CN106755528A - A kind of SNP marker related to Seed of Camellia oleifera oil content and its application - Google Patents
A kind of SNP marker related to Seed of Camellia oleifera oil content and its application Download PDFInfo
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Abstract
The present invention provides a kind of SNP marker related to Seed of Camellia oleifera oil content and its application.Primer of the SNP marker of the invention by nucleotide sequence as shown in SEQ ID NO.1 2 is expanded and obtained, and amplified production contains the site at the 493bp of Cofad2 1A gene open reading frames, and the polymorphism in the site is G/A.Oil tea breeding material is detected using the molecular labeling, Seed of Camellia oleifera oil content can be predicted in seedling stage, substantially increase the efficiency of selection of oil oil tea breeding high.
Description
Technical field
The present invention relates to biological technical field, belong to oil tea molecular biology and Biotechnology in Genetic Breeding field, and in particular to
A kind of pleomorphism site molecular labeling of Seed of Camellia oleifera oil content screening, also relates to the molecular labeling and contains Seed of Camellia oleifera is high
Application in oil cut rate breeding.
Background technology
Oil tea (Camellia oleifera Abel.), is subordinate to Theaceae (Theaceae) Camellia (Camellia
L.), it is the distinctive woody oil tree species of China, is also the important woody edible oil material kind of south China.Camellia seed oil nutrition
Health value is higher, and its quality can compare favourably with olive oil, is a kind of edible oil of high-quality, and its unsaturated fatty acid content reaches
More than 90%, based on oleic acid (more than 80%) and linoleic acid (about 8%), and the effects such as with antitumor, reducing blood lipid.Last decade
Come, in the case where national policy is guided and is helped, China's camellia oleiferaindustry has obtained tremendous development, and national cultivated area is up to more than 6,000 ten thousand
Mu, year more than 60 ten thousand tons of oil-producing.According to《National camellia oleiferaindustry development plan (2009~2020)》, to the year two thousand twenty, China's oil tea kind
Plant area and be up to 93,000,000 mu, therefore oil tea breeding nursery stock is in great demand.At present, oil tea breeding is selecting and crossbreeding
It is main breeding objective for Main Means, with fruit yield, and has achieved impressive progress, but be mesh to improve tea seed oil content
Breeding research carry out it is less.Simultaneously as oil tea juvenile phase biological nature long causes the oil tea breeding time limit more long, new varieties choosing
Educate slow, fine-variety breeding speed is not met by the demand of industry development, it has also become hinder the key factor of camellia oleiferaindustry development
One of.Can be selected since seedling stage using molecular labeling auxiliary (MAS) breeding technique, significantly shorten the process of breeding, to fruit
Actually the economic forest breeding advantage of main purpose is especially apparent.Therefore, the MAS breedings for carrying out oil tea oil content high will effectively be shortened
Oil tea breeding cycle, and with huge application potential.
The history of the molecular mark research existing decades of oil tea, various points including RAPD, ISSR, SRAP etc.
Sub- labelling technique, achieves certain achievement.But the drawbacks of these technologies show certain, the polymorphism mark position for being obtained
Point is difficult really to be used for oil tea assistant breeding.Major drawbacks include：1st, these marks belong to dominant marker, it is impossible to accurate body
The genotype of existing pleomorphism site；2nd, these labelling techniques are higher to experiment operator and environmental requirement, and experimental result is unstable
It is fixed；3rd, these labelling techniques are that whole gene group sequence is analyzed, and workload is big, it is impossible to which pleomorphism site is precisely fixed
Position, is also difficult to screen the mark with purpose proterties close linkage；4th, traditional quantitative trait locus (quantitative
Trait loci, QTL) mapping needs have akin mapping population, oil tea juvenile phase biological nature long so that create big
Time-consuming for the oil tea hybridization mapping population of scale, and difficulty is big and need to take large area forest land.Therefore, it is right to study with natural population
As, marked using codominance SNPs, mapped by linkage disequilibrium and develop the pleomorphism site associated with Seed of Camellia oleifera oil content,
Screening can stablize the mark for being used for early stage assisted Selection, as the effective molecular mark strategy of oil tea.
