CN112126705A - MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in apple and application thereof in prediction of apple fruit acidity - Google Patents
MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in apple and application thereof in prediction of apple fruit acidity Download PDFInfo
- Publication number
- CN112126705A CN112126705A CN202011155395.4A CN202011155395A CN112126705A CN 112126705 A CN112126705 A CN 112126705A CN 202011155395 A CN202011155395 A CN 202011155395A CN 112126705 A CN112126705 A CN 112126705A
- Authority
- CN
- China
- Prior art keywords
- apple
- snp
- mdmyb44
- gene
- malic acid
- 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.)
- Pending
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
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biology, in particular to an MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in an apple and application thereof in prediction of apple fruit acidity. The nucleotide sequence of the MdMYB44 gene promoter is shown as SEQ IN NO: 1, a T/-SNP variation site exists at a position of-536 bp of an ATG upstream promoter region of a promoter initiation coder of the gene, and an A/G SNP variation site exists at a position of-1010 bp. The different combinations of the variation sites SNPA/G and SNPT/-can be applied to early prediction screening and molecular assisted breeding of the acidity character of apple fruits. The method can be used for early screening of the acidity of the apple fruits at the seedling stage of the apple, and can screen the acidity phenotype of offspring at the early stage of crossbreeding, thereby meeting the requirements of scientific research, fresh food, fruit juice processing and the like. Meanwhile, the method can also improve the screening efficiency of apple breeding and shorten the breeding period.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in an apple and application thereof in prediction of apple fruit acidity.
Background
The apple (Malus domestica Borkh.) is rich in nutrition and high in economic value, and is one of important economic crops in the world. Malic acid is the main organic acid in apple fruits and accounts for more than 90% of the total acid content. The organic acid participates in each metabolic process of the fruit, is one of important factors influencing the flavor quality and the processing quality of the fruit, and has important influence on the fresh food quality, the fruit juice processing quality, the market consumption and the like. Therefore, the breeding and demonstration popularization of new apple varieties are enhanced, the genetic improvement is carried out on the quality and the characters of the apples by means of genetics and molecular biology, various requirements of different areas, different markets and different crowds on the apples are met, the global development of the apple industry is enhanced, and the method is a necessary trend of future development of apple breeding.
However, the characteristics of the apple such as complex genetic background, long childhood period, incompatibility of self-crossing and the like seriously restrict the genetic breeding of the apple and the breeding of new varieties. Therefore, by developing the markers related to phenotypic characters and applying molecular markers to assist breeding, the defects of conventional crossbreeding are overcome, directional selective breeding is facilitated, the breeding period is shortened, and the early selection of apple breeding is laid.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides an SNP (single nucleotide polymorphism) variation site of a promoter of MdMYB44 gene in apple and application thereof in predicting acidity of apple fruit.
The technical scheme of the invention is as follows:
the main organic acid of apple pulp is malic acid, and the method of the invention applies MapQTL and BSA-seq method to carry out QTL (quantitative trait Locus) positioning on malic acid content of apple fruits. According to the resequencing SNP marker, the apple reference genome gene function annotation and the expression condition of the gene in the fruit, the gene function is combined, and finally the gene related to the apple pulp malic acid content, namely the transcription factor MdMYB44, is cloned. A T/-SNP variant site (meaning T deletion) exists at-536 bp of the upstream promoter region of the gene initial encoder ATG; an A/G SNP variation site exists at a position of-1010 bp. The MdMYB44 gene promoter sequence is shown in SEQ ID NO. 1. Genotype analysis shows that different combinations of the MdMYB44 promoter region variation site SNP T/-and SNP A/G are closely related to the malic acid content of fruits.
