CN108517374A - A kind of SNP marker and its application - Google Patents
A kind of SNP marker and its application Download PDFInfo
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Abstract
The invention discloses a kind of SNP marker, the SNP marker is to be located at the nucleotide of the exons 1 the 58th of BrFLC1 genes on Chinese cabbage group A10 chromosomes, wild type A, saltant type C.The bolting flowering time of the AA genotype individuals of the SNP marker is later than the individual of AC and CC genotype.The SNP marker can be used for Genetic identification and the breeding of Chinese cabbage group.
Description
Technical field
The invention belongs to crop molecular breeding technology fields, and in particular to a kind of SNP marker and its application.
Background technology
Chinese cabbage group includes that a plurality of types of vegetables such as Chinese cabbage, pakchoi, tender flower stalk, turnip, purple tsai-tai, Wuta-tsai make
Object and turnip type rape as oil crops, it is full of nutrition, there is long cultivation history all over the world.Bolting is bloomed
It is important one of agronomic shape in Chinese cabbage group production, and influences its crucial biological character to produce offspring.No
The Chinese cabbage group of same type, bolting flowering time are variant：Spring rape, husky inferior rape and cabbage heart type are bloomed very early, are pole
Precocious type；Chinese cabbage, purple tsai-tai and Italian cabbage heart etc. belong to mid-early maturity type；Chinese cabbage, Wuta-tsai and Japanese water
Dish etc. is bloomed later, belongs to late-maturing type；Under the conditions of non-vernalization, turnip is difficult that bolting is bloomed, and belongs to extremely late-maturing biennial
Type.Bolting refers to that plant is also not up to commodity picking time with regard to early bolting in advance, reduces the phenomenon that even losing commodity.First
Phase bolting generally results in yield decline, and quality reduces, and becomes a difficult problem of puzzlement Chinese cabbage group production.It can be seen that
Research Chinese cabbage group bolting is bloomed mechanism, prevent in advance bolting bloom, be of great importance for improving Chinese cabbage quality.
FLC is inhibiting factor of blooming, and takes part in vernalization path in flowering of plant regulated and control network and from main path, in nutrition
It grows and plays an important role into the regulated and control network that reproductive growth is converted.Low temperature can inhibit the expression of FLC genes, to release
FLC is to the inhibiting effect bloomed so that flowering of plant.The loss of function of FLC genes can cause requirement of the plant to low temperature to reduce, no
It needs to undergo the process that low temperature inhibits FLC gene expressions, to show as prematurity.
Chinese cabbage is the species for living through genome polyploidization, and there are tri- subunits broken up of LF, MF1 and MF2
Because of group.There is 1 FLC gene for coming from tripling duplication respectively in these three subgenomes, in addition to this there are one come
From the FLC genes for replicating reservation in α.Four FLC genes are the BrFLC2 on A02, BrFLC3 and BrFLC5 on A03 respectively,
And the BrFLC1 on A10.Wherein BrFLC1, BrFLC2, BrFLC3 are respectively present in 3 common ancestral gene group sequence moulds
In block R, the tripling from Chinese cabbage genome replicates.BrFLC5 be located at non co-linearity region (I block and J block it
Between), be arabidopsis and Chinese cabbage common ancestor's producer group α replicate as a result, be lost this copy in arabidopsis, but
It is remained in Chinese cabbage.
BrFLC1(http://brassicadb.org, Bra009055) clear its regulates and controls with flowering time in Chinese cabbage
It is related.
Production cultivation for Chinese cabbage group, bolting, which is bloomed, in advance means that plant switchs to reproduction from nutrient growth in advance
Growth, influences the quality and yield of its product organ, thus the generation of bolting in advance peasant economy can be taken in cause it is prodigious
It influences.Therefore, the kind for cultivating Tolerance to immature bolting is always a vital task of Chinese cabbage breeding.Exploitation can identify Chinese cabbage pumping
The morning and evening relevant SNP that a kind of sedge is bloomed is of great significance to Chinese cabbage breeding and production.And in the prior art about Chinese cabbage bolting
The morning and evening relevant SNP bloomed has found deficiency, is formed and is restricted to Chinese cabbage breeding.
