CN103627780B - Establishing method of fingerprintings of muskmelon hybrid 'Hongyou' - Google Patents

Establishing method of fingerprintings of muskmelon hybrid 'Hongyou' Download PDF

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Publication number
CN103627780B
CN103627780B CN201210300585.XA CN201210300585A CN103627780B CN 103627780 B CN103627780 B CN 103627780B CN 201210300585 A CN201210300585 A CN 201210300585A CN 103627780 B CN103627780 B CN 103627780B
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dna
muskmelon
hongyou
srap
issr
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CN103627780A (en
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张永平
陈幼源
杨少军
陈绯翔
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Shanghai Keyuan Seed Co ltd
Shanghai Academy of Agricultural Sciences
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Shanghai Keyuan Seed Co ltd
Shanghai Academy of Agricultural Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

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Abstract

The invention discloses an establishing method and applications of DNA fingerprintings of muskmelon hybrid 'Hongyou' and parents of the muskmelon hybrid 'Hongyou'. The establishing method comprises following steps: 1) muskmelon DNA is extracted and purified; 2) the obtained high-purity muskmelon DNA is taken as a template for ISSR and SRAP analysis; and 3) polymorphism amplification bands with representativeness are selected for establishment of the DNA fingerprintings of the samples, wherein in the DNA fingerprintings, the muskmelon hybrid 'Hongyou' and the parents all possess specific DNA fingerprints, so that the muskmelon hybrid 'Hongyou' and the parents can be distinguished from each other. The DNA fingerprintings are represented in forms of graphs, so that it is visualized for reading, and is simple for understanding; and the DNA fingerprintings are transformed into digital representation forms, so that it is convenient for computers to read and analyze. The establishing method is capable of realizing purity identification of seeds of the muskmelon hybrid 'Hongyou' based on genetic nature, results are accurate and reliable, and detection is rapid.

