CN104032024B - The SNP marker primer in a kind of pear fruit juice content main effect QTL site and application thereof - Google Patents
The SNP marker primer in a kind of pear fruit juice content main effect QTL site and application thereof Download PDFInfo
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Abstract
The invention provides a kind of SNP marker primer with pear fruit juice content main effect QTL site and application thereof.Close linkage SNP marker Pyd05_008 in pear juice content main effect QTL site, forward primer sequence is as shown in SEQ ID NO.1, and reverse primer sequences is as shown in SEQ ID NO.2.Utilize this special primer to identify juice content based on high-resolution solubility curve, juice content can be carried out the polymorphism on good typing, i.e. detection QTL site qJuice and express the difference of juice content.The pear fruit juice content main effect QTL molecular marker that the present invention provides can be used for the molecular marker assisted selection breeding of pear fruit juice content character, for accelerating the genetic improvement process of Pear varieties, improve breeding selection efficiency and there is important theory and practice directive significance.
Description
Technical field
The invention belongs to molecular genetic breeding field, relate to the SNP molecule in a kind of pear fruit juice content main effect QTL site
Labeled primer and application thereof.
Background technology
China is one of world's pear (Pyrus L.) topmost area of origin of plant, and genetic diversity enriches.The cultivation of pears
Cultivate widely distributed, except Hainan Province and Taiwan Province, have cultivation.The tender and crisp succulence of pear flesh, sweet and sour taste, deeply liked by consumer
Like.The content of melonidum juice not only affects the local flavor of fresh fruit, possibly together with the abundant nutritional labeling being conducive to health, such as
Sucus Pyri cool in nature and can heat clearing away calm, in summer, there is the effect relieved summer heat of quenching one's thirst, the composition such as glycocide contained in Sucus Pyri and tannic acid,
Energy expelling phlegm for arresting cough, has maintenance action to throat；Sucus Pyri also contains relatively polysaccharose substance and vitamin, is easily absorbed by the body, increase
Feed is intended to, and liver is had protective effect；Pectin content in Sucus Pyri is the highest, contributes to digestion, tonneau stool.Therefore, fruit juice
One of important evaluation index when the number of content is selection-breeding New pear variety.
Owing to most Pear varieties are self-incompatible, the genetic constitution apparent altitude heterozygosity of pears；Simultaneously as
The economical character majority of perennial fruit tree shows as Inheritance of Quantitative Characters feature, about the important character genetic development research phase of pears
To delayed, it is also difficult to carry out the genetic improvement of objective trait.In recent years, along with the developing rapidly of molecular marking technique, high density
The appearance of molecular genetic linkage map, and quantitative trait drawing method is the most perfect so that quantitative trait locus (QTL) is fixed
Position becomes reality, and the probability, accuracy and the predictability that also select for improving destination number character superior genotypes are established
Basis.Utilizing Molecular mapping quantitative trait QTL, its essence is exactly the company between analyzing molecules labelling and objective trait QTL
Lock relation, i.e. utilizes the molecular marker at known seat to position the QTL at unknown seat, by calculating between molecular marker and QTL
Exchange rate, determine the particular location of QTL.The pass between quantitative trait phenotypes is separated based on the marker genetype obtained
System, can directly determine that character site of controlling the size, exploitation can be applicable to the technology of molecular marker assisted selection breeding, and this is being permitted
Successful Application is achieved on more important crops.
QTL Position Research about pears quantitative trait at present still belongs to the starting stage, and existing research is primarily directed to
Disease or growth traits, position to the QTL of pear juice content character and detect the polymorphism expression juice content in QTL site poor
The exploitation applied research of different SNP marker not yet has been reported that.Therefore, carry out the QTL location of pear juice content character, based on
Corresponding sequence information development SNP marker, and set up filial generation assisted Selection technical system in early days, for improving pear fruit
Quality, improves breeding efficiency, saves production cost and is particularly important.
Summary of the invention
It is an object of the invention to position pear fruit juice content main effect QTL, according to contributing sites sequence information exploitation based on
High-resolution solubility curve identifies the SNP specific mark Pyd05_008 of pear fruit juice content, in detection QTL site qJuice
Polymorphism express juice content difference.The height of pear juice content can be predicted, for realizing fruit juice by this molecular marker
The early stage of content character is identified and screening provides molecule assisted Selection technical support.
