CN113502293B

CN113502293B - Camellia oleifera self-incompatibility related gene, SNP molecular marker and application

Info

Publication number: CN113502293B
Application number: CN202110978700.8A
Authority: CN
Inventors: 江南; 谭晓风; 周俊琴
Original assignee: Hunan University of Technology; Central South University of Forestry and Technology
Current assignee: Hunan University of Technology; Central South University of Forestry and Technology
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2022-07-22
Anticipated expiration: 2041-08-25
Also published as: CN113502293A; ZA202204301B

Abstract

The invention discloses a self-incompatibility related gene of camellia oleifera, an SNP marker and application. SNP missense mutation exists at a SNP071031 position in OFR of a genome sequence of CoMAPK9, a base is changed from C to A, the SNP missense mutation exists at a SNP181968 position, the base is changed from A to G, and remarkable correlation exists between SNP mutation at two positions and the self-incompatibility character of camellia oleifera. The SNP combined molecular marker can be used for identifying the fruit setting rate type seedling stage of the oil tea, and effectively improves the variety allocation and selection efficiency of new oil tea forestation areas and the unit area yield of the oil tea.

Description

Camellia oleifera self-incompatible associated gene, SNP molecular marker and application

The technical field is as follows:

the invention belongs to the technical field of molecular markers, and relates to a new gene associated with the self-incompatibility character of camellia oleifera, and an SNP molecular marker and application thereof.

The background art comprises the following steps:

the self-incompatibility phenomenon exists widely in the flowering plants. Selfing refers to the combination of male and female gametes from the same individual or the mating between individuals with the same genotype or the mating between individuals from the same clonal line. Self-incompatibility refers to the phenomenon that a hermaphrodite plant which can produce hermaphrodite gametes with normal functions and mature in the same period can not complete fertilization when self-pollinated or cross-pollinated with the same genotype. The gene is an important genetic mechanism for preventing the close-relative propagation and the degeneration of the plants, and has important significance in the aspects of plant reproductive biology, heterosis utilization and the like. Because of the self-incompatibility of plants, pollination trees with pollination affinity are usually configured for main cultivars in cultivation or mutual pollination fructification among the selected main cultivars is ensured, in addition, when new varieties are hybridized and cultivated, the pollination affinity characters among hybridization combinations also need to be known, otherwise, the cultivars cannot be pollinated normally in production to fructification, so that the yield and the quality are not high, and the breeding working process is influenced.

The Camellia oleifera (Camellia oleifera) is a unique woody edible oil tree species in China, and plays a very important role in the construction of forestry industries in China, the safety guarantee of edible vegetable oil and the like. During the long-term production process of the oil tea, the oil tea is full of trees and flowers, but the natural fruit setting rate is extremely low (less than 5%). In recent years, researchers of camellia oleifera have studied the reproductive characteristics of camellia oleifera, and the main reason that the camellia oleifera seed setting rate is low is caused by the self-incompatibility genetic characteristic of camellia oleifera and the improper configuration of pollinated varieties caused by the self-incompatibility genetic characteristic. In order to ensure pollination, fertilization and normal fructification among different varieties of the oil tea, the traditional method is to plant more than 6 oil tea varieties simultaneously in the same new oil tea forestation land, so that the series of problems of complex oil tea production process, high production cost, low production efficiency, difficulty in exerting the high quality and high yield efficiency of the best good varieties and the like are caused, and the development and the upgrade of the oil tea industry in China are seriously hindered.

Because the heterozygosity of the camellia oleifera is high and the genome is complex, early camellia oleifera science and technology personnel mainly search for the self-incompatibility of the camellia oleifera from flowering habits, flowering-period ecology, pollination ways and breeding rules. In recent years, through field pollination, fluorescence microscopic observation and statistical analysis, researchers of the camellia oleifera have made some important progresses on the study of the self-incompatibility of the camellia oleifera. Super high-class (2017) by using an aniline blue dyeing method, the fact that after oil tea self-crossing and cross-crossing pollination are carried out for 40-48 hours, the cross-crossing pollen tube does not stay at the base of a flower column and continues to grow downwards to reach an ovule, but the self-crossing pollen tube grows and slows down at the base of the flower column, only can grow downwards to enter an ovary, but finally still stagnates at the ovary, and incompatible phenomena such as expansion, bifurcation, curling, folding and waveness still occur at the tail end of the stagnant pollen tube. The observation of the super-microstructure shows that the wall of the camellia selfing pollen tube is locally thickened, the content is unclear, the organelles are disintegrated and can not be identified, the characteristic of Programmed Cell Death (PCD) is presented (Wang et al, 2008; Gao super et al, 2015, 2017), the polar growth of the selfing pollen tube is inhibited, and the growth is stopped. Systematic study on the growth rule of pollen tubes in the selfing and outcrossing processes of the camellia oleifera at the cellular level can determine that the camellia oleifera belongs to the later self-incompatible plants in the early stage of the zygote. However, the research on the molecular level of the self-incompatibility of the camellia oleifera is slow.

The self-incompatibility character of the camellia oleifera seriously affects the fruit setting rate of the camellia oleifera, and indexes such as the fruit setting rate, the fruit yield, the fruit size and the like directly affect the yield of the camellia oleifera per unit area, so that the development of the compatibility research on the self-incompatibility character of camellia oleifera varieties and the hybridization among the varieties is very important for the development and the upgrading of the camellia oleifera industry.

The effective molecular marker can identify the camellia oleifera variety in the seedling stage, so that the camellia oleifera new forestation land cultivation variety is scientifically configured, the self-incompatibility phenomenon of pollination is reduced, the fruit setting rate of camellia oleifera is improved, the configuration quantity of camellia oleifera main cultivation varieties is reduced, the production cost of camellia oleifera cultivation and tending management is reduced, and the unit area yield and the product quality of camellia oleifera are improved. Therefore, the development of molecular markers related to the phenotype of the self-incompatibility character of the camellia oleifera has important practical application value in the aspects of improving the comprehensive benefit of the camellia oleifera industry and the like.