The content of the invention
First purpose of the invention is to provide the SNP marker related to Seed of Camellia oleifera oil content.
Second object of the present invention is to provide the method for detecting the SNP marker.
Third object of the present invention is to provide the application of above-mentioned SNP marker.
The purpose of the present invention is achieved through the following technical solutions：
(1) Camellia oleifera Germplasms are collected extensively in oil tea super distributed area, the oil tea that seed oil content is widely separated is set up
Natural population.
(2) tender leaf using 500 individual plants of KAC methods (TaKaRa kit Code No.9768) extraction natural populations is total
DNA, and agarose gel electrophoresis with 0.8%~1% and nucleic acid determination instrument determine the quality for extracting DNA, it is desirable to DNA is without drop
Solution, without the pollution of the impurity such as protein, polysaccharide, concentration reaches more than 100ng/ μ L.
(3) 500 parts of full maturity seeds of oil tea germplasm of collection association colony, seed oil-containing is determined with soxhlet extraction methods
Rate.
(4) according to oil tea Cofad2-1A genes (Tan Xiaofeng, Chen Hongpeng, Zhang Dangquan etc., oil tea FAD2 full length genes cDNA
Cloned culturing, forest-science, 2008,44 (3)：70-75) sequence, synthetic primer P1 and P2, sequence is respectively：5’-
ATGGGTGCTGGTGGACGAATG-3 ' (SEQ ID NO.1) and 5 '-TTGCATCAGAATCAATACGTG-3 ' (SEQ ID
NO.2 performing PCR amplification), and to sample DNA is entered, the amplified production length is 1160 ± 3bp.Amplified production Ago-Gel is reclaimed
Afterwards, nucleotide sequence is determined using generation sequencing technologies.During use software kit primer5 (http://
Www.Premier5BioSoft.com it is) free disclosed；Main agents include Taq enzyme, dNTP, agarose, AxyPrep
DNA gel QIAquick Gel Extraction Kit.
(5) Multiple Sequence Alignment method is used, according to the principle of minimum genotype frequency >=5%, SNP in screening sequence
Point, analysis sequencing peak figure determines SNP site genotype.
(6) by the genotype data input Structure2.3.4 (http of colony://
Pritchardlab.stanford.edu/structure.html) software, carries out population genetic variations analysis.
(7) by the genotype data of colony, genetic makeup data, the phenotypic data of seed oil content and Kinship squares
Battle array data input TASSEL5.0 (http://www.maizegenetics.net/tassel) in software, using unified hybrid guided mode
The linkage disequilibrium of type method (MLM) analysis SNPs marks and Seed of Camellia oleifera oil content proterties, detects SNP01493 sites
Extremely significantly associated with Seed of Camellia oleifera oil content and (use Bonferroni multiple testing adjustments, P<5.49×10-5), to phenotypic variation
Contribution rate be 11.69%.
Using abovementioned technology, the present invention is finally obtained the molecular labeling extremely significantly associated with Seed of Camellia oleifera oil content
SNP01493, the mark is located at oil tea Cofad2-1A gene open reading frames 493bp, and base is G/G or G/A.For oil tea
Seed oil content proterties, if assuming the gene effect of G/A genotype for the gene effect of 0, G/G is 12.81634.
Specifically, a kind of SNP marker related to Seed of Camellia oleifera oil content that the present invention is provided, positioned at oil tea
At Cofad2-1A gene open reading frames 493bp, the loci gene type is G/A.
Further, the SNP marker related to Seed of Camellia oleifera oil content of the invention can be by nucleotide sequence such as
Primer pair shown in SEQ ID NO.1-2 is expanded through PCR as template with oil tea genomic DNA and obtained.
The SNP marker related to Seed of Camellia oleifera oil content of the invention passes through nucleotide sequence such as SEQ ID NO.1-
Primer pair shown in 2 is expanded through PCR as template with oil tea genomic DNA and obtained, and amplified production contains positioned at oil tea Cofad2-1A
Base at gene open reading frame 493bp, its polymorphism is G/A.
The invention provides application of the above-mentioned SNP marker in Seed of Camellia oleifera oil content phenotype is identified, if SNP molecules
When the genotype of mark is G/G, oil tea to be identified is oil content high, to be identified if the genotype of SNP marker is G/A
Oil tea is low oil content or the low oil content of candidate.
Specific method is：(1) oil tea material tender leaf to be identified is extracted into genomic DNA, using P1 and P2 primer pairs (SEQ
ID NO.1-2) enter performing PCR amplification, resulting PCR primer is detected and reclaimed by agarose gel electrophoresis；
(2) base sequence of PCR primer is determined, and identifies the genotype in SNP01493 sites, if the genotype in the site
It is G/G, then oil tea to be identified is oil content oil tea high or candidate oil content oil tea high；If the genotype in the site is G/A, treat
Identify that oil tea is low oil content oil tea or the low oil content oil tea of candidate.
The oil tea to be identified can be any breeding material, including individual and sexual colony of natural population individuality.
In the above method, extract oil tea genomic DNA and use KAC methods (TaKaRa kit Code NO.9768).
In the above method, the PCR programs are：95 DEG C, 3min, 1 circulation predegeneration；95 DEG C, 15s denaturation, 68 DEG C,
45s extends, 40 circulations；68 DEG C, 5min, 1 circulation thoroughly extends.
In the agarose gel electrophoresis, the concentration of agarose cohesion is 1.2%.Glue reclaim is solidifying using AxyPrep DNA
Glue reclaim kit.The above method determines the base sequence of PCR primer, with P1 and P2 as sequencing primer, skill is sequenced using a generation
Art.
The invention provides application of the above-mentioned SNP marker in Camellia oleifera Germplasms improvement.
The invention provides application of the above-mentioned SNP marker in Seed of Camellia oleifera oil content early prediction.
The invention provides application of the above-mentioned SNP marker in oil content oil tea high is screened.
The invention provides the primer pair of the genotype for detecting above-mentioned SNP marker, its nucleotide sequence difference
As shown in SEQ ID NO.1-2.
Kit containing primer pair shown in SEQ ID NO.1-2 belongs to protection scope of the present invention.
Contain in identification height the invention provides the primer pair shown in SEQ ID NO.1-2 or the kit containing the primer pair
Application in oil cut rate oil tea.
Above-mentioned application is to detect oil tea genomic DNA to be measured using PCR, and (amplified production nucleotide sequence is such as amplified production
Shown in SEQ ID NO.3) the 493rd bit base, if genotype be G/G, oil tea to be identified be oil content oil tea high.
In above-mentioned application, the response procedures of PCR are：95℃3min；95 DEG C of 15s, 68 DEG C of 45s, totally 40 circulations；68℃
5min, 1 circulation thoroughly extends.
The beneficial effects of the present invention are：The present invention develops SNP of one and oil tea oil content highlights correlations first
Point, can explain 11.69% oil content phenotypic variance.Assisted Selection is carried out to sexual oil tea colony using the mark, has been tied
Fruit shows, the site in the individual plant of G/G, 64.39% individual its seed oil content is higher than colony's seed oil content average value,
The site is in the individual plant of G/A, 77.78% individual its seed oil content is less than community average.This shows that the mark is used for
Assisted Selection is effective.With the development approach that Seed of Camellia oleifera oil content associates site, its principle is that oil tea is typical
Outcrossing species, linkage disequilibrium (LD) is generally cut down rapidly in the range of a gene, therefore it is crucial to carry out important character
Intragenic LD mappings.The key gene for regulating and controlling oil tea unsaturated fatty acid content is separated, and is opened as present invention mark
The main region of hair.On the premise of possessing the oil tea natural population for generating a large amount of obvious hereditary variations, can effectively carry out
With the significantly correlated marker development of Economic Character Variation such as oil tea oil content, fatty acid composition content.
In oil tea conventional selection breeding, the identification of seed oil content proterties needs the seedling 5-6 that afforests could to identify, takes
When it is laborious.SNP site locality specific in the present invention, detection method fast and easy, not affected by environment, purpose is stronger, work
Work amount is small, in hgher efficiency, low cost.Therefore, by detecting the SNP site, can be identified and assisting sifting in seedling stage, significantly
Save production cost and improve efficiency of selection.In oil tea breeding, molecular labeling of the invention and the method for detecting it may be selected
Identifying oil content oil tea high carries out breeding, can improve the efficiency of selection of oil tea breeding, accelerates breeding process.
Brief description of the drawings
Fig. 1 oil teas natural population seed oil content sample size distribution map (abscissa be Seed of Camellia oleifera oil content (%),
Ordinate represents sample number of individuals).Result shows that Seed of Camellia oleifera oil content phenotype, in normal distribution, belongs to quantitative character.
Fig. 2 extracts tender leaf DNA electrophoretograms, and each swimming lane is a sample, it is seen that the DNA sample extracted is without degraded, nothing
The impurity such as protein, polysaccharide pollute, and quality is higher, can be used for subsequent experimental.
Fig. 3 amplified productions are sequenced peak figure, and because oil tea belongs to outcrossing species, heterozygosity is higher, and many sites belong to heterozygosis
Site, the SNP site detected in the present invention is also heterozygous sites.Part is detection site in dotted line frame, and left figure is G/A bases
Because of type (heterozygous sites), right figure is G/G genotype.
Fig. 4 sample subgroup group structure effect schematic diagrames.Result shows all individualities of natural population used by the present invention, according to
Its nucleotide polymorphisms can be divided into 4 subgroups.
Specific embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of the invention and essence, the modification or replacement made to the inventive method, step or condition belong to the present invention
Scope.
500 parts of individual plants of natural population's material used in this research, by China Forestry Science Research Institute's subtropical zone forestry
Woody oleiferous plants seminar of research institute collects, evaluates, and is stored in Jinhua, Zhejiang Wucheng District Dongfanghong forest farm Germplasm Resources.
If not specializing, the conventional meanses that technological means used is well known to those skilled in the art in embodiment.
The structure and property determination of the Seed of Camellia oleifera oil content segregating population of embodiment 1
500 parts of natural populations of germ plasm resource in garden are collected using C. olelfera resource in the present embodiment, its area of origin is contained
The major part of lid China oil tea main producing region, including Zhejiang Province, Hunan Province, Jiangxi Province, Guangxi District, Fujian Province, Guangdong Province etc..500
Individuality gathers seed after (5% cracking of fruit) after fruit full maturity, and seed oil content is determined using soxhlet extraction methods.Its behaviour
Make step as follows：
(1) Medium speed filter paper bag is prepared, and is put into aluminium box, 105 DEG C are dried to permanent quality, record aluminium box and filter paper bag quality
(W1)。
(2) appropriate Seed of Camellia oleifera peels off sclerotesta, and 105 DEG C are dried to permanent quality, after being crushed with pulverizer, loads in filter paper bag
Bandage, write down the gross mass (W of aluminium box, filter paper bag and sample2)。
(3) Switzerland Buchi surname extraction instrument B-811LSV, the sample filter paper bag that quality will be weighed up is used to be put into extraction flask
In, about 100ml ether is added, 6h is extracted, ether is reclaimed, filter paper bag (inside having residue) is put into aluminium box and is dried to perseverance in 105 DEG C
Quality, writes down the quality (W of aluminium box, filter paper bag and residue3)。
Dry seed oil content=[(W2-W3)/(W2-W1)] × 100%
Seed of Camellia oleifera oil content measurement result shows：Natural population's seed oil content is in normal distribution (see Fig. 1), illustrates this
Proterties has quantitative character feature.
