CN102140455A - Molecular marker for single fruit weight main-effect quantitative trait loci (QTL) of Dangshansu pear fruit and application thereof - Google Patents
Molecular marker for single fruit weight main-effect quantitative trait loci (QTL) of Dangshansu pear fruit and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of inheritance breeding, and relates to a molecular marker for a single fruit weight main-effect quantitative trait loci (QTL) of Dangshansu pear fruit and application thereof. A genetic linkage map of Dangshansu pear is constructed by utilizing sequence-related amplified polymorphism (SRAP), simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP), phenotypic identification on the single fruit weight of the fruit is combined, a group is analyzed through interval mapping, the presence of the main-effect QTL is detected on the 13th linkage group of the Dangshansu pear, and the 48.8 percent of single fruit weight can be interpreted. A molecule closest to the main-effect QTL Pfw-1 is marked as fe3em4-242p* and the distance is 0cM. The obtained molecular marker for the single fruit weight main-effect QTL has important theoretical and practical guidance significance for quickening the genetic improvement progress of pear varieties and improving breeding selection efficiency.
Description
Technical field
The invention belongs to molecular genetic breeding field, the molecule marker and the application thereof of ' Dangshan pear ' fruit single fruit weight main effect QTL are provided, can be used for the early molecule assisted Selection of pear fruit single fruit weight proterties, to improve breeding efficiency.
Background technology
Pears (Pyrus L.) originate in China, are the third-largest fruit tree species of China.Pears are high because of its fruits nutrition is worth, fragrant and sweet succulence, and liked by the human consumer.A operatic circle size is usually said single fruit weight, is one of important indicator of pear fruit exterior quality and commodity property, also is the important factor that constitutes pears variety yield and economic benefit, and therefore, the size of pear fruit single fruit weight is most important to the pears kind.The kind that cultivation has desirable single fruit weight has become one of important goal of pears breeding.
Because pears are the perennial woody fruit tree crop, the proterties relevant with fruit quality mostly is that the offspring shows as phenotypic variation widely in separation by controlled by multiple genes, easy quantitative character affected by environment, and the corresponding relation between its genotype and phenotype is difficult to determine.And traditional breeding method in the past is based on classical quantitative genetics theory, and a plurality of genes of a certain proterties of control are studied as a whole and selected, and improvement objective trait difficulty is come bigger more on existing basis.In recent years, appearance along with the developing rapidly of molecular marking technique, highdensity molecular genetic linkage map, and the quantitative character drawing method is constantly perfect, make quantitative trait locus (Quantitative trait loci, QTL) location has become reality, also lays a good foundation for the possibility, accuracy and the foresight that improve the selection of destination number proterties excellent genes type.Utilize molecule marker location quantitative character QTL, its essence is exactly the linkage relationship between analyzing molecules mark and the objective trait QTL, promptly utilize the molecule marker at known seat to locate the QTL at unknown seat,, determine the particular location of QTL by calculating the exchange rate between molecule marker and the QTL.Based on the relation between the amount of marker gene type separation that obtains and quantitative character, can directly hold the quantitative character gene, and exploitation can be applicable to molecular marker assisted selection (Molecular-assisted selection, MAS) technology of breeding, this has obtained successful Application on many important farm crop.
The QTL Position Research of at present relevant pears quantitative character still belongs to the starting stage, and existing research mainly is at some diseases or growth traits, and the QTL location and the Study on Molecular Marker of important character pears single fruit weight are not still carried out.Therefore, carry out the QTL location of pear fruit single fruit weight major gene, screen closely linked molecule marker, set up the early stage assisted Selection technical system of filial generation, for improving the operatic circle single fruit weight quality-improving efficient, shorten breeding process, saving production cost seems particularly important.
Summary of the invention
The object of the present invention is to provide the molecule marker of a kind of ' Dangshan pear ' fruit single fruit weight main effect QTL.
Another object of the present invention is to provide the molecule marker primer of a kind of ' Dangshan pear ' fruit single fruit weight main effect QTL.