According to the invention, the CoMAPK9 is selected from the transcriptome database of the pistil of the oil-tea camellia, and is a unique sequence of the oil-tea camellia, and is specifically expressed in pistil of 24 hours of self-pollination of the oil-tea camellia and reaches a peak value, so that the CoMAPK9 is presumed to participate in the regulation and control of the self-incompatibility process of the oil-tea camellia, thereby influencing the agronomic traits related to the yield of the oil-tea camellia, such as fruit setting rate, and the research on related contents is not reported so far.

The invention content is as follows:

when varieties are selected and configured conventionally in a new camellia oleifera forestation land, the varieties are configured according to cultivation experience, fruit bearing is carried out only 3-5 years after seedling forestation, and high yield is achieved in 5-7 years. If the variety is not properly configured, time and labor are wasted, and huge economic loss is caused. The SNP molecular marker sites in the invention are clear, and the detection method is simple, convenient, rapid and accurate, and has strong targeting property and low cost. By detecting the SNP locus, the pollination affinity condition of the tea-oil tree forest land configuration variety can be predicted, so that scientific selective configuration of a new tea-oil tree forest land variety is assisted, waste of manpower, material resources and time in the production process of the tea-oil tree forest land is avoided, the production cost of the tea-oil tree is greatly saved, and stable and high-yield development of the tea-oil tree forest land is ensured.

The invention aims to provide a tea-oil tree self-incompatibility related gene, an SNP molecular marker thereof and application thereof, aiming at the bottleneck encountered in the process of researching the self-incompatibility of the tea-oil tree in the prior art. The invention firstly provides 2 SNP molecular markers related to the self-incompatibility of the oil tea, and the combined markers can be used for identifying the fruit setting rate type seedling stage of the oil tea, thereby effectively improving the variety allocation and selection efficiency of the new oil tea forestation land and the unit area yield of the oil tea.

The cDNA sequence of the self-incompatibility related gene CoMAPK9 of the camellia oleifera is as follows: SEQ ID No.1, the genomic sequence is: SEQ ID NO. 2.

The self-incompatible associated gene CoMAPK9 of the camellia oleifera is specifically expressed in pistils of the camellia oleifera, and the expression of the self-incompatible associated gene CoMAPK9 of the camellia oleifera reaches a peak value after 24 hours of self-pollination of the camellia oleifera. The protease formed by the gene can irreversibly inhibit pollen germination and pollen tube growth along with the increase of concentration, so that the phenomenon of self-incompatibility of the oil tea is caused, and finally, the oil tea cannot normally form fruits and seeds, so that the fruit setting rate of the oil tea is extremely low (for example, < 5%).

The basis of the SNP molecular marker related to the self-incompatibility of the camellia oleifera provided by the invention is that a SNP missense mutation exists at a SNP071031 position in an open reading frame of a gene CoMAPK9 genome sequence, a base is changed from C to A, a corresponding amino acid is changed from Pro to Gln, a SNP missense mutation exists at a SNP181968 position, the base is changed from A to G, and the corresponding amino acid is changed from Thr to Ala.

Further, the SNP polymorphism of the two sites has obvious correlation with the self-incompatibility character of the camellia oleifera, and the genotype of the SNP071031 site in the zygote formed after pollination is CC or CA, the self-compatibility character phenotype is shown, the genotype is AA, and the self-incompatibility character phenotype is shown; SNP181968 locus genotype AA or AG in the zygote formed after pollination is a self-compatible character phenotype; genotype GG, is a self-incompatible trait phenotype.

Further, primer pairs for 2 SNP molecular markers in the coomapk 9:

primer pair of SNP polymorphic site SNP 071031: the sequence of the upstream primer is as follows: shown in SEQ ID NO.3, the sequence of the downstream primer is as follows: SEQ ID NO. 4;

the primer pair of SNP polymorphic site SNP181968 has the upstream primer sequence as follows: shown in SEQ ID NO.5, the sequence of the downstream primer is as follows: shown as SEQ ID NO. 6.

Furthermore, if the genotypes of 2 SNP polymorphic sites of the zygote after pollination are all the genotypes corresponding to the self-compatible trait phenotype, or the genotype containing only 1SNP polymorphic site is the genotype corresponding to the self-compatible trait phenotype, the camellia oleifera plant is of the self-compatible trait phenotype and corresponds to a high fruit set rate; if the genotypes of the 2 SNP polymorphic sites are all the genotypes corresponding to the self-incompatible trait phenotype, the oil tea plants are of the self-incompatible trait phenotype and correspond to low fruit set percentage.

Furthermore, the self-compatibility of the genotypes of the 2 SNP polymorphic sites of the zygote after pollination, which are all the genotypes corresponding to the self-compatibility trait phenotype, is higher than that of the camellia oleifera plant containing only 1SNP polymorphic site, which is the genotype corresponding to the self-compatibility trait phenotype, namely the fruit set rate of the plant containing the genotypes of the 2 SNP polymorphic sites of the zygote after pollination, which is the genotype corresponding to the self-compatibility trait phenotype, is higher than that of the plant containing only 1SNP polymorphic site, which is the genotype corresponding to the self-compatibility trait phenotype.

The invention also provides application of the SNP molecular marker associated with the self-incompatibility of the camellia oleifera, which comprises at least one of the following components:

(1) identifying pollination affinity character phenotype of the camellia oleifera woodland configured variety;

(2) early-stage prediction of pollination affinity phenotype of mutual configuration of oil tea varieties in the new oil tea forestation land;

(3) scientifically preparing varieties of the new tea-oil trees in the forestation areas;

(4) and (3) identifying and improving the germplasm resources of the oil tea.

Further, the application selects the camellia oleifera variety with the zygote SNP071031 polymorphic site genotype of CC or CA and/or the SNP181968SNP polymorphic site genotype of AA or AG after pollination as the configured variety with high fruit set percentage in the camellia oleifera forest land.

