CN104593517B - The closely linked molecular labeling with cucumber trichome development gene M ict - Google Patents
The closely linked molecular labeling with cucumber trichome development gene M ict Download PDFInfo
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- CN104593517B CN104593517B CN201510088071.6A CN201510088071A CN104593517B CN 104593517 B CN104593517 B CN 104593517B CN 201510088071 A CN201510088071 A CN 201510088071A CN 104593517 B CN104593517 B CN 104593517B
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Abstract
The invention provides two kinds and the closely linked molecular labeling of cucumber trichome development gene M ict, a kind of named Flanking SNP 01, another kind of named Flanking SNP 02.In the present invention, two molecular labelings closely linked with cucumber trichome development gene M ict are more helpful to the final clone of cucumber trichome development gene and the foundation of Molecular Marker-Assisted Breeding of Cucumber system；The molecular labeling of the present invention easy, quick, high-throughout can be applied to breed cucumber practice.
Description
Technical field
The present invention relates to technique for gene engineering, particularly to closely linked with cucumber trichome development gene M ict
Molecular labeling.
Technical background
Plant epidermal hair (Trichome) is the biology organ of a kind of structure height specialization, is sent out by procuticle cell
Educate, be from individual cell level study plant cell differentiation, including cell fate, the cell cycle, cell polarity,
Cellular morphology such as builds up at the ideal model of aspect.Epidermal hair is widely present in most plant surface, is divided into slender
Born of the same parents or many cells, have body of gland or Non-gland body, has branch or without a few class of branch.Epidermal hair plant grow and
The aspects such as the adaptation to environment, including biological or abiotic, play an important role.As increased skin layer thickness
To regulate body temperature, reduce transpiration moisture loss, reduce mechanical damage, reduce ultraviolet light harms, protect plant
From the infringement of insect, herbivore and pathogen, contribute to collection and the propagation etc. of pollen.
Cucumber (Cucumis sativus L.) belong to Magnoliatae (Magnoliopsida), Curcurbitaceae (Cucurbitaceae),
Cucumis (Cucumis), is 1 year herbaceous plant that overgrows, one of world's important vegetable, because its flower property type is many
Sample, by the model plant as flower property type research.Epidermal hair be distributed widely in the stem of cucumber plant, leaf, tendril,
Petal, sepal, ovary surface, cucumber fruits belongs to Peponidium, is formed by ovary and holder symbiotic developmental, fruit surface
Hair form eggcase, is commonly referred to as " fruit thorn ".
Cao Chenxing was in discovery nothing in the colony of North China type cucumber local varieties " smalt handle " self progeny in 1997
The natural mutant of hairs type, it shows as stem, leaf, tendril, petal, sepal, ovary surface all without epidermal hair,
This mutant is that cucumber epidermal hair formation mechanism study provides preferable material, also provides for cucumber genetic breeding
Stingless parent.The cucumber of smooth in appearance pollutes few, easy to clean, is the preferable kind of pollution-free vegetable.Few without knurl
The cucumber pulp persticide residue of thorn is lower by 27% than spinosity cucumber, and pericarp persticide residue is low by 18%.
Cucumber epidermal hair is different from arabidopsis and cotton, is that typical many cells are without branch epidermal hair.At present for planting
The research of thing epidermal hair focuses primarily upon the single cell type of arabidopsis, and the research to many cells epidermal hair is not yet deep
Entering, the map based cloning of cucumber epidermal hair related gene and regulatory mechanism research also have no report.Cucumber is without chalaza variant
Fruit is had no result knurl, and reproductive growth shifts to an earlier date, and female flower calyx is loose, illustrate that cucumber epidermal hair gene also take part in really knurl
Formation and other growth courses.The Primary Study to cucumber epidermal hair related gene map based cloning for the present invention, not only
Can reveal that the molecule mechanism of plant many cells trichome development, and the clone of more epidermal hair related genes can be
Research with function lays the foundation, and accelerates cucumber quality breeding process.
Map based cloning (Map-based cloning), also known as positional cloning (Positional cloning), can be at gene
In the case that product is unknown, utilize the genetic linkage analysis of segregating population or the exception of chromosome, genes of interest is fixed
Position, to a kind of particular location of chromosome, is found and its closely linked molecular labeling, is constantly reduced candidate region and enter
And clone gene.The most frequently used mark screening method is chorista packet Mixed method (Bulked segregant
Analysis, BSA), the method in there is for a pair the segregating population constructed by the parent of genes of interest phenotypic difference,
According to the phenotype of genes of interest, choose a number of individuality respectively, constitute two subgroups, by each subgroup
The DNA mixed in equal amounts of body, forms " gene pool " (the Gene pool) of two relativity, then with suitably
Two gene pools are analyzed by molecular labeling, show molecular labeling and the genes of interest seat of polymorphism between two ponds
Position is mutually chain, and recycling segregating population detects the linkage degree of gained molecular labeling and genes of interest further, thus
Determine its position on known molecular collection of illustrative plates or chromosome.Employ specific separation group owing to building gene pool
Body, and only genes of interest phenotype is selected when packet, this ensure that the genetic background of other proterties is basic
Identical, mainly should there are differences at genes of interest section in theory between two gene pools, eliminate environment and artificial
The impact of factor, makes result of study more accurately and reliably.
