CN106434906B - A kind of and cauliflower bouquet hair floral formation chain SNP site and its detection method and primer sets - Google Patents
A kind of and cauliflower bouquet hair floral formation chain SNP site and its detection method and primer sets Download PDFInfo
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- CN106434906B CN106434906B CN201610823124.9A CN201610823124A CN106434906B CN 106434906 B CN106434906 B CN 106434906B CN 201610823124 A CN201610823124 A CN 201610823124A CN 106434906 B CN106434906 B CN 106434906B
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Abstract
The invention belongs to vegetables field of molecular biotechnology, and in particular to chain SNP site and its detection method and primer sets with cauliflower bouquet hair floral formation.SNP site of the present invention and cauliflower bouquet hair flower phenotype are chain, in conjunction with the detection method of the SNP site, can carry out assisted Selection by hair floral formation of the genotype detection to cauliflower material.The advantages that invention has high specificity, easy to detect, low-cost, can be obviously shortened breeding cycle, improve efficiency.
Description
Technical field
The invention belongs to vegetables field of molecular biotechnology, and in particular to the chain SNP with cauliflower bouquet hair floral formation
Site and its detection method and primer sets.
Background technique
Cauliflower is one of main foreign exchange earning vegetables of one of important vegetable crop in China and China, plantation
Region almost covers all parts of the country.It is counted according to Food and Agricultural Organization of the United Nations (FAO) and United States Department of Agriculture (USDA), China is spent
Cabbage cultivated area and total output arrange No. 1 in the world, in recent years respectively it is stable annual 400000 hectares and 9,000,000 tons with
On, middle aged about 100,000,000 dollars of volume of foreign exchange earning.
The commodity organ bouquet of cauliflower be the surface that is collectively formed by many cripetura flower buds branch and inflorescence meristem more
Rounding, smooth sphere.And in cauliflower production, bouquet surface often will appear a large amount of developmental hairy buds, this
Phenomenon is known as " hair flower ".Hair is spent very big to bouquet quality and yield effect, will cause bouquet commodity when serious and completely loses, is
One of the most serious problem that cauliflower industry faces.Although influence of the environmental factor (especially temperature) to hair flower phenomenon is not allowed to neglect
Depending on, but between different cultivars/genotype in terms of resistance to/Yi Maohua there are significant difference, it is heritable for imply the character.With
It is past studies have shown that hair floral formation genetic force with higher and significant selection effect, and environmental effect is then very limited.Thus
The genetic improvement of hair floral formation is also one of the important topic of cauliflower breeding for a long time.Therefore, pass through identification and bouquet hair
The relevant sequence variations of floral formation are developed the molecular labeling of close linkage and practical detection method therewith, and are applied to
In breed breeding, it is substantially shorter breeding cycle, improves breeding efficiency.
Summary of the invention
Strong for not high to the efficiency of selection of bouquet hair floral formation in cauliflower traditional breeding method, environment and experience dependence
Problem, the first purpose of the invention is to provide a kind of and chain SNP sites of cauliflower bouquet hair floral formation, are used for cauliflower
The resistance to hair flower molecular mark of bouquet.A second object of the present invention is to provide a kind of above-mentioned colored with cauliflower bouquet hair
The detection method of the SNP site of the linkage of characters, this method is quick, stablizes.A second object of the present invention is to provide a kind of detections
The Specific primer pair of the above-mentioned SNP site chain with cauliflower bouquet hair floral formation.
The first purpose of this invention has the technical scheme that
A kind of and chain SNP site of cauliflower bouquet hair floral formation, it is characterised in that: corresponding curd trait is resistance to hair
Flower, the site and its flanking sequence are 5 '-CCAAAACAAAAGTGCAAAATCTTATATATGCGCGCGGCTT GTATGTATT
TATGTGATTTTTTCAAATGTAAACGTGTTTA-3'；Corresponding curd trait is Yi Maohua, the site and its flanking sequence
For 5 '-CCAAAACAAAAGTGCAAAATCTT ATATATGCACGCGGCTTGTATGTATTTATGTGATTTTT
TCAAATGTAAACGTGTTTA-3’。
Second object of the present invention has the technical scheme that
A kind of detection method with the chain SNP site of cauliflower bouquet hair floral formation, this method includes below in step
It is rapid:
1) cauliflower extracting genome DNA to be detected: cauliflower material to be detected is mentioned respectively according to conventional CTAB method
Genomic DNA is taken, -20 DEG C of preservations are spare；
2) PCR amplification of sample to be tested genomic DNA: using Specific primer pair to the genome of sample to be tested
DNA carries out PCR amplification, Specific primer pair middle and upper reaches primer: 5 '-CCAAAACAAAAGTGCAAAATCT -3 ' respectively;Under
Swim primer: 5'- AAGGCCCTATTGGTA ACTTG -3 '；20 μ L of total volume is reacted, specific system is as follows: 2.5 mmol/L
Above-mentioned primer, 0.2 mmol/L dNTPs, 1 U Taq enzyme, 2mmol/L MgCl2,1 × amplification buffer, genomic DNA 50
Ng adds dd H2O to 20 μ L；Thermocycling program are as follows: 94 DEG C of 2 min of initial denaturation, 1 circulation；94 DEG C of denaturation 1min, 55 DEG C of renaturation
30 s, 72 DEG C of 45 s of extension, 35 circulations；72 DEG C of extension 10min；4 DEG C of preservations；
3) endonuclease reaction and electrophoresis detection of PCR product: 2) PCR product in is existed using restriction enzyme BseP I
3 h of digestion under the conditions of 50 DEG C, 71 μ L of μ L, 10 × buffer of digestion system product containing PCR, 1 UBseP I enzyme add dd
H2O to 10 μ L；Its product is separated by electrophoresis on 1.2% Ago-Gel after digestion, ethidium bromide colour developing；
4) identification of sample to be tested genotype: according to gel-tape mode in 3), the sample of 160 bp of display be containing
The material of Yi Maohua genotype, corresponding curd trait are Yi Maohua；And show that the sample of 128 bp is then containing resistance to Mao Huaji
Because of the material of type；Corresponding curd trait is resistance to hair flower.
Third object of the present invention has the technical scheme that
The Specific primer pair of the SNP site chain with cauliflower bouquet hair floral formation described in a kind of detection, this is special
Primer combines middle and upper reaches primer: 5 '-CCAAAACAAAAGTGCAAAATCT -3 ';Downstream primer: 5'-
AAGGCCCTATTGGTA ACTTG -3’。
The beneficial effects of the present invention are:
(1) SNP site and its detection method chain with cauliflower bouquet hair floral formation provided by the invention can be applied to
The assisted Selection of bouquet hair floral formation, it is easy to detect, it is low-cost.Breeding material can be carried out in seedling stage using this method
Detection, so as to the material in forepart elimination largely containing Yi Maohua genotype, field planting needed for the later period is greatly reduced, field
Between management, phenotypic evaluation workload.In addition, the label in the present invention is developed based on SNP, this is high to realize from now on
The automation genotype detection of flux provides possibility.
(2) traditional breeding way, which places one's entire reliance upon, selects the phenotype investigation of bouquet hair floral formation.And Yi Maohua pairs
Resistance to hair flower be it is dominant, the individual that phenotype is Yi Maohua, which does not represent, does not carry resistance to hair flower gene loci, therefore is selected using phenotype
It generally requires additionally to be selfed a generation when selecting resistance to hair flower, need to take considerable time.The gene of corresponding site can be directed to using this method
Whether type is identified, directly determine in sample containing resistance to hair flower genotype.
Specific embodiment
By following embodiment, the present invention is described in further detail, but the contents of the present invention are not limited thereto.
The genotype identification in embodiment 1:(cauliflower germplasm resource resistance to hair flower site)
(1) by the sowing of cauliflower germplasm resource, nursery, single plant is divided to number.In 2-3 piece leaf period take each single-strain blade according to
Conventional CTAB method extracts genomic DNA respectively, and -20 DEG C of preservations are spare.
(2) Specific primer pair (upstream primer: 5 '-CCAAAACAAAAGTGCAAAATCT -3 ' is utilized;Downstream primer:
5'- AAGGCCCTATTGGTA ACTTG -3 ') PCR amplification is carried out respectively to the DNA sample in (1).React 20 μ of total volume
L, specific system are 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, 50 ng of genomic DNA, adds dd H2O to 20 μ L.Thermocycling program are as follows: 94 DEG C of initial denaturation 2 min, 1
Circulation；94 DEG C of denaturation 1min, 55 DEG C of 30 s of renaturation, 72 DEG C of 45 s of extension, 35 recycle；72 DEG C of extension 10min；4 DEG C of preservations.
(3) PCR product in (2) is utilized into restriction enzymeBsePI 3 h of digestion, digestion system under the conditions of 50 DEG C
71 μ L of μ L, 10 × buffer of product containing PCR, 1 U BseP I enzyme add dd H2O to 10 μ L.Its product is existed after digestion
It is separated by electrophoresis on 1.2% Ago-Gel, ethidium bromide colour developing.
