CN105296472A - Molecular marker of peel full-brown trait gene locus of Chinese 'Qingxiang'-variety pears and screening method of molecular marker - Google Patents
Molecular marker of peel full-brown trait gene locus of Chinese 'Qingxiang'-variety pears and screening method of molecular marker Download PDFInfo
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Abstract
The invention relates to the field of molecular genetics and discloses a molecular marker of a peel full-brown trait gene locus of Chinese 'Qingxiang'-variety pears and a screening method of the molecular marker of the peel full-brown trait gene locus of the Chinese 'Qingxiang'-variety pears. A peel full-brown trait and a genotype of each F1 individual plant obtained by hybridization of Chinese 'Qingxiang'-variety pears and Chinese 'Guancui'-variety pears are subjected to genetic linkage analysis to obtain the molecular marker of the peel full-brown trait gene locus Rus.zaas-8 of the Chinese 'Qingxiang'-variety pears. Whether the gene locus is contained in the 'Qingxiang' variety or other derived varieties (strains) or not is detected by means of the molecular marker of the peel full-brown trait gene locus, peel colors of fruits can be predicated, and accordingly selecting efficiency of fruit peel colors of Chinese pears can be greatly improved.
Description
Technical field
The present invention relates to molecular genetics field, relate to the molecule marker in the complete brown character gene site of a kind of Sand Pear ' delicate fragrance ' pericarp, particularly relate to the screening method of the molecule marker in the complete brown character gene site of this kind of Sand Pear ' delicate fragrance ' pericarp.
Background technology
Chinese pear is the important Southern Fruit Trees of China, belongs to perennial woody plant, and the virgin phase is longer.Pericarp color is one of most important peel character, and fruit colour is consistent, be the important indicator of best buy pears without fruit russeting.Pericarp brown belongs to fruit properties, and the conventional hybridization breeding selection cycle is long, difficulty is large.Preliminary evaluation could be carried out after general needs 3 ~ 5 annual bearing.Carry out assistant breeding by location pericarp brown proterties gene locus can effectively address this problem.
Genetic research shows, the complete brown proterties of the operatic circle skin controls by single or several major gene loci, and its gene locus controlling the complete brown proterties of pericarp of different materials may there are differences.
' delicate fragrance ' is by ' new millennium ' (♀) and ' three flower pears ' (♂) selection cross forms, pericarp brown proterties is from ' three flower pears '.' three flower pears ' belong to the excellent local variety of China, cultivation history is long, best in quality, is one of important parent of China's Chinese pear breeding.' delicate fragrance ' (♀) carries out hybridization obtain in Hybrids F1 with ' emerald green be preced with ' (♂), and pericarp is the brown segregation ratio meeting 1:1 with the brown individual plant of non-fully than through Chi-square statistic entirely, and the complete brown proterties of the pericarp that this shows ' delicate fragrance ' is Dominant gene.
Summary of the invention
The present invention is directed to existing deficiency, disclose the molecule marker in the complete brown character gene site of a kind of Sand Pear delicate fragrance pericarp, also disclose the screening method of the molecule marker in the complete brown character gene site of this kind of Sand Pear ' delicate fragrance ' pericarp.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals.
The molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and molecule marker closely linked with it is Zaasp867 primer, and its sequence is:
Left end primer sequence CCACCTCACCTTATCCCTCA,
Right-hand member primer sequence GCGTTTGCTCCTCGTACTTC.
The amplification complete brown pericarp kind of Chinese pear or breeding material DNA, the amplified fragments obtained is 145bp, be the molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, this gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, utilize PCR to hybridize with ' kingfisher is preced with ' F1 generation obtained to ' delicate fragrance ' to detect, the complete brown individual plant accuracy rate 96.3% of pericarp, utilizing Excel software to record the incoherent probability P value of complete brown proterties with pericarp is 0.004.
The molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, gene locus is positioned at Sand Pear delicate fragrance No. 8 karyomit(e)s, and molecule marker closely linked with it is Zaasp868 primer, and its sequence is:
Left end primer sequence AAAAAGTGATGTGTGGATCGC,
Right-hand member primer sequence CTGGTGACTTGCAAGCTAGG.
