CN105331615B - Identify InDel molecular labelings and its primer and the application of watermelon blight - Google Patents
Identify InDel molecular labelings and its primer and the application of watermelon blight Download PDFInfo
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Abstract
Present invention relates particularly to a kind of InDel molecular labelings for identifying watermelon blight and its primer and the application in Watermelon Resistances To Fusarium Wilt breeding.The nucleotides sequence of the InDel molecular labelings is classified as：TTTATTTTTTATTTTTTATTT, its primer sequence：InDel1_fon1F：TTCCAAAAGTGCAGATTTC；InDel1_fon1R：CACATGGGGATTGACTAAG.The present invention is just positioned by QTL and has equally arrived anti-fon1 main effect QTL in No. 1 chromosome mapping, and resurvey sequence with reference to parent and be prepared into and the InDel molecular labelings of watermelon blight resistance close linkage and its primer and the application in Watermelon Resistances To Fusarium Wilt breeding, new tool can be provided for watermelon blight resistance, accelerate the improvement process of watermelon blight resistance trait, improve the accuracy and efficiency of selection of breeding.
Description
Technical field
The invention belongs to biology field, and in particular to it is a kind of identify watermelon blight InDel molecular labelings and
Its primer and application.
Background technology
Droop（Fusarium wilt, FW）, by Deuteromycotina Fusarium bacterium watermelon specialized form（fon）Parasitism draws
Rise, be a kind of fungi soil-borne disease for causing yield and quality to reduce most serious in world wide in watermelon production.At present, reported
The fon biological strains in road have 0,1,2 and 3 totally four kinds, and except fon3, different commercial watermelon varieties are whole to a certain extent
Biological strain fon0, fon1 and fon2 resistance are closed.But anti-blight, germ can be in watermelon seeds for most of kind
The diffusion and sprawling of growing area quickly.Therefore, the exploitation molecule mark cheap with the easily operated of fusarium wilt disease resistance close linkage, cost
Note, it not only can shorten breeding cycle in seedling stage Rapid identification anti-blight kind/be, also help the withered of improvement watermelon
Sick resistance, technical support is provided for watermelon breeding for disease resistance.
Recently, Lambel etc.（2014）The main effect QTL site of a watermelon fon1 resistance, position are located using SNP marker
In 2.3~8.4cM of No. 1 chromosomal inheritance collection of illustrative plates, corresponding to 56050~6541994bp of No. 1 physical chromosomal map spectrum.Perhaps
Brave seminar develops also according to SNP site to be marked with two CAPs/dCAPs of fon1 close linkages, respectively 7716_fon
And 502124_fon, corresponding to 459624 and 502124bp of No. 1 physical chromosomal map spectrum, and 502124_fon and fon1 connects
The degree of lock is closer.
The molecular labeling of the existing watermelon fon1 resistance close linkages developed marks for CAPS/dCAPs, detection process
Digestion is needed, cost is higher, complex steps.The insertion/deletion of sequence is resurveyed based on full-length genome（InDel）Labeled as codominance mark
Note, detection is easy, and cost is cheap, but remains in some parts that do not comply with one's wishes, and such as making a variation, less, stability is inadequate.
The content of the invention
The present invention passes through QTL（Quantitative trait locus, quantitative trait locus）Just it is positioned at No. 1 dyeing
Body has navigated to anti-fon1 main effect QTL, and resurveys sequence with reference to parent and be prepared into and watermelon blight resistance close linkage
InDel molecular labelings and its primer and the application in Watermelon Resistances To Fusarium Wilt breeding, new hand can be provided for watermelon blight resistance
Section, accelerate the improvement process of watermelon blight resistance trait, improve the accuracy and efficiency of selection of breeding.
To achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of InDel molecular labelings for identifying watermelon blight are designed, its nucleotides sequence is classified as：
TTTATTTTTTATTTTTTATTT。
The primer of above-mentioned InDel molecular labelings, including following nucleotide sequence：
InDel1_fon1F：TTCCAAAAGTGCAGATTTC；
InDel1_fon1R：CACATGGGGATTGACTAAG.
