CN117165718B - Application of SNP molecular marker related to fructose content of papaya pulp - Google Patents
Application of SNP molecular marker related to fructose content of papaya pulp Download PDFInfo
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Abstract
The invention provides an application of SNP molecular markers related to the fructose content of papaya pulp, which belongs to the field of biotechnology, wherein the nucleotide sequence of the SNP molecular markers is shown as SEQ ID NO.1, the 458 th base from the 5' end of the sequence shown as SEQ ID NO.1 is an SNP locus, the SNP locus is positioned on the 26522070 th nucleotide of chromosome 2 of the papaya genome, and the SNP locus is C or T. The molecular marker is used for detecting papaya breeding materials, so that the fruit flesh fructose content of papaya plants can be accurately and efficiently predicted under the condition of not planting and waiting for flowering and fruiting of the papaya plants, and the selection efficiency of papaya breeding is greatly improved.
Description
Technical Field
The invention relates to an SNP molecular marker Cpa02g015040 of amino acid variation in a cds region of a gene related to high sugar content of papaya flesh and fruits, which comprises the following steps: 12 development and application of a kit, belonging to the field of biotechnology.
Background
Papaya (Carica papaya L.) is also called papaya, melon, papaya of papaya genus of papaya family, and is used as a fruit for treating tropical and subtropical evergreen soft woody large perennial herbaceous plants, evergreen soft woody small arbor. Papaya peel is smooth and beautiful, has thick and fine pulp, rich fragrance, rich juice, sweet and delicious taste and rich nutrition, has the elegant names of 'all-benefit fruits', 'Huang of fruits' and 'Wanshou melon', and is one of four fruits in the south of Ling. Papaya is rich in more than 17 amino acids, calcium, iron, papain, etc. Half a medium-sized papaya is sufficient for the whole day of adult vitamin C. The fruit of the guava claw not only can be used as fruits and vegetables, but also has various medicinal values. The milk of immature papaya can be used for extracting papain, is a superior raw material for preparing cosmetics, and has effects of caring skin and whitening. Papain contained in papaya has antitumor effect, antibacterial and antiparasitic effects, and blood pressure lowering effects; the papain can help protein digestion, and can be used for treating chronic dyspepsia, gastritis, etc., and papain also has anticoagulant effect.
Fructose contains 6 carbon atoms, also a monosaccharide, is an isomer of glucose, and is present in free form in large amounts in fruit juices and honey, and fructose can also combine with glucose to form sucrose. Pure fructose is colorless crystals, has a melting point of 103-105 ℃, is not easy to crystallize, is usually a viscous liquid, and is easy to dissolve in water, ethanol and diethyl ether. D-fructose is the sweet monosaccharide. The fructose content of papaya pulp is an important index for evaluating the quality of papaya, so that the fructose content of fruits is required to be measured for screening good plants after the plants bloom and bear fruits in the current selection of the index, and the selection time is long and the selection cost is high.
In traditional molecular breeding, farmers or breeders mainly select plant individuals with excellent characters and fix the excellent characters through hybridization or backcrossing. With the development of molecular biology, breeders have realized that the essence of selecting superior traits is to select individuals carrying superior genes or genotypes. Because the difficulty of determining that plants carry genes related to traits is high, breeders detect whether plants carry excellent genes or genotypes by detecting genetic markers linked with target genes, and plant phenotype character trends (marker-assisted breeding/molecular marker breeding) can be predicted without sowing and field character investigation. First generation molecular markers (RALP, AFLP, etc.) are gradually eliminated due to various problems and defects, and SSR and SNP molecular markers are currently the most commonly used molecular markers. The SNP molecular marker has the advantages of wide distribution and large quantity on the genome of an individual, easy genotyping (the binary nature of SNP), suitability for rapid and large-scale screening and the like. Along with the continuous reduction of sequencing cost of the progress of sequencing technology, more and more crops develop SNP markers through the sequencing technology. At present, SNP molecular markers related to the fructose content of papaya pulp are not developed or reported.
Disclosure of Invention
Aiming at the problems, the invention provides the SNP molecular marker related to the fructose content of papaya pulp and the application thereof, the fructose content of the selected papaya is expected in advance, the breeding period is shortened, and the breeding cost is reduced.