Content of the invention
The purpose of the present invention, it is simply that in order to provide two kinds and closely linked point of cucumber trichome development gene M ict
Son mark.
In order to realize the purpose of the present invention, present invention employs techniques below scheme:
One and the closely linked molecular labeling of cucumber trichome development gene M ict, named
Flanking-SNP-01, by shown in nucleotide fragments shown in SEQ ID NO.1 in sequence table and SEQ ID NO.2
Nucleotide fragments composition, wherein nucleotide fragments shown in SEQ ID NO.1 is tight with trichome development gene M ict
Close chain, nucleotide fragments shown in SEQ ID NO.2 and hairless gene mict close linkage.
Described molecular labeling Flanking-SNP-01 is by upstream primer shown in SEQ ID NO.5 and SEQ ID NO.6
Shown downstream primer amplification obtains.
One and the closely linked molecular labeling of cucumber trichome development gene M ict, named
Flanking-SNP-02, by shown in nucleotide fragments shown in SEQ ID NO.3 in sequence table and SEQ ID NO.4
Nucleotide fragments composition, wherein nucleotide fragments shown in SEQ ID NO.3 is tight with trichome development gene M ict
Close chain, nucleotide fragments shown in SEQ ID NO.4 and hairless gene mict close linkage.
Described molecular labeling Flanking-SNP-02 is by upstream primer shown in SEQ ID NO.7 and SEQ ID NO.8
Shown downstream primer amplification obtains.
The present invention is the method by map based cloning, utilizes cucumber without chalaza variant (mict) and hairiness (Mict)
Wild type (S77) preparing hybrid combines, F1Produce F for selfing2For colony, totally 7936 strains are individual, and identify
Blade hairiness and without hair phenotype, wherein individual 5904 strains of hairiness, without hair individuality 2032 strains, every strain takes a piece of tender leaf
Extract genomic DNA, in conjunction with the screening of BSA method and the closely linked molecular labeling of Mict gene, through double deoxidations
Chain termination method order-checking obtains.
2 molecular labeling Flanking-SNP-01 and Flanking-SNP-02 in the present invention and Mict gene position
Point is chain more tight, final clone and the Molecular Marker-Assisted Breeding of Cucumber system to cucumber trichome development gene
Foundation more helpful；It is real that the molecular labeling of the present invention easy, quick, high-throughout can be applied to breed cucumber
Trample.
Specific implementation method
F2Structure for segregating population
Cucumber is utilized to combine without chalaza variant (mict) and hairiness (Mict) wild type (S77) preparing hybrid,
F1Produce F for selfing2For colony, totally 7936 strains are individual, and identify blade hairiness and without hair phenotype, wherein hairiness
Individual 5904 strains, without individual 2032 strains of hair, chi-square analysis method verifies segregation ratio.Because of when plant rough leaf
Expansion can carry out phenotype judgement period, and segregating population is planted in hole tray.
Cucumber germplasm DNA extracts
Extract parent and F by CTAB method2Genomic DNA for segregating population blade.Take just launched a piece of
True leaf is in 1.5mL centrifuge tube and add liquid nitrogen, and tissue abrasion is powdered, adds 500 μ L CTAB cracking
Buffer solution, acutely vibration 30 seconds, 60 DEG C of water-baths 30 minutes, period turns upside down and fully mixes.To above-mentioned homogenate
Lysate adds 500 μ L chloroform isoamyl alcohol (24:1, v/v), turns upside down and fully mix, 13200rpm 4 DEG C
Centrifugal 15 minutes.Aspirate supernatant 500 μ L, in new centrifuge tube, adds equal-volume isopropanol, turns upside down
Fully mix, stand 30 minutes in-20 DEG C, centrifugal 15 minutes of 13200rpm 4 DEG C.Abandoning supernatant, edge is centrifugal
Tube wall adds 1mL 75% ethanol, and turn upside down washing centrifuge tube tube wall, and 13200rpm 4 DEG C is after centrifugal 5 minutes
Discard ethanol.Drying at room temperature precipitates 30 minutes, adds 100 μ L RNase TE buffer solution (7:993, v/v)
Dissolving, 37 DEG C of water-baths 30 minutes, nucleic acid instrument dilutes final concentration of 30 with TE buffer solution after measuring DNA concentration
Ng/ μ L, standby in-20 DEG C.