Another object of the present invention is to provide the molecule marking method of a kind of ' Dangshan pear ' fruit single fruit weight main effect QTL.
Another purpose of the present invention is to provide the application of molecule marker in the pears molecular breeding of a kind of ' Dangshan pear ' fruit single fruit weight main effect QTL.The present invention locatees pear fruit single fruit weight main effect QTL, and provide and the closely linked molecule marker in QTL site, can predict the pear fruit single fruit weight by detecting these molecule markers, for realizing providing the technical support of molecule assisted Selection to the early stage evaluation and the screening of this proterties.
Purpose of the present invention is achieved through the following technical solutions:
The molecule marker of a kind of ' Dangshan pear ' fruit single fruit weight main effect QTL is to obtain through the following steps:
A) utilize pears kind ' BAYUEHONG ' pears and ' Dangshan pear ' hybridization to obtain F
1Offspring's individual plant;
B) analyze two parents and F thereof with SSR, SRAP and three kinds of molecule marking methods of AFLP
1Colony, statistical study is the hereditary form in progeny population in the site that has polymorphism between the parent.Obtain the polymorphism mark site, then it is carried out linkage analysis, make up the genetic linkage maps of ' Dangshan pear '.Can adopt the Joinmap3.0 mapping software that linkage analysis is carried out in the polymorphism mark site that is obtained.
C) to the F of ' BAYUEHONG ' and ' Dangshan pear ' filial generation
1The fruit single fruit weight of colony's individual plant is measured, and utilizes interval graphing method (can utilize the MapQTL5.0 mapping software), with F
1Molecule marker in the fruit single fruit weight of each individual plant of colony and ' Dangshan pear ' genetic linkage maps carries out chain and qtl analysis, with LOD value 〉=2.5 is standard, there is a QTL site greater than 2.5 explanations, on the 7th linkage group of maternal ' Dangshan pear ', detect the QTL site Pfw-1 of single fruit weight, its LOD value is 4.24, it is main effect QTL, a nearest SRAP molecule marker is fe3em4-242p*, size is 242bp, distance apart from Pfw-1 is 0cM, this mark can be used as the mark of pear fruit single fruit weight, and the SRAP primer of the molecule marker fe3em4-242p* that is screened is fe3:5 '-GTGCTTTACTGTTTGCTCC-3 ' and em4:5 '-GACTGCGTACGAATTTGA-3 '.
The molecule marker of above-mentioned ' Dangshan pear ' fruit single fruit weight main effect QTL, its described QTL site Pfw-1 is positioned on ' Dangshan pear ' the 13rd linkage group, and it explains 48.8% single fruit weight feature.
The SRAP molecule marker primer of a kind of ' Dangshan pear ' fruit single fruit weight main effect QTL is characterized in that this primer sequence is: fe3:5 '-GTGCTTTACTGTTTGCTCC-3 ' and em4:5 '-GACTGCGTACGAATTTGA-3 '.
The molecule marking method of a kind of ' Dangshan pear ' fruit single fruit weight main effect QTL, use the pears genomic dna, adopt SRAP primer fe3:5 '-GTGCTTTACTGTTTGCTCC-3 ' and em4:5 '-GACTGCGTACGAATTTGA-3 ' amplification, amplified production is electrophoretic separation on 8% non-denaturing polyacrylamide gel, be 242bp if obtain a molecule marker size, then show the molecule marker existence linked with the main effect QTL site of pear fruit single fruit weight, this QTL site Pfw-1 is positioned on ' Dangshan pear ' the 13rd linkage group, and it explains 48.8% single fruit weight feature.Its pcr amplification system is cumulative volume 20ul: wherein comprise 0.2mmolL
-1DNTP, 0.3 μ molL
-1The upstream and downstream primer, 2.0mmolL
-1MgCl
2, the rTaq enzyme of 1 unit, dna profiling 50ng, 1 * Buffer; The PCR response procedures is: 94 ℃ of 3min; 35 circulations: 94 ℃ of 40s, 55 ℃ of 50s, 72 ℃ of 1min; 72 ℃ of 10min, 10 ℃ of 10min.
