CN107513564A - A kind of molecular labeling isolated with the soft thorn gene Ts of cucumber fruits and its application - Google Patents

A kind of molecular labeling isolated with the soft thorn gene Ts of cucumber fruits and its application Download PDF

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CN107513564A
CN107513564A CN201710779750.7A CN201710779750A CN107513564A CN 107513564 A CN107513564 A CN 107513564A CN 201710779750 A CN201710779750 A CN 201710779750A CN 107513564 A CN107513564 A CN 107513564A
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molecular labeling
cucumber
soft thorn
gene
primer
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郭春立
蔡润
潘俊松
王刚
孙敬贤
吕铎
张克岩
张琪
肖婷婷
张乐钰
朱文莹
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Shanghai Jiaotong University
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    • C12Q2600/13—Plant traits
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The present invention relates to a kind of molecular labeling isolated with the soft thorn gene Ts of cucumber fruits and its application.The molecular labeling is named as SSR Ts1, and molecular labeling is made up of the anti-sense primer shown in the sense primer and EM4 shown in ME3.The molecular labeling has 103 nucleotide sequence fragments in sequence table SEQ ID NO.1, and 101 nucleotide sequence fragments in sequence table SEQ ID NO.2.Nucleotide sequence in wherein SEQ ID NO.1 isolates with soft thorn gene, and the nucleotide sequence in SEQ ID NO.2 isolates with hard Ci Jiyin.Compared with prior art, the molecular labeling has high stability, can be applied to cucumber all over the world easy, quick, with high throughput and pierce/the screening of soft thorn type firmly.

Description

A kind of molecular labeling isolated with the soft thorn gene Ts of cucumber fruits and its application
Technical field
The present invention relates to the molecular labeling of gene engineering technology field, the soft thorn gene Ts of more particularly to a kind of and cucumber fruits The SSR molecular marker isolated.
Background technology
Cucumber (Cucumis sativus L.) is that Curcurbitaceae (Cucurbitaceae) Cucumis (Cucumis) 1 year is climing Raw herbaceous plant, cucumber is as one of big important vegetable crop in the world ten, and one of main cultivation vegetable crop in China.
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 it is 71.13kb's in No. 3 chromosomal inheritance of cucumber distance using BSA methods In section.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 Cucumis sativus.》(microtriche gene is as the homologous slide fastener gene regulation cucumber many cells epidermis hair of I type leucines Educate) text.1058 plants of F that the same year, Wang Y L etc. is obtained using the hairless mutant of tril and wild type crosses2Colony, pass through BSA methods will control the Tril assignments of genes gene mapping of epidermal hair initial development in No. 6 chromosome UW007183 and UW007211 marks of cucumber Between, genetic distance 37.4kb, detailed results refer to：Wang Y L etc. exist《Theoretical and Applied Genetics》(theoretical applied genetics) 2015 year 305-316 pages of volume 129 is delivered entitled《Identification and mapping of Tril,a homeodomain-leucine zipper gene involved in multicellular trichome initiation in Cucumis sativus》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.
As 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, easy to clean, sanitary edible, is the preferable kind of pollution-free vegetable.Through inspection Survey shows：Pierce cucumber pulp persticide residue lower than spinosity cucumber 27%, pericarp persticide residue low 18% less without knurl.Cucumber is soft Thorn softness does not stimulate, easy to fall off, enhances 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, accelerate the process of breeding.
The content of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of soft with cucumber fruits The molecular labeling and its application that thorn gene Ts is isolated.The present invention utilizes molecular labeling and BSA (BulkedSegregant Analysis) method, there is provided the molecular labeling isolated with the soft thorn gene Ts of cucumber.The molecular labeling of the present invention can be easy, fast Speed, it is applied to breeding practice with high throughput.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of molecular labeling isolated with the soft thorn gene Ts of cucumber fruits, is named as SSR-Ts1, the molecular labeling has 103 nucleotide sequences shown in sequence table SEQ ID NO.1, and 101 nucleotides shown in sequence table SEQ ID NO.2 Nucleotide sequence in sequence, wherein SEQ ID NO.1 isolates with soft thorn gene, the nucleotide sequence in SEQ ID NO.2 Isolated with hard Ci Jiyin.The molecular labeling is characterised by isolating with soft thorn gene, and the molecular labeling has high stability, Cucumber all over the world can be applied to easy, quick, with high throughput and pierce/the screening of soft thorn type firmly.
