CN105734057B - With the SSR marker and its application of muskmelon downy mildew resistance main effect QTL linkage - Google Patents
With the SSR marker and its application of muskmelon downy mildew resistance main effect QTL linkage Download PDFInfo
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Abstract
The invention discloses the SSR markers and its application with muskmelon downy mildew resistance main effect QTL linkage.Genetic linkage map is established by the soft carry out linkage disequilibrium value of known heredity for group by constructing the anti-sense F2 of muskmelon downy mildew with highly resistance resource PI390452 and susceptible farm variety " OK a karaoke club gram match ", LOD interpretation of result peak value, LOD > 3.0 occurs in b290.According to b290, the site SSS is found again with known SSR search software, and redesign SSR primer with known PCR primer design software.The molecular labeling SSR-pc with muskmelon downy mildew resistance main effect QTL linkage is finally found with known Genetics Software according to phenotype and genotype283, genetic distance 0.5CM can be used for molecular marking supplementary breeding, reaches breeding downy mildew resistance strain process and shortens breeding cycle, improves the selection good technical effect of accuracy rate.
Description
Technical field
The present invention relates to molecular genetic breeding fields.Specifically, the present invention relates to muskmelon downy mildew resistance main effect QTL
The technical field of chain SSR marker and its application.
Background technique
Muskmelon (Cucumis melo L.,2n=24) it is a kind of important industrial crops, the cultivated area of China's muskmelon
It ranks first in the world with yield.Muskmelon downy mildew [Pseudoperonospora cubensis(Berk.etCurt.)Rostov.] under conditions of high temperature and humidity, prevalence is rapidly, harm is serious, prevents and treats difficulty, yield, the sugar content of fruit to muskmelon
Direct influence is all caused with commodity rate, is a kind of destructive disease.Chemical prevention downy mildew disease not only low efficiency is also easy
Cause environmental pollution, and cultivate disease-resistant variety be prevent and treat muskmelon downy mildew harm most safely, effectively and save the cost is arranged
One of apply.
The excellent variety of some better resistances usually can be also obtained using the method for conventional breeding, conventional method breeding is disease-resistant to educate
It when kind material is identified using artificial inoculation on seedling, is screened according to the anti-sense feature of the phenotype of plant, sometimes because of inoculation
Insufficient or onset condition is not suitable for and influences efficiency of selection, it is difficult to accurately and rapidly filter out the individual with disease-resistant gene
Plant.In order to accurately and rapidly filter out the individual plant with disease-resistant gene, the molecular labeling auxiliary to grow up in recent years
Breeding technique, using Molecular mapping quantitative character QTL, essence is exactly between analyzing molecules label and objective trait QTL
Linkage relationship, i.e., the QTL at unknown seat is positioned using the molecular labeling at known seat, by calculate molecular labeling with
Exchange rate between QTL, to determine the specific location of QTL.The amount of marker genetype separation and quantitative character based on acquisition
Between relationship, can directly hold Quantitative Trait Genes, and develop and can be applied to molecular marker assisted selection (Molecular-
Assisted selection, MAS) breeding technology.Traditional Phenotypic Selection can be turned by molecular marker assisted selection
Become directly selecting genotype, disease-resistant plant can be selected in early generation, all from reduce cost, improve selection etc.
Have great importance.
SSR points that QTL loci polymorphism expresses disease-resistant sex differernce are positioned and detected at present to the QTL of muskmelon downy mildew resistance
The research of sub- marker development application is substantially at blank, carries out molecular mark without effectively label.Therefore, carry out
The positioning of muskmelon downy mildew resistance character main effect QTL, finds the SSR molecular marker of close linkage, and establishes filial generation early stage
Assisted Selection technical system saves production cost with important for improving the genetic improvement efficiency of muskmelon downy mildew resistance character
Meaning.
Summary of the invention
The purpose of the present invention is in view of the deficiencies of the prior art, there are ambiguities for resistant gene, it was found that and utilize new resistance source
PI390452 simultaneously has found SSR molecular marker with muskmelon downy mildew resistance main effect QTL compact linkage.The prior art is overcome to select
It educates that the disease-resistant strain difficulty of stable muskmelon downy mildew is larger, period longer defect, provides mark for the downy mildew breeding for disease resistance of muskmelon
Remember the technology of assisted Selection, to reduce cost, improve efficiency of selection, is established for the excellent downy mildew resistance melon variety of quickly breeding
Basis.