Specifically, the method comprises the following steps:
determining the malic acid content in mature apple pulp of a mapping population by High Performance Liquid Chromatography (HPLC), carrying out QTL positioning on the malic acid content of apple fruits by applying a MapQTL and BSA-seq method according to the malic acid content data, successfully positioning the acidic state in a 12.4-12.8Mbp interval of No. 8 chromosome, and determining MdMYB44 as a candidate gene by combining gene functions according to reference genome gene function annotation of apples and the expression condition of genes in fruits with the assistance of resequencing SNP marker data. Cloning and sequencing the gene, and having a T/-SNP variation site (-meaning T deletion) at-536 bp of the upstream promoter region of the ATG of the initial coding seed of the gene; an A/G SNP variation site exists at the-1010 bp position (figure 1). KASP genotypic analysis showed: the SNP A/G and SNP T/-site combinations have 9 genotypes in apple: g1, G2, G3, G4, G5, G6, G7, G8, G9 (fig. 4). The experiment carries out correlation analysis on the malic acid content and the 9 genotypes in 246 'Hongyu' × 'gold crown' F1 hybrid individuals, 123 'zisaiminzhu' × 'Hongfishi' F1 hybrid individuals and 135 apple germplasm resources, and the result shows that the 9 genotypes are obviously related to the malic acid content (P < 0.001). The differences in malic acid content among the genotyped G1-G9 individuals were found in 246 'jasper' (AG) × 'gold crown' (AG) F1 hybrid individuals (P ═ 6.3894E-36) (fig. 5), 123 'zisenzhu' (GG) × 'red fuji' (AG) F1 hybrid individuals (P ═ 5.5797E-08) (fig. 6), and 135 apple germplasm resources (P ═ 2.3328E-14) (fig. 7): g1> G4, G6> G2, G7, G8> G5, G9> G3. The results showed that SNP G and SNP-were closely associated with high acid, SNP A and SNP T were closely associated with low acid, and that the combination of variation sites SNP T/-and SNP A/G together affected malic acid content variation (FIG. 8). For example, 'zisenzhu' is of the G7 genotype, SNP G (high acid) combined with SNP T (low acid) appears as medium acid. Therefore, the marker combination can be used for early screening of fruit acidity in apple crossbreeding and apple molecular marker assisted breeding.
The invention achieves the following beneficial effects:
the invention provides an auxiliary screening marker for apple fruit acidity, which can be used for early screening of apple fruit acidity in the seedling stage of apples and screening the acidity phenotype of offspring in the early stage of crossbreeding, thereby meeting the requirements of various requirements (scientific research, fresh food and fruit juice processing). Meanwhile, in view of the characteristics of long childhood period, complicated genetic background and the like of the apples, the method can also improve the screening efficiency of apple breeding and shorten the breeding period.
The invention can identify the genotypes of various apple varieties by a KASP typing (microfluidic SNP chip detection system) method and preliminarily deduce the acid content of future fruits by the genotypes, and is simple, rapid, convenient, economic and effective.
Drawings
FIG. 1, the BSA-seq method is used to identify and screen candidate gene MdMYB 44.
Figure 2, KASP typing: SNP T/-typing results are presented.
Figure 3, KASP typing: and displaying the SNP A/G typing result.
FIG. 4, genotypes of different combinations of the MdMYB44 promoter region variant sites SNP T/-and SNP A/G (G1-G9).
FIG. 5, 246 strain ` Hongyu ` X ` golden crown ` F1 hybrid individuals SNP T/-and SNP A/G variation site analysis.
FIG. 6, 123 strains of ` Zisenzhu ` × ` Hongfush ` F1 individual SNP T/-and SNP A/G variation site analyses.
FIG. 7, SNP T/-and SNP A/G mutation site analysis in 135 parts of apple germplasm resources.
FIG. 8 is a schematic diagram showing the correlation between the genotype of different combinations of the SNP T/-and SNP A/G at the mutation site of the MdMYB44 promoter region and the acidity of fruits.
FIG. 9 is a chromatogram of a part of apple varieties when malic acid content in fruits is detected.
Figure 10, KASP typing: and (4) identifying the genotype.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. The main organic acid of apple pulp is malic acid, and generally, the malic acid accounts for more than 90% (mass percentage content) of the organic acid in the apple pulp.
Example 1 identification of molecular markers
Determining the malic acid content in the mature apple pulp of the mapping population by High Performance Liquid Chromatography (HPLC), carrying out QTL positioning by applying MapQTL and BSA-seq methods based on high density genetic maps according to the malic acid content data, successfully positioning the acidic shape in the 12.4-12.8Mbp interval of chromosome 8, and determining MdMYB44 as a candidate gene by combining gene functions according to the functional annotation of reference genome genes of the apple and the expression condition of the genes in the fruit with the assistance of resequencing SNP marker data.
