CN106939352A - Quick screening cucumber pierces firmly/the InDel molecular labelings of soft bur real type - Google Patents
Quick screening cucumber pierces firmly/the InDel molecular labelings of soft bur real type Download PDFInfo
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- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical class [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 1
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Abstract
/ InDel the molecular labelings of soft bur real type are pierced the invention provides a kind of quick screening cucumber firmly, InDel Ts1 are named as, the mark has 334 nucleotide sequences shown in sequence table SEQ ID NO.1.The InDel molecular labelings are isolated with soft thorn gene Ts.The InDel molecular labelings have high stability, and cucumber all over the world can be applied to easy, quick, with high throughput and pierces/the screening of soft thorn type firmly.
Description
Technical field
The present invention relates to the molecular labeling of gene engineering technology field, more particularly to one and the soft thorn gene Ts of cucumber fruits
The InDel molecular labelings isolated.
Background technology
Cucumber (Cucumissativus L.) is that Curcurbitaceae (Cucurbitaceae) Cucumis (Cucumis) is overgrow for 1 year
Herbaceous plant, cucumber is as one of big important vegetable crop in the world ten, and being also that China is main plants one of vegetable crop.
Fruit is the most important part of cucumber economic characters, and cucumber fruits belong to Peponidium, by ovary and holder symbiotic developmental
Form.It is fruit thorniness to have an important character in cucumber fruits, and fruit thorniness is the epidermal hair of cucumber specialization.Up to the present, it is yellow
The research of melon epidermis hair gene is relatively fewer.2015, Zhao J L etc. were obtained using mict microtriches mutant and wild type crosses
The 7936 plants of F obtained2Colony, by the Mict assignments of genes gene mapping is 71.13kb's in No. 3 chromosomal inheritance of cucumber distance using BSA methods
In interval.Detailed results refer to：Zhao J L etc. exist《Journal of integrative plant biology》(plant
Journal of physiology) 2015 years 925-935 pages of volume 57 deliver it is entitled《Micro-trichome as a class I
homeodomain-leucine zipper gene regulates multicellular trichome development
in Cucumissativus.》(microtriche gene is used as the homologous slide fastener gene regulation cucumber many cells epidermis hair of I type leucines
Educate) text.The same year, 1058 plants of F that Wang Y L etc. are obtained using the hairless mutant of tril and wild type crosses2Colony, passes through
BSA methods will control the Tril assignments of genes gene mapping of epidermal hair initial development to be marked in cucumber No. 6 chromosomes UW007183 and UW007211
Between, genetic distance is 37.4kb, and detailed results refer to：Wang Y L etc. exist《Theoretical and Applied
Genetics》It is entitled that (theoretical applied genetics) 2015 year 305-316 pages of volume 129 is delivered《Identification and
mapping of Tril,a homeodomain-leucine zippergene involved in multicellular
trichome initiation inCucumissativus》One text.
Mict genes (micro-trichomes) and Tril genes (trichome-less) control cucumber microtriche respectively
Shape and hairless character, because fruit thorniness is the specialization of epidermal hair, therefore the fruit thorniness characteristic trait of mict mutant is small fruit thorniness, tril
The fruit thorniness characteristic trait of mutant is no fruit thorniness.Tril genes recessive epistasis is in Mict genes.CsTTG1 genes are parallel and direct
Tril genes are acted on, to regulate and control the formation of cucumber epidermal hair.Therefore, the soft thorn gene Ts of cucumber fruits research will be other works
The research of the trichome development mechanism of thing and cucumber lays the foundation.