What the invention is realized by the following technical scheme:
It is high frost-resistant by selecting by seedling stage to the principle and prior art basis of the Resistance Identification screening of resource downy mildew
The muskmelon resource PI390452 of mildew is highly resistance resource, with highly resistance resource PI390452 and susceptible farm variety " OK a karaoke club gram match " structure
The anti-sense F2 of muskmelon downy mildew is built for group, linkage disequilibrium value is carried out using well known Genetics Software, establishes genetic linkage map,
There are peak value, LOD > 3.0 in b290 in LOD interpretation of result.2kb sequence up and down is searched according to the physical location where b290, with public affairs
The SSR search software known finds the site SSR again, and redesigns SSR primer with known PCR primer design software.According to table
Type and genotype finally find the molecular labeling SSR- with muskmelon downy mildew resistance main effect QTL linkage with well known Genetics Software
pc283, genetic distance 0.5CM can be used for molecular marking supplementary breeding, reaches the shortening of breeding downy mildew resistance strain process and educates
The kind period improves the selection good technical effect of accuracy rate.
The present invention specifically provides a kind of SSR marker of muskmelon downy mildew resistance main effect QTL linkage, comprising the following steps:
It (1) is quantity according to genetics of resistance qualification result resistant gene using muskmelon resource PI390452 as the anti-source of downy mildew
Heredity.
(2) by the susceptible farm variety of muskmelon downy mildew " OK a karaoke club gram match " to be maternal with Resistant germplasm PI390452 be male parent into
Row hybridization, obtains Hybrids F1.
(3) F2 is obtained for group by Hybrids F1 self-pollination, F1 hybridizes with " OK a karaoke club gram match " obtains BCS, F1 with
PI390452 hybridization obtains BCr.
(4) respectively in seedling stage to PI390452, " OK a karaoke club gram match ", F1、BCS, BCr and F2Disease Resistance Identification is carried out, sweet tea is inoculated with
Disease incidence is investigated after melon Pseudoperonospora cubensis conidial suspension, it is of different sizes to account for leaf area according to muskmelon scab, and disease is drawn
It is divided into 0-5 grade, and according to disease scale situation, classification number is converted to the disease-resistant or susceptible phenotype of single plant.
(5) from F2It is anti-for the genomic DNA building of 5 disease-resistant single plant equivalent of 0 grade of phenotype for disease series in group, is taken
Ospc gene mixing pit, taking disease series is that the genomic DNA of 5 susceptible single plant equivalent of 5 grades of phenotypes constructs susceptible gene mixing
Pond.
(6) using disease-resistant gene pond and susceptible gene pool DNA as template, the information announced using known SSR primer, screening is expanded
Increase the screening amplification that stable core SSR primer carries out polymorphism, SSR amplified production electrophoresis point on 6% polyacrylamide gel
From rear, final acquisition polymorphism SSR primer.
(7) have polymorphism primer to F between disease-resistant gene pond and susceptible gene pool to acquired2For group's single plant
Augmentation detection, and genetic linkage analysis is carried out with known Genetics Software, wherein having linkage relationship between 15 pairs of primers, it is divided into 3
There are peak value, LOD > 3.0 in b290 in a linkage group, LOD interpretation of result.
(8) 2kb sequence up and down is searched according to the physical location where b290 in existing well-known technique, is searched with well known SSR
Rope software finds the site SSS again, and redesigns SSR-pc with well known PCR primer design software283sPrimer.
(9) with acquired molecular labeling SSR-pc283s, to F2For group's single plant augmentation detection, genetic linkage point is carried out
Analysis；By F2The genotype of disease-resistant, susceptible phenotype and molecular labeling for group's single plant is lost with existing known Genetics Software
It passes linksystem analysis and determines SSR-pc with 3. 0 for minimum LOD threshold values283sWith muskmelon downy mildew Major resistance gene it is chain away from
From.