The amplification complete brown pericarp kind of Chinese pear or breeding material DNA, the amplified fragments obtained is 129bp, be the molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, this gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, utilize PCR to hybridize with ' kingfisher is preced with ' F1 generation obtained to ' delicate fragrance ' to detect, the complete brown individual plant accuracy rate 97.0% of pericarp, utilizing Excel software to record the incoherent probability P value of complete brown proterties with pericarp is 0.003.
The molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and molecule marker closely linked with it is Zaasp873 primer, and its sequence is:
Left end primer sequence CGCTTTCGAACACAAAACAA,
Right-hand member primer sequence CTGGCACTTTGAATTCGCTT.
The amplification complete brown pericarp kind of Chinese pear or breeding material DNA, the amplified fragments obtained is 242bp, be the molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, this gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, utilize PCR to hybridize with ' kingfisher is preced with ' F1 generation obtained to ' delicate fragrance ' to detect, the complete brown individual plant accuracy rate 97.0% of pericarp, utilizing Excel software to record the incoherent probability P value of complete brown proterties with pericarp is 0.003.
The molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and molecule marker closely linked with it is Zaasp876 primer, and its sequence is:
Left end primer sequence CGCTTTCGAACACAAAACAA,
Right-hand member primer sequence CTGGCACTTTGAATTCGCTT.
The amplification complete brown pericarp kind of Chinese pear or breeding material DNA, the amplified fragments obtained is 241bp, be the molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, this gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, utilize PCR to hybridize with ' kingfisher is preced with ' F1 generation obtained to ' delicate fragrance ' to detect, the complete brown individual plant accuracy rate 97.0% of pericarp, utilizing Excel software to record the incoherent probability P value of complete brown proterties with pericarp is 0.003.
As preferably, gene locus is Rus.zaas-8.
The screening method of the molecule marker in the complete brown character gene site of above-mentioned Chinese pear ' delicate fragrance ' pericarp, comprises following methods:
A. with Sand Pear ' emerald green hat ' for male parent and Sand Pear ' delicate fragrance ' are hybridized, obtain Hybrids F1;
B. the DNA of each individual plant of F1 colony is extracted by CTAB method, the mark SSR of simple sequence repeats is adopted to carry out polymorphism primer screening to two parents, increased by polymerase chain reaction, amplified production is that the polyacrylamide gel of 0.08g/ml carries out electrophoretic separation analysis in density, according to molecular marker screening result, filter out between parent and have polymorphic primer, polymorphic primer is had to analyze in F1 colony between parent, polymerase chain reaction (PCR) amplification program is the same, obtains colony's genotype data;
C. according to chain exchange rule, colony's genotype data is utilized to build the genetic linkage map of Chinese pear;
D. the fructescence carries out Fruit skin color qualification to each individual plant of F1, obtains the Fruit skin color proterties of each individual plant of F1;
E. according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, by analyzing the probability P value determination molecule marker relevant to the complete brown proterties of pericarp, the molecule marker of P<0.05, is molecule marker.
As preferably, in step B, polymerase chain reaction is carried out on amplification instrument.
As preferably, in step C, use software Mapmaker2.0, minimum LOD value is set to 3, obtains linkage map.
As preferably, in step e, according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, analyze and record the relevant probability P value of brown proterties complete in pericarp, the molecule marker of P<0.05, is molecule marker.The position in the complete brown character gene site of pericarp is determined by the chromosome position of molecule marker: in ' delicate fragrance ', find Zaasp867 (P=0.004), Zaasp868 (P=0.003), Zaasp873 (P=0.003), Zaasp876 (P=0.003) mark and Chinese pear pericarp complete brown character gene site close linkage, and Zaasp867 mark, Zaasp868 mark, Zaasp873 mark and Zaasp876 mark are the molecule marker of the complete brown character gene site Rus.zaas-8 of Chinese pear ' delicate fragrance ' pericarp of acquisition.