In order to achieve the above purpose, the technical scheme of the invention is as follows: papaya SNP molecular marker Cpa02g015040:12, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, the 458 th base from the 5' end of the sequence shown as SEQ ID NO.1 is a SNP locus, the SNP locus is positioned at 26522070 th nucleotide of chromosome 2 of the papaya genome, and the SNP locus is C or T.
Furthermore, papaya with the base of C at the SNP locus has high fructose content, and papaya with the base of T at the SNP locus has low fructose content.
Papaya SNP molecular marker Cpa02g015040 described above: use of a molecular marker of papaya for identifying high fructose content and low fructose content of papaya in assisted selective breeding.
A primer pair for detecting SNP molecular markers related to the fructose content of papaya pulp, wherein the upstream primer of the primer pair is designed according to the sequence upstream of the 26522070 nucleotide of the chromosome 2 of the papaya genome, and the downstream primer is designed according to the sequence downstream of the 26522070 nucleotide of the chromosome 2 of the papaya genome; the primer pair consists of two single-stranded DNA, and the upstream primer is 5'-TTGCAGACATGCCATGGTGG-3' as shown in SEQ ID NO. 2; the downstream primer is 5'-GCCATAAAAACGACGCTTGC-3', as shown in SEQ ID NO. 3.
Further, the application of the primer pair for detecting SNP molecular markers related to the fructose content of papaya pulp in papaya molecular marker assisted selection breeding is used for identifying the high fructose content and the low fructose content of papaya.
A kit for identifying the fructose content of papaya pulp, the kit comprising the primer pair described above.
The SNP molecular marker Cpa02g015040 of the amino acid variation in the cds region of the gene related to the high sugar content of papaya flesh and fruit: the development and application of the kit 12 have the beneficial effects that:
the method obtains the molecular marker which is extremely obviously related to the fructose content of papaya pulp, and the molecular marker is used for detecting the papaya breeding material, so that the high or low fructose content of papaya plants can be accurately and efficiently predicted under the condition of not planting and waiting for flowering and fruiting of the papaya plants, and the selection efficiency of papaya breeding is greatly improved;
the primer pair for detecting the SNP molecular marker related to the fructose content of papaya pulp has strong specificity, can accurately amplify and obtain a sequence containing the SNP molecular marker locus, and can assist in selective breeding and efficiently identify the high fructose content and the low fructose content of papaya by utilizing the primer pair to prepare a kit;
the SNP molecular marker Cpa02g015040 of the amino acid variation in the cds region of the gene related to the high sugar content of papaya flesh and fruit: the development and application of the kit can efficiently assist the breeding selection of papaya and have extremely high economic value.
Drawings
The invention will be described in further detail with reference to the drawings and the detailed description.
FIG. 1 is a Manhattan diagram of SNP markers highly correlated with the fructose content of papaya pulp in the present invention;
FIG. 2 is a Manhattan diagram and a linkage disequilibrium haplotype block diagram of a partial interval of a chromosome of Chr02 in which SNP markers extremely significantly related to the fructose content of papaya pulp are located, wherein arrow pointing sites in the interval are molecular markers screened by the invention;
FIG. 3 is a graph showing that the C allele is significantly correlated with the high fructose content in comparison with the difference in fructose content between different genotypes of SNP markers which are significantly correlated with the fructose content of papaya pulp in the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Papaya genome-wide SNP marker development
340 papaya collected from areas such as mexico, south africa, guangxi of China and Hainan of China are sown in Wenchang base of Tropical agricultural academy of sciences of China, soil fertility is medium, diseases and insect pests are avoided, phenotypic character data are planted and collected for many years in multiple points, and the phenotypic character data are used for subsequent analysis after being processed by Excel 2016.
And respectively taking 1-2 g of fresh and tender leaves from each papaya plant, grinding with liquid nitrogen, and extracting papaya material DNA according to a selected root plant genome DNA extraction kit (DP 305). Detecting the quality and concentration of the DNA sample by using an ultra-micro spectrophotometer and 1% agarose gel electrophoresis, and selecting the DNA sample with clear electrophoresis strip and no obvious protein residue in a gel hole, wherein the OD260/OD280 is 1.8-1.9, and the concentration is more than 100 ng/mu L, so as to carry out library construction and sequencing.