In the present invention, have polymorphism 2 to primer between disease-resistant gene pond and susceptible gene pool according to acquired
B290 and SSR-pc283In, the polymorphic molecular marker, primer sequence are as follows:
b290 5' GCTCCTCCTTAACTCTATAC' 3;
5' GCATTATTACCCATGTACGAG' 3;
SSR-pc283 5' TCGAACCTAGTTATCAGAAGTC' 3;
5' CAACCGACATTGAAAACCAAC' 3;
SSR-pc283DNA sequence dna
TCGAACCTAGTTATCAGAAGTCTCCTTACTTAAATAAAACAATTTCACTTTGTTGTGAATTAGTTGAT
TTTGTTGTT 77
GTTGAATGTTGAATTGATATAAAAAGCAATTAAGAATTTATTGATTTGCACGTGCTTAAGTAAACATG
TCTATATAT 154
ATATATATATATATGTATGTATGTATTTAATAATAATAAATTGATGATAGACATTTAGTATATATTAG
ATGATCATCGAC 234
AAAAAGTAGGTGTAAAACCAATGTTGAAGTTGGTTTTCAATGTCGGTTG 283
Further, according to the present invention with the chain molecular labeling of muskmelon downy mildew Major resistance gene Pc-7 and its to answer
With by molecular labeling SSR-pc283For melon variety or the genotype detection of strain, for judging whether are the kind or strain
Anti- muskmelon downy mildew, comprising the following steps:
(1) hybridized with Resistant germplasm PI390452 with other muskmelons and multiplied to F2It is more than generation.
(2) to the single plant of muskmelon obtained by step (1), genomic DNA is extracted, SSR-pc is used283This mark into
Row PCR amplification has detected whether that 283bp specific band occurs, has occurred if any specific band, then predict that Muskmelon Plants are frost-resistant
Mildew.
The present invention is announced and the newest hair of A. DiaZ using disease-resistant gene pond and susceptible gene pool DNA as template using ICuGI
The article and https of table: the SSR primer of the //website melonomics.net filters out 300 couples of stable core SSR of amplification
Primer carries out the screening amplification of polymorphism, and after SSR amplified production is separated by electrophoresis on 6% polyacrylamide gel, final acquisition is more
State property SSR primer 22 is right.
The present invention is according to the newest article delivered of A. DiaZ and https: in the //website melonomics.net where b290
Physical location search 2kb sequence up and down, find the site SSS again with SSRHunter, and again with Array Designer 4
Design SSR primer SSR-pc283.It is finally found with QTL IciMapping Genetics Software according to phenotype and genotype anti-with muskmelon
The molecular labeling SSR-pc of downy mildew main effect QTL linkage283, genetic distance 0.5CM can be used for molecular marking supplementary breeding,
Reach breeding downy mildew resistance strain process to shorten breeding cycle, improve the selection good technical effect of accuracy rate.
In the present invention, by 200 plants of F2Disease-resistant, the susceptible phenotype and molecular marker gene type of single plant pass through Genetics Software
QTL IciMapping carries out linksystem analysis, with 3. 0 for minimum LOD threshold values, determines muskmelon downy mildew Major resistance gene
Pc-7 and molecular labeling SSR-pc283Close linkage, genetic linkage distance are 0.5cM.
The Disease investigation that the present invention uses uses common investigation disease incidence, classification identification standard method is referring to often
The assessment resistance standards of (1989) such as the father-in-law ancestral letter seen simultaneously make the appropriate adjustments, and are statistics mark with the area coverage of the mould layer of vacuum side of blade
Standard, 0 grade, 1 grade and 2 grades of disease is set to disease-resistant, and 3 grades, 4 grades and 5 grades of disease are set to susceptible, and concrete condition is as follows:
Disease investigation: investigating disease incidence after 10-14d, and it is of different sizes to account for leaf area according to muskmelon scab, by disease
It is divided into 0-5 grade, and according to disease scale situation, classification number is converted to the disease-resistant or susceptible phenotype of single plant,
Specific grade scale is as follows:
0 grade without any illness；
1 grade of lesion area accounts for the < 1/10 of entire blade area;
2 grades of lesion areas account for the 1/10-1/4 of entire blade area;
3 grades of lesion areas account for the 1/4-1/2 of entire blade area;
4 grades of lesion areas account for the 1/2-3/4 of entire blade area;
Scab is covered with substantially on 5 grades of leaves, and lesion area accounts for the > 3/4 of entire blade area;
In the present invention, according in seedling stage to PI390452, " OK a karaoke club gram match ", F1、BCS、BCrAnd F2Disease Resistance Identification is carried out,
As a result PI390452 shows highly resistance, F1Also show disease-resistant, resistance is weaker than PI390452, and " OK a karaoke club gram match " shows susceptible, BCrIn resist
Induction reactance sense separates presentation diversification, and disease-resistant plant is more, and disease plant is few, BCSWith BCrOpposite disease-resistant plant is few, and disease plant is more,
F2In anti-sense separation be in normal distribution, it can be seen that resistant gene is controlled by multiple genes.
