CN106868132B - SNP molecular marker related to contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil and application thereof - Google Patents
SNP molecular marker related to contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil and application thereof Download PDFInfo
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Abstract
The invention provides an SNP molecular marker related to the contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil and application thereof. The SNP molecular marker is obtained by amplifying a primer with a nucleotide sequence shown as SEQ ID NO.1-2, an amplification product contains a locus at 474bp of an open reading frame of Cofad2-1A gene, and the polymorphism of the locus is A/G. The molecular marker is used for detecting the oil-tea camellia breeding material, so that the content of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil can be predicted in the seedling stage, and the selection efficiency of oil-tea camellia oil quality breeding is greatly improved.
Description
Technical Field
The invention relates to the technical field of biology, belongs to the technical field of camellia oleifera molecular biology and genetic breeding, and particularly relates to a polymorphic site molecular marker for jointly screening the fatty acid component content of camellia oleifera seed oil, and an application of the molecular marker in camellia oleifera seed oil quality breeding.
Background
Camellia oleifera (Camellia oleifera Abel.), belonging to the genus Camellia (Camellia L.) of the family Theaceae, is a unique woody oil tree species in China and is also an important woody edible oil species in the south of China. The camellia seed oil has high nutrition and health care value, the quality of the camellia seed oil can be compared with that of olive oil, the camellia seed oil is high-quality edible oil, the content of unsaturated fatty acid reaches more than 90%, oleic acid (more than 80%) and linoleic acid (about 8%) are used as main materials, and the camellia seed oil has the effects of resisting tumors, reducing blood fat and the like. In the last decade, under the guidance and support of national policies, the oil tea industry in China has been developed greatly, the planting area in China reaches 6000 to ten thousand mu, and 60 to ten thousand tons of oil are produced annually. According to the national oil tea industry development plans (2009-2020), by 2020, the planting area of oil tea in China reaches 9300 ten thousand mu, so that the demand of improved oil tea seedling is large. At present, oil tea breeding takes selection and cross breeding as main means and fruit yield as a main breeding purpose, and has made important progress, but few breeding researches are carried out aiming at improving the quality of oil tea seeds. Meanwhile, due to the long childbearing period of the camellia oleifera, the camellia oleifera breeding period is long, the new variety breeding is slow, and the improved variety breeding speed cannot meet the requirement of industrial development, so that the camellia oleifera breeding method becomes one of important factors for hindering the development of the camellia oleifera industry. The molecular Marker Assisted (MAS) breeding method can be used for selecting from the seedling stage, greatly shortens the breeding process, and has particularly obvious advantages on economic forest breeding which mainly aims at fruits. Therefore, the development of MAS breeding of the oil-tea camellia seed oil quality can effectively shorten the breeding period of the oil-tea camellia and has great application potential.
Molecular marker assisted breeding research of the oil tea has been carried out for decades, and certain achievements are achieved by various molecular marker technologies such as RAPD, ISSR, SRAP and the like. However, the technologies all show certain disadvantages, and the obtained polymorphic marker loci are difficult to be really used for the auxiliary breeding of the camellia oleifera. The main disadvantages include: 1. the markers belong to dominant markers, and the genotype of the polymorphic sites cannot be accurately reflected; 2. the marking technologies have high requirements on experiment operators and environment, and the experiment result is unstable; 3. these marking techniques are all to analyze the whole genome sequence, the workload is large, the polymorphic sites can not be accurately positioned, and it is difficult to screen the markers closely linked with the target characters; 4. the traditional Quantitative Trait Locus (QTL) mapping needs mapping populations with genetic relationship and the biological characteristics of the camellia oleifera have long childbearing period, so that the establishment of large-scale camellia oleifera hybrid mapping populations is long in time and difficult and needs to occupy large-area forest land. Therefore, by taking natural populations as research objects, co-dominant SNPs markers are adopted, polymorphic sites related to the contents of palmitic acid, oleic acid and linolenic acid in the oil tea seed oil are developed through linkage disequilibrium mapping, and markers which can be stably used for early auxiliary selection are screened, so that the molecular marker-assisted breeding strategy for the oil tea is effective.