The DNA is randomly broken into fragments of about 300bp by adopting an ultrasonic breaking (or enzyme cutting) method, and the construction of a sequencing library is completed by end repair, addition of A at the 3' end, addition of a sequencing adapter pair, purification and PCR amplification of the DNA fragments. And sequencing the library through an illuminea platform after the library is qualified by quality inspection. After the sequencing data is taken off the machine, the quality control of the original data (Raw reads) is required according to a certain standard, and the filtering standard is as follows: (1) removing the sequence with the adapter, (2) removing the pair of sequences with the single-ended sequence having a nitrogen content of >10%, and (3) removing the pair of sequences with the low-quality number of bases exceeding 50%. And (5) removing the low-quality sequence, the linker sequence and the inaccurate sequence, and performing next sequence alignment on the clear reads. Reference genome selection the papaya susup genome (GenBank gca_ 021527605.1) newly released in 2022 was compared with the reference genome sequence using BWA-mem software, clear reads were sequenced using samtools software comparison, mutation detection was performed after PCR repeat removal by GATK4.0 software, and mutation site datasets were retained with statistical significance using hard criteria of QD > 2.0, qual > 30.0, fs < 60.0, mq > 40.0. The mutation sites were filtered again with MAF (minor allele frequency) > = 0.05 and mass (deletion rate) <= 0.2 as criteria to obtain a high quality collection of mutation sites.
(II) SNP molecular markers Cpa02g015040 of amino acid variation in the cds region of the gene related to the high sugar content of papaya flesh and fruit: 12 screening and use
Whole genome association analysis (Genome wide association studies, GWAS) finds a new strategy for genetic variation affecting complex traits by using genotype information at SNP sites in the genome as genetic markers. GWAS were first applied to human disease research, and they play an important role in genetic basis analysis of complex quantitative traits, and have absolute advantages in mining true major genes or key mutation sites. In 2010, popularization of second-generation sequencing technology and mass development of high-throughput molecular markers, a whole genome association analysis method is applied to research on complex characters of crops on a large scale, and complete use methods and experience are provided in rice, wheat, corn, rape and cotton. Carrying out genome-wide association analysis by combining phenotypic character data, and screening on chromosome 2 to obtain SNP locus Cpa02g015040:12 (-log) which is obviously related to the fructose content of papaya pulp 10 (P)>=6 ）。
The method for measuring the content of the fruit pulp fructose comprises the following steps: after 340 papaya varieties are planted, a representative plant is selected according to the phenotype characteristics of the varieties (generally, 340 papaya is planted in each plant, one plant with better growth vigor in plants with consistent phenotype characteristics is selected from the plants in each plant according to the external morphological characteristics of the plants to serve as the representative plant), mature fruits are selected on the representative plant, 5-10g pulp samples are taken, liquid nitrogen is frozen and then sent to a Chinese tropical agricultural academy of sciences variety test center, the fructose content in the pulp of the papaya varieties is measured by adopting a kit detection method, and the kit specifically adopts the product number produced by Shanghai pullus biotechnology Co as follows: kit of ZC-S0693, the kit instructions are as follows:
i kit description
Spectrophotometry 50 tubes/48 samples
Before formal measurement, 2-3 samples with larger expected differences are taken as prediction measurement significance:
fructose is one of the most common hexitoses, and is an isomer of glucose, which exists in free form in large quantities in fruit juices and honey, and can combine with glucose to produce sucrose. Fructose is the sweet monosaccharide and is widely applied to the production of foods, medicines and health products.
Fructose (FT) content kit description principle:
fructose reacts with resorcinol under acidic conditions to form a colored material with a characteristic absorption peak at 480 nm.
