CN113604593A - DNA fragment related to linoleic acid content in oil tea seed oil and application thereof - Google Patents
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Abstract
The invention relates to the technical field of molecular markers, in particular to a DNA fragment related to the linoleic acid content in oil tea seed oil and application thereof. The DNA fragment related to the linoleic acid content in the oil-tea camellia seed oil provided by the invention is yys.11-1 located at 163.975cM position of No. 11 linkage group of an oil-tea camellia linkage map. The invention also provides an SNP molecular marker Chr11-114235680 closely linked with the linoleic acid content site in the oil tea seed oil, which is a nucleotide sequence containing the 203 th polymorphism of the sequence shown as SEQ ID NO.1 as C/T and can explain the phenotype variance of the linoleic acid content in the oil tea seed oil of 9.6 percent. According to the invention, by detecting the SNP molecular marker, the linoleic acid content in the oil-tea camellia seed oil is identified and screened in an auxiliary manner in the seedling stage, so that the production cost is greatly saved, and the selection efficiency of oil-tea camellia oil character selective breeding is improved.
Description
Technical Field
The invention belongs to the technical field of molecular markers and genetic breeding of oil tea. In particular to a DNA fragment related to the content of linoleic acid in oil tea seed oil, a closely linked SNP 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.
The oleic acid content in the camellia oleosa seed oil is relatively stable, but the linoleic acid content is within the range of 2% -17%, and is related to the genetic characteristics of varieties (lines). Linoleic acid (18: 2, n-6) is an unsaturated fatty acid (EFA) which is not synthesized by human body but is essential, it is the main raw material for synthesizing prostaglandin, and it has special functions of reducing cholesterol and blood fat, improving blood rheological property and preventing coronary heart disease.
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 linoleic acid content in oil and fat of camellia oleifera seeds and a molecular marker closely linked with the gene locus, and the other purpose of the invention is to provide application of the molecular marker in phenotypic identification and breeding of high linoleic acid content of camellia oleifera.
The development of the genetic locus of the linoleic acid content in the oil tea seed oil and the molecular marker tightly linked with the genetic locus is realized by developing the construction of a high-density genetic linkage map and the QTL positioning of the linoleic acid content character based on the established oil tea 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 linoleic acid content gene locus and the linked SNP molecular marker in the oil tea seed oil is basically as follows:
(1) the pollination is controlled by using the camellia oleifera clone Changbai No. 53 (11.23%) and Changbai No. 81 (6.73%) with obvious linoleic acid content difference, and camellia oleifera F1 generation hybrid populations with widely separated linoleic acid content are created.
(2) And collecting the fully mature seeds of 180 single plants of the hybrid population, and determining the content of linoleic 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, 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 linoleic acid content in the seed oil of the F1 population is normally distributed, which shows that the characteristic of the linoleic acid content in the seed oil 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 yys.11-1 of the linoleic acid content in the camellia seed oil positioned on No. 11 linkage group (LG11), the contribution rate of the gene locus is 9.6%, the SNP marker closely linked with the gene locus is Chr11-114235680, and the polymorphism of the gene locus 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 |
| yys.11-1 | LG11 | 163.975cM | Chr11-114235680 | CC*CT | 3.07 | 9.6％ |
Specifically, the invention provides the following technical scheme:
in a first aspect, the invention provides a DNA fragment (gene locus) yys.11-1 related to the linoleic acid content in oil tea seed oil, wherein yys.11-1 is located at 163.975cM of number 11 linkage group (LG11) of an oil tea linkage map. The contribution rate of the DNA fragment to the linoleic acid content in the oil-tea camellia seed oil is 9.6%, and the DNA fragment can be used for map-based cloning and molecular marker assisted selection.
The SNP molecular marker closely linked with the DNA fragment is Chr11-114235680, the Chr11-114235680 is located at 114235680bp of the 11 # chromosome of the oil tea genome, and the polymorphism is C/T.
In a second aspect, the invention provides an SNP molecular marker closely linked with the linoleic acid content site in the oil tea seed oil, which comprises an SNP molecular marker Chr11-114235680, wherein Chr11-114235680 is located at position 114235680bp of chromosome 11 of an oil tea genome, and the polymorphism is C/T. The Chr11-114235680 is closely linked with the linoleic acid content gene locus yys.11-1 in the oil tea seed oil.
Specifically, the SNP molecular marker Chr11-114235680 contains a nucleotide sequence with the polymorphism of C/T at the 203 nd position of the sequence shown as SEQ ID NO. 1.
Preferably, the nucleotide sequence of the SNP molecular marker Chr11-114235680 is shown as SEQ ID NO.1, the SNP site is located at the 203 th site of the sequence shown as SEQ ID NO.1, and the polymorphism is C/T.
