CN113584203A - DNA fragment related to quality of single fruit of camellia oleifera, SNP molecular marker closely linked with same and application thereof - Google Patents
DNA fragment related to quality of single fruit of camellia oleifera, SNP molecular marker closely linked with same and application thereof Download PDFInfo
- Publication number
- CN113584203A CN113584203A CN202110837844.1A CN202110837844A CN113584203A CN 113584203 A CN113584203 A CN 113584203A CN 202110837844 A CN202110837844 A CN 202110837844A CN 113584203 A CN113584203 A CN 113584203A
- Authority
- CN
- China
- Prior art keywords
- fruit
- quality
- oil
- molecular marker
- tea
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of molecular markers and genetic breeding of oil tea, in particular to a DNA fragment related to the quality of single fruit of oil tea, a closely linked SNP molecular marker and application thereof. The invention provides a DNA fragment related to the quality of single fruit of camellia oleifera abel, which is wsf.10-1 of a linkage group No. 10 of a camellia oleifera abel linkage map, and has a confidence interval of 95.61cM-95.62 cM. The invention also provides an SNP molecular marker chr10-122184778 closely linked with the quality locus of the single fruit of the oil-tea camellia, which is a nucleotide sequence with the polymorphism A/G at the 174 th site of the sequence shown as SEQ ID NO.1 and can explain the phenotypic variance of the single fruit quality of the oil-tea camellia of 55.2 percent. By detecting the SNP molecular marker, the quality phenotype of the single fruit of the camellia oleifera can be identified and screened in an auxiliary manner in the seedling stage, the production cost is greatly saved, and the efficiency of selection breeding of the fruit size of the camellia oleifera is effectively improved.
Description
Technical Field
The invention relates to the technical field of molecular markers and genetic breeding of oil tea, in particular to a DNA fragment related to the quality of single fruit of oil tea, a closely linked SNP molecular marker and application thereof.
Background
Tea-oil tree (Camellia oleifera) is widely planted in subtropical regions of china as one of four Chinese oil plants (rape, peanut, soybean and tea-oil tree). The content of unsaturated fatty acid in the oil-tea camellia seed oil is more than 90%, and the oil-tea camellia seed oil is rich in nutrient components such as squalene, vitamin E and the like, is high-quality edible oil and is called as 'east olive oil'. The breeding of high-yield (oil) quality oil tea fine varieties is always the basis and guarantee of healthy development of the oil tea industry. The oil tea breeding work mainly by means of selection and cross breeding has been developed greatly, but the conventional breeding period of the oil tea is long, the new variety breeding is slow, and the improved variety breeding speed cannot meet the requirement of industrial development, which becomes one of the important factors for limiting the development of the oil tea industry.
Compared with the traditional breeding technology, the molecular marker assisted breeding can be selected from the seedling stage, the breeding period is greatly shortened, and the advantages of the molecular marker assisted breeding on economic forests mainly aiming at fruits are particularly obvious. The molecular marker assisted breeding cannot separate effective molecular markers, so that the development of the molecular markers related to the fruit size, the oil-tea camellia fruit yield and the oil quality phenotype has important significance for molecular marker assisted breeding of the oil-tea camellia oil yield and quality and genetic improvement of related characters.
The oil tea yield (oil) per unit area is directly determined by indexes such as fruit size, fruit yield, kernel oil content and the like, and meanwhile, the breeding of new varieties of large fruit types has important significance for improving picking efficiency and reducing labor cost. Therefore, the development of the breeding research of the new variety of the big fruit type of the oil tea is an important way for improving the yield of the oil tea and reducing the labor cost, and has very important significance for the promotion and the healthy development of the oil tea industry.
Disclosure of Invention
One of the purposes of the invention is to provide a DNA fragment (gene locus) related to the quality of single fruit of oil-tea camellia and an SNP molecular marker closely linked with the DNA fragment. The invention also aims to provide application of the molecular marker in phenotype identification and breeding of the camellia oleifera large fruit type germplasm.
The development of the DNA fragments related to the quality of the single fruit of the oil-tea camellia and the molecular markers closely linked with the DNA fragments is realized by carrying out high-density genetic linkage map construction and QTL positioning of the quality characters of the single fruit based on the established F1 generation hybrid population of the oil-tea camellia. The simplified genome sequence of the camellia oleifera is a region for the marker development of the invention.
