CN106434906A - SNP locus linked with broccoli ball flower lanatoside character and detecting method and primer set thereof - Google Patents
SNP locus linked with broccoli ball flower lanatoside character and detecting method and primer set thereof Download PDFInfo
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- CN106434906A CN106434906A CN201610823124.9A CN201610823124A CN106434906A CN 106434906 A CN106434906 A CN 106434906A CN 201610823124 A CN201610823124 A CN 201610823124A CN 106434906 A CN106434906 A CN 106434906A
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Abstract
The invention belongs to the technical field of vegetable molecule biology, and particularly relates to an SNP locus linked with the broccoli ball flower lanatoside character and a detecting method and primer set thereof. The SNP locus is linked with the broccoli ball flower lanatoside phenotype; through the combination of the detecting method of the SNP locus, the lanatoside character of a broccoli material can be selected in an assistant mode through genotype detection. The SNP locus has the advantages of being high in specificity, convenient to detect, low in cost and the like, the breeding cycle can be remarkably shortened, and efficiency is improved.
Description
Technical field
The invention belongs to vegetable field of molecular biotechnology is and in particular to the SNP chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation
Site and its detection method and primer sets.
Background technology
Brassica oleracea L. var. botrytis L. is one of important vegetable crop of China, is also one of main foreign exchange earning vegetable of China, its plantation
Region almost covers all parts of the country.According to Food and Agricultural Organization of the United Nations（FAO）And United States Department of Agriculture (USDA)（USDA）Statistics, China spends
Broccoli cultivated area and total output all arrange No. 1 in the world, in recent years respectively stable annual 400000 hectares and 9,000,000 tons with
On, about 100,000,000 dollars of its middle aged foreign exchange earning volume.
The commodity organ bouquet of Brassica oleracea L. var. botrytis L. be the surface being collectively formed by many cripetura flower bud branches and inflorescence meristem more
Rounding, smooth spheroid.And in Brassica oleracea L. var. botrytis L. produces, bouquet surface often occurs developmental hairy alabastrum in a large number, this
Phenomenon is referred to as " hair flower ".Hair is spent very big to bouquet quality and yield effect, bouquet commodity can be caused to completely lose, be when serious
One of most serious problem that Brassica oleracea L. var. botrytis L. industry faces.Although environmental factorss（Especially temperature）Impact to hair flower phenomenon is not allowed to neglect
Depending on, but there is significant difference in terms of resistance to/easy hair flower between different cultivars/genotype, it is heritable for imply this character.With
Show, hair floral formation has higher heritability and significant selection effect, and environmental effect is then very limited toward research.Thus
The genetic improvement of hair floral formation is also one of important topic of Brassica oleracea L. var. botrytis L. breeding for a long time.Therefore, by identification and bouquet hair
The related sequence variations of floral formation, the exploitation molecular marker of close linkage and practical detection method therewith, and be applied to
In breed breeding, it is substantially shorter breeding cycle, improve breeding efficiency.
Content of the invention
Strong for not high to the efficiency of selection of bouquet hair floral formation in Brassica oleracea L. var. botrytis L. traditional breeding method, environment and experience dependency
Problem, first purpose of the present invention is to provide a kind of SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation, for Brassica oleracea L. var. botrytis L.
The resistance to hair of bouquet spends molecular mark.Second object of the present invention is to provide a kind of above-mentioned spending with Brassica oleracea L. var. botrytis L. bouquet hair
The detection method of the SNP site of the linkage of characters, the method is quick, stable.Second object of the present invention is to provide a kind of detection
The Specific primer pair of the above-mentioned SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation.
First purpose of the present invention technical scheme is that：
A kind of SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation it is characterised in that：Corresponding curd trait is resistance to hair flower,
This site and its flanking sequence are 5 '-CCAAAACAAAAGTGCAAAATCTTATATATGCGCGCGGCTT
GTATGTATTTATGTGATTTTTTCAAATGTAAACGTGTTTA-3’；Corresponding curd trait be Yi Maohua, this site and
Its flanking sequence is 5 '-CCAAAACAAAAGTGCAAAATCTT ATATATGCACGCGGCTTGTATGTATTTATGTGATTTTT
TCAAATGTAAACGTGTTTA-3’.