The purpose of the present invention can be achieved through the following technical solutions:
One and pear fruit juice content main effect QTL site closely linked SNP marker primer, wherein:
Forward primer JUICE F:5 ' ACACGCTGAATAGTTGGACTT 3 ' (SEQ ID NO.1),
Reverse primer JUICE R:5 ' GGTACGACTGAGAAGCTTTAAGT 3 ' (SEQ ID NO.2).
SNP marker primer of the present invention is to the application in pears molecular breeding, and the primer described in utilization is to based on height
Resolution solubility curve identification and detection accession number is the of the pears genome sequence scaffold171.0 of AJSU00000000
Whether there is QTL site qJuice relevant to pear fruit juice content at 123191 bases, detect QTL site simultaneously
Polymorphism on qJuice expresses the difference of fruit juices content, it was predicted that the height of pear fruit juice content, to realize pear fruit
The early stage of juice content character is identified and screening.
Wherein, described pear fruit juice content refers to that the juice weight ratio that every 100g sarcocarp squeezes is more than or equal to more
40%, pear fruit juice content refers to that the juice weight ratio that every 100g sarcocarp squeezes is less than 20% less.
SNP marker primer of the present invention is at detection pear fruit juice content main effect QTL site qJuice and SNP
Application in polymorphism.
Primer of the present invention is for detecting the SNP marker method in pear fruit juice content main effect QTL site: exploitation right
Profit requires that the SNP marker primer described in 1 carries out HRM reaction to pears genomic DNA, if amplified production sequence size is at 215bp,
Show to there is QTL site qJuice relevant to pear juice content；PCR cycle melts after terminating, finally,In the Gene Scanning software of 480II, 1.5version automatically generates the melting curve of amplified production, respectively
To express the many kinds of juice content in QTL site qJuice that known pear juice content is relevant and to express the product that juice content is few
Plant as comparison, the difference of the polymorphism expression juice content in detection QTL site qJuice.If the amplification that unknown kind obtains
The melting curve of product is identical with the color of comparison, line style is similar, then it represents that on juice content main effect QTL site qJuice
The juice content that polymorphism is expressed is similar with comparison.
Wherein, in described known QTL site qJuice relevant to pear juice content, polymorphism expression juice content is many
Kind be ' Dangshan pear ', expressing the few kind of juice content is ' BAYUEHONG '.
The reaction system of described HRM reaction according to480High Resolution Melting
Description in Master test kit is carried out, HRM analyze beCarry out on 480II quantitative real time PCR Instrument；10μL
Reaction system: containing 2ng μ L‐1Pears genomic DNA template, 1 × Master Mix, 2.0mmol L‐1MgCl2、0.2mmol·
L‐1Primer described in claim 1；Amplification program uses PCR:95 DEG C of denaturation 10min of landing-type, then 95 DEG C of degeneration 10s,
60～55 DEG C, often circulation declines 0.5 DEG C, and annealing 15s, 72 DEG C of programs extending 12s carry out 45 circulations；After PCR cycle terminates
The program carrying out melting is: 95 DEG C of 1min, 40 DEG C of 1min, 65 DEG C of 1s, then from 65 DEG C of continuous warmings to 95 DEG C, often raises 0.04
DEG C, collect fluorescence 1 time, be finally cooled to 40 DEG C.
One and the pear fruit juice content main effect QTL closely linked SNP marker of site qJuice, this molecular marker is by drawing
Thing pair: forward primer JUICE F:SEQ ID NO.1 and reverse primer JUICE R:SEQ ID NO.2 expands pears genomic DNA
Obtain.
The SNP marker of the present invention application in pears molecular breeding, this molecular marker is positioned at the 5th linkage group of pears
At 11.5cM, Kruskal-Wallis statistical test value is 16.238, notable under the significant level of α=0.001, utilizes interval to make
The LOD value of figure method is 3.15, explains the hereditary variation of 15.6%.
SNP marker of the present invention is in detection pear fruit juice content main effect QTL site qJuice and SNP polymorphism thereof
In application.
SNP marker of the present invention is for detecting the SNP marker side of pear fruit juice content main effect QTL site qJuice
Method: utilize the SNP marker primer described in claim 1 that pears genomic DNA is carried out HRM reaction, if amplified production sequence is big
Little at 215bp, show to there is QTL site qJuice relevant to pear juice content；PCR cycle melts after terminating,
Finally, existIn the Gene Scanning software of 480II, 1.5version automatically generates the melting song of amplified production
Line, expresses the many kinds of juice content and expression fruit juice contains so that QTL site qJuice that known pear juice content is relevant is upper respectively
Measuring few kind is comparison, and the polymorphism in detection QTL site qJuice expresses the difference of juice content.If unknown kind obtains
The melting curve of the amplified production arrived is identical with the color of comparison, line style is similar, then it represents that in juice content main effect QTL site
The juice content that polymorphism on qJuice is expressed is similar with comparison；Wherein, the described known QTL relevant to pear juice content
The kind that on the qJuice of site, polymorphism expresses juice content many is ' Dangshan pear ', and the kind expressing juice content few is ' eight
The moon is red '.