The application of molecule marker in the pears molecular breeding of above-mentioned ' Dangshan pear ' fruit single fruit weight main effect QTL.
Above-mentioned application, its application method is: use the pears genomic dna, adopt SRAP primer fe3:5 '-GTGCTTTACTGTTTGCTCC-3 ' and em4:5 '-GACTGCGTACGAATTTGA-3 ' amplification, amplified production is after electrophoretic separation on 8% non-denaturing polyacrylamide gel, be 242bp if obtain a molecule marker size, then show the molecule marker existence linked with the main effect QTL site of pear fruit single fruit weight, measurable this pears kind fruit has lower single fruit weight.The pcr amplification system is cumulative volume 20ul: wherein comprise 0.2mmolL
-1DNTP, 0.3 μ molL
-1The upstream and downstream primer, 2.0mmolL
-1MgCl
2, the rTaq enzyme of 1 unit, dna profiling 50ng, 1 * Buffer; The PCR response procedures is: 94 ℃ of 3min; 35 circulations: 94 ℃ of 40s, 55 ℃ of 50s, 72 ℃ of 1min; 72 ℃ of 10min, 10 ℃ of 10min.
Beneficial effect of the present invention:
(1) the present invention has carried out the QTL location to the quantitative trait locus of decision ' Dangshan pear ' fruit single fruit weight first, and this has established important basis and necessary precondition for realizing based on the controlled by multiple genes character improvement of molecular breeding.
(2) determined the main effect QTL site of ' Dangshan pear ' fruit single fruit weight among the present invention, higher to the contribution rate of this quantitative character decision, the contribution rate in site is 48.8%.Therefore, the accuracy of objective trait being judged based on this site screening linkage molecule mark improves greatly.
(3) generally believe the distance need≤5cM of the linked marker that can be applicable to the molecule assisted Selection and objective trait at present, be 0cM with the chain marking path in QTL site among the present invention, show as close linkage with target site, this is significant for the accuracy and the efficient that improve molecular marker assisted selection.Therefore, above mark has excellent application value, can accelerate the progress to pear fruit single fruit weight character improvement.
Description of drawings
Fig. 1 is the position of ' Dangshan pear ' single fruit weight QTL site Pfw-1 on BYH13.
What BYH represented is ' Dangshan pear ' linkage group, the digitized representation linkage group number behind the BYH.
The numeral of linkage group upper left side is the genetic distance between the mark, and unit is cM, and what the right side was then represented is the title of molecule marker.Have 4 kinds of molecule markers, " E-M-" is the AFLP mark, and E and M refer to that respectively restriction enzyme Eco I and Mse I enzyme cut, and numeral thereafter is the numbering of this mark polymorphic bands; Other is not sterile S site of SRAP, SSR mark and selfing.
The solid rectangle indication QTL on the linkage group right side interval Pfw-1 that maps.
Fig. 2 is that SRAP combination of primers fe3em4 detects figure to ' BAYUEHONG ' pears and ' Dangshan pear ' F1 offspring pcr amplification result at native polyacrylamide gel electrophoresis.
The band of arrow indication is fe3em4-242p*, and M is pBR322DNA/Msp I ladder.
Embodiment
Embodiment 1:
A) utilize two pears kinds ' BAYUEHONG ' pears of China and ' Dangshan pear ' cross combination, obtain its 97 strain F
1Colony.
B) analyze two parents and F thereof with SSR (Simple sequence repeat), SRAP (Sequence related amplified polymorphism) and three kinds of molecule marking methods of AFLP (Amplified fragment length polymorphism)
1Colony, statistical study is the hereditary form in progeny population in the site that has polymorphism between the parent.Totally 732 polymorphism mark sites such as 23 SSRs, S gene locus, 226 SRAPs and 482 AFLPs have finally been obtained, utilize the Jionmap3.0 mapping software that it is carried out linkage analysis then, made up a genetic linkage maps that comprises ' Dangshan pear ' of 240 polymorphism marks altogether.