Embodiment 2Cofad2-1A gene fragment amplifications
1st, blade Genome DNA extraction：
Using in TaKaRa MiniBEST Plant Genomic DNA Extraction Kit rich in polysaccharide, polyphenol and
Oil and fatty plant material cracking system extracts blade STb gene, comprises the following steps that：
(1) the Buffer HS II of 500 μ l are added in 1.5ml centrifuge tubes first.Taking the fresh blades of 0.1g adds liquid nitrogen to fill
Divide grinding, ground blade powder is added in centrifuge tube fully mixes rapidly, be subsequently adding the RNaseA of 10 μ l
(10mg/ml), fully vibration were mixed, in 56 DEG C of water-bath warm bath 10 minutes；
(2) the Buffer KAC of 62.5 μ l are added, is fully mixed.Place 5 minutes on ice, 12000rpm is centrifuged 5 minutes.Take
Supernatant, adds and the isometric Buffer GB of supernatant, fully mixing.
(3) Spin Column are placed on collecting pipe, (solution is excessive, can be divided to two during solution moves to Spin Column
Secondary to cross post, each volume for crossing post does not exceed 700 μ l), 12000rpm is centrifuged 1 minute, abandons filtrate.
(4) the Buffer WA of 500 μ l are added into Spin Column, 12000rpm is centrifuged 1 minute, abandons filtrate.
(5) the Buffer WB of 700 μ l are added into Spin Column, 12000rpm is centrifuged 1 minute, abandons filtrate.
(6) step (5) is repeated.
(7) Spin Column are placed on collecting pipe, 12000rpm is centrifuged 2 minutes.
(8) Spin Column are positioned on the centrifuge tube of new 1.5ml, are added in the centre of Spin Column films
The Elution Buffer of 30~50 μ l, are stored at room temperature 5 minutes, and 12000rpm is centrifuged 2 minutes eluted dnas.Use ultraviolet spectrometry light
Degree meter determines DNA concentration, is saved backup in -20 DEG C of refrigerators (Fig. 2).
2nd, the exploitation and synthesis of primer：
The primer be according to Tan Xiaofeng etc. (Tan Xiaofeng, Chen Hongpeng, Zhang Dangquan etc., oil tea FAD2 full length genes cDNA's
Cloned culturing, forest-science, 2008,44 (3)：The cDNA sequence of oil tea Cofad2-1A genes 70-75) cloned
Design.Specific development approach is to utilize Primer5 softwares (http according to the cDNA sequence of the gene://
Www.Premier5BioSoft.com) separately designed near initiation codon and terminator codon primer P1 (5 '-
ATGGGTGCTGGTGGACGAATG-3 ') and P2 (5 '-TTGCATCAGAATCAATACGTG-3 ') and transfer to company to synthesize.
3rd, Cofad2-1A gene fragment amplifications, its flow is as follows：
It is template with all individual DNA for extracting, P1 and P2 is amplimer, enters performing PCR amplification, reaction system：
PCR amplification programs are：
4th, the gel detection of amplified fragments and purifying reclaim and be sequenced, Genotyping, according to AxyPrep DNA gels reclaim
Kit specification is carried out, and its flow is as follows：
(1) 1.2% Ago-Gel is prepared, by 50 μ l amplified productions whole loading, electrophoretic voltage is 5V/cm, electrophoresis
Stop electrophoresis when being reached at the 1cm of gel front end to dimethylbenzene green grass or young crops in sample-loading buffer within about 20 minutes.
(2) Ago-Gel containing target DNA is cut under uviol lamp, the liquid of gel surface is exhausted with paper handkerchief and is cut
It is broken.Calculated for gel weight, the weight is used as a gel volume (such as 100mg=100 μ l volumes).
(3) 3 Buffer DE-A of gel volume are added, is well mixed after 75 DEG C of heating, interruption is mixed within every 2~3 minutes
Close, until gel piece is completely melt.
(4) 0.5 Buffer DE-B of Buffer DE-A volumes is added, is well mixed.
(5) above-mentioned solution is transferred to during DNA prepares pipe, 12000rpm is centrifuged 1 minute, abandons filtrate.
(6) 500 μ l Buffer W1,12000rpm centrifugation 30 seconds is added, filtrate is abandoned.
