CN105002167B - The application of the method and primer pair of identification section cucurbit wilt - Google Patents
The application of the method and primer pair of identification section cucurbit wilt Download PDFInfo
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Abstract
The present invention relates to one kind the present invention relates to molecular biology DNA marker detection technique field, in particular for the SRAP molecular labeling primers and its methods and applications of section cucurbit wilt identification.Present invention firstly provides a kind of SRAP molecular labelings, described molecular labeling is to be used to identify section cucurbit wilt.The present invention additionally provides a kind of primer pair, the nucleotide sequence of the primer pair is respectively as shown in SEQ ID No.1 and No.14.The method that section cucurbit wilt is identified using SRAP molecular marking techniques, using the genomic DNA of the amplification section cucurbit wilt in PCR amplification system, gained PCR primer is subjected to electrophoresis detection.The present invention saves cucurbit wilt using the identification of SRAP molecular marking techniques, as a result not protected from environmental, as a result accurately and reliably；Meanwhile SRAP molecular marking techniques provided by the invention operation is quick and easy, testing result is stable, cost is cheap.
Description
Technical field
The present invention relates to molecular biology DNA marker detection technique field, in particular for section cucurbit wilt identification
SRAP molecular labeling primers and its methods and applications.
Background technology
Save melon also known as hairy squash, Curcurbitaceae is annual climbs up by holding on to herbaceous plant for category, is a mutation for wax gourd, is South China's weight
Want one of vegetable crop.
The plant disease that droop is led to by fungi or bacterium, morbidity is unexpected, symptom include serious stigma, it is wilting or
The death of leaf, flower, fruit, stem or whole plant.Rapid tender tissue is grown often to be attacked.
Cucurbits fusarium wilt is also known as wilt disease, by Deuteromycotina point Fusarium Fusarium oxysporum (Fusarium
Oxysporum) cause, it produces a large amount of sickle shaped conidiums and locust with caused cotton-shaped mycelia invaded plantses body after ripe
Ovate conidium carries out propagation and infects (peace weight jade-like stone etc., 2009), and spread speed is soon, spread scope is wide, crushing big,
Lose big, harm is unmanageable；And (She little Man etc., 2011) according to the literature, Fusarium oxysporum at least have 8
Specialized form, i.e. Fusarium oxysporum f. sp. niveum (F.oxyspoorum Schl.f.sp.niveum (E.F.Smith) Snyder＆
Hansen), Fusarium oxysporum cucumber specialized form (F.oxyspoorum Schl.f.sp.cucumerinum Owen), sharp spore sickle
Knife bacterium muskmelon specialized form (F.oxyspoorum Schl.f.sp.melonis Snyder＆Hansen), Fusarium oxysporum cucurbit are special
Change type (F.oxyspoorum Schl.f.sp.lagenariae Matuo＆Yamamoto), Fusarium oxysporum wax gourd specialized form
(F.oxyspoorum Schl.f.sp.benincasae Sun S.K.＆Huang J.W.), Fusarium oxysporum balsam pear specialized form
(F.oxyspoorum Schl.f.sp.momodicase Sun＆Huang), Fusarium oxysporum sponge gourd specialized form
(F.oxyspoorum Schl.f.sp.luffae Kwai Suzuki＆Kawai) and the melon specialized form of Fusarium oxysporum
(F.oxyspoorum Schl.f.sp.cucurbitacearum Gerlagh M.＆Block W.J.), and each specialized form is to melon
There is notable difference in the pathogenic of class crop, only it is to be understood that melon crop belongs to any specialized form, just there is a corresponding defence
And remedy measures, but do not have Fusarium oxysporum section melon specialized form in this 8 specialized forms, so carrying out anti-blight point to section melon
Sub- marker research, the generation for epidemic situation carry out look-ahead, it appears particularly important.
In view of this, it is special to propose the present invention.
The content of the invention
For can not fundamentally understand pathogenic factor using biological control and chemical prevention in production at present, from basic
Upper understanding pathogenic property, the present invention provide one kind and section cucurbit wilt can be early diagnosed, timely, quick, accurate early warning
Save the authentication method that cucurbit wilt occurs.
Present invention firstly provides a kind of SRAP molecular labelings, described molecular labeling is to be used to identify section cucurbit wilt.