The provided by the present invention and chain molecular labeling SSR-pc of muskmelon PI390452 downy mildew resistance gene283In, with drawing
Object SSR-666 expands the DNA of the anti-source PI390452 of muskmelon downy mildew, only amplifies the band that 1 length is 283bp specificity,
Show to carry disease-resistant gene.
The present invention relates to various genetic resources by purchase approach or approach can be granted can obtain.
Selected new resistance source PI390452 derives from United States Department of Agriculture's National Botanical germ plasm resource system in the present invention
(USDA NPGS), quoted from United States Department of Agriculture's National Botanical germ plasm resource system, existing Hami Melon Research Center of Xinjiang Academy of Agricultural Sciences
It saving, anti-source PI390452 material those of ordinary skill in the art can introduce approach by the website USDA NPGS and obtain, or
It is introduced by Hami Melon Research Center of Xinjiang Academy of Agricultural Sciences.
Selected farm variety " OK a karaoke club gram match " in the present invention, in the research of Xinjiang Agricultural Sciences institute "Hami" melon
The heart, Xinjiang Seed Market are commercially available.
Selected F in the present invention1, obtained from disease-resistant material and susceptible hybridization between selfed lines, i.e., by with
PI390452 male parent, " OK a karaoke club gram match " are female parent, are obtained by common hybridization technique means, those of ordinary skill in the art are logical
Crossing conventional hybridization approach can obtain.
Selected F in the present invention2, derive from F1Selfing generates, and those of ordinary skill in the art are selfed approach by conventional
It can obtain.
The present invention using PI390452 and its derived varieties or strain as male parent or it is maternal hybridize with other muskmelons and multiply to
F2It is more than generation.The PI390452 and its derived varieties or strain, refer to PI390452 for parent, by conventional hybridization or
Using tissue cultures or using Anther Culture or with other either physically or chemically hybridized induction monoploid, then use colchicine
And other plant chromosomes are doubled reagent and double the melon variety or product that obtain dihaploid or obtained using genetic transforming method
System, these technological means are well known to those of ordinary skill in the art.
The sequence information of the SSR primer of screening of the present invention has been published from this development in laboratory and ICuGI
(http://www. icugi.org/cgi-bin/ICuGI/index.cgi).
By the specific summary of the invention of the implementation present invention, following effect can achieve:
(1) it obtains and the chain SSR molecular marker SSR-pc of muskmelon downy mildew resistance major gene resistance for the first time through the invention283。
(2) by detection and the chain molecular labeling of downy mildew resistance major gene resistance, the downy mildew of Muskmelon Plants can be predicted
Resistance, quickly screens disease-resistant variety or strain is used for muskmelon downy mildew resistance molecular breeding.Disease-resistant major gene resistance fast and easy is detected,
And it is not affected by environment.
(3) molecular labeling provided through the invention, assistant breeding selection target is clear, save the cost.In traditional breeding method
In method, first have to collect parent and the cultivar with disease-resistant gene carry out it is a series of hybridize to be returned establish group, and
Single-plant selection is carried out to downy mildew resistance in group.Artificial inoculation on seedling identification is carried out to muskmelon downy mildew, because inoculation is not filled
Divide or onset condition is not suitable for and influences efficiency of selection and accuracy rate.Therefore breeding for disease resistance is not only time-consuming, but also difficulty is big, cost
It is high.Pass through SSR-pc283Label detects downy mildew resistance major gene resistance, can just identify the single plant of downy mildew resistance in seedling stage, washes in a pan
Other plant are eliminated, not only save production cost but also greatly improve the efficiency of selection of downy mildew resistance muskmelon.