(7) 700 μ l Buffer W2,12000rpm centrifugation 30 seconds is added, filtrate is abandoned.In the same way again with 700 μ l
Buffer W2 washed once, and 12000rpm is centrifuged 1 minute, abandons filtrate.
(8) pipe will be prepared to put back in centrifuge tube, 12000rpm is centrifuged 1 minute.
(9) pipe will be prepared to be placed in the 1.5ml centrifuge tubes of cleaning, Jia 25~30 μ l deionized waters, room film center is prepared
Temperature stands 1 minute.12000rpm is centrifuged 1 minute eluted dna.
(10) gel reclaims DNA, with P1 and P2 as sequencing primer, amplified production nucleotides sequence is determined using generation sequencing
Row, the genotype (Fig. 3) of each SNP site in peak figure is sequenced with the interpretation of Chromas softwares.
The screening of the SNP site related to Seed of Camellia oleifera oil content of embodiment 3
Group structure is analyzed and linkage disequilibrium value, and its step is as follows：
(1) the SNPs sites data of all samples are imported in Structure2.3.4 softwares, K=2~9 is set, per K values
Operation 5 times, burnin5000 times, repeats 50000 times.When LnP (D) and α values keep stabilization, and during α ＜ 0.2, determine colony
The K values (Fig. 4) of structure, K=4 in the present invention, and determine the individual subgroup effect values (table 1) of K (4) of each sample.
4 subgroup effect values of the natural population's some individuals of table 1
(2) by the SNPs sites data of all samples, K subgroup effect Value Data, phenotypic data (see embodiment 1) and
Kinship matrix datas are imported in TASSEL5.0 softwares, using MLM methods analysis SNPs and the linkage disequilibrium of oil content proterties
Property, the molecular labeling that screening is significantly associated with seed oil content, using Bonferroni multiple testing adjustments, detects one
There is the extremely significantly site SNP01493 of association with oil content, its genotype is G/G or G/A, and the mark associates F inspections with oil content
The P values tested are 4.66 × 10-5, the contribution rate to oil content difference is 11.69%.
Application of the molecular marker SNP 01493 of embodiment 4 in oil tea breeding for high oil content
(1) it is material to select an oil tea first familiar generation family colony, and collection tender leaf extracts STb gene (see embodiment 2).
(2) performing PCR amplification is entered (see embodiment 2) using P1 and P2 primer pair STb genes.
(3) pcr amplification product carries out gel purification and sequencing analysis (see embodiment 2).
(4) genotype in all individual SNP01493 sites is identified.If the genotype in the site is G/G, oil tea is individual
It is oil-containing oil tea high or candidate oil-containing oil tea high；If the genotype in the site is G/A, it is low oil-containing oil tea or time that oil tea is individual
Select low oil-containing oil tea.
(5) all F1 generation individuality full maturity seeds are gathered, the oil content (see embodiment 1) of its seed is determined.As a result table
Bright, the site is in the individual plant of G/G, 64.39% individual its seed oil content is higher than colony's seed oil content average value
(36.20%), the site is in the individual plant of G/A, 77.78% individual its seed oil content is less than community average
(36.20%) (table 2).This shows that the mark is effective for assisted Selection, can be used for Early Identification or auxiliary differentiates,
Production cost can be greatlyd save, efficiency of selection is improved, accelerates oil tea breeding for high oil content process.
The seed oil content data of the F1 individual plants that table 2 is obtained using SNP01493 sites assisted Selection
Although having used general explanation, specific embodiment and experiment above, the present invention is described in detail,
But on the basis of the present invention, some modifications or improvement can be made to it, this is to those skilled in the art apparent
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Scope.
