CN106167798B - The molecular labeling of the anti-anthracnose main effect QTL of grape and application - Google Patents
The molecular labeling of the anti-anthracnose main effect QTL of grape and application Download PDFInfo
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Abstract
The invention discloses a kind of molecular labeling of the anti-anthracnose main effect QTL of grape and applications, are related to fruit tree genetics breeding field.The present invention has carried out QTL positioning to the quantitative trait locus of the decision anti-anthracnose of grape for the first time, it is determined that the main effect QTL site RA of the anti-anthracnose of grape, the contribution rate in site are 71.5%, and the 37.7% anti-anthracnose feature of grape can be explained.Linkage molecule label M1E12-1500-m based on the screening of this site improves the accuracy to objective trait judgement, to realize that the improvement of the controlled by multiple genes character based on molecular breeding is laid a good foundation.In addition, molecular labeling M1E12-1500-m distance chain with main effect QTL site RA in the present invention is 0.96cM, close linkage is shown as with target site.Therefore, the label of acquisition has good application value, and the accuracy and efficiency of molecular marker assisted selection can be improved, and accelerates the progress improved to grape to resistance toanthracnose.
Description
Technical field
The present invention relates to fruit tree genetics breeding field more particularly to a kind of molecular labelings of the anti-anthracnose main effect QTL of grape
And application.
Background technique
Grape occupies an important position in China's production of fruit trees, and China's vinegrowing area in 2014 is 76.7 ten thousand hm2, produces
12,500,000 t or more are measured, second place of the world and first place (FAO, 2014) is occupied respectively, is the weight of China's agricultural industry and increasing peasant income
Want pillar of the economy.With the fast development of grape industry, disease becomes the major obstacle of grape high-efficiency high-quality production.
Bitter rot or anthracnose of grape is to endanger a kind of Major Diseases of grape production, can cause grape underproduction 10%-20%, anthracnose
Cause fruit to generate scab, grape quality is caused to seriously endanger.Currently, the prevention and treatment to bitter rot or anthracnose of grape mostly uses chemical agent
Prevention and treatment, but chemical agent can remain in grape fruit and soil, adversely affect to human health and environment.
Therefore, cultivating the anti-anthracnose new varieties of grape is one of important goal of grape breeding.
Currently, for bitter rot or anthracnose of grape being infected and posting for the pathogenicity of bitter rot or anthracnose of grape infective pathogen bacterium, germ mostly
The research of the gene that key enzyme, regulation bacterial strain in main procedure cause a disease etc..And to bitter rot or anthracnose of grape gene anti-in grape
Research, which yet there are no, clearly to be reported.Breeding is carried out using bitter rot or anthracnose of grape gene anti-in grape also to have not been reported.
Summary of the invention
The purpose of the present invention is to provide a kind of molecular labeling of the anti-anthracnose main effect QTL of grape and applications, to solve
Foregoing problems existing in the prior art.
To achieve the goals above, The technical solution adopted by the invention is as follows:
A kind of molecular labeling M1E12-1500-m of grape anti-anthracnose main effect QTL site RA, the molecular labeling
The forward primer sequence of M1E12-1500-m is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2.
Preferably, the length of the molecular labeling M1E12-1500-m is 1500bp, molecular labeling M1E12-1500-m
Genetic distance between grape anti-anthracnose main effect QTL site RA is 0.96cM.
Preferably, comprising: forward primer sequence is as shown in SEQ ID NO.1, reverse primer sequences such as SEQ ID NO.2 institute
Show.
A kind of molecule labelling method for identifying grape anti-anthracnose main effect QTL site RA, utilizes above-mentioned molecular labeling
The primer pair of M1E12-1500-m is primer, carries out PCR amplification to grape material to be identified, amplified production carries out electrophoresis inspection
It surveys, such as amplifies the DNA fragmentation of corresponding size, then indicate that grape anti-anthracnose main effect QTL site RA exists.
Preferably, the reaction system of the PCR are as follows:
10 × buffer: 2.0 μ L, dNTP:2.5mmol L-1× 1.6 μ L, Mg2+: 25mmol L-1× 1.2 μ L, DNA moulds
Plate: 40ng μ L-1× 2.0 μ L, ExTaq enzymes: 5U μ L-1× 0.2 μ L, forward primer: 10mmol L-1× 1.0 μ L, reverse primer:
10mmol L-1× 1.0 μ L are mended with distilled water to 20 μ L.
