阅读说明：本技术 与油茶种子出仁率相关的dna片段、其紧密连锁的snp分子标记及其应用 (DNA fragment related to kernel-out rate of camellia oleifera seeds, SNP molecular marker closely linked with same and application thereof ) 是由 林萍 王开良 姚小华 常君 于 2021-07-23 设计创作，主要内容包括：本发明涉及油茶分子标记及遗传育种技术领域,具体涉及与油茶种子出仁率相关的DNA片段、其紧密连锁的SNP分子标记及其应用。本发明提供与油茶种子出仁率相关的DNA片段,其为位于油茶连锁图谱6号连锁群的rkf.06-2,置信区间为53.7cM-64.5cM。本发明还提供与油茶种子出仁率位点紧密连锁的SNP分子标记chr06-88890750,其为含有如SEQ ID NO.1所示序列第156位的多态性为C/T的核苷酸序列,可以解释14.9％的油茶种子出仁率的表型方差。通过检测该SNP分子标记,可在苗期进行油茶种子出仁率表型的鉴定和辅助筛选,大大节约生产成本,显著提高了油茶种子出仁率选择育种的效率。(The invention relates to the technical field of camellia oleifera molecular markers and genetic breeding, in particular to a DNA fragment related to the seed kernel-out rate of camellia oleifera seeds, a closely linked SNP molecular marker and application thereof. The invention provides a DNA fragment related to the kernel-out rate of oil-tea camellia seeds, which is rkf.06-2 of a linkage group No. 6 of an oil-tea camellia linkage map, and has a confidence interval of 53.7cM-64.5 cM. The invention also provides an SNP molecular marker chr06-88890750 closely linked with the kernel-out rate locus of the oil-tea camellia seeds, which is a nucleotide sequence containing the polymorphism C/T at the 156 th site of the sequence shown as SEQ ID NO.1 and can explain the phenotypic variance of the kernel-out rate of the oil-tea camellia seeds of 14.9 percent. By detecting the SNP molecular marker, the identification and auxiliary screening of the kernel-out rate phenotype of the camellia oleifera seeds can be carried out in the seedling stage, the production cost is greatly saved, and the efficiency of selective breeding of the kernel-out rate of the camellia oleifera seeds is remarkably improved.)

1. The DNA fragment related to the seed kernel-out rate of the camellia oleifera seeds is characterized in that the DNA fragment is rkf.06-2 of the linkage group No. 6 of the camellia oleifera linkage map, and the confidence interval is 53.7cM-64.5 cM.

2. The SNP molecular marker closely linked with the seed kernel-out rate site of the camellia oleifera seed is characterized by comprising the SNP molecular marker chr06-88890750, wherein chr06-88890750 contains a nucleotide sequence with the polymorphism of C/T at the 156 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 having the polymorphism in the SNP molecular marker chr06-88890750 is C/C, corresponding to a high outing rate; genotype is C/T, corresponding to low kernel rate.

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 applications of the DNA fragment related to the kernel-out rate of the camellia oleifera seed of claim 1, or the SNP molecular marker of any one of claims 2 to 4, or the primer of claim 5 or 6, or the reagent or kit of claim 7:

(1) the application in identifying the kernel-out rate phenotype of the camellia oleifera seeds;

(2) the application in the improvement of the kernel-out rate of the oil-tea camellia seeds or the molecular marker assisted breeding of the kernel-out rate of the oil-tea camellia seeds;

(3) the application in early prediction of the kernel-out rate of the camellia oleifera seeds;

(4) the application in screening the oil tea with high kernel yield.

9. A method for identifying the kernel-out rate phenotype of oil-tea camellia seeds or screening oil-tea camellia with high seed kernel-out rate 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 a primer with a sequence 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 ℃ 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 kernel-out rate phenotype of the camellia oleifera seeds to be identified comprises the following steps:

if the genotype of the site with the polymorphism of the SNP molecular marker chr06-88890750 is C/C, the kernel-out rate of 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 kernel-out rate.

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 seed kernel-out rate of camellia oleifera seeds, a closely linked SNP molecular marker and application thereof.

Background

Tea-oil tree (Camellia oleifera) is used as four major oil plants in China together with rape, peanut and soybean, is an important economic crop and is widely planted in subtropical regions in China. The camellia seed oil is high-quality edible oil, wherein the content of unsaturated fatty acid can reach more than 90%, and the camellia seed oil is rich in nutrient components such as squalene, vitamin E and the like. 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. For a long time, the oil tea breeding work which takes selection and cross breeding as main means is greatly developed, but the conventional breeding of the oil tea has the problems of long period, slow new variety breeding and the like, and the speed of fine variety breeding still cannot meet the development requirement of the oil tea industry, and 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 molecular marker assisted breeding method has particularly obvious breeding advantages of economic forests mainly aiming at fruits. The effective molecular marker is the key of molecular marker assisted breeding, so that the development of the molecular marker related to the phenotypes of the oil-tea camellia fruit yield, the seed kernel yield, the kernel oil content and the oil quality has important significance for molecular marker assisted breeding of the oil-tea camellia oil yield and quality and genetic improvement of related characters.

