CN109536638A - The molecular labeling and its application that a kind of and rape pattern character is closely related - Google Patents
The molecular labeling and its application that a kind of and rape pattern character is closely related Download PDFInfo
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- 235000007516 Chrysanthemum Nutrition 0.000 claims abstract description 27
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- 240000002791 Brassica napus Species 0.000 abstract description 5
- 235000004977 Brassica sinapistrum Nutrition 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 239000000463 material Substances 0.000 description 14
- 238000001514 detection method Methods 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 9
- 101100166427 Arabidopsis thaliana CCD4 gene Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
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- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 235000006008 Brassica napus var napus Nutrition 0.000 description 2
- 101150098356 CCD4 gene Proteins 0.000 description 2
- 241001674939 Caulanthus Species 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
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- 101150084750 1 gene Proteins 0.000 description 1
- 235000011293 Brassica napus Nutrition 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 108020005120 Plant DNA Proteins 0.000 description 1
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- 101150036080 at gene Proteins 0.000 description 1
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- 230000002068 genetic effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- LGZXYFMMLRYXLK-UHFFFAOYSA-N mercury(2+);sulfide Chemical compound [S-2].[Hg+2] LGZXYFMMLRYXLK-UHFFFAOYSA-N 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
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- 210000000056 organ Anatomy 0.000 description 1
- 238000009400 out breeding Methods 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
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Abstract
The invention belongs to rapeseed breeding and technical field of molecular biology, disclosing a kind of molecular labeling being closely related with rape white flower character and its application, the molecular labeling includes forward primer and reverse primer.Using total DNA as template, molecular labeling carries out PCR amplification, and electrophoretic separation the single plant of the single band of 180bp occurs only for homozygous chrysanthemum individual, the single plant of the single band of 177bp occurs only for homozygous white flower individual, while the single plant for two kinds of bands occur is heterozygous individual.The molecular labeling that the present invention develops can be used for the identification and prediction of rape white flower and chrysanthemum, easy to operate, easy to implement the method, can effectively solve the problems, such as the identification of heterozygous genotypes and homozygous genotype, and progeny selection is made to have more specific aim, improve efficiency of selection.
Description
Technical field
The invention belongs to rapeseed breedings and molecular biology field field, relate generally to a kind of close with rape pattern character
Relevant molecular labeling and its application.
Background technique
Pattern is important one of the character of plant, has and attracts and indicate pollinator, protection floral organ, maintains flower tissue energy
The functions such as amount balance.The rape planted extensively is mainly chrysanthemum, but breeder has had developed white, red, Chinese red, powder at present
A variety of colored rape flowers such as red, white powder, pink, yield and common chrysanthemum rape are close.Guaranteeing rapeseed yield
Except, colored rape flower more improves the ornamental value, economic value and social value of rape.Colored rape flower is by remote
The modes such as edge hybridization, recurrent selection, orientation backcrossing carry out breeding, the cultivation of colour rape flower at present still in development phase, because
This is badly in need of the available molecular labeling of a batch and carries out early stage assisted Selection, to improve efficiency of selection, reduce field planting scale and
Later period identification work shortens breeding time., there is white flower and chrysanthemum two in nature in parent kind one of of the wild cabbage as rape
The white flower character of wild cabbage, can be transferred in rape by distant hybridization means, create white flower rape by kind variation type.However mesh
Before until there are no the breedings that functional label is applied to white flower rape.
Summary of the invention
To solve the above problems, inventor is miscellaneous using a white flower artificial synthesis Brassica napus and chrysanthemum cabbage type rape
DH group is handed over and developed, QTL positioning is carried out to pattern character and candidate gene is identified, is obtained 5 in candidate gene BnC03.CCD
Locate indel difference at SNP difference and one, since the Land use systems of indel label are most simple, inventor is for above-mentioned
Indel devises 4 labels and is detected, it is thus identified that an indel specific mark and the rape pattern of BnC03.CCD4 is divided into
From.The rape flower color sorting that may be directly applied to of the label educates field, carries out early stage identification and assisted Selection to pattern, can as early as possible really
Recognize homozygous individual, and select heterozygous individual and enter next round selection, the planting scale and later period for mitigating field material identify work
Make, effectively improves the efficiency and accuracy of selection.