Invention content
The first technical problem to be solved by the present invention be for Chinese cabbage group there are multicopy FLC genes the case where,
The allelic variation for different BrFLC genes is needed to be detected, the present invention, which provides, is based on KASP (Kompetitive Allele
Specific PCR, competitive ApoE gene) function on technical appraisement Chinese cabbage group BrFLC1 genes
The SNP marker detection technique system of variant sites, the SNP marker is easy to operate, can identify that Chinese cabbage group controls bolting
It blooms the functional variants site of Pe1+58 (A/C) existing for related gene B rFLC1, in Chinese cabbage group bolting resistant property
Effectively carry out marker assisted selection in molecular breeding.
More specifically, first aspect present invention provides a kind of relevant SNP marks of Chinese cabbage group bolting flowering time
Note, the SNP marker are to be located at the nucleotide of the exons 1 the 58th of BrFLC1 genes on Chinese cabbage group A10 chromosomes,
For A or C.
In the present invention, the wild-type coding sequence such as SEQ ID NO of the BrFLC1 genes:Shown in 1, the BrFLC1 bases
The wild type protein sequence of cause such as SEQ ID NO:Shown in 2, the position of the SNP marker is the Chinese cabbage gene based on 1.5 versions
Group sequence and determination, accession number of the BrFLC1 genes in Btassica database Brassica Database be
The entry address of Bra009055, the Btassica database are http://brassicadb.org.
In certain embodiments of the present invention, the allelotype of the BrFLC1 genes of the SNP marker includes AA bases
Because of type, AC genotype and CC genotype, wherein the bolting and/or flowering time of AA genotype Chinese cabbage group individual are later than AC
With the individual of CC genotype, when the site of the SNP marker sports C by A and can shorten Chinese cabbage group bolting and/or bloom
Between.
The second aspect of the present invention provides a kind of method of prediction Chinese cabbage group bolting flowering time, the method packet
The SNP marker as described in the first aspect of the invention in detection Chinese cabbage group sample is included, to determine Chinese cabbage group
The allelotype of BrFLC1 genes.
In certain embodiments of the present invention, using selected from allele specific pcr, Single base extension method, DNA sequencing
One or more in method, mass spectrography and KASP methods detect the genome, genomic fragment and/or transcript profile of Chinese cabbage group
In the SNP marker.
Allele specific pcr, Single base extension method, PCR sequencing PCR, mass spectrography, KASP methods are that can determine SNP mutation position
Set the method with genotype.
PCR sequencing PCR can also use gene order-checking, transcript profile sequencing means, can be surveyed to individual gene reverse transcription product
Sequence, individual chip is sequenced after can also using PCR amplification, and genetic chip screening can be used.
In some preferred embodiments of the present invention, the method is KASP methods.
In further preferred embodiment of the present invention, by KASP methods detect Chinese cabbage group sample genome/
It realizes in the site of SNP marker in genomic fragment.
In other embodiments of the present invention, the KASP methods include：
Step A extracts the genome of Chinese cabbage group sample, obtains DNA profiling；
Step B mixes DNA profiling with KASP Primer mix and KASP Master mix, obtains PCR reaction systems
And expanded, obtain pcr amplification product；
Step C determines that BrFLC1 exists in Chinese cabbage group sample according to the type of the fluorescence signal of pcr amplification product
(A/C) variant sites genotype, Chinese cabbage group sample is determined by the genotype of (A/C) variant sites existing for BrFLC1
The bolting and/or flowering time of product.
In some specific embodiments of the invention, in stepb, the KASP Primer mix include three spies
Specific primer, the respectively two positive fluorescent primers and a reverse primer for carrying different fluorescent markers；
Wherein, the sequence of the fluorescent marker is located at the ends 5' of positive fluorescent primer, the ends 3' of the forward direction fluorescent primer
The variant sites of the SNP marker for identification；
The reverse primer can be with the sequence complementary pairing of BrFLC1 allele, and reverse primer sequences can make institute
The fragment length for stating amplified production is 60-120bp.
In some preferred embodiments of the present invention, the sequence of the fluorescent marker be FAM fluorescent markers sequence or
VIC fluorescent marker sequences.