Mark scan F2Segregating population
Utilize the above-mentioned F of two molecular labeling scannings that the present invention develops2Segregating population, finds marking type and proterties table
The difference individual plant of existing type, it is thus achieved that the exchange individual plant of mark and Mict gene.PCR system: 1 × Taq Buffer, 1.5
mM MgCl2, 200 μM of dNTPs, each 0.2 μM of primer, 30ng template DNA, 0.5U Taq DNA
Polymerase, overall reaction system is 10 μ L.PCR program: 94 DEG C 5 minutes；35 circulations, 94 DEG C 30
Second, 55 DEG C 30 seconds, 72 DEG C 30 seconds；72 DEG C 5 minutes.BioTeke SYBR Green is added in PCR primer
I nucleic acid dye 3 μ L, 4 DEG C of standings make dyestuff be combined with DNA in 15 minutes, divide with 1.5% agarose gel electrophoresis
From cutting purpose fragment under uviol lamp, use TaKaRa MiniBEST Agarose Gel DNA Extraction
Kit reclaims purifying DNA fragment, concrete steps reference reagent box specification, production code member 9762.Piece will be purified
Section is sent by the order-checking of Sani bio tech ltd, Shanghai, and sequencing result passes through Chromas 2.3 software analysis.
Above 2 molecular labelings are through F2Segregating population is verified, all with Mict gene loci close linkage and special
Opposite sex PCR has high stable feature.Utilize these molecular labelings to build high density heredity and physical map, will have
Help the final clone of cucumber trichome development gene.
Claims (4)
1. with the closely linked molecular labeling of cucumber trichome development gene M ict, named
Flanking-SNP-01, by shown in nucleotide fragments shown in SEQ ID NO.1 in sequence table and SEQ ID NO.2
Nucleotide fragments composition, wherein nucleotide fragments shown in SEQ ID NO.1 is tight with trichome development gene M ict
Close chain, nucleotide fragments shown in SEQ ID NO.2 and hairless gene mict close linkage.
2. as claimed in claim 1 with the closely linked molecular labeling of cucumber trichome development gene M ict, its spy
Levying and being, described molecular labeling Flanking-SNP-01 is by upstream primer and SEQ ID shown in SEQ ID NO.5
Downstream primer amplification shown in NO.6 obtains.
3. with the closely linked molecular labeling of cucumber trichome development gene M ict, named
Flanking-SNP-02, by shown in nucleotide fragments shown in SEQ ID NO.3 in sequence table and SEQ ID NO.4
Nucleotide fragments composition, wherein nucleotide fragments shown in SEQ ID NO.3 is tight with trichome development gene M ict
Close chain, nucleotide fragments shown in SEQ ID NO.4 and hairless gene mict close linkage.
4. as claimed in claim 3 with the closely linked molecular labeling of cucumber trichome development gene M ict, its spy
Levying and being, described molecular labeling Flanking-SNP-02 is by upstream primer and SEQ ID shown in SEQ ID NO.7
Downstream primer amplification shown in NO.8 obtains.
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| CN201510088071.6A CN104593517B (en) | 2015-02-26 | 2015-02-26 | The closely linked molecular labeling with cucumber trichome development gene M ict |
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| CN112322769B (en) * | 2020-11-19 | 2022-02-18 | 南京农业大学 | SNP molecular marker related to cucumber multi-epidermal hair traits and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250523A (en) * | 2008-04-03 | 2008-08-27 | 上海交通大学 | Molecule marker tightly linked with cucumber pubescence gene G1 |
| CN102757959A (en) * | 2012-07-25 | 2012-10-31 | 天津科润农业科技股份有限公司 | Gene fragment tightly linked with cucumber hairiness gene and application of gene fragment |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250523A (en) * | 2008-04-03 | 2008-08-27 | 上海交通大学 | Molecule marker tightly linked with cucumber pubescence gene G1 |
| CN102757959A (en) * | 2012-07-25 | 2012-10-31 | 天津科润农业科技股份有限公司 | Gene fragment tightly linked with cucumber hairiness gene and application of gene fragment |
Non-Patent Citations (1)
| Title |
|---|
| Transcriptome analysis in Cucumis sativus identifies genes involved in multicellular trichome development;Jun-Long Zhao等;《Genomics》;Elsevier Inc.;20150207;第105卷;296-303 * |
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Inventor after: Cai Run Inventor after: Pan Jian Inventor after: Pan Junsong Inventor after: He Huanle Inventor after: Zhao Junlong Inventor after: Nie Jingtao Inventor after: Guo Chunli Inventor after: Qu Meiling Inventor before: Cai Run Inventor before: Pan Junsong Inventor before: He Huanle Inventor before: Zhao Junlong Inventor before: Nie Jingtao Inventor before: Guo Chunli Inventor before: Yang Junjun Inventor before: Qu Meiling |
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Granted publication date: 20161102 Termination date: 20190226 |