(4) according to gel-tape mode in (3), the sample of 128 bp of display is then the material containing resistance to hair flower genotype.
The progress for selecting other economical characters good in these resources is continuously selfed and is screened, and the resistance to hair flower of a batch and economical character are obtained
Excellent breeding material.
The molecular labeling of ' 4251 ' hair floral formation of embodiment 2:(cauliflower self-mating system assists improvement)
' 4251 ' economical character of cauliflower self-mating system is excellent, but bouquet Yi Maohua in some environments, needs to utilize bouquet
The self-mating system ' 4229 ' for being not easy hair flower is oriented genetic improvement to it.Specific molecular marker amplified production in the present invention exists
There are the polymorphisms of 160bp/128bp between the two, therefore carry out molecular labeling auxiliary choosing in backcross process using the present invention
It selects.
(1) ' 4229 ' and ' 4251 ' hybridization and backcrossing: ' 4229 ' and ' 4251 ' seeds are sowed respectively, nursery, field planting
In vinyl house.It carries out broadcasting flower bud hybridization manually for maternal florescence with ' 4229 ' for male parent, ' 4251 ', be collected after kind of pod is mature
Seed, as F1.Next year is by F1Seed and ' 4251 ' seed of recurrent parent are sowed respectively, nursery, are colonized in vinyl house.With F1
It carries out broadcasting flower bud hybridization manually for maternal florescence for male parent, ' 4251 ', collects seed (200 or more) after kind of pod is mature, i.e.,
For BC1F1。
(2) BC1F1Seedling stage generation molecular marker assisted selection: by the BC of collection1F1Generation seed randomly selects 200, cave
Disk sowing, nursery, and single plant is divided to number.Each single-strain blade is taken to extract base respectively according to conventional CTAB method in 2-3 piece leaf period
Because of a group DNA, -20 DEG C of preservations are spare.Utilize Specific primer pair (upstream primer: 5 '-CCAAAACAAAAGTGCAAAATCT-
3';Downstream primer: 5'- AAGGCCCTATTGGTAACTTG -3 ') PCR amplification is carried out respectively to above-mentioned DNA.React total volume
20 μ L, specific system are 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, 50 ng of genomic DNA add dd H2O to 20 μ L.Thermocycling program are as follows: 94 DEG C of initial denaturations 2
Min, 1 circulation；94 DEG C of denaturation 1min, 55 DEG C of 30 s of renaturation, 72 DEG C of 45 s of extension, 35 recycle；72 DEG C of extension 10min；4℃
It saves.PCR product is utilized into restriction enzymeBsePI 3 h of digestion under the conditions of 50 DEG C, 7 μ of digestion system product containing PCR
1 μ L of L, 10 × buffer, 1 U BseP I enzyme add dd H2O to 10 μ L.By its product in 1.2% Ago-Gel after digestion
On be separated by electrophoresis, ethidium bromide colour developing.In the BC1F1In generation, the single plant for being shown as 128 bp, which is considered as, contains the Hua Weidian of resistance to hair
The single plant that the material of genotype is retained, and is shown as 160 bp is then considered as the material containing Yi Maohua loci gene type and gives
It eliminates.The plant and recurrent parent of other agronomy, economic characters closest to recurrent parent ' 4251 ' are selected in selected single plant
' 4251 ' hybridization, obtain BC2F1From generation to generation.
(3) selection of subsequent backcross generations: to the BC of acquisition2F1It is selected from generation to generation according to above-mentioned (2), (3) step,
And then it is returned and obtains BC3F1From generation to generation；And it is in kind selected and is returned until BC6F1From generation to generation, then to it is selfed,
Obtain the BC of inheritance stability6F2From generation to generation, the individual of resistance to hair flower site homozygosis is therefrom screened, as hair floral formation is significantly improved
' 4251 '.