Cloning and sequencing MdMYB44 gene promoter sequence
1. Extraction of apple genomic DNA
The experiment uses a CTAB method to extract the genome DNA of the young apple leaves, and comprises the following specific steps:
(1) adding the apple leaf sample ground into powder, and transferring the apple leaf sample to a 2mL centrifuge tube;
(2) adding 800uL of CTAB extracting solution preheated at 65 ℃ into a centrifugal tube containing blade powder, and adding 2% of beta-mercaptoethanol before preheating the CTAB extracting solution;
(3) shaking with vortex to mix the sample and extractive solution thoroughly, and turning over 3-5 times in 65 deg.C water bath for 30 min;
(4) adding equal volume of CI (chloroform: isoamyl alcohol 24:1), reversing, mixing for 3-5min, centrifuging at 12000rpm for 10min at 4 deg.C;
(5) taking the supernatant, and extracting for 1-2 times by using CI;
(6) taking the supernatant, putting the supernatant into a 1.5mL centrifuge tube, adding 2 times of anhydrous ethanol (after precooling, adding), gently mixing uniformly, and precipitating for 1-2h at-20 ℃;
(7) the white flocculent precipitate is gently transferred into 1ml of 75% ethanol by a gun head, and after being reversed and cleaned, the white flocculent precipitate is centrifuged at 6000rpm for 5 min;
(8) removing waste liquid, washing the precipitate with 75% ethanol once, and centrifuging at 6000rpm for 5 min;
(9) removing supernatant and residual liquid on the tube wall, and blow-drying in a fume hood for 5-10 min;
(10) adding 70-100 μ L of sterilized ddH2O dissolves the DNA. And adding 10 mu l of 3 thousandth RNase into the dissolved DNA solution, digesting for 0.5-1h at 37 ℃, and removing RNA mixed in the sample.
2. Cloning of genes
The promoter sequence of the MdMYB44 gene was cloned by PCR using the extracted DNA as a template. The PCR reaction system is shown in Table 1 below:
TABLE 1
The nucleotide sequence of the primer F is as follows: 5'-CCTCCCAACTAAACCATACCGT-3'
(SEQ IN NO：2)
The nucleotide sequence of the primer R is as follows: 5'-AGAAATCAATCCAAAATCTTC-3'
(SEQ IN NO：3)
The PCR reaction was started in a PCR amplification apparatus (Bio-Rad). After the reaction was completed, the amplified band was checked for the band of interest by electrophoresis on 1.0% to 2.0% agarose gel. The correct strip is then cut and recovered.
3, recovery and purification of PCR amplification product
The PCR product purification gel recovery and purification kit (Beijing holotype gold biology, Inc.) comprises the following specific operation steps:
(1) dissolving the cut gel block containing the target strip by using 3 times of sol buffer solution;
(2) transferring the sol solution which is completely dissolved and cooled into a centrifugal adsorption column, and centrifuging to discard the waste liquid;
(3) adding 650-fold 700 mu L WB rinsing liquid, centrifuging and discarding the waste liquid, repeating the steps once;
(4) centrifuging the empty column for 1-2min, and removing rinsing liquid;
(5) adding a proper amount (30-50 mu L) of elution buffer EB (Epstein-Barr) on a centrifugal adsorption column;
(6) centrifuging and removing the adsorption column to obtain a PCR purified product.
4. Target fragment ligation, transformation and sequencing
The target gene was ligated to the cloning vector pEASY-blunt, as shown in Table 2 below:
TABLE 2
Mixing, and connecting at 25 deg.C for 10-30 min. The specific connection time was operated according to the general gold company specifications. The ligation products were transformed into E.coli competent DH5a, spread on the corresponding resistance selection medium, and cultured in an inverted state at 37 ℃ for 12-16 h. Single clones that were PCR-identified as positive for colony sequencing were picked. Various sequencing analyses were performed by Shanghai Biotechnology services, Inc.
II, MdMYB44 gene promoter region site SNPT/-and SNPA/G analysis and genotype verification
A T/-SNP variant site (meaning T deletion) exists at-536 bp of the upstream promoter region of the gene initial encoder ATG; an A/G SNP variation site exists at the-1010 bp position (figure 1). KASP genotypic analysis showed: the SNP A/G and SNP T/-site combinations have 9 genotypes in apple: g1, G2, G3, G4, G5, G6, G7, G8, G9 (fig. 4). The experiment carries out correlation analysis on the malic acid content and the 9 genotypes in 246 'Hongyu' × 'gold crown' F1 hybrid individuals, 123 'zisaiminzhu' × 'Hongfishi' F1 hybrid individuals and 135 apple germplasm resources, and the result shows that the 9 genotypes are obviously related to the malic acid content (P < 0.001). The differences in malic acid content among the genotyped G1-G9 individuals were found in 246 'jasper' (AG) × 'gold crown' (AG) F1 hybrid individuals (P ═ 6.3894E-36) (fig. 5), 123 'zisenzhu' (GG) × 'red fuji' (AG) F1 hybrid individuals (P ═ 5.5797E-08) (fig. 6), and 135 apple germplasm resources (P ═ 2.3328E-14) (fig. 7): g1> G4, G6> G2, G7, G8> G5, G9> G3.