Compared with prior art, beneficial effect of the present invention is:
(1) the invention provides the molecule marker in the complete brown character gene site of Chinese pear ' delicate fragrance ' pericarp, by detecting the molecule marker chain with the complete brown character gene site of pericarp, Chinese pear Fruit skin color proterties can be predicted, accelerate the selection progress of Chinese pear Fruit skin color proterties.
(2) located the complete brown character gene site of pericarp obtained in Sand Pear ' delicate fragrance ' in the world first by molecule marker of the present invention, they can obtain the pericarp brown recall rate of 96.3% ~ 97.0%.Chinese pear is Self-Incompatibility seeds, and tree body is F1 hybrid, and genome height heterozygosis, occupy same domain prostatitis to the accurate positioning work in the complete brown character gene site of Chinese pear pericarp.
(3) by the gene locus locality specific of Molecular mapping of the present invention, qualification is convenient.By detecting the chain molecule marker in these and the complete brown character gene site of pericarp, namely the pericarp brown proterties of Chinese pear plant can be predicted, for the genotype detection of Sand Pear or strain, to judge whether this kind or strain have the complete brown proterties of pericarp, and then rapid screening objective fruit color of the leather kind or strain are used for Chinese pear breeding.Qualitative trait gene site fast easy to detect, not affected by environment.
(4) assistant breeding select target is clear and definite, cost-saving.In traditional breeding way, first will wait until that plant spends the virgin phase, just can carry out the brown skin proterties investigation of fruit after result, not only the cycle is very long, also occupies land resources.Therefore traditional breeding method is not only time-consuming, and cost is high.By detecting the complete brown character gene site of pericarp, can just identify the individual plant of complete brown pericarp in seedling stage, carrying out morning for examination, not only save production cost but also the efficiency of selection of raising Chinese pear Fruit skin color proterties greatly.
Embodiment
Embodiment 1
The screening method of the molecule marker in the complete brown character gene site of Chinese pear ' delicate fragrance ' pericarp, comprises following methods,
A. with Sand Pear ' emerald green hat ' for male parent and Sand Pear ' delicate fragrance ' are hybridized, obtain Hybrids F1;
B. the DNA of each individual plant of F1 colony is extracted by CTAB method, the mark SSR of simple sequence repeats is adopted to carry out polymorphism primer screening to two parents, increased by polymerase chain reaction, amplified production is that the polyacrylamide gel of 0.08g/ml carries out electrophoretic separation analysis in density, according to molecular marker screening result, filter out between parent and have polymorphic primer, polymorphic primer is had to analyze in F1 colony between parent, polymerase chain reaction (PCR) amplification program is the same, obtains colony's genotype data;
C. according to chain exchange rule, colony's genotype data is utilized to build the genetic linkage map of Chinese pear;
D. the fructescence carries out Fruit skin color qualification to each individual plant of F1, obtains the Fruit skin color proterties of each individual plant of F1;
E. according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, by analyzing the probability P value determination molecule marker relevant to the complete brown proterties of pericarp, the molecule marker of P<0.05, is molecule marker.
In step B, polymerase chain reaction is carried out on amplification instrument.
In step C, use software Mapmaker2.0, minimum LOD value is set to 3, obtains linkage map.
In step e, according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, analyze and record the relevant probability P value of brown proterties complete in pericarp, the molecule marker of P<0.05, is molecule marker.Find Zaasp867 (P=0.004), Zaasp868 (P=0.003), Zaasp873 (P=0.003), Zaasp876 (P=0.003) mark and Chinese pear pericarp complete brown character gene site close linkage, Zaasp867 mark, Zaasp868 mark, Zaasp873 mark and Zaasp876 mark are the molecule marker in the complete brown character gene site of Chinese pear ' delicate fragrance ' pericarp of acquisition.