Detailed description of the invention
Fig. 1 is to screen 15 pairs of polymorphism primers according to anti-sense pond, carries out genetic linkage analysis using WinQTLCart2.0,
Establish the genetic linkage map of 3 linkage groups.
Fig. 2 is to carry out the analysis of linkage disequilibrium LOD value to polymorphism primer using WinQTLCart2.0, is drawn as the result is shown
The corresponding LOD of object b290 is greater than 3.0.
Fig. 3 is to carry out dominant performance analysis using WinQTLCart2.0.
Fig. 4 is to be shown as primer SSR-pc283To F2The augmentation detection of single plant.
Specific embodiment
In the following, illustrating the present invention for embodiment, still, the present invention is not limited to following embodiments.
Equipment and material have in the present invention: TC-512 type PCR amplification instrument (Techne company, Britain) .DYY-12 type computer
Three permanent multi-purpose electrophoresis apparatus power supply (6 1 Biotechnology Co., Ltd of Beijing), JY-SPE type electrophoresis tank (Beijing east Jun Yi electrophoresis
Equipment Co., Ltd), JY04S-3C type number gel imager (Beijing east Jun Yi electrophoresis equipment Co., Ltd), SSR primer,
Taq
Enzyme, PCR reaction kit and other reagents are purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The breeding material being related in the present invention includes: muskmelon downy mildew resistance resource PI390452, is studied through genetic development
Analysis PI390452 disease resistance is quantitative inheritance, carries downy mildew main effect Resistance QTL temporary designations Pc-7, and anti-source PI390452 draws
From United States Department of Agriculture's National Botanical germ plasm resource system.Anti- source PI390452 material those of ordinary skill in the art can be by drawing
It is obtained into approach.Muskmelon farm variety " OK a karaoke club gram match " is common farm variety " OK a karaoke club gram match " selected in the present invention, is
Xinjiang thick-skinned melon, susceptible downy mildew derive from Hami Melon Research Center of Xinjiang Academy of Agricultural Sciences, and Xinjiang Seed Market is commercially available
It obtains.
In the present invention, 6% polyacrylate hydrogel: 30% polyacrylamide (29:1) 24mL, 10 × TBE 8mL, distilled water
40mL, APS 650uL, TEMED 80uL.
All reagents and instrument selected in the present invention are all well known in the art selection, but do not limit reality of the invention
It applies, other some reagents well known in the art and equipment are applied both to the implementation of following implementation of the present invention.
Embodiment one: the SSR marker with muskmelon downy mildew resistance main effect QTL linkage
A kind of SSR marker with muskmelon downy mildew resistance main effect QTL linkage, specifically includes the following steps:
It (1) is quantity according to genetics of resistance qualification result resistant gene using muskmelon resource PI390452 as the anti-source of downy mildew
Heredity.
(2) by the susceptible farm variety of muskmelon downy mildew " OK a karaoke club gram match " to be maternal with Resistant germplasm PI390452 be male parent into
Row hybridization, obtains hybrid F1。
(3) by hybrid F1Self-pollination obtains F2For group, F1Hybridize with " OK a karaoke club gram match " and obtains BCS,F1With PI390452
Hybridization obtains BCr.
(4) respectively in seedling stage to PI390452, " OK a karaoke club gram match ", F1、BCS, BCr and F2Disease Resistance Identification is carried out, sweet tea is inoculated with
Disease incidence is investigated after melon Pseudoperonospora cubensis conidial suspension, it is of different sizes to account for leaf area according to muskmelon scab, and disease is drawn
It is divided into 0-5 grade, and according to disease scale situation, classification number is converted to the disease-resistant or susceptible phenotype of single plant.