Second object of the present invention technical scheme is that：
A kind of detection method of the SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation, the method include following in step：
1）Brassica oleracea L. var. botrytis L. extracting genome DNA to be detected：Brassica oleracea L. var. botrytis L. material to be detected is extracted base respectively according to conventional CTAB method
Because organizing DNA, -20 DEG C preserve, standby；
2）The PCR amplification of detected sample genomic DNA：Using Specific primer pair, the genomic DNA of detected sample is divided
Do not enter performing PCR amplification, Specific primer pair middle and upper reaches primer：5’- CCAAAACAAAAGTGCAAAATCT -3’;Downstream primer
:5'- AAGGCCCTATTGGTA ACTTG -3’；Reaction cumulative volume 20 μ L, concrete system is as follows：2.5 mmol/L are above-mentioned to be drawn
Thing, 0.2 mmol/L dNTPs, 1 U Taq enzyme, 2mmol/L MgCl2,1 × amplification buffer, genomic DNA 50 ng, plus
Dd H2O to 20 μ L；Thermocycling program is：94 DEG C of denaturation 2 min, 1 circulation；94 DEG C of degeneration 1min, 55 DEG C of renaturation 30 s,
72 DEG C of extension 45 s, 35 circulations；72 DEG C of extension 10min；4 DEG C of preservations；
3）The endonuclease reaction of PCR primer and electrophoresis detection：By 2）In PCR primer utilize restricted enzyme BseP I at 50 DEG C
Under the conditions of enzyme action 3 h, enzyme action system product containing PCR 7 μ L, 10 × buffer 1 μ L, 1 U MaeI enzyme, plus dd H2O is extremely
10μL；After enzyme action, its product is separated by electrophoresis on 1.2% agarose gel, ethidium bromide develops the color；
4）The identification of detected sample genotype：According to 3）Middle gel-tape pattern, the sample of display 160 bp is containing easy hair
The material of flower genotype, corresponding curd trait is Yi Maohua；And show 128 bp sample be then containing resistance to hair spend genotype
Material；Corresponding curd trait is resistance to hair flower.
Third object of the present invention technical scheme is that：
A kind of Specific primer pair of the described SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation of detection, this special primer
Combination middle and upper reaches primer：5’- CCAAAACAAAAGTGCAAAATCT -3’;Downstream primer:5'- AAGGCCCTATTGGTA
ACTTG -3’.
The invention has the beneficial effects as follows：
（1）The SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation that the present invention provides and its detection method can be applicable to bouquet
The assisted Selection of hair floral formation, easy to detect, low cost.Breeding material can be detected in seedling stage using this method,
Thus the material of easy hair flower genotype can be contained in a large number in forepart elimination, be greatly reduced field planting needed for the later stage, field management,
The workload of phenotypic evaluation.In addition, the labelling in the present invention be based on SNP exploitation, this be from now on realize high-throughout oneself
Dynamicization genotype detection provides possibility.
（2）Traditional breeding way places one's entire reliance upon and the investigation of the phenotype of bouquet hair floral formation is selected.And Yi Maohua pair
Resistance to hair flower is dominant, and the individuality for Yi Maohua for the phenotype does not represent and do not carry resistance to hair flower gene locis, hence with phenotype choosing
Generally require an extra selfing generation when selecting resistance to hair flower, need to take considerable time.The gene of corresponding site can be directed to using this method
Type is identified, directly determines that whether containing resistance to hair in sample spends genotype.
Specific embodiment
By following examples, the present invention is described in further detail, but present disclosure is not limited thereto.
Embodiment 1：（The genotype identification in cauliflower germplasm resource resistance to hair flower site）
（1）By the sowing of cauliflower germplasm resource, nursery, point individual plant numbering.Take each single-strain blade in 2-3 piece leaf period according to routine
CTAB method extracts genomic DNA respectively, and -20 DEG C preserve, standby.
（2）Using Specific primer pair（Forward primer：5’- CCAAAACAAAAGTGCAAAATCT -3’;Downstream primer
:5'- AAGGCCCTATTGGTA ACTTG -3’）Right（1）In DNA sample enter respectively performing PCR amplification.Reaction cumulative volume 20 μ
L, concrete system is as follows：The above-mentioned primer of 2.5 mmol/L, 0.2 mmol/L dNTPs, 1 U Taq enzyme, 2mmol/L MgCl2, 1
× amplification buffer, genomic DNA 50 ng, plus dd H2O to 20 μ L.Thermocycling program is：94 DEG C of denaturation 2 min, 1
Circulation；94 DEG C of degeneration 1min, 55 DEG C of renaturation 30 s, 72 DEG C of extension 45 s, 35 circulations；72 DEG C of extension 10min；4 DEG C of preservations.
（3）Will（2）In PCR primer utilize restricted enzymeBsePI enzyme action 3 h under the conditions of 50 DEG C, enzyme action system
Product containing PCR 7 μ L, 10 × buffer 1 μ L, 1 UMaeI enzyme, plus dd H2O to 10 μ L.After enzyme action, its product is existed
It is separated by electrophoresis on 1.2% agarose gel, ethidium bromide develops the color.