Embodiment 2
The screening method of the molecule marker in the complete brown character gene site of Chinese pear ' delicate fragrance ' pericarp, comprises following methods,
A. with Sand Pear ' emerald green hat ' for male parent and Sand Pear ' delicate fragrance ' are hybridized, obtain Hybrids F1;
B. the DNA of each individual plant of F1 colony is extracted by CTAB method, the mark SSR of simple sequence repeats is adopted to carry out polymorphism primer screening to two parents, increased by polymerase chain reaction, amplified production is that the polyacrylamide gel of 0.08g/ml carries out electrophoretic separation analysis in density, according to molecular marker screening result, filter out between parent and have polymorphic primer, polymorphic primer is had to analyze in F1 colony between parent, polymerase chain reaction (PCR) amplification program is the same, obtains colony's genotype data;
C. according to chain exchange rule, colony's genotype data is utilized to build the genetic linkage map of Chinese pear;
D. the fructescence carries out Fruit skin color qualification to each individual plant of F1, obtains the Fruit skin color proterties of each individual plant of F1;
E. according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, by analyzing the probability P value determination molecule marker relevant to the complete brown proterties of pericarp, the molecule marker of P<0.05, is molecule marker.In ' delicate fragrance ', find Zaasp867 (P=0.004), Zaasp868 (P=0.003), Zaasp873 (P=0.003), Zaasp876 (P=0.003) mark and Chinese pear pericarp complete brown character gene site close linkage, Zaasp867 mark, Zaasp868 mark, Zaasp873 mark and Zaasp876 mark are the molecule marker of the complete brown character gene site Rus.zaas-8 of Chinese pear ' delicate fragrance ' pericarp of acquisition.
In step B, polymerase chain reaction is carried out on amplification instrument.
Embodiment 3
The screening method of the molecule marker in the complete brown character gene site of Chinese pear ' delicate fragrance ' pericarp, comprises following methods,
A. with Sand Pear ' emerald green hat ' for male parent and Sand Pear ' delicate fragrance ' are hybridized, obtain Hybrids F1;
B. the DNA of each individual plant of F1 colony is extracted by CTAB method, the mark SSR of simple sequence repeats is adopted to carry out polymorphism primer screening to two parents, increased by polymerase chain reaction, amplified production is that the polyacrylamide gel of 0.08g/ml carries out electrophoretic separation analysis in density, according to molecular marker screening result, filter out between parent and have polymorphic primer, polymorphic primer is had to analyze in F1 colony between parent, polymerase chain reaction (PCR) amplification program is the same, obtains colony's genotype data;
C. according to chain exchange rule, colony's genotype data is utilized to build the genetic linkage map of Chinese pear;
D. the fructescence carries out Fruit skin color qualification to each individual plant of F1, obtains the Fruit skin color proterties of each individual plant of F1;
E. according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, by analyzing the probability P value determination molecule marker relevant to the complete brown proterties of pericarp, the molecule marker of P<0.05, is molecule marker.In delicate fragrance, find Zaasp867 (P=0.004), Zaasp868 (P=0.003), Zaasp873 (P=0.003), Zaasp876 (P=0.003) mark and Chinese pear pericarp complete brown character gene site close linkage, Zaasp867 mark, Zaasp868 mark, Zaasp873 mark and Zaasp876 mark are the molecule marker of the complete brown character gene site Rus.zaas-8 of Chinese pear ' delicate fragrance ' pericarp of acquisition.
In step B, polymerase chain reaction is carried out on amplification instrument.
In step C, use software Mapmaker2.0, minimum LOD value is set to 3, obtains linkage map.