Further, if the genotypes of the 2 SNP polymorphic sites of the zygote after pollination are all the genotypes corresponding to the self-compatibility character phenotype, or the genotype only containing 1SNP polymorphic site is the genotype corresponding to the self-compatibility character phenotype, the camellia oleifera plant is of the self-compatibility character phenotype; and if the genotypes of the 2 SNP polymorphic sites are all the genotypes corresponding to the self-incompatible trait phenotype, the oil tea plant is of the self-incompatible trait phenotype.

Furthermore, the genotypes of the 2 SNP polymorphic sites of the zygote after pollination are all the genotypes corresponding to the self-compatible character phenotype, and the self-compatibility of the genotypes is higher than that of the camellia oleifera plant which only contains 1SNP polymorphic site and is the genotype corresponding to the self-compatible character phenotype; corresponding to the fruit setting rate of the camellia oleifera individuals, the fruit setting rate of the plants of which the genotypes of the 2 SNP polymorphic sites of the zygotes are all the genotypes corresponding to the self-compatible trait phenotype after pollination is higher than the fruit setting rate of the plants of which the genotypes containing only 1SNP polymorphic site are the genotypes corresponding to the self-compatible trait phenotype.

For the applications (1) and (2), the formed tea-oil tree forest land is predicted, the SNP molecular markers of the configured varieties can be identified and cultivated in the seedling stage, and the fruit setting rate can be predicted according to the SNP locus genotypes of the configured varieties, so that intervention can be performed as early as possible, and loss can be reduced.

For the application (3), scientific variety allocation is carried out on the camellia oleifera woodland to be subjected to afforestation; the method can be used for identifying SNP molecular markers of alternative configured cultivars in the seedling stage, and selecting the camellia oleifera cultivars with high fruit setting rate in camellia oleifera forests, wherein the genotype of SNP071031SNP polymorphic site is CC or CA and/or the genotype of SNP181968SNP polymorphic site is AA or AG on the zygote probability after pollination as much as possible according to the genotype of the alternative configured cultivars.

For the application (4), germplasm resources can be identified and improved according to the SNP molecular markers of the cultivars, so as to obtain the good varieties of oil-tea trees with high fruit setting rate.

The invention has the beneficial effects that:

1. the invention relates to a gene CoMAPK9 obviously related to the self-incompatibility character of oil-tea camellia, and also provides 2 SNP molecular markers which are positioned in the ORF of the CoMAPK9 genome sequence and are obviously related to the self-incompatibility character of oil-tea camellia, so that the configured variety of the oil-tea camellia new forestation land can be identified in the seedling stage, and great convenience is provided for accurately screening and scientifically configuring the pollinated variety of the oil-tea camellia new forestation land.

2. The 2 SNP molecular markers obviously associated with the self-incompatibility character of the camellia oleifera provided by the invention are suitable for application in screening and scientifically configuring new pollination varieties of camellia oleifera forestland. According to the invention, the molecular marker is used for identifying and assisting in selection of a sexual oil tea group, and the result shows that the single plant with high fruit set rate genotype at the same time of the SNP site 071031 and the SNP site 181968 of the oil tea kernel accounts for 47.9%, wherein the fruit set rate of 96.8% of the single plant (57-100%) is higher than the average fruit set rate (54.50%); the single plant containing any 1 genotype with high fruit setting rate accounts for 35.2 percent, wherein the fruit setting rate (54.58-64.6 percent) of 43.48 percent of the single plant is higher than the average fruit setting rate (54.50 percent); the above shows pollination affinity character. 16.9% of single plants have two SNP loci of low fruit set rate genotypes at the same time, the single plant fruit set rate is 0-3.51%, and the pollination incompatibility character is represented. The molecular marker is shown to be very effective when applied to pollination variety identification and variety configuration selection of the camellia oleifera new forestation land.

Description of the drawings:

FIG. 1 electrophoresis detection of PCR amplification products;

lanes 1-4 correspond to the amplification products of primers 1FP1, 2F2R, 3F3R, 4F1R in Table 1.

FIG. 2A is the expression pattern of the CoMAPK9 gene at different sites in flowers selfed for 24 h; FIG. 2B is the expression pattern of the CoMAPK9 gene for 2-84h in the various treated flower columns.

FIG. 3A shows prokaryotic expression of CoMAPK9 gene (in the figure, Lane: A is total bacterial protein without IPTG induction, B is total bacterial protein of transformation empty vector, C-I is total recombinant bacterial protein induced by IPTG for 2h, 4h, 6h, 8h, 10h, 12h and 14 h); FIGS. 3B and C are the inhibition of pollen tube growth by the CoMAPK9 protein; figure 3D is the subcellular localization of the coapk 9 gene.

The specific implementation mode is as follows:

the technical means used in the following examples, if not specifically indicated, are conventional techniques and methods well known to those skilled in the art for illustrating the present invention, but do not limit the scope of the present invention.

Example 1 Total DNA extraction and fruit set percentage calculation of Camellia oleifera kernels, roots or leaves

The total DNA of a single plant is extracted by using a DNA secure novel plant genome DNA extraction kit (TIANGEN kit Code No. DP320, centrifugal column type) and combining a laboratory improved CTAB method. The specific operation steps are as follows:

(1) taking 600 mu L of 2 xCTAB liquid into a 1.5mL centrifuge tube, uniformly mixing, and keeping the temperature in an air heater at 65 ℃ for later use;

(2) respectively and quickly taking out kernels, roots or leaves of the camellia oleifera seeds from liquid nitrogen, and quickly grinding the kernels, the roots or the leaves into fine powder in a precooling mortar containing the liquid nitrogen;

(3) transferring the ground powder into a centrifuge tube, uniformly mixing the powder and the solution on a vortex instrument, and carrying out water bath at 65 ℃ for 10min, wherein the powder and the solution are uniformly stirred every 2 min;

(4) adding 600 μ L chloroform to isoamyl alcohol (24:1), vortexing, and centrifuging at room temperature of 10,000r/min for 10 min;

(5) taking the supernatant to another sterile enzyme-free 1.5mL centrifuge tube, adding equal volume of chloroform and isoamylol (V/V is 24:1), reversing, mixing, and centrifuging at 10,000r/min at 4 ℃ for 10 min;

(6) taking the supernatant to another sterile and enzyme-free 1.5mL centrifuge tube, and extracting the total DNA of the kernels according to the instruction of the TIANGEN plant genome DNA extraction kit;

(7) finally dissolving and collecting the extracted total DNA by using 30 mu L of ultrapure water;

(8) the extracted genomic DNA was subjected to 1% agarose gel electrophoresis to examine the presence or absence of degradation, Nanodrop ND2000 and

2100Bioanalyzer measures DNA concentration and purity, diluted to 30 ng/. mu.L, stored at-20 ℃.