Beneficial effect
(1) quantitative trait locus determining ' BAYUEHONG ' and ' Dangshan pear ' fruit juices content is carried out by the present invention first
QTL location, the QTL site of the location extremely notable incidence relation (α=0.0001) of existence to this quantitative trait, and contribution rate is relatively
Height, the contribution rate in site is 15.6%.This has established important for realizing the improvement of marker assisted selection controlled by multiple genes character inheritance
Basis and necessary premise.
(2) the distance need≤5cM of linked marker and the objective trait that can be applicable to molecule assisted Selection is generally believed at present,
In the present invention, juice content main effect QTL location is directly targeted in SNP marker, and Kruskal Wallis test value is
16.238, notable under the significant level of α=0.0001, Interval mapping detects that LOD value is 3.15, and linksystem and reliability are high,
This is significant for the accuracy and efficiency improving molecular marker assisted selection.
(3) present invention is according to the SNP mark of the pear juice content character main effect QTL site exploitation detection pear juice content of location
Note primer Pyd05_008, is used for predicting in QTL site qJuice that polymorphism expresses juice content difference, contains for pear fruit fruit juice
Being pre-selected of amount provides reliable molecular marker source.
(4) the SNP marker primer of exploitation, carries out QTL position to ' BAYUEHONG ' and ' Dangshan pear ' 37 strain hybrid Population
On some qJuice, polymorphism expresses the detection of juice content Differential genotype, hybrid Population can be divided into two groups, sight actual with fruit
The juice content examined is many and juice content has higher coincidence rate less.Population experiment shows, the SNP marker special primer of exploitation
Offspring's juice content can be carried out good typing (Fig. 2), therefore, there is good using value, can realize pear fruit fruit
Being pre-selected and assistant breeding of juice content character.
Accompanying drawing explanation
Fig. 1 is pear juice content main effect QTL interval and SNP marker site Pyd05_008 position in the 5th linkage group.
What LG5 represented is the 5th linkage group of ' BAYUEHONG ' and ' Dangshan pear ' merging genetic linkage maps.SNP marker title is initiated with
The representative of ' Pyb ' is marked from the SNP marker of ' BAYUEHONG ', the representative of initial entitled ' Pyd ' from the SNP of ' Dangshan pear '
Note, the representative of initial entitled ' Pybd ' is simultaneously from ' BAYUEHONG ' and the SNP marker of ' Dangshan pear '.
Numeral on the left of linkage group is the genetic distance between labelling, and unit is cM.Solid rectangle on the right side of linkage group
Instruction QTL mapping interval.The LOD scattergram that curve chart is QTL on the right.In fruit juices content QTL site correspondence linkage group
Pyd05_008 labelling, it is positioned at the 5th linkage group 11.5cM, and LOD value is 3.15.
Fig. 2 is the HRM specific mark primer according to the exploitation of SNP marker Pyd05_008, in ' BAYUEHONG ' and ' Dangshan pear '
The solubility curve of detection in 37 individualities of offspring, can good typing.Melting curve: line style 1 red curve, 21 strains are individual, its
In 19 strain juice contents many；Line style 2 blue curve, 16 strains are individual, and wherein 12 strain juice contents are few.The coincidence rate of two groups is respectively
It is 90% and 75%.
Detailed description of the invention
Embodiment 1:
The molecular marker of pear fruit juice content main effect QTL linkage, is prepared by the following:
A) (kind is public, sees document: Zhang Ruiping etc., pears AFLP labelling to utilize ' BAYUEHONG ' and ' Dangshan pear '
The QTL location of genetic map construction and fruit correlated traits, gardening journal, 2011,38 (10): 1,991 1998) hybridization obtains it
102 strain F1Offspring's individual plant.
B) utilize RADseq method high-flux sequence, ' BAYUEHONG ' and ' Dangshan pear ' and offspring are analyzed, and unite
Pleomorphism site hereditary form in progeny population analyzed by meter, utilizes χ2The each labelling of test Analysis separate whether meet 3:1 or
The mendelian inheritance segregation ratio of 1:1.
