CN113430298A - DNA fragment related to content of linolenic acid in camellia seed oil, SNP molecular marker closely linked with DNA fragment and application of SNP molecular marker - Google Patents
DNA fragment related to content of linolenic acid in camellia seed oil, SNP molecular marker closely linked with DNA fragment and application of SNP molecular marker Download PDFInfo
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Abstract
The invention relates to the technical field of camellia oleifera molecular markers and genetic breeding, in particular to a DNA fragment related to the content of linolenic acid in camellia oleifera seed oil, a closely linked SNP molecular marker and application thereof. The invention provides a DNA fragment related to the content of linolenic acid in oil-tea camellia seed oil, which is yms.11-1 located at 85.508cM of an oil-tea camellia linkage map LG 11. The invention also provides an SNP molecular marker closely linked with the content of the linolenic acid in the oil tea seed oil, which comprises the SNP molecular marker chr11-27083024 and a nucleotide sequence with the polymorphism of C/T at the 227 th site of the sequence shown as SEQ ID NO. 1. The molecular marker can explain the phenotypic variance of linolenic acid content in the oil-tea camellia seed oil of 9.7 percent. By detecting the SNP molecular marker, identification and auxiliary screening can be carried out in the seedling stage, the production cost is greatly saved, and the selection efficiency of oil-tea camellia oil property selective breeding is improved.
Description
Technical Field
The invention relates to the technical field of camellia oleifera molecular markers and genetic breeding, in particular to a DNA fragment related to the content of linolenic acid in camellia oleifera seed oil, a closely linked SNP molecular marker and application thereof.
Background
Camellia oleifera (Camellia oleifera) as one of four kinds of oil plants (rape, peanut, soybean and Camellia oleifera) is widely planted in subtropical regions, and the planting area reaches over 6500 ten thousand mu. The content of unsaturated fatty acid in the oil-tea camellia seed oil is more than 90%, and the oil-tea camellia seed oil is rich in nutrient components such as squalene, vitamin E and the like, is high-quality edible oil and is called as 'east olive oil'. The breeding of high-yield (oil) quality oil tea fine varieties is always the basis and guarantee of healthy development of the oil tea industry. The oil tea breeding work mainly by means of selection and cross breeding has been developed greatly, but the conventional breeding period of the oil tea is long, the new variety breeding is slow, and the improved variety breeding speed cannot meet the requirement of industrial development, which becomes one of the important factors for limiting the development of the oil tea industry.
Compared with the traditional breeding technology, the molecular marker assisted breeding can be selected from the seedling stage, the breeding period is greatly shortened, and the advantages of the molecular marker assisted breeding on economic forests mainly aiming at fruits are particularly obvious. The molecular marker assisted breeding cannot be separated from effective molecular markers, so that the development of the molecular marker related to the content of fatty acid in the oil-tea camellia oil has important significance for molecular marker assisted breeding of the oil-tea camellia oil quality and genetic improvement of related characters.
Disclosure of Invention
One purpose of the invention is to provide a DNA fragment related to the linolenic acid content in oil-tea camellia seed oil and a molecular marker closely linked with the DNA fragment, and the other purpose of the invention is to provide application of the molecular marker in phenotypic identification and breeding of the linolenic acid content of the oil-tea camellia.
The development of the DNA fragments related to the content of linolenic acid in the oil-tea camellia seed oil and the molecular markers closely linked with the DNA fragments is realized by developing the construction of a high-density genetic linkage map and the QTL positioning of the linolenic acid content character based on the established oil-tea camellia F1 generation hybrid population. The simplified genome sequence of the camellia oleifera is a region for the marker development of the invention.
The development process of the key DNA fragments (gene loci) and the linked SNP molecular markers is basically as follows:
(1) pollination is controlled on the camellia oleifera clone Chang Lin No. 53 (0.31%) and Chang Lin No. 81 (0.22%) with obvious linolenic acid content difference, and camellia oleifera F1 generation hybrid population with widely separated linolenic acid content is created.