The molecular labeling is expanded to obtain as the anti-sense primer shown in the sense primer and EM4 shown in ME3, and primer is by Shanghai Hua Jin bio tech ltd synthesizes.
The primer ME3 sequences, it is specially：ME3：5 '-TGAAGGTTGATTGGACAAAT-3 ',
The primer EM4 sequences, it is specially：EM4：5’-TCATAACGTCATCCCTAAAA-3’.
The present invention obtains F using a variety of hard thorn parents and soft thorn parents1, F1Individual plant is hard thorn；F1Acquisition is selfed 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 Ts is positioned in 109.7kb sections.In the 109.7kb, SSR-Ts1 shows polymorphism between parent, and with Ts genes Isolate.
With the preparation method of the soft thorn gene Ts of the cucumber fruits molecular labelings isolated, the molecular labeling is as shown in ME3 Sense primer and EM4 shown in anti-sense primer expand to obtain,
The primer ME3 sequences, it is specially：ME3：5 '-TGAAGGTTGATTGGACAAAT-3 ',
The primer EM4 sequences, it is specially：EM4：5’-TCATAACGTCATCCCTAAAA-3’.
Reaction system is genomic DNA 30ng, primer 0.5 μm of ol/L, 200 μm of ol/L dNTPs, 2mmol/L MgCl2, 1 μ l 10 × PCR reactions buffer, 0.6U Taq DNA polymerases, overall reaction system are 10 μ l, and primer is shown in ME3 Anti-sense primer shown in sense primer and EM4.
PCR amplification programs are：94℃5min；94℃30s 55℃30s；72℃30s；32 circulations.Amplified production is with 8% Native polyacrylamide gel electrophoresis separates, and electrophoretic buffer is 0.5 × TBE, 160W invariable powers, electrophoresis 3h.
With the molecular labeling that the soft thorn gene Ts of cucumber fruits is isolated and cucumber it is soft thorn character isolate, it is all over the world Cucumber variety can be pierced firmly with this molecular marker screening/it is soft thorn kind (removing stingless no knurl cucumber variety gl).
Traditional breeding method is selected according to the form of plant, because the soft thorn character of cucumber needs plant knot melon When just can determine that, expend that the time is longer, molecular labeling of the invention can be during vegetable seeds or early stage that cotyledon is grown It can identify, i.e., it is time saving also accurate, so can be used for Molecular Marker-Assisted Breeding of Cucumber, accelerate the process of cucumber quality breeding.
Compared with prior art, molecular labeling of the present invention has high stability, can it is easy, quick, apply with high throughput / the screening of soft thorn type is pierced in cucumber all over the world firmly.
Brief description of the drawings
Fig. 1 is molecular labeling SSR-Ts1 PCR expanding effect figures.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.These embodiments are merely to illustrate the present invention and not had to In limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as 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 1
The identification of the structure of hereditary segregating population and the soft thorn gene of cucumber
More than 1. kinds of F2The structure of colony
Build F2Soft thorn kind used in colony is：NC073.Hard thorn kind has：S77, SA419-2.
NC073 is obtained by known NC072, wherein NC072, i.e. Csa1G056960-072 gDNA sequences such as sequence Shown in list SEQ ID NO.6.Csa1G056960-072 CDS sequences are as shown in sequence table SEQ ID NO.7.
Wherein it is soft thorn kind NC073 biological properties be：NC073 fruit thorniness softnesses are flexible, and touching to come off, and do not have Tingling sensation.Compared with wild type cucumber SA419-2, the maximum difference of biological structure is basal cell.The base of NC073 fruit thorniness is thin Born of the same parents are by many cells irregular alignment and are in extended bars chondritic, and SA419-2 fruit thorniness is crisp and hard, touches and easily snaps off, and has Tingling sensation, basal cell is then by the chondritic of many cells arrangement form.
Genetic analysis show that NC073 genomic DNAs sport GC in second introne splice site GT, cause Second Exon deletion of CDNA.
Wherein, SA419-2 (Csa1G056960-SA419-2) gDNA sequences are as shown in sequence table SEQ ID NO.3. NC073 (Csa1G056960-NC073) gDNA sequences are as shown in sequence table SEQ ID NO.4.NC073(Csa1G056960- NC073) CDS sequences are as shown in sequence table SEQ ID NO.5.According to NC073gDNA sequences and NC073CDS sequences, use General Biology method can obtain soft thorn kind NC073.
Hard thorn kind S77, SA419-2 is known commercially available prod.
