CN105349538B - With the short fruit of cucumber two SNP markers of close linkage and its application - Google Patents
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Abstract
The present invention relates to a kind of molecular labeling with the short fruit of cucumber gene close linkage of gene engineering technology field, there is provided two SNP markers with the short fruit of cucumber QTL close linkages, is respectively designated as Chr4SNP fs01 and Chr4SNP fs02.Chr4SNP fs01 are located at cucumber rice chromosome 15,735,913, and Chr4SNP fs02 are located at cucumber rice chromosome 16,650,897.The present invention obtains the SNP marker with the short fruit of cucumber gene close linkage using the method for variation group and Population Genetics, cucumber gene type is detected by the method for being sequenced and being converted into dCAPS marks, otherness of the cucumber fruit length can determine whether according to genotype, so as to accelerate the short fruit of cucumber breed breeding process.Meanwhile the two SNP markers also lay a good foundation the clone of gene and Innovation Germplasm, the short fruit of seed selection new breeds of cucumbers for the short fruit of cucumber.
Description
Technical field
The invention belongs to genetic engineering and biology field, is related to a kind of (two) and the short fruit of cucumber is close gene
Chain SNP marker.
Background technology
Cucumber is one of staple vegetable crop that China's cultivated area is maximum, planting range is most wide.With living standard
Improve, requirement of the consumer to cucumber quality increasingly increase, and cucumber fruit is important exterior quality and edible quality length, is
An important factor for causing consumer's desire to purchase.The national regulatory documents for successively having put into effect correlation are, it is specified that export special cucumber
Indicator of product variety is：Stingless or few thorn, fruit are free of bitter principle, and fruit grows 12~15 centimetres, and size is neat, and fruit length is consistent, mark
Quasi- difference is not more than the 1/5 of length, fruit surface smooth, is easy to wash；Fruit is sweet short, fragrant in taste；It is downy mildew resistance, angular leaf spot, white
More than three kinds diseases in the diseases such as powder disease, droop；Yield is with external suitable with veriety, suitable for Long term cultivation.Fruit handle
Length is the important quality trait of cucumber, while is also one of important character for influenceing cucumber commodity.We determine 12
The total sugar content with flesh chamber pulp fraction, discovery are had 10 kind fruits by the fruit of 9 days normal development fruits after cucumber variety pollination
Total sugar content be substantially less than the total sugar content of flesh chamber pulp, it is clear that fruit is the quality at position not as good as flesh chamber pulp position
Quality.
With the application and development of molecular biotechnology and genetic marker, molecular mark enters in quickening breeding
Journey, the effect to become more and more important is played in terms of improving breeding efficiency, the direct choosing to genotype can be realized according to molecular labeling
Select.In recent years, the research in terms of cucumber genetic mapping and the assignment of genes gene mapping makes remarkable progress, and has obtained cucumber bitter taste, morning
The linkage molecule mark of the characters such as ripe, fruit shape, fruit weight, sugared content, fruit colour and pulp colour, but for the short fruit of cucumber base
The Position Research of cause is relatively weak, and there has been no SNP marker to be used for report of the short fruit of cucumber character assisted Selection.Li Xiaozun
(2007) detect 7 cucumber fruits the related QTL of length (fsl1.1, fsl1.2, fsl1.3, fsl2.1, fsl2.2,
Fsl3.1, fsl3.2) (mark used：EST-SSR、SCAR)；Wang Guiling etc. (2008) detects a fruit length correlation
QTL, the mark are located between CSWGATT01B-Qchl1-CSWGATT01C marks, apart from the genetic distance difference of both sides mark
For 3.4cM and 18.0cM (marks used：SSR)；Zhao Peng (2011) detect 2 cucumber fruits the related QTL of length (Qcl1,
Qcl2) (mark used：SRAP).
The content of the invention
The invention provides a kind of (two) and the short fruit of cucumber the SNP marker of QTL close linkages and to provide for expanding
The primer pair of above-mentioned SNP marker.
The two of the present invention SNP markers with the short fruit of cucumber close linkage, be Chr4SNP-fs01 and
Chr4SNP-fs02, wherein Chr4SNP-fs01 are located at cucumber rice chromosome 15,735,913, and Chr4SNP-fs02 is located at
At cucumber rice chromosome 16,650,897.The cucumber that base is A at Chr4SNP-fs01 is short fruit handle, is herein G cucumber
For long fruit handle.The cucumber that base is G at Chr4SNP-fs02 is short fruit handle, and the cucumber for A is long fruit handle herein.