(2) And collecting the complete mature seeds of 180 single plants of the hybrid population, and determining the content of the linolenic acid in the grease. 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) Crushing the kernels by a crusher, wrapping the kernels by medium-speed filter paper, adding a proper amount of petroleum ether, soaking and extracting the kernels overnight.
3) And after the petroleum ether is completely volatilized, determining 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 content of the linolenic acid in the seed oil of the F1 colony is normally distributed, which shows that the character has the characteristic of quantitative character.
(3) Collecting 180 single plants of a hybrid population and young leaves of two parents, extracting DNA by using a TaKaRa MiniBEST plant genome DNA extraction kit (TaKaRa, Dalian, China), constructing a simplified genome (ddRAD) sequencing library by using EcoRI and NlaIII (Hin1II) for each sample after double enzyme digestion, and sequencing by using an Illumina HiSeqXten platform.
(4) And (3) analyzing the SNP loci of 180 samples and 2 parent simplified genomes obtained in the step (3) by taking the diploid oil tea genome as a reference sequence. SNP data were filtered according to the following principles: the sequencing depth of the parent is more than or equal to 10X, and the sequencing depth of the offspring is more than or equal to 8X; the genotype deletion rate is less than or equal to 30 percent; the SNP mass value is more than or equal to 30. The use of the software BWA in the process is open for free.
(5) Constructing a linkage map by using Joinmap4.0 software, wherein the parameters are set as follows: rec is less than or equal to 0.4, LOD is more than or equal to 3.0, Jump is 5, and Kosambi is used as a plotting function; analyzing the arrangement sequence of the markers in the linkage group, and calculating the genetic distance between adjacent markers.
(6) QTL Isimulping software is used for data analysis, and a complete interval mapping method (ICIM) is used for QTL positioning. The scanning step is set to 1 cM; the probability of stepwise regression labeling entry (PIN) is 0.002(POUT 2 PIN 0.002); the LOD value was 2.5.
By utilizing the technical measures, the invention obtains a gene locus ym.11-1 of the content of the linolenic acid in the camellia seed oil positioned on No. 11 linkage group (LG11), the contribution rate of the gene locus is 9.7%, the SNP marker closely linked with the gene locus is chr11-27083024, and the genotype is C/T (Table 1).
TABLE 1 Gene locus and linkage SNP molecular marker information
| Gene locus | Linkage group | Position of | Linkage SNP | Parents genotype | LOD | Rate of contribution |
| yms.11-1 | LG11 | 85.508cM | chr11-27083024 | CC*CT | 3.93 | 9.7％ |
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides a DNA fragment associated with the content of linolenic acid in camellia seed oil, which is yms.11-1 located at 85.508cM of camellia oleifera linkage map LG 11.
The contribution rate of the DNA fragment to the phenotype of the linolenic acid content in the oil-tea camellia seed oil is 9.7%, and the DNA fragment can be used for map-based cloning and molecular marker assisted selection.
The SNP molecular marker closely linked to the above DNA fragment is chr 11-27083024.
In a second aspect, the invention provides an SNP molecular marker closely linked with the content of the linolenic acid in the oil tea seed oil, which comprises an SNP molecular marker chr11-27083024, wherein the SNP molecular marker is located at 27083024bp position of the oil tea genome No. 11 chromosome, and the polymorphism is C/T.
The SNP molecular marker is closely linked with the gene locus ym.11-1 of the content of the linolenic acid in the oil-tea camellia seed oil.
Specifically, the SNP molecular marker chr11-27083024 contains a nucleotide sequence with the polymorphism of C/T at the 227 th site of the sequence shown as SEQ ID NO. 1.
Preferably, the nucleotide sequence of the SNP molecular marker chr11-27083024 is shown as SEQ ID NO.1, the SNP site is located at the 227 th position shown as SEQ ID NO.1, and the polymorphism is C/T.