The present embodiment has prepared cross combination using this 3 parents, obtains F1Generation, F1Generation is hard thorn individual plant；F1In generation, is certainly Hand over, backcrossing produces F2For colony and BC1Colony.In F2And BC1Occur hard thorn and soft thorn trait segregation phenomenon in colony, identification is simultaneously The hard thorn of statistics/soft thorn phenotype, hard thorn and soft thorn are analyzed in F2And BC1In segregation ratio, verified using chi-square analysis method, most Show that the soft thorn character of cucumber belongs to the recessive character of nucleus Dominant gene afterwards.
The screening of 2.Ts gene close linkage SSR markers
2.1 Cucumber germplasm DNA extraction
With CTAB methods extraction parent, BC1And 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, 750uL CTAB extractions cushioning liquid (60 DEG C of preheatings), grinder 60HZ/90s is ground, 60 DEG C 0.5-1h is placed, centre slowly shakes up 1 time.Add isometric chloroform, slowly rock 5min, 12000r/min centrifugation 10min, inhale 500uL supernatants are taken, add two volumes absolute ethyl alcohol, -20 DEG C of placement 30min, supernatant is removed in centrifugation, and 70% alcohol is washed twice.Dry in the air It is dry, 1 × TE dissolvings.Add 10ug/mL RNase.0.8% agarose gel electrophoresis detects its purity, and it is dense that nucleic acid instrument detects its Degree.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 soft each 10 plants of thorn strain individual are randomly selected in segregating population respectively, establishes hard thorn 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.Reaction system is genomic DNA 30ng, primer 0.5 μm of ol/L, 200 μm of ol/L dNTPs, 2mmol/L MgCl2, 1 μ 10 × PCR of l reactions Buffer, 0.6U Taq DNA polymerase, overall reaction system are 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, and electrophoretic buffer is 0.5 × TBE, 160W invariable power, electrophoresis 3h.
Primer includes ME3 primer sequences and EM4 primer sequences, and primer is synthesized by Shanghai Hua Jin bio tech ltd.
The primer ME3 sequences, it is specially：ME3:5’-TGAAGGTTGATTGGACAAAT-3’.
The primer EM4 sequences, it is specially：EM4：5’-TCATAACGTCATCCCTAAAA-3’.
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, statistical result is simultaneously taken pictures.Wherein developer solution component is：30g is added in 1L distilled water NaOH and 4ml formaldehyde is uniformly mixed so as to obtain.
Pierce between two hard thorn, soft thorn parents and firmly and show identical polymorphic bandses, such as Fig. 1 between pond, soft thorn pond It is shown, molecular labeling SSR-Ts1 PCR expanding effects：M represents Marker DL500, and SA419-2 is that cucumber fruits pierce parent firmly This；NC073 is the soft thorn parent of cucumber fruits；F1Represent two parent filial generations；F2Soft thorn individual plant and F2Hard thorn individual plant represents respectively In F2The 10 plants of soft thorns selected at random in colony and 10 plants of hard thorn plant.In i.e. firmly thorn parent SA419-2 and hard thorn gene pool Band is consistent, and soft thorn parent NC073 is consistent with soft thorn gene pool band；And pierce firmly in parent SA419-2 and hard thorn gene pool Band is different from soft thorn parent NC073 and soft thorn gene pool band.By 721 group of hill bodies carry out genetic linkage analysis, will with it is soft The individual that thorn parent NC073 banding pattern is consistent is designated as a, and the individual consistent with hard thorn parent's SA419-2 banding patterns is designated as into b, heterozygosis Banding pattern is designated as h.As a result this pleomorphism site isolates with Ts genes.According to 9930 Cucumber germplasm website (http:// Www.icugi.org/cgi-bin/gb2/gbrowse/cucumber_v2/) show the SSR marker apart from the soft thorn gene of cucumber Ts physical distance is 13325bp.
The above-mentioned description to embodiment is understood that for ease of those skilled in the art and using invention. Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general Principle is applied in other embodiment without by performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability Field technique personnel do not depart from improvement that scope made and modification all should be the present invention's according to the announcement of the present invention Within protection domain.
Sequence table
<110>Shanghai Communications University
<120>A kind of molecular labeling isolated with the soft thorn gene Ts of cucumber fruits and its application
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<213>Cucumber (Cucumis sativus L.)