Embodiment 4
The screening method of the molecule marker in the complete brown character gene site of Chinese pear ' delicate fragrance ' pericarp, comprises following methods,
A. with Sand Pear ' emerald green hat ' for male parent and Sand Pear ' delicate fragrance ' are hybridized, obtain Hybrids F1;
B. the DNA of each individual plant of F1 colony is extracted by CTAB method, the mark SSR of simple sequence repeats is adopted to carry out polymorphism primer screening to two parents, increased by polymerase chain reaction, amplified production is that the polyacrylamide gel of 0.08g/ml carries out electrophoretic separation analysis in density, according to molecular marker screening result, filter out between parent and have polymorphic primer, polymorphic primer is had to analyze in F1 colony between parent, polymerase chain reaction (PCR) amplification program is the same, obtains colony's genotype data;
C. according to chain exchange rule, colony's genotype data is utilized to build the genetic linkage map of Chinese pear;
D. the fructescence carries out Fruit skin color qualification to each individual plant of F1, obtains the Fruit skin color proterties of each individual plant of F1;
E. according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, by analyzing the probability P value determination molecule marker relevant to the complete brown proterties of pericarp, the molecule marker of P<0.05, is molecule marker.In ' delicate fragrance ', find Zaasp867 (P=0.004), Zaasp868 (P=0.003), Zaasp873 (P=0.003), Zaasp876 (P=0.003) mark and Chinese pear pericarp complete brown character gene site close linkage, Zaasp867 mark, Zaasp868 mark, Zaasp873 mark and Zaasp876 mark are the molecule marker of the complete brown character gene site Rus.zaas-8 of Chinese pear ' delicate fragrance ' pericarp of acquisition.
In step B, polymerase chain reaction is carried out on amplification instrument.
In step C, use software Mapmaker2.0, minimum LOD value is set to 3, obtains linkage map.
In step e, according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, analyze and record the relevant probability P value of brown proterties complete in pericarp, the molecule marker of P<0.05, is molecule marker.
Embodiment 5
Materials and methods:
(1) structure of ' delicate fragrance ' F1 colony and phenotypic evaluation:
(1) south China Sand Pear ' delicate fragrance ' (female parent) and Sand Pear ' kingfisher is preced with ' (male parent) are carried out to hybridization and obtain F1 individual plant 135.
(2) F1 single-strain planting is in base, Haining, Zhejiang Academy of Agricultural Science, is positioned at Zhejiang Province's Haining City and is permitted Yang Du village, villages and small towns.Fructescence carries out Fruit skin color macroscopical identification to each F1 individual plant.During qualification, Fruit skin color is divided into full brown and non-fully brown 2 type, wherein non-fully brown includes middle shade and green.
(2) molecular marker analysis of recombinant inbred lines
(1) each strain DNA of recombinant inbred lines is extracted by CTAB method;
(2) DNA polymorphism of 977 pairs of SSR primer pairs ' delicate fragrance ' and ' emerald green hat ' parent is first used to carry out initial analysis.PCR reaction volume is 25 microlitres, wherein 10 × buffer2.5 microlitre, 25mMMgCl21.5 microlitre, 2.5mMdNTPs2 microlitre, and Taq enzyme (5 units/microlitre) 0.2 microlitre, template DNA 20 nanogram, adds water to 25 microlitres.SSR reaction system is after DNA94 DEG C of denaturation 3min, 94 DEG C of sex change 1min, 56 DEG C of annealing 1min, and 72 DEG C extend exhibition 1min, circulate 35 times, and last 72 DEG C extend 7min.In the enterprising performing PCR amplification of PE9600 amplification instrument, amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel (containing 7.6 grams of acrylamides and 0.4 gram of methylene diacrylamide in 100ml polyacrylamide solution), then take a picture on ultraviolet transilluminator, record result, there is polymorphic primer to analyze in recombinant inbred lines between parent, obtain colony's genotype data;
(3) according to chain exchange rule, utilize colony's genotype data to build the genetic map of Chinese pear, software used is Mapmaker2.0, and minimum LOD value is set to 3, obtains linkage map;
(4) Excel software is utilized to carry out linkage analysis, gene location site to colony's genotype data of each molecule marker and the Fruit skin color of each individual plant corresponding to it.