Further, the SNP molecular marker chr11-27083024 can be obtained by PCR amplification of a primer pair with a nucleotide sequence shown as SEQ ID NO.2-3 and the genome DNA of the camellia oleifera as a template.
SEQ ID NO.2：5’-TTGAGAAATGAGATAACATAC-3’；
SEQ ID NO.3：5’-TGATGATTCAGATATGGCCG-3’。
In the SNP molecular marker chr11-27083024, the genotype of a site with the polymorphism is C/C, which corresponds to high linolenic acid content; the genotype is C/T, corresponding to low linolenic acid content.
The linolenic acid content is the percentage of the linoleic acid in the oil-tea camellia seed oil in the total fatty acid.
In a third aspect, the present invention provides primers for amplifying the SNP molecular markers.
As an embodiment of the present invention, the primer includes a primer shown in SEQ ID NO. 2-3.
The invention also provides a reagent or a kit containing the primer.
In a fourth aspect, the invention provides any one of the following applications of the DNA fragment related to the content of linoleic acid in camellia seed oil or the SNP molecular marker or the primer or the reagent or the kit:
(1) the application in identifying the phenotype of the linolenic acid content in the oil tea seed oil;
(2) the application in the improvement of linolenic acid content in oil-tea camellia seed oil or molecular marker-assisted breeding;
(3) the application in the early prediction of the linolenic acid content in the oil-tea camellia seed oil;
(4) the application in screening the oil tea with high linolenic acid content.
In a fifth aspect, the present invention provides a method for identifying a phenotype of the linolenic acid content in camellia seed oil, comprising the steps of:
(1) extracting the genomic DNA of the camellia oleifera to be identified;
(2) taking genome DNA as a template, and carrying out PCR amplification by using primers shown in SEQ ID NO. 2-3;
(3) analyzing the genotype of the SNP molecular marker chr11-27083024 in the PCR amplification product, and judging the content phenotype of the linolenic acid in the seed oil of the camellia oleifera to be identified according to the genotype.
In step (1) of the above method, the Camellia oleifera to be identified is specifically selected from individuals of hybrid generation F1 of Changlin No. 53 x Changlin No. 81.
Extraction of Camellia oleifera genomic DNA A TaKaRa MiniBEST plant genomic DNA extraction kit (TaKaRa, Dalian, China) was used.
In the step (2), the reaction procedure of the PCR amplification is as follows: 94-95 ℃ for 3-5 min; 94-95 ℃, 15-30 s, 65-69 ℃, 40-60 s and 38-45 cycles; 67-70 ℃ for 3-6 min. Preferably, the pre-denaturation is carried out 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 step (2), after the amplification, the resulting PCR product is detected and recovered by agarose gel electrophoresis.
In one embodiment, the agarose gel electrophoresis is performed at an agarose gel concentration of 1.2%. Gel recovery Using AxyPrep DNA gel recovery kit (AxyGEN, Code No. AP-GX-50).
In the step (3), the genotype of the SNP molecular marker can be analyzed by adopting the conventional technical means in the field, such as sequencing and the like, and sequencing can be carried out by taking SEQ ID NO.2-3 as a sequencing primer.
The method for judging the phenotype of the content of the linolenic acid in the seed oil of the camellia oleifera to be identified in the step (3) comprises the following steps:
if the genotype of the site with the polymorphism of the SNP molecular marker chr11-27083024 is C/C, the content of linolenic acid in the oil tea to be identified is high; and if the genotype is C/T, determining that the oil tea to be identified has low linolenic acid content.
The invention provides a method for identifying/screening oil tea with high linolenic acid content, which comprises the following steps:
(1) extracting the genomic DNA of the camellia oleifera to be identified;
(2) using DNA as a template and utilizing the primer to carry out PCR amplification;
(3) analyzing the genotype of the SNP molecular marker in the PCR amplification product, and judging whether the oil tea to be identified is the oil tea with high linolenic acid content according to the genotype.
If the genotype of the site with the polymorphism of the SNP molecular marker chr11-27083024 is C/C, the content of linolenic acid in the oil tea to be identified is high; and if the genotype is C/T, determining that the oil tea to be identified has low linolenic acid content.
