CN109182588A - The molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene - Google Patents
The molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene Download PDFInfo
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- 238000002372 labelling Methods 0.000 title claims abstract description 41
- 244000299906 Cucumis sativus var. sativus Species 0.000 title 1
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- 239000002773 nucleotide Substances 0.000 claims abstract description 12
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 12
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- 235000013311 vegetables Nutrition 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 2
- 235000010071 Cucumis prophetarum Nutrition 0.000 description 2
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Abstract
The present invention relates to the molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene.Molecular labeling Indel-de2 has 1492 nucleotide sequence fragments in sequence table SEQ ID NO.1, and 189 nucleotide sequence fragments in sequence table SEQ ID NO.2, wherein the nucleotide sequence in SEQ ID NO.1 is isolated with one dimensional infinite wall gene, and the nucleotide sequence in SEQ ID NO.2 is isolated with determinate growth gene.Downstream primer shown in evaluation of markers upstream primer and EM4 as shown in ME3 expands to obtain.ME3:5 '-CCTCTCGATCCCAACACCAC-3 ', EM4:5 '-CAGGCAGGCAAGTTTTGGAC-3 '.Molecular labeling Indel-de2 has high stability, can it is easy, quickly, be applied to cucumber determinate growth/one dimensional infinite wall type screening all over the world with high throughput.
Description
Technical field
The present invention relates to the molecular labeling of gene engineering technology field, in particular to one and cucumber one dimensional infinite wall genes
The Indel molecular labeling Indel-de2 isolated.
Background technique
Cucumber (Cucumis sativus L.) is Curcurbitaceae (Cucurbitaceae) Cucumis (Cucumis) 1 year climing
Herbaceous plant is climbed up by holding on in life.One of cucumber vegetable crop important greatly as the world ten and one of main cultivation vegetable crop in China,
The cucumber variety of the main cultivation in China is sprawling species.
The length of cucumber plant main stem is plant height, can be divided into sprawling species and Dwarf cultivar according to vines characteristic, this stem
Climing characteristic also influences one of an important factor for cucumber economic benefit.Having a characteristic in cucumber Dwarf cultivar is colored topping, i.e.,
Determinate growth.Cucumber determinate growth gene possesses long research history.1940, Hutchins reported limited life for the first time
Long gene de, after this, there are also multidigit scholars to carry out a series of research to this gene, it is indicated that dominant gene De control is yellow
The character that do not bind of melon, and recessiveness de is the character that binds, in addition there is also some modifier In-de.2003, Fazio etc.
Using limited short raw cucumber kind G421 and cucumber variety H-19 building group of overgrowing, de is located in linkage group, with label
CSWCT28 is chain, and distance is 5.0cM, and detailed results may refer to: Fazio etc. is in " Theoretical and Applied
Genetics " (theoretical applied genetics) the 5th 875-883 pages of the phase entitled " Comparative delivered of volume 107 in 2003
analysis of response to phenotypic and marker-assisted selection for multiple
Ateral branching in cucumber (Cucumis sativus L.) " text.2005, from cucumber cultivation kind
Two libraries BAC are constructed in self-mating system, de gene and label AJ6SCAR are chain, and detailed results may refer to: Nam etc. exists
150-161 pages of volume 111 of " Theoretical and Applied Genetics " (theoretical applied genetics) 2005 are delivered
Entitled " Construction of two BAC libraries from cucumber (Cucumis sativus L.) and
Identification of clones linked to yield component quantitative trait loci " one
Text.2010, father-in-law benefit group is equal to construct map with Gy7 × H-19 for parent, and determinate growth gene de is located in No. 6 chromosomes
It marks between XCSWCTT14b and XSSR13251, is located at 80.7-86.3cM, detailed results may refer to: Weng Y Q etc.
In " HortScience " (horticultural science) the 6th 882-886 pages of the phase entitled " An extended delivered of volume 45 in 2010
Intervarietal microsatellite linkage map of cucumber, Cucumis sativus L. " text.