(3) results and analysis:
Molecular marker screening result shows, has 406 pairs of SSR primers variant between parents.The Fruit skin color proterties of DataExcel software to colony's genotype data each individual plant corresponding to it of each molecule marker is utilized to carry out linkage analysis, it is namely chain with a complete brown character gene site of pericarp to the molecule marker of the contribution rate R2 (table 2) of the complete brown proterties of pericarp, P<0.05 that One-way ANOVA records complete brown proterties incoherent probability P value and site with pericarp.The genotype of gained molecule marker in colony presses the genotypic categorization of parent's ' delicate fragrance ' and ' emerald green hat ', if the strain phenotype that genotype is identical with ' delicate fragrance ' is the full brown of pericarp, then the complete brown character gene site of pericarp of this mark location is from ' delicate fragrance ': the Zaasp867 mark finding P=0.004 in ' delicate fragrance ', the Zaasp868 mark of P=0.003, the Zaasp876 of Zaasp873 and P=0.003 of P=0.003 and pericarp complete brown character gene site close linkage, namely the molecule marker of the complete brown character gene site Rus.zaas-8 of Chinese pear ' delicate fragrance ' pericarp is obtained.
Predict the complete brown proterties of Chinese pear pericarp by above-mentioned molecular markers for identification gene locus, estimate the breeding process that can improve rapidly China's Sand Pear Fruit skin color proterties.
The sequence of table 1. labeled primer and amplified fragments size
The One-way ANOVA in the complete brown character gene site of table 2. Chinese pear ' delicate fragrance ' pericarp
P: represent and the incoherent probability of the complete brown proterties of pericarp, P<0.05 shows this mark and the complete brown proterties close linkage of pericarp; R
2: site is to the contribution rate of the complete brown proterties of pericarp, and value shows that more greatly brown proterties complete in pericarp is more relevant.
Embodiment 6
The molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and gene locus is Rus.zaas-8.Molecule marker closely linked with it is Zaasp867 primer, and its sequence is:
Left end primer sequence CCACCTCACCTTATCCCTCA,
Right-hand member primer sequence GCGTTTGCTCCTCGTACTTC.
Embodiment 7
The molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and gene locus is Rus.zaas-8.Molecule marker closely linked with it is Zaasp868 primer, and its sequence is:
Left end primer sequence AAAAAGTGATGTGTGGATCGC,
Right-hand member primer sequence CTGGTGACTTGCAAGCTAGG.
Embodiment 8
The molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and gene locus is Rus.zaas-8.Molecule marker closely linked with it is Zaasp873 primer, and its sequence is:
Left end primer sequence CGCTTTCGAACACAAAACAA,
Right-hand member primer sequence CTGGCACTTTGAATTCGCTT.
Embodiment 9
The molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and gene locus is Rus.zaas-8.Molecule marker closely linked with it is Zaasp876 primer, and its sequence is:
Left end primer sequence CGCTTTCGAACACAAAACAA,
Right-hand member primer sequence CTGGCACTTTGAATTCGCTT.
Embodiment 10
The molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s.Molecule marker closely linked with it is Zaasp876 primer, and its sequence is:
Left end primer sequence CGCTTTCGAACACAAAACAA,
Right-hand member primer sequence CTGGCACTTTGAATTCGCTT.
In a word, the foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to the covering scope of patent of the present invention.
Claims (9)
1. the molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, is characterized in that: gene locus is positioned at Sand Pear delicate fragrance No. 8 karyomit(e)s, and molecule marker closely linked with it is Zaasp867 primer, and its sequence is:
Left end primer sequence CCACCTCACCTTATCCCTCA,
Right-hand member primer sequence GCGTTTGCTCCTCGTACTTC.
2. the molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, is characterized in that: gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and molecule marker closely linked with it is Zaasp868 primer, and its sequence is:
Left end primer sequence AAAAAGTGATGTGTGGATCGC,
Right-hand member primer sequence CTGGTGACTTGCAAGCTAGG.
3. the molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, is characterized in that: gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and molecule marker closely linked with it is Zaasp873 primer, and its sequence is:
Left end primer sequence CGCTTTCGAACACAAAACAA,
Right-hand member primer sequence CTGGCACTTTGAATTCGCTT.