The results showed that SNP G and SNP-were closely associated with high acid, SNP A and SNP T were closely associated with low acid, and that the combination of variation sites SNP T/-and SNP A/G together affected malic acid content variation (FIG. 8). For example, 'zisenzhu' is of the G7 genotype, SNP G (high acid) combined with SNP T (low acid) appears as medium acid. Therefore, the marker combination can be used for early screening of fruit acidity in apple crossbreeding and apple molecular marker assisted breeding.
Example 2 identification of malic acid content traits in progeny of Cross population Using molecular markers
First, preparation of hybrid population
1. Pollen of the 'golden crown' apple is taken, pollination is carried out on the castrated 'Hongyu' apple, and hybrid seeds are harvested.
2. And (5) cultivating the hybrid seeds to obtain apple seedlings.
The planting place is Chang Ping district in Beijing city, and 1600 apple seedlings are obtained.
Secondly, measuring the malic acid content of the fruits
And (4) collecting mature fruits for identification when the seedlings start to bear fruits.
1. After the fruit on the apple plant is ripe, the peel and the kernel are removed, and the pulp is taken.
2. Weighing 5g (fresh weight) of the pulp obtained in the step 1, adding 10ml of double distilled water, grinding into homogenate, then carrying out water bath at 75 ℃ for 30min, then centrifuging at 12000rpm for 10min, collecting supernatant, and carrying out residual precipitation.
3. To the precipitate of step 2 was added 10ml of double distilled water, followed by a water bath at 75 ℃ for 30min, followed by centrifugation at 12000rpm for 10min, and the supernatant was collected.
4. And (3) combining the supernatant obtained in the step (2) and the supernatant obtained in the step (3), then using double distilled water to fix the volume to 25ml, then using a 0.45 mu m filter membrane for filtration, and collecting the filtrate.
5. And (4) taking the filtrate obtained in the step (4), and detecting the malic acid content by using a Waters 600 chromatograph and a Waters 2487 ultraviolet lamp detector.
The column used was a reversed C18 column, 4.6mm by 150 mm.
The mobile phase was a 0.01M aqueous solution of K2HPO4 adjusted to pH 2.6 with phosphoric acid.
The column temperature was 30 ℃. The flow rate of the mobile phase was 0.5 ml/min.
A malic acid standard product: DL-Malicacid (240176-50G, Sigma-ALDRICH, USA).
The peak position of the malic acid standard product is as follows: the retention time is 6.5-7.0 min.
The standard curve equation is: 2132881.2915 x-10004.2136; r2＝0.9988；
X represents the concentration (mg/mL) and Y represents the peak area.
Calculating the malic acid content of the pulp, wherein the unit is mg/g, mg is the unit of the malic acid content, and g is the unit of the fresh weight of the pulp.
For each apple plant, 5 ripe fruits randomly sampled were tested and the results averaged to determine the malic acid content of the fruit of the apple plant (fig. 9).
Third, genotype identification
1. Extracting the genome DNA of the leaves of the apple plants.
2. SNP T/-and SNP A/G are genotyped by KASP typing (microfluidic SNP chip detection).
And (2) taking the genomic DNA extracted in the step (1) as a template, extracting the genomic DNA of the material to be detected, taking 100bp base sequences of the upstream and downstream of the mutation site, and designing a specific primer to carry out detection work.
The specific primers are as follows:
KASP verified SNP A/G site primers:
Primer_AlleleFAM(F):5’-CCATGGAATTTTATGGAGTTTAATTGATTTA-3’(SEQ IN NO：4)
Primer_AlleleHEX(F):5’-CATGGAATTTTATGGAGTTTAATTGATTTG-3’(SEQ IN NO：5)
Primer_Common(R):5’-AGCATTCACAAATCTTATCTCTCCTTTTT-3’(SEQ IN NO：6)
KASP verified SNP T/-site primers:
Primer_AlleleFAM(F):5’-AATGGACGGTGATAAGAATTATCCACTT-3’(SEQ IN NO：7)
Primer_AlleleHEX(F):5’-TGGACGGTGATAAGAATTATCCACTG-3’
(SEQ IN NO：8)
Primer_Common(R):5’-GATGATCAATGGTGGAGCCCTCAA-3’
(SEQ IN NO：9)。
the method is based on the principle of competitive allele specific PCR, carries out accurate double allele judgment on SNPs and InDels on specific sites on a microfluidic chip, and realizes SNP/InDel typing, as shown in (figure 10). KASP typing detection was entrusted to Beijing Boo classical Biotechnology Limited.