SEQUENCE LISTING
<110>Inst. of Subtropical Forestry, Chinese Academy of Forestry Sciences
<120>A kind of SNP marker related to Seed of Camellia oleifera oil content and its application
<130> KHP171110815.5
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
atgggtgctg gtggacgaat g 21
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
ttgcatcaga atcaatacgt g 21
<210> 3
<211> 1160
<212> DNA
<213>Oil tea
<400> 3
atgggtgctg gtggacgaat gcctgtccca gcaaccaaac atgaacagca gattaccccc 60
cacagggccc ctcactcaaa gccaccattc actctcggtg aaatcaagaa agccatccca 120
ccccactgct ttgaacgttc tctcctccgc tcattctcct acattgttta tgacttctct 180
ctcgtctttc ttttctacta cgtcaccacc tcttacatcc acctccttcc acagcacttc 240
cgttatcttg tgtggcccat ctactgggca cttcaaggtt gtgtcctcac tggtgtgtgg 300
gtcattgctc atgaatgtgg tcaccatgca ttcagtgatt accaatgggt cgatgacacg 360
gttggtctca tccttcactc caccctttta gttccctact tctcatggaa atacagtcac 420
cgccgtcacc actccaacac cagttccctt gagcatgatg aagtttttgt cccgaaaccc 480
aaatccaaac tcgcatggta ttccaaatac ttgaacaacc cggtgggtcg tgttgtcaca 540
cttgtgatca cactcactct tggctggccc tcttacttgg ccttcaatgt atcagggaga 600
ccttatgatc gttttgcatg tcactacgac ccatatggcc cgatctacaa caaccgtgaa 660
aggctccaga tttacatctc tgatgttggt atcatcacta tagtttatgt tctctgtcgc 720
cttgcttttg caaaagggct ggcttggctt gtttgtgttt atggggttcc gttactgatt 780
gtgaacgggt tccttgtctt gatcacattc ctgcagcaca ctcatcctgc tctgcctcat 840
tatgactcat cggaatggga ctggctgagg ggagctctgt caaccatgga tagggattat 900
ggagtgctga acaaggtgtt ccataatatc acagatactc atgttgctca ccacctcttc 960
tctacaatgc cacattacca tgcaatggag gccacaaagg cgattaagcc tattctcggt 1020
gagtattacc tgtttgatgg tactgcattt tacaaggcga tgtggaggga ggcaagagag 1080
tgtctctacg tggaatcaga tgacgatacc accaccaaag gtgtattttg gtataaaaac 1140
acgtattgat tctgatgcaa 1160
Claims (10)
1. a kind of SNP marker related to Seed of Camellia oleifera oil content, it is characterised in that Cofad2-1A gene opens are read
Site at the 493bp of frame, the genotype in the site is G/A.
2. SNP marker as claimed in claim 1, it is characterised in that as nucleotide sequence as shown in SEQ ID NO.1-2
Primer pair with oil tea genomic DNA be template through PCR expand obtain.
3. application of the SNP marker described in claim 1 or 2 in Seed of Camellia oleifera oil content phenotype is identified, if SNP molecules
When the genotype of mark is G/G, oil tea to be identified is oil content high, to be identified if the genotype of SNP marker is G/A
Oil tea is low oil content or the low oil content of candidate.
4. application of the SNP marker described in claim 1 or 2 in Camellia oleifera Germplasms improvement.
5. application of the SNP marker described in claim 1 or 2 in Seed of Camellia oleifera oil content early prediction.
6. application of the SNP marker described in claim 1 or 2 in oil content oil tea high is screened.
7. the primer pair of the genotype of SNP marker described in test right requirement 1 or 2 is used for, it is characterised in that its nucleotides
Sequence is respectively as shown in SEQ ID NO.1-2.
8. the kit of primer pair described in claim 7 is contained.
9. application of the kit described in the primer pair or claim 8 described in claim 7 in oil content oil tea high is identified.