Description

The foundation of the red excellent finger printing of muskmelon cross-fertilize seed
One, technical field:
The present invention relates to muskmelon seeds Purity technology, specifically the establishment method of the DNA fingerprinting of muskmelon and application.
Two, background technology:
Muskmelon (Cucumis melo L.) is the cultivar in Curcurbitaceae Cucumis, and be one of important cash crop, the first generation of hybrid is at home and abroad extensively cultivated.But because emasculation is not in time or thorough in artificial hybridization production of hybrid seeds process, be often mixed with maternal self-mating system seed, melon variety is comparatively numerous and diverse simultaneously, and between some kinds, phytomorph closely, is difficult to differentiate.Traditional authentication method is judged by phenotypic characteristic, take time and effort, undertaken differentiating also to affect by the aspect such as environment or etap by isozyme and Seed Storage Protein electrophoretic technique, there is larger shortcoming, and molecular marking technique detection is the difference in genomic dna level, not by the impact of external environment, therefore result is highly stable.In the molecular marking technique set up, ISSR (simple sequence repeats interval) mark is a kind of highly effective molecule marker grown up based on microsatellite sequence, has that masterplate requirement is few, without the need to test kit, outcome record is convenient, experimentation cost is low, simple to operate, test stability comparatively advantages of higher; SRAP (sequence be correlated with amplification polymorphism) is a kind of Mk system of novel PCR-based, has easy, stable, moderate yield, can produce codominant marker and be convenient to the features such as cloning and sequencing target fragment, and the rich polymorphism of amplification.
Three, summary of the invention:
The object of this invention is to provide a kind of DNA fingerprinting for muskmelon seeds authenticate technology and establishment method thereof and application, to overcome the deficiency that prior art exists.
Technical conceive of the present invention is as follows:
Seed is molecule marking method based on the polymorphism of DNA to utilize DNA fingerprinting to identify, can make up and overcome in many defects of the Morphological Identification of seed purity and isozyme, Seed Storage Protein electroresis appraisal and a difficult problem.The present invention utilizes the DNA fingerprinting of ISSR and SRAP technique construction southern areas muskmelon facility cultivation kind " red excellent " such as Shanghai and its parent; the qualification of kind and purity detecting can provide scientific basis for this reason, and then the legitimate rights and interests of the protection producer and user.
The DNA fingerprinting of muskmelon cross-fertilize seed of the present invention " red excellent " and parent thereof, is made up of " 1 " and " 0 ", and ISSR primer I-19 male parent is 110011, and female parent is 111010, and " red excellent " is 1111111; SRAP primer Me15-Em8 male parent is 111011, and female parent is 110111, and " red excellent " is 111111.Wherein said numeral " 1 " represents in collection of illustrative plates, and certain position has amplified band to exist, and digital " 0 " represents do not have amplified band on certain position.
The product of said " red excellent " muskmelon cross-fertilize seed to be Shanghai be Academy of Agricultural Sciences's Horticultural Research Institute public offering.
The establishment method of muskmelon DNA fingerprinting, comprises the steps:
1) extraction and the genomic DNA of purifying muskmelon;
2) with obtain muskmelon DNA be masterplate carry out ISSR and SRAP analyze;
3) representational polymorphism amplified band is selected, having, without building for examination material DNA fingerprinting according to selected band.
The DNA fingerprint that wherein said " red excellent " muskmelon cross-fertilize seed and parent thereof have it special, can make a distinction mutually.Primer I-19 base sequence adopted in described ISSR amplification is (TC) 7CC; The primer Me15-Em8 adopted in SRAP amplification, its base sequence is 5 ' TGAGTCCAAACCGGTAC-3 ', 5 ' GACTGCGTACGAATTCTG-3 ', is synthesized by Shanghai Sheng Gong biotechnology company limited.
The qualification of muskmelon seeds purity that DNA fingerprinting of the present invention can be applied to " red excellent ".
Tool of the present invention has the following advantages:
1. invention creates the DNA fingerprinting of " red excellent " muskmelon cross-fertilize seed and parent thereof, disclose result of study muskmelon seeds identified by DNA fingerprint analytical technology, the method set up can identify therefore accurately, reliably, have legal effect from inheritance to muskmelon seeds.
2. adopt the present invention, when truly detecting " red excellent " muskmelon seeds sample, extracting the DNA of testing sample by quick, the minim DNA extraction method of current widespread use, with the DNA extracted as masterplate, carrying out ISSR or SRAP and analyzing,
In several hours, just can know this DNA fingerprint, therefore, his verity can be judged rapidly.
3., because DNA fingerprinting of the present invention represents by the form of figure, seem more directly perceived, understandable; And owing to being converted to digital form, being convenient to computer recognition and analyzing.
Four, accompanying drawing illustrates:
Fig. 1 is the finger printing that the present invention sets up SRAP primer Me15-Em8, and Fig. 2 is the finger printing that the present invention sets up ISSR primer I-19.Wherein 1 is " red excellent " male parent; 2 is that " red excellent " is maternal; 3 is " red excellent "; M is Mark.
Five, embodiment:
Embodiment 1
1 material
Muskmelon cross-fertilize seed " red excellent " and parent thereof provided by Academy of Agricultural Sciences, Shanghai City gardening.
2 reagent
Test extraction DNA, PCR reagent and agarose used all purchased from Shanghai Sheng Gong biotechnology company limited.
3 methods
The isolation and determination of 3.1DNA.
DNA adopts CTAB micromethod extraction step:
(1) 2ml centrifuge tube adds 1 ~ 2, the fresh blade of centrifuge tube lid size.
(2) add 700ulCTAB damping fluid, carry out grinding 5min with shredder.
(3) 65 DEG C of water-bath 1h.
(4) 4 DEG C of refrigerators are placed in or room temperature is cooled to less than 15 DEG C.
(5) add 700 μ l 24: 1 chloroforms/primary isoamyl alcohol, mix 5min up and down, ensure that sample and chloroform fully mix.
(6) the centrifugal 10min of 13000rpm.
(7) get supernatant liquor 500 μ l, add in the 1.5ml centrifuge tube having added 500 μ l Virahols in advance, mixing of turning upside down gently.
(8) 4 DEG C of refrigerators leave standstill more than 30min.
(9) the centrifugal 10min of 13000rpm, abandons supernatant liquor.
(10) dry up DNA, allow Virahol volatilize clean.
(11) 100 μ l ddH are added 2o (RNase containing 1 μ l10mg/ml) dissolving DNA.
(12) 37 DEG C of water-baths or room temperature remove RNA in 1 hour.
(13) sepharose configuring % (takes lg agarose, after adding 100mlTBE Buffer heating for dissolving, be cooled to 50 ~ 60 DEG C), add an EB (0.5 μ g/ml) and pour in glue groove, insert comb, detect after gelling is solid.Get the DNA of 5 μ l, add 1 μ l Lodading Buffer, detect, click and enter 4 μ l, 5 μ l respectively 3 loading wells, 6 μ l λ DNA (45ng/ μ l) in contrast, carry out the detection of sample DNA concentration.General concentration is between 50 ~ 100ng.
Attached: CTAB solution composition:
100ml 1M TRIS pH7.5140ml 5M NaCl
20ml 0.5M EDTA pH8.0740ml MiliQ H 2O
20g CTAB (65 DEG C of water-baths are dissolved) (noting: must guard against the supernatant that sample size is too much and absorption is excessive)
3.2PCR amplification and product detect
SRAP amplified reaction adopts the reaction system of 20 μ L, wherein 25mmol/L MgCl 22.0 μ L, 10 × PCR Buffer 2.0 μ L, 10mmol/L dNTP 0.4 μ L, 5U/ μ L Taq archaeal dna polymerase 0.2 μ L, 0.1 μm of ol/L primer each 3.0 μ L, 10ng/ μ L template DNA 3.0 μ L, sterilizing distilled water 6.4 μ L.Response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 35 DEG C of annealing 1min, 72 DEG C extend 1.5min, 5 circulations; 94 DEG C of sex change 1min, 50 DEG C of annealing 1min, 72 DEG C extend 1.5min, 35 circulations; 4 DEG C of preservations after 72 DEG C of extension 10min.
ISSR amplified reaction adopts the reaction system of 20 μ L, wherein comprises 25mmolL -1mgCl 22 μ l, 10 × PCR Buffer2.0 μ l, 10mmolL -1dNTP0.4 μ l, 5UL -1taq E 0.2 μ l, 10ngL -1template DNA 3 μ l, 0.1 μm of olL -1primer3 μ l, sterilizing distilled water 9.4 μ l.Amplification program: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 52 DEG C of annealing 1min, 72 DEG C extend 1.5min, 35 circulations, 4 DEG C of preservations after 72 DEG C of extension 10min.
Pcr amplification product is in 2.0% sepharose (containing E B) electrophoretic separation, and voltage 120V, the time is about 1.5h, Gel Doc tMeQ 170-8060 gel imaging instrument carries out observation and photographic analysis.
4 primer screenings and amplification analysis
Pcr amplification is carried out for examination material with 40 couples of SRAP and 40 ISSR primer pairs, wherein 40 pairs of SRAP primers and 15 ISSR primers all can obtain comparatively stable AFLP system, but the amplification of most of primer is consistent between parents and hybrid, discriminating effect can not be played.Finishing screen selects a pair SRAP special primer Me15-Em8 (Fig. 1) and 1 ISSR primer I-19 (Fig. 2) can be separated cross-fertilize seed and its Parent.
The foundation of 5 digital finger-prints
According to above-mentioned amplification, the presence or absence of the DNA band that certain allelotrope site amplifies is represented respectively with 1 and 0, according to from top to bottom namely by the tape reading direction of small segment to large fragment, finger printing is converted to the character string be made up of 1 and 0, namely forms digital finger-print.ISSR primer I-19 male parent is 110011, and female parent is 111010, and " red excellent " is 1111111; SRAP primer Me15-Em8 male parent is 111011, and female parent is 110111, and " red excellent " is 111111.Wherein said numeral " 1 " represents in collection of illustrative plates, and certain position has amplified band to exist, and digital " 0 " represents do not have amplified band on certain position.
Embodiment 2
DNA fingerprinting of the present invention is used for the qualification of " red excellent " purity of hybrid: the cross-fertilize seed produced from seed breeding base randomly draws 100, germinate, DNA is extracted by micro-CTAB method, with the DNA extracted as masterplate, carry out ISSR and SRAP amplification respectively with primer I-19 and Me15-Em8, amplified production carries out electrophoretic analysis on the agarose of 2.5%.If the individual plant DNA fingerprint utilizing ISSR primer I-19 labeled analysis to find is 1111111, be " red excellent " true cross-fertilize seed, DNA fingerprint is 110011 or 111010, is " red excellent " parental autocopulation kind, namely false cross-fertilize seed.If the individual plant DNA fingerprint utilizing SRAP primer Me15-Em8 labeled analysis to find is 111111, be " red excellent " true cross-fertilize seed, DNA fingerprint is 111011 or 110111, is " red excellent " parental autocopulation kind, namely false cross-fertilize seed.Then use false cross-fertilize seed number divided by 100, the purity of the muskmelon cross-fertilize seed that seed breeding base is produced can be obtained, not only save time but also convenient compared with field planting investigation purity, purity information can be provided timely for the sale of muskmelon cross-fertilize seed.If the purity of qualification, lower than 95%, just can not be sold as commodity seed, just may avoid the generation of pseudosperm event.

Claims (2)

1. the establishment method of muskmelon DNA fingerprinting, is characterized in that, comprises the steps:
1) extraction and the genomic DNA of purifying muskmelon;
2) with obtain muskmelon DNA be masterplate carry out ISSR and SRAP analyze;
3) representational polymorphism amplified band is selected, having, without building for examination material DNA fingerprinting according to selected band;
The DNA fingerprint that described muskmelon cross-fertilize seed " red excellent " and parent thereof have it special, can mutually make a distinction, concrete finger print data is made up of " 1 " and " 0 ", and ISSR primer I-19 male parent is 110011, and female parent is 111010, and " red excellent " is 1111111; SRAP primer Me15-Em8 male parent is 111011, and female parent is 110111, and " red excellent " is 111111; Wherein said numeral " 1 " represents in collection of illustrative plates, and certain position has amplified band to exist, and digital " 0 " represents do not have amplified band on certain position;
The I-19 base sequence adopted in described ISSR amplification is (TC) 7cC; The primer Me15-Em8 adopted in SRAP amplification, its base sequence is 5 ' TGAGT CCAAACCGGTAC-3 ', 5 ' GACTGCGTACG AATTCTG-3 '.
2. the method according to claim 1 application of DNA fingerprinting of setting up, is characterized in that, for the qualification of muskmelon cross-fertilize seed " red excellent " seed purity.
CN201210300585.XA 2012-08-22 2012-08-22 Establishing method of fingerprintings of muskmelon hybrid 'Hongyou' Active CN103627780B (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN102134584A (en) * 2010-01-22 2011-07-27 新疆大学 Xinjiang honey melon amplified fragment length polymorphism (AFLP) fingerprint

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
CN102134584A (en) * 2010-01-22 2011-07-27 新疆大学 Xinjiang honey melon amplified fragment length polymorphism (AFLP) fingerprint

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甜瓜分子标记纯度鉴定研究;张红等;《中国瓜菜》;20060131(第1期);第7-10页 *
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