(1) Designing primers, namely respectively designing two upstream PCR primers and 1 universal downstream primer aiming at two alleles of a target SNP locus.
(2) SNP identification, template denaturation, pairing of an upstream PCR primer and template DNA, pairing of a downstream primer and another strand, annealing and extension to complete the identification of SNP sites.
(3) Introducing a label sequence, combining and extending downstream primers, and amplifying a PCR product with the label sequence, thereby completing the introduction of the universal label sequence into the PCR product corresponding to the SNP.
(4) Fluorescence signals are generated, and the fluorescent probe is annealed to a newly synthesized complementary strand having no quenching group more by PCR amplification, separated from the quenching group, and fluoresced to generate fluorescence signals.
(5) And (6) data acquisition. After the PCR amplification program is operated, the chip is placed into a LuxScan-10K/D scanner for scanning to generate tif files, the tif files are converted into data signal values through software, and then typing is carried out through typing software SNPTyper.
(6) And (5) parting results. And (4) importing the original data of the scanned picture into SNPTyper software, and outputting the SNP typing result of each site.
3. KASP typing results.
(1) SNP T/-typing results presentation
The typing result of SNP T/-in the apple filial generation is obtained by KASP typing, -/-represents that the site is homozygous and deleted, T/-represents that the site is heterozygous and deleted, and TT represents that the site is homozygous and T (figure 3).
(2) SNP A/G typing results presentation
The typing result of SNP A/G in the apple hybridization offspring is obtained by KASP typing, GG represents that the site is homozygous G, AG represents that the site is heterozygous A/G, and AA represents that the site is homozygous A (figure 4).
Verification of malic acid content and genotype of filial generation of four and different populations
If the malic acid content of the fruit of an apple plant is more than 8mg/g, the apple plant is an extremely high-acid plant; if the malic acid content of the fruit of an apple plant is below 4mg/g, the apple plant is an extremely low-acid plant.
(1) Verification of malic acid content and genotype of progeny of 'Hongyu' × 'golden crown' hybrid population 246 plants were randomly selected from 1600 apple seedlings of 'Hongyu' × 'golden crown' hybrid population.
The 246 plants are divided into three groups according to genotypes, and the average value of the malic acid content of fruits in each group is counted, and the results are as follows:
the malic acid content of the fruit of the plant with G1 genotype, 63 plants, is 10.04 +/-3.50 mg/G.
The malic acid content of the fruits of the plants with the G2 genotype, 105 plants, is 7.82 +/-3.10 mg/G.
78 plants of G3 genotype, with malic acid content of 3.23 + -1.11 mg/G.
The results are shown (FIG. 5). The p-value of the 246 plant population is 6.3894E-36 and less than 0.001, which indicates that the combined genotype of SNP T/-and SNP A/G site polymorphism (G1, G2 and G3, and FIG. 5) found in example 1 shows very significant correlation with fruit malic acid content.
(2) Verification of malic acid content and genotype of progeny of 'Zisaiming bead' (GG) × 'Hongfisha' hybrid population
123 plants were randomly selected from 1200 apple seedlings of a ` Ziseneming' (GG) × ` Red Fuji ` hybrid population.
The 123 plants are divided into two groups according to genotypes, and the average value of the malic acid content of fruits in each group is counted, and the result is as follows:
the malic acid content of the fruits of the plants with the G4 genotype, 68 plants, is 7.30 +/-3.98 mg/G.
The malic acid content of the fruit of the plant with G5 genotype, 55 plants, is 3.76 +/-2.39 mg/G.
The results are shown (FIG. 6). The p-value of the 123 plant population is 5.5797E-08 and less than 0.001, which indicates that the combined genotype of SNP T/-and SNP A/G site polymorphism (G4, G5, FIG. 6) found in example 1 shows very significant correlation with fruit malic acid content.
Combining the above results, the results show that SNP G and SNP-are closely associated with high acid, and SNP A and SNP T are closely associated with low acid. The combination of SNP T/-and SNP A/G site polymorphism found in example 1 can be used for early screening of fruit acidity in apple cross breeding.