With the improvement of people ' s living standards, quality breeding has mentioned critical positions.Cucumber fruit thorniness character belongs to sense organ product
Matter category.The cucumber of American-European greenhouse is have no result knurl, the pericarp that pierces less, is called Fruit Cucumber, its market price is common
2-3 times of cucumber.The pollution of smooth in appearance cucumber is few, and easy to clean, sanitary edible is the preferable kind of pollution-free vegetable.Through inspection
Survey shows：Pierce cucumber pulp persticide residue less without knurl lower than spinosity cucumber by 27%, pericarp persticide residue is low by 18%.Cucumber is soft
Thorn softness is not stimulated, easy to fall off, is enhanced the resistance of plant, is the more preferably selection of consumer.Gene Ts controls the shape of soft thorn
Into its research will promote quality breeding process.Auxiliary choosing is marked with the molecular labeling with purpose character close linkage
It is highly effective method to select in cucumber genetic breeding, and molecular labeling is that objective trait is selected on DNA level, tool
Have the advantages that it is efficient, quick, do not limited by environmental condition, can be selected in seedling stage, be accelerated the process of breeding.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, molecular labeling and BSA (Bulked Segregant are utilized
Analysis) method pierces the/InDel molecular labelings of soft bur real type, the mark stability there is provided a kind of quick screening cucumber firmly
It is high, highly reliable, it just can quickly be screened in seedling stage, identify the molecular labeling of hard thorn/soft thorn knurl cucumber fruits type, and then contracted
Short breeding cycle, accelerate cucumber pierce firmly/it is soft thorn character breeding process.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of quick screening cucumber pierces firmly/the InDel molecular labelings of soft bur real type, InDel-Ts1 is named as, the mark
Note is with 334 nucleotide sequences shown in sequence table SEQ ID NO.1.The InDel molecular labelings are total to soft thorn gene Ts
Separation.The InDel molecular labelings have high stability, cucumber all over the world can be applied to easy, quick, with high throughput hard
The screening of thorn/soft thorn type.
Above-mentioned screening pierces firmly/the InDel molecular labelings that are isolated with soft thorn gene Ts of soft thorn cucumber fruits type, by SEQ
Anti-sense primer amplification shown in sense primer and SEQ ID NO.3 shown in ID NO.2 is obtained.Primer is synthesized by Shanghai life work.
Its amplification program：94℃5min；32cycles；94℃25s；55℃25s；72℃25s；72℃5min.
InDel molecular labelings in the present invention are isolated with soft thorn gene Ts, are had for final clone Ts genes very big
Help, for study fruit thorniness character formation lay a solid foundation.
The present invention obtains F using a variety of hard thorn parents and soft thorn parents1, F1Individual plant is hard thorn；F1Selfing is obtained again
A variety of F2Segregating population, by F2In segregating population firmly thorn, it is soft lunge row genetic analysis, determine cucumber it is soft thorn character belong to
Single-gene recessive inheritance character.From F2Random each 10 plants of hard thorn individual plants of picking and 10 plants of soft thorn individual plants in colony, and build hard thorn
Pond and soft thorn pond.Using SSR molecular marker combination BSA methods, the soft thorn linkage molecule mark of screening cucumber.Most soft thorn gene at last
It is interval interior that Ts is positioned at 109.7kb.300 pairs of InDel marks are developed in the 109.7kb, chain mark is further screened with reference to BSA methods
Note, finally wherein InDel molecular labelings InDel-Ts1 shows polymorphism between parent, and is isolated with Ts genes.
Compared with prior art, the present invention has the advantages that：Traditional breeding method is the form according to plant
To be selected, because the soft thorn character of cucumber needs just to can determine that during plant knot melon, consuming time longer, of the invention molecule mark
Note can be that is, time saving also accurate during vegetable seeds or early stage that cotyledon is grown can be identified, so available for cucumber point
Sub- marker-assisted breeding, accelerates the process of cucumber quality breeding.The molecular labeling of the present invention is isolated with the soft thorn character of cucumber, institute
The cucumber variety for having all over the world can firmly pierce with this molecular marker screening/and soft thorn kind is (except stingless without knurl cucumber variety
gl)。
Brief description of the drawings
Fig. 1：Molecular labeling InDel-Ts1 PCR expanding effects：SA419-2 pierces parent firmly for cucumber fruits；NC073 is
The soft thorn parent of cucumber fruits；F1Represent two parent filial generations；F2Hard thorn individual plant and F2Soft thorn individual plant is represented in F respectively2In colony
The 10 plants of hard thorns selected at random and 10 plants of soft thorn plant.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.These embodiments be merely to illustrate the present invention and without
In limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, for example
Sambrook equimoleculars are cloned：Laboratory manual (New York:Cold Spring Harbor Laboratory Press,
1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment
The identification of the structure of one hereditary segregating population and the soft thorn gene of cucumber
1. many kinds of F2The structure of colony
Build F2Soft thorn kind used in colony is：NC073.Hard thorn kind has：S77, SA419-2.The present embodiment profit
Cross combination has been prepared with this 3 parents, F has been obtained1Generation, F1In generation, is hard thorn individual plant；F1F is produced for selfing, backcrossing2For colony
And BC1Colony.In F2And BC1Occur hard thorn and soft thorn trait segregation phenomenon in colony, identify and count hard thorn/soft thorn phenotype, point
The hard thorn of analysis and soft thorn are in F2And BC1In segregation ratio, verified using chi-square analysis method, finally draw cucumber it is soft thorn character category
In the recessive character of nucleus Dominant gene.