Further, described molecular labeling is primer pair.
Another purpose of the invention is to provide a kind of primer pair, and described primer pair is from described SRAP molecular labelings
Upper screening, save cucurbit wilt for identifying.
Third object of the present invention is to provide a kind of specificity for being used to identify the SRAP molecular labelings of section cucurbit wilt
Amplimer pair, the nucleotides sequence number of columns of the reverse primer are right more than the nucleotides sequence number of columns 9 of forward primer for 11 pairs.
Further, the nucleotide sequence of the forward primer and reverse primer is selected from one of following combinations：Em9me8,
Em7me6, em2me5, em10me5, em5me1, em1me5, specific primer sequence table are as shown in table 1；
Further, the nucleotides sequence of the forward primer and reverse primer is classified as em5me1 combination, respectively such as SEQ
Shown in ID No.1 and No.14.
Above-described specificity amplification primer is to the application in terms of identification section cucurbit wilt.
Fourth object of the present invention is to provide the method for being identified section cucurbit wilt using SRAP molecular marking techniques, adopted
With em5me1 primer pairs noted earlier, the genomic DNA of section cucurbit wilt is expanded with PCR amplification system, gained PCR primer is entered
Row electrophoresis detection, if occurring 309bp characteristic strip in pcr amplification product, the section melon identified is infected with droop.
Wherein, it is preferred that the PCR amplification system is 25ul, and PCR-buffer is 2.5ul, Mg2+It is 2.0ul, dNTPs
It is 0.5ul, tTaq archaeal dna polymerases are 0.3ul, and DNA is 4ul, and primer pair is 2ul, H2O is 13.7ul；
Preferably, the primer pair concentration is 0.5-2.5 μM, and Taq archaeal dna polymerases concentration is 0.5-1.5U/25ul, Mg2+
Concentration is 0.5-2.5mM, and dNTPs concentration is 0.1-1.0mM, DNA concentration 5-40ng/25ul.
Preferably, the primer pair concentration is 1.0 μM, and Taq archaeal dna polymerases concentration is 1.0U/25ul, Mg2+Concentration is
2.5mM, dNTPs concentration are 0.5mM, DNA concentration 20ng/25ul.
Specific PCR amplification programs are 94 DEG C of 5min, and 1 circulates；94 DEG C of 1min, 35 DEG C of 1min, 72 DEG C of 1.5min, 5 are followed
Ring；94 DEG C of 1min, 50 DEG C of 1min, 72 DEG C of 1.5min, 35 circulations；72 DEG C of extension 8min, after amplification terminates, it is in firm power
50W, weight/mass percentage composition be 4% polyacrylamide gel under electrophoresis 1.5h, be then fixed, silver staining, colour developing, take pictures, obtain
To electrophoretogram.
Compared with prior art, the present invention has advantages below：The present invention is using SRAP molecular marking techniques identification section melon
Droop, it is as a result not protected from environmental, as a result accurately and reliably；Meanwhile SRAP molecular marking techniques operation provided by the invention
Quickly and easily, testing result is stable, cost is cheap.
Brief description of the drawings
Fig. 1 is the electrophoretogram of pcr amplification products of the primer em5me1 in microcommunity；
Fig. 2 is the electrophoretogram of pcr amplification products of the primer em5me1 in big colony；
Fig. 3 is the electrophoretogram of the pcr amplification product of embodiment 4.
Embodiment
The present invention is described in further detail with specific embodiment below in conjunction with the accompanying drawings.