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 Chang Ling No. 53 (20.38g) and Chang Ling No. 81 (14.31g) with remarkable fruit size difference, and camellia oleifera F1 generation hybrid population with widely separated fruit sizes is created.
(2) Fully mature fruits of 180 individuals of the hybrid population are collected, and the weight of each fruit is determined.
(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), performing double enzyme digestion by using EcoRI and NlaIII (Hin1II)) to construct a simplified genome (ddRAD) sequencing library for each sample, and performing 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 a major gene site wsf.10-1 of the quality of the single fruit of the camellia oleifera located on the No. 10 linkage group (LG10), wherein the contribution rate of the gene site to the quality of the single fruit of the camellia oleifera is 55.2%, the SNP marker tightly linked with the gene site is chr10-122184778, and the genotype of the gene site is A/G (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 quality of single fruit of camellia oleifera, which is wsf.10-1 of linkage group No. 10 of camellia oleifera linkage map, and has a confidence interval of 95.61cM-95.62 cM.
The contribution rate of the DNA fragment to the quality phenotype of the single fruit of the camellia oleifera abel is 55.2%, 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 chr10-122184778, the chr10-122184778 is located at position 122184778bp of No. 10 chromosome of the oil tea genome, and the polymorphism is A/G.
In a second aspect, the invention provides an SNP molecular marker closely linked with the single fruit mass locus of the oil tea, which comprises an SNP molecular marker chr10-122184778, wherein the SNP molecular marker is located at 122184778bp position of the No. 10 chromosome of the oil tea genome, and the polymorphism is A/G.
The SNP molecular marker is closely linked with a single fruit quality major gene site wsf.10-1 of the oil tea.
Specifically, the SNP molecular marker chr10-122184778 contains a nucleotide sequence with polymorphism A/G at position 174 of the sequence shown in SEQ ID NO. 1.
Preferably, the nucleotide sequence of the SNP molecular marker chr10-122184778 is shown as SEQ ID NO.1, the SNP site is located at the 174 th site of the sequence shown as SEQ ID NO.1, and the polymorphism is A/G.
Further, the SNP molecular marker chr10-122184778 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’-GGACGGTGAGGATGAGACTAG-3’;
SEQ ID NO.3:5’-AATGGCCCTTGCCTTGGTCA-3’。
In the SNP molecular marker chr10-122184778, the genotype of a site with the polymorphism is A/A, which corresponds to high single fruit quality; genotype is A/G, corresponding to low single fruit quality.
The mass of the single fruit is the mass (weight) of the completely mature fresh fruit, and the unit is gram.
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 single fruit quality of camellia oleifera or the SNP molecular marker or the primer or the reagent or the kit:
(1) the application in identifying the high-low phenotype of the single fruit of the camellia oleifera or the size phenotype of the fruit of the camellia oleifera;
(2) the application in the quality improvement of the single oil tea fruit or the molecular marker-assisted breeding of the single oil tea fruit is realized;
(3) the application in early prediction of the quality of single fruit of the camellia oleifera or the size of the fruit;
(4) the application in screening large fruit type oil tea.
In a fifth aspect, the invention provides a method for identifying high and low phenotype of single fruit of oil-tea camellia or screening large fruit type oil-tea camellia, 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 in the PCR amplification product, and judging the high-low phenotype of the single fruit 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 PCR 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 high-low phenotype of the single fruit quality of the oil tea to be identified in the step (3) comprises the following steps:
if the genotype of the polymorphic site of the SNP molecular marker chr10-122184778 is A/A, the oil tea to be identified is high-single-fruit-quality oil tea; and if the genotype is A/G, determining that the oil tea to be identified is low single-fruit quality oil tea.
The invention provides a method for identifying high-single-fruit-quality oil tea, 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 high single fruit quality oil tea or not according to the genotype.
If the genotype of the SNP molecular marker chr10-122184778 at the polymorphic site is A/A, the quality of the single fruit of the oil tea to be identified is high; and if the genotype is A/G, determining that the oil tea to be identified has low single fruit quality.
The invention has the beneficial effects that: the invention provides a main effect gene locus of single fruit quality of oil-tea camellia, the contribution rate of the main effect gene locus to the single fruit quality phenotype is 55.2%, and SNP loci closely linked with the gene loci are developed. The SNP marker is used for carrying out auxiliary selection on hybrid F1 generation individuals of Changlin No. 53 multiplied by Changlin No. 81, and the result shows that in individuals of which the locus is a genotype with high single fruit quality, 86.42 percent of individuals have single fruit quality higher than the average mass value (19.21g) of the population single fruit; among individuals with low single fruit quality genotypes, 85.71% of individuals have a single fruit quality lower than the population mean (19.21g), indicating that this marker is practical and effective for use in assisted selection of the camellia oleifera single fruit quality phenotype.
