anemophilous plant.Bacles etc. (2008) utilize microsatellite marker to carry out the Paternity analysis of the pollen gene flow of Scotland F.excelsior colony under the big scale view, and group size is depended in the exchange of pollen gene flow, rather than the geographical position.Goto etc. (2006) utilize 5 microsatellite locus to the parent of the F.mandshurica var.japonica of Japan northern strand forest and offspring's gene type assay, and parent's ontoanalysis can provide quantitative analysis for anemophilous plant in the result showed among a small circle.Relevant dna marker sequence to Fraxinus velutina (F.Velutina) fails to retrieve at GenBank, and the genetics fundamental research is very weak, the genetic background absence of information, and this has limited to a great extent selects carrying out smoothly of breeding work.Therefore carry out corresponding genetics fundamental research, location important economical trait and be and at first need the problem that solves in China's Chinese wax seed selection work.In addition, traditional Chinese wax classification mainly relies on the different of fruit shape and fruit wing number, and this sorting technique can't effectively be used treelet in early days.Therefore utilize the molecular biology way, set up effectively, the germplasm discrimination method has great importance to solving China's Chinese ash fine-variety breeding efficiently.
The molecular marker assisted selection breeding is a kind of novel breeding mode, uses the molecular marker assisted selection breeding, can effectively detect genotype, carries out the QTL location, directly contains the individuality of target gene type by the marker gene assisted Selection.Compare with traditional breeding method, the molecular marker assisted selection breeding method directly occurs with dna form, all can detect at each tissue, each developmental stage of plant materials, is not subjected to season, environmental limit, does not have expression whether problem; Quantity is many, spreads all over whole genome; Polymorphism is high, utilizes a large amount of primers, probe can finish the analysis of covering gene group; Neutral mark does not namely affect the expression of objective trait, with bad proterties without inevitable chain codominant marker, can identify the genotype of isozygotying and the genotype of heterozygosis, complete genetic information is provided.RFLP mark, AFLP marking operation are comparatively loaded down with trivial details, and RAPD mark Stability and veracity is relatively poor, all is not suitable for the evaluation work of large-scale field material; The SCAR marking operation is easy, accuracy is high, the high shortcoming that overcomes above-mentioned mark of stability, is the molecule marking method of first-selection.Because the forest genetics cycle is long, change the backward situation on Fraxinus velutina breeding field, key is extensively to collect as early as possible variety source, walks the road that modern molecular biology technique combines with conventional breeding, shortening the breeding cycle, accelerate the research that molecular marker assisted selection is utilized.Therefore, obtain Salt And Alkali Tolerance Fraxinus velutina SCAR mark, have great importance for the Fraxinus velutina fine-variety breeding, for the seed selection research of China's Salt And Alkali Tolerance forest germplasm is provided fundamental basis and technical system; Enrich the genetic diversity of Salt And Alkali Tolerance green tree species, control the soil salinification, improve fertility; Increase ecology, society and economic benefit and all have reality and profound significance.
Summary of the invention
The purpose of this invention is to provide a kind of Salt And Alkali Tolerance Fraxinus velutina SCAR mark and be used for the application of assisted selection.
Salt And Alkali Tolerance Fraxinus velutina SCAR mark of the present invention is characterized in that, the specific fragment S20-592 that this SCAR mark is total length 592bp, and its nucleotide sequence is shown in SEQ ID NO.1.
For realizing above-mentioned task, the present invention takes the technical scheme of following steps: (1), be respectively R36, R39 and the common Fraxinus velutina of 5 strains as material take 2 plant height salt-resistant type Fraxinus velutinas; (2), adopt the CTAB method to extract the total DNA of Fraxinus velutina blade; (3), set up and optimization Fraxinus velutina RAPD reaction system the RAPD amplification of acquisition stability and high efficiency; (4), use the RAPD Analysis and Screening and go out a molecule marker S20-592 who is associated with Salt And Alkali Tolerance; (5), the RAPD molecule marker S20-592 with saline-resisting and alkaline-resisting gene clones, checks order and is converted into the SCAR mark.Wherein:
The described high salt-resistant type Fraxinus velutina R36 of step (1), R39 are that the seed selection that the Liu De of Shandong Forest Science Academy imperial or royal seal passed through 20 years obtains.