Example 3 identification of existing varieties by molecular markers
The following operations were performed on 135 existing apple varieties (natural population) respectively:
the method for detecting the malic acid content of the fruits is the same as the second step in the example 2. The chromatogram when the malic acid content of the fruit is detected by part of varieties is shown in figure 9.
Genotyping was performed in the same manner as in step three of example 2.
Dividing 135 varieties into three groups according to genotypes, and counting the average value of malic acid content of fruits in each group, wherein the results are as follows:
9 varieties of G1 genotype, wherein the malic acid content of the fruits is 8.59 +/-2.73 mg/G.
57 varieties of G2 genotype, the malic acid content of the fruit is 4.72 +/-1.97 mg/G.
11 varieties of G3 genotype, the malic acid content of the fruit is 2.59 +/-0.65 mg/G.
30G 4 genotype varieties, the malic acid content of the fruit is 7.23 +/-0.88 mg/G.
15 varieties of G5 genotype, the malic acid content of the fruits is 4.91 +/-1.73 mg/G.
1 variety of G6 genotype, with malic acid content of 8.27 mg/G.
9 varieties of G7 genotype, the malic acid content of the fruit is 5.30 +/-1.57 mg/G.
2 varieties of G8 genotype, the malic acid content of the fruits is 4.84 +/-0.45 mg/G.
1 variety of G9 genotype, with malic acid content of 4.17 mg/G.
The results are shown (FIG. 7). The p-value of 2.3328E-14, which is less than 0.001, among the natural population of 92 apples indicates that the combined genotype of SNP T/-and SNP A/G site polymorphism found in example 1 (G1-G9, FIG. 7) appears to be very significantly related to fruit malic acid content. Some results are shown in table 1.
The differences in malic acid content among individuals of genotypes G1-G9 were: g1> G4, G6> G2, G7, G8> G5, G9> G3. The results showed that SNP G and SNP-were closely associated with high acid, SNP A and SNP T were closely associated with low acid, and that the combination of variation sites SNP T/-and SNP A/G together affected malic acid content variation (FIG. 8). Therefore, through verification in apple planting resource groups, SNP T/-and SNP A/G locus polymorphism G1-G9 can be used for early screening of fruit acidity in apple crossbreeding and apple molecular marker-assisted breeding.
TABLE 3
The above-described embodiments of the present invention do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Sequence listing
<110> Qingdao agricultural university
<120> MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in apple and application thereof in prediction of apple fruit acidity
<130> 2020
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1563
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tattatttta agattttgag attattttgt ctcatcatat tatattagtg cgttattgat 60
aaaatgtgat tgctttgatt gcgacactca catatatccg ttatctaaaa ttctacattt 120
aattttaatt taattattaa agaaaatgag ttttttgttg ttgtaatatt taacaaaata 180
agttgaacga aaaagaaatt ctaataacac cacatttctg gtccttgtct aacttccaaa 240
acggtcgttc aacaattaaa tgccgcggtt ttagacgtca cacatctcaa ctgcttactg 300
ttacagtttc cacctctcac ttcccattta aagttactca aataaatatt tatcttgaat 360
gtttatgtta aaatttatat taaaaacaaa agaaaaaaag taattaaaaa catatagtga 420
aattataccc gctgtattca caaaaatact aaaattatac aaaataaaat ttaggaaggt 480
gtattcaatt gagaatttta gagattttga tggatttata aatccatgga attttatgga 540
gtttaattga tttrtagaga ttccatgtaa aattttgatt caattccctc aaaaagggag 600
agataagatt tgtgaatgct taaaatacac tacgaaatct ctcaaagtcc ctcgaatttc 660
ttaacttttt aaaattcttt aaaatcagtt tttaattgaa tacactcaga atgttataaa 720
ctgctttaaa atcctaagtc aatacactcg aatttctaag gattttaatt tactatctta 780
aaattatgat tgaatacact tgaaatttta gagaatcact tacaatccta attaaatacc 840
tctacattta ttaaaagaat taaaatatct caaaatctca tttgaataca accccttaaa 900
tttagatata aaaaacgaaa aacgatctta atcaactaat acaactcatg tttatttgtt 960
aaaaaaaaaa aaatacaact cacgtttaaa acatcagtaa aatggacggt gataagaatt 1020
atccactrga gcggttgagg gctccaccat tgatcatcat ctcgtagcct atcgttcaaa 1080
tttgagatga ctttgtaagg ttcttactac aaacagtagg gcaaagagta gccgaagcta 1140
atttgttttt cacttttatt ttaattttcc atttgttttt tttcacatat aaaaaaataa 1200
aagggcaaaa cagatggact gattatattt ttctgcgtgc aagttagagg gaaaaagaag 1260
cgacgcgtgt tagttataca gctgtatgct agtaactgcc tttgcctgga agaatcccta 1320
ctcgcaacgg cttagaccgg ttactcagac cggctatccc cggtcaccgc tatccccttc 1380
ctcgtatact atatatactc acccacacct gcatgaccct cttctctctc agaacacggt 1440