Preferably, the reaction condition of the PCR are as follows:
94 DEG C of initial denaturation 5min；Then 94 DEG C of denaturation 1min, 35 DEG C of renaturation 1min, 72 DEG C of extension 1min, totally 5 recycle；
Then 94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, totally 35 recycle；Last 72 DEG C extend 7min eventually.
A kind of screening technique of molecular labeling M1E12-1500-m, comprising the following steps:
S1 obtains F1 filial generation group using grape Eurasian kind of ' Rizamat ' and wild Vitis davidii Foex ' black pearl '；
S2 utilizes the true of the primer pair filial generation that can amplify male parent specific band using SSR molecular marker method
Puppet is identified, building and QTL positioning of the true hybrid identified for next step grape molecular genetic linkage map；
S3 analyzes two parents and its F1 group with two kinds of molecule labelling methods of SRAP, SSR, statisticallys analyze between parent
Hereditary form of the site with polymorphism in progeny population obtains polymorphism mark site, then carries out chain point to it
Analysis, constructs the genetic linkage maps of grape；
S4 reflects to the resistance toanthracnose of the filial generation of ' Rizamat ' and ' black pearl ' using bacterium solution inocalation method
It is fixed；
S5 selects Interval Mapping, by the resistance and genetic map of each single plant of hybrid Population using MapQTL5.0 software
Molecular labeling in spectrum carries out chain and qtl analysis, is standard with LOD value >=3.0, and being greater than 3.0 explanations, there are one QTL
Point detects the QTL site RA of anti-anthracnose in No. 12 linkage group of grape, and LOD value 3.03 is main effect QTL, away from
A SRAP molecular labeling nearest from main effect QTL is M1E12-1500-m, size 1500bp, and the distance away from RA is
0.96cM, the label can be used as the label of the anti-anthracnose of grape, and the forward direction of molecular labeling M1E12-1500-m obtained is drawn
Object sequence as shown in SEQ ID NO.1, are as follows: 5 '-TGAGTCCAAACCGGATA-3 ', reverse primer sequences SEQ ID NO.2 institute
Show, are as follows: 5 '-GACTGCGTACGAATTCTC-3 '.
Preferably, the main effect QTL site RA is located at Eurasian kind of ' Rizamat ' and wild Vitis davidii Foex ' black pearl ' joint
In 12 linkage groups of map, which is 71.5%.
It is a kind of for screening the kit of anti-anthracnose grape, including above-mentioned primer pair.
Application of the above-mentioned primer pair in anti-anthracnose grape breeding.
The beneficial effects of the present invention are: the molecular labeling of the anti-anthracnose main effect QTL of grape provided in an embodiment of the present invention and
Using, for the first time to determine the anti-anthracnose of grape quantitative trait locus carried out QTL positioning, it is determined that the master of the anti-anthracnose of grape
QTL site RA is imitated, the contribution rate in site is 71.5%, and the 37.7% anti-anthracnose feature of grape can be explained.It is sieved based on this site
The linkage molecule label M1E12-1500-m of choosing improves the accuracy to objective trait judgement, to realize based on molecular breeding
The improvement of controlled by multiple genes character is laid a good foundation.In addition, molecular labeling chain with main effect QTL site RA in the present invention
M1E12-1500-m distance is 0.96cM, shows as close linkage with target site.Therefore, the label of acquisition has good answer
With value, the accuracy and efficiency of molecular marker assisted selection can be improved, shorten breeding process, accelerate anti-to anthracnose to grape
Property improvement progress.
Detailed description of the invention
Fig. 1 is the position of the anti-anthracnose QTL site RA of grape on chromosome.
In figure, what LG was represented is the linkage group of grape, and the number after LG represents linkage group number, the number of linkage group upper left side
It is the genetic distance between molecular labeling, unit cM, what right side then indicated is the title of molecular labeling, shares SRAP, SSR two
Kind molecular labeling, the number after marking "-" are the sizes of the polymorphic bands of label amplification, the solid rectangular on the right side of linkage group
Shape indicates QTL mapping section RA.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with attached drawing, to the present invention into
Row is further described.It should be appreciated that the specific embodiments described herein are only used to explain the present invention, it is not used to
Limit the present invention.
In the original amur grape in China, Vitis davidii Foex, V. amurensis, downy grape, East China grape, Ying Azulene etc. have anthracnose
Very strong resistance.But these disease-resistant amur grape low qualities should not be utilized directly.And Eurasian kind of China's Main Cultivation and
Franco-american grape, it is weaker to the resistance of bitter rot or anthracnose of grape but best in quality.Therefore the disease resistance of Chinese wild grape is utilized
New resistance excellent variety is cultivated with the fine quality of vitis vinifera, is the basic solution route for preventing and treating bitter rot or anthracnose of grape,
It is most economical, effective, safe method.