Utilize SSR, SRAP, AFLP molecule marking method, ' BAYUEHONG ' pears and ' Dangshan pear ' and offspring analyzed, and statistical study in the site that has polymorphism between the parent hereditary form in progeny population.
(Euphytica, 2002, methods 124:129-137) such as the experimental implementation procedure reference Yomamoto T. of SSR mark; (Theor Appl Genet, 2001, methods 103:455-461) such as the experimental implementation procedure reference Li of SRAP mark; The AFLP mark uses Eco I and two kinds of restriction endonucleases of Mes I respectively, (Nucleic Acids Res, 1995, methods 23:4407-4414) such as concrete experimental implementation procedure reference Vos.The primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.S SR and SRAP mark detect with 8% non-denaturing polyacrylamide gel highly basic argentation, and the AFLP mark dyes detection with 6% denaturing polyacrylamide gel silver.
The polymorphic bands of clear appearance on SSR, SRAP, the AFLP electrophoretogram is designated as " 1 ", do not have band and be designated as " 0 ", unclear band or disappearance are designated as "-", calculate the molecular weight of each polymorphic bands according to the Marker (100bp DNA Ladder) of known molecular amount.To separate band and press the parental source classification, determine its source and hereditary form.Utilize χ
2Each mark of test Analysis separates the Mendelian's segregation ratio that whether meets 3: 1 or 1: 1, isolatingly partially marks with " * " at the mark end.
C) use the Joinmap3.0 analysis software to make up the molecular genetic linkage collection of illustrative plates of ' Dangshan pear '.
With b) the polymorphism mark site that obtains in the step imports Joinmap3.0 by the form that is suitable for cp colony composition in the Joinmap3.0 analysis software, get rid of too much site of missing data and remarkable isolating partially site, with LOD=4.0～7.0, recombination fraction=0.4, select the Kosambi mapping function to make up genetic linkage map (Van Ooijen, 2001).
D) to this F
1The fruit single fruit weight of colony's individual plant is measured.The phenotypic number of single fruit weight and the associated documents of ' Dangshan pear ' linkage map are imported the MapQTL5.0 mapping software, select interval graphing method, with LOD value 〉=2.5 is standard, and the single fruit weight of pears is carried out qtl analysis and location on the linkage map of ' Dangshan pear '.
The result shows, detect the QTL site Pfw-1 (Fig. 1) of single fruit weight on the 7th linkage group of ' Dangshan pear ', the LOD value is 4.24, is the main effect QTL site, with the linkage distance of nearest SRAP molecule marker fe3em4-242p* be 1cM, be 48.8% to the contribution rate of this proterties.The distance of this QTL site mark nearest with it is less than the desired ultimate range of linked marker that can be applicable to the molecule assisted Selection and objective trait.
Wherein the SRAP molecule marking method is, is template with pears DNA, adopts the SRAP primer:
fe3：5′-GTGCTTTACTGTTTGCTCC-3′
em4：5′-GACTGCGTACGAATTTGA-3′
The pcr amplification system that amplification condition: SRAP analyzes is cumulative volume 20ul: wherein comprise 0.2mmolL
-1DNTP, 0.3 μ molL
-1The upstream and downstream primer, 2.0mmolL
-1MgCl
2, the rTaq enzyme of 1 unit, dna profiling 50ng, 1 * Buffer.The PCR response procedures is: 94 ℃ of 3min; 35 circulations: 94 ℃ of 40s, 55 ℃ of 50s, 72 ℃ of 1min; 72 ℃ of 10min, 10 ℃ of 10min;
Amplified production is after electrophoretic separation on 8% non-denaturing polyacrylamide gel, can amplify 14 polymorphic bandses, wherein one of the 242bp size is exactly target stripe fe3em4-242p*, then show the molecule marker existence linked with QTL site Pfw-1 (Fig. 1), the linkage distance of this QTL site and SRAP molecule marker fe3em4-242p* is 0cM, is 48.8% to the contribution rate of this proterties.