The concrete grammar that the described employing of step (2) CTAB method is extracted the total DNA of Fraxinus velutina blade is: 1) get 0.1～0.2g Fraxinus velutina blade, grind in liquid nitrogen.Add 800 μ l DNA extraction damping fluids: contain 2% (w/v) CTAB, 20mmol/LEDTA (pH=8.0), 100mmol/L Tris-HCl (pH=8.0), 1.4mol/LNaCl and 2% beta-mercaptoethanol, put upside down gently mixing; 2) incubation 30min in 65 ℃ of water-baths, every 10min puts upside down gently mixing, 12000rpm, centrifugal 10min; 3) get supernatant 700 μ L, and adding equal-volume chloroform/primary isoamyl alcohol (24: 1, v: v), put upside down gently mixing, 4 ℃, 12000rpm, centrifugal 10min; 4) get supernatant 600 μ l, and adding equal-volume chloroform/primary isoamyl alcohol (24: 1, v: v), put upside down gently mixing, 4 ℃, 12000rpm, centrifugal 10min; 5) get supernatant 500 μ l, add the ice-cold dehydrated alcohol of 2 times of volumes, put upside down mixing ,-20 ℃ leave standstill 30min; 6) 4 ℃, 12000rpm, centrifugal 10min; 7) remove supernatant, use 75% washing with alcohol of precooling to precipitate 2 times, 4 ℃, 10000rpm, centrifugal 5min; 8) at the drying precipitated 30min of Bechtop, water (ddH is steamed in the sterilization that adds 30 μ l
2O) dissolution precipitation after concentration and purity detecting, is put-20 ℃ or-70 ℃ of Refrigerator stores for subsequent use.
The concrete grammar of the described RAPD amplification of step (3) is: PCR reaction cumulative volume 25 μ l, 10 * reaction buffer, 2.5 μ l, 25mM/L MgCl
21.8 μ l, 10mM/L dNTPs 1.5 μ l, 30ng/ μ l primer P 2 μ l, template DNA 2 μ l, 2u/ul TaqDNA polysaccharase 0.25 μ l, ddH
2O 14.95 μ l; The PCR reaction conditions: 95 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 36 ℃ of annealing 1min, 72 ℃ are extended 2min, circulate 44 times, and 72 ℃ are extended 10min.Electrophoresis Marker and reagent are that TAKARA company produces, after the amplification with 1.0% agarose gel electrophoresis, GeneGenius full automatic gel imaging analysis systematic observation result and photograph.
The described concrete grammar of step (4) is: randomly draw the common Fraxinus velutina of 5 strains and 2 plant height Salt And Alkali Tolerance plant R36, R39, set up respectively 3 gene pools of the high Salt And Alkali Tolerance plant of 5 strains common Fraxinus velutina mixing gene pool and R36, R39, utilize 150 of the synthetic S series primers of Shanghai bio-engineering corporation, high Salt And Alkali Tolerance and common Fraxinus velutina mixing gene pool have been carried out the RAPD analysis, found to only have the amplified production of S20 primer in high salt-tolerant plants and common plant, to show polymorphism.Fig. 1 is the RAPD amplification of primer S20, can observe the molecule marker S20-592 that a molecular weight is about the specific band of 600bp in the Salt And Alkali Tolerance plant, revision test 3 times, and this band still can be stablized appearance.