The screening technique of the InDel molecular labelings of watermelon blight is identified, is comprised the following steps：
（1）Utilize maternal " ZXG01478 "（High resistance to wilt good）With male parent " 14CB11 "（Height sense droop）The F of hybridization2Group
Body builds highdensity genetic linkage mapses, and to Parent, F1And F293 individual plants of colony carry out fusarium wilt disease resistance identification；
（2）Identified with reference to genetic linkage mapses and fusarium wilt disease resistance, using WinQTL cartographer 2.5
software (http://statgen.ncsu.edu/qtlcart/WQTLCart.htm) QTL just positioning is carried out, reflected in LG1
The related QTL site fon1 of fixed to one fusarium wilt disease resistance, its LOD peak value are 26.05, explain 80.18% phenotypic variation, put
Believe 193331~2775577bp that physical location corresponding to section is No. 1 chromosome；
（3）Sequence is resurveyed with reference to two parents, finds 19 insertion and deletion fragments more than 20bp's in QTL sections
InDels, it is further 5 sections QTL region divisions, according to the insertion positioned at endpoint location to reduce the scope of candidate gene
Missing design InDel molecular labelings, InDel primers corresponding to design；
（4）Using corresponding InDel primers in Parent, F1And F2Genotype identification is carried out in colony；
（5）Utilized plus the genotype identification result and other SNP markers on LG1 of InDel molecular labelings
JoinMap4.0 carries out map construction, and carries out sweeping again for QTL using the software of WinQTL cartographer 2.5
Retouch；As a result one of molecular labeling is shown, InDel1_fon1 appears in the peak dot of the QTL, and LOD value is 31.65, solution
Release 91.46% phenotypic variation, physical location corresponding to QTL confidential intervals be similarly No. 1 chromosome 193331~
2775577bp。
Using abovementioned technology, applicant is finally obtained the insertion and deletion mark with watermelon blight resistance close linkage
Remember InDel1_fon1, the position of its corresponding watermelon reference gene group physical map is the 464377bp of No. 1 chromosome, sequence
For TTTATTTTTTATTTTTTATTT, primer sequence is,
InDel1_fon1F: TTCCAAAAGTGCAGATTTC;
InDel1_fon1R: CACATGGGGATTGACTAAG。
LOD value corresponding to marker site InDel1_fon1 is 31.65, explains 91.46% phenotypic variation.
Application process of the InDel molecular labelings of above-mentioned identification watermelon blight in watermelon blight resistance breeding, bag
Include following steps：
（1）To F293 individual plants of colony carry out genotype identification using the molecular labeling InDel1_fon1；
（2）Using above-mentioned（1）Analysis result is carried out according to molecular labeling InDel1_fon1 genotype to different individual plants
Classification, and identified and verified using fusarium wilt disease resistance appraising datum.
The present invention has following positive beneficial technique effect：
InDel molecular labelings variation of the present invention is stable, detection is easy, it is not necessary to the cumbersome step such as digestion, and cost
It is cheap（Marked relative to the CAPS/dCAPs developed）, improve the efficiency of selection of watermelon blight resistance breeding and accurate
Rate, so as to accelerate breeding process to greatest extent；In addition, this mark explains very big phenotype contribution rate positioned at QTL peak value
（91.46%）, accelerate clone and the functional verification of watermelon blight resistant gene.
Brief description of the drawings
Fig. 1 F2The fusarium wilt disease resistance qualification result distribution map of colony.
QTL scanning results compare before and after Fig. 2 screening InDel1_fon1 molecular labelings.Font-weight and underscore are
QTL fon1 peak molecular labeling；The mark of the upper overstrikings of Chromosome1 is the InDel molecular labelings newly screened.
Fig. 3 is using molecular labeling InDel1_fon1 primer in part F2Amplification in Meta-genomic DNA.Father
Band is purpose band in maternal square frame.Title be all variety name latter two/tri- of code name.