Embodiment 2
Utilization screen with the linked molecule marker of ' Dangshan pear ' fruit single fruit weight main effect QTL to the 97 strain Fs of ' BAYUEHONG ' pears with ' Dangshan pear '
1Filial generation carries out Preliminary detection, to detect the practical value of this method in pear fruit single fruit weight molecular marker assisted selection.Carry out the PCR reaction with this 97 strain offspring's genomic dna and SRAP primer fe3 and em4, the pcr amplification system that SRAP analyzes is cumulative volume 20ul: wherein comprise 0.2mmolL
-1DNTP, 0.3 μ molL
-1The upstream and downstream primer, 2.0mmolL
-1MgCl
2, the rTaq enzyme of 1 unit, dna profiling 50ng, 1 * Buffer.The PCR response procedures is: 94 ℃ of 3min; 35 circulations: 94 ℃ of 40s, 55 ℃ of 50s, 72 ℃ of 1min; 72 ℃ of 10min, 10 ℃ of 10min.The pcr amplification result is at 8% native polyacrylamide gel electrophoresis (result such as Fig. 2), determine whether to exist corresponding mark according to having or not of fe3em4-242p* band behind the electrophoresis, if there is mark, the fruit single fruit weight that this plant is described is low, do not exist then explanation high, verify with actual fruit single fruit weight result who records and Markers for Detection result simultaneously.
The result shows, at the 97 strain Fs of ' BAYUEHONG ' pears with ' Dangshan pear '
1In the filial generation, the fe3em4-242p* band appears in the DNA cloning result who has 80 strains, and the fruit single fruit weight that 35 strains are arranged in the middle of this 80 strain is less than 160g, and the fruit single fruit weight mean value of all these 80 strains is 176.46g; The fe3em4-242p* band does not appear in the DNA cloning result who has 12 strain offsprings, and wherein the single fruit weight of 10 strains is greater than 160g, and the mean value of the fruit single fruit weight of all these 12 strains is 184.06g; 5 individual plant band disappearances.With fe3em4-242p* prediction single fruit weight the better prediction effect is arranged.
Claims (8)
1. the molecule marker of ' Dangshan pear ' fruit single fruit weight main effect QTL is characterized in that it being to obtain through the following steps:
A) utilize pears kind ' BAYUEHONG ' pears and ' Dangshan pear ' hybridization to obtain F
1Offspring's individual plant;
B) analyze two parents and F thereof with SSR, SRAP and three kinds of molecule marking methods of AFLP
1Colony, statistical study is the hereditary form in progeny population in the site that has polymorphism between the parent.Obtain the polymorphism mark site, then it is carried out linkage analysis, make up the genetic linkage maps of ' Dangshan pear '.
C) to the F of ' BAYUEHONG ' pears and ' Dangshan pear ' filial generation
1The fruit single fruit weight of colony's individual plant is measured, and utilizes interval graphing method, with F
1Molecule marker in the fruit single fruit weight of each individual plant of colony and ' Dangshan pear ' genetic linkage maps carries out chain and qtl analysis, with LOD value 〉=2.5 is standard, there is a QTL site greater than 2.5 explanations, on the 7th linkage group of maternal ' Dangshan pear ', detect the QTL site Pfw-1 of single fruit weight, its LOD value is 4.24, it is main effect QTL, a nearest SRAP molecule marker is fe3em4-242p*, size is 242bp, distance apart from Pfw-1 is 0cM, this mark can be used as the mark of pear fruit single fruit weight, and the SRAP primer of the molecule marker fe3em4-242p* that is screened is fe3:5 '-GTGCTTTACTGTTTGCTCC-3 ' and em4:5 '-GACTGCGTACGAATTTGA-3 '.
2. the molecule marker of ' Dangshan pear ' according to claim 1 fruit single fruit weight main effect QTL, it is characterized in that: described QTL site Pfw-1 is positioned on ' Dangshan pear ' the 13rd linkage group, and it explains 48.8% single fruit weight feature.