The described molecule marker S20-592 of step (5) clone, check order and the concrete grammar that is converted into the SCAR mark is: the special segment S20-592DNA that pcr amplification is gone out is behind 1.0% agarose gel electrophoresis, the glass milk test kit of producing with vast Tyke reclaims, and operation is carried out according to the test kit specification sheets.Dna segment after the recovery is connected with PMD-18T Vector, transforms bacillus coli DH 5 alpha, and recombinant plasmid obtains positive colony through T7, Sp6PCR electrophoresis detection, serves the order-checking of marine life engineering corporation, and its nucleotide sequence total length 592bp is specially:
ggacccttac caaatgacaa tcaatttcta tgtgcttggt ttgttcgtgg aaaactggat 60
tacttgctat gcctatagca aatgactatt acagaacaac aaagcaggtc tggtgtgatc 120
aatttgcaag ttattcaata tagtttttgg ccatgtaaac tcacggacgg ctgcagccat 180
tgatctgtat tcagcttcag ctgaagatct tgatacagtg tgttgcttct tggacttcca 240
tgacacaagg gaattgccca aaaagataca atatcctgta atcgactgtc ttgtatccga 300
acatgctgcc cgatcagcat cacaacaacc tttgagcaga agatcagatt tgctagaaat 360
gaataaccct gtcttgatga gttctacaga taccttaggc gtgataagct gcctccagat 420
gagactgtct tggggactga aggaattgac tcagaacttg tacaggatta aataaatctg 480
gcctggtaat agttaggtac aaaatcctcc caacaagcct tttgtaggca ttaagatcat 540
ctacaggctc ttcttcatcc tttctcaact ttaggttgca cggtaagggt cc 592
Sequencing result is analyzed with information biology SMS, and carry out homology analysis by the international gene database of Internet and GenBank, do not inquire relevant gene order and this segment homology, illustrate that this segment is the new molecule marker that is associated with the Salt And Alkali Tolerance Fraxinus velutina.
According to the dna sequence dna of clone's segment, synthesized SCAR mark special primer (Shanghai bio-engineering corporation is synthetic), its sequence is:
Forward primer: FS20-592:5 ' TGCTTGGTTTGTTCGTGG 3 '
Reverse primer: RS20-592:5 ' CGTGCAACCTAAAGTTGAG 3 '
Increase respectively in 5 individual plants of high Salt And Alkali Tolerance R36, R39 and common Fraxinus velutina with above-mentioned primer, PCR reaction system cumulative volume is 25 μ l, 2 * Taq PCR MasterMix, 12.5 μ l (day root biochemical technology company limited), FS20-592 (10 μ mol/L) 1 μ l, Rs20-592 (10 μ mol/L) 1 μ l, template DNA (20ng) 2 μ l, ddH
2O8.5 μ l; Amplification reaction condition is: 94 ℃ of denaturation 4min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ of 1.5min circulate 30 times, and 72 ℃ are extended 10min.Electrophoresis Marker is the DL2000 that TAKARA company produces, and with 1% agarose gel electrophoresis, the rear observation under ultraviolet lamp of EB dyeing taken pictures after the amplification.Amplification shows, amplifies the 592bp specific band in high Salt And Alkali Tolerance Fraxinus velutina, and do not amplify specific band in common Fraxinus velutina individual plant, as shown in Figure 2.
In order to verify the reliability of SCAR mark, the high salt-tolerant plants F1 of R36 is analyzed for the segregating population salt resistance.The result shows in anti-salt plant individuality and has detected the SCAR mark, and do not detect the SCAR mark in perceptual plant.
The SCAR of Salt And Alkali Tolerance Fraxinus velutina of the present invention is marked at the application for assisted selection.
The present invention adopts the RAPD technology, seek the RAPD molecule marker that is associated with the Salt And Alkali Tolerance proterties, be further converted to stability, repeated strong SCAR mark, lay a good foundation for carrying out Salt And Alkali Tolerance Fraxinus velutina molecular mark, provide technical guarantee for shortening breeding cycle.
The present invention has obtained a stable SCAR mark of Fraxinus velutina Salt And Alkali Tolerance genes involved, points out this Fraxinus velutina Salt And Alkali Tolerance SCAR to be marked in the assist-breeding Fraxinus velutina Salt And Alkali Tolerance new lines and has widespread use.
Beneficial effect of the present invention: the present invention utilizes molecule marking method to obtain the RAPD molecule marker of new and the anti-saline and alkaline gene-correlation connection of Fraxinus velutina, and by the clone, order-checking, design of primers, again the RAPD mark is converted to stable SCAR mark, for Fraxinus velutina molecular marker assisted selection breeding system is laid a good foundation, compare with present technology, its advantage is: 1) originally be labeled as stable SCAR mark, can be widely used in the assisted selection of Salt And Alkali Tolerance Fraxinus velutina molecule marker, be simultaneously clone's Fraxinus velutina saline-resisting and alkaline-resisting gene, gene sequencing is had laid a good foundation, and for further understanding the anti-saline and alkaline molecular genetics mechanism of Fraxinus velutina positive effect is arranged.2) molecular marker assisted selection is easy and simple to handle, saves cost; Fraxinus velutina is the perennial woody plant, the conventional herd breeding method, and the cycle is long, and cost is high, wastes time and energy.Only need carry out simple pcr amplification by the present invention, get final product the salt tolerance of effective expert evidence, avoid conventional herd breeding method required time cycle length and the loaded down with trivial details shortcomings such as screening process.3) the present invention can carry out the evaluation in seedling stage to individuality by the SCAR mark, has greatly improved breeding efficiency, has shortened breeding process.