The technical solution of the present invention is as follows: a kind of molecular labeling being closely related with rape pattern comprising forward primer and
Reverse primer, the forward primer sequence are 5'-AACGGCATTGGTTTGGC-3', reverse primer sequences 5'-
GGTTTTCGGGTGAGCTGTC-3'。
Application of the above-mentioned molecular labeling in identification rape pattern.
The application method that above-mentioned molecular labeling is applied in identification rape pattern, comprising the following steps:
(1) it using rape single plant DNA to be identified as template, is carried out using above-mentioned molecular labeling (forward primer and reverse primer)
PCR amplification obtains amplified production；
(2) amplified production for obtaining step (1) is through being separated by electrophoresis, if only obtaining 180bp band, rape list to be identified
Strain is homozygous chrysanthemum individual, if only obtaining 177bp band, single plant to be identified is homozygous all sorts of flowers individual, if occurring 177- simultaneously
Two kinds of bands of 180bp, then single plant to be identified is heterozygous individual (present age shows as white flower, and offspring has separation).
The beneficial effects of the present invention are: compared with forefathers are for the transposons detection label of BnC03.CCD4 design, this
The PCR label detection mode of invention is simple；The positive effect of the present invention is extremely can early to judge rapeseed plants pattern, can be by heterozygosis base
Because type plant (showing as white flower) and homozygous white flower plant distinguish, to retain it into next round selection.The present invention can
Enhance the purpose of selection, improves efficiency of selection.
Detailed description of the invention
Fig. 1 is rape pattern QTL (HS), the position of white flower character candidate gene and connective marker LJ04 on genetic map
Fig. 2 is that RT-PCR detects expression of the BnC03.CCD4 gene in white flower and chrysanthemum rape
Fig. 3 is detection case schematic diagram of the label L J04 in white flower and chrysanthemum rape.M, P1, P2 and F1 are respectively represented point
Sub- amount standard, rape maternal (white flower), rape male parent (chrysanthemum) and F-1 hybrids (white flower).
Specific embodiment
The present invention is further detailed with reference to embodiments.
Embodiment 1:
A kind of molecular labeling LJ04 being closely related with rape white flower character, is prepared by the following:
The present embodiment is described in detail and obtains by taking a white flower rape and the DH segregating population of a chrysanthemum rape development as an example
Be closely related the method for molecular labeling with rape white flower character, specific as follows:
1. informative population and phenotypic evaluation
Hybridized with a white flower rape W1 with portion chrysanthemum rape Y1 and generate F1 generation, F1 generation passes through microspore culture again
Development is DH group.The petal that plant flowering newly bloomed to the same day piece is scanned, and extracts the B value in RGB as Phenotype Number
According to.
2.SNP chip analysis
To 91 parts of DH systems, young leaflet tablet DNA is extracted in seedling stage and carries out rape 60K SNP chip analysis (Illumina
Inc., CA, USA), result is corresponded into rape with reference to genome (http://brassicadb.org/brad/
Index.php it), and with phenotype is associated analysis, a significant association section, the area are detected from C03 chromosome
Between span 2.49Mb (such as Fig. 1).
3. candidate gene approach:
White flower and chrysanthemum strain each 15 are selected from DH group, petal is taken to be built into white flower pond and chrysanthemum pond respectively, are led to
Cross transcript profile sequencing discovery, in the candidate section on C03, have and only 1 gene there are tables between white flower pond and chrysanthemum pond
Up to difference, which is BnC03.CCD4, and gene number is BnC03g0606760 in ncbi database.RT-PCR detection is true
Recognize the gene high expression in white flower rape, does not express (such as Fig. 2) substantially in chrysanthemum rape.
4. functional label conversion and detection: obtaining indel difference, invention at difference and one in candidate gene BnC03.CCD
People has separately designed 5 corresponding specific marks for above-mentioned difference and has detected, and confirms that the indel of BnC03.CCD4 is specifically marked
Note is isolated with rape pattern.