Disclosure of Invention
The invention aims to provide a molecular marker which is extremely obviously related to the contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil. The marker is positioned in an open reading frame of the camellia Cofad2-1A gene and belongs to a codominant SNP marker, so that the marker is reliable and convenient to use, and great convenience is provided for the breeding of high-quality strains of the camellia oleifera.
The invention also aims to provide application of a molecular marker which is extremely obviously related to the contents of palmitic acid, oleic acid and linolenic acid in camellia seed oil in camellia quality breeding. The invention utilizes the marker to perform auxiliary selection on the sexual oil tea population, and the result shows that in the individual plant with the A/G site, 58.33 percent of individuals have palmitic acid content lower than the average value of the palmitic acid content of the population, 70.83 percent of individuals have linolenic acid content lower than the average value of the linolenic acid content of the population, and 66.67 percent of individuals have oleic acid content higher than the average value of the oleic acid content of the population. This indicates that the marker is useful for aiding selection.
The principle of the development method of the loci related to the fatty acid content of the oil-tea camellia seed oil is that the oil-tea camellia is a typical outcrossing species, and Linkage Disequilibrium (LD) is usually reduced rapidly in a gene range, so that LD mapping in key genes of important traits can be carried out. The tea oil is superior to other oil materials and has the main characteristic that the content of unsaturated fatty acid reaches more than 90 percent, wherein the content of oleic acid is 74 to 87 percent, and the content of linoleic acid is 7 to 14 percent. The key gene for regulating the unsaturated fatty acid content of the oil-tea camellia has been separated and is used as a main area for the marker development of the invention. On the premise of having a natural population of the camellia oleifera which generates a large number of obvious genetic variations, the development of markers which are obviously related to the variation of the content of unsaturated fatty acid can be effectively developed.
In order to achieve the purpose, the invention adopts the following technical measures:
(1) oil tea germplasm resources are widely collected in an oil tea full-distribution area, and an oil tea natural population with widely separated seed oil fatty acid content is established as a related population.
(2) The KAC method (TaKaRa kit Code No.9768) is adopted to extract the total DNA of 500 young leaves of single plants in a natural population, and the quality of the extracted DNA is determined by 0.8-1% agarose gel electrophoresis and a nucleic acid determinator, wherein the DNA is required to be free from degradation and impurity pollution such as protein, polysaccharide and the like, and the concentration reaches more than 100 ng/mu L.
(3) Collecting 500 parts of completely mature seeds of oil-tea camellia germplasm of related population, and measuring the content of 5 fatty acid components of mature seed oil by using a gas chromatography, wherein the content comprises stearic acid, palmitic acid, oleic acid, linoleic acid and linolenic acid, and the specific method is implemented according to GB/T17376 animal and vegetable oil fatty acid methyl ester preparation and GB/T17377 gas chromatography analysis of animal and vegetable oil fatty acid methyl ester.
(4) According to the sequence of the camellia Cofad2-1A gene, primers P1 and P2 are synthesized, and the sequences are respectively as follows: 5'-ATGGGTGCTGGTGGACGAATG-3' (SEQ ID NO.1) and 5'-TTGCATCAGAATCAATACGTG-3' (SEQ ID NO.2), and performing PCR amplification on the sample DNA, wherein the length of the amplification product is 1160 +/-3 bp. And (4) after the amplification product is recovered by agarose gel, determining the nucleotide sequence by adopting a first-generation sequencing technology. The software package primer5(http:// www.Premier5BioSoft.com) is used in the process and is freely disclosed; the main reagent comprises Taq enzyme, dNTP, agarose and AxyPrepDNA gel recovery kit.
(5) And screening SNP sites in the sequence by adopting a multi-sequence comparison method according to the principle that the minimum genotype frequency is more than or equal to 5 percent, and analyzing a sequencing peak diagram to determine the genotype of the SNP sites.