Fig. 4 is using molecular labeling fon_7716 primer in part F2Amplification in Meta-genomic DNA.Father and mother
Band is purpose band in this square frame.Title be all variety name latter two/tri- of code name.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
Invention is further described.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.Involved raw material, is commercially available unless otherwise instructed, involved detection method is such as without spy in following examples
Do not mentionlet alone bright, be then conventional method.
Embodiment 1
Watermelon blight Resistance Identification, is comprised the following steps that：
（1）The preparation of bacterium soil：Strain is separately cultured from the watermelon blight plant of incubator culture, wheat is used before use
Expand after numerous culture with sterilizing sandy soil by 1：50 ratios are well mixed, standby；
（2）Seed preparation：Kind 100 is taken per kind, room temperature is soaked seed 24 hours, and 33 DEG C of vernalization are sowed for 36 hours；
（3）Inoculated identification：Using wheat sand bacterium local method, ready bacterium soil is installed with nutritive cube, seedling is heated in greenhouse
Sowing in bed kind, per nutritive cube 5, repeat twice, if susceptible and disease-resistant control and protection row；
（4）Disease investigation：Sowing starts to fall ill after 10 days, records emergence and incidence, treats within 4 weeks or so susceptible and disease-resistant
After check variety respectively reaches expected sick level, the number of live vaccine of each kind is counted for the last time, calculates the state of an illness；
（5）State of an illness computational methods：
Death rate（%）=(number of emerging-number of live vaccine)/number of emerging × 100%；
（6）Disease resistance is classified：
Height is anti-(HR)：0≤death rate≤20%；
Disease-resistant (R)：20% ＜ death rate≤40%；
Moderate resistance (MR)：40% ＜ death rate≤60%；
Susceptible (S)：60% ＜ death rate≤80%；
Height sense (HS)：Death rate ＞ 80%.
Embodiment 2
The development approach of watermelon blight InDel molecular labelings is identified, is comprised the following steps that：
（1）Fusarium wilt disease resistance full-length genome QTL is just positioned
Utilize maternal " ZXG01478 "（High resistance to wilt good）With male parent " 14CB11 "（Height sense droop）Hybridize the F obtained2
Colony has had been built up highdensity genetic linkage mapses（Chain point of group has been carried out by the mLOD values between molecular label, has been shown in Table
1）, and to Parent, F1And F293 individual plants of colony carry out fusarium wilt disease resistance identification；With reference to genetic linkage mapses and F2Colony
Fusarium wilt disease resistance qualification result（Fig. 1）, utilize the software (http of WinQTL cartographer 2.5://
Statgen.ncsu.edu/qtlcart/WQTLCart.htm) carry out QTL just to position, identifying one in LG1 linkage groups withers
The related QTL site fon1 of sick resistance, its LOD peak value are 26.05, explain 80.18% phenotypic variation, corresponding to confidential interval
Physical location is 193331~2775577bp of No. 1 chromosome（Table 2）.
The genetic linkage mapses of table 1
The high density collection of illustrative plates of table 2 and QTL scanning informations after being marked plus two InDel
。
（2）Parent resurveys sequence
Utilize maternal " ZXG01478 "（High resistance to wilt good）With male parent " 14CB11 "（Height sense droop）22x depth is carried out
High flux resurvey sequence.Two parents detect 45134 small InDel altogether.
（3）The exploitation of QTL regions InDel marks
Find that 19 insertion and deletion fragments are more than 20bp InDels in the section of QTL positioning.In order to reduce candidate gene
Region, be 5 sections QTL region divisions, InDel primers designed according to the insertion and deletion positioned at endpoint location/neighbouring, design altogether
6 pairs of InDel primers.Using the sequence of each 500bp before and after Program extraction insertion and deletion relevant position, InDel corresponding to design points
Son mark.