The screening of 2.Ts gene close linkage SSR markers and InDel marks
2.1 Cucumber germplasm DNA extraction
Parent, BC are extracted with CTAB methods1And F2The blade STb gene of segregating population.Method is：Take individual plant spire be put in 2mL from
In heart pipe, two steel balls are added, 750uLCTAB extracts cushioning liquid (60 DEG C of preheatings), and grinder 60HZ/90s is ground, and 60 DEG C put
.5-1h is set to 0, centre slowly shakes up 1 time.Plus isometric chloroform, 5min, 12000r/min centrifugation 10min are slowly rocked, is drawn
500uL supernatants, add two volumes absolute ethyl alcohol, and supernatant is removed in -20 DEG C of placement 30min, centrifugation, and 70% alcohol is washed twice.Dry,
1 × TE dissolves.Add 10ug/mL RNase.0.8% agarose gel electrophoresis detects its purity, and nucleic acid instrument detects its concentration.
The final concentration for being diluted to 50ng/uL, -20 DEG C store for future use.
The foundation of 2.2 hard thorns/soft thorn gene pool
BSA methods are from F2Hard thorn strain and each 10 plants of soft thorn strain individual are randomly selected in segregating population respectively, hard thorn is set up and soft
Pierce the gene pool of cucumber.
The screening of 2.3 marks
Two parents and two gene pools are screened with 551 pairs of SSR primers combinations and 300 pairs of InDel marks.Reaction
System is genomic DNA 30ng, primer 0.5 μm of ol/L, 200 μm of ol/L dNTPs, 2mmol/LMgCl2, 1 μ l10 × PCR
Reactions buffer, 0.6U Taq DNA polymerases, overall reaction system is 10 μ l.PCR amplification programs are：94℃5min；94
℃30s 55℃30s；72℃30s；32 circulations.Amplified production is separated with 8% native polyacrylamide gel electrophoresis, electrophoresis
Buffer solution is 0.5 × TBE, 160W invariable powers, electrophoresis 3h.
Silver staining is carried out after electrophoresis.Silver staining method is：Glass plate with glue is put into fixer, gently shaken on shaking table
To indicator color fade, the composition of wherein fixer is：Glacial acetic acid, absolute ethyl alcohol, the volume ratio of distilled water are 1:10:100；
With ultrapure washing 1min-3min；Offset plate after flushing is put into dyeing liquor and shakes half an hour, the component of wherein dyeing liquor is
2g/L silver nitrates；Offset plate after dyeing is put into after being rinsed in ultra-pure water and be put into the plastic casing equipped with nitrite ion, gently shaken
It is clear to band, it is put into running water and rinses, statistics is simultaneously taken pictures.Wherein developer solution component is：Added in 1L distilled water
30gNaOH and 4ml formaldehyde is uniformly mixed so as to obtain.