Demand with the continuous expansion of Chinese cucumber in greenhouse cultivated area and market to processing type cucumber, for cucumber
Plant height is it is also proposed that different breedings requires.The general plant of the cucumber that overgrows is tall and big, and breeding time is long, and yield is high.And limited dwarfing
Cucumber plant is shorter and smaller, precocious, and in result set, growth period is short, is more suitable mechanized harvesting and the cultivation in facility, can
To significantly improve planting density, to improve yield, human and material resources and financial resources are saved.Therefore how to select that plant height is suitable, strain
The cucumber variety that type rationally, convenient for different cultivations requires is necessary.And with the molecule with purpose character close linkage
It is highly effective method in cucumber genetic breeding that assisted Selection, which is marked, in label, and molecular labeling is right on DNA level
Objective trait is selected, and is had many advantages, such as not limited by environmental condition efficiently, quickly, can be selected in seedling stage, accelerate to educate
The process of kind.
Summary of the invention
The purpose of the present invention selects the requirement of the plant type of suitable plant, provides one under being that meeting different cultivations requires
The Indel molecular labeling isolated with cucumber indeterminate growth gene is named as Indel-de2, and this label utilizes agarose
Gel electrophoresis can clearly indicate difference, convenient to be applied to determinate growth marker assisted selection.
The present invention is sent out using the sequencing of molecular labeling, BSA (Bulked Segregant Analysis) between method and parent
Existing, the deletion mutation of a 20kb in determinate growth parent causes the afunction of determinate growth gene.So as to cause
Determinate growth character, is mutated using insertion/deletion, and the present invention develops the molecule isolated with cucumber indeterminate growth gene
Indel-de2, molecular labeling Indel-de2 is marked to be characterized in that isolating with one dimensional infinite wall gene.It provides and Huang
The molecular labeling Indel-de2 that melon one dimensional infinite wall gene isolates.Molecular labeling Indel-de2 of the invention can be easy, fast
Speed 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 cucumber one dimensional infinite wall gene, is named as Indel-de2, the molecular labeling
Indel-de2 has 1492 nucleotide sequence fragments and sequence table SEQ ID shown in sequence table SEQ ID NO.1
189 nucleotide sequence fragments shown in NO.2, wherein the nucleotide sequence in SEQ ID NO.1 and one dimensional infinite wall gene are total
It separates, the nucleotide sequence in SEQ ID NO.2 is isolated with determinate growth gene.
Molecular labeling Indel-de2 has high stability, can be applied to all over the world easy, quickly, with high throughput
The screening of cucumber determinate growth/one dimensional infinite wall.
The above-mentioned Indel molecular labeling Indel-de2 isolated with one dimensional infinite wall gene de, by ME3 primer sequence and
EM4 primer sequence expands to obtain.Primer is synthesized by Shanghai Hua Jin Biotechnology Co., Ltd.
The ME3, sequence specifically: ME3:5 '-CCTCTCGATCCCAACACCAC-3 '；
The EM4, sequence specifically: EM4:5 '-CAGGCAGGCAAGTTTTGGAC-3 '.
The present invention obtains F using a variety of determinate growth parents and one dimensional infinite wall hybridization1, F1Single plant is indeterminate growth
Type；F1It is returned again with determinate growth parent and obtains BC1Group, by BC1One dimensional infinite wall, determinate growth in segregating population
Genetic analysis is carried out, determines that cucumber determinate growth character belongs to single-gene recessive inheritance character.From BC1It is random each in group
10 plants of determinate growth single plants of picking and 10 plants of one dimensional infinite wall single plants, and construct one dimensional infinite wall pond and determinate growth pond.
Using Indel molecular labeling combination BSA method, cucumber indeterminate growth linkage molecule label is screened, finally by indeterminate growth gene
De is located in the section 468.8kb.In the 468.8kb, Indel-de2 shows polymorphism between parent, and with de base
Because isolating.
The present invention provides the preparation method of the molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene, institute
Downstream primer shown in molecular labeling Indel-de2 upstream primer and EM4 as shown in ME3 is stated to expand to obtain,
The ME3, sequence specifically: ME3:5 '-CCTCTCGATCCCAACACCAC-3 '；
The EM4, sequence specifically: EM4:5 '-CAGGCAGGCAAGTTTTGGAC-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 polymerase, overall reaction system is 10 μ l.
PCR amplification program are as follows: 94 DEG C of 5min；94℃30s 55℃30s；72℃30s；32 circulations.
Amplified production is separated with 1% agarose gel electrophoresis, and electrophoretic buffer is 1 × TAE, 120W invariable power, electrophoresis
20min。
The molecular labeling Indel-de2 of the present invention isolated with cucumber one dimensional infinite wall gene is for the limited life of cucumber
The screening of elongated/one dimensional infinite wall type.
The molecular labeling Indel-de2 is for during cucumber seeds or early stage that cotyledon is grown is identified.
Traditional breeding method is selected according to the form of plant, since cucumber determinate growth character needs plant
Occur just can determine that when self sealing top, it is longer to expend the time.Compared with prior art, molecular labeling of the invention and cucumber are infinitely raw
Long character isolates, and all cucumber varieties all over the world can use this molecular marker screening indeterminate growth/determinate growth
Kind.The early stage that molecular labeling Indel-de2 of the invention can be during vegetable seeds or cotyledon is grown can identify, i.e.,
It is time saving also accurate, so can be used for cucumber molecular labeling Indel-de2 assistant breeding, accelerate the process of cucumber quality breeding.
Detailed description of the invention
Fig. 1 is the PCR amplification effect of molecular labeling Indel-de2.
Specific embodiment
In the following with reference to the drawings and specific embodiments, the present invention is further explained.These embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sambrook etc.
Molecular cloning: described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989)
Condition, or according to the normal condition proposed by manufacturer.
Embodiment 1
The building and the identification of cucumber indeterminate growth gene of hereditary segregating population
1.F2The building of group
Construct F2Determinate growth kind used in group are as follows: WI1983H.Indeterminate growth kind has: H38.The present embodiment
Cross combination has been prepared using this 2 parents, has obtained F1Generation, F1Generation is indeterminate growth single plant；F1Generation selfing and WI1983H parent
This backcrossing generates F2For group and BC1Group.In F2And BC1Occur indeterminate growth and determinate growth trait segregation phenomenon in group,
Indeterminate growth/determinate growth phenotype is identified and counted, analyzes indeterminate growth and determinate growth in F2And BC1In segregation ratio, utilize
Chi-square analysis method is verified, and finally show that cucumber indeterminate growth character belongs to the recessive character of nucleus Dominant gene.
The screening of 2.de gene close linkage Indel label
The extraction of 2.1 Cucumber germplasm DNA
Parent, BC are extracted with CTAB method1And F2The blade total DNA of segregating population.Method are as follows: take single plant spire be put in 2mL from
In heart pipe, two steel balls are added, 750uL CTAB is extracted buffer solution (60 DEG C of preheatings), grinder 60HZ/90s grinding, and 60 DEG C
0.5-1h is placed, centre slowly shakes up 1 time.Add isometric chloroform, slowly rock 5min, 12000r/min is centrifuged 10min, inhales
500uL supernatant is taken, two volumes dehydrated alcohol, -20 DEG C of placement 30min is added, supernatant is removed in centrifugation, and 70% alcohol is washed twice.It dries in the air
It is dry, 1 × TE dissolution.The RNA enzyme of 10ug/mL is added.0.8% agarose gel electrophoresis detects its purity, and it is dense that nucleic acid instrument detects its
Degree.It is finally diluted to the concentration of 50ng/uL, -20 DEG C store for future use.
The foundation of 2.2 determinate growths/indeterminate growth gene pool
BSA method is from F2Indeterminate growth strain and each 10 plants of individual of determinate growth strain are randomly selected in segregating population respectively, is established
The gene pool of indeterminate growth and determinate growth cucumber.
The screening of 2.3 labels
Two parents and two gene pools are screened with 551 pairs of Indel primer combinations.Reaction system is genome
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 program are as follows: 94 DEG C of 5min；94℃30s 55℃
30s；72℃30s；32 circulations.Amplified production is separated with 1% agarose gel electrophoresis, and electrophoretic buffer is 1 × TAE, 120W
Invariable power, electrophoresis 20min.