The grape molecular genetic linkage map that domestic and international worker constructs has been directed to the riverside grape of Eurasian kind, America kind
And downy grape and V. amurensis in sand grape and Chinese wild grape, and have detected that and anti-grape Pearls, frost-resistant
The relevant QTL of mildew, black rot resistance, mildew-resistance, botrytis resistant, winter resistance and yet there are no and Chinese Wild Vitis davidii Foex phase
The molecular genetic linkage map of pass.
As shown in Figure 1, the embodiment of the invention provides the molecular labelings of grape anti-anthracnose main effect QTL site RA a kind of
The forward primer sequence of M1E12-1500-m, the molecular labeling M1E12-1500-m are as shown in SEQ ID NO.1, reverse primer
Sequence is as shown in SEQ ID NO.2.
SRAP (Sequence Related Amplified Polymorphism, related sequence amplified polymorphism) technology
It is a kind of molecular marking technique of based on PCR, is also known as based on sequence amplification polymorphism (Sequence based Amplified
Polymorphism, SBAP), it was proposed by Li the and Quiros doctor of California, USA university vegetable crop system in 2001, mainly
The region opening code-reading frame (ORFs) for detecting gene has many advantages, such as that simple, efficient, repeatability is high, easily sequencing, is suitable for gene
Positioning with clone etc. molecular biology research.The primer sequence (table 1) that primer sequence is announced referring to Li and Quiros, primer
It is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The SRAP primer sequence of 1 detection of table
The length of the molecular labeling M1E12-1500-m is 1500bp, and molecular labeling M1E12-1500-m and grape are anti-
Genetic distance between anthracnose main effect QTL site RA is 0.96cM.
The screening technique of molecular labeling M1E12-1500-m, comprising the following steps:
S1 obtains F1 filial generation group using grape Eurasian kind of ' Rizamat ' and wild Vitis davidii Foex ' black pearl '；
S2 utilizes the true of the primer pair filial generation that can amplify male parent specific band using SSR molecular marker method
Puppet is identified, building and QTL positioning of the true hybrid identified for next step grape molecular genetic linkage map；
S3 analyzes two parents and its F1 group with two kinds of molecule labelling methods of SRAP, SSR, statisticallys analyze between parent
Hereditary form of the site with polymorphism in progeny population obtains polymorphism mark site, then carries out chain point to it
Analysis, constructs the genetic linkage maps of grape；
S4 reflects to the resistance toanthracnose of the filial generation of ' Rizamat ' and ' black pearl ' using bacterium solution inocalation method
It is fixed；
S5 selects Interval Mapping, by the resistance and genetic map of each single plant of hybrid Population using MapQTL5.0 software
Molecular labeling in spectrum carries out chain and qtl analysis, is standard with LOD value >=3.0, and being greater than 3.0 explanations, there are one QTL
Point detects the QTL site RA of anti-anthracnose in No. 12 linkage group of grape, and LOD value 3.03 is main effect QTL, away from
A SRAP molecular labeling nearest from main effect QTL is M1E12-1500-m, size 1500bp, and the distance away from RA is
0.96cM, the label can be used as the label of the anti-anthracnose of grape, and the forward direction of molecular labeling M1E12-1500-m obtained is drawn
Object sequence as shown in SEQ ID NO.1, are as follows: 5 '-TGAGTCCAAACCGGATA-3 ', reverse primer sequences SEQ ID NO.2 institute
Show, are as follows: 5 '-GACTGCGTACGAATTCTC-3 '.
Wherein, the reaction system of the PCR are as follows:
10 × buffer: 2.0 μ L, dNTP:2.5mmol L-1× 1.6 μ L, Mg2+: 25mmol L-1× 1.2 μ L, DNA moulds
Plate: 40ng μ L-1× 2.0 μ L, ExTaq enzymes: 5U μ L-1× 0.2 μ L, forward primer: 10mmol L-1× 1.0 μ L, reverse primer:
10mmol L-1× 1.0 μ L are mended with distilled water to 20 μ L.
The reaction condition of the PCR are as follows:
94 DEG C of initial denaturation 5min；Then 94 DEG C of denaturation 1min, 35 DEG C of renaturation 1min, 72 DEG C of extension 1min, totally 5 recycle；
Then 94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, totally 35 recycle；Last 72 DEG C extend 7min eventually.