(5) from F2It is anti-for the genomic DNA building of 5 disease-resistant single plant equivalent of 0 grade of phenotype for disease series in group, is taken
Ospc gene mixing pit, taking disease series is that the genomic DNA of 5 susceptible single plant equivalent of 5 grades of phenotypes constructs susceptible gene mixing
Pond.
(6) using disease-resistant gene pond and susceptible gene pool DNA as template, the information announced using known SSR primer, screening is expanded
Increase the screening amplification that stable core SSR primer carries out polymorphism, SSR amplified production electrophoresis point on 6% polyacrylamide gel
From rear, final to obtain polymorphism SSR primer 22 right.
(7) have polymorphism 22 to primer pair F between disease-resistant gene pond and susceptible gene pool to acquired2Dai Qun
Body single plant augmentation detection, and genetic linkage analysis is carried out with known Genetics Software, wherein having linkage relationship, quilt between 15 pairs of primers
It is divided into 3 linkage groups, LOD interpretation of result peak value, LOD > 3.0 occurs in b290.
(8) 2kb sequence up and down is searched according to the physical location where b290 in existing well-known technique, is searched with well known SSR
Rope software finds the site SSR again, and redesigns SSR-pc with well known PCR primer design software283Primer.
(9) with acquired molecular labeling SSR-pc283, to F2For group's single plant augmentation detection, genetic linkage point is carried out
Analysis；By F2The genotype of disease-resistant, susceptible phenotype and molecular labeling for group's single plant is lost with existing known Genetics Software
It passes linksystem analysis and determines SSR-pc with 3. 0 for minimum LOD threshold values283With muskmelon downy mildew Major resistance gene it is chain away from
From.
In the present invention, have polymorphism 2 to primer between disease-resistant gene pond and susceptible gene pool according to acquired
B290 and SSR-pc283In, the polymorphic molecular marker, primer sequence are as follows:
b290 5' GCTCCTCCTTAACTCTATAC' 3;
5' GCATTATTACCCATGTACGAG' 3;
SSR-pc283 5' TCGAACCTAGTTATCAGAAGTC' 3;
5' CAACCGACATTGAAAACCAAC' 3;
SSR-pc283DNA sequence dna
TCGAACCTAGTTATCAGAAGTCTCCTTACTTAAATAAAACAATTTCACTTTGTTGTGAATTAGTTGAT
TTTGTTGTT 77
GTTGAATGTTGAATTGATATAAAAAGCAATTAAGAATTTATTGATTTGCACGTGCTTAAGTAAACATG
TCTATATAT 154
ATATATATATATATGTATGTATGTATTTAATAATAATAAATTGATGATAGACATTTAGTATATATTAG
ATGATCATCGAC 234
AAAAAGTAGGTGTAAAACCAATGTTGAAGTTGGTTTTCAATGTCGGTTG 283
The present invention carries out linkage disequilibrium value, genetic linkage using well known Genetics Software tool WinQTLCart2.0
Analysis, establishes genetic linkage map.
The present invention finds the site SSSR using well known SSR search software SSRHunter again, and with known PCR primer
Design software Array Designer 4 redesigns SSR primer.
The present invention is finally found with well known QTL IciMapping Genetics Software anti-with muskmelon according to phenotype and genotype
The molecular labeling SSR-pc283 of downy mildew main effect QTL linkage.
The present invention finds the site SSS using well known SSR search software SSRHunter again, and with well known PCR primer
Design software Array Designer 4 redesigns SSR-pc283 primer.
Embodiment two: the application of chain molecular labeling with muskmelon downy mildew Major resistance gene
The application of chain molecular labeling with muskmelon downy mildew Major resistance gene, passes through provide above-described embodiment one
Molecular labeling SSR-pc283For melon variety or the genotype detection of strain, for judging the kind or the whether anti-muskmelon of strain
Downy mildew, comprising the following steps:
(1) hybridized with Resistant germplasm PI390452 with other muskmelons and multiplied to F2It is more than generation;
(2) to the single plant of muskmelon obtained by step (1), genomic DNA is extracted, SSR-pc is used283This mark into
Row PCR amplification has detected whether that 283bp specific band occurs, has occurred if any specific band, then predict that Muskmelon Plants are frost-resistant
Mildew.