The yield (oil) of the camellia oleifera per unit area is directly determined by indexes such as fruit yield, fresh fruit seed yield, seed kernel yield, kernel oil content and the like. Therefore, the research and improvement on the seed kernel yield character of the oil-tea camellia seeds are one of the important ways for improving the yield of the oil-tea camellia, and have very important significance on the promotion and the healthy development of the oil-tea camellia industry.

Disclosure of Invention

One of the purposes of the invention is to provide a DNA fragment (gene locus) related to the kernel-out rate of oil-tea camellia seeds and a SNP molecular marker closely linked with the DNA fragment, and the other purpose of the invention is to provide the application of the molecular marker in phenotypic identification and breeding of oil-tea camellia high kernel-out rate.

The development of the camellia seed kernel-out rate genetic locus and the molecular marker closely linked with the genetic locus provided by the invention is realized by developing high-density genetic linkage map construction and QTL positioning of kernel-out rate characters based on the established camellia F1 generation hybrid population. The simplified genome sequence of the oil tea is a region for developing the molecular marker.

The development process of the key gene locus and the linked SNP molecular marker is basically as follows:

(1) pollination is controlled on the camellia oleifera clone No. 53 (47.66%) and No. 81 (58.14%) with obvious seed kernel-out rate difference, and a camellia oleifera F1 generation hybrid population with widely separated seed kernel-out rate is created.

(2) And collecting the fully mature seeds of 180 single plants in the hybrid population, and determining the kernel outing rate of the seeds.

(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 software BWA used in the above 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 means, the invention obtains the camellia oleifera seed kernel-out rate gene locus rkf.06-2 positioned on the No. 6 linkage group (LG06), the contribution rate of the gene locus to the camellia oleifera seed kernel-out rate is 14.9%, the SNP marker closely linked with the gene locus is chr06-88890750, and the genotype is C/T (Table 1).

TABLE 1 Gene locus and linkage SNP molecular marker information

Specifically, the invention provides the following technical scheme:

in a first aspect, the invention provides a DNA fragment related to the kernel-out rate of camellia oleifera seeds, which is rkf.06-2 of the linkage group No. 6 of the camellia oleifera linkage map, and has a confidence interval of 53.7cM-64.5 cM.

The contribution rate of the DNA fragment to the kernel-out rate phenotype of the camellia oleifera seeds is 14.9%, 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 chr06-88890750, the chr06-88890750 is located at 88890750bp of No. 6 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 kernel-out rate locus of oil-tea camellia seeds, which comprises an SNP molecular marker chr06-88890750, wherein the SNP molecular marker is located at 88890750bp position of the No. 6 chromosome of the oil-tea camellia genome and has C/T polymorphism.

The SNP molecular marker is closely linked with the kernel-out rate gene locus rkf.06-2 of the oil-tea camellia seeds.

Specifically, the SNP molecular marker chr06-88890750 contains a nucleotide sequence with polymorphism C/T at position 156 of the sequence shown as SEQ ID NO. 1.

Preferably, the nucleotide sequence of the SNP molecular marker chr06-88890750 is shown as SEQ ID NO.1, the SNP site is located at 156 th site shown as SEQ ID NO.1, and the polymorphism is C/T.

Further, the SNP molecular marker chr06-88890750 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’-AAGGAAGTTCAGGGAATAAGT-3’；

SEQ ID NO.3：5’-ATTAGCTACAACTAACTAATA-3’。

In the SNP molecular marker chr06-88890750, the genotype of the site with the polymorphism is C/C, which corresponds to high birth rate; genotype is C/T, corresponding to low kernel rate.

The kernel yield of the camellia oleifera seeds is the kernel yield of the camellia oleifera seeds which are dried to constant weight.

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 kernel-out rate of camellia oleifera seeds or the SNP molecular marker or the primer or the reagent or the kit:

(1) the application in identifying the kernel-out rate phenotype of the camellia oleifera seeds;

(2) the application in the improvement of the kernel-out rate of the oil-tea camellia seeds or the molecular marker assisted breeding of the kernel-out rate of the oil-tea camellia seeds;

(3) the application in early prediction of the kernel-out rate of the camellia oleifera seeds;

(4) the application in screening the oil tea with high kernel yield.