The fruit setting rate of the camellia oleifera is calculated according to the following formula: fruit setting rate is 100% of fruit tree real number/flowering number

Example 2 third Generation sequencing + second Generation sequencing samples and Annotation analysis

1. Preparation and collection of sequencing samples:

selecting national approved oil tea seed oil 'Huaxin' and 'Huajin' (the seed numbers are respectively: national S-SC-CO-009 and national S-SC-CO-010-, removing petals and stamens to expose pistils, performing artificial pollination, bagging with a sulfuric acid paper bag, and marking with a tag. Taking off pistils 48h and 72h after non-pollination, selfing and cross-pollination respectively, immediately wrapping with tin foil paper, quick freezing with liquid nitrogen, and storing at-80 deg.C. A total of 7 sets of samples (SP48, SP72, CP48, CP72, NP48, NP72 and Pn) were sequenced, with 3 replicates per set, and the first 6 sets of samples contained 60 pistils per replicate for a total of 21 samples.

2. Third and second generation transcriptome sequencing

1g of pistils are taken, and the extraction of 21 sample RNAs is completed according to the requirements of the instruction of a total RNA extraction kit of an OMEGA company. Using 1% agarose gel, Nanodrop ND2000 and

2100Bioanalyzer measures RNA concentration and integrity. RNA with the RNA integrity value RIN more than or equal to 7 and 28S/18S more than or equal to 0.7 can be used for library construction. 10 mu L of each sample RNA is taken, mixed evenly and then a Camellia pistil ISO-Seq library is constructed by adopting Clontech SMARTer PCR cDNA Synthesis Kit, and then the sequencing of the full-length transcription group (ISO-Seq) is completed by a Pacbio three-generation sequencer based on Single Molecule Real-Time sequencing technology (Single Molecule, Real-Time, SMRT). Separately taking 6 mu g of total RNA of each Sample, uniformly mixing, enriching mRNA by using a Truseq RNA Sample Prep Kit, and adding an interrupting reagent to break the mRNA into short fragments. The first strand cDNA was synthesized using the Super Script Double-Stranded cDNA Synthesis Kit using the fragmented mRNA as a template, and the Double strand cDNA was further synthesized to complete the construction of the sequencing library. Using Illumina HiSeq ^TM2500 platform for second generation sequencing (RNA-seq) of the constructed library)。

3. Transcriptome sequencing data processing and annotation analysis

The second generation sequencing technology RNA-Seq can generate sequencing sequences with short length, higher sequencing depth and base correctness, but is not beneficial to sequence assembly. The third generation sequencing technology, full-length transcriptome sequencing (Iso-Seq), can obtain relatively complete transcripts, but the base error rate is high. Since Camellia oleifera has no whole genome sequence, the combination of the third generation (ISO-seq) and the second generation transcriptome (RNA-seq) is adopted to carry out base error correction on the third generation sequencing data by using short and high-precision RNA-seq (Hackl T, Hedrich R, Schultz J, et al. Not only can obtain high-quality transcripts, but also can obtain the expression quantity of each transcript in different samples.

First, third generation sequencing data was processed using the SMRTlink 5.0 software to form a CCS (circular Consensus sequence) by self-error correction and pooling. Clustering and correcting the full-length CCS sequence by using an ICE (iterative Clustering and Error correction) tool of an SMRTlink software Cluster module to obtain a polar iso-sequences, further correcting the polar iso-sequences by using prooverread Error correction software through second-generation sequencing data, and then Clustering and de-redundancy the Error-corrected iso-sequences by using cd-hit-est software (Li W, Godzik A.Cd-hit: a fast program for Clustering and matching of the polar iso-sequences of the protein or nuclear sequences. New Photologist, 2006,22(13): 1658.).

To show the advantage of ISO-seq and RNA-seq binding, we assembled the second generation sequencing data using Trinity software, using the redundantly removed ISO-seq data as the reference sequence, and aligned the RNA-seq parametrically analyzed data after each sample assembly with ISO-seq sequence using TopHat software (Ryan K, Geo P, Steven LS, et al. TopHat2: acquisition alignment of transactions in the presence of interactions, deletions and genes fusion. genome biology,2013,14(4): 621-. FPKM (fragments Per. Kilobase of transcript Per Million fragments mapped) was used as a measure of transcript or gene expression levels, while correlation between samples was analyzed using a Person correlation coefficient. Transcript expression differential analysis was performed using DESeq2(Love MI, Huber W, Anders S. modeled evaluation of field change and dispersion for RNA-seq data with DESeq2.genome Biology,2014, 15(12): 31-36).

The software PLEK (Li, A.J.Zhang, and Z.Zhou.PLEK: A tool for predicting the coding of non-coding RNAs and messenger RNAs based on an improved K-mer scheme.BMC Bioinformatics,2014,15(1): NO.311.), CPC (Kong Lei, et al. "CPC: associated with the protein-coding site of transcription using sequence utilities and subset vector machinery," Nucleic Acids Research, 2007,35: W345-W349.) and ANGEL (Shimizu K, Adachi Open J, Muraoka Y, et al. Angles: a. reagent coding Frame for coding of protein and promoter Reading Frame 664: (journal Reading Frame of coding for protein 643, prediction of coding of protein) was used. Transcript annotations were then made in databases NR, GO, KOG, KEGG, and SWISS-prot, among others.

