CN102115791B - Molecular marker of content of soluble sugar in August red pear fruit - Google Patents
Molecular marker of content of soluble sugar in August red pear fruit Download PDFInfo
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- CN102115791B CN102115791B CN201010596658A CN201010596658A CN102115791B CN 102115791 B CN102115791 B CN 102115791B CN 201010596658 A CN201010596658 A CN 201010596658A CN 201010596658 A CN201010596658 A CN 201010596658A CN 102115791 B CN102115791 B CN 102115791B
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Abstract
The invention relates to a molecular marker for the content of soluble sugar in an 'August red' pear fruit, which belongs to the field of inheritance breeding. A genetic linkage map of the 'August red' pear fruit is constructed by using SRAP (Sequence-Related Amplified Polymorphism), SSR (Simple Sequence Repeats) and AFLP (Amplified Fragment Length Polymorphism), a colony is analyzed by using an interval mapping method in combination with phenotypic identification of the content of the soluble sugar in the fruit, and the existence of major QTL (Quantitative Trait Loci) on a seventh linkage group of the 'August red' pear fruit is detected, so that the characteristic of the soluble sugar content of 12.4 percent can be explained. A marker which is closest to the locus Psg-1 of the major QTL is me6pm19-1300, wherein the distance is 0 centimeter. The obtained major QTL molecular marker of the soluble sugar plays an important theoretical and practical guide role in accelerating the genetic improvement process of a pear variety and improving the breeding selection efficiency.
Description
One, technical field
The invention provides the molecule marker of BAYUEHONG pear fruit soluble sugar content, belong to molecular genetic breeding field, can be used for the early molecule assisted Selection of pear fruit soluble sugar content proterties, to improve breeding efficiency.
Two, background technology
Pears (
PyrusL.) originating in China, is 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.Soluble sugar content is one of important factor that influences the operatic circle quality and flavor; It is the primary factor of decision sugariness mouthfeel; And China human consumer generally likes the pears of high sugariness mouthfeel, and therefore cultivating the high kind of fruit soluble sugar content 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 by controlled by multiple genes, is prone to quantitative character affected by environment, and the offspring shows as phenotypic variation widely in separation, and the corresponding relation between its genotype and phenotype is difficult to confirm.And that traditional breeding method in the past is based on classical quantitative genetics is theoretical, studies a plurality of genes of a certain proterties of control as a whole and selects, and on existing basis, improves the objective trait difficulty and comes bigger more.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 that (Quantitative trait loci, QTL) location has become reality to quantitative trait locus, 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,, confirm the particular location of QTL through 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 to some diseases or growth traits, and the QTL location and the Study on Molecular Marker of important quality trait pears soluble sugar content are not still carried out.Therefore, carry out the QTL location of pear fruit soluble sugar content major gene, screen closely linked molecule marker; Set up the early stage assisted Selection technical system of filial generation; For improving the operatic circle soluble sugar content quality-improving efficient, shorten breeding process, saving production cost seems particularly important.
Three, summary of the invention
Technical problem
The objective of the invention is to locate pear fruit soluble sugar content QTL; And provide and the closely linked molecule marker in QTL site; Can predict the pear fruit soluble sugar content through detecting these molecule markers, for realizing the technical support of molecule assisted Selection being provided to the early stage evaluation and the screening of this proterties.
Technical scheme
The chain molecule marker of a kind of pear fruit soluble sugar content QTL is to obtain through the following step:
A) utilize pears kind ' BAYUEHONG ' and ' Dangshan pear ' hybridization to obtain F
1Offspring's individual plant;
B) adopting SRAP primer me6:5'-TGAGTCCAAACCGGTAG-3' and pm19:5'-CTTCAAGCGGTGCATTCC-3' is that template is carried out pcr amplification with the pears genomic dna; Amplified production is electrophoretic separation on 8% non-denaturing polyacrylamide gel; ' BAYUEHONG ' and ' Dangshan pear ' and filial generation genomic dna thereof are analyzed; Statistical study is the hereditary form in progeny population in the site that has polymorphum between the parent; The polymorphism mark site that obtains is imported the Joinmap3.0 mapping software, make up the genetic linkage maps of ' BAYUEHONG ' pears;
C) to the F of ' BAYUEHONG ' and ' Dangshan pear ' filial generation
1The fruit soluble sugar content of colony's individual plant is measured, and utilizes the interval graphing method of MapQTL5.0 mapping software, with F
1Molecule marker in the fruit soluble sugar content of each individual plant of colony and ' BAYUEHONG ' pears genetic linkage maps carries out chain and qtl analysis; With LOD value>=2.5 is standard, has a QTL site greater than 2.5 explanations, detects the QTL site Psg-1 that on the 4th linkage group of maternal ' BAYUEHONG ', detects soluble sugar content; Its LOD value is 2.56; Be main effect QTL, a nearest molecule marker is me6pm19-1300, and size is 150bp; Distance apart from Psg-1 is 0cM, and this mark can be used as the mark of pear fruit soluble sugar content.Said QTL site Psg-1 is positioned on ' BAYUEHONG ' pears the 7th linkage group, and it explains 12.4% soluble sugar content characteristic.