The invention has the beneficial effects that:
the invention provides a DNA fragment related to the content of linolenic acid in oil-tea camellia seed oil, the contribution rate of the DNA fragment to the content of linolenic acid in the seed oil is 9.7%, and SNP loci closely linked with the DNA fragment are developed. The SNP marker is used for carrying out auxiliary selection on hybrid F1 generation individuals of Changlin No. 53 multiplied by Changlin No. 81, and the result shows that in a single plant of which the site is a genotype with high linolenic acid content, 69.05 percent of individuals have seeds with linolenic acid content higher than the average value (0.245 percent) of the linolenic acid content of the population; among individuals with a low linolenic acid content genotype, 76.09% of individuals had a linolenic acid content below the population mean (0.245%). This indicates that the marker is useful for aiding selection.
In the conventional selection breeding of the oil tea, the identification of the content character of the linolenic acid in the seed oil requires 5 to 6 years of seedling afforestation, and 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, and the selection efficiency is improved. In the oil tea breeding, the molecular marker and the detection method thereof can be selected to identify the oil tea with high linolenic acid content for breeding, so that the selection efficiency of the oil tea breeding can be improved, and the breeding process can be accelerated.
Drawings
FIG. 1 is a schematic diagram of the position of a DNA fragment (gene locus) yms.11-1 related to the content of linolenic acid in oil-tea camellia seed oil in example 3 of the invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The individual hybrid F1 of Changlin No. 53X Changlin No. 81 used in the following examples was collected and evaluated by woody oil breeding and cultivating research group of subtropical forestry research institute of China forestry science research institute, and stored in germplasm resource garden of Oriental Red forest farm in Wuhuawu City, Zhejiang.
Example 1 construction and Property measurement of isolated population of linolenic acid content in Camellia seed oil
In the embodiment, Changlin No. 53 and Changlin No. 81 are used as female parent and male parent respectively, and a hybrid F1 generation group with widely separated economic characters is created by adopting a controlled pollination technology. The F1 colony is stored in Wuchen east Honglin farm in Zhejiang province by 3 times of repeated design in random block. And collecting seeds after the 180 generations of individuals have completely matured fruits (5 percent of fruits are cracked), and measuring the content of the linolenic acid in the seed oil. 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) Crushing the kernels by a crusher, wrapping the kernels by medium-speed filter paper, adding a proper amount of petroleum ether, soaking and extracting the kernels overnight.
(3) And after the petroleum 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 determination result of the linolenic acid content in the oil tea seeds shows that: the content of the linolenic acid in the seed oil of the hybrid population is obviously separated, which shows that the character has the characteristic of quantitative character.
Example 2 construction of Camellia oleifera linkage map
1. Genomic DNA extraction
180 individuals of Changlin No. 53X Changlin No. 81 family and spring young leaves of the parents thereof were collected in 3 months, and the total genomic DNA was extracted by the KAC method (TaKaRa kit Code No. 9768). The method comprises the following specific steps:
(1) firstly, 500 mul of Buffer HS II is added into a 1.5ml centrifuge tube; accurately weighing 100mg of tea-oil tree tender leaves and grinding by liquid nitrogen; the ground powder was quickly added to the centrifuge tube and mixed well, then 10. mu.l of RNase A (10mg/ml) was added thereto, mixed well with shaking, and incubated in a 56 ℃ water bath for 10 minutes.
(2) Add 62.5. mu.l of Buffer KAC and mix well. The mixture was left on ice for 5 minutes and centrifuged at 12,000rpm 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 set in a Collection Tube, the solution was transferred to the Spin Column (generally two Column passes were required because of the large amount of solution, the volume of each pass did not exceed 700. mu.l), centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded.
(4) Mu.l of Buffer WA WAs added to the Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate WAs discarded.
(5) Mu.l of Buffer WB was added to Spin Column, centrifuged at 12,000rpm for 1 min, and the filtrate was discarded.