Example 3: CoMAPK gene acquisition and SNP site analysis

1. Analysis of characteristics of CoMAPK9 gene associated with self-incompatibility

And performing annotation analysis on the transcripts and trend analysis and Pearson correlation analysis on the differential transcripts and differential proteins, and screening to find that the CoMAPK9 is a unique sequence of the camellia oleifera and strongly responds to self-pollination and cross-pollination. The functional characteristics of the CoMAPK9 gene in the camellia oleifera self-incompatibility process are researched by sequence analysis expression pattern analysis, prokaryotic expression, subcellular localization and other experiments.

The expression pattern of the CoMAPK9 was analyzed at different sites in the flower (FIG. 2A) and in different treated flower columns for 2-84h using real-time fluorescent quantitative PCR (FIG. 2B).

The accuracy of the CoMAPK9 gene sequence was verified by inducing CoMAPK9 protein expression through prokaryotic expression experiments (FIG. 3A), and recombinant protein was purified by using a Ni-NTA affinity chromatography column. And (3) carrying out isolated culture on camellia oleifera 'Huashuo' pollen by using the control group and the experimental group with the final concentrations of 0.01, 0.05, 0.1, 0.2 and 0.5mmol/L of CoMAPK9 protein, and counting the pollen germination rate and the pollen tube length of the control group and the experimental group. Compared with a control group, CoMAPK9 can obviously inhibit pollen germination and pollen tube growth. Moreover, the higher the inhibitor concentration, the more significant the inhibition effect on pollen germination rate and pollen tube length (FIGS. 3B and C). Construction of a green fluorescent protein fusion expression vector for transformation of tobacco, subcellular localization of the CoMAPK9 gene was observed, and expression of the CoMAPK9 gene in the endoplasmic reticulum was found (FIG. 3D).

2. CoMAPK9 gene SNP site screening

A CoMAPK9 transcript cb10019_ c17495/F1P0/3256 sequence is selected to design a primer (Table 1P1 and Table 1F1R) for amplifying the full length of cDNA, a primer (Table 1 amplification success primer) for amplifying the full length of CoMAPK9 genome is designed according to the full length of the cDNA, and amplification adjacent PCR products are partially overlapped to ensure effective amplification of an intron and ensure screening of all SNP sites on CoMAPK 9.

Collecting 20 oil tea varieties with different self-incompatibility degrees in the oil tea distribution area, extracting total DNA according to the method of the embodiment 1, mixing, and respectively amplifying by using primers 1FP1, 2F2R, 3F3R and 4F1R in the table 1, wherein the amplification conditions are as follows: the total volume of the PCR system was 50uL, 25. mu.L of Novozam 2 XTaq Plus Master Mix II (Dye Plus) enzyme, 2. mu.L each of the upstream and downstream primers (10. mu. mol/L), 3. mu.L of the mixed template, and 18. mu.L of deionized water. Reaction procedure: 5min at 94 ℃; 30s at 94 ℃, 30-70 ℃ lmin and 40s at 72 ℃ for 35 cycles; 7min at 72 ℃. And (3) detecting the PCR product by gel electrophoresis (figure 1), recovering and sequencing, and splicing the product by the ontigExpress to obtain a CoMAPK9 full-length sequence. And (4) screening 19 potential SNP sites of CoMAPK9 according to the spliced sequence and the position where a peak appears in a peak diagram of a reverse sequencing result (the specific information of the SNP is shown in Table 2).

TABLE 1 successful amplification primers

The sequences in table 1 are shown in SEQ ID NOs: 7-14 Table 2 specific information on SNP sites

3. Correlation analysis of SNP locus of CoMAPK gene and self-incompatibility

Selecting main camellia oleifera cultivars with different pollination affinities in camellia oleifera distribution areas to establish camellia oleifera germplasm resource natural groups, wherein the camellia oleifera germplasm origins of the groups are from most camellia oleifera main production areas in China, including main production areas in Hunan, Jiangxi, Guangxi, Fujian, Guangdong, Hainan and other provinces, and the camellia oleifera germplasm resources are collected and stored in a Tansha city camellia oleifera planting experimental base of China, China national forestry science and technology university, China and China forestry bureau key laboratories.

Randomly taking 400 oil tea individual plants in the experimental base, measuring the fruit setting rate of the individual plants in the fruit expanding period, collecting the individual fruits in the high-speed oil synthesis period, and immediately peeling kernels for preservation by liquid nitrogen after the fruits are collected. Statistical results obtained an average fruit set rate of 28.83%, 178 parts fruit set rate > 50%, 156 parts fruit set rate < 20%, 66 parts 50% < fruit set rate < 20%. Using genome DNA of single plant kernel as a template, carrying out SNP typing on 300 single plants in total of 150 samples with fruit setting rate of > 50% and 150 samples with fruit setting rate of < 20% by using a fluorescent quantitative PCR growth curve and dissolution curve analysis method (Shisqin, Tantang rainbow, Yusimian, and Linqiong 2005. SNP assay method based on fluorescent quantitative PCR amplification reaction 06: 110-.