The application of said molecule marker in the pears molecular breeding; It is characterized in that: use the pears genomic dna; Adopt SRAP primer me6:5'-TGAGTCCAAACCGGTAG-3' and pm19:5'-CTTCAAGCGGTGCATTCC-3' to carry out pcr amplification; Amplified production is 1300bp if obtain a molecule marker size after electrophoretic separation on 8% non-denaturing polyacrylamide gel, show that then the main effect QTL site of pear fruit soluble sugar content exists; This QTL site Psg-1 is positioned on ' BAYUEHONG ' pears the 4th linkage group, and it explains 12.4% soluble sugar content characteristic.
Beneficial effect
(1) the present invention has carried out the QTL location to the quantitative trait locus of decision ' BAYUEHONG ' pear fruit soluble sugar content 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) confirmed the QTL site of ' BAYUEHONG ' fruit soluble sugar content among the present invention, higher to the contribution rate of this quantitative character decision, the contribution rate in site is 12.4%.Therefore, the accuracy of based on this site screening linkage molecule mark objective trait being judged 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 soluble sugar character improvement.
Four, description of drawings
Fig. 1 is the position of ' BAYUEHONG ' soluble sugar content QTL site Psg-1 on BYH4.
What BYH represented is ' BAYUEHONG ' 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 restriction enzyme respectively
EcoI with
MseThe I enzyme is 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 Psg-1 that maps.The graphic representation on the right is the LOD distribution plan of QTL, and dotted line is 2.5 threshold values.
Fig. 2 is that SRAP combination of primers me6pm19 detects figure at native polyacrylamide gel electrophoresis.The band of arrow indication is me6pm19-1300, and M is pBR322 DNA/ Msp I ladder.
Five, embodiment
Embodiment 1:
A) utilize two pears kinds ' BAYUEHONG ' and ' Dangshan pear ' cross combination of China, 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 polymorphum 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 ' BAYUEHONG ' pears of 240 polymorphism marks altogether.
Utilize SSR, SRAP, AFLP molecule marking method, ' BAYUEHONG ' and ' Dangshan pear ' and offspring analyzed, and statistical study in the site that has polymorphum 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 respectively
EcoI with
MesTwo kinds of restriction endonucleases of I, (Nucleic Acids Res, 1995, methods 23:4407-4414) such as concrete experimental implementation procedure reference Vos.SSR 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, confirm 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, isolating partially " * " mark of using at the mark end.
C) make up the molecular genetic linkage collection of illustrative plates of ' BAYUEHONG ' pears with the Joinmap3.0 analysis software.
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 selects the Kosambi mapping function to make up genetic linkage map (Van Ooijen, 2001).
D) to this F
1The fruit soluble sugar content of colony's individual plant is measured, and measuring method adopts improvement DNS method to measure (Feng Ji etc., 1989).The phenotypic number of soluble sugar content and the associated documents of ' BAYUEHONG ' linkage map are imported the MapQTL5.0 mapping software; Select interval graphing method; With LOD value >=2.5 is standard, and the soluble sugar content of pears is carried out qtl analysis and location on the linkage map of ' BAYUEHONG '.
The result shows, on the 7th linkage group of ' BAYUEHONG ', detects the QTL site Psg-1 (Fig. 1) of soluble sugar content, and the LOD value is 2.56, with the linkage distance of nearest SRAP molecule marker me6pm19-1300 be 1cM, be 12.4% 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 does, is template with pears DNA, adopts the SRAP primer:
me6：?5'-TGAGTCCAAACCGGTAG-3'
pm19：5'-?CTTCAAGCGGTGCATTCC-3'
The pcr amplification system that amplification condition: SRAP analyzes is TV 20ul: wherein comprise 0.2mmolL-1 dNTP, 0.3 μ molL-1 upstream and downstream primer, 2.0mmolL-1 MgCl2, 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 1300bp size is exactly target stripe me6pm19-1300; Show that then QTL site Psg-1 (Fig. 1) exists, with the linkage distance of SRAP molecule marker me6pm19-1300 be 0cM, be 12.4% to the contribution rate of this proterties.
Utilization screen with the linked molecule marker of ' BAYUEHONG ' fruit soluble sugar content main effect QTL to ' BAYUEHONG ' 97 strain F with ' Dangshan pear '
1Filial generation carries out Preliminary detection, to detect the practical value of this method in pear fruit soluble sugar content molecular marker assisted selection.This 97 strain of usefulness offspring's genomic dna and SRAP primer me6 and pm19 carry out the PCR reaction; Determine whether to exist corresponding mark according to having or not of me6pm19-1300 band behind the electrophoresis; If there is mark; The fruit soluble sugar content that this plant is described is high, does not exist then to explain lowly, verifies with actual fruit soluble sugar content result who records and Markers for Detection result simultaneously.
The result shows; In the 97 strain F1 filial generations of ' BAYUEHONG ' and ' Dangshan pear '; The me6pm19-1300 band appears in the DNA cloning result who has 51 strains; The fruit soluble sugar content that 37 strains are arranged in the middle of this 51 strain is less than 9.0%, and the fruit soluble sugar content MV of all these 51 strains is 8.3 %; The me6pm19-1300 band does not appear in the DNA cloning result who has 38 strain offsprings, and wherein the soluble sugar content of 16 strains is greater than 9.0%, and the MV of the fruit soluble sugar content of all these 38 strains is 9.2%; 8 individual plant band disappearances.With me6pm19-1300 prediction soluble sugar content the better prediction effect is arranged.
Claims (2)
1. the molecule marker of ' BAYUEHONG ' pear fruit soluble sugar content is to obtain through the following step:
A) utilize pears kind ' BAYUEHONG ' and ' Dangshan pear ' hybridization to obtain F
1Offspring's individual plant;
B) adopting SRAP primer me6:5'-TGAGTCCAAACCGGTAG-3' and pm19:5'-CTTCAAGCGGTGCATTCC-3' is that template is carried out pcr amplification with the pears genomic dna; Amplified production is electrophoretic separation on 8% non-denaturing polyacrylamide gel; ' BAYUEHONG ' and ' Dangshan pear ' and filial generation genomic dna thereof are analyzed; Statistical study is the hereditary form in progeny population in the site that has polymorphum between the parent; The polymorphism mark site that obtains is imported the Joinmap3.0 mapping software, make up the genetic linkage maps of ' BAYUEHONG ' pears;
C) to the F of ' BAYUEHONG ' and ' Dangshan pear ' filial generation
1The fruit soluble sugar content of colony's individual plant is measured, and utilizes the interval graphing method of MapQTL5.0 mapping software, with F
1Molecule marker in the fruit soluble sugar content of each individual plant of colony and ' BAYUEHONG ' pears 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; Detect a QTL site that on the 7th linkage group of maternal ' BAYUEHONG ', detects soluble sugar content, its LOD value is 2.56, and a nearest molecule marker size is 1300bp; Distance apart from this QTL site is 0cM, and this is labeled as the mark of pear fruit soluble sugar content.
2. molecule marker according to claim 1 is characterized in that: said QTL site is positioned on ' BAYUEHONG ' pears the 7th linkage group, and 12.4% soluble sugar content characteristic is explained in this site.
1. claim 1 or 2 application of said molecule marker in the pears molecular breeding.
4. according to the said application of claim 3; It is characterized in that: use the pears genomic dna; Adopt SRAP primer me6:5'-TGAGTCCAAACCGGTAG-3' and pm19:5'-CTTCAAGCGGTGCATTCC-3' amplification; Amplified production is 1300bp if obtain a molecule marker size after electrophoretic separation on 8% non-denaturing polyacrylamide gel, show that then the main effect QTL site of pear fruit soluble sugar content exists; This QTL site is positioned on ' BAYUEHONG ' pears the 7th linkage group, and it explains 12.4% soluble sugar content characteristic.
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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 |
| CN116179746B (en) * | 2022-11-29 | 2024-06-28 | 上海市农业科学院 | Primer group of KASP molecular marker for identifying early sugar accumulation amount of pears and application of primer group |
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| Candidate genes and QTLs for sugar and organic acid content in peach [Prunus persica (L.) Batsch];C. Etienne et al.;《Theoretical and Applied Genetics》;20020731;第105卷(第1期);145-159 * |
| Genetic Analysis, Expression and Molecular Characterization of BoGSL-ELONG, a Major Gene Involved in the Aliphatic Glucosinolate Pathway of Brassica Species;Genyi Li and Carlos F. Quiros;《Genetics》;20021201;第162卷(第4期);1937-1943 * |
| Genyi Li and Carlos F. Quiros.Genetic Analysis, Expression and Molecular Characterization of BoGSL-ELONG, a Major Gene Involved in the Aliphatic Glucosinolate Pathway of Brassica Species.《Genetics》.2002,第162卷(第4期),1937-1943. |
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