Section cucurbit wilt is identified using SRAP molecular marking techniques, is broadly divided into following steps：
(1) separation, identification and the artificial infection of cucurbit wilt bacterium are saved.The present invention separates from the section melon plant of morbidity first
Go out to save cucurbit wilt bacterium, then by the section cucurbit wilt bacterium artificial infection separated to growing on normal plumule, Ran Houji
Record observation counts its incidence of disease；
(2) high anti-and susceptible droop gene pool the establishment of melon is saved.After this step is mainly with artificial infection in step (1)
Plant obtain F after being hybridized for maternal, male parent1Generation, by F1Generation selfing obtains F2Generation, F2Generation selfing obtains F3In generation, it will obtain
F2Generation and F3In generation, as research colony, in order to ensure the accuracy of research, disease resistance was further filtered out from this research colony
Good and susceptible serious plant, these plant are defined as to the base for the high resistance to wilt good and susceptible droop for meeting this test requirements document
Because of pond, then these plant progress DNA extractions through obtaining；
(3) the SRAP molecular marker screenings molecular labeling related to droop is utilized.This step is divided into 3 big steps：It is first
With 9 pairs of forward primers and 11 pairs of reverse primers, totally 99 primer pairs enter performing PCR expansion to the female parent in step (2) and male parent respectively
Increase, it can be seen that there are 6 pairs of primer pairs that there is characteristic strip；Secondly, the disease-resistant F that selecting step (2) obtains2-F3Generation, susceptible F2-F3
Generation, maternal, male parent, enter performing PCR respectively with 6 pairs of primer pairs and expand, it is the primer pair with characteristic strip to filter out em5me1；Most
Afterwards, by em5me1 characteristic strips in F2Generation and F3Expanded in the mixing gene pool of generation composition, by amplification with by manually identifying
Plant type is compared, it is found that result matches, so as to further determined that the characteristic strip of em5me1 amplifications and section melon variable rate technology
Significant correlation, similitude 97.4% be present in droop；
(4) field will be applied to using the SRAP molecular marker screenings molecular labeling em5me1 primer pair related to droop
The kind of More female lines material and in recent years seed selection (is all the material and product that early stage is manually accredited as anti-blight in daily breeding
Kind) droop identification is carried out, as a result it is shown in the material for examination, does not find droop.
Embodiment 1：Save separation, identification and the inoculation test of cucurbit wilt bacterium；
(1) section cucurbit wilt bacterium is chosen from the section melon plant of morbidity；
(2) culture medium is prepared.By 200g potatoes it is well-done after filter to take liquid, by its liquid and 20g agar, 18g glucose
1L solution is made into, 121 DEG C sterilize 20 minutes in autoclave, are cooled to room temperature and obtain solid medium；200g potatoes are boiled
Liquid is filtered to take after rotten, its liquid and 18g glucose are made into 1L solution, 121 DEG C sterilize 20 minutes in autoclave, are cooled to
Room temperature obtains fluid nutrient medium；
(3) solid medium, fluid nutrient medium are dispensed into stand-by on culture dish；
(4) germ of selection is cultivated on solid culture ware；
(5) germ after being cultivated 7-10 days on solid culture ware is chosen on purer wilt liquid medium within
240r/min shakes bacterium 16-18h, observes bacterial concentration under the microscope, and regulation concentration is diluted with water to 4 × 106Individual microspore/
ml；
(6) artificial infection, inoculation temperature are controlled at 25 DEG C or so, and vernalization, seed are carried out to the hairy squash seed Jing Guo vernalization
Radicle when growing 0.5cm, selection develops the intact seed with radicle 4 × 106Soaked in individual microspore/ml bacterium solution
It is half an hour, the soil in seedlings nursing plate is sterile-processed, on the record inoculation date, the statistical observation incidence of disease is carried out after emerging.