In some specific embodiments of the invention, the sequence of two positive fluorescent primers is respectively：
Sequence 1：5'-GAAGGTGACCAAGTTCATGCTGAGAACAAAAGTAGCCGAC AAGTTA-3', sequence is such as
SEQ ID NO:Shown in 3；
Sequence 2：5'-GAAGGTCGGAGTCAACGGATTGAGAACAAAAGTAGCCGAC AAGTTC-3', sequence is such as
SEQ ID NO:Shown in 4；
The reverse primer sequences are：5'-GAGACCGTTGCGTCGTTTRGAGAA-3', sequence such as SEQ ID NO:5
It is shown.
The length in two forward direction ends fluorescent primer 3' SNP marker site for identification can adjust, the ends 3' alkali
Base is respectively A and C.
The position of the reverse primer and length can adjust, if ensure sequence-specific, and reverse primer sequence
The fragment length for selecting PCR amplification to be ensured to obtain of row is 60-120bp.
Can equally detect SNP genotype in view of the base by detecting antisense strand related locus, the forward direction of primer and
It is reversely opposite, as long as variant sites can be distinguished.
In certain embodiments of the present invention, in stepb, the KASP Master mix include：General FRET
Cassette fluorescent primers, ROX internal reference dyestuffs, KlearTaq archaeal dna polymerases and dNTPs.
In general FRET cassette fluorescent primers, it is separately selected from two kinds that fluorescence spectrum does not interfere
Fluorescent molecular is not limited to FAM and VIC, and two kinds of fluorescence can be interchanged into another primer.
As needed, ROX internal references dyestuff can be omitted.
In other specific embodiments of the invention, in stepb, the PCR reaction systems are：
；
The step of PCR amplification is：
。
In some preferred embodiments of the present invention, the PCR amplification is further comprising the steps of：
。
In certain embodiments of the present invention, in step C, fluorescence that the pcr amplification products of the KASP methods is sent out
For single fluorescence signal when, Chinese cabbage group sample be SNP marker AA/CC genotype individuals；The PCR amplification of the KASP methods
When the fluorescence that product is sent out is mixing fluorescence signal, Chinese cabbage group sample is the AC genotype individuals of SNP marker.
Third aspect present invention provides a kind of SNP marker or second aspect of the present invention institute as described in the first aspect of the invention
The method for the prediction Chinese cabbage group bolting flowering time stated is identified, in advance sooner or later in Chinese cabbage group bolting and/or flowering time
Survey, monitoring or the application in Chinese cabbage group breeding or purposes.
The purposes is included in breeding early stage when predicting the bolting of the plant in advance by detecting the SNP marker or bloom
Between, to determine the processing to plant.Also include by the SNP and BrFLC1 gene difference SNP or with BrFLC1 gene linkages
Other difference SNP association analysis.
For BrFLC1 genes as inhibiting factor of blooming, SNP marker site sports C by A will cause the site to encode
Amino acid becomes proline, the wild type protein sequence such as SEQ ID NO of BrFLC1 genes from threonine:Shown in 2.Gene order
Change cause the to bloom protein sequence of inhibiting factor change, because the amino acid of SNP marker site coding is located at BrFLC1 eggs
White Core Feature area, change can influence regulating and controlling effect of the BrFLC1 albumen to gene downstream, then influence plant bolting
With the approach bloomed.Inventor has found that the site of the SNP marker sports C by A and can shorten Chinese cabbage group bolting and/or hold
It takes time.The allelotype of the BrFLC1 genes of SNP marker includes AA genotype, AC genotype and CC genotype, wherein AA
The bolting and/or flowering time of genotype Chinese cabbage group individual are later than the individual of AC and CC genotype.Therefore, detection can be passed through
Genotype of the BrFLC1 genes in the site determines the bolting and/or flowering time of Chinese cabbage group sample, this is to scientific research
There is directive significance with agricultural production application.
Beneficial effects of the present invention are：SNP marker of the present invention being capable of high throughput identification Chinese cabbage group control pumping
A kind of sedge is bloomed functional variants site existing for related gene B rFLC1, and the Detection accuracy through test-target loci gene type can be high
Up to absolutely, there is high efficiency and accuracy.