Primer includes ME3 primer sequence and EM4 primer sequence, and primer is synthesized by Shanghai Hua Jin Biotechnology Co., Ltd.
The ME3, sequence specifically: ME3:5 '-CCTCTCGATCCCAACACCAC-3 '；
The EM4, sequence specifically: EM4:5 '-CAGGCAGGCAAGTTTTGGAC-3 '.
It is shown between indeterminate growth pond, determinate growth pond between two indeterminate growth, determinate growth parents identical
Polymorphic bands, as shown in Figure 1, the PCR amplification effect of molecular labeling Indel-De2: M represents Marker DL2000,83H, i.e.,
WI1983H, cucumber determinate growth parent；H38 is cucumber indeterminate growth parent；BC1 group represents and selects at random in BC1 group
Plant；Indeterminate growth kind is respectively c-8-6,50,30, G38, S06, and 422,9930, S52,61, GY14,419-2 and Xia Mei
Human relations.
Indeterminate growth parent H38 is consistent with the band in indeterminate growth gene pool, determinate growth parent WI1983H and limited
Growth hormone gene pond band is consistent.Genetic linkage analysis is carried out by 340 group of hill bodies, by the band with determinate growth parent WI1983H
The consistent individual of type is denoted as a, will be denoted as b with the consistent individual of indeterminate growth parent's H38 banding pattern, heterozygosis banding pattern is denoted as h.As a result this
A polymorphic site is isolated with De gene.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Sequence table
<110>Shanghai Communications University
<120>the molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1492
<212> DNA
<213>cucumber (Cucumis sativus L.)
<400> 1
cctctcgatc ccaacaccac ttattactat cagtgcagct cgaactcggc ccggaatttc 60
agtttcaaaa ccccacctgc tcagctcccc atcaaatttg ttgtcattgg tatgtataga 120
aggaaacttc tgctacacaa gaaaaacttg ttaatggctt caagtttttg tacaaataac 180
ccatttccag aggtgtttat gtggaatgac ccattcctaa aaaacctatg aaaatcgacc 240
tattgttgac attatagagc actaatgtcc actgtgaaat aatcgaaggt cttatgcaat 300
atcgttttaa ttgagatttt taaaatcaac agttggccaa ccggtgatta aaaatgaata 360
tcgcgttaga gctttgatca tctcgcagtg ggtgaggcat ctctagcacg ataatgcatc 420
tatgacttag gcaaaaattt ggttttgcat taattttcaa gggttgggtc attctgcatt 480
aatgtcgcta cgatggagtc atttatatct ctagaaacta agtcgtgtgt taattcttat 540
gatcaatgtt ttggtaggtg atcttgggca gacagaatgg acggaaacaa ctcttaaaaa 600
tgtagcgaaa tcagactatg atgtgctcct acttcccggt gacttgtcgt atgctgatta 660
cattcagtct ctatgggact cttttggcag gctcgtcgag ccactggcca gccaaaggcc 720
atggatggtg actcatggca atcatgaagt agagcgcatc ccattgattc atcccctccc 780
tttcacagca tacaatgcaa gatggcacat gcctttcgaa cagagctctt caagttccaa 840
cctatactat tcattcaaca cggctggtgt gcacgttatc atgttgggat cgtataccga 900
tttcgacaag agttcagctc agtacgaatg gctcgtagcg gacttgaaga agattgatcg 960
tgcaacaacc ccatggattg tggtgcttct tcatgctccg tggtataact ccaacactgc 1020
tcaccaggga gagaaagagt ctgttgacat gaaggcagcc atggaagatc tgctctatca 1080
agctcgagta gacgtggtgt ttgctgggca tgtccatgct tatgagcgct tcgtatgttt 1140
ctctaacaat caagatcact ttttatttac atttaagcga agttgatata tctttcctta 1200
cctgaataac ccttttaaat cattttatta ttgcagactc gtgtttataa tggggaagca 1260
aacaattgtg caccaatata tatcacaatc ggcgacgggg gcaaccgtga aggccttgcg 1320
agcaagtaat acttcaaact tgtttttcaa tatataacat tttacaagag ttcaaacata 1380
ctttatttag atactgtgcg ttaaactact actagactaa aaagtttaat cttatgcagg 1440
tttatggacc cgacgccaac gatttcatta ttcaggcagg caagttttgg ac 1492
<210> 1
<211> 189
<212> DNA
<213>cucumber (Cucumis sativus L.)