The embodiment of the invention provides the primers of the molecular labeling M1E12-1500-m of the anti-anthracnose main effect QTL of grape a kind of
Right, the forward primer sequence of the primer pair is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2.
The embodiment of the invention provides a kind of molecule labelling method for identifying grape anti-anthracnose main effect QTL site RA, benefits
With the primer pair of molecular labeling M1E12-1500-m, (for forward primer sequence as shown in SEQ ID NO.1, reverse primer sequences are such as
Shown in SEQ ID NO.2) it is primer, PCR amplification is carried out to grape material to be identified, amplified production carries out electrophoresis detection, such as
The DNA fragmentation of corresponding size is amplified, then indicates that grape anti-anthracnose main effect QTL site RA exists.
In the embodiment of the present invention, main effect QTL site RA is located at Eurasian kind of ' Rizamat ' and wild Vitis davidii Foex ' black pearl '
In 12 linkage groups of joint map, which is 71.5%.
The embodiment of the invention provides a kind of primer pair (forward primer sequence as shown in SEQ ID NO.1, reverse primer sequence
Arrange as shown in SEQ ID NO.2) application in anti-anthracnose grape breeding.
Specific embodiment 1: the anti-anthracnose main effect QTL of grape and its discovery with the molecular labeling of main effect QTL linkage.
1) using the wild Vitis davidii Foex of anti-anthracnose and Eurasian kind of the Rizamat sensitive to anthracnose, it is miscellaneous to carry out remote edge
It hands over, obtains 203 plants of hybrid Populations.
2) SSR molecular marker is utilized, the true or false of Vitis davidii Foex and Rizamat Interspecific Hybrids is identified.Sieve
The primer that can amplify male parent specific band is selected, filial generation is identified, there are 92 plants of offsprings to have the specificity of male parent
Band analyses in conjunction with field shape credit, is confirmed as true hybrid.The true hybrid identified can be used for constructing molecular genetic in next step
The QTL of map and anti-anthracnose.
3) two parents and its F1 group are analyzed with two kinds of molecule labelling methods of SRAP, SSR, statistical analysis has between parent
There is hereditary form of the site of polymorphism in progeny population.It is finally obtained 128 SSRs and 62 SRAPs etc. totally 190
Then polymorphism mark site carries out linkage analysis to it using Jionmap4.0 mapping software, construct one altogether comprising 109
The grape genetic linkage maps of a polymorphism mark.
Using SRAP, SSR molecular marker method, ' Rizamat ' and ' black pearl ' and offspring are analyzed, and counted
Analyze hereditary form of the site between parent with polymorphism in progeny population.
Side of the experimental procedures of SRAP label with reference to Li et al. (Theor Appl Genet, 2001,103:455-461)
Method；The experimental procedures of SSR marker are with reference to Gabriele D.G. etc. (Molecular Breeding, 2005,15:11-20)
Method.The primer is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.SRAP and SSR amplified production is with 8%
The detection of non-denaturing polyacrylamide gel highly basic argentation.
The polymorphic bands clearly occurred on SRAP, SSR electrophorogram are denoted as " 1 ", no band is denoted as " 0 ", it is unclear that
Band or missing are denoted as "-", and point of each polymorphic bands is calculated according to the Marker (100bp DNA Ladder) of known molecular amount
Son amount.Separated bands are sorted out by parental source, determine its source and hereditary form.Separation is respectively marked using 2 test Analysis of χ is
It is no meet 3: 1 or 1: 1 Mendelian segregation ratio, separate partially label end with " * " mark.
4) it is composed with the Genetic Linkage Map of Joinmap4.0 analysis software building grape.
By polymorphism mark band obtained in 3) step according to the separation type of parent and offspring, according to CP model into
Row statistic of classification.The data that label generates separation in offspring are imported under mapping software Joinmap 4.0Dataset menu and are selected
Create new dataset is selected, checks whether data format is correct, detects correct nothing under High light error order
After accidentally, data analysis state is gone under Create population node window.Under Calculate options window
LOD value, maximum recombination fraction etc. are set.Under data analysis state, successively individual genot.freq,
The lower individual or label for excluding uncomfortable collaboration diagram of similarity of loci and similarity of individual order,
Then each label is detected using Locus genot.freq and analyzes its separation situation partially.Make under Grouping menu
Map linkage group is obtained with Create groups for mapping order, Calculate map is clicked, is created that each company
The linkage map for locking group finally selects the Map of each linkage group, and the Combine maps under Join menu is selected to merge map.