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tttgctgtgc agatgtttaa cgagtcagga attgggtatt cggttgtttc cagaaaggaa 240
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attgaaggaa aaagaaaaca gtttagggtt ttttcaaatg tctttaatat ttaggggaaa 360
aaatgctccc ttcgttctta ttatttatgt agcgtttcct atttagtttt ttagcttact 420
ttcttcttcc ttgcattcaa gtatactaca ccccatatga gaagttgtct caatactcgg 480
tttgtcattg ttatgctatg gttataatgc ggcatcctca aattctatta ttttctctgt 540
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tcatgtccag ccctaaatca tggaatgatt cagagaccca atgcaatagt tttggtggga 660
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ctcttggtgg ctgttgggtt ggtggcagag cgttcaactc tttgaatgat ttcgtttgga 780
agtggtcaga taatgtttca aaatggaacg attccatttt tcccagcgca actttgcaat 840
cgaactgcaa aaatgcctct tgcctccgga atgattcggt tgaaacatgt acattaatat 900
ttggtggacc agcaacacca ttccttaggg acgagaaatg caacagttct caccctttta 960
tatgcatgat aaacctaggt atgttatcat cttcgaagtt tttgatacat tattgatcat 1020
agttgactat gtattttgtt tccctcggcc catttccaca cacatagatt tgaaaataaa 1080
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aaggagtatc tagttattct agctgttgtg agtgggttga tattttgcac aactttagct 1320
gtggtgattt ggcttctagc ccacaagagg agcaaaaagc ggagaaggtc gcggaagcca 1380
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ctaagatcaa tgacaaaaaa cttcagtgag ggaaaccgtc ttctcggaga tgcaaaaaca 1500
ggcggtacat acagtggact tctacctgat ggttcaagag ttgcaattaa aaggttgaag 1560
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cgtcacccaa atttagttgc cctgaaagga tgctgctatg accatggtga ccgctacatt 1680
gtttatgaat tcattgttaa tgggccctta gatagatggc ttcatcatgt accaagaggt 1740
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gatggcagga ggaacctatg gataccttgc tcctgaattt gtctacagaa acgagctaac 2100
aacaaagagt gatgtctaca gttttggggt cctattgctt gaaattgtaa ccggacgaag 2160
gcctgcacag gcagttgatt cagttggttg gcagagcatt tttgaatggg caaccccgct 2220
cgtgcaggct caccgttacc tagatctatt ggatcctcat ataacagcca cttcaacttc 2280
tgaaatccca gaggctggca ttgttcaaaa ggttgtggac cttgtttatg cttgcacaca 2340
gcatgtcccg tccatgcgac ctagaatgtc acacgtcgtt catcaactgc aacaattggc 2400
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tgcttggtgt tatattcttg ttttggctgt cccttttgaa attgtgtctt gaagttttct 180
tttgctgtgc agatgtttaa cgagtcagga attgggtatt cggttgtttc cagaaaggaa 240
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attgaaggaa aaagaaaaca gtttagggtt ttttcaaatg tctttaatat ttaggggaaa 360
aaatgctccc ttcgttctta ttatttatgt agcgtttcct atttagtttt ttagcttact 420
ttcttcttcc ttgcattcaa gtatactaca ccccatatga gaagttgtct caatactcgg 480
tttgtcattg ttatgctatg gttataatgc ggcatcctca aattctatta ttttctctgt 540
ggctaggacg gtgcccacat ggttggatta ttagcccaag caagactaag tgctttggtt 600
tcatgtccag ccctaaatca tggaatgatt cagagaccca atgcaatagt tttggtggga 660
acttagcagc tctggtaaca tatcaagagt ttagctatgc acaaaatctg tgtaatggaa 720
ctcttggtgg ctgttgggtt ggtggcagag cgttcaactc tttgaatgat ttcgtttgga 780
agtggtcaga taatgtttca aaatggaacg attccatttt tcccagcgca actttgcaat 840
cgaactgcaa aaatgcctct tgcctccgga atgattcggt tgaaacatgt acattaatat 900
ttggtggacc agcaacacca ttccttaggg acgagaaatg caacagttct caccctttta 960