(6) And (5) repeating the operation step.
(7) Spin Column was mounted on the Collection Tube and centrifuged at 12,000rpm for 2 minutes.
(8) The Spin Column was placed in a new 1.5ml centrifuge tube, and 30-50. mu.l of Elution Buffer or sterile distilled water was added to the center of the Spin Column membrane, followed by standing at room temperature for 5 minutes.
(9) DNA was eluted by centrifugation at 12,000rpm for 2 minutes.
2. dd-RAD simplified genomic sequencing
The optimal enzyme digestion combination EcoRI and NlaIII of each sample genome DNA is subjected to double enzyme digestion and then connected with a joint, and the joint comprises 3 parts, namely a sequencing primer, a molecular recognition sequence (barcode) and a sequence which is complementary with a sticky end generated after the endonuclease digests the genome. Each sample was then subjected to PCR amplification. And (3) amplification procedure: 2min at 98 ℃; 30s at 98 ℃, 30s at 60 ℃, 15s at 72 ℃ and 13 cycles; 5min at 72 ℃. And (3) carrying out electrophoresis on the PCR product by using 2% agarose gel, and recovering and purifying the fragment with the length of 300-500 bp from the gel by using an AxyPrep DNA gel recovery kit. And (3) constructing a dd-RAD sequencing library by taking purified DNA samples with different barcode in each 12 individual mixed pools as a sample, and sequencing by using an Illumina HiSeqXten platform.
3. SNP site recognition and genotyping
(1) Filtering sequencing data, wherein the raw sequence data obtained by sequencing is firstly filtered according to the following steps:
1) according to the barcode on the sequence, quickly separating the mixed data of 12 samples according to individuals by using a self-defined Perl script;
2) sequences with a barcode followed by a recognition site of an endonuclease are retained, and the rest of sequences are discarded;
3) the sequences with the number of nucleotides being deleted being more than 3 are discarded;
4) other low Quality, contaminating sequences were further filtered using the NGS QC Toolkit software package (Patel R K, Jain M. NGS QC Toolkit: APlatform for Quality Control of Next-Generation Sequencing Data [ M ]. Springer US, 2015.).
(2) SNP identification and filtration: the high quality reads of each sample were aligned to the reference genomic sequence (diploid Camellia oleifera genome). Sequences not aligned were deleted and the remaining sequences recognized SNP sites. The identified SNP sites are strictly filtered to obtain SNPs data with high quality. The software Tophat v2.1.1, bcftools v1.9 and BWA used in the process are open and free. The SNPs filtration criteria were as follows:
1) the sequencing depth of the parent is more than or equal to 10X, and the sequencing depth of the offspring is more than or equal to 8X;
2) the genotype deletion rate is less than or equal to 30 percent;
3) the SNP mass value is more than or equal to 30.
4. Genetic map construction
(1) Detection of marker isolation mode: all detected SNPs were analyzed by using the CP function in the JoinMap4.0 software, the separation ratio of the markers was calculated by Chi-square test, the separation pattern of each marker, e.g., ab × cd, ef × eg, hk × hk, lm × ll, nn × np, cc × ab, ab × cc, etc., was determined, and the markers significantly separated abnormally (P <0.05) or containing abnormal bases were filtered and removed. The four types of markers, ef × eg, hk × hk, lm × ll and nn × np, are used for subsequent linkage map construction.
(2) Construction of genetic linkage map: the linkage diagram is constructed by using JoinMap4.0 software, and the parameters are set as follows: rec is less than or equal to 0.4, LOD is more than or equal to 3.0, Jump is 5, and Kosambi is used as a plotting function; analyzing the arrangement sequence of the markers in the linkage group, and calculating the genetic distance between adjacent markers. The constructed linkage map has 15 linkage groups, the upper symbol is 2780, the total coverage is 3327cM, and the average distance is 1.20 cM.