Utilize detectable 1536 samples of PCR of the label (when experiment is using 1536 microwell plate) or 384 samples
(when experiment is using 384 microwell plate) or 96 samples (when experiment is using 96 microwell plate), the testing cost of each sample is only 0.3
Member/sample (uses 1536 orifice plates), 0.8-1.0 members/sample (384 orifice plate), 1.8 yuan/sample (96 orifice plates), easy to operate, valence
Lattice are cheap, can effectively carry out marker assisted selection in Chinese cabbage group bolting resistant property molecular breeding.
Description of the drawings
Below in conjunction with attached drawing, the present invention will be described in detail.
Fig. 1 is that the result of the pcr amplification product progress fluoroscopic examination to 236 parts of Chinese cabbage materials in embodiment 1 counts signal
Figure.
Specific implementation mode
To make the present invention be readily appreciated that, the present invention is described more detail below.But before describing the present invention in detail, it should be understood that
The present invention is not limited to the specific implementation modes of description.It is also understood that term used herein is embodied only for description
Mode, and be not offered as restrictive.
1. the determination of candidate SNP
The present inventor passes through whole-genome association (GWAS), it was found that new is used for Chinese cabbage group bolting
It blooms the SNP marker of identification, which is to be located at the exons 1 the 58th of BrFLC1 genes on Chinese cabbage group A10 chromosomes
Position nucleotide, is A or C, wild type A, saltant type C, is named as Pe1+58 (A/C).A genotype codes amino acid is Soviet Union
Propylhomoserin, C genotype codes amino acid are proline.Report is yet there are no about the variation in the site and its with the correlation of flowering time
Road.The present invention converts Pe1+58 (A/C) variations to functional indicia, for use in the mark of Tolerance to immature bolting in Chinese cabbage breeding
Remember assisted Selection.
2. reference gene
Chinese cabbage genome sequence (Bra009055 (the B.rapa reference genome sequence of 1.5 versions
V1.5) 2013-05-17) disclose the sequence of BrFLC1, network address：http://brassicadb.org.SNP (the Pe1 of the present invention
+ 58 (A/C)) it is to be determined based on the Chinese cabbage genome sequences of 1.5 versions.Wild type BrFLC1 gene coded sequences such as SEQ ID
NO:Shown in 1.Its corresponding protein sequence such as SEQ ID NO:Shown in 2.
3. detection technique
Competitive ApoE gene (Kompetitive Allele Specific PCR, KASP) technology is one
The SNP classifying methods of high-throughput, inexpensive, the low false detection rate of kind, it has also become one of the main stream approach of snp analysis in the world,
It has been widely used in terms of crop molecular breeding.The SNP marker of BrFLC1 of the exploitation based on KASP technologies will be used for
The earlier evaluations of Chinese cabbage bolting resistant property effectively improve the efficiency for the molecular breeding of resistance to bolting.
4. experiment material
Chinese cabbage group kind of the present invention is provided by Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science.
5. the design of primer
According to SNP mutation information feature, the special set of competitive ApoE gene primer of design, including two
Forward primer (positive FAM fluorescence homologous primer and forward direction VIC fluorescence homologous primer), a reverse primer；Its sequence information
For：
Positive FAM fluorescence homologous primer：5'-GAAGGTGACCAAGTTCATGCTGAGAACAAA
AGTAGCCGACAAGTTA-3', sequence such as SEQ ID NO:Shown in 3；
Positive VIC fluorescence homologous primer：5'-GAAGGTCGGAGTCAACGGATTGAGAACAAA
AGTAGCCGACAAGTTC-3', sequence such as SEQ ID NO:Shown in 4；
Reverse primer：5'-GAGACCGTTGCGTCGTTTRGAGAA-3', sequence such as SEQ ID NO:Shown in 5.This is anti-
It is a kind of degenerate primer to primer, degenerate primer refers to the different sequences for representing coding all different bases possibilities of single amino acids
The mixture of row.R=A in this primer or G, the nucleotide matched with it can be T or C, specifically, the reverse primer
For the mixture of 5'-GAGACCGTTGCGTCGTTTAGAG AA-3' and 5'-GAGACCGTTGCGTCGTTTGGAGAA-3'.