tatgcatgat aaacctaggt atgttatcat cttcgaagtt tttgatacat tattgatcat 1020
agttgactat gtattttgtt tccctcggcc catttccaca cacatagatt tgaaaataaa 1080
aacaaaggag acagagacag aggatcttta tagcaagagg attggaaaga atctaacatg 1140
tttgatattc catttcctta ttctttggta gtttagtcat tgagagatgt tgtgatccac 1200
gctgagatct ttattctttt atttgtatca gatgatagat gccaccgaat gcactgtcac 1260
aaggagtatc tagttattct agctgttgtg agtgggttga tattttgcac aactttagct 1320
gtggtgattt ggcttctagc ccacaagagg agcaaaaagc ggagaaggtc gcggaagcca 1380
tccaaccctg cagcttctgc actggtaccg cctttgtgga gggtgtttac caaagaagag 1440
ctaagatcaa tgacaaaaaa cttcagtgag ggaaaccgtc ttctcggaga tgcaaaaaca 1500
ggcggtacat acagtggact tctacctgat ggttcaagag ttgcaattaa aaggttgaag 1560
aagtccagct ttcagaggaa aaaggagttt cattcagaaa ttgcaagagt tgctaggctt 1620
cgtcacccaa atttagttgc cctgaaagga tgctgctatg accatggtga ccgctacatt 1680
gtttatgaat tcattgttaa tgggccctta gatagatggc ttcatcatgt accaagaggt 1740
ggtcgaagct tagattggac tatgagaatg aaaattgcca cgactcttgc ccaagggatt 1800
gcgtatgtac aattttcatt tgtgtcttta agatgcaaaa gaacgtgctc ttgttagaga 1860
ttatcagcta actcatgaaa atgtctttta caggttcctt cacgacaagg tcaaaccaca 1920
cgtggtacat cgtgatatcc gtgcgagtaa tgtgcttctc gatgaagaat ttggagcaca 1980
tctgatggga gttggcctat cgaaattggt tgcatatgaa gtgatgcatg agaggacagt 2040
gatggcagga ggaacctatg gataccttgc tcctgaattt gtctacagaa acgagctaac 2100
aacaaagagt gatgtctaca gttttggggt cctattgctt gaaattgtaa ccggacgaag 2160
gcctgcacag gcagttgatt cagttggttg gcagagcatt tttgaatggg caaccccgct 2220
cgtgcaggct caccgttacc tagatctatt ggatcctcat ataacagcca cttcaacttc 2280
tgaaatccca gaggctggca ttgttcaaaa ggttgtggac cttgtttatg cttgcacaca 2340
gcatgtcccg tccatgcgac ctagaatgtc acacgtcgtt catcaactgc aacaattggc 2400
tccctctcct ctcactacga agtaa 2425
<210> 5
<211> 1916
<212> DNA
<213>Cucumber (Cucumis sativus L.)
<400> 5
tagggggggt tgtggtcaga gaaggcagac cgcattaact tccgaacaac cactactact 60
ctcactattg acccattcca aatccttctc cttcctctca ccccacctca cctcagctac 120
tccttcattt ctctgattcc taccacttcc tgaacttcat ggaaatggag accaacacta 180
acaccttctt tatctaatcc tcaaccattt ccattttcaa gtctacccct tctcgatgtt 240
taacgagtca ggaattgggt attcggttgt ttccagaaag gaaatttcaa aaggacggtg 300
cccacatggt tggattatta gcccaagcaa gactaagtgc tttggtttca tgtccagccc 360
taaatcatgg aatgattcag agacccaatg caatagtttt ggtgggaact tagcagctct 420
ggtaacatat caagagttta gctatgcaca aaatctgtgt aatggaactc ttggtggctg 480
ttgggttggt ggcagagcgt tcaactcttt gaatgatttc gtttggaagt ggtcagataa 540
tgtttcaaaa tggaacgatt ccatttttcc cagcgcaact ttgcaatcga actgcaaaaa 600
tgcctcttgc ctccggaatg attcggttga aacatgtaca ttaatatttg gtggaccagc 660
aacaccattc cttagggacg agaaatgcaa cagttctcac ccttttatat gcatgataaa 720
cctagatgat agatgccacc gaatgcactg tcacaaggag tatctagtta ttctagctgt 780
tgtgagtggg ttgatatttt gcacaacttt agctgtggtg atttggcttc tagcccacaa 840
gaggagcaaa aagcggagaa ggtcgcggaa gccatccaac cctgcagctt ctgcactggt 900
accgcctttg tggagggtgt ttaccaaaga agagctaaga tcaatgacaa aaaacttcag 960
tgagggaaac cgtcttctcg gagatgcaaa aacaggcggt acatacagtg gacttctacc 1020
tgatggttca agagttgcaa ttaaaaggtt gaagaagtcc agctttcaga ggaaaaagga 1080
gtttcattca gaaattgcaa gagttgctag gcttcgtcac ccaaatttag ttgccctgaa 1140
