CN105803083B - The molecular labeling of pear fruit single fruit weight main effect QTL site MSF and application - Google Patents
The molecular labeling of pear fruit single fruit weight main effect QTL site MSF and application Download PDFInfo
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Abstract
The invention belongs to pears field of molecular breeding, molecular labeling and the application in a kind of pear fruit single fruit weight main effect QTL site are provided.Utilize SSR marker building ' red eggplant pearss ' and ' evening show pears ' joint genetic linkage maps, in conjunction with pear fruit single fruit weight phenotypic evaluation, and qtl analysis is carried out using Interval Mapping, the presence in main effect QTL site is detected in the 11st linkage group of joint linkage map, which is 51.9%.SSR marker with the main effect QTL site close linkage is QS1122-135, genetic distance 4.37cm.Present invention pear fruit single fruit weight main effect QTL molecular labeling obtained can be used for this fruit properties of single fruit weight and carry out Seedling selection, have important theory and practice directive significance for improving breeding efficiency.
Description
Technical field
The invention belongs to pears molecular genetic breeding fields, and in particular to a kind of pear fruit single fruit weight main effect QTL site MSF's
Molecular labeling and its screening technique and application.
Background technique
Pears (Pyrus L.) are the important industrial crops in China, and cultivated area and yield occupy first place in the world.Pear fruit
Nutritive value is high, crisp succulence, sour and sweet palatability, and has certain medical value, is liked by the majority of consumers.Single fruit weight
An important factor for being pear fruit exterior quality, fruit is big or the small yield and economic benefit for directly determining Pear varieties.Therefore, pears
Fruit single fruit weight is one of Pear varieties Major Economic.It is pears breeding objective that the single fruit weight of different consumer demands is catered in cultivation
One of.
Pears are perennial woody plants, and the generation cycle is long, as a result late.It is to utilize fruit phenotype character pair in traditional breeding method
Pears genotype is selected, and this method is not only influenced by pears juvenile phase, but also the left and right vulnerable to external environment.With point
The development of sub- breeding technique constructs high density genetic linkage maps by molecular labeling and carries out QTL to fruit single fruit weight
(Quantitative trait loci, quantitative trait locus) analysis and positioning become a reality, and are also raising destination number
The accuracy and foresight of shape excellent genes selection are laid a good foundation.Using Molecular mapping quantitative character QTL, essence is just
It is the exchange rate between analyzing molecules label and objective trait QTL, to determine the specific location of QTL.Label base based on acquisition
Because of the relationship between type separation and quantitative character, the site for character of controlling the size can be directly determined, exploitation can be applied to molecule mark
Remember the technology of assisted selection, this achieves successful application on many important crops.
The current QTL Position Research in relation to pears quantitative character still belongs to the starting stage, for disease, growth traits and fruit portion
Point character QTL positioning and the research of molecular labeling have been reported.But ' August is only limited to for the QTL of pear fruit single fruit weight positioning
It is red ' this parent, the practicality is still limited by the F of ' August is red ' and ' Dangshan pear '1Hybrid Population.Therefore, carry out distinct group
The building of body genetic linkage maps, and the QTL positioning for carrying out pears single fruit weight is the necessary means for widening molecular labeling application range.This
Invention uses the filial generation F of ' red eggplant pearss ' and ' evening show pears '1The QTL of genetic linkage maps building and single fruit weight is carried out for group
Positioning is the raising and supplement of single fruit weight molecular marker assisted selection breeding technique.
Summary of the invention
The object of the present invention is to provide the molecular labelings and the molecule of a kind of pear fruit single fruit weight main effect QTL site MSF
The primer pair of label.
It is a further object of the present invention to provide the molecule labelling methods of pear fruit single fruit weight main effect QTL site MSF a kind of.
It is yet another object of the invention to provide a kind of screenings of the molecular labeling of pear fruit single fruit weight main effect QTL site MSF
Method.
The molecular labeling that another object of the present invention is to provide pear fruit single fruit weight main effect QTL site MSF is educated in pears molecule
Application in kind.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of the molecular labeling QS1122-135 of pear fruit single fruit weight main effect QTL site MSF, molecular labeling QS1122-
135 forward primer sequence is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2.