gaggctccgc ctcaaccacc gccggcgccg cttcctcctc ctccttgaac cctccagcct 1500
ctctcaagaa tctaatttat ctttagtatt tatttttaat ttgaagattt tggattgatt 1560
tct 1563
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cctcccaact aaaccatacc gt 22
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agaaatcaat ccaaaatctt c 21
<210> 4
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccatggaatt ttatggagtt taattgattt a 31
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
catggaattt tatggagttt aattgatttg 30
<210> 6
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
agcattcaca aatcttatct ctccttttt 29
<210> 7
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aatggacggt gataagaatt atccactt 28
<210> 8
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tggacggtga taagaattat ccactg 26
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gatgatcaat ggtggagccc tcaa 24
Claims (5)
1. An apple MdMYB44 gene promoter SNP variation site is characterized in that: the nucleotide sequence of the MdMYB44 gene promoter is shown as SEQ IN NO: 1, a T/-SNP variation site exists at a position of-536 bp of an ATG upstream promoter region of a promoter initiation coder of the gene, and an A/G SNP variation site exists at a position of-1010 bp.
2. The SNP mutation site of the MdMYB44 gene promoter in apple according to claim 1, wherein the SNP mutation site is characterized in that: the MdMYB44 gene promoter determines the malic acid content in mature apple pulp of a mapping population through high performance liquid chromatography, QTL positioning is carried out on the malic acid content of apple fruits by applying a MapQTL and BSA-seq method according to malic acid content data, the acidic state is successfully positioned in a 12.4-12.8Mbp interval of No. 8 chromosome, and MdMYB44 is determined to be a candidate gene by combining gene functions according to functional annotation of reference genome genes of the apples and the expression condition of the genes in the fruits through resequencing SNP marker data assistance.
3. The SNP mutation site of the MdMYB44 gene promoter in apple according to claim 1, wherein the SNP mutation site is characterized in that: the mutation site can be applied to early prediction screening and molecular assisted breeding of the acidity character of the apple fruit.
4. The SNP mutation site of the MdMYB44 gene promoter in apple according to claim 3, wherein: the different combinations of the variation sites SNPA/G and SNPT/-can be applied to early prediction screening and molecular assisted breeding of the acidity character of apple fruits.
5. The SNP mutation site of the MdMYB44 gene promoter in apple according to claim 4, wherein: the combination of the variation sites SNPA/G and SNP T/-sites has 9 genotypes in apple.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011155395.4A CN112126705A (en) | 2020-10-26 | 2020-10-26 | MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in apple and application thereof in prediction of apple fruit acidity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011155395.4A CN112126705A (en) | 2020-10-26 | 2020-10-26 | MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in apple and application thereof in prediction of apple fruit acidity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112126705A true CN112126705A (en) | 2020-12-25 |
Family
ID=73852779
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011155395.4A Pending CN112126705A (en) | 2020-10-26 | 2020-10-26 | MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in apple and application thereof in prediction of apple fruit acidity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112126705A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112662683A (en) * | 2021-01-26 | 2021-04-16 | 中国科学院武汉植物园 | Dicarboxylic acid transporter gene for controlling citric acid content in apple fruits and application thereof |
| CN112795574A (en) * | 2021-01-26 | 2021-05-14 | 中国科学院武汉植物园 | Sugar transporter gene for controlling sorbitol content of apple fruits and application thereof |
| CN116121259A (en) * | 2022-10-31 | 2023-05-16 | 西北农林科技大学 | Gene MdMYB21 for regulating and controlling apple fruit acid content and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728436A (en) * | 2017-11-16 | 2018-11-02 | 中国农业大学 | A kind of molecular labeling and its application in the screening of Apple malic acid content |
-
2020