Embodiment 3
Watermelon F2Colony's molecular marker analysis, comprises the following steps：
（1）Blade STb gene is extracted using CTAB methods, is comprised the following steps that：
Take the fresh blades of 1 g to be put into mortar, add liquid nitrogen grind into fine powder, be transferred to extracted added with 1 ml CTAB immediately
In the centrifuge tube of liquid, the two is fully mixed, be subsequently placed in 65 DEG C of min of water bath with thermostatic control 60, overturn mixing 2-3 times therebetween；
After water-bath taking-up, 8000 rpm centrifuge 1 min；
Take supernatant to be placed in another centrifuge tube, add isometric chloroform：Isoamyl alcohol（24：1, V/ V）, gently overturn
It is set fully to mix；
10000 rpm centrifuge 5 min, take supernatant（Middle level precipitation is not got as far as possible）；
Add the isopropanol of 0.7 times of volume（The min of precooling 30 need to be shifted to an earlier date）, -20 °C of freezings are placed in after mixing and are no more than 30
Min allows DNA to separate out；10000 rpm centrifuge 5 min after taking-up, stay precipitation to abandon supernatant；
With washes of absolute alcohol precipitation for several times, soak is outwelled, opens centrifuge tube lid and dry；
Add 200 μ l distilled water dissolving DNA；DNA concentration is determined with ultraviolet specrophotometer, in -20 °C of refrigerators
In save backup.
（2）PCR reaction systems and program
Reaction system is as follows：
PCR response procedures are：94 DEG C 5 minutes, 35 circulation 94 DEG C 20 seconds, 55 DEG C 1 minute, 72 DEG C 30 seconds, 72 DEG C
5 minutes.
（3）8% polyacrylamide gel electrophoresis, development, dyeing and banding pattern interpretation are carried out to PCR primer.
Preparation of reagents：
A.5 × TBE：
53.9 grams of Tris-base
3.72 grams of EDTA
27.5 grams of boric acid
1 liter is settled to distilled water.
B. 40% polyacrylamide solution：
193.34 grams of polyacrylamide
6.66 grams of methylene diacrylamide
500 milliliters are settled to distilled water.
C. 8% polyacrylamide gel（It is now with the current）：
40% 10 milliliters of polyacrylamide solution
5 milliliters of 5 × TBE
10% ammonium persulfate（APS）200 microlitres
Tetramethylethylenediamine（TEMED）80 microlitres
22 milliliters of distilled water.
D. silver staining liquid：
1 gram of silver nitrate
5 milliliters of glacial acetic acid
50 milliliters of absolute ethyl alcohol
500 milliliters are settled to deionized water.
E. developer solution：
15 grams of sodium hydroxide
Formaldehyde（37%）2.5 milliliter
500 milliliters are settled to deionized water.
Gel slab prepares：
Glass plate cleaned with distilled water, dry after, wiped, dried with the rayon balls for soaking absolute ethyl alcohol.By notch board
The clip that its both sides is compressed and buckled well in gel maker is put into after close with flat board overlapping（One gel maker can make two plate gels）.
8% polyacrylamide gel solution is configured in wash bottle（Two parts）, it is rapidly injected after mixing in the gap among two plates, after filling
Insert comb with teeth（The lower section of comb tooth is not allowed to produce bubble）；If now liquid level has declined, can be drawn with pipettor
Not solidified solution supplements.Solution is waited fully to solidify.
Electrophoresis：
Gel maker support is removed from base, is directly placed into supporting electrophoresis tank, on electrophoresis trench bottom and support
1 appropriate × tbe buffer liquid is poured among two pieces of glass plates.6 × DNA Loading of 0.2 times of volume are added in PCR primer
Buffer, 0.8 microlitre is taken to add in loading wells after mixing, 260 volts of electrophoresis 35 minutes.
Dyeing and development：
After the completion of electrophoresis, take out glass plate from electrophoresis tank, sled removes notch board, and gel can be attached on flat board, gel towards
On flat board is put into silver staining liquid, be placed in jog 15 minutes on decolorization swinging table, gel can Automatic-falling get off；Silver staining finishes, will
Gel takes out to be put into deionized water and cleaned 10 seconds；Gel is transferred in developer solution after cleaning, jog shaking table, treats that band is clear
Gel is taken out after clear, is placed on the position difference that band is visually observed on diagosis device, and preservation of taking pictures.