In a fifth aspect, the invention provides a method for identifying the kernel-out rate phenotype of oil-tea camellia seeds or screening oil-tea camellia with high seed kernel-out rate, which comprises 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 a primer with a sequence shown as SEQ ID NO. 2-3;

(3) analyzing the genotype of the SNP molecular marker in the PCR amplification product, and judging the kernel-out rate phenotype of the seeds of the oil tea 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.

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 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 kernel-out rate phenotype of the seeds 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 chr06-88890750 is C/C, the kernel-out rate of 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 kernel-out rate.

The invention provides a method for identifying oil tea with high kernel yield, 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 a primer shown in SEQ ID NO.2-3 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 kernel-out rate according to the genotype.

If the genotype of the site with the polymorphism of the SNP molecular marker chr06-88890750 is C/C, the kernel-out rate of 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 kernel-out rate.

The invention has the beneficial effects that: the invention provides a gene locus for the kernel-out rate of camellia oleifera seeds, the contribution rate to the phenotype of the kernel-out rate of the seeds is 14.9%, and SNP molecular markers closely linked with the gene locus are developed. The SNP molecular 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 70.42 percent of the individual seeds in the individual plants with the locus of the genotype with high kernel-out rate have the kernel-out rate higher than the average seed kernel-out rate (59.16 percent) of the population; the seed outing rate was lower than the population mean (59.16%) in 69.10% of individuals with low outing rate genotypes, indicating that this marker is practical for use in assisted selection of the seed outing rate phenotype.

In the conventional selection breeding of the camellia oleifera, the identification of the seed kernel yield character 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, and the selection efficiency is improved. In the camellia oleifera breeding, the molecular marker and the detection method thereof can be selected to identify the camellia oleifera with high kernel-out rate for breeding, so that the selection efficiency of the camellia oleifera breeding can be improved, and the breeding process can be accelerated.

Drawings

FIG. 1 is a schematic diagram showing the location of the kernel-out rate gene site rkf.06-2 of Camellia Oleifera 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.

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 of Camellia oleifera seed kernel-out rate segregation population and determination of traits

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 fruits of 180 generations of individuals are completely ripe (5 percent of fruits are cracked), and measuring the kernel yield of the seeds. The operation steps are as follows:

(1) taking 10-15 seeds from each sample, baking at 105 deg.C to constant mass, and recording seed mass (W)₁)。

(2) Removing hard seed coat from oil tea seed, and recording total weight (W) of kernel₂)。

(3) Calculate seed kernel rate for each sample:

the kernel yield of the seeds is W₂/W₁×100％。

The kernel-yielding rate determination result of the camellia oleifera seeds shows that: the kernel-yielding rate of the seeds 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: at 98 deg.C for 2 min; 13 cycles of 98 deg.C, 30s, 60 deg.C, 30s, 72 deg.C, 15 s; 72 deg.C, 5 min. 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: 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 seed kernel-out rate gene locus and linkage SNP locus of Camellia oleifera seed

QTL Isimulping software is used for data analysis, and a complete interval mapping method (ICIM) is used for positioning the QTL of the seed kernel rate. 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 kernel-out rate gene locus rkf.06-2 of the camellia oleifera seeds is located on the chromosome 6 of the camellia oleifera, the contribution rate is 14.9%, and the SNP molecular marker closely linked with the gene locus is chr06-88890750 (table 1, figure 1).

Example 4 application of kernel-out rate gene locus and linkage SNP molecular marker in camellia oleifera breeding

1. 124 individuals are randomly selected as materials from the Changlin No. 53 multiplied by No. 81 camellia oleifera hybrid F1 generation family population, 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 reaction System for PCR amplification

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 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 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) 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 chr06-88890750 loci of all individuals were identified separately. Comparing the genotype of each site with the relationship between the kernel-out rate, if the genotype of the site is C/C, the oil tea individual is the oil tea with high kernel-out rate; if the genotype is C/T, the oil tea individual is the oil tea with low kernel-out rate.

5. 124 individuals of F1 generations and parents of fully mature seeds were collected and the seed kernel rate was determined (see example 1). The results show (table 3) that 70.42% of individuals with chr06-88890750 locus as high-kernel-rate genotype have kernel rate higher than average kernel rate (59.16%) of seeds of the population; of the individuals with genotypes with low kernel-out rates, 69.10% had lower seed-out rates than the population mean (59.16%). The marker is practical and effective when used for auxiliary selection of the seed kernel-out rate phenotype, can be used for early identification or auxiliary identification of the seed kernel-out rate phenotype of the camellia oleifera seeds, can greatly save the production cost, improves the selection efficiency and accelerates the camellia oleifera breeding process.

TABLE 3 seed kernel yields 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

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aaggaagttc agggaataag t 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

attagctaca actaactaat a 21