- 2020-10-26 CN CN202011155395.4A patent/CN112126705A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728436A (en) * | 2017-11-16 | 2018-11-02 | 中国农业大学 | A kind of molecular labeling and its application in the screening of Apple malic acid content |
Non-Patent Citations (2)
| Title |
|---|
| DONGJIE JIA ET AL: "Apple fruit acidity is genetically diversified by natural variations in three hierarchical epistatic genes: MdSAUR37,MdPP2CH and MdALMTII", 《THE PLANT JOURNAL》 * |
| 王刚刚等: "苹果果实酸/低酸性状的SSR标记", 《西北农林科技大学学报(自然科学版)》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112662683A (en) * | 2021-01-26 | 2021-04-16 | 中国科学院武汉植物园 | Dicarboxylic acid transporter gene for controlling citric acid content in apple fruits and application thereof |
| CN112795574A (en) * | 2021-01-26 | 2021-05-14 | 中国科学院武汉植物园 | Sugar transporter gene for controlling sorbitol content of apple fruits and application thereof |
| CN112662683B (en) * | 2021-01-26 | 2022-07-29 | 中国科学院武汉植物园 | Dicarboxylic acid transporter gene for controlling citric acid content in apple fruits and application thereof |
| CN112795574B (en) * | 2021-01-26 | 2022-07-29 | 中国科学院武汉植物园 | Sugar transporter gene for controlling sorbitol content of apple fruits and application thereof |
| CN116121259A (en) * | 2022-10-31 | 2023-05-16 | 西北农林科技大学 | Gene MdMYB21 for regulating and controlling apple fruit acid content and application thereof |
| CN116121259B (en) * | 2022-10-31 | 2024-05-14 | 西北农林科技大学 | Gene MdMYB21 for regulating and controlling apple fruit acid content and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112126705A (en) | MdMYB44 gene promoter SNP (Single nucleotide polymorphism) variation site in apple and application thereof in prediction of apple fruit acidity | |
| CN108893551B (en) | Molecular marking method for detecting high oleic acid content of peanuts and application | |
| CN108728436B (en) | Molecular marker and application thereof in malic acid content screening of apple fruits | |
| CN105256035B (en) | Molecular labeling for differentiating Jinhua both ends crow and duroc genetic background and its application | |
| CN111500763B (en) | SNP molecular marker related to palmitoleic acid content in oil tea seed oil and application thereof | |
| CN110894542A (en) | Primer for identifying types of GS5 gene and GLW7 gene of rice and application of primer | |
| CN111500764B (en) | SNP molecular marker related to oleic acid and linoleic acid content in oil tea seed oil and application thereof | |
| CN110747288A (en) | Rice large grain gene function marker and application | |
| CN113637787B (en) | DNA fragment related to quality of single oil tea fruit and application thereof | |
| CN113637786B (en) | DNA fragment and SNP molecular marker related to linoleic acid content in oil tea seed oil and application thereof | |
| CN108728565B (en) | InDel marker for apple fruit acidity and application of InDel marker in molecular assisted selection | |
| CN112011640B (en) | KASP molecular marker, primer and application for identifying pH of watermelon fruit | |
| CN115948600B (en) | Grape powdery mildew resistance dCAPS molecular marker and application thereof | |
| CN111378781A (en) | Molecular marker primer for quickly and efficiently identifying salt-tolerant gene SKC1 of rice and application | |
| CN113584204B (en) | DNA fragment related to kernel yield of camellia seeds, SNP molecular marker closely linked with DNA fragment and application of DNA fragment | |
| 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 | |
| CN107287210B (en) | Rice appearance quality gene qAQ7 and molecular marking method and application thereof | |
| CN114457091B (en) | Gene Taxip affecting quality of wheat grains and application thereof | |
| CN113881801B (en) | Molecular marker composition closely linked with deep green stem traits of cucurbita moschata and application of molecular marker composition | |
| EP3772909A1 (en) | Genes associated with resistance to wheat yellow rust | |
| CN112680460B (en) | Male sterile gene ZmTGA9 and application thereof in creating male sterile line of corn | |
| 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 | |
| CN106480057B (en) | Recombinant nucleic acid fragment RecCR012083 and detection method thereof | |
| CN110106270B (en) | Molecular marker coseparated from melon yellow seed coat and application thereof | |
| CN111733278A (en) | Rice sodium and potassium ion absorption QTL (quantitative trait loci) linked SNP (Single nucleotide polymorphism) molecular marker and application thereof |
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 |