The base of positive FAM fluorescence homologous primer end is A, and the base of positive VIC fluorescence homologous primer end is C, instead
Selection to primer sequence will ensure the fragment length of amplification in 60-120bp, to ensure the integrality of amplification.Two forward directions are drawn
The ends 5' of object are connected with the public sequence label homologous with the universal primer of fluorescent marker, wherein FAM fluorescence is homologous draws for forward direction
The ends 5' of object connect the sequence 5'-GAAGGTGACCAAGTTCATGCT-3' homologous with the universal primer of FAM fluorescent markers, positive
The ends 5' of VIC fluorescence homologous primers connect the sequence 5'- homologous with the universal primer of VIC fluorescent markers
GAAGGTCGGAGTCAACGGATT-3'。
When subsequent detection, if the fluorescence that PCR product is sent out is FAM fluorescence, Chinese cabbage group to be measured is the AA of SNP marker
Genotype individuals；If the fluorescence that PCR product is sent out is VIC fluorescence, Chinese cabbage group to be measured is the CC genotype of SNP marker
Body；If the fluorescence that PCR product is sent out is the mixing fluorescence of FAM and VIC, Chinese cabbage group to be measured is the AC genes of SNP marker
Type individual.
The sequence of " two forward primers " of the present invention is not limited to positive FAM fluorescence homologous primer sequence and forward direction
VIC fluorescence homologous primer sequences；Positive special primer 5'-GAGAACAAAAGTAGCCGAC AAGTTA-3' and 5'-
The ends 5' of GAGAACAAAAGTAGCCGACAAGTTC-3' can connect the homologous sequence of other public fluorescent marker sequences；Example
Such as, the universal primer that VIC fluorescent markers can also be connected at the ends 5' of 5'-GAGAACAAAAGTAGCCG ACAAGTTA-3' is same
The sequence in source, the universal primer that FAM fluorescent markers are connected at the ends 5' of 5'-GAGAACAAAAGTAGCCGACAAGTTC-3' are homologous
Sequence, form two new forward primer sequences.
Primer employed in the present invention synthesizes acquisition by Invitrogen (Shanghai) Trading Co., Ltd..
6.KASP is detected
(1) extraction of Chinese cabbage group kind complete genome DNA:
The leaf tissue (totally 236 parts of Chinese cabbage group materials described above) of Chinese cabbage group kind to be measured is taken, is used
CTAB methods extract complete genome DNA, and the complete genome DNA of each Chinese cabbage group kind is diluted to 15ng/ μ l.
(2) primer is diluted：
The primer dilution information provided according to synthetic primer company is diluted, and is first centrifuged dry powder primer, is prevented
Dry powder primer when only uncapping near nozzle splashes, and information is diluted according to primer, by positive FAM fluorescence homologous primer, forward direction VIC
The concentration of fluorescence homologous primer and reverse primer is diluted to 10 μM.
(3) KASP Primer mix are prepared：
Take the positive FAM fluorescence homologous primer diluted, each 12 μ l of forward direction VIC fluorescence homologous primers, 30 μ l of reverse primer,
Sterile ultra-pure water (ddH is added2O) 46 μ l obtain the KASP Primer mix of 100 μ l；
The KASP Master mix include following each component：General FRET cassette fluorescent primers, ROX internal references
Dyestuff, KlearTaq archaeal dna polymerases and dNTPs are purchased from LGC companies (Laboratory of the Government
Chemist British governments chemist laboratory, http://www.lgcgroup.com/) KASP Master mix kits：
The Mix containing 2 × Master in the kit, MgCl2And DMSO, wherein 2 × Master Mix are what the present invention mentioned
KASPMaster mix；MgCl2It is in the detection and genotyping signal that cannot succeed caused by high or low G/C contents with DMSO
In the case of be added and use in KASP Master mix, the present invention does not use MgCl2And DMSO, it can be according to reality in experiment
The actual conditions selection tested is added said components or is added without.