3. the SRAP molecule marker primer of ' Dangshan pear ' fruit single fruit weight main effect QTL is characterized in that this primer sequence is: fe3:5 '-GTGCTTTACTGTTTGCTCC-3 ' and em4:5 '-GACTGCGTACGAATTTGA-3 '.
4. the molecule marking method of ' Dangshan pear ' fruit single fruit weight main effect QTL, it is characterized in that: use the pears genomic dna, adopt SRAP primer fe3:5 '-GTGCTTTACTGTTTGCTCC-3 ' and em4:5 '-GACTGCGTACGAATTTGA-3 ' amplification, amplified production is electrophoretic separation on 8% non-denaturing polyacrylamide gel, be 242bp if obtain a molecule marker size, then show the molecule marker existence linked with the main effect QTL site Pfw-1 of pear fruit single fruit weight, Pfw-1 is positioned on ' Dangshan pear ' the 13rd linkage group, and it explains 48.8% single fruit weight feature.
5. the molecule marking method of ' Dangshan pear ' according to claim 4 fruit single fruit weight main effect QTL is characterized in that the pcr amplification system is cumulative volume 20ul: wherein comprise 0.2mmolL
-1DNTP, 0.3 μ molL
-1The upstream and downstream primer, 2.0mmolL
-1MgCl
2, the rTaq enzyme of 1 unit, dna profiling 50ng, 1 * Buffer; The PCR response procedures is: 94 ℃ of 3min; 35 circulations: 94 ℃ of 40s, 55 ℃ of 50s, 72 ℃ of 1min; 72 ℃ of 10min, 10 ℃ of 10min.
6. the application of molecule marker in the pears molecular breeding of any described ' Dangshan pear ' fruit single fruit weight main effect QTL in the claim 1 or 2.
7. according to the described application of claim 6, it is characterized in that: use the pears genomic dna, adopt SRAP primer fe3:5 '-GTGCTTTACTGTTTGCTCC-3 ' and em4:5 '-GACTGCGTACGAATTTGA-3 ' amplification, amplified production is after electrophoretic separation on 8% non-denaturing polyacrylamide gel, be 242bp if obtain a molecule marker size, then show the molecule marker existence linked with the main effect QTL site of pear fruit single fruit weight, measurable this pears kind fruit has lower single fruit weight.
8. according to the described application of claim 7, it is characterized in that: the pcr amplification system is cumulative volume 20ul: wherein comprise 0.2mmolL
-1DNTP, 0.3 μ molL
-1The upstream and downstream primer, 2.0mmolL
-1MgCl
2, the rTaq enzyme of 1 unit, dna profiling 50ng, 1 * Buffer; The PCR response procedures is: 94 ℃ of 3min; 35 circulations: 94 ℃ of 40s, 55 ℃ of 50s, 72 ℃ of 1min; 72 ℃ of 10min, 10 ℃ of 10min.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740828A (en) * | 2014-01-14 | 2014-04-23 | 南京农业大学 | SNP (Single Nucleotide Polymorphism) molecular marking method for major QTL (Quantitative Trait Locus) in fruit stem length of pear fruit and application thereof |
| 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 |
| CN103773864A (en) * | 2014-01-14 | 2014-05-07 | 南京农业大学 | SNP (Single Nucleotide Polymorphism) marking method of pear fruit transverse diameter main-effect QTL (Quantitative Trait Locus) and application thereof |
| CN105296472A (en) * | 2015-08-12 | 2016-02-03 | 浙江省农业科学院 | Molecular marker of peel full-brown trait gene locus of Chinese 'Qingxiang'-variety pears and screening method of molecular marker |
| CN105803083A (en) * | 2016-04-26 | 2016-07-27 | 中国农业科学院郑州果树研究所 | Molecular marker of pear fruit single fruit weight main-effect QTL (quantitative trait loci) site MSF, and application thereof |
| CN106167798A (en) * | 2016-08-30 | 2016-11-30 | 中国农业科学院郑州果树研究所 | The molecular marker of Fructus Vitis viniferae anti-anthrax main effect QTL and application |
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