Above-mentioned molecular labeling QS1122-135 is to carry out PCR amplification as substrate using the genomic DNA of Pear varieties evening elegant pears
Resulting length is the DNA fragmentation of 135bp, and the genetic distance between molecular labeling QS1122-135 and MSF is 4.37cM.
A kind of primer pair of the molecular labeling QS1122-135 of pear fruit single fruit weight main effect QTL site MSF, the primer pair
Forward primer sequence as shown in SEQ ID NO.1, reverse primer sequences are as shown in SEQ ID NO.2.
A kind of molecule labelling method for identifying pear fruit single fruit weight main effect QTL site MSF, the method is with pears genome
DNA is as template, and the primer pair with above-mentioned molecular labeling QS1122-135 is that primer carries out PCR amplification, and amplified production carries out electricity
Swimming detection such as amplifies the DNA fragmentation that length is 135bp, then indicates that pear fruit single fruit weight main effect QTL site MSF exists.
A kind of screening technique of the molecular labeling QS1122-135 of pear fruit single fruit weight main effect QTL site MSF, including it is following
Step:
(1) Pear varieties ' red eggplant pearss ' and ' evening show pears ' hybridization, acquisition F are utilized1For group's single plant；
(2) two parents and its F are analyzed using SSR molecular marker method1Group, statistical analysis have polymorphic between parent
Property hereditary form of the site in progeny population, obtain polymorphism mark site, linkage analysis then carried out to it, construct
' red eggplant pearss ' and ' evening show pears ' joint genetic linkage maps, use when wherein carrying out linkage analysis to polymorphism mark site
Jionmap4.0 mapping software；
(3) to ' red eggplant pearss ' and ' F of evening show pears ' filial generation1The fruit single fruit weight of group's single plant is measured, and is utilized
The Interval Mapping (MapQTL5.0 mapping software) of MapQTL5.0 mapping software, by F1The single fruit weight and step of each single plant of group
Suddenly the molecular labeling that ' the red eggplant pearss ' of (2) building and ' evening show pears ' combine in genetic linkage maps carries out chain and qtl analysis, with
LOD value >=3 are standard, and being greater than 3 explanations, there are a QTL sites, in ' red eggplant pearss ' and ' evening show pears ' joint genetic linkage maps
The 11st linkage group on detect the QTL site MSF of single fruit weight, LOD value 8.78, contribution rate 51.9% is main effect
QTL；
(4) the main effect QTL site region obtained according to step (3), selection are less than the SSR of 5cM with its linkage distance
Molecular labeling, wherein molecular labeling QS1122-135 can be to F1Group's fruit single fruit weight character carries out good parting, therefore, this
Invention selects QS1122-135 as the molecular labeling of pear fruit single fruit weight main effect QTL site MSF.The molecular labeling
Genetic distance of the length of QS1122-135 between 135bp, with MSF is 4.37cM, which can be used as pear fruit
The label of single fruit weight；The forward primer sequence of the molecular labeling QS1122-135 is as shown in SEQ ID NO.1, reverse primer sequence
Column are as shown in SEQ ID NO.2.
According to the screening technique of the molecular labeling QS1122-135 of above-mentioned pear fruit single fruit weight main effect QTL site MSF, institute
It states main effect QTL site MSF to be located in the 11st linkage group of ' red eggplant pearss ' and ' evening show pears ' joint genetic map, the site contribution rate
It is 51.9%.
The molecular labeling QS1122-135 of above-mentioned pear fruit single fruit weight main effect QTL site MSF is in pears molecular breeding
Using.
The screening technique of the molecular labeling QS1122-135 of above-mentioned pear fruit single fruit weight main effect QTL site MSF is in pears point
Application in sub- breeding.
Beneficial effects of the present invention:
(1) two parents ' red eggplant pearss ' and ' evening show pears ' fruit single fruit Traits change is big, ' red eggplant pearss ' that the present invention uses
Mean fruit weight 200g, ' late show pears ' mean fruit weight 400g or so, filial generation fruit single fruit weight character has carried out very well
Separation, therefore, it can be ensured that pear fruit single fruit weight character QTL site detection accuracy and repeatability.