SEQ ID NO.1：
TCAGCTAGAGAGAGTTTAAACCCACTTGTCATTCAACCAATCCTGATGAATCCTCA
ACTGAGCCAGTGGGTAAGCGATCTCATCCCCTGTTTTCTCAAAATACACATAGTAT
TTGGGACCCAAGTCTCCCAGTAATCTTCCCAACCCACCAACCATCGTTGTCGAAT
GCATCGACTCTCTCGTACAGACCAAACCCAGTTGCCGGAATTTCTGGTGGCACCG
GCCGGACCTCGTCGGCGGCGACAATCTCTCTCAAGGGTCCGGCCTCGTCTTCCTT
CAACAGAGTCTTGTACTGAACCATGTAGTGCTTCTTCATGAGTTCTGAAATCACTGTTGCTGAGTAG。
Further, the SNP molecular marker Chr11-114235680 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’-TCAGCTAGAGAGAGTTTAAAC-3’；
SEQ ID NO.3：5’-CTACTCAGCAACAGTGATTTC-3’。
In the SNP molecular marker Chr11-114235680, the genotype of the site with the polymorphism is CC, and the content of linoleic acid is correspondingly high; genotype is CT, corresponding to low linoleic acid content.
The linoleic acid content is the percentage of 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 present invention provides any one of the following applications of the SNP molecular marker or the primer or the reagent or the kit:
(1) the application in identifying the linoleic acid content phenotype in the oil tea seed oil;
(2) the application in molecular marker assisted breeding of linoleic acid content improvement in oil tea seed oil or linoleic acid content in oil tea seed oil;
(3) the application in early prediction of linoleic acid content in oil tea seed oil;
(4) the application in screening the oil tea with high linoleic acid content.
In a fifth aspect, the invention provides a method for identifying the linoleic acid content in oil-tea camellia seed oil, which comprises the following steps:
(1) extracting the genomic DNA of the camellia oleifera to be identified;
(2) using genome 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 the linoleic acid content phenotype 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 deg.C for 3-5 min; 94-95 ℃, 15-30s, 65-69 ℃, 40-60s, 38-45 cycles; 67-70 deg.C, 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 content of the linoleic 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 polymorphic site of the SNP molecular marker Chr11-114235680 is CC, the content of linoleic acid in the oil tea to be identified is high; if the genotype is CT, the oil tea to be identified has low linoleic acid content.
The invention provides a method for identifying camellia oleifera seeds with high linoleic 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 linoleic acid content according to the genotype.
If the genotype of the polymorphic site of the SNP molecular marker Chr11-114235680 is CC, the content of linoleic acid in the oil tea to be identified is high; if the genotype is CT, the oil tea to be identified has low linoleic acid content.
The invention has the beneficial effects that: the invention provides a gene locus of linoleic acid content in oil tea seed oil, the contribution rate of the gene locus to the linoleic acid content in the oil tea seed oil is 9.6%, and an SNP marker tightly linked with the gene locus is 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 (CC) with high linoleic acid content, 56.45% of individuals have the linoleic acid content of seeds higher than the average value (8.15%) of the linoleic acid content of a population; of the individuals with the low linoleic acid content genotype (CT), 63.63% of the individuals had linoleic acid content below the population mean (8.15%). This indicates that the marker is useful for aiding selection.
In the conventional selective breeding of the camellia oleifera, the identification of the linoleic acid content character in the seed oil requires 5-6 years for seedling afforestation, and wastes time and labor. The SNP molecular marker in the invention has definite position, convenient and quick detection method, no environmental influence, stronger purpose, less workload, higher efficiency and low cost. Therefore, by detecting the SNP molecular marker loci, identification and auxiliary screening can be carried out in the seedling stage, so that 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 linoleic 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 position diagram of a linoleic acid content site yys.11-1 in camellia oleosa seed oil in example 3 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
Unless otherwise specified, the experimental materials, reagents, instruments and the like used in the examples of the present invention are commercially available; unless otherwise specified, all technical means in the examples of the present invention 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 linoleic acid content segregation population in Camellia oleifera 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 linoleic 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 linoleic acid content determination result in the camellia oleifera seeds shows that: the linoleic acid content 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 obtained in example 1 and spring shoot young leaves of its parents were collected in 3 months, and total genomic DNA was extracted by 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 ℃. The PCR product was electrophoresed on 2% agarose gel, and the 300-and 500-bp fragment was recovered and purified from the gel using 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: A Platform for Quality Control of Next-Generation Sequencing Data [ M ]. Springer US, 2015.).
(2) SNP identification and filtration: the high quality reads for each sample were aligned to the reference genomic sequence. 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 linoleic acid content Gene site and linkage 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 linoleic 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 locus yys.11-1 of the linoleic acid content gene of the camellia oleifera is positioned on the chromosome 5 of the camellia oleifera, the contribution rate is 9.6%, and the SNP molecular marker closely linked with the locus yys is Chr11-114235680 (Table 1, figure 1).