<110>Zhejiang Academy of Agricultural Science
<120>a kind of and cauliflower bouquet hair floral formation chain SNP site and its detection method and primer sets
<160>4
<210>1
<211>80
<212>DNA
<213>cauliflower
<400>1
CCAAAACAAA AGTGCAAAAT CTTATATATG CGCGCGGCTT GTATGTATTT ATGTGATTTT 60
TTCAAATGTA AACGTGTTTA 80
<210>2
<211>80
<212>DNA
<213>cauliflower
<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 (2)
1. a kind of and chain SNP site of cauliflower bouquet hair floral formation detection method,
Corresponding curd trait is resistance to hair flower, and the SNP site and its flanking sequence are 5 '-CCAAAACAAAAGTGCAAAATCTT
ATATATGCGCGCGGCTTGTATGTATTTATGTGATTTTTTCAAATGTAAACGTGTTTA-3'；
Corresponding curd trait is Yi Maohua, and the SNP site and its flanking sequence are 5 '-CCAAAACAAAAGTGCAAAATCTT
ATATATGCACGCGGCTTGTATGTATTTATGTGATTTTTTCAAATGTAAACGTGTTTA-3'；
It is characterized in that, method includes the following steps:
1) cauliflower material to be detected cauliflower extracting genome DNA to be detected: is extracted into base according to conventional CTAB method respectively
Because of a group DNA, -20 DEG C of preservations are spare；
2) PCR amplification of sample to be tested genomic DNA: using primer pair sample to be tested genomic DNA respectively into
Row PCR amplification, special primer middle and upper reaches primer: 5 '-CCAAAACAAAAGTGCAAAATCT -3 ';Downstream primer: 5'-
AAGGCCCTATTGGTA ACTTG -3'；20 μ L of total volume is reacted, specific 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, 50 ng of genomic DNA adds dd H2O is extremely
20μL；Thermocycling program are as follows: 94 DEG C of 2 min of initial denaturation, 1 circulation；94 DEG C of denaturation 1min, 55 DEG C of 30 s of renaturation, 72 DEG C extend
45 s, 35 circulations；72 DEG C of extension 10min；4 DEG C of preservations；
3) PCR product in the endonuclease reaction and electrophoresis detection of PCR product: 2) is utilized into restriction enzymeBsePI is at 50 DEG C
Under the conditions of 3 h of digestion, digestion system product containing PCR 7 μ L, 10 × buffer 1 μ L, 1 UBsePI enzyme adds dd H2O is extremely
10μL；Its product is separated by electrophoresis on 1.2% Ago-Gel after digestion, ethidium bromide colour developing；
4) identification of sample to be tested genotype: according to gel-tape mode in 3), the sample of 160 bp of display is to contain easy hair
The material of flower genotype, corresponding curd trait are Yi Maohua；And show that the sample of 128 bp is then containing resistance to hair flower genotype
Material；Corresponding curd trait is resistance to hair flower.
2. a kind of special primer of detection and the chain SNP site of cauliflower bouquet hair floral formation, corresponding curd trait are resistance to
Mao Hua, the SNP site and its flanking sequence are 5 '-CCAAAACAAAAGTGCAAAATCTTATATATGCGCGCGGCTTGTATG
TATTTATGTGATTTTTTCAAATGTAAACGTGTTTA-3'；
Corresponding curd trait is Yi Maohua, and the SNP site and its flanking sequence are 5 '-CCAAAACAAAAGTGCAAAATCTT
ATATATGCACGCGGCTTGTATGTATTTATGTGATTTTTTCAAATGTAAACGTGTTTA-3'；It is characterized in that, the spy
Different primer middle and upper reaches primer: 5 '-CCAAAACAAAAGTGCAAAATCT -3 ';Downstream primer: 5'- AAGGCCCTATTGGTA
ACTTG -3’。
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| CN111793710B (en) * | 2020-07-01 | 2021-04-09 | 浙江省农业科学院 | SNP marker linked with cauliflower ball-bottom flower stalk branch angle, method and application |
| CN111733274B (en) * | 2020-07-01 | 2022-09-16 | 浙江省农业科学院 | SNP locus, primer set and method for identifying cauliflower Zhe nong piny flower 80-day variety |
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| CN104946732A (en) * | 2014-03-31 | 2015-09-30 | 上海市农业科学院 | Broccoli flower trait identification method and application thereof |
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| CN102643913A (en) * | 2012-04-16 | 2012-08-22 | 温州市农业科学研究院 | Fast cauliflower genetic purity detection method based on polymerase chain reaction (PCR) technology |
| CN104946732A (en) * | 2014-03-31 | 2015-09-30 | 上海市农业科学院 | Broccoli flower trait identification method and application thereof |
| CN104561338A (en) * | 2015-01-22 | 2015-04-29 | 上海交通大学 | Fingerprint spectrum of cauliflower hybrid and construction method thereof |
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| 花椰菜花球和植株性状的遗传与相关分析;杨加付等;《福建农林大学学报(自然科学版)》;20100718(第04期);361-365 * |
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