aggatgctgc tatgaccatg gtgaccgcta cattgtttat gaattcattg ttaatgggcc 1200
cttagataga tggcttcatc atgtaccaag aggtggtcga agcttagatt ggactatgag 1260
aatgaaaatt gccacgactc ttgcccaagg gattgcgttc cttcacgaca aggtcaaacc 1320
acacgtggta catcgtgata tccgtgcgag taatgtgctt ctcgatgaag aatttggagc 1380
acatctgatg ggagttggcc tatcgaaatt ggttgcatat gaagtgatgc atgagaggac 1440
agtgatggca ggaggaacct atggatacct tgctcctgaa tttgtctaca gaaacgagct 1500
aacaacaaag agtgatgtct acagttttgg ggtcctattg cttgaaattg taaccggacg 1560
aaggcctgca caggcagttg attcagttgg ttggcagagc atttttgaat gggcaacccc 1620
gctcgtgcag gctcaccgtt acctagatct attggatcct catataacag ccacttcaac 1680
ttctgaaatc ccagaggctg gcattgttca aaaggttgtg gaccttgttt atgctgcaca 1740
cagcatgtcc cgtccatgcg acctagaatg tcacacgtcg ttcatcaact gcaacaattg 1800
gctccctctc ctctcactac gaagtaaagg tattcatcag acttcaagat gatttcctat 1860
tgcaagattc taacttggca agaagccagt taatgtagga taggaaccaa ggtaga 1916
<210> 6
<211> 2425
<212> DNA
<213>Cucumber (Cucumis sativus L.)
<400> 6
atggctccga cagaggtgaa aacgctattt ctttctcttt tcgtgttaat ttggttgtcc 60
agaatgggcg tctctgatac tgtaagtagc tcgaattcga ctgattttta tcttgtttgc 120
tgcttggtgt tatattcttg ttttggctgt cccttttgaa attgtgtctt gaagttttct 180
tttgctgtgc agatgtttaa cgagtcagga attgggtatt cggttgtttc cagaaaggaa 240
atttcaaaag gtatgaagtt aactcttaga attgctactt tgcctccaag agtttggtaa 300
attgaaggaa aaagaaaaca gtttagggtt ttttcaaatg tctttaatat ttaggggaaa 360
aaatgctccc ttcgttctta ttatttatgt agcgtttcct atttagtttt ttagcttact 420
ttcttcttcc ttgcattcaa gtatactaca ccccatatga gaagttgtct caatactcgg 480
tttgtcattg ttatgctatg gttataatgc ggcatcctca aattctatta ttttctctgt 540
ggctaggacg gtgcccacat ggttggatta ttagcccaag caagactaag tgctttggtt 600
tcatgtccag ccctaaatca tggaatgatt cagagaccca atgcaatagt tttggtggga 660
acttagcagc tctggtaaca tatcaagagt ttagctatgc acaaaatctg tgtaatggaa 720
ctcttggtgg ctgttgggtt ggtggcagag cgttcaactc tttgaatgat ttcgtttgga 780
agtggtcaga taatgtttca aaatggaacg attccatttt tcccagcgca actttgcaat 840
cgaactgcaa aaatgcctct tgcctccgga atgattcggt tgaaacatgt acattaatat 900
ttggtggacc agcaacacca ttccttaggg acgagaaatg caacagttct caccctttta 960
tatgcatgat aaacctaggt atgttatcat cttcgaagtt tttgatacat tattgatcat 1020
agttgactat gtattttgtt tccctcggcc catttccaca cacatagatt tgaaaataaa 1080
aacaaaggag acagagacag aggatcttta tagcaagagg attggaaaga atctaacatg 1140
tttgatattc catttcctta ttctttggta gtttagtcat tgagagatgt tgtgatccac 1200
gctgagatct ttattctttt atttgtatca gatgatagat gccaccgaat gcactgtcac 1260
aaggagtatc tagttattct agctgttgtg agtgggttga tattttgcac aactttagct 1320
gtggtgattt ggcttctagc ccacaagagg agcaaaaagc ggagaaggtc gcggaagcca 1380
tccaaccctg cagcttctgc actggtaccg cctttgtgga gggtgtttac caaagaagag 1440
ctaagatcaa tgacaaaaaa cttcagtgag ggaaaccgtc ttctcggaga tgcaaaaaca 1500
ggcggtacat acagtggact tctacctgat ggttcaagag ttgcaattaa aaggttgaag 1560
aagtccagct ttcagaggaa aaaggagttt cattcagaaa ttgcaagagt tgctaggctt 1620
cgtcacccaa atttagttgc cctgaaagga tgctgctatg accatggtga ccgctacatt 1680
gtttatgaat tcattgttaa tgggccctta gatagatggc ttcatcatgt accaagaggt 1740
ggtcgaagct tagattggac tatgagaatg aaaattgcca cgactcttgc ccaagggatt 1800
gcgtatgtac aattttcatt tgtgtcttta agatgcaaaa gaacgtgctc ttgttagaga 1860