Description of drawings
Fig. 1: the RAPD molecule marker S20-592 screening electrophoretic band figure of high Salt And Alkali Tolerance Fraxinus velutina R36, R39 and the common Fraxinus velutina mixing of 5 strains gene pool DNA.M-DL2000Mark; The RAPD result of the common Fraxinus velutina mixing of 0:1-5 strain gene pool DNA; R36, R39-are respectively the RAPD result of high Salt And Alkali Tolerance Fraxinus velutina.
Fig. 2: SCAR is marked at the PCR electrophoretic band figure in high Salt And Alkali Tolerance Fraxinus velutina and the common Fraxinus velutina individual plant.M-DL2000Mark; R36, R39-are respectively high Salt And Alkali Tolerance Fraxinus velutina; 1-5: common Fraxinus velutina individual plant.
Fig. 3: the present invention uses the anti-electrophoretogram of feeling plant genomic dna of SCAR mark to having identified.M:DL2000Mark; The F1 of 1-10:R36 is for resistant plant 10 strains; The F1 of 11-20:R36 is for perceptual plant 10 strains.
Embodiment
Embodiment 1: the acquisition of the RAPD polymorphism mark that is associated with saline-resisting and alkaline-resisting gene
One, material:
High Salt And Alkali Tolerance Fraxinus velutina R36, R39 and common Fraxinus velutina are provided by Shandong Forest Science Academy's Liu De of Dongying branch imperial or royal seal.
Two, a small amount of method DNA extraction:
1) gets 0.2g Fraxinus velutina blade, in liquid nitrogen, grind.Add 800 μ l DNA extraction damping fluids: contain 2% (w/v) CTAB, 20mmol/LEDTA (pH=8.0), 100mmol/L Tris-HCl (pH=8.0), 1.4mol/LNaCl and 2% beta-mercaptoethanol, put upside down gently mixing;
2) incubation 30min in 65 ℃ of water-baths, every 10min puts upside down gently mixing, 12000rpm, centrifugal 10min; 3) get supernatant 700 μ l, and adding equal-volume chloroform/primary isoamyl alcohol (24: 1, v: v), put upside down gently mixing, 4 ℃, 12000rpm, centrifugal 10min;
4) get supernatant 600 μ l, and adding equal-volume chloroform/primary isoamyl alcohol (24: 1, v: v), put upside down gently mixing, 4 ℃, 12000rpm, centrifugal 10min;
5) get supernatant 500 μ l, add the ice-cold dehydrated alcohol of 2 times of volumes, put upside down mixing ,-20 ℃ leave standstill 30min; 6) 4 ℃, 12000rpm, centrifugal 10min;
7) remove supernatant, use 75% washing with alcohol of precooling to precipitate 2 times, 4 ℃, 10000rpm, centrifugal 5min;
8) at the drying precipitated 30min of Bechtop, add the sterilization ddH of 30 μ l
2The O dissolution precipitation after concentration and purity detecting, is put-20 ℃ or-70 ℃ of Refrigerator stores for subsequent use.
Three, RAPD amplification polymorphism labeled analysis:
The RAPD primer is S series S1-S150, totally 150, and available from Shanghai bio-engineering corporation.
The RAPD amplification system of optimizing is:
(1) the PCR reaction system is:
10 * reaction buffer, 2.5 μ l, 25mM/L MgCl
21.8 μ l, 10mM/L dNTPs 1.5 μ l, 30ng/ μ l primer P 2 μ l, template DNA (20ng) 2 μ l, 2u/ul Taq archaeal dna polymerase 0.25 μ l, ddH
2O 14.95 μ l cumulative volumes 25 μ l;
(2) the PCR response procedures is:
95 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 36 ℃ of annealing 1min, 72 ℃ are extended 2min, circulate 44 times, and 72 ℃ are extended 10min.
Get 8 μ L amplified productions with 1.0% agarose gel electrophoresis, GeneGenius full automatic gel imaging analysis systematic observation result also takes a picture, and electrophoresis Marker and reagent are that TAKARA company produces.
With 150 random primers, respectively high Salt And Alkali Tolerance and common low Fraxinus velutina gene pool are carried out the RAPD amplification, discovery only has the amplified production of S20 primer to show polymorphism in high saline-resisting and alkaline-resisting gene pond and common low saline-resisting and alkaline-resisting gene pond, through 3 repeat amplification protcols, stable, the consistent difference of performance between two gene pools, S20 amplifies specific band S20-592 in high saline-resisting and alkaline-resisting gene pond, and lack this band at common gene pool, obtained to be associated with the Salt And Alkali Tolerance proterties special segment S20-592, molecular size range is 592bp.
Embodiment 2: acquisition and the evaluation of the SCAR mark that is associated with saline-resisting and alkaline-resisting gene
One, the recovery of special segment S20-592:
The special segment S20-592DNA that pcr amplification is gone out is behind 1.0% agarose gel electrophoresis, and the glass milk test kit of producing with vast Tyke reclaims, and operation is carried out according to the test kit specification sheets.
Two, ligation:
The ligation volume is 10 μ l, wherein contains DNA 5 μ l (30～50ng), PMD-18T Vector, 1 μ l, T4 ligase enzyme 1 μ l, connection damping fluid 1 μ l, the sterilization ddH of recovery
2O1 μ l.Behind the abundant mixing of reaction mixture, connect 12 hours at 16 ℃.
Three, transform:
1, from-70 ℃ of refrigerators, get under 200 μ l bacillus coli DH 5 alphas (this laboratory preparation preserve) the competent cell room temperature it thawed, after thawing immediately as on ice.
2, add 10 μ l and connect mixture, place 30min after shaking up on ice.
3,42 ℃ of thermal shock mixture 90s, fast transfer is to cooled on ice 2～3min behind the thermal shock.
4, add 800 μ l liquid LB substratum in centrifuge tube, behind 37 ℃ of 180rpm vibration 1h, concentrated bacterium liquid removes supernatant 800 μ l, gets 200 μ l transformation mixtures and coats on the flat board of solid LB substratum.With coated flat board at room temperature face up place 30min after, be inverted dull and stereotypedly, put into 37 ℃ of constant incubators and cultivate 16-24h.After manifesting white colony, the picking white colony places the LB liquid nutrient medium of 10ml, and 37 ℃ of 180rpm overnight incubation are identified with PCR method to mid-log phase, and the bacterium liquid of positive colony is delivered to the order-checking of Shanghai bio-engineering corporation.
Four, the acquisition of SCAR mark and detection:
1, obtained the special segment SCAR mark of length 592bp, its nucleotide sequence is:
ggacccttac caaatgacaa tcaatttcta tgtgcttggt ttgttcgtgg aaaactggat 60
tacttgctat gcctatagca aatgactatt acagaacaac aaagcaggtc tggtgtgatc 120
aatttgcaag ttattcaata tagtttttgg ccatgtaaac tcacggacgg ctgcagccat 180
tgatctgtat tcagcttcag ctgaagatct tgatacagtg tgttgcttct tggacttcca 240
tgacacaagg gaattgccca aaaagataca atatcctgta atcgactgtc ttgtatccga 300
acatgctgcc cgatcagcat cacaacaacc tttgagcaga agatcagatt tgctagaaat 360
gaataaccct gtcttgatga gttctacaga taccttaggc gtgataagct gcctccagat 420
gagactgtct tggggactga aggaattgac tcagaacttg tacaggatta aataaatctg 480
gcctggtaat agttaggtac aaaatcctcc caacaagcct tttgtaggca ttaagatcat 540
ctacaggctc ttcttcatcc tttctcaact ttaggttgca cggtaagggt cc 592
2, the PCR primer sequence of SCAR mark acquisition:
According to the SCAR flag sequence, by the Primer software design special primer sequence be:
Forward primer: FS20-592:5 ' TGCTTGGTTTGTTCGTGG 3 '
Reverse primer: RS20-592:5 ' CGTGCAACCTAAAGTTGAG 3 ', synthetic by Shanghai bio-engineering corporation.