10. application as claimed in claim 9, it is characterised in that the 493rd bit base of amplified production, if genotype is G/G
When, then oil tea to be identified is oil content oil tea high.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643729A (en) * | 2019-09-04 | 2020-01-03 | 广东省农业科学院茶叶研究所 | SNP molecular marker locus linked with content of gallocatechin gallate of tea tree and application thereof |
| CN111455091A (en) * | 2020-05-22 | 2020-07-28 | 中国林业科学研究院亚热带林业研究所 | SNP molecular marker related to content of linolenic acid in oil-tea seed kernel oil and application thereof |
| CN111518943A (en) * | 2020-05-26 | 2020-08-11 | 中国林业科学研究院亚热带林业研究所 | SNP molecular marker related to content of eicosenoic acid in camellia seed oil and application thereof |
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| CN113430297A (en) * | 2021-07-23 | 2021-09-24 | 中国林业科学研究院亚热带林业研究所 | DNA fragment related to content of palmitic acid in oil-tea camellia seed oil, SNP molecular marker closely linked with DNA fragment and application of SNP molecular marker |
| CN113584204A (en) * | 2021-07-23 | 2021-11-02 | 中国林业科学研究院亚热带林业研究所 | DNA fragment related to kernel-out rate of camellia oleifera seeds, SNP molecular marker closely linked with same and application thereof |
| CN113604593A (en) * | 2021-07-26 | 2021-11-05 | 中国林业科学研究院亚热带林业研究所 | DNA fragment related to linoleic acid content in oil tea seed oil and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014062607A1 (en) * | 2012-10-15 | 2014-04-24 | Commercial Marine Biology Institute, Llc | Marine extract compositions and methods of use |
| CN104004584A (en) * | 2014-06-17 | 2014-08-27 | 中国林业科学研究院亚热带林业研究所 | Appropriate processing method for squeezing camellia oleosa seed oil |
| CN104087576A (en) * | 2014-06-18 | 2014-10-08 | 中国农业科学院蔬菜花卉研究所 | Molecular marker related to color property of tomato fruit and application thereof |
| CN106676102B (en) * | 2017-02-23 | 2019-02-19 | 中国林业科学研究院亚热带林业研究所 | One kind SNP marker relevant to arachidonic acid content in Seed of Camellia oleifera grease and its application |
-
2017
- 2017-02-23 CN CN201710100586.2A patent/CN106755528B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014062607A1 (en) * | 2012-10-15 | 2014-04-24 | Commercial Marine Biology Institute, Llc | Marine extract compositions and methods of use |
| CN104004584A (en) * | 2014-06-17 | 2014-08-27 | 中国林业科学研究院亚热带林业研究所 | Appropriate processing method for squeezing camellia oleosa seed oil |
| CN104087576A (en) * | 2014-06-18 | 2014-10-08 | 中国农业科学院蔬菜花卉研究所 | Molecular marker related to color property of tomato fruit and application thereof |
| CN106676102B (en) * | 2017-02-23 | 2019-02-19 | 中国林业科学研究院亚热带林业研究所 | One kind SNP marker relevant to arachidonic acid content in Seed of Camellia oleifera grease and its application |
Non-Patent Citations (6)
| Title |
|---|
| EN-HUA XIA ET AL.: "Transcriptome Analysis of the Oil-Rich Tea Plant,Camellia oleifera, Reveals Candidate Genes Related to Lipid Metabolism", 《PLOS ONE》 * |
| PING LIN ET AL.: "Association Genetics Identifies Single Nucleotide Polymorphisms Related to Kernel Oil Content and Quality in Camellia oleifera", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| TAN,X ET AL.: "Camellia oleifera cultivar Huashuo fatty acid desaturase-2 (FAD-2) mRNA, complete cds，GenBank: KJ995981.1", 《GENBANK》 * |
| 周长富等: "油茶果实发育特性及水分、油脂含量动态分析", 《扬州大学学报（农业与生命科学版）》 * |
| 谭晓风等: "油茶FAD2基因全长cDNA的克隆和序列分析", 《林业科学》 * |
| 陈鸿鹏等: "油茶FAD2基因的植物表达载体和ＲNA干扰载体构建及其功能分析", 《植物研究》 * |
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| CN113604593B (en) * | 2021-07-26 | 2023-08-15 | 中国林业科学研究院亚热带林业研究所 | DNA fragment related to linoleic acid content in oil tea seed oil and application thereof |
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