Banding pattern interpretation：
The glass plate spontaneously dried after development is placed on diagosis platform, visually observes the position difference of two parent's bands.
Embodiment 4
F2The reconstruct of population genetic linkage map is built to be relocated with QTL, and its step is as follows：
（1）Plus InDel molecular labelings in F2Other SNP markers utilize business in the genotype identification and LG1 of colony
The JoinMap4.0 that approach obtains carries out map construction.After being marked plus InDel, LG1 order end to end is overturned, still
The SNP marker of the mark and surrounding is both positioned on same scaffold, and with the synteny one of physical map of genome
Cause（Fig. 2）.
（2）QTL is repositioned according in embodiment 2 using the collection of illustrative plates newly built（1）Carry out.As a result as shown in table 2,
A molecular labeling InDel1_fon1 wherein newly developed appears in the peak value of the QTL, and LOD value is 31.65, is explained
91.46% phenotypic variation, physical location corresponding to QTL confidential intervals are similarly 193331~2775577bp of No. 1 chromosome.
The 21bp insertions for corresponding to No. 1 chromosome 464377bp positioned at the molecular labeling InDel1_fon1 of summit lack
Lose, its sequence is TTTATTTTTTATTTTTTATTT, and its primer sequence is InDel1_fon1F: TTCCAAAAGTGCAGATTT
C; InDel1_fon1R: CACATGGGGATTGACTAAG.The primer is in female parent（High resistance to wilt good kind）In amplified production
For 335bp, in male parent（Susceptible droop kind）In amplified production be 356bp.After being marked plus the InDel, droop resists
Property QTL fon1 LOD peak values are higher, and the phenotype contribution rate of explanation is bigger, illustrates that the mark and QTL linkage degree are very close.
Embodiment 5
A kind of embodiment 4 screens application of the InDel molecular labelings in breeding, and its applying step is as follows：
（1）As embodiment 3 carries out InDel1_fon1 molecular labelings in F2Genotype identification in colony（Fig. 3）.
（2）As embodiment 3 carries out F2The fusarium wilt disease resistance identification of colony.
（3）Check two kinds of genotype of InDel1_fon1 molecular labelings（A represents maternal banding pattern, and B represents male parent banding pattern,
AB represents heterozygous genotypes）In 93 F2Distribution situation in colony（Table 3）.
As a result show, molecular labeling InDel1_fon1 genotype is disease-resistant at 71 parts（Death rate<60%）22 parts in strain
For A（335bp）, 1 part is B（356bp）, 49 parts are AB.And in 20 parts of susceptible strains, it is B to have 18 parts（356bp）, 2 parts are
AB, and all 1 B gene type individual plants are all high senses（Dead seedling>80%）Strain.
Molecular labeling InDel1_fon1 genotype is that A 22 strains are all Resistant variants, and is B's in genotype
19 strains 18 are high sense strains, and 1 is Resistant variants（Death rate=32.6%）.The rate of accuracy reached of its genotype identification arrives
97.56%。
（4）Also marked using the CAPs delivered in fon_7716 such as embodiment 3 to F2The each individual plant of colony enters performing PCR expansion
Increase, then using restriction enzyme Tag1 carry out digestion, finally as used in Example 3 8% polyacrylamide gel electrophoresis,
Development, dyeing and banding pattern interpretation（Fig. 4）.As a result show（Table 3 ~ 6）, fon_7716 and InDel1_fon1 are in F293 lists of colony
The genotype of strain is completely the same, further demonstrates the qualification result of the present invention.
Result above is enough to illustrate to predict watermelon blight resistance, Ke Yiti by molecular labeling InDel1_fon1
The efficiency of selection of high watermelon blight resistance breeding, so as to accelerate breeding process.