<400> 1
cctctcgatc ccaacaccac ttattactat cagtgcagct cgaactcggc ccggaatttc 60
agtttcaaaa ccccacctgc tcagctcccc atcaaatttg ttgtcattgg tatgtataga 120
aggaaacttc tgctacacaa gaaaaacttg ttaatggctt caagtttttg tacaaataac 180
ccatttcca 189
Claims (8)
1. a kind of molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene, which is characterized in that the molecular labeling
Indel-de2 has 1492 nucleotide sequence fragments and sequence table SEQ ID shown in sequence table SEQ ID NO.1
189 nucleotide sequence fragments shown in NO.2, wherein the nucleotide sequence in SEQ ID NO.1 and one dimensional infinite wall gene are total
It separates, the nucleotide sequence in SEQ ID NO.2 is isolated with determinate growth gene.
2. a kind of molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene according to claim 1,
It being characterized in that, downstream primer shown in molecular labeling Indel-de2 upstream primer and EM4 as shown in ME3 expands to obtain,
The ME3, sequence specifically: ME3:5 '-CCTCTCGATCCCAACACCAC-3 '；
The EM4, sequence specifically: EM4:5 '-CAGGCAGGCAAGTTTTGGAC-3 '.
3. a kind of preparation method of the molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene, which is characterized in that
Downstream primer shown in molecular labeling Indel-de2 upstream primer and EM4 as shown in ME3 expands to obtain,
The ME3, sequence specifically: ME3:5 '-CCTCTCGATCCCAACACCAC-3 '；
The EM4, sequence specifically: EM4:5 '-CAGGCAGGCAAGTTTTGGAC-3 '.
4. a kind of molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene is obtained according to claim 3
The method of obtaining, which is characterized in that 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 polymerase, overall reaction system is 10 μ
l。
5. a kind of molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene is obtained according to claim 3
The method of obtaining, which is characterized in that PCR amplification program are as follows: 94 DEG C of 5min；94℃30s55℃30s；72℃30s；32 circulations.
6. a kind of molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene is obtained according to claim 3
The method of obtaining, which is characterized in that amplified production is separated with 1% agarose gel electrophoresis, and electrophoretic buffer is 1 × TAE, 120W perseverance function
Rate, electrophoresis 20min.
7. a kind of application of the molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene as described in claim 1,
It is characterized in that, the molecular labeling Indel-de2 is used for the screening of cucumber determinate growth/one dimensional infinite wall type.
8. a kind of molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene is answered according to claim 7
With, which is characterized in that the molecular labeling Indel-de2 is used for the early stage progress that during cucumber seeds or cotyledon is grown
Identification.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811536A (en) * | 2017-03-16 | 2017-06-09 | 上海交通大学 | The InDel molecular labelings isolated with cucumber ZYMV resistant genes |
| CN106868156A (en) * | 2017-03-16 | 2017-06-20 | 上海交通大学 | The novel I nDel molecular labelings isolated with cucumber ZYMV resistant genes |
| 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 |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811536A (en) * | 2017-03-16 | 2017-06-09 | 上海交通大学 | The InDel molecular labelings isolated with cucumber ZYMV resistant genes |
| CN106868156A (en) * | 2017-03-16 | 2017-06-20 | 上海交通大学 | The novel I nDel molecular labelings isolated with cucumber ZYMV resistant genes |
| 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 |
| 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 |
Non-Patent Citations (2)
| Title |
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| YIQUN WENG等: "An Extended Intervarietal Microsatellite Linkage Map of Cucumber,Cucumis sativus L.", 《HORTSCIENCE》 * |
| 苗晗等: "利用永久群体在不同环境下定位黄瓜株高QTL", 《中国农业科学》 * |
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