DNA is extracted to white flower pond and chrysanthemum pond, carries out resurveying sequence point using 2500 microarray dataset of Illumina HiSeq
Analysis, the BnC03.CCD4 gene both found SNP at code area is there are five (are located at gene C DS reference sequences
At 343,360,384,609,636bp) and one at 3bp indel (positioned at 778~780bp of gene C DS reference sequences).
Gene order is referred to according to rape BnC03.CCD4, special indel labeled primer is designed for 3bp, designs amplifiable above-mentioned out
The PCR label of 3bp indel 4 is successively named as LJ01~LJ04, and carries out PCR amplification according to following steps:
1) using rape single plant total DNA as template；
2) PCR process is carried out using following primer:
3) PCR reaction system: total volume 10ul, specific ingredient are as follows:
4) PCR amplification program: 94 DEG C of 5min, [94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 1min] × 35 are recycled, 72 DEG C of 10min.
It is saved under the conditions of 4 DEG C after operation.
5) detection of pcr amplification product: using 13% polyacrylamide gel carry out electrophoresis, 100 volts of voltage, electrophoresis time
8 hours.
According to above-mentioned steps, using LJ01~LJ04 totally 4 labeled primers chrysanthemum and white flower rape parent, F1 plant with
And PCR amplification is carried out in 91 DH systems, find LJ01 and LJ02 label since amplified production is too long (respectively 480 and 600bp)
And can not be by the difference of electrophoresis detection 3bp, LJ03 label is only capable of amplifying band in some materials, and LJ04 label can be
Specific band is smoothly amplified in all material, it is single which in white flower parent W1 and 46 parts of white flower DH systems 177bp only occurs
Band；Only there is the single band of 180bp in chrysanthemum parent Y1 and 45 parts of chrysanthemum DH systems；F1 plant (white) occur simultaneously 177bp and
Two kinds of bands of 180bp determine that the label can successfully distinguish heterozygous genotypes plant.
Embodiment 2
The present embodiment the difference from embodiment 1 is that, rape parent genotype is different in the present embodiment, other with implementation
Example a kind the 4th " functional label converts and detection " is identical.
Using 101 parts of DH systems of white flower rape W2 and chrysanthemum rape Y2 development as research material in the present embodiment, which plants
There are 56 parts of performance white flowers, 45 parts of performance chrysanthemums in strain.It is identified using the label L J04 developed in embodiment 1, PCR amplification
: after the separation of 13% polyacrylamide gel electrophoresis, only there are 57 parts of materials of the single band of 177bp in the detection of product, is white
Flower parent W2 and 56 parts of white flower DH systems；180bp specific band only occur has 46 parts, is chrysanthemum parent Y2 and 45 parts of chrysanthemum DH
System；Occur only 1 part of material of two kinds of bands of 177bp and 180bp simultaneously, is F1, pattern shows as white.
Embodiment 3
The present embodiment the difference from example 2 is that, rape segregating population is different in the present embodiment, other are and embodiment
2 is identical.
F2 generation is generated using the F1 selfing in embodiment 2 in the present embodiment, and according to identical detection method in embodiment 2
255 F2 individuals are detected.As a result as follows: the single band of 177bp only occur has 66 parts of materials, shows as white flower；
180bp specific band only occur has 59 parts, shows as chrysanthemum；Occur two kinds of bands of 177bp and 180bp simultaneously has 120
Part material, is F1, and pattern shows as white (such as Fig. 3).
Select that 5 parts of the material for the single band of 177bp only occur (white flower), 180bp only occur special in above-mentioned F2 material
5 parts of material (chrysanthemum) of band occur 12 parts of material (white) of two kinds of bands of 177bp and 180bp simultaneously, and selfing generates respectively
F2:3 family, plantation visually observe pattern phenotype behind crop field, it is found that the pattern of preceding 2 class material does not separate, and with it is upper
It is consistent to represent type；The previous generation shows as 12 parts of materials for occurring two kinds of bands of 177bp and 180bp simultaneously, and F2:3 family occurs
The segregation phenomenon of table pattern character.
Above-mentioned qualification result shows the identification and screening that pass through label L J04 in breeding, according to 177bp and 180bp item
The appearance situation of band can effectively prejudge plant pattern, and the efficiency and accuracy of next round selection can be improved, and mitigate later period screening
The workload of identification accelerates breeding process.
Above-described specific embodiment has carried out further the purpose of the present invention, technical scheme and beneficial effects
It is described in detail, it should be understood that being not intended to limit the present invention the foregoing is merely a specific embodiment of the invention
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
Claims (3)
1. a kind of molecular labeling being closely related with rape pattern, including forward primer and reverse primer, the forward primer sequence
It is classified as 5'-AACGGCATTGGTTTGGC-3', reverse primer sequences 5'-GGTTTTCGGGTGAGCTGTC-3'.
2. application of the molecular labeling described in claim 1 in identification rape pattern.
3. the application method applied described in claim 1, comprising the following steps:
(1) it using rape single plant total DNA to be identified as template, is carried out using above-mentioned molecular labeling (forward primer and reverse primer)
PCR amplification obtains amplified production；
(2) amplified production for obtaining step (1) is through being separated by electrophoresis, if only obtaining 180bp band, rape single plant to be identified is
Homozygous chrysanthemum individual, if only obtaining 177bp band, single plant to be identified is homozygous white flower individual, if occurring 177-180bp simultaneously
Two kinds of bands, then single plant to be identified is heterozygous individual.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424103A (en) * | 2020-01-13 | 2020-07-17 | 浙江省农业科学院 | Rape flower color character detection method |
| CN111944921A (en) * | 2020-08-26 | 2020-11-17 | 中国农业科学院油料作物研究所 | Application of brassica napus BnaA08.PDS3 gene in breeding of color traits of brassica napus petals |
| CN113106164A (en) * | 2020-01-13 | 2021-07-13 | 中国医学科学院药用植物研究所 | SNP molecular marker for predicting red sage root flower color and application thereof |
| CN114875171A (en) * | 2022-06-24 | 2022-08-09 | 华中农业大学 | Gene closely related to rape reddish orange flower character, molecular marker and application thereof |
-
2019
- 2019-01-31 CN CN201910097676.XA patent/CN109536638B/en not_active Expired - Fee Related
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| BAO ZHANG ET AL: "Disruption of a CAROTENOID CLEAVAGE DIOXYGENASE 4 gene converts flower colour from white to yellow in Brassica species", 《NEW PHYTOLOGIST》 * |
| CRUZ, VON MARK V ET AL: "Molecular genetic analyses of oilseed Brassica germplasm: determination of life forms and germplasm management strategies by using microsatellite markers and FLOWERING LOCUS-C (FLC1 and FLC3) gene sequences", 《RETROSPECTIVE THESES AND DISSERTATIONS》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424103A (en) * | 2020-01-13 | 2020-07-17 | 浙江省农业科学院 | Rape flower color character detection method |
| CN113106164A (en) * | 2020-01-13 | 2021-07-13 | 中国医学科学院药用植物研究所 | SNP molecular marker for predicting red sage root flower color and application thereof |
| CN113106164B (en) * | 2020-01-13 | 2022-08-09 | 中国医学科学院药用植物研究所 | SNP molecular marker for predicting red sage root flower color and application thereof |
| CN111944921A (en) * | 2020-08-26 | 2020-11-17 | 中国农业科学院油料作物研究所 | Application of brassica napus BnaA08.PDS3 gene in breeding of color traits of brassica napus petals |
| CN111944921B (en) * | 2020-08-26 | 2022-08-05 | 中国农业科学院油料作物研究所 | Application of brassica napus BnaA08.PDS3 gene in breeding of color traits of brassica napus petals |
| CN114875171A (en) * | 2022-06-24 | 2022-08-09 | 华中农业大学 | Gene closely related to rape reddish orange flower character, molecular marker and application thereof |
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