4. the molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp, is characterized in that: gene locus is positioned at Sand Pear ' delicate fragrance ' No. 8 karyomit(e)s, and molecule marker closely linked with it is Zaasp876 primer, and its sequence is:
Left end primer sequence CGCTTTCGAACACAAAACAA,
Right-hand member primer sequence CTGGCACTTTGAATTCGCTT.
5. the molecule marker in the complete brown character gene site of Sand Pear ' delicate fragrance ' pericarp according to claim 1 or 2 or 3 or 4, is characterized in that: gene locus is Rus.zaas-8.
6. the screening method of the molecule marker in claim 1 or 2 or the complete brown character gene site of Chinese pear ' delicate fragrance ' pericarp described in 3 or 4, is characterized in that: comprise following methods,
A. with Sand Pear ' emerald green hat ' for male parent and Sand Pear ' delicate fragrance ' are hybridized, obtain Hybrids F1;
B. the DNA of each individual plant of F1 colony is extracted by CTAB method, the mark SSR of simple sequence repeats is adopted to carry out polymorphism primer screening to two parents, increased by polymerase chain reaction, amplified production is that the polyacrylamide gel of 0.08g/ml carries out electrophoretic separation analysis in density, according to molecular marker screening result, filter out between parent and have polymorphic primer, polymorphic primer is had to analyze in F1 colony between parent, polymerase chain reaction (PCR) amplification program is the same, obtains colony's genotype data;
C. according to chain exchange rule, colony's genotype data is utilized to build the genetic linkage map of Chinese pear;
D. the fructescence carries out Fruit skin color qualification to each individual plant of F1, obtains the Fruit skin color proterties of each individual plant of F1;
E. according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, by analyzing the probability P value determination molecule marker relevant to the complete brown proterties of pericarp, the molecule marker of P<0.05, is molecule marker.
7. the screening method of the molecule marker in the complete brown character gene site of Chinese pear according to claim 6 ' delicate fragrance ' pericarp, it is characterized in that: in step B, polymerase chain reaction is carried out on amplification instrument.
8. the screening method of the molecule marker in the complete brown character gene site of Chinese pear according to claim 6 ' delicate fragrance ' pericarp, it is characterized in that: in step C, use software Mapmaker2.0, minimum LOD value is set to 3, obtains linkage map.
9. the screening method of the molecule marker in the complete brown character gene site of Chinese pear according to claim 6 ' delicate fragrance ' pericarp, it is characterized in that: in step e, according to colony's genotype data of each molecule marker and the Fruit skin color proterties of each individual plant corresponding to it, the molecule marker of P<0.05, is molecule marker.
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| CN105838800A (en) * | 2016-05-04 | 2016-08-10 | 中国农业科学院郑州果树研究所 | Molecular marker linked with pear fruit peel color traits and application thereof |
| CN106701911A (en) * | 2016-08-15 | 2017-05-24 | 浙江省农业科学院 | Molecular marker of pear PpAIV2 gene locus and screening method of molecular marker |
| CN112695133A (en) * | 2021-02-05 | 2021-04-23 | 青岛农业大学 | Method for screening molecular markers associated with pear brown skin by using BSA-seq |
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| CN105838800B (en) * | 2016-05-04 | 2019-05-31 | 中国农业科学院郑州果树研究所 | A kind of and the operatic circle skin color trait chain molecular labeling and its application |
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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 |
| CN112695133A (en) * | 2021-02-05 | 2021-04-23 | 青岛农业大学 | Method for screening molecular markers associated with pear brown skin by using BSA-seq |
| CN112695133B (en) * | 2021-02-05 | 2023-08-22 | 青岛农业大学 | Method for screening molecular marker related to pear brown skin by using BSA-seq |
| CN113388015A (en) * | 2021-06-25 | 2021-09-14 | 浙江省农业科学院 | Pear protein fragment PyDwarf1-462 and coding sequence and application thereof |
| CN113388015B (en) * | 2021-06-25 | 2024-05-10 | 浙江省农业科学院 | Pear protein fragment PyDwarf-462 and coding sequence and application thereof |
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