Example 3 excavation of Gene site of linolenic acid content and Linked SNP site in Camellia seed oil
QTL Isimulping software is used for data analysis, and a complete interval mapping method (ICIM) is used for QTL positioning of linolenic acid content. The scanning step is set to 1 cM; the probability of stepwise regression labeling entry (PIN) is 0.002(POUT 2 PIN 0.002); the LOD value was 2.5. The LOD significance threshold was determined by running 1000 permatation tests. The gene site ym s.11-1 of linolenic acid content of the camellia oleifera is located on the 11 th chromosome of the camellia oleifera, the contribution rate is 9.7%, and the SNP molecular marker closely linked with the gene site is chr11-27083024 (table 1, figure 1). The molecular marker contains a nucleotide sequence with the polymorphism of C/T at the 227 th site of the sequence shown in SEQ ID NO. 1.
Example 4 application of linolenic acid content gene locus and linkage SNP molecular marker in camellia oleifera breeding
(1) 175 individuals are randomly selected as materials from the Changlin No. 53 multiplied by No. 81 camellia oleifera hybrid F1 generation family group, and tender leaves are collected to extract total DNA (see example 2). The DNA solution was diluted 100-fold to prepare a working solution.
(2) The primer pair shown in SEQ ID NO.2-3 is used for carrying out PCR amplification on the DNA working solution, and the reaction system is shown in Table 2:
TABLE 2
The PCR amplification procedure was:
(3) and carrying out gel detection, purification, recovery, sequencing and genotyping on the PCR amplification product. Gel detection and purification recovery were performed according to AxyPrep DNA gel recovery kit (AxyGEN, Code No. AP-GX-50) instructions, and the procedure was as follows:
preparing 1.2% agarose gel, completely loading 50 mul of amplification products, wherein the electrophoresis voltage is 5V/cm, and stopping electrophoresis after electrophoresis for about 20 minutes until xylene in loading buffer solution reaches a position 1cm away from the front end of the gel.
② cutting the agarose gel containing the target DNA under an ultraviolet lamp, completely absorbing the liquid on the surface of the gel by a paper towel and cutting. The gel weight is calculated as the volume of one gel (e.g. 100mg to 100 μ l volume).
And thirdly, adding 3 buffers DE-A with gel volume, uniformly mixing, heating at 75 ℃, and intermittently mixing every 2-3 minutes until the gel block is completely melted.
And fourthly, adding 0.5 Buffer DE-B with the volume of the Buffer DE-A and mixing evenly.
Fifthly, transferring the solution into a DNA preparation tube, centrifuging at 12000rpm for 1 minute, and discarding the filtrate.
Sixthly, adding 500 mu l of Buffer W1, centrifuging at 12000rpm for 30 seconds, and removing the filtrate.
Seventhly, 700 mu l of Buffer W2 is added, and the mixture is centrifuged at 12000rpm for 30 seconds, and the filtrate is discarded. The cells were washed once with 700. mu.l Buffer W2 in the same manner, centrifuged at 12000rpm for 1 minute, and the filtrate was discarded.
The prepared tube is put back into a centrifuge tube and centrifuged at 12000rpm for 1 minute.
Ninthly, placing the preparation tube in a clean 1.5ml centrifugal tube, adding 25-30 mu l of deionized water in the center of the preparation film, and standing for 1 minute at room temperature. DNA was eluted by centrifugation at 12000rpm for 1 minute.
And recovering DNA from the gel at the red (r) gel, taking a corresponding amplification primer as a sequencing primer, determining the nucleotide sequence of an amplification product by adopting first-generation sequencing, and judging the genotype of the SNP site on a sequencing peak map by using Chromas software.
(4) The genotypes of the chr11-27083024 loci of all individuals were identified separately. Comparing the genotype of each site with the relationship between the linolenic acid content, if the genotype of the site is C/C, the oil tea individual is the oil tea with high linolenic acid content; if the genotype is C/T, the oil tea individual is the oil tea with low linolenic acid content.