(4) PCR reaction systems are prepared：
According to the addition of each component shown in table 1, PCR reaction systems are prepared；
Table 1：The addition of each component in PCR reaction systems
| Component | 96 orifice plates | 384 orifice plates | 1536 orifice plates |
| DNA profiling | 5μl | 2.5μl | 0.625μl |
| KASP Master Mix | 5μl | 2.5μl | 0.625μl |
| KASP Primer Mix | 0.14μl | 0.07μl | 0.0175μl |
| React total volume | 10.14μl | 5.07μl | 1.2675μl |
The blank control (NTC) of DNA profiling is not added in setting simultaneously, one or more blank controls are arranged in each reaction plate；
(5) PCR amplification：
After microwell plate installs, pcr amplification reaction is followed the steps below；
Table 2-1 reaction conditions are arranged
In the step 2 of above-mentioned PCR amplification, the amplification of the 1st cycle is 94 DEG C of holdings 20s, 61 DEG C of holding 60s；2nd is followed
The amplification of ring is 94 DEG C of holdings 20s, 60.4 DEG C of holding 60s；……；10th cycle amplification be 94 DEG C holding 20s, 55.6 DEG C
Keep 60s.
(6) PCR amplification is supplemented：
If by above-mentioned steps carry out PCR amplification after, fluorimetric analysis show genotyping result it is not satisfactory, can continue into
The PCR amplification that row following steps carry out,
Table 2-2 reaction conditions are arranged
(7) fluoroscopic examination：
Fluorimetric analysis is carried out to PCR product, the fluorescence sent out according to PCR product blooms to the bolting of Chinese cabbage group
It is identified；
Fluoroscopic examination can utilize (Laboratory of the Government Chemist British governments of LGC companies
Chemist laboratory, http://www.lgcgroup.com/) SNPline be detected, other, which can also be used, to detect
The instrument of FAM fluorescence and VIC fluorescence is detected；
If the fluorescence that PCR product is sent out is FAM fluorescence, Chinese cabbage group to be measured is the AA genotype individuals of SNP marker；
If the fluorescence that PCR product is sent out is VIC fluorescence, Chinese cabbage group to be measured is the CC genotype individuals of SNP marker；
If the fluorescence that PCR product is sent out is the mixing fluorescence of FAM and VIC, Chinese cabbage group to be measured is the AC of SNP marker
Genotype individuals, label is in table 3.
PCR after reaction, uses the Quant Studio 12K Flex of Applied Biosystems companies production
Real-Time PCR System machines are scanned pcr amplification product, carry out fluorimetric analysis；And utilize software
Allelic Discrimination modules in Quant Studio 12K Flex Software carry out parting, FAM fluorescence
Excitation wavelength and launch wavelength be respectively (492nm and 518nm), the excitation wavelength and launch wavelength of VIC fluorescence are respectively
(538nm and 554nm), is detected using above-mentioned instrument, according to the design of primer that fluorescence signal is related to SNP bases
The genotype of all detection samples is labeled in two-dimensional coordinate by connection, and testing result is as shown in Fig. 1.
7. experimental result
Fluoroscopic examination result
Testing result according to following standard as shown in Figure 1, according to testing result, determine in Chinese cabbage group kind to be measured
The genotype of (A-C) variant sites existing for BrFLC1.
The standard is specially：The fluorescence signal of Chinese cabbage group kind pcr amplification product to be measured passes through Quant
Studio 12K Flex Software softwares are analyzed, and display fluorescence signal gathers for following 3 class, including is aggregated in close to Y-axis
Dot, representative sample have FAM fluorescence, then the 58th nucleosides of the exons 1 of the BrFLC1 of the Chinese cabbage group kind
The genotype of sour (A-C) variant sites is AA；It is aggregated in the cross form point close to X-axis, representative sample has VIC fluorescence, then should
The genotype of variant sites is CC；The triangle point being aggregated near coordinate diagonal line, representative sample have there are two types of fluorescence values,
Then the genotype of the variant sites is heterozygous AC.It is distributed near the origin in the coordinate lower left corner, legend is solid black box
Sample be blank control, blank control should both not have FAM fluorescence values, without VIC fluorescence values yet.