(2) present invention employs the sieves that SSR marker method carries out pear fruit single fruit weight character QTL positioning and linked marker
Choosing, SSR marker operated in molecular mark it is more easy, repeatability more preferably, technical requirements are low, accuracy is high, be
Codominance and anchor marker are more easier to apply in molecular mark.
(3) ' red eggplant pearss ' and ' late show pears ' hybrid Population F have been determined in the present invention1For the main effect of single plant pear fruit single fruit weight
QTL site, higher to the quantitative character contribution rate, the contribution rate in site is 51.9%, therefore, screens chain point based on this site
The accuracy that son label judge single fruit weight is preferable, especially single fruit weight greatly with it is minimum between identification accuracy it is higher.
(4) at present the universally recognized linked marker applied to molecule assisted Selection at a distance from objective trait need≤
5.0cM, the genetic distance between molecular labeling QS1122-135 of the invention and main effect QTL site are 4.37cM, chain very tight
It is close, effectively increase accuracy and pears large fruit kind using Marker-assisted selection pear fruit single fruit weight main effect QTL site
Breeding Efficiency, therefore, the pear fruit single fruit weight main effect QTL site and molecular labeling that the present invention develops have good using valence
Value, can accelerate the improvement to pear fruit single fruit weight character.
Detailed description of the invention
Fig. 1 is pears single fruit weight QTL site MSF in ' red eggplant pearss ' and ' evening show pears ' joint the 11st linkage group of genetic linkage maps
On position.
Wherein, what LG was represented is linkage group, and what the number after LG represented is linkage group number；The number of linkage group upper left side is
Genetic distance between label, unit cM, what right side indicated is the title of SSR molecular marker；Solid length on the right side of linkage group
Rectangular instruction QTL mapping section MSF.
The primer pair that Fig. 2 is molecular labeling QS1122-135 is to ' red eggplant pearss ' and ' late show pears ' hybrid Population F1Expand for PCR
The electrophoresis detection figure of increasing.Wherein, band, that is, QS1122-135 of arrow meaning, filial generation can amplify showing as greatly for this band
Fruit.
Fig. 3 is molecular labeling QS1122-135 in ' red eggplant pearss ' and ' evening show pears ' hybridization F1For the property of group's fruit single fruit weight
(each vertical bar band indicates 1 plant of F to the parting effect of shape in figure1For single plant, vertical bar band from left to right successively indicates fruit single fruit weight
The F arranged from small to large1For single plant).Wherein, vertical black band band indicates the F of no 135bp amplified band1Single plant；Vertical white band band
Indicate the F of 135bp band1Single plant；Vertical bar band among two black vertical lines indicates the F of single fruit weight type placed in the middle1For single plant.
Specific embodiment
Embodiment 1: the acquisition of pear fruit single fruit weight main effect QTL site MSF and its molecular labeling QS1122-135
(1) two Pear varieties ' red eggplant pearss ' (female parent) are utilized and ' evening show pears ' (male parent) cross combination, obtains 81 plants of F1Group
Body；
(2) two parents and its F are analyzed with SSR molecular marker method1Group, statistical analysis have polymorphism between parent
Hereditary form of the site in progeny population, be finally obtained 207 polymorphism mark sites.Then Jionmap4.0 is utilized
Mapping software carries out linkage analysis to polymorphism mark site, construct one ' red eggplant pearss ' comprising 187 SSR markers and
' evening show pears ' joint genetic linkage maps.Wherein, the test operation procedure reference Yomamoto T. etc. of SSR marker
The method of (Euphytica, 2002,124:129-137), the primer are synthesized by Jin Weizhi biotechnology company.SSR marker is used
The detection of 8% polyacrylamide highly basic argentation.