ttatcagcta actcatgaaa atgtctttta caggttcctt cacgacaagg tcaaaccaca 1920
cgtggtacat cgtgatatcc gtgcgagtaa tgtgcttctc gatgaagaat ttggagcaca 1980
tctgatggga gttggcctat cgaaattggt tgcatatgaa gtgatgcatg agaggacagt 2040
gatggcagga ggaacctatg gataccttgc tcctgaattt gtctacagaa acgagctaac 2100
aacaaagagt gatgtctaca gttttggggt cctattgctt gaaattgtaa ccggacgaag 2160
gcctgcacag gcagttgatt cagttggttg gcagagcatt tttgaatggg caaccccgct 2220
cgtgcaggct caccgttacc tagatctatt ggatcctcat ataacagcca cttcaacttc 2280
tgaaatccca gaggctggca ttgttcaaaa ggttgtggac cttgtttatg cttgcacaca 2340
gcatgtcccg tccatgcgac ctagaatgtc acacgtcgtt catcaactgc aacaattggc 2400
tccctctcct ctcactacga agtaa 2425
<210> 7
<211> 2008
<212> DNA
<213>Cucumber (Cucumis sativus L.)
<400> 7
tatggggggt tgtggtcaga gaaggcagac cgcattaact tccgaacaac cactactact 60
ctcactattg acccattcca aatccttctc cttcctctca ccccacctca cctcagctac 120
tccttcattt ctctgattcc taccacttcc tgaacttcat ggaaatggag accaacacta 180
acaccttctt tatctaatcc tcaaccattt ccattttcaa gtctacccct tctcgggttt 240
gggtaatggc tccgacagag gtgaaaacgc tatttctttc tcttttcgtg ttaatttggt 300
tgtccagaat gggcgtctct gatactatgt ttaacgagtc aggaattggg tattcggttg 360
tttccagaaa ggaaatttca aaaggacggt gcccacatgg ttggattatt agcccaagca 420
agactaagtg ctttggtttc atgtccagcc ctaaatcatg gaatgattca gagacccaat 480
gcaatagttt tggtgggaac ttagcagctc tggtaacata tcaagagttt agctatgcac 540
aaaatctgtg taatggaact cttggtggct gttgggttgg tggcagagcg ttcaactctt 600
tgaatgattt cgtttggaag tggtcagata atgtttcaaa atggaacgat tccatttttc 660
ccagcgcaac tttgcaatcg aactgcaaaa atgcctcttg cctccggaat gattcggttg 720
aaacatgtac attaatattt ggtggaccag caacaccatt ccttagggac gagaaatgca 780
acagttctca cccttttata tgcatgataa acctagatga tagatgccac cgaatgcact 840
gtcacaagga gtatctagtt attctagctg ttgtgagtgg gttgatattt tgcacaactt 900
tagctgtggt gatttggctt ctagcccaca agaggagcaa aaagcggaga aggtcgcgga 960
agccatccaa ccctgcagct tctgcactgg taccgccttt gtggagggtg tttaccaaag 1020
aagagctaag atcaatgaca aaaaacttca gtgagggaaa ccgtcttctc ggagatgcaa 1080
aaacaggcgg tacatacagt ggacttctac ctgatggttc aagagttgca attaaaaggt 1140
tgaagaagtc cagctttcag aggaaaaagg agtttcattc agaaattgca agagttgcta 1200
ggcttcgtca cccaaattta gttgccctga aaggatgctg ctatgaccat ggtgaccgct 1260
acattgttta tgaattcatt gttaatgggc ccttagatag atggcttcat catgtaccaa 1320
gaggtggtcg aagcttagat tggactatga gaatgaaaat tgccacgact cttgcccaag 1380
ggattgcgtt ccttcacgac aaggtcaaac cacacgtggt acatcgtgat atccgtgcga 1440
gtaatgtgct tctcgatgaa gaatttggag cacatctgat gggagttggc ctatcgaaat 1500
tggttgcata tgaagtgatg catgagagga cagtgatggc aggaggaacc tatggatacc 1560
ttgctcctga atttgtctac agaaacgagc taacaacaaa gagtgatgtc tacagttttg 1620
gggtcctatt gcttgaaatt gtaaccggac gaaggcctgc acaggcagtt gattcagttg 1680
gttggcagag catttttgaa tgggcaaccc cgctcgtgca ggctcaccgt tacctagatc 1740
tattggatcc tcatataaca gccacttcaa cttctgaaat cccagaggct ggcattgttc 1800
aaaaggttgt ggaccttgtt tatgcttgca cacagcatgt cccgtccatg cgacctagaa 1860
tgtcacacgt cgttcatcaa ctgcaacaat tggctccctc tcctctcact acgaagtaaa 1920
ggtattcatc agacttcaag atgatttcct attgcaagat tctaacttgg caagaagcca 1980
gttaatgtag gataggaacc aaggtaga 2008

Claims (7)