（4）Foundation（3）Middle gel-tape pattern, the sample of display 128 bp is then the material containing resistance to hair flower genotype.
These resources select other economical characters good carry out continuous selfing and screening, obtain a collection of resistance to hair flower and economical character
Excellent breeding material.
Embodiment 2：（The molecular marker auxiliary improvement of Brassica oleracea L. var. botrytis L. selfing line ' 4251 ' hair floral formation）
Brassica oleracea L. var. botrytis L. selfing line ' 4251 ' economical character is excellent, but bouquet easy hair flower in some environments, need to be difficult using bouquet
The selfing line ' 4229 ' of hair flower is oriented genetic improvement to it.Specific molecular marker amplified production in the present invention is between the two
There is the polymorphism of 160bp/128bp, therefore can be utilized the present invention to carry out molecular marker assisted selection in backcross process.
（1）' 4229 ' and ' 4251 ' hybridization and backcrossing：Will ' 4229 ' and ' 4251 ' seeds sow respectively, nursery, field planting
In hot house.With ' 4229 ' as male parent, ' 4251 ' is maternal florescence manually to be broadcast flower bud hybridization, collects after kind of pod maturation
Seed, as F1.Next year is by F1Seed and recurrent parent ' 4251 ' seed are sowed respectively, nursery, be colonized in hot house.With F1
For male parent, ' 4251 ' is maternal florescence manually to be broadcast flower bud hybridization, collects seed after kind of pod maturation（More than 200）, that is,
For BC1F1.
（2）BC1F1Seedling stage generation molecular marker assisted selection：The BC that will collect1F1Generation seed randomly selects 200, cave
Disk is sowed, nursery, and point individual plant numbering.Each single-strain blade is taken to extract base respectively according to conventional CTAB method in 2-3 piece leaf period
Because organizing DNA, -20 DEG C preserve, standby.Using Specific primer pair（Forward primer：5’- CCAAAACAAAAGTGCAAAATCT -
3’;Downstream primer:5'- AAGGCCCTATTGGTAACTTG -3’）Above-mentioned DNA is entered respectively with performing PCR amplification.Reaction cumulative volume
20 μ L, concrete system is as follows：The above-mentioned primer of 2.5 mmol/L, 0.2 mmol/L dNTPs, 1 U Taq enzyme, 2mmol/L
MgCl2,1 × amplification buffer, genomic DNA 50 ng, plus dd H2O to 20 μ L.Thermocycling program is：94 DEG C of denaturations 2
Min, 1 circulation；94 DEG C of degeneration 1min, 55 DEG C of renaturation 30 s, 72 DEG C of extension 45 s, 35 circulations；72 DEG C of extension 10min；4℃
Preserve.PCR primer is utilized restricted enzymeBsePI enzyme action 3 h under the conditions of 50 DEG C, enzyme action system product containing PCR 7 μ
L, 10 × buffer 1 μ L, 1 UMaeI enzyme, plus dd H2O to 10 μ L.By its product in 1.2% agarose gel after enzyme action
On be separated by electrophoresis, ethidium bromide develop the color.In this BC1F1In from generation to generation, the individual plant being shown as 128 bp is considered as spending site containing resistance to hair
The material of genotype is retained, and the individual plant being shown as 160 bp is then considered as the material containing easy hair flower loci gene type and gives
Eliminate.Select other agronomy, economic characters closest to the plant of recurrent parent ' 4251 ' and recurrent parent in selected individual plant
' 4251 ' hybridization, obtain BC2F1From generation to generation.
（3）The selection of follow-up backcross generations：To the BC obtaining2F1From generation to generation according to above-mentioned the（2）、（3）Step is selected,
And then it is returned acquisition BC3F1From generation to generation；And in kind selected and be returned until BC6F1From generation to generation, then to it carry out selfing,
Obtain the BC of inheritance stability6F2From generation to generation, therefrom screen the individuality of resistance to hair flower site homozygosis, as hair floral formation is significantly improved
' 4251 '.
<110>Zhejiang Academy of Agricultural Science
<120>A kind of SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation and its detection method and primer sets
<160>4
<210>1
<211>80
<212>DNA
<213>Brassica oleracea L. var. botrytis L.
<400>1
CCAAAACAAA AGTGCAAAAT CTTATATATG CGCGCGGCTT GTATGTATTT ATGTGATTTT 60
TTCAAATGTA AACGTGTTTA 80
<210>2
<211>80
<212>DNA
<213>Brassica oleracea L. var. botrytis L.
<400>2
CCAAAACAAA AGTGCAAAAT CTTATATATG CACGCGGCTT GTATGTATTT ATGTGATTTT 60
TTCAAATGTA AACGTGTTTA 80
<210>3
<211>22
<212>DNA
<213>Artificial sequence
<400>3
CCAAAACAAA AGTGCAAAAT CT 22
<210>4
<211>20
<212>DNA
<213>Artificial sequence
<400>4
AAGGCCCTAT TGGTAACTTG 20
Claims (3)
1. a kind of SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation it is characterised in that：Corresponding curd trait is resistance to hair
Flower, this site and its flanking sequence are 5 '-CCAAAACAAAAGTGCAAAATCTTATATATGCGCGCGGCTT
GTATGTATTTATGTGATTTTTTCAAATGTAAACGTGTTTA-3’；Corresponding curd trait be Yi Maohua, this site and
Its flanking sequence is 5 '-CCAAAACAAAAGTGCAAAATCTT ATATATGCACGCGGCTTGTATGTATTTATGTGATTTTT
TCAAATGTAAACGTGTTTA-3’.
2. the detection method of the SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation described in a kind of claim 1, its feature exists
In the method include following in step：
1）Brassica oleracea L. var. botrytis L. extracting genome DNA to be detected：Brassica oleracea L. var. botrytis L. material to be detected is extracted base respectively according to conventional CTAB method
Because organizing DNA, -20 DEG C preserve, standby；
2）The PCR amplification of detected sample genomic DNA：Using Specific primer pair, the genomic DNA of detected sample is divided
Do not enter performing PCR amplification, Specific primer pair middle and upper reaches primer：5’- CCAAAACAAAAGTGCAAAATCT -3’;Downstream primer
:5'- AAGGCCCTATTGGTA ACTTG -3’；Reaction cumulative volume 20 μ L, concrete system is as follows：2.5 mmol/L are above-mentioned to be drawn
Thing, 0.2 mmol/L dNTPs, 1 U Taq enzyme, 2mmol/L MgCl2, 1 × amplification buffer, genomic DNA 50 ng, plus
dd H2O to 20 μ L；Thermocycling program is：94 DEG C of denaturation 2 min, 1 circulation；94 DEG C of degeneration 1min, 55 DEG C of renaturation 30 s,
72 DEG C of extension 45 s, 35 circulations；72 DEG C of extension 10min；4 DEG C of preservations；
3）The endonuclease reaction of PCR primer and electrophoresis detection：By 2）In PCR primer utilize restricted enzymeBsePI is at 50 DEG C
Under the conditions of enzyme action 3 h, enzyme action system product containing PCR 7 μ L, 10 × buffer 1 μ L, 1 UMaeI enzyme, plus dd H2O is extremely
10μL；After enzyme action, its product is separated by electrophoresis on 1.2% agarose gel, ethidium bromide develops the color；
4）The identification of detected sample genotype：According to 3）Middle gel-tape pattern, the sample of display 160 bp is containing easy hair
The material of flower genotype, corresponding curd trait is Yi Maohua；And show 128 bp sample be then containing resistance to hair spend genotype
Material；Corresponding curd trait is resistance to hair flower.
3. a kind of test right requires the Specific primer pair of the SNP site chain with Brassica oleracea L. var. botrytis L. bouquet hair floral formation described in 1,
It is characterized in that this Specific primer pair middle and upper reaches primer：5’- CCAAAACAAAAGTGCAAAATCT -3’;Downstream primer:
5'- AAGGCCCTATTGGTA ACTTG -3’.
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| CN201610823124.9A CN106434906B (en) | 2016-09-14 | 2016-09-14 | A kind of and cauliflower bouquet hair floral formation chain SNP site and its detection method and primer sets |
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| CN111733274A (en) * | 2020-07-01 | 2020-10-02 | 浙江省农业科学院 | SNP locus, primer set and method for identifying cauliflower Zhe nong piny flower 80-day variety |
| CN111793710A (en) * | 2020-07-01 | 2020-10-20 | 浙江省农业科学院 | SNP marker linked with cauliflower ball-bottom flower stalk branch angle, method and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733274A (en) * | 2020-07-01 | 2020-10-02 | 浙江省农业科学院 | SNP locus, primer set and method for identifying cauliflower Zhe nong piny flower 80-day variety |
| CN111793710A (en) * | 2020-07-01 | 2020-10-20 | 浙江省农业科学院 | SNP marker linked with cauliflower ball-bottom flower stalk branch angle, method and application |
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| CN106434906B (en) | 2019-09-13 |
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