(3) by F1The separation type of each label of group according to " CP " mode be converted to lm × ll, nn × np, hk × hk,
5 kinds of ef × eg, ab × cd separation types, no amplified band or amplification is unsharp is denoted as "-" count and meet SSR marker
In F1Mask data in mapping population temporarily first removes inclined separation marking through Chi-square Test, chooses LOD=4.0-10.0, most
Big recombination fraction r=0.4 constructs genetic linkage maps with Kosambi mapping function, then the label separated partially is inserted into one by one
In map, does not change the positional relationship of other labels, otherwise remove.With the linkage inheritance that MapChart2.0 Software on Drawing is final
Map.
(4) in fructescence, ' red eggplant pearss ' and ' late show pears ' filial generation F are measured1For fruit single fruit weight.According to arrangement
The fruit single fruit weight data of investigation, using MapQTL5.0 software by permutation test (Permutation Test) (number >
1000) threshold value detects the marker site of LOD peak value using Interval mapping (Interval Mapping).
(5) it the result shows that, ' detects in ' red eggplant pearss ' and in the 11st linkage group of evening show pears ' joint genetic linkage maps
The QTL site MSF (Fig. 1) of single fruit weight, LOD value 8.78, contribution rate 51.90%.The QTL site is main effect QTL.
Wherein, SSR molecular marker method are as follows:
Using pears genomic DNA as template, PCR amplification is carried out by primer of the primer pair of molecular labeling QS1122-135, is expanded
Increase production object after the separation of 8% polyacrylamide gel electrophoresis, the band that length is 135bp can be amplified, shown and QTL
The chain molecular labeling of point MSF phase exists, and the linkage distance between MSF and molecular labeling QS1122-135 is 4.37cM.
The forward primer sequence (SEQ ID NO.1) of the primer pair of the molecular labeling QS1122-135 are as follows:
CGCCTTCTCGATCTCTTC；Reverse primer sequences (SEQ ID NO.2) are as follows: CGGTTGCCAGATTCCATA；
Specific PCR amplification system is (20 μ L): 10 × Buffer (Mg2+Plus) 2 μ L, 2.5mmol/L dNTP1.6 μ L,
0.6 μ L, 10mmol/L reverse primer of 10mmol/L forward primer, 0.6 μ L, Taq archaeal dna polymerase 0.5U, DNA profiling 50ng, it is double
Steaming water and adding to total volume is 20 μ L.
The response procedures of PCR amplification are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend
45s, totally 32 recycle；72 DEG C of extension 10min.
Embodiment 2: the molecular labeling QS1122-135 of the invention application test in pears molecular breeding
It (1) is female parent with ' red eggplant pearss ', ' evening show pears ' are that male parent constructs F1For hybrid Population, 81 plants of F are obtained1Group's single plant；
(2) to 81 plants of F obtained1Single plant carries out QS1122-135 label detection, method particularly includes: with pears genome
DNA is template, carries out PCR amplification, 8% polyacrylamide of amplified production by primer of the primer pair of molecular labeling QS1122-135
Amine gel electrophoresis separates (result such as Fig. 2)；Phase is determined whether there is according to the presence or absence of molecular labeling QS1122-135 after electrophoresis
The label answered illustrates that single plant fruit single fruit weight is high if there is label, low there is no illustrating, while with actually measured fruit
Real single fruit weight result is mutually verified with Markers for Detection result.
Wherein, the forward primer sequence (SEQ ID NO.1) of the primer pair of the molecular labeling QS1122-135 are as follows:
CGCCTTCTCGATCTCTTC；Reverse primer sequences (SEQ ID NO.2) are as follows: CGGTTGCCAGATTCCATA；
PCR amplification system is (20 μ L): 10 × Buffer (Mg2+Plus) 2 μ L, 2.5mmol/L dNTP, 1.6 μ L,
0.6 μ L, 10mmol/L reverse primer of 10mmol/L forward primer, 0.6 μ L, Taq archaeal dna polymerase 0.5U, DNA profiling 50ng, it is double
Steaming water and adding to total volume is 20 μ L；
The response procedures of PCR amplification are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend
45s, totally 32 recycle；72 DEG C of extension 10min.