The InDel1_Fon1 of table 3 and fon_7716 are in F2Identification and checking in colony
The InDel1_Fon1 of table 4 and fon_7716 are in F2Identification and checking in colony（It is continuous）
The InDel1_Fon1 of table 5 and fon_7716 are in F2Identification and checking in colony（It is continuous）
The InDel1_Fon1 of table 6 and fon_7716 are in F2Identification and checking in colony（It is continuous）
。
SEQUENCE LISTING
<110>Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy
<120>Identify InDel molecular labelings and its primer and the application of watermelon blight
<130> /
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
tttatttttt attttttatt t 21
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
ttccaaaagt gcagatttc 19
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
cacatgggga ttgactaag 19
Claims (6)
1. a kind of InDel molecular labelings for identifying watermelon blight, its nucleotides sequence are classified as：TTTATTTTTTATTTTTTATTT.
2. the primer of InDel molecular labelings described in a kind of claim 1, it is characterised in that the nucleotides sequence of the primer is classified as：
InDel1_fon1F：TTCCAAAAGTGCAGATTTC；
InDel1_fon1R：CACATGGGGATTGACTAAG.
3. the screening technique of InDel molecular labelings, comprises the following steps described in a kind of claim 1：
（1）Utilize the F of the maternal and high sense droop type paternal hybrid of high resistance to wilt good type2The highdensity genetic linkage of informative population
Collection of illustrative plates, and to Parent, F1And F2The individual plant of colony carries out fusarium wilt disease resistance identification；
（2）Identified with reference to genetic linkage mapses and fusarium wilt disease resistance, carry out QTL and just position, resisted in LG1 linkage groups identification droop
Property related QTL site fon1；
（3）Sequence is resurveyed with reference to two parents, is more than 20bp InDels in QTL range lookup insertion and deletions fragment, in order to contract
The region of small candidate gene, it is 5 sections by QTL region divisions, InDel molecule marks is designed according to the insertion and deletion positioned at endpoint location
Note, InDel primers corresponding to design；
（4）Using corresponding InDel primers in Parent, F1And F2Genotype identification is carried out in colony；
（5）Entered plus other SNP markers in the genotype identification result and LG1 of InDel molecular labelings using JoinMap4.0
Row map construction, and carry out rescaning for QTL using the software of WinQTL cartographer 2.5.
4. molecular labeling of the primer in watermelon blight resistance described in InDel molecular labelings described in claim 1 or claim 2
Application in assisted Selection.
5. application side of the InDel molecular labelings in the molecular marker assisted selection of watermelon blight resistance described in claim 1
Method, comprise the following steps：
（1）To F2The individual plant of colony carries out genotype identification using the molecular labeling；
（2）Using above-mentioned（1）Analysis result is classified according to the genotype of molecular labeling to different individual plants, and utilizes droop
Resistance Identification data are identified and verified.
6. primer described in InDel molecular labelings described in claim 1 or claim 2 is in watermelon blight resistant gene figure position gram
Application in grand.
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| CN106191249B (en) * | 2016-07-13 | 2019-05-14 | 河南牧业经济学院 | A kind of identification watermelon pericarp covers the InDel molecular marker and primer thereof and application of line feature |
| CN107254542B (en) * | 2017-08-09 | 2020-05-19 | 中国农业科学院郑州果树研究所 | Watermelon flesh color character major gene locus and InDel molecular marker and application thereof |
| CN107881252B (en) * | 2017-11-29 | 2020-07-14 | 湖南省农业生物技术研究中心 | dCAPS marker and primer for identifying watermelon fusarium wilt, and acquisition method and application thereof |
| CN110622852A (en) * | 2019-09-10 | 2019-12-31 | 黑龙江省农业科学院园艺分院 | Method for cultivating watermelon variety with high fusarium wilt resistance |
| CN112195263B (en) * | 2020-10-21 | 2021-05-14 | 北京市农林科学院 | SNP (Single nucleotide polymorphism) locus and primer set for identifying purity of watermelon hybrid and application |
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| CN103146691A (en) * | 2013-02-18 | 2013-06-12 | 北京市农林科学院 | SNP loci linked with blight resistant gene Fon-1 in watermelon, and markers thereof |
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