Inputting SNP genotype data and fruit setting rate phenotype data into SPSS software for chi square test, analyzing the correlation between the genotyping result and the fruit setting rate of 19 SNP sites (Table 3), and finally obtaining 2 SNP markers which are significantly related to the fruit setting rate of the camellia oleifera and are positioned in the ORF of the CoMAPK9 genome sequence, are respectively positioned in the SNP 031 site and the SNP 03181968 site which are significantly related to the fruit setting rate phenotypic variation (P <0.01, Table 3)

TABLE 3 Association analysis of SNP sites and fruit setting rate traits of Camellia oleifera

Example 4 application of 2 SNP molecular markers in selection of high fruit setting rate pollinated variety configuration in camellia oleifera forest land

(1) Selecting a single plant of a national approved oil tea fine variety 'Huaxin' (the fine variety number is national S-SC-CO-009-. Collection of ` Xin ` and ` Xinjin ` young leaves extracted total DNA (example 1). Respectively carrying out PCR amplification on the DNA by using primers of SEQ ID NO.3-4 and SEQ ID NO.5-6, wherein the amplification system is as follows: total volume of PCR system is 50uL, Novozan 2

Max Master Mix (Dye Plus) high fidelity enzyme Mix 25. mu.L, 2. mu.L each of the upstream and downstream primers (10. mu. mol/L), 1. mu.L of DNA template, and 20. mu.L of deionized water. Reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing and extension at 63 ℃ for l min, and extension at 72 ℃ for 40s for 35 cycles; total extension at 72 ℃ for 7 min. Detecting PCR product by Gel electrophoresis, purifying and recovering PCR product by Gel Extraction Kit (OMEGA, Code No. D2500-02), and performing the experimental process by Kit instruction.

(2) Performing first-generation sequencing on the PCR product by using a corresponding amplification primer, analyzing a sequencing peak map by using Chromas software to determine genotypes of 'Huaxin' and 'Huajin' SNP071031 loci and SNP0181968 loci, wherein the genotypes of the 'Huaxin' and 'Huajin' SNP071031 loci and the SNP0181968 loci are respectively as follows: 'Huaxin' CA/GG, 'Huajin' CA/AG. According to the genetic combination calculation of the genetic rule, 75% of seeds of the single plants of fruits after pollination contain genotypes with high fruit setting rate, 25% of seeds of the single plants contain genotypes with low fruit setting rate, and the high fruit setting rate phenotype is mainly shown after the single plants in the group are pollinated.

(3) After artificial pollination is carried out on the population, the fruit setting rate of each individual plant is recorded in the expanding period of the camellia oleifera fruits, and the seeds of each individual plant in the population are collected in the high-speed fruit oil synthesis period to extract DNA (example 1). And (3) respectively carrying out PCR amplification on the DNA by using primers of SEQ ID NO.3-4 and SEQ ID NO.5-6 according to the method in (1), and determining the genotype of the SNP site of each single-plant kernel according to the sequencing method in (2).

(4) After identifying the genotype of the SNP071031 locus and the SNP0181968 locus of each single plant kernel in the population, contrasting the relationship between the genotype of each locus and the fruit setting rate phenotype: the genotypes of the 2 SNP molecular markers in the fruit kernels are all high-fruit-setting rate types or only contain 1 high-fruit-setting rate type, so that the camellia oleifera plant presents a high-fruit-setting rate phenotype, and pollination is an affinity character phenotype; if the genotypes of the 2 SNP molecular markers in the fruit kernels are all low fruit setting rate types, the camellia oleifera plant is of the low fruit setting rate phenotype, and the pollination is of the incompatible character phenotype.

The result shows that (table 4), the SNP site SNP071031 and the SNP site SNP181968 of the camellia oleifera seed kernel are 47.9% of single plants with genotypes with high fruit set rate at the same time, wherein 96.8% of the fruit set rate (57-100%) of the single plants is higher than the average fruit set rate (54.50%); the single plant with the genotype with high fruit setting rate in any 1 of the two sites of SNP site 071031 and SNP site 181968 accounts for 35.2%, wherein the fruit setting rate of 43.48% of the single plant (54.58% -64.6%) is higher than the average fruit setting rate (54.50%). The above shows pollination affinity character. 16.9% of two SNP loci of a single plant are simultaneously of low fruit setting rate genotypes (AA/GG), the fruit setting rate of the single plant is 0-3.51%, and the single plant shows a pollination incompatibility character. The fruit set rate phenotype of the single plant is consistent with the judgment result according to the genotype. The molecular markers of the SNP071031 site and the SNP0181968 site are proved to be practical and effective when used for auxiliary selection and identification of high fruit setting rate pollination variety configuration in the camellia oleifera forest land, can be used for early pollination variety identification or auxiliary selection in the camellia oleifera forest land, greatly saves the production cost, and promotes the high-yield and stable-yield development of the camellia oleifera forest land.

TABLE 4 fruit set percentage and genotype data in kernels of individual plants of Camellia oleifera population

Description of the invention: in the table ". -" indicates a genotype deletion.

Sequence listing

<110> industrial university in Hunan

CENTRAL SOUTH University OF FORESTRY AND TECHNOLOGY

<120> camellia oleifera self-incompatibility associated gene, SNP molecular marker and application

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1764

<212> DNA

<213> Camellia oleifera (Camellia oleifera)