In the conventional selection breeding of the camellia oleifera, the identification of the fruit size and character requires 5-6 years of 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 oil tea breeding, the molecular marker and the detection method thereof can be selected to identify the oil tea with high single fruit quality for breeding, so that the selection efficiency of the oil tea breeding can be improved, and the breeding process can be accelerated.
Drawings
FIG. 1 is a schematic diagram showing the position of the site wsf.10-1 of the primary gene for the quality of Camellia Oleifera Linne 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 and Property measurement of Single fruit quality segregating population of Camellia oleifera
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. After the fruits of 180 generations are completely ripe (5% of fruits are cracked), collecting 20-30 fresh fruits per sample, measuring the mass of the fresh fruits, and calculating the average single fruit mass. The result shows that the quality of single fruit 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) 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) rapidly separating the mixed data of 12 samples according to the barcode on the sequence 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 (RK, Jain M. NGS QC Toolkit: APlatform for Quality Control of Next-Generation Sequencing Data [ M ]. Springer US, 2015.).
(2) SNP identification and filtration: the high quality reads 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 major gene loci and linkage SNP loci of single fruit quality of camellia oleifera are mined by performing data analysis by using QTL Isimulping software and performing QTL positioning of single fruit quality by using a complete interval mapping method (ICIM). 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 major gene site wsf.10-1 of the quality of the single fruit of the oil-tea camellia is positioned on the chromosome 10 of the oil-tea camellia, the contribution rate is 55.2 percent, and the SNP molecular marker closely linked with the major gene site is chr10-122184778 (table 1, figure 1).
Example 4 application of Single fruit quality major gene locus and linkage SNP molecular marker in Camellia oleifera breeding
1. 158 individuals are randomly selected as materials from the family group of the Camellia oleifera hybrid F1 generation of Changlin No. 53X Changlin No. 81, and the tender leaves are collected to extract the 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. performing gel detection, purification, recovery, sequencing and genotyping on PCR amplification products
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 chr10-122184778 loci of all individuals were identified separately. Comparing the relationship between the genotype of each site and the quality of single fruit, if the genotype of the site is A/A, the oil tea individuals are the oil tea with high single fruit quality; if the genotype is A/G, the oil tea individual is low single-fruit quality oil tea.
5. 158 individuals of F1 generation and the parent fully mature fruit were collected and their individual fruit quality was determined (see example 1). The results show (table 3) that, in the individual plants with chr10-122184778 loci of high single fruit quality genotypes, 86.42 percent of individuals have higher fruit quality than the average mass value (19.21g) of the single fruit of the population; of the individuals with genotypes of low single fruit mass, 85.71% had a single fruit mass below the population mean (19.21 g). The molecular marker chr10-122184778 is practical and effective when being used for auxiliary selection of single fruit quality, can be used for early identification or auxiliary identification of single fruit quality phenotype, can greatly save production cost, improves selection efficiency and accelerates the oil tea breeding process.
TABLE 3 Single fruit quality and genotype data for female parent Changlin No. 53, male parent Changlin No. 81 and F1 individuals
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> subtropical forestry research institute of China forestry science research institute
<120> DNA fragment related to single fruit quality of camellia oleifera, SNP molecular marker closely linked with same and application thereof
<130> KHP211117128.4
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 359
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggacggtgag gatgagacta gccccactgc cttctttacg gaatgatcca tcgacattga 60
gggtccagga gtggggatct tctgccttag tgttgaattg cttgggcgga gggagcgtgg 120
ttggggtggg attggtgaat tccaagataa aatcagcgac agcctgtcat ttgatcactg 180
tttgaggtcg gtaagagatt tcaaattagt tgagctcgac agaccattgc ataagtttgt 240
cggacaactc tggttgttgc attaccttct ttaaaggttg gtcagtgagg accactatgg 300
agtgagcttg gaaataaggt tggagttgtc ttgaggctat gaccaaggca agggccatt 359
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggacggtgag gatgagacta g 21
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aatggccctt gccttggtca 20
Claims (10)
1. The DNA fragment related to the quality of the single fruit of the oil-tea camellia is characterized in that the DNA fragment is wsf.10-1 of a linkage group No. 10 of the oil-tea camellia linkage map, and the confidence interval is 95.61cM-95.62 cM.