Interpretation of result：Using the bolting time of above-mentioned testing result and the Chinese cabbage group kind (from being seeded into bolting
Time) and flowering time (from the time spent is seeded into out), analyze the correlation between three；The present embodiment uses 236 altogether
Part Chinese cabbage carries out above-mentioned analysis, and the calculating of correlation is calculated by software I BM SPSS Statistics22.
There are 4 BrFLC genes, the A-C function variant sites of the exons 1 position of wherein BrFLC1 for Chinese cabbage group
(hereinafter referred to as " BrFLC1 target sites ") is significantly correlated with flowering time.
The relationship of genotype and phenotype
In the present embodiment when the above-mentioned target site genotype information of 236 parts of Chinese cabbage group kinds, variety type, bolting
Between (from the time for being seeded into bolting) and the details of flowering time (from the time spent is seeded into out) are as shown in table 3.Table
In " FLC1 " to represent in the genotype of Pe1+58 (A/C) variant sites of the exons 1 position of gene BrFLC1 (be by above-mentioned
The genotype of case step detection)；" C " represents the allelotype of the BrFLC1 genes of SNP marker as CC genotype in table
Body；" A " represents the allelotype of the BrFLC1 genes of SNP marker as AA genotype individuals in table；" H " is heterozygote in table
" hyterozygote's " writes a Chinese character in simplified form, and represents the allelotype of the BrFLC1 genes of SNP marker as AC genotype individuals.
The correspondence of table 3 bolting time and flowering time and genotype
3. interpretation of result
As shown in Fig. 1 and table 3, analysis result shows that BrFLC1 target site genotype is the Chinese cabbage group kind of AA
Average bolting time (129.76 days) and Chinese cabbage group kind that flowering time (152.89 days) and genotype are CC pumping
A kind of sedge time (84.83 days) and flowering time (96.59 days) all have pole significant difference (bolting time p<0.001, flowering time
p<0.001) individual that, genotype is AA is obviously later than the individual that genotype is CC；BrFLC1 target site genotype is that AA is white
The bolting time (129.76 days) of vegetables kind and flowering time (152.89 days) and genotype are the bolting of AC Chinese cabbage verieties
Time (77.69 days) and flowering time (86.85 days) all have extremely pole significant difference (bolting time p<0.001 when blooming
Between p<0.001), the individual of frequency of genotypes AA is significantly later than the individual that genotype is AC.
By to randomly selected 236 parts of Chinese cabbage groups (including Chinese cabbage, little Bai from 11 different cultivation populations
Dish, Italian cabbage heart, cabbage heart, water dish, oil are with Chinese cabbage, a kind of sedge dish, Wuta-tsai, small Chinese cabbage, turnip, purple tsai-tai) carry out molecular labeling
Screening, the results showed that, this label can detect the genotype in all 236 parts of material object sites, and carry different
The storeroom flowering time of BrFLC1 allele and bolting time, there are significant differences.
It follows that the different genotype of the Pe1+58 (A/C) of BrFLC1 genes is bloomed and bolting time difference
Extremely significantly, can be used for the marker-assisted breeding of Chinese cabbage group, can be used for identify, prediction Chinese cabbage group bolting and
Flowering time.
It should be noted that embodiment described above is only used for explaining the present invention, do not constitute to any of the present invention
Limitation.By referring to exemplary embodiments, invention has been described, it should be appreciated that word used in it is descriptive
With explanatory vocabulary, rather than limited vocabulary.The present invention can be made within the scope of the claims by regulation
Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it relates to
And specific method, material and embodiment, it is not intended that the present invention is limited to particular case disclosed in it, on the contrary, this hair
It is bright to can be extended to other all methods and applications with the same function.
Claims (12)
1. a kind of relevant SNP marker of Chinese cabbage group bolting flowering time, which is characterized in that the SNP marker is to be located in vain
The nucleotide of the exons 1 of BrFLC1 genes the 58th on cole crop A10 chromosomes is A or C.
2. SNP marker according to claim 1, which is characterized in that the wild-type coding sequence of the BrFLC1 genes is such as
SEQ ID NO:Shown in 1, the position of the SNP marker is the Chinese cabbage genome sequence based on 1.5 versions and determination.