Identical polymorphic bandses are shown between hard thorn, two parents of soft thorn and firmly between thorn pond, soft thorn pond, i.e., hard thorn
Parent SA419-2 is consistent with the band in hard thorn gene pool, and soft thorn parent NC073 is consistent with soft thorn gene pool band；And pierce firmly
Band in parent SA419-2 and hard thorn gene pool is different from soft thorn parent NC073 and soft thorn gene pool band.Pass through 721 plants
Colony carries out genetic linkage analysis, and the individual consistent with soft thorn parent NC073 banding pattern is designated as into a, will be with hard thorn parent
The consistent individual of SA419-2 banding patterns is designated as b, and heterozygosis banding pattern is designated as h.As a result InDel marks are isolated with Ts genes, according to
9930 Cucumber germplasm website (http://www.icugi.org/cgi-bin/gb2/gbrowse/cucumber_v2/) display
The soft thorn gene Ts of InDel marking paths physical distance is 254bp.
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Sequence table
<110>Jiangsu Normal University
<120>Quick screening cucumber pierces firmly/the InDel molecular labelings of soft bur real type
<160>3
<210> 1
<211>334
<212> DNA
<213>Cucumber（Cucumissativus L.）
<400> 1
tcggagagacaatgagaaccgtttcggagatagcgtcaaaacgcaaatgtcgcaaaacga 60
aaatggatagttgcaaatttagcaattaaattcaaattaattaagtatataaaacaattt 120
taaaaaatttgcaaatatagcaaaatttgtcaagtcctatcaatgatataagtctatatc 180
actgatatatcatatgaagtctatcagtgatagttttgctatatttgcaatttttttaaa 240
atattgctatatccttaattattattgctaaaatggtcattcattgcaatttctcaaacg 300
aaaatgggtttcaacaatagggacataaaatcgg 334
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
tcggagagacaatgagaacc20
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
ccgattttatgtccctattgt 21
Claims (4)
1. a kind of quick screening cucumber pierces firmly/the InDel molecular labelings of soft bur real type, it is characterised in that：It is named as
InDel-Ts1, the mark has 334 nucleotide sequences shown in sequence table SEQ ID NO.1.
2. quick screening cucumber according to claim 1 pierces firmly/the InDel molecular labelings of soft bur real type, its feature
It is：The InDel molecular labelings are isolated with soft thorn gene Ts.
3. quick screening cucumber according to claim 1 pierces firmly/the InDel molecular labelings of soft bur real type, its feature
It is：The InDel molecular labelings are as the anti-sense primer shown in the sense primer shown in SEQ ID NO.2 and SEQ ID NO.3
Amplification is obtained.
4. quick screening cucumber according to claim 1 pierces firmly/the InDel molecular labelings of soft bur real type, its feature
It is：The amplification program is：94℃5min；32cycles；94℃25s；55℃25s；72℃25s；72℃5min.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107513564A (en) * | 2017-09-01 | 2017-12-26 | 上海交通大学 | A kind of molecular labeling isolated with the soft thorn gene Ts of cucumber fruits and its application |
| CN107574258A (en) * | 2017-09-21 | 2018-01-12 | 上海交通大学 | With the soft thorn gene Ts of the cucumber fruits InDel Ts2 molecular labelings isolated and its application |
| CN107586869A (en) * | 2017-09-21 | 2018-01-16 | 上海交通大学 | With the soft thorn gene Ts of the cucumber fruits InDel Ts3 molecular labelings isolated and its application |
| CN107586870A (en) * | 2017-09-21 | 2018-01-16 | 上海交通大学 | InDel molecular labelings and its application with the soft thorn gene Ts close linkages of cucumber fruits |
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| CN107586870A (en) * | 2017-09-21 | 2018-01-16 | 上海交通大学 | InDel molecular labelings and its application with the soft thorn gene Ts close linkages of cucumber fruits |
| CN109182588A (en) * | 2018-10-31 | 2019-01-11 | 上海交通大学 | The molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene |
| CN109234438A (en) * | 2018-10-31 | 2019-01-18 | 上海交通大学 | The molecular labeling Indel-De3 isolated with cucumber one dimensional infinite wall gene |
| CN116064911A (en) * | 2022-11-10 | 2023-05-05 | 上海农林职业技术学院 | InDel molecular marker co-separated from cucumber dense thorn and less thorn genes, detection primer and application thereof |
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