2. The SNP molecular marker closely linked with the single fruit mass locus of the oil-tea camellia is characterized by comprising a SNP molecular marker chr10_122184778, wherein chr10_122184778 contains a nucleotide sequence with the polymorphism of A/G at the 174 th site of the sequence shown in SEQ ID NO. 1.
3. The SNP molecular marker according to claim 2, wherein the primer set having the sequence shown in SEQ ID No.2-3 is obtained by PCR amplification using Camellia oleifera genomic DNA as a template.
4. The SNP molecular marker according to claim 2 or 3, wherein the genotype of the site with the polymorphism in the SNP molecular marker chr10-122184778 is A/A, corresponding to high single fruit quality; genotype is A/G, corresponding to low single fruit quality.
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 quality of the single fruit of Camellia oleifera as claimed in claim 1, or the SNP molecular marker as claimed in any one of claims 2 to 4, or the primer as claimed in claim 5 or 6, or the reagent or kit as claimed in claim 7:
(1) the application in identifying the high-low phenotype or the fruit size phenotype of the single fruit of the camellia oleifera;
(2) the application in the quality improvement of the single oil tea fruit or the molecular marker-assisted breeding of the single oil tea fruit is realized;
(3) the application in early prediction of the quality of single fruit of the camellia oleifera or the size of the fruit;
(4) the application in screening large fruit type oil tea.
9. A method for identifying the high-low phenotype of single fruit of oil-tea camellia or screening large fruit type oil-tea camellia 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 high-low phenotype of the single fruit of the oil tea 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 high-low phenotype of the single fruit of the oil-tea camellia to be identified comprises the following steps:
if the genotype of the polymorphic site of the SNP molecular marker chr10-122184778 is A/A, the oil tea to be identified is high-single-fruit-quality oil tea; and if the genotype is A/G, determining that the oil tea to be identified is low single-fruit quality oil tea.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110837844.1A CN113584203B (en) | 2021-07-23 | 2021-07-23 | DNA fragment related to single fruit quality of camellia oleifera, SNP molecular marker closely linked with DNA fragment and application of DNA fragment |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110837844.1A CN113584203B (en) | 2021-07-23 | 2021-07-23 | DNA fragment related to single fruit quality of camellia oleifera, SNP molecular marker closely linked with DNA fragment and application of DNA fragment |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113584203A true CN113584203A (en) | 2021-11-02 |
CN113584203B CN113584203B (en) | 2023-06-23 |
Family
ID=78249316
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110837844.1A Active CN113584203B (en) | 2021-07-23 | 2021-07-23 | DNA fragment related to single fruit quality of camellia oleifera, SNP molecular marker closely linked with DNA fragment and application of DNA fragment |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113584203B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114410668A (en) * | 2022-01-29 | 2022-04-29 | 中南林业科技大学 | Camellia oleifera self-incompatible gene S-RNase, SNP locus and application |
Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060135758A1 (en) * | 2004-08-31 | 2006-06-22 | Kunsheng Wu | Soybean polymorphisms and methods of genotyping |
CN102321768A (en) * | 2011-10-21 | 2012-01-18 | 南京林业大学 | Method for identifying camellia oleifera cultivar and special primer and kit thereof |
CN103233065A (en) * | 2013-04-10 | 2013-08-07 | 浙江省林业科学研究院 | Molecular specific marker primers for No. 4 and No.32 of an improved variety Changlin of Camellia oleifera and an identification method |
JP2015006197A (en) * | 2014-09-03 | 2015-01-15 | 独立行政法人農業・食品産業技術総合研究機構 | Single nucleotide repeat polymorphism analyzing method and single nucleotide polymorphism analyzing method |
CN106834477A (en) * | 2017-04-17 | 2017-06-13 | 中国林业科学研究院亚热带林业研究所 | The method for identifying oil content oil tea high |