3. the SNP marker according to claims 1 or 2, which is characterized in that the BrFLC1 genes of the SNP marker etc.
Position genotype include AA genotype, AC genotype and CC genotype, wherein the bolting of AA genotype Chinese cabbage group individual with/
Or flowering time is later than the individual of AC and CC genotype.
4. it is a kind of prediction Chinese cabbage group bolting flowering time method, including detection Chinese cabbage group sample according to right
It is required that the SNP marker described in any one of 1-3, to determine the allelotype of the BrFLC1 genes of Chinese cabbage group.
5. according to the method described in claim 4, it is characterized in that, using selected from allele specific pcr, Single base extension
One or more in method, DNA sequencing method, mass spectrography and KASP methods detect genome, the genomic fragment of Chinese cabbage group
And/or the SNP marker in transcript profile.
6. method according to claim 4 or 5 comprising：
Step A extracts the genome of Chinese cabbage group sample, obtains DNA profiling；
Step B mixes DNA profiling with KASP Primer mix and KASP Master mix, obtains PCR reaction systems and goes forward side by side
Row amplification, obtains pcr amplification product；
Step C determines (A/ existing for BrFLC1 in Chinese cabbage group sample according to the type of the fluorescence signal of pcr amplification product
C) the genotype of variant sites determines Chinese cabbage group sample by the genotype of (A/C) variant sites existing for BrFLC1
Bolting and/or flowering time.
7. according to the method described in claim 6, it is characterized in that, in stepb, the KASP Primer mix include three
Specific primer, the respectively two positive fluorescent primers and a reverse primer for carrying different fluorescent markers；
Wherein, the sequence of the fluorescent marker is located at the ends 5' of positive fluorescent primer, and the ends 3' of the forward direction fluorescent primer are used for
Identify the variant sites of the SNP marker；
The reverse primer can be with the sequence complementary pairing of BrFLC1 allele, and reverse primer sequences can make the expansion
The fragment length for increasing production object is 60-120bp.
8. the method according to the description of claim 7 is characterized in that the sequence of the fluorescent marker is FAM fluorescent marker sequences
Or VIC fluorescent marker sequences.
9. method according to claim 7 or 8, which is characterized in that the sequence of two positive fluorescent primers is respectively：
Sequence 1：5'-GAAGGTGACCAAGTTCATGCTGAGAACAAAAGTAGCCGACAAGTTA-3', sequence such as SEQ ID
NO:Shown in 3；
Sequence 2：5'-GAAGGTCGGAGTCAACGGATTGAGAACAAAAGTAGCCGACAAGTTC-3', sequence such as SEQ ID
NO:Shown in 4；
The reverse primer sequences are：5'-GAGACCGTTGCGTCGTTTRGAGAA-3', sequence such as SEQ ID NO:5 institutes
Show.
10. according to the method described in any one of claim 6-9, which is characterized in that in stepb, the PCR reactants
System is：
；
The step of PCR amplification is：
；
Preferably, the PCR amplification is further comprising the steps of：
。
11. according to the method described in claim 6, it is characterized in that, in step C, fluorescence that the pcr amplification product is sent out
For single fluorescence signal when, Chinese cabbage group sample be SNP marker AA/CC genotype individuals；The pcr amplification product is sent out
Fluorescence be mixing fluorescence signal when, Chinese cabbage group sample be SNP marker AC genotype individuals.
12. as described in a kind of any one of SNP marker or claim 4-11 as described in any one of claim 1-3
Application of the method in the identification sooner or later of Chinese cabbage group bolting and/or flowering time, prediction, monitoring or Chinese cabbage group breeding or
Purposes.
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| CN109609671A (en) * | 2018-11-09 | 2019-04-12 | 北京市农林科学院 | A kind of detection Vegetable Crops of Brassica vegetable leaf marginal slit carves the SNP marker and its application of character |
| CN117344045A (en) * | 2023-09-13 | 2024-01-05 | 广东省农业科学院蔬菜研究所 | Cabbage core KASP molecular marker developed based on whole genome resequencing and application thereof |
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Application publication date: 20180911 |