5) the F1 group single plant identifies the resistance of anthracnose.It will be in resistance toanthracnose grade and linkage map
Associated documents to import MapQTL5.0 mapping software using Interval Mapping be standard with LOD value >=3.0, it is anti-to anthracnose
Property qtl analysis and positioning are carried out on the linkage map of grape.
The result shows that detecting the QTL site RA (referring to Fig. 1) of anti-anthracnose, LOD value in the 12nd linkage group of grape
It is 3.03, the linkage distance with nearest SRAP molecular labeling M1E12-1500-m is 0.96cM, and the contribution rate to the character is
71.5%, the 37.7% anti-anthracnose feature of grape can be explained.Therefore, the QTL site be main effect QTL, mark recently away from
From the required maximum distance also much smaller than the linked marker and objective trait that can be applied to molecule assisted Selection.
By taking SRAP therein label as an example:
The PCR reaction amplification system total volume of SRAP is 20 2.0 μ L, dNTP (2.5mmolL of μ L, 10 × Buffer-1)
1.6 μ L, Mg2+(25mmol·L-1) 1.2 μ L, DNA profiling (40ng μ L-1) 2.0 μ L, ExTaq enzyme (5U μ L-1) 0.2 μ L, upper,
Downstream primer (10mmolL-1) each 1.0 μ L, finally mended with distilled water to 20 μ L.
Amplimer:
Me1:5 '-TGAGTCCAAACCGGATA-3 '；
Em12:5 '-GACTGCGTACGAATTCTC-3 '.
SRAP primer PCR response procedures are as follows: 94 DEG C of initial denaturation 5min；Then 94 DEG C of denaturation 1min, 35 DEG C of renaturation 1min, 72
DEG C extend 1min, totally 5 circulation；Then 94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, totally 35 recycle；Most
72 DEG C of extension 7min afterwards, 4 DEG C of preservations.
The product of PCR amplification is detected with 8% native polyacrylamide gel electrophoresis, and coamplification has gone out 10 rules
Band, it is exactly target stripe M1E12-1500-m that wherein size, which is the band of 1500bp,.
Specific embodiment 2: grape anti-anthracnose main effect QTL site RA and molecular labeling M1E12-1500-m are in grape breeding
In application
Using the molecular labeling mutually chain with the anti-anthracnose main effect QTL of grape screened to ' black pearl ' and ' Li Zhama
It is special ' 92 plants of F1 filial generations carry out Preliminary detection, to detect this method in bitter rot or anthracnose of grape resistance molecule marker assisted selection
In practical value.It is anti-that PCR is carried out with the genomic DNA of 92 plants of offsprings and SRAP selective amplification primer combination Me1Em12
It answers, the operating process of reaction is with the SRAP molecule labelling method in embodiment 1, and PCR amplification result is in non-denaturing polyacrylamide
Gel electrophoresis (result as scheme) determines whether there is corresponding label according to the presence or absence of M1E12-1500-m band after electrophoresis,
If there is label, illustrate that the single plant is high to the resistance of anthracnose, it is low to the resistance of anthracnose there is no then illustrating, while with reality
The bitter rot or anthracnose of grape resistance of border identification is verified with Markers for Detection result.
The results show that sharing 73 plants of DNA cloning knot in 92 plants of F1 filial generations of ' black pearl ' and ' Rizamat '
There is M1E12-1500-m band in fruit, has 28 plants of resistance to belong to highly resistance in this 73 plants, has 33 plants of resistance to belong to anti-, there is 12
The resistance of strain resists in belonging to；The DNA cloning result for sharing 19 plants of offsprings does not occur M1E12-1500-m band, wherein 14 plants anti-
Property belongs to sensitivity, and 5 plants of resistance belongs to high sense.There is preferable prediction effect with M1E12-1500-m prediction bitter rot or anthracnose of grape resistance
Fruit.
By using above-mentioned technical proposal disclosed by the invention, obtained following beneficial effect: the present invention fights to the finish for the first time
The quantitative trait locus for determining the anti-anthracnose of grape has carried out QTL positioning, it is determined that the main effect QTL site of the anti-anthracnose of grape, position
The contribution rate of point is 71.5%, and the 37.7% anti-anthracnose feature of grape can be explained.Therefore, chain point based on the screening of this site
Son label improves the accuracy to objective trait judgement, to realize that the improvement of the controlled by multiple genes character based on molecular breeding is established
Basis is determined.In addition, in the present invention with the marking path of main effect QTL linkage be 0.96cM, show as closely connecting with target site
Lock.Therefore, the label of acquisition has good application value, and the accuracy and efficiency of molecular marker assisted selection can be improved, and contracts
Short breeding process accelerates the progress improved to grape to resistance toanthracnose.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
Depending on protection scope of the present invention.