(6) Genotype data for the population was imported into Structure2.3.4(http:// handed.
(7) Genotype data, genetic structure data, phenotype data of fatty acid component content and Kinship matrix data of the population are input into TASSEL5.0(http:// www.maizegenetics.net/TASSEL) software, linkage imbalance of SNPs markers and 5 fatty acid content characters is analyzed by a uniform mixed model method (MLM), and 3 SNPs which are obviously related to the fatty acid content are detected. The SNP01474 site is very obviously related to the contents of palmitic acid, oleic acid and linolenic acid in oil tea seed oil at the same time (Bonferroni multiple test correction, P)<5.49×10-5) And the contribution rate to phenotypic variation is larger, and the contribution rate is respectively responsible for 13.04%, 10.22% and 31.28% of the variation of the contents of palmitic acid, linoleic acid and linolenic acid, so that the site is one of the main effective sites of the content of linolenic acid.
By utilizing the technical measures, the applicant finally obtains a molecular marker SNP01474 which is extremely obviously related to the contents of palmitic acid, oleic acid and linolenic acid in the oil tea seed oil, wherein the marker is positioned at an open reading frame 474bp of an oil tea Cofad2-1A gene, and the basic group is A/A or A/G. If the gene effect of the A/A genotype is assumed to be 0, the gene effect of the A/G is-0.23272, 1.26192 and-0.028249 respectively relative to the 3 traits of the oil tea seed oil palmitic acid content, the oleic acid content and the linolenic acid content.
Specifically, the SNP molecular marker related to the contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil provided by the invention is located at an open reading frame 474bp of an oil-tea camellia Cofad2-1A gene, and the polymorphism of the site is A/G.
Further, the SNP molecular marker related to the contents of palmitic acid, oleic acid and linolenic acid in the oil tea seed oil can be obtained by PCR amplification of a primer pair with a nucleotide sequence shown as SEQ ID NO.1-2 by taking oil tea genome DNA as a template.
The invention provides the application of the SNP molecular marker in identifying the oil tea producing high-quality grease, if the genotype of the SNP molecular marker is A/A, the oil tea to be identified is the oil tea with high palmitic acid, high linolenic acid and low oleic acid content or candidate oil tea with high palmitic acid, high linolenic acid and low oleic acid content; if the genotype of the SNP molecular marker is A/G, the oil tea to be identified is the oil tea with low palmitic acid, low linolenic acid and high oleic acid content or the candidate oil tea with low palmitic acid, low linolenic acid and high oleic acid content.
The specific method comprises the following steps:
(1) extracting genome DNA from tender leaves of the oil tea material to be identified, carrying out PCR amplification by adopting a primer pair P1 and a primer pair P2, detecting through agarose gel electrophoresis and recovering the obtained PCR product;
(2) determining the base sequence of the PCR product, and identifying the genotype of the SNP01474 site, wherein if the genotype of the site is A/A, the oil tea to be identified is the oil tea with high palmitic acid, high linolenic acid and low oleic acid content or candidate oil tea with high palmitic acid, high linolenic acid and low oleic acid content; if the genotype of the site is A/G, the oil tea to be identified is the oil tea with low palmitic acid, low linolenic acid and high oleic acid content or the candidate oil tea with low palmitic acid, low linolenic acid and high oleic acid content.
The camellia oleifera to be identified can be any breeding material, including natural population individuals and sexual population individuals.
In the above method, KAC method (TaKaRa kit Code No.9768) is used for extracting genomic DNA of Camellia oleifera.
In the above method, the PCR procedure is: pre-denaturation at 95 ℃ for 3min for 1 cycle; denaturation at 95 ℃ for 15s, elongation at 68 ℃ for 45s, and 40 cycles; at 68 ℃ for 5min, 1 cycle was run through.
In the agarose gel electrophoresis, the concentration of agarose gel aggregation was 1.2%. The recovery of gel used AxyPrep DNA gel recovery kit.