C) the Genetic Linkage Map spectrum of software building ' BAYUEHONG ' and ' Dangshan pear ' is analyzed with Joinmap4.0.By
B) the form importing being suitable to CP colony composition in software is analyzed in the polymorphism mark site obtained in step by Joinmap4.0
Joinmap4.0, gets rid of the too much site of missing data and the notable site partially separated, and the P value of Chi-square statistic is 0.05, selects
Kosambi mapping function builds genetic linkage map.
D) to ' BAYUEHONG ' and ' Dangshan pear ' and F thereof1The fruit juices content of colony's individual plant is evaluated.Will experiment fruit
Peeling chopping, uniformly mixes, and uses quartering to take the sarcocarp of constant weight, weighs juice weight after squeezing into juice with juice extractor.
Juice content (%)=juice weight/sarcocarp weight.Pear fruit juice content refers to the juice weight that every 100g sarcocarp squeezes more
Ratio is more than or equal to 40%, and juice content refers to that the juice weight ratio that every 100g sarcocarp squeezes is less than 20% less.
E) phenotypic number of juice content and the associated documents of label information are imported MapQTL5.0 software, select interval to make
Figure method, with LOD value >=3.0 as standard, carries out qtl analysis and location to the juice content of pears.Result shows, in ' BAYUEHONG ' and
Main effect QTL site qJuice (Fig. 1) of juice content, the contribution to this character is detected in 5th linkage group of ' Dangshan pear '
Rate is 15.6%, and SNP marker Pyd05_008 of its correspondence genetic distance in linkage group is 11.5cM, and LOD value is 3.15.
F) SNP marker site Pyd05_008 is utilized to develop HRM special primer.
By pears full-length genome data base (http://peargenome.njau.edu.cn/), from accession number it is
The pears genome sequence scaffold171.0 of AJSU00000000 searches out SNP marker Pyd05_008 in the 5th linkage group
DNA sequence, the sequence of 300bp before and after this site chosen, according to design of primers principle, exploitation design SNP marker primer.Forward
Primer sequence JUICE F is ' 5 ACACGCTGAATAGTTGGACTT 3 '；Reverse primer JUICE R be 5 '
GGTACGACTGAGAAGCTTTAAGT‐3’.Amplified production sequence size 215bp, utilizes the SNP marker primer of design in ' August
Red ' and the genomic DNA enterprising performing PCR amplification of ' Dangshan pear ', primer all expands normally, and PCR primer meets prediction size.Cause
This, this primer can be as the detection labelling of pear juice content character.
G) utilize HRM technology that pear fruit juice content is carried out typing.
HRM reaction system according toIn 480High Resolution Melting Master test kit
Description is carried out, HRM analyze beCarry out on 480II quantitative real time PCR Instrument.
10 μ L reaction systems: containing 2ng μ L‐1Pears genomic DNA template, 1 × Master Mix, 2.0mmol L‐ 1MgCl2, 0.2mmol L‐1Primer JUICE F/JUICE R；Amplification program uses landing-type PCR (touchdown PCR): 95
DEG C denaturation 10min, then 95 DEG C of degeneration 10s, 60～55 DEG C of (often circulation declines 0.5 DEG C) annealing 15s, 72 DEG C extend 12s'
Program carries out 45 circulations.
PCR cycle melts after terminating, and its program is: 95 DEG C of 1min, 40 DEG C of 1min, 65 DEG C of 1s, then from 65 DEG C continuously
It is warming up to 95 DEG C, often raises 0.04 DEG C, collect fluorescence 1 time, be finally cooled to 40 DEG C.
Finally, existIn the Gene Scanning software of 480II, 1.5version automatically generates amplified production
Melting curve
Utilize the SNP marker primer obtained in g) step 37 filial generation colonies to ' BAYUEHONG ' × ' Dangshan pear '
Carry out HRM analysis (Fig. 2).
Line style 1 red curve, 21 individual line styles are similar, and wherein 19 individualities are that juice content is many；
Line style 2 blue curve, 16 individual line styles are similar, and wherein 12 individualities are that juice content is few.
