CN106868132A - Palmitic acid, oleic acid, linolenic acid content are related in a kind of grease to Seed of Camellia oleifera SNP marker and its application - Google Patents
Palmitic acid, oleic acid, linolenic acid content are related in a kind of grease to Seed of Camellia oleifera SNP marker and its application Download PDFInfo
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Abstract
Palmitic acid, oleic acid, linolenic acid content are related in a kind of grease to Seed of Camellia oleifera of present invention offer SNP marker 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 474bp of Cofad2 1A gene open reading frames, and the polymorphism in the site is A/G.Oil tea breeding material is detected using the molecular labeling, can seedling stage prediction Seed of Camellia oleifera grease in palmitic acid, oleic acid, linolenic acid content height, substantially increase the efficiency of selection of oil tea oil quality breeding.
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 fatty acid oil component content joint screening, also relates to the molecular labeling
Application in Seed of Camellia oleifera oil quality 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 camellia seed oil quality
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, carrying out camellia seed oil quality MAS breedings will effectively shorten 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 to develop by linkage disequilibrium is contained with Seed of Camellia oleifera oil palmitic acid, oleic acid and leukotrienes
The pleomorphism site of association is measured, screening can stablize the mark for being used for early stage assisted Selection, auxiliary as the effective molecular labeling of oil tea
Help Breeding Strategies.
The content of the invention
Extremely significantly associated with Seed of Camellia oleifera oil palmitic acid, oleic acid and linolenic acid content it is an object of the invention to provide one
Molecular labeling.The mark is located in oil tea Cofad2-1A gene open reading frames, belongs to codominant SNP marker, thus can
By and it is easy to use, this is provided a great convenience for oil tea high-quality strain breeding.
Extremely show with Seed of Camellia oleifera oil palmitic acid, oleic acid and linolenic acid content another object of the present invention is to provide one
Write application of the molecular labeling of association in oil tea quality breeding.The present invention has been carried out auxiliary using the mark to sexual oil tea colony
Selection is helped, is as a result shown, the site is in the individual plant of A/G, 58.33% individual its palmitic acid content contains less than colony's palmitic acid
Amount average value, 70.83% individual its linolenic acid content is less than colony's linolenic acid content average value, 66.67% individual its oil
Acid content exceedes colony's oleic acid content average value.This shows that the mark is effective for assisted Selection.
With the development approach that Seed of Camellia oleifera fatty acid oil content associates site, its principle is that oil tea is typical outcrossing thing
Kind, linkage disequilibrium (LD) is generally cut down rapidly in the range of a gene, therefore can be carried out in important character key gene
LD mapping.Tea oil is mainly characterized by unsaturated fatty acid content up to more than 90% better than other oil plants, and wherein oleic acid contains
74%~87%, linoleic acid content is 7%~14% to amount.Regulate and control oil tea unsaturated fatty acid content key gene by
Separate, as the main region of marker development of the present invention.The significantly oil tea natural group of hereditary variations is generated largely possessing
On the premise of body, can effectively carry out the marker development significantly correlated with unsaturated fatty acid content variation.
To achieve these goals, the present invention uses following technical measures：
(1) Camellia oleifera Germplasms are collected extensively in oil tea super distributed area, seed grease content of fatty acid is set up and is divided extensively
From oil tea natural population, as association colony.
(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, mature seed oil is determined with gas chromatography
5 kinds of fatty acid composition contents of fat, including stearic acid, palmitic acid, oleic acid, linoleic acid, leukotrienes, specific method is according to GB/T
17376《It is prepared by animal and plant fat fatty acid methyl ester》With GB/T 17377《The gas-chromatography of animal and plant fat Fatty acid methyl ester point
Analysis》Perform.
(4) according to oil tea Cofad2-1A gene orders, synthetic primer P1 and P2, sequence are 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 fatty acid composition content and
Kinship matrix datas input TASSEL5.0 (http://www.maizegenetics.net/tassel) in software, use
Unified method with mixed model (MLM) analysis SNPs marks and 5 linkage disequilibriums of content of fatty acid proterties, detect altogether
3 SNPs significantly associated with content of fatty acid.Wherein SNP01474 sites simultaneously with Seed of Camellia oleifera oil palmitic acid, oleic acid and Asia
Extremely significantly association (uses Bonferroni multiple testing adjustments, P to numb acid content<5.49×10-5), and to the tribute of phenotypic variation
Offer that rate is larger, be each responsible for palmitic acid, 13.04%, 10.22% and the 31.28% of the variation of linoleic acid plus linolenic acid content, therefore
The site is one of main effect site of linolenic acid content.
Using abovementioned technology, applicant is finally obtained and Seed of Camellia oleifera oil palmitic acid, oleic acid and linolenic acid content
The molecular marker SNP 01474 for extremely significantly associating, the mark is located at oil tea Cofad2-1A gene open reading frames 474bp, alkali
Base is A/A or A/G.If the gene effect for assuming A/A genotype is 0, relative to Seed of Camellia oleifera oil palmitic acid content, oleic acid content
With 3 proterties of linolenic acid content, the gene effect of A/G is respectively -0.23272,1.26192 and -0.028249.
Specifically, the related SNP of the present invention is provided a kind of oil palmitic acid with Seed of Camellia oleifera, oleic acid and linolenic acid content divides
Son mark, at oil tea Cofad2-1A gene open reading frames 474bp, the loci polymorphism is A/G.
Further, the oil palmitic acid to Seed of Camellia oleifera of the invention, oleic acid and the related SNP marker of linolenic acid content
Can be obtained through PCR amplifications by template of oil tea genomic DNA as primer pair of the nucleotide sequence as shown in SEQ ID NO.1-2
.
The invention provides application of the above-mentioned SNP marker in the oil tea that high-quality grease is produced in identification, if SNP molecules
When the genotype of mark is A/A, oil tea to be identified is palmitic acid high, high linolenic, low oleic acid content oil tea or candidate's palmitin high
Acid, high linolenic, low oleic acid content oil tea；If the genotype of SNP marker be A/G, oil tea to be identified be low palmitic acid,
Low linolenic, high oleic acid content oil tea or the low palmitic acid of candidate, low linolenic, high oleic acid content oil tea.
Specific method is：
(1) oil tea material tender leaf to be identified is extracted into genomic DNA, entering performing PCR using P1 and P2 primer pairs expands, and passes through
Agarose gel electrophoresis is detected and reclaims resulting PCR primer；
(2) base sequence of PCR primer is determined, and identifies the genotype in SNP01474 sites, if the genotype in the site
Be A/A, then oil tea to be identified be palmitic acid high, high linolenic, low oleic acid content oil tea or candidate's palmitic acid high, high linolenic,
Low oleic acid content oil tea；If the genotype in the site is A/G, oil tea to be identified is low palmitic acid, low linolenic, high oleic acid contain
Gauging tea or the low palmitic acid of candidate, low linolenic, high oleic acid content oil tea.
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 as sequencing primer, using generation sequencing technologies.
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 quality early prediction.
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.
Produced in identification the invention provides the primer pair shown in SEQ ID NO.1-2 or the kit containing the primer pair high
Application in the oil tea of quality grease.
Above-mentioned application is to detect oil tea genomic DNA to be measured, amplified production (nucleotide sequence such as SEQ ID using PCR
Shown in NO.3) the 474th bit base, if genotype be A/A, oil tea to be identified for high linolenic grease oil tea；If base
During because of type for A/G, then oil tea to be identified is the oil tea of high oleic acid grease.
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 present invention develop first one with oil tea palmitic acid, oleic acid and leukotrienes and meanwhile highlights correlations SNP site, can
To explain 13.04% palmitic acid content phenotypic variance, 10.22% oleic acid content phenotypic variance and 31.28% leukotrienes
Content phenotypic variance.In oil tea conventional selection breeding, the identification of seed lubricant component proterties needs seedling afforestation 5-6 ability
Identification, wastes time and energy.SNP site locality specific in the present invention, detection method fast and easy, not affected by environment, purpose
Stronger, workload is small, in hgher efficiency, low cost.Therefore, by detecting the SNP site, can be identified in seedling stage and be aided in sieve
Choosing, greatlys save production cost and improves efficiency of selection, accelerates to produce the breeding process of high quality oils fatty oil tea.
Brief description of the drawings
Figure 1A-Fig. 1 C are respectively palmitic acid, oleic acid and linolenic acid content distribution map (horizontal seat in oil tea natural population seed oil
Mark represents palmitic acid, oleic acid and linolenic acid content (%) in Seed of Camellia oleifera grease respectively, and ordinate represents sample number of individuals).Knot
Palmitic acid content, oleic acid content, linolenic acid content phenotype belong to quantitative character in normal distribution in really showing Seed of Camellia oleifera oil.
Fig. 2 extract tender leaf STb gene electrophoretogram, each swimming lane be a sample, it is seen that the DNA sample extracted without degraded,
Without the pollution of the impurity such as protein, polysaccharide, 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 A/A bases
Because of type, right figure is A/G genotype (heterozygous sites).
Fig. 4 sample subgroup group structure effect schematic diagrames, as a result show 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 fatty acid oil component 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, extracts grease and determines fatty acid composition and content.Its behaviour
Make step as follows：
(1) appropriate Seed of Camellia oleifera 80 DEG C of bakings of baking box peel off sclerotesta overnight to constant weight.
(2) after benevolence is crushed with pulverizer, wrapped with Medium speed filter paper, add appropriate ether immersion extracting overnight.
(3) after ether volatilizees completely, using Agilent6890N gas chromatographs according to GB/T17376-2008, GB/
T17377-2008 methods determine fatty acid composition and content.
Fatty acid composition assay result shows：Palmitic acid, oleic acid and linolenic acid content are equal in natural population's seed oil
In normal distribution (Figure 1A, Figure 1B and Fig. 1 C), illustrate that these three proterties all have quantitative character feature.
The Cofad2-1A gene fragment amplifications of embodiment 2
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 '), with colony individuality DNA as mould
Plate, expands the genome sequence of Cofad2-1A genes.
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 fatty acid 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, and the correlation between SNPs and 5 phenotypic character is analyzed using MLM methods
Property, the linkage disequilibrium of analysis SNPs and content of fatty acid, the molecule mark that screening is significantly associated with unsaturated fatty acid content
Note, using Bonferroni multiple testing adjustments, detects a site that there is significantly association with multiple proterties
SNP01474 (table 2).The mark is associated with the presence of palmitic acid, stearic acid, oleic acid, linoleic acid plus linolenic acid content, but according to
Bonferroni multiple testing adjustments, the level of signifiance is not up to stearic acid and associating for linoleic acid content, with palmitic acid, oleic acid
Extremely notable interrelation level is reached with linolenic acid content.
The phenotypic character of table 2 and SNPs association analysis partial results
Application of the molecular marker SNP 01474 of embodiment 4 in Seed of Camellia oleifera oil quality breeding
(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 SNP01474 sites is identified.If the genotype in the site is A/A, oil tea is individual
It is palmitic acid high, high linolenic, low oleic acid content oil tea or candidate's palmitic acid high, high linolenic, low oleic acid content oil tea；If should
The genotype in site is A/G, then oil tea it is individual for low palmitic acid, low linolenic, high oleic acid content oil tea or the low palmitic acid of candidate,
Low linolenic, high oleic acid content oil tea.
(5) all F1 generation individuality full maturity seeds are gathered, the fatty acid composition and content of its seed oil is determined (see implementing
Example 1).Result shows (table 3), and the site is in the individual plant of A/G, 58.33% individual its palmitic acid content is less than colony's palmitin
Acid content average value (8.65%), 70.83% individual its linolenic acid content is less than colony's linolenic acid content average value
(0.28%), 66.67% individual its oleic acid content exceedes colony's oleic acid content average value (78.18%).This shows the mark
It is effective for assisted Selection, can be used for Early Identification or auxiliary differentiates, production cost can be greatlyd save, improves selection
Efficiency, accelerates oil tea tea oil quality breeding process.
The content of fatty acid data of the F1 individual plants that table 3 is obtained using SNP01474 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 palmitic acid, oleic acid, linolenic acid content are related in grease to Seed of Camellia oleifera SNP marker and its
Using
<130> KHP171110819.9
<160> 3
<170> PatentIn version 3.5
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atgggtgctg gtggacgaat g 21
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ttgcatcaga atcaatacgt g 21
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<213>Artificial sequence
<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 (9)
1. the SNP marker that palmitic acid, oleic acid, linolenic acid content are related in a kind of grease to Seed of Camellia oleifera, it is characterised in that
Site at the 474bp of Cofad2-1A gene open reading frames, the polymorphism in the site is A/G.
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 the oil tea that high-quality grease is produced in identification, if SNP molecules
When the genotype of mark is A/A, oil tea to be identified is palmitic acid high, high linolenic, low oleic acid content oil tea or candidate's palmitin high
Acid, high linolenic, low oleic acid content oil tea；If the genotype of SNP marker be A/G, oil tea to be identified be low palmitic acid,
Low linolenic, high oleic acid content oil tea or the low palmitic acid of candidate, low linolenic, high oleic acid content oil tea.
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 quality early prediction.
6. 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.
7. the kit of primer pair described in claim 6 is contained.
8. the kit described in the primer pair or claim 7 described in claim 6 is in the oil tea that high-quality grease is produced in identification
Application.
9. application as claimed in claim 8, it is characterised in that the 474th bit base of amplified production, if genotype is A/A,
Then oil tea to be identified is the oil tea of high linolenic；If genotype is A/G, oil tea to be identified is the oil tea of high oleic acid.
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| CN201710100615.5A CN106868132B (en) | 2017-02-23 | 2017-02-23 | SNP molecular marker related to contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil and application thereof |
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| 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 |
| CN111455090A (en) * | 2020-05-22 | 2020-07-28 | 中国林业科学研究院亚热带林业研究所 | Key SNP molecular marker related to content of linolenic acid in oil-tea seed kernel oil and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154529A (en) * | 2015-07-23 | 2015-12-16 | 浙江省林业科学研究院 | Molecular specific marker primer and identification method for fine varieties of camellia oleifera |
| US20160228352A1 (en) * | 2012-10-15 | 2016-08-11 | I Commercial Marine Biology Institute, LLC | Marine extract compositions and methods of use |
| 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 CN201710100615.5A patent/CN106868132B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160228352A1 (en) * | 2012-10-15 | 2016-08-11 | I Commercial Marine Biology Institute, LLC | Marine extract compositions and methods of use |
| CN105154529A (en) * | 2015-07-23 | 2015-12-16 | 浙江省林业科学研究院 | Molecular specific marker primer and identification method for fine varieties of camellia oleifera |
| 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 (8)
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