The method is used for determining the base sequence of the PCR product, and adopts a first-generation sequencing technology by taking P1 as a sequencing primer.
The invention provides application of the SNP molecular marker in the improvement of oil tea germplasm resources.
The invention provides application of the SNP molecular marker in early prediction of oil quality of oil-tea camellia seeds.
The invention provides a primer pair for detecting the genotype of the SNP molecular marker, and the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO. 1-2.
The kit containing the primer pair shown in SEQ ID NO.1-2 belongs to the protection scope of the invention.
The invention provides application of a primer pair shown in SEQ ID NO.1-2 or a kit containing the primer pair in identifying oil tea for producing high-quality grease.
The application is that PCR is adopted to detect the genome DNA of the oil-tea camellia to be detected, the 474 th base of an amplification product (the nucleotide sequence is shown as SEQ ID NO. 3) is amplified, and if the genotype is A/A, the oil-tea camellia to be identified is the oil-tea camellia with high linolenic acid grease; and if the genotype is A/G, identifying the oil tea as the oil tea with high oleic acid grease.
In the above application, the reaction procedure of PCR is as follows: 3min at 95 ℃; at 95 ℃ for 15s and at 68 ℃ for 45s for 40 cycles; at 68 ℃ for 5min, 1 cycle was completely extended.
The SNP locus highly related to the palmitic acid, the oleic acid and the linolenic acid of the camellia oleifera is developed for the first time, and the phenotypic variance of the palmitic acid content of 13.04%, the phenotypic variance of the oleic acid content of 10.22% and the phenotypic variance of the linolenic acid content of 31.28% can be explained. In the conventional selection breeding of the camellia oleifera, the identification of the oil and fat component character of the seeds needs to be carried out for 5-6 years after seedling afforestation, which wastes time and labor. The SNP locus position in the invention is definite, the detection method is convenient and quick, is not influenced by the environment, and has stronger purpose, less workload, higher efficiency and low cost. Therefore, by detecting the SNP locus, identification and auxiliary screening can be carried out in the seedling stage, the production cost is greatly saved, the selection efficiency is improved, and the breeding process of producing high-quality oil tea is accelerated.
Drawings
Fig. 1A to fig. 1C are graphs showing the content distribution of palmitic acid, oleic acid and linolenic acid in the oil-tea camellia natural population seed oil, respectively (the abscissa shows the content (%) of palmitic acid, oleic acid and linolenic acid in the oil-tea camellia seed oil, respectively, and the ordinate shows the number of sample individuals). The results show that the phenotype of the palmitic acid content, the oleic acid content and the linolenic acid content in the oil-tea camellia seed oil is normally distributed, and the oil-tea camellia seed oil belongs to quantitative traits.
FIG. 2 is a diagram of the total DNA electrophoresis of the tender leaves, each lane is a sample, and it can be seen that the extracted DNA sample has no degradation, no impurity pollution such as protein and polysaccharide, and high quality, and can be used in the subsequent experiment.
FIG. 3 is a peak diagram of sequencing of amplified products, in which the Camellia oleifera is a heterozygote, the heterozygosity is high, many sites belong to heterozygous sites, and the SNP site detected in the invention is also a heterozygous site. The part in the dotted line is the detection site, the left panel is the A/A genotype, and the right panel is the A/G genotype (heterozygous site).
FIG. 4 is a schematic diagram showing the structural effect of the population subgroups, and the results show that all individuals in the natural population used in the present invention can be divided into 4 subgroups according to their nucleotide polymorphisms.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
500 individual plants of natural population materials used in the research are collected and evaluated by woody oil research groups of subtropical forestry research institute of China forestry science research institute, and are stored in germplasm resource gardens of east Hongling farms in Wuhua Wuzhou areas in Zhejiang.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 construction and Property measurement of isolated population of fatty acid component content of Camellia oleifera seed oil
In the embodiment, natural groups of 500 germplasm resources in a common oil tea resource collection garden are used, and the origin of the natural groups covers most of the main production areas of oil tea in China, including Zhejiang province, Hunan province, Jiangxi province, Guangxi province, Fujian province, Guangdong province and the like. After the fruits of 500 individuals are completely ripe (5% of fruits are cracked), collecting seeds, extracting oil and determining the fatty acid content and the content. The operation steps are as follows:
(1) baking appropriate amount of oil tea seeds in oven at 80 deg.C overnight to constant weight, and peeling off hard seed coat.