3, SCAR marker detection
Increase respectively in 5 individual plants of high Salt And Alkali Tolerance R36, R39 and common Fraxinus velutina with the SCAR-PCR primer.
The PCR reaction system is: cumulative volume is 25 μ l, 2 * Taq PCR MasterMix, 12.5 μ l (day root biochemical technology company limited), FS20-592 (10 μ mol/L) 1 μ l, RS20-592 (10 μ mol/L) 1 μ l, template DNA (20ng) 2 μ l, ddH
2O8.5 μ l.
The PCR reaction conditions is: 94 ℃ of denaturation 4min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ of 1.5min circulate 30 times, and 72 ℃ are extended 10min.
Electrophoresis result: amplified production is observed under ultraviolet lamp after the EB dyeing and is taken pictures with 1% agarose gel electrophoresis.
Amplification shows, amplifies the 592bp specific band in high Salt And Alkali Tolerance Fraxinus velutina, and do not amplify specific band (Fig. 2) in common Fraxinus velutina individual plant.
Pcr amplification reagent and Marker are available from TAKARA company.
Embodiment 3: the SCAR of Salt And Alkali Tolerance Fraxinus velutina is marked at the application for assisted selection
Get the F1 of the stable anti-salt plant R36 that has identified for resistant plant 10 strains and 10 strains of perceptual plant, extract respectively the DNA of each plant with the CTAB method, then carry out SCAR take the DNA that extracts as template and analyze.
Concrete, respectively take above-mentioned genomic dna as template, utilize two primers (primer 1:5 ' TGCTTGGTTTGTTCGTGG 3 ' of SCAR mark; Primer 2: 5 ' CGTGCAACCTAAAGTTGAG 3 ') carry out pcr amplification, the reaction cumulative volume is 25 microlitres, 2 * Taq PCR MasterMix, 12.5 μ l (day root biochemical technology company limited), primer 1 (10 μ mol/L) 1 μ l, primer 2 (10 μ mol/L) 1 μ l, 20ng genomic dna 2 μ l, ddH
2O 8.5 μ l.
Under 55 ℃ annealing temperature, carry out pcr amplification, reaction parameter is 94 ℃ of denaturation 4min, 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ of 1.5min circulate 30 times, and 72 ℃ are extended 10min.Pcr amplification product separates at 1.0% agarose gel electrophoresis, and GeneGenius full automatic gel imaging analysis systematic observation result also takes a picture.
Electrophoresis Marker DL2000 and reagent are TAKARA company.
Analytical results such as Fig. 3.As can be seen from Fig. 3, antagonism plant 1-10 increases with the SCAR Auele Specific Primer respectively, and the result has amplified respectively the specificity bands of a spectrum.And in the amplified production of perceptual plant 11-20, all do not amplify spawn.This has confirmed the reliability of SCAR marker detection and has used seedling stage that the molecule marker that is associated with the salt resistance shape is as the feasibility of breeding for salt resistance assisted Selection again.
Claims (3)
1. a Salt And Alkali Tolerance Fraxinus velutina SCAR mark is characterized in that, described SCAR mark is the nucleotide sequence shown in the 33-582bp among the SEQ ID NO.1.
2. primer that is used for the described Salt And Alkali Tolerance Fraxinus velutina of pcr amplification claim 1 SCAR mark, it is characterized in that: described primer is:
Forward primer: FS20-592:5 ' TGCTTGGTTTGTTCGTGG3 '
Reverse primer: RS20-592:5 ' CGTGCAACCTAAAGTTGAG3 '.
3. the SCAR of the described Salt And Alkali Tolerance Fraxinus velutina of claim 1 is marked at the application for assist-breeding Salt And Alkali Tolerance Fraxinus velutina.
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| CN103205424B (en) * | 2013-04-10 | 2014-07-23 | 浙江省林业科学研究院 | Molecule specificity marker primer and identification method of improved variety of camellia oleifera Changlin Number 55 |
| CN104046697B (en) * | 2014-07-04 | 2015-08-12 | 中国农业大学 | Based on the Fraxinus velutina transcript profile order-checking SSR primer sets of information development and the application in Idioplasm identification thereof |
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