<400> 1

atggggagtg gaacattcgt ggatggtgtt cgtcgctggt ttcaacgtcg tccttcgtcg 60

ctacaaccaa acaacactca aaacctcaat aatagccaac cgttggtctc cgaatttcaa 120

ggagaggacc acctcaacgt tgttgaagac tttgatttct ctggcttgaa gatcatcaaa 180

gtccccaaac gcatcaattt tcatcccacc tctatggatc ctcacaaaaa gaccactctg 240

gacacagaat tcttcacaga gtatggggag gcgagcaggt accaagttca ggaagttgtt 300

ggcaaaggga gttatggaat tgttggttcc gccactgata cccacactgg ggagagggtt 360

gcaatcaaga agatcaatga tgtctttgat catgtctctg atgccacaag gatcctcaga 420

gaaatcaagc tccttcgact gctccgtcat ccagatattg tagaaataaa gcatatcatg 480

cttcctcctt ctcgaagaga atttaaagat atatatgttg tatttgagtt gatggaatct 540

gaccttcatc aagtgattaa ggcgaatgat gatcttactc ctgaacatta tcagtttttc 600

ttgtatcagc ttcttcgtgg cctaaaattt atacatactg caaatgtgtt tcatcgagat 660

ttaaaaccaa aaaatatact tgctaatgcg gattgcaagt tgaagatttg cgattttggg 720

cttgctcgtg tatcctttaa tgaagcgcca tcagctattt tctggactga ctatgttgct 780

actcgatggt atcgtgctcc tgaactctgt ggttcttttt tctccaaata tacgcccgca 840

attgatatat ggagcattgg atgcattttt gcagaaatgc ttacaggaaa accattgttt 900

cctgggaaaa atgtggtgca ccagttagat ctcatgactg atttccttgg cactcctgct 960

ccagaatcca ttgctaggat taggaatgaa aaagcaagaa gatatctaag tagcatgcgt 1020

aaaaaatccc aagttccatt ttcacagaag ttccccaatg cagatccatt agctcttcgc 1080

cttctggaac gcttgatagc atttgatcct aaagatcgac catctgctga agacgcatta 1140

acggatcctt actttactgg tttggcaaat gcggaccgtg aaccagcatc tgctcaaccc 1200

atatcaaaac tggagtttga atttgaaagg agaaaactaa caaaagatga tgttagagag 1260

ttgatttatc gagagatttt agaatatcat ccccagatgc tccaggagta tcttcgtggg 1320

ggagatcaga ctagcttcat gtacccaagt ggtattgatc gatttaagcg acagtttgcc 1380

catcttgagg agcattatgg taaaggtgaa aagagtactc cgcttcaaag gcagcatgct 1440

tcattgccta gagaacgggt ttgtgggcag aaggatgaaa cgatttccca aaatgatgat 1500

cttgagaagc gaactgtggc atctgttgct acaactattc agagttctcc caaagaatcc 1560

gaggaatcag aaaatgcaaa tacaaatgca caaagtggac taaacaagcc gaactacagt 1620

gctcgtaccc tattaaagag tgctagtatt agtggttcca agtgtgtagt tgtccaagca 1680

aaaaaagatt caaaggaaga accaattgct gagcatgagg aggttgatga gttgacacaa 1740

aaattagcag ccatcaattc gtga 1764

<210> 2

<211> 2296

<212> DNA

<213> Camellia oleifera (Camellia oleifera)

<400> 2

aaagccaaaa taataaaaaa atcaccgcta cgtttgtaga tgatcgttat aattacacaa 60

aacaaaaaca taaaaacata agaccatctc caacgctgta tcaaatatga tatcaaatgg 120

ggagtggaac attcgtggat ggtgttcgtc gctggtttca acgtcgtcct tcgtcgctac 180

aaccaaacaa cactcaaaac ctcaataata gccaaccgtt ggtctccgaa tttcaaggag 240

aggaccacct caacgttgtt gaagactttg atttctctgg cttgaagatc atcaaagtcc 300

ccaaacgcat caattttcat cccacctcta tggatcctca caaaaagacc actctggaca 360

cagaattctt cacagagtat ggggaggcga gcaggtacca agttcaggaa gttgttggca 420

aagggagtta tggaattgtt ggttccgcca ctgataccca cactggggag agggttgcaa 480

tcaagaagat caatgatgtc tttgatcatg tctctgatgc cacaaggatc ctcagagaaa 540

tcaagctcct tcgactgctc cgtcatccag atattgtaga aataaagcat atcatgcttc 600

ctccttctcg aagagaattt aaagatatat atgttgtatt tgagttgatg gaatctgacc 660

ttcatcaagt gattaaggcg aatgatgatc ttactcctga acattatcag tttttcttgt 720

atcagcttct tcgtggccta aaatttatac atactgcaaa tgtgtttcat cgagatttaa 780

aaccaaaaaa tatacttgct aatgcggatt gcaagttgaa gatttgcgat tttgggcttg 840

ctcgtgtatc ctttaatgaa gcgccatcag ctattttctg gactgactat gttgctactc 900

gatggtatcg tgctcctgaa ctctgtggtt cttttttctc caaatatacg cccgcaattg 960

atatatggag cattggatgc atttttgcag aaatgcttac aggaaaacca ttgtttcctg 1020

ggaaaaatgt ggtgcaccag ttagatctca tgactgattt ccttggcact cctgctccag 1080

aatccattgc taggattagg aatgaaaaag caagaagata tctaagtagc atgcgtaaaa 1140

aatcccaagt tccattttca cagaagttcc ccaatgcaga tccattagct cttcgccttc 1200

tggaacgctt gatagcattt gatcctaaag atcgaccatc tgctgaagac gcattaacgg 1260

atccttactt tactggtttg gcaaatgcgg accgtgaacc agcatctgct caacccatat 1320

caaaactgga gtttgaattt gaaaggagaa aactaacaaa agatgatgtt agagagttga 1380

tttatcgaga gattttagaa tatcatcccc agatgctcca ggagtatctt cgtgggggag 1440

atcagactag cttcatgtac ccaagtggta ttgatcgatt taagcgacag tttgcccatc 1500

ttgaggagca ttatggtaaa ggtgaaaaga gtactccgct tcaaaggcag catgcttcat 1560

tgcctaggta aggtgttgaa cataaaaatg cacatttttg tacaagtatt attgattgca 1620

cataacgtaa acaatctcaa ttaattaatt agtttttccc atttattttc ctaacaatcc 1680

aaaagatttc tgtgtttgct gaacctgatt tgtgcaattc tgttcgctat catagtaagc 1740

tagtctgcat atttggttta tcttgtgagc ttctagttcc actaggttga tggtatcttt 1800

atactatcac catgcttcat tgcctagaga acgggtttgt gggcagaagg atgaaacgat 1860

ttcccaaaat gatgatcttg agaagcgaac tgtggcatct gttgctacaa ctattcagag 1920

ttctcccaaa gaatccgagg aatcagaaaa tgcaaataca aatgcacaaa gtggactaaa 1980

caagccgaac tacagtgctc gtaccctatt aaagagtgct agtattagtg gttccaagtg 2040

tgtagttgtc caagcaaaaa aagattcaaa ggaagaacca attgctgagc atgaggaggt 2100

tgatgagttg acacaaaaat tagcagccat caattcgtga ttttatactt agctctaata 2160

tctcaaattg cttcagatat ggcttcattt ggtgccctga tttgaaataa tagtatgatg 2220

tgtagaaacg gaccaccttg tgtgtgatgc caagtaacaa ttacatgagg caaggatgtc 2280

tttttaaaat caagtc 2296

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gctccagaat ccattgctag gat 23