Statistical analysis shows, 37 individual typings are two kinds of genotype, and segregation ratio is 21:16.According to QTL site
The upper polymorphism of qJuice expresses juice content differential gene genotyping result by 37 individual juice content phenotypic data cards
Side's inspection (P=0.00005), test result display juice content is the most relevant with genotype, and the coincidence rate of two groups is respectively
90% and 75%.Therefore, by the comparative analysis to the phenotypic measurements of juice content character with HRM genotyping result, it was demonstrated that
This special SNP marker can detect the polymorphism in QTL site qJuice and express the difference of juice content, to pear fruit juice content
Good typing.
Claims (5)
1. one kind and pear fruit juice content main effect QTL site qJuice closely linked SNP marker primer pair, its feature exists
In forward primer JUICE F:SEQ ID NO.1, reverse primer JUICE R:SEQ ID NO.2, described with pear fruit fruit juice
Content main effect QTL site qJuice is positioned at the of the pears genome sequence scaffold171.0 that accession number is AJSU00000000
At 123191 bases, the SNP marker primer described in utilization carries out HRM reaction to pears genomic DNA, if amplified production sequence
Row size, at 215bp, shows to there is QTL site qJuice relevant to pear juice content.
2. the SNP marker primer described in claim 1 is to the application in pears molecular breeding, it is characterised in that drawing described in utilization
Thing is to based on the pears genome sequence that high-resolution solubility curve identification and detection accession number is AJSU00000000
A QTL site relevant to pear fruit juice content whether is there is at 123191st base of scaffold171.0
QJuice, detects the polymorphism in this QTL site simultaneously and expresses the difference of fruit juices content, it was predicted that pear fruit juice content
Just, to realize early stage qualification and the screening of pear fruit juice content character；Described pears are selected from ' Dangshan pear ' or ' August
Red '.
3. the SNP marker primer described in claim 1 to detection pear fruit juice content main effect QTL site qJuice and
Application in SNP polymorphism；Described pears are selected from ' Dangshan pear ' or ' BAYUEHONG '；Described with pear fruit juice content main effect
QTL site qJuice is positioned at the 123191st of the pears genome sequence scaffold171.0 that accession number is AJSU00000000
At base, the SNP marker primer described in utilization carries out HRM reaction to pears genomic DNA, if amplified production sequence size exists
215bp, shows to there is QTL site qJuice relevant to pear juice content.
4. the primer described in claim 1 is for detecting the SNP marker side of pear fruit juice content main effect QTL site qJuice
Method, described is positioned at, with pear fruit juice content main effect QTL site qJuice, the pears genome that accession number is AJSU00000000
At 123191st base of sequence scaffold171.0, it is characterised in that: utilize the SNP marker primer described in claim 1
Carry out HRM reaction to pears genomic DNA, if amplified production sequence size is at 215bp, show that having one contains with pear juice
QTL site qJuice that amount is relevant；PCR cycle melts after terminating, finally,The Gene of 480II
Scanning software 1.5version automatically generates the melting curve of amplified production, is correlated with known pear juice content respectively
Expressing the many kinds of the juice content kind few with expressing juice content in QTL site qJuice is comparison, detects QTL site
Polymorphism on qJuice expresses the difference of juice content, if the melting curve of amplified production that obtains of unknown kind with compare
Color identical, line style is similar, then it represents that the juice content that polymorphism on juice content main effect QTL site qJuice is expressed
Similar with comparison；Described pears are selected from ' Dangshan pear ' or ' BAYUEHONG '；Described known relevant to pear juice content QTL position
The kind that on some qJuice, polymorphism expresses juice content many is ' Dangshan pear ', and the kind expressing juice content few is ' August
Red '.
SNP marker method the most according to claim 4, it is characterised in that: the reaction system of described HRM reaction according toDescription in 480High Resolution Melting Master test kit is carried out, HRM analyze beCarry out on 480II quantitative real time PCR Instrument；10 μ L reaction systems: containing 2ng μ L‐1Pears genomic DNA template, 1
×Master Mix、2.0mmol·L‐1MgCl2、0.2mmol·L‐1Primer described in claim 1；Amplification program uses landing
Formula PCR:95 DEG C denaturation 10min, then 95 DEG C of degeneration 10s, 60～55 DEG C, often circulation declines 0.5 DEG C, annealing 15s, 72 DEG C prolong
The program stretching 12s carries out 45 circulations；The program that PCR cycle carries out melting after terminating is: 95 DEG C of 1min, 40 DEG C of 1min, 65 DEG C
1s, then from 65 DEG C of continuous warmings to 95 DEG C, often raise 0.04 DEG C, collect fluorescence 1 time, be finally cooled to 40 DEG C.
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