Example 4 application of linoleic acid content gene locus and linkage SNP molecular marker in camellia oleifera breeding
1. 128 individuals were randomly selected as materials from the Camellia oleifera hybrid F1 generation family population of Changlin No. 53X Changlin No. 81 obtained in example 1, and young leaves were collected to extract total DNA (see example 2 for DNA extraction method). 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:
(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 gel volumes of Buffer DE-A, mixing well, heating at 75 deg.C, and mixing intermittently every 2-3 minutes until the gel mass 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) The preparation tube was placed in a clean 1.5ml centrifuge tube, 25-30. mu.l of deionized water was added to the center of the preparation membrane, and the membrane was allowed to stand at room temperature for 1 minute. DNA was eluted by centrifugation at 12000rpm for 1 minute.
(10) And recovering DNA by gel, taking the 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 locus on a sequencing peak map by using Chromas software.
4. The genotypes of the Chr11-114235680 sites of all individuals were identified separately. Comparing the genotype of each site with the relationship between the linoleic acid content, if the genotype of the site is CC, the oil tea individual is the oil tea with high linoleic acid content; if the genotype is CT, the oil tea individual is oil tea with low linoleic acid content.
5. 128F 1 generation individuals and complete mature seeds of parents are collected, and the linoleic acid content in the seed oil is determined (the method for determining the linoleic acid content in the seed oil is shown in example 1). The results in Table 3 show that in the individual strains with the Chr11-114235680 locus CC, the linoleic acid content of 56.45 percent of individuals is higher than the average value (8.15 percent) of the linoleic acid content in the grease of the seeds of the group; of the individuals with genotype CT, 63.63% had linoleic acid content below the population mean (8.15%). 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 linoleic acid content and genotype data for individual plants of female parent Changlin No. 53, male parent Changlin No. 81 and F1
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
<110> subtropical forestry research institute of China forestry science research institute
<120> DNA fragment related to linoleic acid content in oil tea seed oil and application thereof
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agccagtggg taagcgatct catcccctgt tttctcaaaa tacacatagt atttgggacc 120
caagtctccc agtaatcttc ccaacccacc aaccatcgtt gtcgaatgca tcgactctct 180
cgtacagacc aaacccagtt gccggaattt ctggtggcac cggccggacc tcgtcggcgg 240
cgacaatctc tctcaagggt ccggcctcgt cttccttcaa cagagtcttg tactgaacca 300
tgtagtgctt cttcatgagt tctgaaatca ctgttgctga gtag 344
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Claims (10)
1. The DNA fragment related to the linoleic acid content in the oil-tea camellia seed oil is characterized in that the DNA fragment is yys.11-1 located at 163.975cM position of No. 11 linkage group of the oil-tea camellia linkage map; the SNP molecular marker closely linked with yys.11-1 is located at 114235680 th site of the No. 11 chromosome of the oil tea genome.
2. The SNP molecular marker closely linked with the linoleic acid content site in the oil tea seed oil is characterized by comprising the SNP molecular marker Chr11-114235680, wherein the Chr11-114235680 contains a nucleotide sequence with the polymorphism of C/T at the 203 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 any one of claims 2 to 3, wherein the genotype of the site with said polymorphism in the SNP molecular marker Chr11-114235680 is CC, corresponding to high linoleic acid content; genotype is CT, corresponding to low linoleic 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. Any one of the following uses of the DNA fragment related to linoleic acid content in oil tea seed oil and fat of claim 1, or the SNP molecular marker of any one of claims 2 to 4, or the primer of any one of claims 5 to 6, or the reagent or kit of claim 7:
(1) the application in identifying the linoleic acid content phenotype in the oil tea seed oil;
(2) the application in molecular marker assisted breeding of linoleic acid content improvement in oil tea seed oil or linoleic acid content in oil tea seed oil;
(3) the application in early prediction of linoleic acid content in oil tea seed oil;
(4) the application in screening the oil tea with high linoleic acid content.
9. A method for identifying the linoleic acid content in oil and fat of camellia oleifera seeds or screening camellia oleifera seeds with high linoleic 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 as SEQ ID NO. 2-3;
(3) analyzing the genotype of the SNP molecular marker of any one of claims 2 to 4 in the PCR amplification product, and judging the kernel-out rate phenotype of the oil-tea camellia seeds to be identified according to the genotype;
preferably, in step (2), the reaction procedure of the PCR amplification is: 94-95 deg.C for 3-5 min; 94-95 ℃, 15-30s, 65-69 ℃, 40-60s, 38-45 cycles; 67-70 deg.C, 3-6 min.
10. The method according to claim 9, wherein in the step (3), the method for judging the content of linoleic acid in the oil-tea camellia seed oil to be identified comprises the following steps:
if the SNP molecular marker Chr11-114235680 has the genotype of the polymorphic site CC, the oil tea seed oil to be identified has high linoleic acid content; and if the genotype is CT, the oil and fat of the oil tea seeds to be identified is low in linoleic acid content.
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