(2) Pulverizing kernel with pulverizer, wrapping with medium-speed filter paper, adding appropriate amount of diethyl ether, soaking and extracting overnight.
(3) And after the ether is completely volatilized, measuring the components and the content of the fatty acid by using an Agilent6890N gas chromatograph according to GB/T17376-2008 and GB/T17377-2008 methods.
The content determination result of the fatty acid component shows that: the contents of palmitic acid, oleic acid and linolenic acid in the seed oil of the natural population are normally distributed (figure 1A, figure 1B and figure 1C), which shows that the three traits are all characterized by quantitative traits.
Example 2 Cofad2-1A Gene fragment amplification
1. Extracting total DNA of leaves:
the method comprises the following steps of extracting total DNA of leaves by utilizing a Plant material cracking system rich in polysaccharide, polyphenol and grease in a TaKaRa MiniBEST Plant Genomic DNA Extraction Kit:
(1) first, 500. mu.l of Buffer HS II was added to a 1.5ml centrifuge tube. Adding 0.1g of fresh leaves into liquid nitrogen, fully grinding, quickly adding the grinded leaf powder into a centrifuge tube, fully mixing, then adding 10 mu l of RNaseA (10mg/ml), fully shaking, uniformly mixing, and carrying out warm bath for 10 minutes in 56 ℃ water bath;
(2) add 62.5. mu.l of Buffer KAC and mix well. The mixture was left on ice for 5 minutes and centrifuged at 12000rpm for 5 minutes. And taking the supernatant, adding Buffer GB with the same volume as the supernatant, and fully and uniformly mixing.
(3) The Spin Column was mounted on a collection tube, the solution was transferred to the Spin Column (excess solution, two Column passes were made, the volume of each pass did not exceed 700. mu.l), centrifuged at 12000rpm for 1 minute, and the filtrate was discarded.
(4) Mu.l of Buffer WA WAs added to Spin Column, centrifuged at 12000rpm for 1 min, and the filtrate WAs discarded.
(5) Mu.l of Buffer WB was added to Spin Column, centrifuged at 12000rpm for 1 min, and the filtrate was discarded.
(6) And (5) repeating the operation step.
(7) Spin columns were mounted on collection tubes and centrifuged at 12000rpm for 2 minutes.
(8) The Spin Column was placed in a new 1.5ml centrifuge tube, 30-50. mu.l of precipitation Buffer was added to the center of the Spin Column membrane, and the resulting mixture was left to stand at room temperature for 5 minutes and centrifuged at 12000rpm for 2 minutes to elute DNA. The DNA concentration was determined by UV spectrophotometer and stored in a freezer at-20 deg.C (FIG. 2).
2. Development and synthesis of primers:
the primers are designed according to the cDNA sequence of the cloned camellia Cofad2-1A gene of the camellia oleifera after the full-length cDNA of the camellia oleifera FAD2 gene is cloned and subjected to sequence analysis, forestry science 2008,44 (3): 70-75. The specific development method is to design primers P1 (5'-ATGGGTGCTGGTGGACGAATG-3') and P2 (5'-TTGCATCAGAATCAATACGTG-3') near the start codon and the stop codon respectively by using Primer5 software (http:// www.Premier5BioSoft.com) according to the cDNA sequence of the gene, and amplify the genome sequence of the Cofad2-1A gene by using population individual DNA as a template.