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggttgagcag atgctggttc ac 22

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cattgcctag agaacgggtt tgt 23

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtgtcaactc atcaacctcc tca 23

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgagaaggag gaagcatgat atgct 25

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gaccatctcc aacgctgtat caa 23

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atcacgaatt gatggctgct a 21

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gactgctccg tcatccagat attgt 25

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctcctggagc atctggggat gatat 25

<210> 12

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gaaccagcat ctgctcaacc catatc 26

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ctatcaccat gcttcattgc cta 23

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cgctatcata gtaagctagt ctgca 25

Claims

1. The two SNP molecular markers are characterized in that the molecular marker SNP071031 is established based on the fact that one SNP missense mutation exists at the 1031bp position in an open reading frame of a CoMAPK9 genome sequence, a base is changed from C to A, and a corresponding amino acid is changed from Pro to Gln, the molecular marker SNP181968 is established based on the fact that one SNP missense mutation exists at the 1968bp position in the open reading frame of a CoMAPK9 genome sequence, the base is changed from A to G, and the corresponding amino acid is changed from Thr to Ala; the genome sequence of the coapk 9 is: SEQ ID NO. 2; the 1031bp position corresponds to 1147bp position of the sequence shown in SEQ ID NO.2, and the 1968bp position corresponds to 2084bp position of the sequence shown in SEQ ID NO. 2.

2. The SNP molecular marker related to the self-incompatibility of the oil tea as claimed in claim 1, wherein the SNP polymorphism of two loci has significant correlation with the self-incompatibility of the oil tea, and the SNP polymorphism of two loci is expressed as CC or CA at the SNP071031 locus genotype of the zygote formed after pollination, the phenotype of the self-incompatibility is the genotype AA, and the phenotype of the self-incompatibility is the phenotype of the self-incompatibility; SNP181968 locus genotype AA or AG in the zygote formed after pollination is a self-compatible character phenotype; genotype GG, is a self-incompatible trait phenotype.

3. The SNP molecular markers related to the self-incompatibility of camellia oleifera as claimed in claim 1, wherein the primer pairs of the 2 SNP molecular markers in the CoMAPK9 are as follows:

4. The oil tea self-incompatibility related SNP molecular marker according to claim 1, wherein if the genotypes of 2 SNP polymorphic sites of the zygote are the genotypes corresponding to the self-compatible trait phenotype after pollination, or the genotype containing only 1SNP polymorphic site is the genotype corresponding to the self-compatible trait phenotype, the oil tea plant is the self-compatible trait phenotype, and the individual exhibits high fruit set percentage; if the genotypes of the 2 SNP polymorphic sites are the genotypes corresponding to the self-incompatible trait phenotype, the oil tea plants are of the self-incompatible trait phenotype, and the individuals have low fruit set rate.

5. The oil tea self-incompatibility correlated SNP molecular marker according to claim 4, wherein the genotypes of 2 SNP polymorphic sites of the zygote after pollination are all the genotypes corresponding to the self-compatible trait phenotype, and the self-compatibility is higher than that of the oil tea plant containing only 1SNP polymorphic site, i.e. the fruit setting rate of the plant containing 2 SNP polymorphic sites of the zygote after pollination is higher than that of the plant containing 1SNP polymorphic site.

6. The use of the SNP molecular markers for the self-incompatibility association of Camellia oleifera Abel as claimed in any one of claims 1 to 5, which comprises at least one of:

(2) early predicting the pollination affinity phenotype of the camellia oleifera variety in the new camellia forestation;

(3) scientifically configuring varieties of the new tea-oil trees in the forestation land;

(4) and (3) identifying and improving oil tea germplasm resources.

7. The application of claim 6, wherein the oil tea variety with the zygote SNP071031 polymorphic site genotype of CC or CA and/or SNP181968 polymorphic site genotype of AA or AG after pollination is selected as a configured variety with high fruit set percentage in the oil tea forest land.

8. The use according to claim 7,

if the genotypes of the 2 SNP polymorphic sites of the zygote are all the genotypes corresponding to the self-compatible character phenotype after pollination, or the genotype containing only 1SNP polymorphic site is the genotype corresponding to the self-compatible character phenotype, the camellia oleifera plant is the self-compatible character phenotype; if the genotypes of the 2 SNP polymorphic sites are all the genotypes corresponding to the self-incompatible trait phenotype, the oil tea plant is in the self-incompatible trait phenotype.

9. The use according to claim 8,

after pollination, the genotypes of the 2 SNP polymorphic sites of the zygote are all the genotypes corresponding to the self-compatible character phenotype, and the self-compatibility of the genotypes is higher than that of the camellia oleifera plant which only contains 1SNP polymorphic site and is the genotype corresponding to the self-compatible character phenotype; the fruit setting rate of the plants corresponding to the individual fruit setting rate of the camellia oleifera and the genotypes of the 2 SNP polymorphic sites of the zygotes after pollination, which are the genotypes corresponding to the self-compatible character phenotype, is higher than the fruit setting rate of the plants which only contain 1SNP polymorphic site and have the genotypes corresponding to the self-compatible character phenotype.

10. The application of the camellia oleifera self-incompatibility associated gene CoMAPK9 is characterized in that the gene is specifically expressed in camellia oleifera pistil; the pollen germination and the pollen tube growth can be irreversibly inhibited, so that the self-incompatibility phenomenon of the camellia oleifera can be caused, and finally, fruits and seeds can not be normally formed on the camellia oleifera, so that the fruit setting rate of the camellia oleifera can be reduced; the cDNA sequence of the tea-oil tree self-incompatibility related gene CoMAPK9 is as follows: SEQ ID No.1, the genomic sequence is: SEQ ID NO. 2.