CN106868132A (en) * | 2017-02-23 | 2017-06-20 | 中国林业科学研究院亚热带林业研究所 | Palmitic acid, oleic acid, linolenic acid content are related in a kind of grease to Seed of Camellia oleifera SNP marker and its application |
CN107164072A (en) * | 2016-03-08 | 2017-09-15 | 北京中天金谷科技股份有限公司 | Camellia oleifera fruit seed peel Intelligent treatment technique |
CN109311952A (en) * | 2015-06-15 | 2019-02-05 | 马来西亚棕榈油委员会 | For controlling the allele in the domain MADS-BOX of palm shell phenotype |
CN111500763A (en) * | 2020-05-26 | 2020-08-07 | 中国林业科学研究院亚热带林业研究所 | SNP molecular marker related to palmitoleic acid content in oil tea seed oil and application thereof |
CN111534632A (en) * | 2020-05-29 | 2020-08-14 | 中国林业科学研究院亚热带林业研究所 | 3 SNP molecular markers related to oil content of oil-tea camellia kernel and application thereof |
CN111534631A (en) * | 2020-05-29 | 2020-08-14 | 中国林业科学研究院亚热带林业研究所 | 2 SNP molecular markers related to oil content of oil-tea camellia kernel and application thereof |
CN111607661A (en) * | 2020-06-29 | 2020-09-01 | 北部湾大学 | Molecular marker primer group based on Camellia oleifera transcriptome hAT transposon and application thereof |
CN111705152A (en) * | 2020-05-26 | 2020-09-25 | 中国林业科学研究院亚热带林业研究所 | SNP molecular marker related to stearic acid content in camellia seed oil and application thereof |
CN112889506A (en) * | 2021-03-16 | 2021-06-04 | 广西壮族自治区林业科学研究院 | Method for grafting new variety of red-feather camellia oleifera plantlet by adopting distant variety as stock |
-
2021
- 2021-07-23 CN CN202110837844.1A patent/CN113584203B/en active Active
Patent Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060135758A1 (en) * | 2004-08-31 | 2006-06-22 | Kunsheng Wu | Soybean polymorphisms and methods of genotyping |
CN102321768A (en) * | 2011-10-21 | 2012-01-18 | 南京林业大学 | Method for identifying camellia oleifera cultivar and special primer and kit thereof |
CN103233065A (en) * | 2013-04-10 | 2013-08-07 | 浙江省林业科学研究院 | Molecular specific marker primers for No. 4 and No.32 of an improved variety Changlin of Camellia oleifera and an identification method |
JP2015006197A (en) * | 2014-09-03 | 2015-01-15 | 独立行政法人農業・食品産業技術総合研究機構 | Single nucleotide repeat polymorphism analyzing method and single nucleotide polymorphism analyzing method |
CN109311952A (en) * | 2015-06-15 | 2019-02-05 | 马来西亚棕榈油委员会 | For controlling the allele in the domain MADS-BOX of palm shell phenotype |
CN107164072A (en) * | 2016-03-08 | 2017-09-15 | 北京中天金谷科技股份有限公司 | Camellia oleifera fruit seed peel Intelligent treatment technique |
CN106868132A (en) * | 2017-02-23 | 2017-06-20 | 中国林业科学研究院亚热带林业研究所 | Palmitic acid, oleic acid, linolenic acid content are related in a kind of grease to Seed of Camellia oleifera SNP marker and its application |
CN106834477A (en) * | 2017-04-17 | 2017-06-13 | 中国林业科学研究院亚热带林业研究所 | The method for identifying oil content oil tea high |
CN111500763A (en) * | 2020-05-26 | 2020-08-07 | 中国林业科学研究院亚热带林业研究所 | SNP molecular marker related to palmitoleic acid content in oil tea seed oil and application thereof |
CN111705152A (en) * | 2020-05-26 | 2020-09-25 | 中国林业科学研究院亚热带林业研究所 | SNP molecular marker related to stearic acid content in camellia seed oil and application thereof |
CN111534632A (en) * | 2020-05-29 | 2020-08-14 | 中国林业科学研究院亚热带林业研究所 | 3 SNP molecular markers related to oil content of oil-tea camellia kernel and application thereof |
CN111534631A (en) * | 2020-05-29 | 2020-08-14 | 中国林业科学研究院亚热带林业研究所 | 2 SNP molecular markers related to oil content of oil-tea camellia kernel and application thereof |
CN111607661A (en) * | 2020-06-29 | 2020-09-01 | 北部湾大学 | Molecular marker primer group based on Camellia oleifera transcriptome hAT transposon and application thereof |
CN112889506A (en) * | 2021-03-16 | 2021-06-04 | 广西壮族自治区林业科学研究院 | Method for grafting new variety of red-feather camellia oleifera plantlet by adopting distant variety as stock |
Non-Patent Citations (5)
Title |
---|
PING LIN等: "Association Genetics Identifies Single Nucleotide Polymorphisms Related to Kernel Oil Content and Quality in Camellia oleifera", J. AGRIC. FOOD CHEM., vol. 67, pages 2547 * |
RUI YAN等: "SNP discovery of Camelliaoleifera based on RNA-seq and its application for identification of genetic relationships and locus for oil content among different cultivars", THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY, pages 1 - 17 * |