3. Cofad2-1A gene fragment amplification, which comprises the following steps:
performing PCR amplification by using all extracted individual DNA as a template and P1 and P2 as amplification primers, wherein the reaction system comprises:
the PCR amplification procedure was:
4. gel detection, purification, recovery, sequencing and genotyping of the amplified fragment are carried out according to the specification of the AxyPrep DNA gel recovery kit, and the process is as follows:
(1) preparing 1.2% agarose gel, loading 50 μ l of amplification product, electrophoresis voltage is 5V/cm, and stopping electrophoresis after electrophoresis for about 20 min until xylene in loading buffer solution reaches 1cm from the front end of gel.
(2) The agarose gel containing the desired DNA was cut under an ultraviolet lamp, and the surface of the gel was blotted with a paper towel and minced. The gel weight is calculated as the volume of one gel (e.g. 100mg to 100 μ l volume).
(3) Adding 3 volumes of Buffer DE-A, uniformly mixing, heating at 75 ℃, and intermittently mixing every 2-3 minutes until the gel block is completely melted.
(4) 0.5 volume of Buffer DE-B was added and mixed well.
(5) The above solution was transferred to a DNA preparation tube, centrifuged at 12000rpm for 1 minute, and the filtrate was discarded.
(6) Mu.l of Buffer W1 was added and centrifuged at 12000rpm for 30 seconds, and the filtrate was discarded.
(7) Mu.l of Buffer W2 was added and centrifuged at 12000rpm for 30 seconds, and the filtrate was discarded. The cells were washed once with 700. mu.l of buffer W2 in the same manner, centrifuged at 12000rpm for 1 minute, and the filtrate was discarded.
(8) The prepared tube was returned to the centrifuge tube and centrifuged at 12000rpm for 1 minute.
(9) And (3) placing the preparation tube into a clean 1.5ml centrifugal tube, adding 25-30 mu l of deionized water into the center of the preparation membrane, and standing for 1 minute at room temperature. DNA was eluted by centrifugation at 12000rpm for 1 minute.
(10) DNA was recovered on a gel, and the nucleotide sequence of the amplified product was determined by one-generation sequencing using P1 and P2 as sequencing primers. The genotype of each SNP site on the sequencing peak map was interpreted by Chromas software (FIG. 3)
Example 3 screening of SNP sites related to fatty acid content of Camellia oleifera seed oil
The method comprises the following steps of group structure analysis and linkage disequilibrium analysis:
(1) and (3) importing the SNPs locus data of all samples into structure2.3.4 software, setting K to be 2-9, running for 5 times per K value, burning for 5000 times, and repeating for 50000 times. When both lnp (d) and alpha values remained stable and alpha < 0.2, K values for the population structure were determined (fig. 4), where K is 4, and K (4) subpopulation effect values were determined for each sample (table 1).
TABLE 1 Effect values of 4 subgroups of partial individuals of the Natural population
(2) SNPs locus data, K subgroup effect value data, phenotype data (see example 1) and Kinship matrix data of all samples are introduced into TASSEL5.0 software, the MLM method is adopted to analyze the correlation between SNPs and 5 phenotypic characters, the linkage imbalance between SNPs and fatty acid content is analyzed, molecular markers which are obviously related to unsaturated fatty acid content are screened, and a SNP01474 locus which is obviously related to a plurality of characters is detected by adopting Bonferroni multiple test correction (Table 2). The marker is associated with the contents of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid, but according to the correction of the Bonferroni multiple test, the association with the contents of stearic acid and linoleic acid does not reach a significant level, and the association with the contents of palmitic acid, oleic acid and linolenic acid reaches a very significant level.
TABLE 2 results of partial analysis of the association of phenotypic traits with SNPs
Example 4 application of molecular marker SNP01474 in oil quality breeding of oil tea seeds
(1) A tea-oil tree hybrid F1 generation family group is selected as a material, and tender leaves are collected to extract total DNA (see example 2).
(2) PCR amplification of total DNA was performed using the P1 and P2 primers (see example 2).
(3) The PCR amplification products were gel recovered for purification and sequencing analysis (see example 2).