It is used to detect the above-mentioned primer pair with the short fruit of cucumber the SNP marker of gene-correlation present invention also offers one group,
- the GGGCATGTATCTCTCTACCATGATT-3 ' of sense primer 5 ' (SEQ ID NO.1) including Chr4SNP-fs01, reversely draws
- the TCCCAAAAGCAAGACAAG-3 ' of thing 5 ' (SEQ ID NO.2)；Chr4SNP-fs02 forward primer 5 '-
ACAAGTCAGTCGATCAAA-3 ' (SEQ ID NO.3) ,-TTTGCAAGCAAACATTTTTCTCTTT-3 ' of reverse primer 5 '
(SEQ ID NO.4)。
The invention also discloses described SNP and derivative dCAPS primers in the short fruit of cucumber molecular mark
In application.
Application of the present invention, specific method are：
1) Cucumber germplasm DNA to be detected is extracted；
2) using Cucumber germplasm DNA to be detected as template, Chr4SNP-fs01 primer pair and Chr4SNP-fs02 are utilized
Primer pair, carry out pcr amplification reaction；
3) PCR primer expanded using the digestion with restriction enzyme Chr4SNP-fs01 primer pairs of Hind III, use
The PCR primer digestion that MseI digestion with restriction enzyme Chr4fs-pm02 primer pairs are expanded；
4) through above-mentioned enzyme digestion rear electrophoresis, as band is consistent with Jin5-508 (i.e. can not be by Hind III and MseI digestions, only
Have a band, stripe size is respectively 224bp and 177bp), then it is long fruit strain；As band is consistent with D8 (by Hind III
Two band sizes after digestion are respectively 221bp and 3bp；By two band sizes after MseI digestions be respectively 175bp and
2bp), then it is short fruit strain.
The present invention is obtained using the method for variation group and Population Genetics and the SNP of close linkage is marked with cucumber short fruit
Note, cucumber gene type is detected by the method for being sequenced and being converted into dCAPS marks, can determine whether cucumber fruit long according to genotype
Otherness is spent, so as to accelerate the short fruit of cucumber breed breeding process.Meanwhile the two SNP markers also for the short fruit of cucumber gene
Clone lay a good foundation.
Brief description of the drawings
Fig. 1 is (P1 between the SSR marker parent of part：Short fruit is parent D8, P2：Long fruit is parent JIN5-508) polyacrylamide
Amine gel electrophoresis polymorphism.
Fig. 2 is SSR marker in F2In polyacrylamide gel electrophoresis polymorphic detection.(P1：Short fruit is parent D8, P2：
For long fruit parent JIN5-508,1 to 22 is F2, M is 20bp marker)
Fig. 3 is agarose gel electrophoresis polymorphic detection of the SNP marker between parent.(P1：Short fruit is parent D8, P2：It is long
For fruit parent JIN5-508, M is 2000bp marker)
Fig. 4 is the Ago-Gel electricity of Chr4SNP-fs01 (Fig. 4 is left) and Chr4SNP-fs02 (Fig. 4 is right) in recombinant strain
Swim polymorphic detection (M is 2000bp marker, from bottom to top respectively 2000bp, 1000bp, 750bp, 500bp, 250bp,
100bp)。
Fig. 5 is to be based on F2Recombinant strain to the short fruit of cucumber QTL carry out finely positioning (×:Recombinant strain, fs are fruit gene)
Fig. 6 is agarose gel electrophoresis polymorphic detection (P1s of the Chr4SNP-fs01 in eight cucumber self-mating systems:Short fruit
Parent D8, P2：Long fruit is parent JIN5-508,1:YN-052:SG-013:MVV 4:PD075:ZGL 6:JZ27:Zaoer
8:NY, M are 2000bp marker)
Fig. 7 is agarose gel electrophoresis polymorphic detection ((P1s of the Chr4SNP-fs02 in eight cucumber self-mating systems:It is short
Fruit is parent D8, P2：Long fruit is parent JIN5-508,1:YN-052:SG-013:MVV 4:PD075:ZGL 6:JZ27:Zaoer
8:NY, M are 2000bp marker).
Embodiment
Embodiment one
First, the identification built with exchanging individual plant of hereditary segregating population
1、F2The structure of colony
D8 (female parent), short fruit handle；Quoted from North Carolina, USA university, cane is sturdy, top when internode grows to 12-14 sections
Bound and stopped growing with a branch of female flower, blade dirty-green, fruit is short, and pulp is thick, pericarp dirty-green.
JIN5-508 (male parent), long fruit handle；For sprawling species, plant is tall and big, internode length, is from domestic variety " Tianjin spring 5
Number " (Tianjin Kerun Agricultural Science ＆ Technology Co., Ltd.) continuously selfing separated for 8 generations, fruit is selected in each filial generation longer
Plant, the long fruit for no longer separating and obtaining is selfed to the 8th generation character self-mating system.
The implementation case is configured with cross combination F using the two parents1, F1Selfing produces F2Colony.In 110 plants of F2Group
In body, the 9d fruit of cucumber fruits is developed after measurement pollination length.Reality of the assay method method with reference to (1994) such as Gu Xingfang
Standard inspection is accurate to be carried out：Cucumber fruits are carried out vertical profile by the same day, with ruler measurement fruit top end to be free of seeds chamber position length,
Its value is recorded as fruit length (cm).
2nd, Primary Location of the short fruit of cucumber gene
1st, Cucumber germplasm DNA extraction
Cucumber parents and F are extracted using CTAB methods2Meta-genomic DNA.
2nd, polymorphism SSR primer screenings
SSR primers come from the article delivered on PLoS ONE in 2009 such as Ren《An Integrated Genetic
And Cytoenetic Map of Cucumber Genome》And Cavagnaro etc. is delivered for 2010 on BMC Genomics
's《Genome-wide characterization of simple sequence repeats in cucumber
(Cucumis sativus L.).》.SSR PCR response procedures are：95 DEG C of pre-degeneration 3min；94 DEG C denaturation 45s, 68-58 DEG C
Anneal 1min (each circulation reduces by 2 DEG C), 72 DEG C of extension 1min, totally 6 circulations；94 DEG C of denaturation 30s, 58-50 DEG C of annealing 1min
(each circulation reduces by 1 DEG C), 72 DEG C of extension 1min, totally 8 circulations；72 DEG C of extension 1min；94 DEG C of denaturation 30s, 50 DEG C of annealing
30s, 72 DEG C of extension 1min, totally 20 circulations；72 DEG C of extension 7min；4 DEG C of preservations.Using 6% non-denaturing polyacrylamide gel
Electrophoresis detection SSR specificity.Polymorphism mark screening is carried out parent from 1335 SSR primers altogether, wherein 172 pairs are drawn
Thing shows specificity, polymorphic rate 12.88% in two parents.Fig. 1 is part SSR primers amplified band electrophoresis between parent
Figure.There are 172 pairs of SSR primers of polymorphism in F parent2Polymorphism checking is carried out in colony, through χ2Examine, remove and divide partially
From mark, remaining 160 pairs of SSR polymorphism primers can be used for building genetic map, and Fig. 2 is SSR marker in F2In polyacrylamide
Amine gel electrophoresis polymorphic detection band.
3rd, the short fruit of cucumber is long QTL Primary Locations
With reference to F2Each individual plant fruit is entered long measured value long gene to cucumber fruit using Interval Mapping (CIM) is met
Row positioning analysis, likelihood ratio LOD >=3, detect 2 QTL.QTL LOD value is respectively 3.0167 in the 4th linkage group, side
The wing is labeled as SSR29712, SSR10368, and two mark map distances be 11cM, and phenotypic variation rate is 14.58%, and additive effect is-
0.6855；QTL LOD value is respectively 3.8225 in the 7th linkage group, flanking marker SSR15322, SSR21743, two
Mark map distance is 16cM, and phenotypic variation rate is 23.34%, and additive effect is -1.0517.
4th, the exploitation and identification of SNP marker
Genomic DNA is carried out to parents (D8 and Jin5-508) using Highseq2500 and resurveys sequence.According to weight sequencing result
And cucumber gene group information, 30 SNPs of candidate are total between two flanking marker SSR29712, SSR10368.Candidate SNP s
It is determined that afterwards, use (the http of dCAPS Finder 2.0://helix.wustl.edu/dcaps/dcaps.htm) and Primer
Premier 5.0 separately designs the derivative mismatched primers of digestion polymorphism extension increasing sequence (dCAPS) primer and drawing for opposite side
Thing, so as to complete the conversion that SNPs marks to dCAPS.Mismatched primers design principle is length 23-25 bp, and base mismatch number is
1.Opposite side design of primers principle is length 18-22bp, target stripe length 150-200 bp.PCR amplification system uses 25
μ L amplification systems, include 1 U Taq enzymes, 1.5ul template DNAs, 2ul dNTP, 2.5ul primers, 10 × PCR of 2.5ul buffer
(containing Mg2+), add ddH2O to 25 μ L.PCR amplification programs are：94 DEG C of 3 min, cyclic process is 94 DEG C of 30s, anneal 30 s, 72
DEG C 1 min, 35 circulations, 10 min of last 72 DEG C of extensions., 52 DEG C of annealing temperature.Enter performing PCR amplification between two parents, through limit
Detected after property endonuclease digestion processed using agarose gel electrophoresis, 30 SNPs show specific totally 6 pairs parent.Fig. 3
The test strip of polymorphism is presented between parent for SNPs.
3rd, the short fruit of cucumber is gene finely positioning
F is identified using above-mentioned 6 pairs of SNP markers2Colony's (1249 plants), obtain totally 4 plants of the recombinant strain of short fruit handle.Used in parent
The middle specific polymorphism SNP marker of performance carries out specific detection in recombinant strain, and most the short fruit of cucumber is fine gene at last
Navigate between two SNP markers, be respectively designated as Chr4SNP-fs01 and Chr4SNP-fs02 (Fig. 4, left side Chr4SNP-
Fs01, right side Chr4SNP-fs02), the physical distance of two marks is 0.873Mbp (Fig. 5).Utilize the two SNP markers
QTL is contributed to final clone of the short fruit of cucumber gene by the short fruit of cucumber of positioning.
Embodiment two：
(1) cucumber self-mating system of 8 known fruits length is chosen, wherein 4 long fruit handle (ZGL, JZ2, Zaozr, NY), 4
Individual short fruit is (YN05, SG01, MVV, PD107)
(2) above-mentioned eight kinds and parental gene group DNA are extracted respectively, use Chr4SNP-fs01 and Chr4SNP-fs02
Derivative dCAPS marks to be expanded into performing PCR, then is expanded with the digestion with restriction enzyme Chr4SNP-fs01 primer pairs of Hind III
The PCR primer of increasing, the PCR primer expanded using MseI digestion with restriction enzyme Chr4SNP-fs02 primer pairs；
(3) agarose gel electrophoresis result shows：Length is identified (Fig. 6, Fig. 7) consistent with double digestion result by fruit, above-mentioned
Two SNP markers can distinguish self-mating system long fruit handle with short fruit.
Claims (5)
1. two SNP markers with the short fruit of cucumber gene close linkage, it is characterised in that be Chr4SNP-fs01 and
Chr4SNP-fs02, wherein Chr4SNP-fs01 are located at Cucumber germplasm rice chromosome 15,735,913, Chr4SNP-
The cucumber that base is A at fs01 is short fruit handle, and the cucumber for being G is long fruit handle；Chr4SNP-fs02 is located at Cucumber germplasm the 4th
At chromosome 16,650,897；The cucumber that base is G at Chr4SNP-fs02 is short fruit handle, and the cucumber for being A is long fruit handle.
2.Chr4SNP-fs01 and Chr4SNP-fs02 as the application with the short fruit of cucumber the SNP marker of gene close linkage,
Wherein Chr4SNP-fs01 is located at Cucumber germplasm rice chromosome 15,735,913, and base is A's at Chr4SNP-fs01
Cucumber is short fruit handle, and the cucumber for being G is long fruit handle；Chr4SNP-fs02 is located at Cucumber germplasm rice chromosome 16,650,
At 897；The cucumber that base is G at Chr4SNP-fs02 is short fruit handle, and the cucumber for being A is long fruit handle.
3. one group is used for the primer pair with the short fruit of cucumber the SNP marker of gene-correlation of test right requirement 1, it is special
Sign is：Primer pair including Chr4SNP-fs01：
- the GGGCATGTATCTCTCTACCATGATT-3 ' of sense primer 5 ',
- the TCCCAAAAGCAAGACAAG-3 ' of reverse primer 5 '；
Chr4SNP-fs02 primer pair：
- the ACAAGTCAGTCGATCAAA-3 ' of forward primer 5 ',
- the TTTGCAAGCAAACATTTTTCTCTTT-3 ' of reverse primer 5 '.
4. the primer described in SNP marker and claim 3 described in claim 1 is in the short fruit of cucumber molecular mark
In application.
5. application as claimed in claim 4, it is characterised in that comprise the following steps：
1) Cucumber germplasm DNA to be detected is extracted；
2) using Cucumber germplasm DNA to be detected as template, using primer pair described in claim 3, pcr amplification reaction is carried out；
3) PCR primer expanded using the digestion with restriction enzyme Chr4SNP-fs01 primer pairs of Hind III, is limited using MseI
The PCR primer digestion that property endonuclease digestion Chr4fs-pm02 primer pairs processed are expanded；
4) through above-mentioned enzyme digestion rear electrophoresis, if band is long fruit not by Hind III and MseI digestions strain；If band quilt
Hind III and MseI digestions are then short fruit strain.
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| CN104521743A (en) * | 2014-09-16 | 2015-04-22 | 扬州大学 | Cultivation method of high-powdery-mildew-resistance, short-fruit-stem, and thick-flesh cucumber chromosome single segment substitution line |
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| CN104521743A (en) * | 2014-09-16 | 2015-04-22 | 扬州大学 | Cultivation method of high-powdery-mildew-resistance, short-fruit-stem, and thick-flesh cucumber chromosome single segment substitution line |
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| 黄瓜RIL群体瓜长和把长的QTL定位分析;王敏 等;《西北植物学报》;20141231;第34卷(第9期);第1764-1770页 * |
| 黄瓜果把长度的遗传分析及QTL定位;瞿文琴 等;《中国园艺学会黄瓜分会第五届会议论文集》;20150401;摘要 * |
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