The required instruments and supplies:
visible spectrophotometer, water bath, adjustable pipettor, 1mL glass cuvette, mortar and distilled water
Composition and formulation of the reagents:
extracting solution: a 100ml×1 bottle of liquid, stored at 4 ℃;
reagent one: 1mL of 5mg/mL standard solution is multiplied by 1, and the mixture is stored at 4 ℃;
and (2) a reagent II: liquid 40ml×1 bottle, stored at 4deg.C;
and (3) a reagent III: 10ml of liquid is stored in a 1 bottle at 4 ℃;
and (3) a reagent IV: powder 0.5g×1 bottle, and storing at normal temperature.
II operation procedure
(1) Fructose extraction:
weighing 0.1g of sample, and grinding at normal temperature; adding 1mL of extracting solution, grinding properly, and transferring to a covered centrifuge tube quickly; placing in a water bath kettle at 80 ℃ for 10min (tightly covered to prevent water loss), oscillating for 3-5 times, cooling, centrifuging at normal temperature for 10min with 4000g, and taking the supernatant; adding a small amount (about 2 mg) of reagent IV, decolorizing at 80deg.C for 30min (tightening to prevent water loss); then 1mL of extract solution, 4000g of the extract solution is added, the mixture is centrifuged for 10min at normal temperature, and the supernatant is taken for measurement.
(2) Measuring the dosage of the operating reagent:
1. the visible spectrophotometer is preheated for more than 30min, the wavelength is regulated to 480nm, and the distilled water is zeroed.
2. Sample assay (the following reagents were added sequentially to a 1.5mL EP tube):
reagent (. Mu.L) blank tube standard tube measuring tube
Sample 100
Reagent one 100
Distilled water 100
Reagent two 700 700 700
Reagent III 200 200 200
Mixing, water bath reaction at 80deg.C for 10min (covering tightly to prevent water loss), cooling, measuring light absorption value at 480nm, counting into A blank tube, A standard tube, A measuring tube, and calculating ΔA measurement=A measuring tube-A blank tube, ΔA standard=A standard tube-A blank tube.
And (3) calculating the fructose content: 1. calculation according to protein concentration
Fructose content (mg/mg prot) =c × Δa assay ≡Δa standard × sample total V ≡ (cpr×sample total) =5× Δa assay ≡Δa standard ≡cpr
2. Calculated according to fresh weight of sample
Fructose content (mg/g fresh weight) =c × Δa measurement × Δa standard × sample total × V =10× Δa measurement × Δa standard ++w
Standard C, standard concentration, 5mg/mL; w: fresh weight of sample, g; cpr: sample protein concentration, mg/mL; sample V total: the volume of the extract was 2mL.
Note that: if the OD of the assay tube is greater than 0.6, the sample is diluted with the extract. FIG. 1 is a Manhattan plot obtained after GWAS analysis, with the x-axis representing the position of SNP markers on the chromosome and the y-axis representing-log 10 And (P) a value, wherein the larger the value is, the higher the correlation between the SNP locus and the trait is. The SNP markers thus selected are located at the arrow in FIG. 1. FIG. 2 is a Manhattan diagram and a linkage disequilibrium haplotype block diagram of a partial interval of a chromosome of Chr02 in which SNP markers extremely significantly relate to the fructose content of papaya pulp in the invention, wherein arrow pointing sites in the interval are molecular markers screened by the invention. 26 parts of the 340 parts of materials carry a T genotype at the SNP locus, the average value of fructose content is lower than that of 233 parts of the materials carrying a C genotype, fig. 3 is a graph of the genotype carried in the 340 parts of materials and the fructose content box, and the rest 81 parts of materials not shown in the graph contain other types of genotypes besides the C/T heterozygous genotype, and the fructose content is lower than that of the CC homozygous genotype.
SNP locus Cpa02g015040:12, which is significantly associated with the fructose content of papaya pulp, carries two genotypes C/T, where the C genotype is associated with high fructose content.
Designing primers for sequences near SNP loci which are obviously related to the fructose content of papaya pulp, taking the total DNA of papaya varieties as a template, amplifying to obtain target fragments through common PCR reaction, sequencing, and detecting genotypes carried by papaya at the SNP loci. By detecting which genotype (C or T) the papaya variety carries at the SNP locus, the high or low fructose content of the papaya variety is predicted under the condition of waiting for flowering and fruiting of plants without planting.