(4) The genotype of all individuals at SNP01474 site was identified. If the genotype of the site is A/A, the oil tea individual is the oil tea with high palmitic acid, high linolenic acid and low oleic acid content or candidate oil tea with high palmitic acid, high linolenic acid and low oleic acid content; if the genotype of the site is A/G, the oil tea individual is the oil tea with low palmitic acid, low linolenic acid and high oleic acid content or candidate oil tea with low palmitic acid, low linolenic acid and high oleic acid content.
(5) All F1 individual fully mature seeds were collected and their seed oils were assayed for fatty acid content and content (see example 1). The results show (Table 3) that 58.33% of individuals with this site A/G had palmitic acid contents lower than the mean value of the palmitic acid content of the population (8.65%), 70.83% of individuals had linolenic acid contents lower than the mean value of the linolenic acid content of the population (0.28%), and 66.67% of individuals had oleic acid contents exceeding the mean value of the oleic acid content of the population (78.18%). The marker is practical and effective when used for auxiliary selection, can be used for early identification or auxiliary identification, can greatly save production cost, improve selection efficiency and accelerate the quality breeding process of the camellia oleifera and camellia oleifera oil.
TABLE 3 fatty acid content data of F1 individuals obtained by SNP01474 site-assisted selection
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain modifications and improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> subtropical forestry research institute of China forestry science research institute
<120> SNP molecular marker related to contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil and fat and SNP molecular marker
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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 (8)
1. An SNP molecular marker related to the contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil is characterized in that the molecular marker is positioned inCofad2-1AThe nucleotide sequence of the molecular marker is shown as SEQ ID NO.3, and the polymorphism of the site at the 474bp position of the sequence is A/G.
2. The use of the SNP molecular marker of claim 1 for identifying the oil tea seed palmitic acid content phenotype, if the genotype of the SNP molecular marker is A/A, the oil tea to be identified is the oil tea with high palmitic acid content or candidate high palmitic acid content; if the genotype of the SNP molecular marker is A/G, the oil tea to be identified is the oil tea with low palmitic acid content or candidate oil tea with low palmitic acid content.
3. The use of the SNP molecular marker of claim 1 for identifying the phenotype of the linolenic acid content of the seeds of the oil tea, wherein if the genotype of the SNP molecular marker is A/A, the oil tea to be identified is the oil tea with high linolenic acid content or the candidate oil tea with high linolenic acid content; if the genotype of the SNP molecular marker is A/G, the oil tea to be identified is the oil tea with low linolenic acid content or the candidate oil tea with low linolenic acid content.
4. The application of the SNP molecular marker of claim 1 in identifying the oleic acid content phenotype of oil tea seeds, wherein if the genotype of the SNP molecular marker is A/A, the oil tea to be identified is the low oleic acid content oil tea or the candidate low oleic acid content oil tea; and if the genotype of the SNP molecular marker is A/G, determining that the oil tea to be identified is the oil tea with high oleic acid content or the candidate oil tea with high oleic acid content.
5. The application of the SNP molecular marker of claim 1 in germplasm resource improvement of Camellia oleifera, wherein the traits of germplasm resource improvement are the contents of palmitic acid, oleic acid and linolenic acid in Camellia oleifera seeds.
6. The use of the SNP molecular marker of claim 1 in early prediction of the content of palmitic acid, oleic acid and linolenic acid in oil tea seeds.
7. Application of a primer pair or a kit containing the primer pair in identifying oil tea producing high palmitic acid, high linolenic acid or high oleic acid content, wherein nucleotide sequences of the primer pair are respectively shown as SEQ ID NO. 1-2.
8. The use according to claim 7, wherein the 474 th base of the amplification product, if the genotype is A/A, is to identify Camellia oleifera with high linolenic acid content or Camellia oleifera with high palmitic acid content; and if the genotype is A/G, identifying the oil tea to be identified as the oil tea with high oleic acid content.
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| CN111455091B (en) * | 2020-05-22 | 2023-05-12 | 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 camellia seed kernel oil and application thereof |
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| CN113430297B (en) * | 2021-07-23 | 2022-03-08 | 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 |
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