1. a kind of molecular labeling isolated with the soft thorn gene Ts of cucumber fruits, it is characterised in that be named as SSR-Ts1, this point Son mark has 103 nucleotide sequences shown in sequence table SEQ ID NO.1, and shown in sequence table SEQ ID NO.2 Nucleotide sequence in 101 nucleotide sequences, wherein SEQ ID NO.1 isolates with soft thorn gene, in SEQ ID NO.2 Nucleotide sequence isolates with hard Ci Jiyin.
A kind of 2. molecular labeling isolated with the soft thorn gene Ts of cucumber fruits according to claim 1, it is characterised in that The molecular labeling is expanded to obtain as the anti-sense primer shown in the sense primer and EM4 shown in ME3,
The primer ME3 sequences, it is specially：ME3：5 '-TGAAGGTTGATTGGACAAAT-3 ',
The primer EM4 sequences, it is specially：EM4：5’-TCATAACGTCATCCCTAAAA-3’.
A kind of 3. preparation method of the molecular labeling isolated with the soft thorn gene Ts of cucumber fruits, it is characterised in that the molecule Mark is expanded to obtain as the anti-sense primer shown in the sense primer and EM4 shown in ME3,
The primer ME3 sequences, it is specially：ME3：5 '-TGAAGGTTGATTGGACAAAT-3 ',
The primer EM4 sequences, it is specially：EM4：5’-TCATAACGTCATCCCTAAAA-3’.
4. a kind of preparation method of the molecular labeling isolated with the soft thorn gene Ts of cucumber fruits according to claim 3, its It is characterised by, reaction system is genomic DNA 30ng, primer 0.5 μm of ol/L, 200 μm of ol/L dNTPs, 2mmol/L MgCl2, 1 μ l 10 × PCR reactions buffer, 0.6U Taq DNA polymerases, overall reaction system is 10 μ l, and primer is Anti-sense primer shown in sense primer shown in ME3 and EM4.
5. a kind of preparation method of the molecular labeling isolated with the soft thorn gene Ts of cucumber fruits according to claim 3, its It is characterised by, PCR amplification programs are：94℃5min；94℃30s 55℃30s；72℃30s；32 circulations.Amplified production is used 8% native polyacrylamide gel electrophoresis separates, and electrophoretic buffer is 0.5 × TBE, 160W invariable powers, electrophoresis 3h.
6. as claimed in claim 1 with the application of the soft thorn gene Ts of the cucumber fruits molecular labelings isolated, it is characterised in that institute State molecular labeling be used for various cucumber pierce firmly/it is soft thorn type screening.
7. according to claim 6 with the application of the soft thorn gene Ts of the cucumber fruits molecular labelings isolated, it is characterised in that The molecular labeling, which is used for the early stage that during vegetable seeds or cotyledon is grown, to be identified.
CN201710779750.7A 2017-09-01 2017-09-01 A kind of molecular labeling isolated with the soft thorn gene Ts of cucumber fruits and its application Pending CN107513564A (en)

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CN106939352A (en) * 2017-05-03 2017-07-11 江苏师范大学 Quick screening cucumber pierces firmly/the InDel molecular labelings of soft bur real type

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Publication number Priority date Publication date Assignee Title
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