Embodiment 2：Save high anti-and susceptible droop gene pool the establishment of melon
(1) plant in embodiment 1 Jing Guo artificial infection is observed, determines the plant of a collection of high resistance to wilt good and susceptible droop
Strain is used as research material, wherein, selection male parent is high resistance to wilt good, and labeled as B-4, female parent is susceptible droop, is labeled as
0001；
(2) male parent B-4 and the hybridization of female parent 0001 are obtained into F1Generation, by F1Generation selfing obtains F2Generation, F2Generation selfing obtains F3Generation,
The F that will be obtained2Generation and F3In generation, as research colony, in order to ensure the accuracy of research, further filters out from this research colony
Disease resistance is good and susceptible serious plant, high resistance to wilt good that these plant are defined as meeting this test requirements document and susceptible withered
The gene pool of disease；
Embodiment 3：Utilize the SRAP molecular marker screenings molecular labeling related to droop
(1) 77 representative plant populations are chosen from the gene pool described in embodiment 2 as genetic material, mother
Originally, male parent carries out DNA extractions respectively.Specific extraction process is as follows：
The plant material that 200mg treats through wilt is chosen, every part of material is pulverized in liquid nitrogen after numbering
End, the CTAB for adding weight/mass percentage composition to be 3%, is extracted in 65 DEG C of water-baths, then 10000 leaves the heart in centrifuge
5min, take supernatant；Again with isometric 24：The chloroform isoamyl alcohol purifying of 1 (volume ratio), is existed with 12000 turns/min speed
Centrifugal treating 10min is carried out in centrifuge；Above-mentioned purification step is repeated, takes supernatant；Add 2/3rds volume supernatants
Pure isopropanol, after mixing subzero 20 DEG C stand 20min, then with 10000 turns/min centrifugation 2min, you can obtain
White precipitate, i.e. DNA, then white precipitate DNA is rinsed inside 70% and 100% alcohol respectively, air-dried, is added
100 μ, 1mo/L TE solution is dissolved, and by electrophoresis detection, concentration is finally determined according to DNA brightness；
In wherein 77 plant populations, first screening feature is carried out using 6 plant populations as microcommunity, then by 77 plant
Verified wherein as big colony, disease-resistant, 2 plant groups are shown as after there are 4 plant population inoculations in 6 plant populations
Susceptible, the resistance level situation such as institute of table 1 after 77 plant populations and maternal, male parent artificial infection droop is shown as after body inoculation
Show：
Resistance level situation after 1 77 parts of plant populations of table and two parent's artificial infection droops
| Material resistance level | Material resistance level | Material resistance level | Material resistance level |
| 1 R | 21 R | 41 R | 61 R |
| 2 R | 22 R | 42 R | 62 R |
| 3 R | 23 s | 43 s | 63 R |
| 4 R | 24 s | 44 R | 64 R |
| 5 s | 25 R | 45 R | 65 s |
| 6 s | 26 R | 46 R | 66 R |
| 7 R | 27 R | 47 R | 67 R |
| 8 R | 28 R | 48 R | 68 R |
| 9 R | 29 s | 49 R | 69 R |
| 10 s | 30 R | 50 R | 70 R |
| 11 R | 31 s | 51 R | 71 R |
| 12 R | 32 R | 52 R | 72 R |
| 13 R | 33 R | 53 R | 73 R |
| 14 R | 34 R | 54 s | 74 R |
| 15 s | 35 R | 55 R | 75 R |
| 16 R | 36 s | 56 R | 76 R |
| 17 R | 37 R | 57 R | 77 R |
| 18 R | 38 s | 58 s | Male parent B4 HR |
| 19 R | 39 R | 59 R | 0001 S of female parent |
| 20 R | 40 R | 60 R |
Wherein, S is represented susceptible, and R represents disease-resistant, and HR represents high anti-.
(2) with 9 pairs of forward primers and 11 pairs of reverse primers, totally 99 primer pairs are carried out to male parent B-4 and female parent 0001 respectively
PCR is expanded, it can be seen that there is characteristic strip, respectively em9me8, em7me6, em2me5, em10me5 in 6 pairs of primer pairs,
Em5me1, em1me5；
(3) performing PCR is entered with female parent 0001 to 6 plant populations in embodiment 2 and male parent B-4 respectively with 6 pairs of primer pairs
Amplification, it is the primer pair with characteristic strip to filter out em5me1, obtains the electrophoretogram of pcr amplification product, is specifically referred to figure
1, M is marker wherein in figure, and K is high anti-parent, and g is susceptible parent, K1, K2, K3, K4Disease-resistant F is showed after being respectively inoculated with2Son
Generation, g1, g2Susceptible F is showed after being respectively inoculated with2Filial generation, in visible maternal 0001 characteristic strips of 309bp or so, K1, K2, K3, K4,
g1, g2Electrophoresis result and the result manually identified be identical, it may be determined that using primer pair em5me1 as molecular labeling.