(5) 175F 1 individuals and parental fully mature seeds were collected and the linolenic acid content in the oil of the seeds was determined (see example 1). The result shows that (table 3), in the individual plant with chr11-27083024 locus as high linolenic acid content genotype, the linolenic acid content of 69.05% of individuals is higher than the average value (0.245%) of the linolenic acid content in the grease of the seeds of the population; of the individuals with genotypes having low linolenic acid content, 76.09% had linolenic acid content below the population mean (0.245%). 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, improves selection efficiency and accelerates the oil tea breeding process.
TABLE 3 linolenic acid content and genotype data for female parent Changlin No. 53, male parent Changlin No. 81 and F1 individuals
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or 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
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<120> DNA fragment related to content of linolenic acid in oil-tea camellia seed oil, SNP molecular marker closely linked with same and application thereof
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taaccaggat cgccccatga tgagattaaa tggcgatggc agcctgacga ccaagaattc 180
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tagattggcg ttgaagctga ataatggata aggaaccgga gagagatccg attctgagaa 300
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Claims (10)
1. A DNA fragment related to the content of linolenic acid in oil tea seed oil, which is characterized in that the DNA fragment is yms.11-1 located at 85.508cM of an oil tea linkage map LG 11.
2. The SNP molecular marker closely linked with the content of the linolenic acid in the oil tea seed oil is characterized by comprising the SNP molecular marker chr11-27083024, which contains a nucleotide sequence with the polymorphism of C/T at the 227 th site of the sequence shown as SEQ ID NO. 1.
3. The SNP molecular marker according to claim 2, wherein the primer set having the sequence shown in SEQ ID No.2-3 is obtained by PCR amplification using Camellia oleifera genomic DNA as a template.
4. The SNP molecular marker according to claim 2 or 3, wherein the genotype of the site with the polymorphism in the SNP molecular marker chr11-27083024 is C/C, corresponding to a high linolenic acid content; the genotype is C/T, corresponding to low linolenic acid content.
5. A primer for amplifying the SNP molecular marker according to any one of claims 2 to 4.
6. The primer according to claim 5, comprising a primer having a sequence shown in SEQ ID NO. 2-3.
7. A reagent or kit comprising the primer of claim 5 or 6.
8. The use of the DNA fragment related to the content of linoleic acid in camellia seed oil of claim 1 or the SNP molecular marker of any one of claims 2-4 or the primer of claim 5 or 6 or the reagent or kit of claim 7 for any one of the following:
(1) the application in identifying the phenotype of the linolenic acid content in the oil tea seed oil;
(2) the application in the improvement of linolenic acid content in oil-tea camellia seed oil or molecular marker-assisted breeding;
(3) the application in the early prediction of the linolenic acid content in the oil-tea camellia seed oil;
(4) the application in screening the oil tea with high linolenic acid content.
9. A method for identifying the phenotype of the linolenic acid content in the oil tea seed oil or screening the oil tea seed with the high linolenic acid content is characterized by comprising the following steps:
(1) extracting the genomic DNA of the camellia oleifera to be identified;
(2) taking genome DNA as a template, and carrying out PCR amplification by using primers shown in SEQ ID NO. 2-3;
(3) analyzing the genotype of the SNP molecular marker of any one of claims 2 to 4 in a PCR amplification product, and judging the content phenotype of the linolenic acid in the seed oil of the camellia oleifera to be identified according to the genotype;
preferably, in step (2), the reaction procedure of the PCR amplification is: 94-95 ℃ for 3-5 min; 94-95 ℃, 15-30 s, 65-69 ℃, 40-60 s and 38-45 cycles; 67-70 ℃ for 3-6 min.
10. The method according to claim 9, wherein in the step (3), the method for judging the phenotype of the content of the linolenic acid in the seed oil of the camellia oleifera to be identified is as follows:
if the genotype of the site with the polymorphism of the SNP molecular marker chr11-27083024 is C/C, the content of linolenic acid in the oil tea to be identified is high; and if the genotype is C/T, determining that the oil tea to be identified has low linolenic acid content.
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