As the result is shown: in ' red eggplant pearss ' and ' 81 plants of F that evening show pears ' hybridization obtains1In group, single fruit weight is at 45-175 grams
30 plants of small fruit type single plants in detect that length is the amplified band (QS1122-135) of 135bp except 5 plants of DNA cloning result
Outside, the band is not detected in other 25 plants of single plants；Single fruit weight is in 242-584 grams of 29 plants of large fruit single plants except 5 plants are not examined
Measure amplified band (QS1122-135) that length is 135bp outside, other 24 plants of single plants detect the band, detect accuracy
Reach 83.3%, therefore, detection accuracy rate is higher.It can thus be seen that predicting pear fruit list with molecular labeling QS1122-135
Fruit has preferable prediction effect again.
Claims (4)
1. a kind of pear fruit single fruit weight main effect QTL siteMSFMolecular labeling QS1122-135, which is characterized in that for expanding
The forward primer sequence of the molecular labeling QS1122-135 is as shown in SEQ ID NO.1, reverse primer sequences such as SEQ ID
Shown in NO.2；The molecular labeling QS1122-135 is to carry out PCR amplification as substrate using the genomic DNA of Pear varieties " evening show pears "
Resulting length be 135bp DNA fragmentation, molecular labeling QS1122-135 withMSFBetween genetic distance be 4.37cM.
2. a kind of for expanding the primer pair of molecular labeling QS1122-135 described in claim 1, which is characterized in that described to draw
The forward primer sequence of object pair is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2.
3. a kind of identification pear fruit single fruit weight main effect QTL siteMSFMolecule labelling method, which is characterized in that the method is
It is to draw with the primer pair as claimed in claim 2 for amplifier molecule label QS1122-135 using pears genomic DNA as template
Object carries out PCR amplification, and amplified production carries out electrophoresis detection, such as amplifies the DNA fragmentation that length is 135bp, then indicates pear fruit
Single fruit weight main effect QTL siteMSFIn the presence of.
4. molecular labeling QS1122-135 described in claim 1 answering in the relevant molecular breeding of pear fruit single fruit weight character
With.
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| CN106701911B (en) * | 2016-08-15 | 2020-11-27 | 浙江省农业科学院 | Molecular marker of pear PpAIV2 gene locus and screening method thereof |
| CN107164368B (en) * | 2017-07-19 | 2020-12-04 | 中国农业科学院郑州果树研究所 | Molecular Marker37152 of pear fruit heart size major QTL site and primer pair and application thereof |
| CN115261504A (en) * | 2022-08-11 | 2022-11-01 | 青岛农业大学 | Molecular marker associated with pear pollen abortion character and screening method thereof |
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| CN102140455A (en) * | 2011-01-29 | 2011-08-03 | 南京农业大学 | Molecular marker for single fruit weight main-effect quantitative trait loci (QTL) of Dangshansu pear fruit and application thereof |
| CN102154273A (en) * | 2011-01-29 | 2011-08-17 | 南京农业大学 | Molecular marker of single fruit weight major quantitative trait loci (QTL) of August red pyrus L. fruits and application of molecular marker |
| CN103740829A (en) * | 2014-01-14 | 2014-04-23 | 南京农业大学 | SNP marking method on main effect QTL site of longitudinal diameter of pear fruit and application thereof |
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|---|---|---|---|---|
| CN102140455A (en) * | 2011-01-29 | 2011-08-03 | 南京农业大学 | Molecular marker for single fruit weight main-effect quantitative trait loci (QTL) of Dangshansu pear fruit and application thereof |
| CN102154273A (en) * | 2011-01-29 | 2011-08-17 | 南京农业大学 | Molecular marker of single fruit weight major quantitative trait loci (QTL) of August red pyrus L. fruits and application of molecular marker |
| CN103740829A (en) * | 2014-01-14 | 2014-04-23 | 南京农业大学 | SNP marking method on main effect QTL site of longitudinal diameter of pear fruit and application thereof |
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| 梨分子遗传图谱的构建及其部分果实性状的定位分析;王文魁;《中国优秀硕士学位论文全文数据库 农业科技辑》;20150515(第05期);D048-85 * |
| 梨遗传连锁图谱的构建及部分果实性状QTL的定位;韩明丽等;《果树学报》;20100430;第27卷(第4期);496–503 * |
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