Wherein, the PCR amplification system is 25ul, and PCR-buffer is 2.5ul, Mg2+It is 2.0ul, dNTPs is 0.5ul,
TTaq archaeal dna polymerases are 0.3ul, and DNA is 4ul, and primer pair is 2ul, H2O is 13.7ul；The primer pair concentration is 1.0 μM,
Taq archaeal dna polymerases concentration is 1.0U/25ul, Mg2+Concentration is 2.5mM, and dNTPs concentration is 0.5mM, DNA concentration 20ng/
25ul。
Specific PCR amplification programs are 94 DEG C of 5min, and 1 circulates；94 DEG C of 1min, 35 DEG C of 1min, 72 DEG C of 1.5min, 5 are followed
Ring；94 DEG C of 1min, 50 DEG C of 1min, 72 DEG C of 1.5min, 35 circulations；72 DEG C of extension 8min, after amplification terminates, in firm power
50W, weight/mass percentage composition are electrophoresis 1.5h under 4% polyacrylamide gel.Dye, take pictures.
(4) by the primer pair em5me1 of determination in the F described in the table 1 of embodiment 32Generation and F377 plant populations of generation composition
Enter performing PCR amplification in mixing gene pool, specific amplification procedure is identical with step (3), and concrete outcome is shown in Fig. 2, comprehensive analysis Fig. 2 with
In table 1 in gene pool material sense/anti-blight situation, in table 1, No. 6 and No. 54 performances are susceptible, in fig. 2 No. 6 not
Show susceptible, No. 54 performances are susceptible, and band and susceptible band are about the same, the material basic expressions one in other gene pools
Cause, it is seen that significant correlation, phase be present in the band and section melon variable rate technology droop of 309bp or so primers em5me1 amplifications
It is 97.4% like property.
Embodiment 4：Droop identification is carried out to More female lines material and the section melon kind of seed selection using the molecular labeling of screening
Field More female lines material and the kind of seed selection in recent years are equally carried out with the primer pair em5me1 that embodiment 3 screens
DNA extractions, PCR amplifications, electrophoresis result are not found as shown in figure 3, as can be seen that in 15 parts of materials for examination from Fig. 3
Droop.15 parts of materials include 9 More female lines of numbering and 6 different cultivars, 9 More female lines be respectively labeled as D-5, J-9, too-
13-5-2-2, G-2-1, D-2-2, J16, D-3-1, H-3-1, B-4,6 kinds be respectively labeled as K section melon, No. 28 section melons, No. 37
Save melon, hat star 2, hat China 4, hat China 6.
The molecular labeling related to droop and the performance of field artificial infection that the present invention obtains are more consistent, can be with auxiliary
The identification of field droop is helped, is a kind of method of fast and effective identification, breeding can provide reference between section melon patch.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (5)
1. application of the specificity amplification primer to em5me1 in terms of identification saves cucurbit wilt, the em5me1 is respectively such as SEQ ID
Shown in No.1 and No.14, it is specially：
No.1：Tgagtccaaa ccggata,
No.14：gactgcgtac gaattaac.
2. using the method for SRAP molecular marking techniques identification section cucurbit wilt, it is characterised in that using specificity amplification primer
Right, the genomic DNA of amplification section cucurbit wilt, electrophoresis detection is carried out by gained PCR primer in PCR amplification system；
Wherein, the specificity amplification primer to for em5me1, the em5me1 it is as claimed in claim 1；
Field More female lines will be applied to using the SRAP molecular marker screenings molecular labeling em5me1 primer pair related to droop
The kind of material and in recent years seed selection carries out droop identification.
3. the method according to claim 2 that section cucurbit wilt is identified using SRAP molecular marking techniques, it is characterised in that
The PCR amplification system is 25ul, wherein, PCR-buffer is 2.5ul, Mg2+It is 2.0ul, dNTPs is 0.5ul, Taq DNA
Polymerase is 0.3ul, and DNA is 4ul, and primer pair is 2ul, H2O is 13.7ul.
4. the method according to claim 3 that section cucurbit wilt is identified using SRAP molecular marking techniques, it is characterised in that
The primer pair concentration is 0.5-2.5 μM, and Taq archaeal dna polymerases concentration is 0.5-1.5U/25ul, Mg2+Concentration is 0.5-
2.5mM, dNTPs concentration are 0.1-1.0mM, DNA concentration 5-40ng/25ul.
5. the method according to claim 3 that section cucurbit wilt is identified using SRAP molecular marking techniques, it is characterised in that
The primer pair concentration is 1.0 μM, and Taq archaeal dna polymerases concentration is 1.0U/25ul, Mg2+Concentration is 2.5mM, and dNTPs concentration is
0.5mM, DNA concentration 20ng/25ul.
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