Claims (6)
1. a kind of method for the molecular labeling for identifying grape anti-anthracnose main effect QTL site RA, which is characterized in that utilize molecule mark
The primer pair for remembering M1E12-1500-m is primer, wherein forward primer sequence is as shown in SEQ ID NO.1, reverse primer sequences
As shown in SEQ ID NO.2, PCR amplification is carried out to grape material to be identified, amplified production carries out electrophoresis detection, such as amplifies
The DNA fragmentation of corresponding size then indicates that grape anti-anthracnose main effect QTL site RA exists.
2. the method for the molecular labeling of identification grape anti-anthracnose main effect QTL site RA, feature exist as described in claim 1
In the reaction system of the PCR are as follows:
10 × buffer: 2.0 μ L, dNTP:2.5mmol L-1× 1.6 μ L, Mg2+: 25mmol L-1× 1.2 μ L, DNA profiling:
40ngμL-1× 2.0 μ L, ExTaq enzymes: 5U μ L-1× 0.2 μ L, forward primer: 10mmol L-1× 1.0 μ L, reverse primer:
10mmol L-1× 1.0 μ L are mended with distilled water to 20 μ L.
3. the method for the molecular labeling of identification grape anti-anthracnose main effect QTL site RA, feature exist as described in claim 1
In the reaction condition of the PCR are as follows:
94 DEG C of initial denaturation 5min；Then 94 DEG C of denaturation 1min, 35 DEG C of renaturation 1min, 72 DEG C of extension 1min, totally 5 recycle；Then
94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, totally 35 recycle；Last 72 DEG C extend 7min eventually.
4. a kind of screening technique of the molecular labeling M1E12-1500-m of grape anti-anthracnose main effect QTL site RA, feature exist
In, comprising the following steps:
S1 obtains F1 filial generation group using grape Eurasian kind of ' Rizamat ' and wild Vitis davidii Foex ' black pearl '；
S2 utilizes the true or false for the primer pair filial generation that can amplify male parent specific band using SSR molecular marker method
Identified, building and QTL positioning of the true hybrid identified for next step grape molecular genetic linkage map；
S3 analyzes two parents and its F1 group with two kinds of molecule labelling methods of SRAP, SSR, and statistical analysis has between parent
Hereditary form of the site of polymorphism in progeny population obtains polymorphism mark site, then carries out linkage analysis, structure to it
Build the genetic linkage maps of grape；
S4 identifies the resistance toanthracnose of the filial generation of ' Rizamat ' and ' black pearl ' using bacterium solution inocalation method；
S5 selects Interval Mapping using MapQTL5.0 software, will be in the resistance and genetic map of each single plant of hybrid Population
Molecular labeling carry out chain and qtl analysis, be standard with LOD value >=3.0, be greater than 3.0 explanations there are a QTL site,
The QTL site RA of anti-anthracnose is detected in No. 12 linkage group of grape, LOD value 3.03 is main effect QTL, and distance is main
Imitating a nearest SRAP molecular labeling of QTL is M1E12-1500-m, and size 1500bp, the distance away from RA is 0.96cM, should
Label can be used as the label of the anti-anthracnose of grape, and the forward primer sequence of molecular labeling M1E12-1500-m obtained is such as
Shown in SEQ ID NO.1, are as follows: shown in 5 '-TGAGTCCAAACCGGATA-3 ', reverse primer sequences SEQ ID NO.2, are as follows: 5 '-
GACTGCGTACGAATTCTC-3′。
5. the screening technique of molecular labeling M1E12-1500-m according to claim 4, which is characterized in that the main effect
QTL site RA is located in 12 linkage groups of Eurasian kind of ' Rizamat ' and wild Vitis davidii Foex ' black pearl ' joint map, the site
Contribution rate is 71.5%.
6. application of the primer pair of molecular labeling M1E12-1500-m in anti-anthracnose grape breeding, which is characterized in that described
The forward primer sequence of primer pair is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2.
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| 葡萄炭疽病菌SRAP遗传多样性分析;李利霞 等;《中国农学通报》;20121231;230-235页 * |
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