Embodiment three: the building of hereditary segregating population and downy mildew inoculation method and disease scale
1, the building of F2 group
(1) by disease-resistant material PI390452(male parent) and Xinjiang farm variety " OK a karaoke club gram match " (female parent) carry out hybridization acquisition
F1, F1Selfing generates F2.By to parents, F1Generation and F2Artificial infection is carried out for group and counts the incidence of each single plant.
(2) downy mildew and disease scale are inoculated with: being inoculated with using conidiospore suspension spray.Natural occurrence is acquired from field
Early stage disease leaf be placed in the beaker for filling sterile water with the sporangium of banister brush swipe leaf back, be inoculated in sense with a connection
On the cotyledon of sick kind, be put into later illumination box expand it is numerous, after the cotyledon back side grows the mould layer of large stretch of black, then with soft
The sporangium of hairbrush swipe leaf back, is placed in the beaker for filling sterile water, is counted after mixing evenly with blood counting chamber mitogenetic
Spore count, ultimate density are 5 × 103/ ml spore.Sub- suspension is embraced with micro-sprayer sprinkling, plant leaf is sprayed onto and starts
Until dripping.Small plastic shed moisturizing is used after inoculation, 90% or more relative humidity opens Small plastic shed after 3d, investigation statistics disease after 10-14d
Feelings.Be classified the simultaneously anti-sense phenotype of recording individual according to plant leaf diseases area.
0 grade without any illness；
1 grade of lesion area accounts for the < 1/10 of entire blade area;
2 grades of lesion areas account for the 1/10-1/4 of entire blade area;
3 grades of lesion areas account for the 1/4-1/2 of entire blade area;
4 grades of lesion areas account for the 1/2-3/4 of entire blade area;
Scab is covered with substantially on 5 grades of leaves, and lesion area accounts for the > 3/4 of entire blade area;
Identification method is system with the area coverage of the mould layer of vacuum side of blade referring to the assessment resistance standard of (1989) such as Weng Zuxin
Meter standard.The individual record that disease rank is 0,1 and 2 be it is disease-resistant, the individual record that disease rank is 3,4 and 5 is susceptible.
Example IV: the QTL positioning of downy mildew resistance gene
(1) extraction of genomic DNA and the building in the pond DNA, take 0.5 ~ 1.0g muskmelon young leaflet tablet, are ground into liquid nitrogen
It is powdered, it is fitted into the centrifuge tube of 2.0mL, is extracted using the plant genome DNA of TIANGEN Biotech (Beijing) Co., Ltd.
Kit extracts DNA, measures DNA concentration final adjustment to 100mgL with Quawell Q3000-1It is spare.Using BSA method, 5 are taken
The DNA mixed in equal amounts of a 0 grade of disease-resistant single plant and 55 grades of susceptible single plants constructs the polymorphism that anti-sense gene pool is used for SSR primer
Screening.
Primer sequence for screening polymorphism is shown in the website ICuGI http://www.icugi.org/cgi-bin/
ICuGI/misc/download.cgi and according to newest article " the Anchoring the consensus delivered of A. DiaZ
ICuGI genetic map to the melon(Cucumis melo L.) genome》
(2) SSR-PCR reaction system are as follows: PCR amplification uses 2 × Easy of Beijing Quanshijin Biotechnology Co., Ltd
Taq PCR SuperMix for PAGE kit, contain in reaction system (20 μ L): template DNA 10ng, positive anti-primer are each
1uL, 2 × Easy Taq PCR SuperMix for PAGE10 μ L, PCR amplification are enterprising at TC-512 amplification instrument (TECHNE)
Row, response procedures are as follows: 94 DEG C of initial denaturation 5min, then 94 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 2min, carry out
35 circulations continue to extend 10min, 4 DEG C of preservations at 72 DEG C.
(3) polyacrylamide gel electrophoresis detects: taking 10uLPCR reaction product and 1.5uL to smell phenol blue (0. 25 %) mixed
It closes, point sample is in 6% polyacrylamide gel, and using 1 X TBE as electrophoretic buffer, 1. 5h of electrophoresis is left under 120V burning voltage
It is right.It is taken a picture with JY04S-3C number gel imager.