刘凯;王东雪;江泽鹏;莫健芳;曾雯;张乃燕;梁国校;: "基于SLAF-seq技术的油茶SNP位点开发及杂交后代早期鉴定", 广西林业科学, no. 01, pages 17 - 21 * |
林萍等: "普通油茶两个 Δ-12 脂肪酸脱氢酶基因序列特征及表达模式研究", 林业科学研究, vol. 29, pages 743 - 751 * |
赵松子;赵攀;: "油茶的驯化及面临的问题", 南方林业科学, no. 06, pages 54 - 58 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114410668A (en) * | 2022-01-29 | 2022-04-29 | 中南林业科技大学 | Camellia oleifera self-incompatible gene S-RNase, SNP locus and application |
CN114410668B (en) * | 2022-01-29 | 2024-06-21 | 中南林业科技大学 | Camellia oleifera self-incompatibility gene S-RNase, SNP locus and application |
Also Published As
Publication number | Publication date |
---|---|
CN113584203B (en) | 2023-06-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106868132B (en) | SNP molecular marker related to contents of palmitic acid, oleic acid and linolenic acid in oil-tea camellia seed oil and application thereof | |
US20170233831A1 (en) | Sidt1 GENE CONTROLLING DETERMINATE GROWTH HABIT IN SESAME AND SNP MOLECULAR MARKER THEREOF | |
CN106755528B (en) | SNP molecular marker related to oil content of camellia oleifera seeds and application thereof | |
CN106834477B (en) | Method for identifying oil tea with high oil content | |
CN111500763B (en) | SNP molecular marker related to palmitoleic acid content in oil tea seed oil and application thereof | |
CN111455090B (en) | Key SNP molecular marker related to content of linolenic acid in oil-tea seed kernel oil and application thereof | |
CN111705152B (en) | SNP molecular marker related to stearic acid content in camellia seed oil and application thereof | |
CN106367496B (en) | kiwi species associated specific single nucleotide molecular marker, detection primer group and application thereof | |
CN114672587A (en) | SNP molecular marker related to fructose content of papaya fruit, amplification primer, detection kit and application thereof | |
CN113637787B (en) | DNA fragment related to quality of single oil tea fruit and application thereof | |
CN111534632B (en) | 3 SNP molecular markers related to oil content of oil-tea camellia kernel and application thereof | |
CN113637786B (en) | DNA fragment and SNP molecular marker related to linoleic acid content in oil tea seed oil and application thereof | |
CN111500764B (en) | SNP molecular marker related to oleic acid and linoleic acid content in oil tea seed oil and application thereof | |
CN112126705A (en) | MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in apple and application thereof in prediction of apple fruit acidity | |
CN110894542A (en) | Primer for identifying types of GS5 gene and GLW7 gene of rice and application of primer | |
CN113584203B (en) | DNA fragment related to single fruit quality of camellia oleifera, SNP molecular marker closely linked with DNA fragment and application of DNA fragment | |
CN113430298B (en) | DNA fragment related to content of linolenic acid in camellia seed oil, SNP molecular marker closely linked with DNA fragment and application of SNP molecular marker | |
CN113584204B (en) | DNA fragment related to kernel yield of camellia seeds, SNP molecular marker closely linked with DNA fragment and application of DNA fragment | |
CN111534631B (en) | 2 SNP molecular markers related to oil content of oil-tea camellia kernel and application thereof | |
CN111534630B (en) | SNP molecular marker related to oil content of camellia seed kernels and application thereof | |
CN113604596A (en) | KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof | |
CN111378781A (en) | Molecular marker primer for quickly and efficiently identifying salt-tolerant gene SKC1 of rice and application | |
CN115896331A (en) | SNP molecular marker and dCAPS molecular marker closely linked with eggplant peel color character and application thereof | |
CN113430297B (en) | DNA fragment related to content of palmitic acid in oil-tea camellia seed oil, SNP molecular marker closely linked with DNA fragment and application of SNP molecular marker | |
CN106399538B (en) | Application of SNP (single nucleotide polymorphism) marker closely linked with peach tree dwarfing gene |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |