CN105838812B - The SSR molecular marker isolated with cucumber powdery mildew resistance main effect QTL - Google Patents
The SSR molecular marker isolated with cucumber powdery mildew resistance main effect QTL Download PDFInfo
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Abstract
The SSR molecular marker isolated the present invention provides one with cucumber powdery mildew resistance main effect QTL, is named as SSR-N2.The nucleotide sequence of SSR-N2 is as shown in SEQ ID NO.1.SSR molecular marker of the invention has high stability, the assisting sifting of cucumber at seedling stage powder mildew resistance single plant can be simply and quickly applied to, it lays a good foundation for the molecular mark of powder mildew resistance, this will greatly speed up the process of cucumber powdery mildew resistance molecular breeding.Meanwhile this molecular labeling isolated is also that the clone of cucumber powdery mildew resistance main effect QTL lays the foundation.
Description
The present invention is entitled " SSR molecular marker isolated with cucumber powdery mildew resistance main effect QTL ", application number
It is 2014103823068, the applying date is the divisional application of the earlier application of 2014-8-6.
Technical field
The present invention relates to technique for gene engineerings, and in particular to the SSR molecule isolated with cucumber powdery mildew resistance main effect QTL
Label.
Background technique
Cucumber (Cucumis sativus L.) overgrows for Curcurbitaceae (Cucurbitaceae) Cucumis annual herb climbs
Edge plant.Cucumber is as one of one of big important vegetable crop in the world ten and the main cultivation vegetable crop in China, Zhan Quanguo vegetables
10% or so of area.Cucumber serves not only as important vegetable crop always by the attention of breeder, and cucumber chromosomal
The less 2n=2x=14 of number, genome is smaller, and especially in 2009, the successful sequencing of Cucumber germplasm is cucumber as cucurbit
The model plant of section carries out molecular biology research and provides great convenience.
In cucumber production, the harm of many diseases is faced, wherein powdery mildew is one of disease the most serious.Cucumber is white
Powder disease is the fungal disease as caused by obligate parasite (Podosphaera xanthii), can be fallen ill in entire cucumber breeding time,
Main harm blade can endanger vines when serious, especially serious in the middle and late growth stage morbidity, so that plant is uprooted plants after their edible portions have been harvested ahead of time, cause
Serious production loss.Field rotating medicine causes pesticide residue, influences fruit quality, jeopardizes food safety, and can pollute ring
Border.Long-term application can also promote Physiological Races of Powdery Mildew to generate resistance, to increase difficulty of prevention and cure, increase farmer and be produced into
This.Cultivating disease-resistant cucumber variety is the best method for solving powdery mildew harm.Time-consuming for conventional breeding for disease resistance, need by
Mostly generation hybridization and backcross process；Disease generation is closely related with environmental condition, and special sick nursery is needed to carry out inoculated identification；These
Both increase the difficulty for cultivating cucumber disease-resistant variety.
Molecular breeding can greatly speed up breeding process, significantly shortening breeding cycle.The fast development of modern biotechnology,
New approach is opened for breeding for disease resistance, is cultivated disease-resistant variety using biotechnology and is had become current hot spot.Molecular breeding
On condition that obtaining the functional gene of correlated traits or the molecular labeling with its close linkage.Using molecular marker analysis system,
Resistant heredity rule is identified in genetic group, is composed in combination with molecular markers linkage map, positioning and clone's powder mildew resistance
Gene studies the molecular regulation mechanism of its function and resistance, can for cucumber resistant gene molecular mark and point
Sub- design and context provides theoretical foundation.Using the molecular labeling with disease-resistant gene close linkage, carry out molecular marker assisted selection
Multiple disease-resistant genes can be integrated into a kind by breeding, significantly improve breeding efficiency, shorten breeding time, and significantly
Disease-resistant dynamics and persistence are improved, this has important meaning for cultivating the cucumber disease-resistant varieties for meeting grower's demand
Justice.
Although the generation of ground family crop powdery mildew of cucumber is commonplace and serious, about its powdery mildew resistance gene
The research of positioning is relatively weak, not yet finds the label isolated with powdery mildew resistance gene/QTL, needless to say gene
The research of clone and Resistance mechnism rests on major gene resistance/QTL positioning stage at present.Since many reports show Huang
Melon powder mildew resistance is controlled by multiple recessive genes, and researcher has carried out the analysis of QTL to it.2006, Sakata etc. was utilized
The combined recombinant inbred lines of the anti-sense of 97 plants of cucumber, located the QTL of anti-cucumber powdery mildew character for the first time；In 4 linkage groups
On detect 6 QTLs relevant to temperature, wherein a main effect QTL on LGII shows to resist at 20 DEG C and 26 DEG C
Property.The F that Liu et al. (2008a) is constructed using cucumber height sense powdery mildew self-mating system S94 and high mildew-resistance self-mating system S062:3Family
QTL positioning has been carried out, has detected 5 powder mildew resistance QTL altogether, has been distributed in linkage group 1,2,5, the contribution rate of single QTL is situated between
Between 3.4%~45%；4 powder mildew resistance QTL are also detected by the recombinant inbred lines that this two parent constructs altogether, respectively
In linkage group 1,2,4,6, the contribution rate of single QTL (Liu et al., 2008b) between 5.2%~21.0%.Shen
Beautiful flat (2009) apply ISSR (inter-simple sequence repeats) and SRAP (sequence-related
Amplified polymorphism) labelling technique, with height sense powdery mildew cucumber variety D8 and high powdery mildew resisting green cucumber variety
The F of JIN52Crowd surveillance is respectively positioned in the 3rd linkage group, contribution rate 7.6% to 2 QTL of control cucumber powdery mildew resistance
With 13.5%.Holy equality (2011) is opened with the F of (susceptible) combination of K8 (disease-resistant) × K182And F2:3Family is research object, is examined altogether
Measure the QTL of 4 powder mildew resistances.Recently, Fukino etc. (2013) detects 9 QTL using recombinant inbred lines, is located at
On chromosome 1,3,4,5,6, the contribution rate of single QTL is between 5%~44%, wherein the effect in 4 sites passes through residue
Heterozygote (residual heterozygous lines, RHLs) is confirmed.He etc. (2013) utilizes F2:3Family is to Huang
The white powder resistance of melon hypocotyl, cotyledon and true leaf has carried out qtl analysis simultaneously, as a result detects 6 on 1,3,4, No. 5 chromosome
A QTL, the contribution rate of single QTL is between 6.1%~74.5%；Wherein 2 main effect QTLs are located at the 40cM of No. 5 chromosome
Section in, explain 21.0-74.5% contribution rate, hypocotyl resistance QTL plays most important work to cucumber white powder resistance
With.
It is above-mentioned most studies have shown that cucumber to the resistance of powdery mildew by multiple gene collective effects, and resistant gene table
It is now recessive effect, these factors increase disease-resistant gene finely positioning and isolated difficulty.Since powdery mildew of cucumber is to cucumber
Influence of production is very big, many to the research of cucumber powdery mildew resistance gene, but there has been no the cloned resistance gene and divides at present
The report of sub- Mechanism Study, acquired linked marker genetic distance farther out, are unfavorable for the development of molecular mark, resistance
The process of disease-resistant variety molecular breeding is hindered.Therefore, find with cucumber powdery mildew resistance gene/QTL close linkage, isolate
Molecular labeling carries out finely positioning, separation and clone to it, this is not only able to provide good technology for its disease-resistant molecular breeding
Support, also lays the foundation to open the molecular mechanism of cucumber powdery mildew resistance.
Summary of the invention
The present invention is application No. is 2014103823068, and the applying date is the divisional application of the earlier application of 2014-8-6.
The purpose of the present invention is to overcome the problems, such as that QTL positioning difficulty is big, provides one and cucumber powdery mildew resistance main effect
The codominance SSR molecular marker that QTL pm5.1 is isolated.The present invention using BSA (Bulked Segregant Analysis) and
QTL positioning mode finds the main effect QTL of a control powder mildew resistance.For this main effect QTL of finely positioning, we, which construct, contains
The chromosome segment substitution line (Chromosome Segment Substitution Lines, CSSL) of main effect QTL and its backcrossing
Segregating population.By the exploitation of finely positioning and label, three SSR isolated with cucumber powdery mildew resistance main effect QTL are obtained
Label, so as to the foundation of molecular mark system.Molecular labeling of the invention can be applied easy, quickly, with high throughput
In breeding practice.
The present invention is achieved by the following technical solutions:
One SSR molecular marker isolated with cucumber powdery mildew resistance main effect QTL, is named as SSR-N2, nucleotide
Sequence is as shown in SEQ ID NO.1.
Above-mentioned SSR-N2 is obtained by upstream primer SSR-N2-F and downstream primer SSR-N2-R PCR amplification, and the upstream is drawn
The sequence of object SSR-N2-F is 5 '-CTTCATTGTTGATTTCCAGGC-3 ', and the sequence of the downstream primer SSR-N2-R is 5 '-
TGTTACGACCTATAACCACAAAAT-3’。
The present invention BSA and QTL positioning mode, utilizes F2Group finds the main effect QTL of a control powder mildew resistance.In order to
This main effect QTL of finely positioning constructs the chromosome segment substitution line containing main effect QTL using the method being constantly returned
(Chromosome Segment Substitution Lines, CSSL) and its backcrossing segregating population.By separating group to backcrossing
Body BC3F1, BC2F2Resistance Identification analysis, powder mildew resistance has turned into the heredity of single Mendelian factor, i.e., by Dominant gene, resists
Property is recessive inheritance.By finely positioning, resistant gene is navigated between label UW065021 and UW065094, physical distance
170kb.By Cucumber germplasm sequence, developing SSR is marked therebetween, finally obtains three and cucumber powdery mildew resistance main effect
The SSR marker that QTL is isolated is named as SSR-N1, SSR-N2, SSR-N3 respectively.It is total that 3, which isolate SSR molecular marker,
Dominant marker can distinguish homozygote and heterozygote, be beneficial to building for the molecular labeling assistant system of powder mildew resistance breeding
It is vertical, can it is easy, quickly, be applied to breeding practice with high throughput.
Compared with prior art, the invention has the following beneficial effects: conventional traditional breeding for disease resistance, time-consuming, need through
Excessive generation hybridization and backcross process；Disease generation is closely related with environmental condition, and special sick nursery is needed to carry out inoculated identification；This
The difficulty for cultivating cucumber disease-resistant variety is both increased a bit.Since Powdery Mildew is obligate parasite, Resistance Identification needs to be inoculated with, and
Plant is easy to die after the onset；And powdery mildew resistance gene shows as recessive effect more, and conventional method back cross breeding needs back
Be selfed a generation again after handing over a generation to confirm the infiltration of resistant gene, these factors both increase powder mildew resistance breeding period and
Difficulty.The SSR molecular marker that isolates of the invention is codominance, can identify plant in cucumber at seedling stage, and can distinguish homozygote
And heterozygote, backcrossing penetrate into during eliminate per generation selfing the step of, resistant gene is tracked with molecular labeling, it is both time saving or
Accurately, so can be used for the molecular mark of cucumber powdery mildew resistance, cucumber powdery mildew resistance breeding is greatly accelerated
Process.The clone that label will also promote powder mildew resistance QTL/ gene is isolated simultaneously, to be formed to disclose powder mildew resistance
Molecular mechanism lay the foundation.
Detailed description of the invention
Fig. 1 is the polyacrylamide gel electrophoresis effect of molecular labeling SSR-N1；
Fig. 2 is the polyacrylamide gel electrophoresis effect of molecular labeling SSR-N2；
Fig. 3 is the polyacrylamide gel electrophoresis effect of molecular labeling SSR-N3.
As shown in the figure, S1003 is disease-resistant parent；S05 is Susceptible parent；F1Represent two parent filial generations；R and S difference
Represent BC2F2The testing result of the disease-resistant and disease plant selected at random in backcrossing segregating population.
Specific embodiment
One, cucumber powdery mildew resistance main effect QTL/gene identification
1. informative population and Resistance Identification
Preliminary Resistance QTL positioning is F2Group, disease-resistant parent are S1003, and Susceptible parent is S1001, they are all
Belong to the cucumber self-mating system of North China type.The F that two parents generate1Generation selfing generates F2For group.Powder mildew resistance identifies institute
The strain used is isolated from the cucumber plant that Shanghai Communications University greenhouse is fallen ill.Randomly choose 148 plants of F2Generation
Body carries out Resistance Identification in seedling stage.Spore suspension is made with sterile water in the Powdery Mildew that purifying is acquired from susceptible seedling, adjusts
It is 1 × 10 to concentration5A mL-1.Deployed spore suspension is uniformly sprayed onto very when cucumber third piece true leaf is just unfolded
Ye Shang can not be subject to drops.Incidence is investigated after inoculated and cultured 12d.The incidence of plant is according to Morishita etc.
(2003) 5 grades are divided into.0 grade and 1 grade be considered as it is disease-resistant, 2 grades or more be considered as it is susceptible.
According to incidence survey, S1003 is highly resistance, and S1001 is high sense, F1Be it is susceptible, be partial to Susceptible parent；F2Group is anti-
Bimodal distribution is presented in characteristic of disease, and incidence trend is partial to susceptible, and intermediate form is less, illustrate to control in the presence of recessive major gene resistance
The disease resistance of white powder.
2.BSA method and qtl analysis determine the chromosome location of major gene resistance
Therefore the chromosomal region where we take BSA method elder generation antagonism major gene resistance is analyzed.From F2Separation
Highly resistance and individual each 10 plants of high sense are randomly selected in group respectively, establishes anti-, sense gene pool.It is average on chromosome with 780 pairs
Two parents of SSR primer pair of distribution and two gene pools screen.
SSR reaction system is genomic DNA 30ng, primer 0.2 μm of ol/L, 200 μm of ol/L dNTPs, 2mmol/L
MgCl2,1 μ l 10 × PCR reactions buffer, 0.5U Taq DNA polymerase, overall reaction system are 10 μ l.PCR amplification
Program are as follows: 94 DEG C of 5min；35 circulations: 94 DEG C of 30s, 55-60 DEG C of 30s, 72 DEG C of 30s；72℃5min.Amplified production is become with 6%
Property polyacrylamide gel electrophoresis separation, electrophoretic buffer be 1 × TBE, 45W invariable power, electrophoresis 1.5h-2h.
Silver staining is carried out after electrophoresis.Silver staining method are as follows: the glass plate with glue is put into fixer, is gently shaken on shaking table
To indicator color fade, the wherein composition of fixer are as follows: glacial acetic acid, dehydrated alcohol, distilled water volume ratio be 1:10:100；
With ultrapure washing 1min-3min；Offset plate after flushing is put into dyeing liquor and shakes half an hour, wherein the group of dyeing liquor is divided into
2g/L silver nitrate；Offset plate after dyeing is put into after rinsing 5s in ultrapure water and is put into the plastic casing equipped with developer solution, gently shaken
It is clear to band to move, and is put into tap water and rinses 3min；It dries, takes pictures after dry at room temperature, wherein developer solution is distilled in 1L
It is added what 15g NaOH and 3ml formaldehyde was uniformly mixed so as to obtain in water.
It is screened by BSA, continuous 7 SSR markers in the 5th chromosome long arm end show as polymorphism between pond, because
This powdery mildew resistance main effect gene is located in this region.In order to further determine the position of resistance main effect gene, we are carried out
Qtl analysis.73 polymorphism SSR markers of average selection are to 148 plants of F from 7 chromosomes of cucumber2It is carried out for individual
Electrophoretic analysis.Linkage map is constructed using JoinMap 3.0, wherein LOD >=5.0, are converted recombination fraction using Kosambi function
For genetic distance.By F2The mean value of three blade grades of the group's single plant disease index disease-resistant as plant carries out QTL mapping.
Qtl analysis uses QTL Cartographer 2.5, carries out QTL mapping using composite interval mapping method (CIM).Pass through QTL points
It is analysing that as a result, finding a main effect QTL pm5.1 in the long-armed end of the 5th chromosome, this coincide with the position that BSA method obtains,
Therefore we determined that this position exist one control powder mildew resistance main effect QTL.
Two, the building of segregating population and the finely positioning of main effect QTL are returned
Since parent S1003 and S1001 are North China type cucumber, affiliation is close, and polymorphism mark is few, therefore constructs
Using the European greenhouse cucumber self-mating system S05 of high sense powdery mildew as donor parents when being returned segregating population.In conjunction with MAS
By being selfed after mostly generation backcrossing, the backcross population BC of only main effect QTL pm5.1 region disconnecting is constructed3F1, BC2F2.Respectively to 2
A BC3F1Group's (114 plants, 480 plants) and BC2F2The Resistance Identification of group's (483 plants), anti-, sense ratio meet by chi-square analysis
1:1 and 1:3, therefore, Resistance QTL have been converted into single Mendelian factor, i.e. Dominant gene, and disease-resistant is recessive inheritance.It is logical
It crosses the developed molecular labeling in main effect QTL region and linkage analysis is carried out to this 1074 single plants, it is as a result that disease-resistant gene is finely fixed
Position is arrived between SSR marker UW065021 and UW065094, physical distance 170kb.By Cucumber germplasm sequence, open therebetween
15 pairs of SSR markers have been sent out, have as a result had polymorphism, i.e. molecular labeling SSR-N1, SSR-N2, SSR-N3 parent for only 3 betweens.It is logical
Group's linkage analysis is crossed, finally show that these three SSR molecular markers are isolated with cucumber powdery mildew resistance main effect QTL, i.e. group
Disease-resistant plant banding pattern it is all consistent with disease-resistant parent's banding pattern, and all Susceptible parents of disease plant banding pattern or F1Band
Type.
Fig. 1 is the polyacrylamide gel electrophoresis effect of molecular labeling SSR-N1, and Fig. 2 is poly- the third of molecular labeling SSR-N2
Acrylamide gel electrophoresis effect, Fig. 3 are the polyacrylamide gel electrophoresis effects of molecular labeling SSR-N3.
Claims (2)
1. a SSR molecular marker isolated with cucumber powdery mildew resistance main effect QTL, is named as SSR-N2, nucleotides sequence
Column are as shown in SEQ ID NO.1.
2. the SSR molecular marker according to claim 1 isolated with cucumber powdery mildew resistance main effect QTL, feature exist
In the SSR-N2 is obtained by upstream primer SSR-N2-F and downstream primer SSR-N2-R PCR amplification, the upstream primer
The sequence of SSR-N2-F is 5 '-CTTCATTGTTGATTTCCAGGC-3 ', and the sequence of the downstream primer SSR-N2-R is 5 '-
TGTTACGACCTATAACCACAAAAT-3’。
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| CN104498484B (en) * | 2014-12-01 | 2018-01-16 | 北京市农林科学院 | The dominant disease-resistant gene pm1 of Pumpkin powdery mildew linkage molecule mark and its application |
| CN104498486B (en) * | 2014-12-02 | 2017-03-01 | 中国农业科学院蔬菜花卉研究所 | The Indel labelling of anti-cucumber powdery mildew gene pm h and its application |
| CN104560983B (en) * | 2015-01-30 | 2017-03-08 | 扬州大学 | Two SNP marker and its application with anti-cucumber powdery mildew close linkage |
| CN105734057B (en) * | 2016-04-29 | 2019-05-10 | 新疆农业科学院哈密瓜研究中心 | With the SSR marker and its application of muskmelon downy mildew resistance main effect QTL linkage |
| CN105969860A (en) * | 2016-05-17 | 2016-09-28 | 青岛市农业科学研究院 | Anti-cucumber-powdery-mildew major QTL (quantitative trait locus) mapping |
| CN108546778B (en) * | 2018-07-19 | 2021-06-01 | 上海市农业科学院 | SNP molecular marker for detecting powdery mildew resistance of cucumber and application thereof |
| CN109234429B (en) * | 2018-08-01 | 2021-07-20 | 扬州大学 | Two SNP markers closely linked with powdery mildew resistance of cucumber and application thereof |
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| CN101240342A (en) * | 2008-03-13 | 2008-08-13 | 上海交通大学 | Cucumber powdery mildew resistance main effect QTL compact linkage molecule labeling method and applying method |
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| CN101240342A (en) * | 2008-03-13 | 2008-08-13 | 上海交通大学 | Cucumber powdery mildew resistance main effect QTL compact linkage molecule labeling method and applying method |
Non-Patent Citations (3)
| Title |
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| "Identification and validation of powdery mildew(Podosphaera xanthii)-resistant loci in recombinant inbred lines of cucumber (Cucumis sativus L.)";N. Fukino等;《Mol Breeding》;20130420;第32卷(第2期);摘要，第271页左栏第2，3段，第274页最后一段，表1 * |
| "黄瓜抗白粉病染色体片段导入系的SSR鉴定";林肖剑;《园艺学报》;20120325;第39卷(第3期);第485-492页 * |
| "黄瓜白粉病抗性基因QTL定位";张圣平;《中国农业科学》;20110901;第44卷(第17期);第3584-3593页 * |
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| Publication number | Publication date |
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| CN105838712B (en) | 2018-11-16 |
| CN104152446A (en) | 2014-11-19 |
| CN105838712A (en) | 2016-08-10 |
| CN105838812A (en) | 2016-08-10 |
| CN104152446B (en) | 2017-03-15 |
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