(4) the screening amplification that 300 pairs of core SSR primers carry out polymorphism is filtered out, it is final to obtain polymorphism SSR primer 22
It is right.
(5) have polymorphism 22 to primer pair F between disease-resistant gene pond and susceptible gene pool to acquired2For group
Single plant augmentation detection, and genetic linkage analysis is carried out with WinQTLCart2.0, wherein having linkage relationship, quilt between 15 pairs of primers
It is divided into 3 linkage groups, referring to attached drawing Fig. 1, there is peak value in b290 in LOD interpretation of result, LOD > 3.0, referring to attached drawing 2, dominant effect
It should be 0.48, referring to attached drawing 3.
(6) according to the newest article delivered of A. DiaZ and https: in the //website melonomics.net where b290
Physical location searches 2kb sequence up and down, finds the site SSS again with SSRHunter, and set again with Array Designer 4
Count SSR-pc283Primer.
(7) with acquired molecular labeling SSR-pc283, to F2For group's single plant augmentation detection, genetic linkage point is carried out
Analysis.Monoseeding F2240 cave of seed is emerged 200 plants, wherein sick series is 0-2 grades and has 150 plants, is shown as disease-resistant；Sick series is
3-5 grades have 50 plants, show as susceptible；In 50 plants of disease plants, 50 plants amplify susceptible polymorphic bands, and 0 plant only amplifies
Disease resistance state property multi-ribbon, i.e., there is no recombinations；In 150 plants of disease-resistant plants, 49 plants only amplify disease-resistant polymorphic bands,
100 plants amplify disease-resistant and susceptible 2 polymorphic bands, and 1 plant amplifies susceptible polymorphic bands, i.e. performance recombination.By F2 generation
Disease-resistant, the susceptible phenotype of group's single plant and the genotype of molecular labeling carry out heredity even with QTL IciMapping Genetics Software
The analysis of lock property determines SSR-pc with 3. 0 for minimum LOD threshold values283There are chain passes with muskmelon downy mildew Major resistance gene
System, genetic distance 0.50cM.Referring to attached drawing 4.
Embodiment five: breeding material disease-resistant gene detection
It is the F of parent with muskmelon resource PI3904522, F3Progeny population after planting, takes plant cotyledon or blade to extract base
Because of a group DNA, with primer SSR-pc283PCR amplification and electrophoresis detection are carried out, downy mildew resistance gene is carried and shows disease-resistant plant energy
About 283bp band is enough amplified, and shaping cannot be expanded in same position without the plant that downy mildew resistance gene shows sense downy mildew
Band.From genomic DNA is extracted to qualification result is obtained, 1-2 working day is only needed to can be completed in laboratory.
Downy mildew resistance gene whether is carried by conventional method identification, then is needed muskmelon and PI390452 to be identified is first
Hybridization obtains F1, then it is selfed and is returned building F2And BC, group, in conjunction with artificial infection muskmelon downy mildew bacterium, according to anti-sense phenotype
Inheritance can just obtain qualification result.Each hybridization, selfing and backcrossing combination are calculated according to 200 single plants, at least
It needs to plant 1000 group of hill body plant and carries out artificial infection didymella bryoniae.It calculates, passes according to 100 days seasons of growth of muskmelon one
After the identification of system method at least needs 200 days, can just identify whether carry downy mildew resistance gene.
Molecular mapping downy mildew resistance gene through the invention is clear, and identification is convenient.Pass through detection and downy mildew resistance
The molecular labeling of gene linkage can predict the downy mildew resistance of Muskmelon Plants, quickly screen disease-resistant variety or strain is used for sweet tea
Melon breeding.Disease-resistant gene fast and easy is detected, and not affected by environment.
Claims (1)
1. a kind of and muskmelon downy mildew resistance main effect QTL linkage SSR marker, which is characterized in that the DNA sequence of the SSR marker
Column are as shown in SEQID NO.5.
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| 甜瓜霜霉病抗性基因的SSＲ标记;贺玉花等;《江苏农业科学》;20140731;第42卷(第7期);第1.1-2.2节 * |
| 甜瓜霜霉病抗性研究进展;贺玉花等;《果树学报》;20140228;第31卷(第2期);全文 * |
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