The upstream primer of the primer pair is designed according to the sequence upstream of the 26522070 nucleotide of the No.2 chromosome of the papaya genome, and the downstream primer is designed according to the sequence downstream of the 26522070 nucleotide of the No.2 chromosome of the papaya genome; the primer pair consists of two single-stranded DNA, and the upstream primer is 5'-TTGCAGACATGCCATGGTGG-3' as shown in SEQ ID NO. 2; the downstream primer is 5'-GCCATAAAAACGACGCTTGC-3', and is shown as SEQ ID NO. 3;
the PCR amplification reaction system is as follows:
2X Rapid Taq Master Mix 12.5.5. Mu.l, 10. Mu.M forward primer 1. Mu.l, 10. Mu.M reverse primer 1. Mu.l, DNA template 1. Mu.l, ddH2O 9.5. Mu.l. (Mix (amplification buffer) was purchased from Novain Biotech Co., ltd., primer commission Synthesis by Beijing Octomy Dingsheng Biotech Co., ltd.)
The reaction procedure of PCR amplification is 95 ℃ for 5min;95 ℃ for 30s, 62 ℃ for 30s,72 ℃ for 30s,35 cycles; and at 72℃for 5min. (after PCR reaction, target fragment sequencing entrusted China large gene company is completed.) the nucleotide sequence of the SNP molecular marker obtained by amplification of the primer is shown as SEQ ID NO.1, the 458 th base from the 5' end of the sequence shown as SEQ ID NO.1 is a SNP locus, the SNP locus is positioned at the 26522070 th base of chromosome 2 of papaya genome, and the SNP locus is C or T; papaya with the base of C at the SNP locus has high fructose content, and papaya with the base of T at the SNP locus has low fructose content;
to verify the practicability of the SNP molecular marker, 48 papaya strains (excluding 340 papaya strains for SNP marker development) are randomly selected in a papaya planting area of Wenchang base of biological technology of national academy of tropical agricultural sciences, and subjected to genotyping and papaya pulp fructose content investigation after sequencing; the primer used for sequencing and the amplification reaction system are the same as those described above. The sequencing results are shown in tables 1 and 2 below.
TABLE 1.48 papaya at Cpa02g015040:12 different genotypes and fructose content (mg/g)
TABLE 2 average fructose content (mg/g) of 48 papaya at Cpa02g015040:12 different genotypes
From tables 1 and 2, it can be seen that the SNP molecular markers of the invention have higher accuracy in identifying papaya with high fructose content and low fructose content, and the fructose content of papaya pulp of CC genotype is much higher than that of TT genotype.
In conclusion, the SNP molecular marker can accurately predict the fructose content of papaya pulp, is rapid and effective, can identify plants without waiting for flowering and fruiting in the seedling stage, greatly shortens the breeding period, and can be applied to mass production.
It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Claims (3)
1. Detecting papaya SNP molecular markers Cpa02g015040:12, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, the 458 th base from the 5' end of the sequence shown as SEQ ID NO.1 is an SNP locus, and the SNP locus is C or T;
the SNP molecular markers are used for identifying the high fructose content and the low fructose content of papaya pulp;
papaya pulp with genotype CC of SNP locus is high in fructose content, and papaya pulp with genotype TT of SNP locus is low in fructose content.
2. Use of a primer pair for detecting the SNP molecular markers as set forth in claim 1 for papaya pulp fructose content molecular marker-assisted selection breeding, characterized in that the primer pair is used for identifying high fructose content and low fructose content of papaya pulp; the primer pair consists of two single-stranded DNA, and the upstream primer is 5'-TTGCAGACATGCCATGGTGG-3' as shown in SEQ ID NO. 2; the downstream primer is 5'-GCCATAAAAACGACGCTTGC-3', and is shown as SEQ ID NO. 3; papaya pulp with CC genotype at the SNP molecular marker has high fructose content, and papaya pulp with TT genotype has low fructose content.
3. Use of a kit for identifying the SNP molecular markers of claim 1 in papaya pulp fructose content molecular marker-assisted selection breeding, characterized in that the kit comprises the primer pair of claim 2; papaya pulp with CC genotype at the SNP molecular marker has high fructose content, and papaya pulp with TT genotype has low fructose content.
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| Discovery of SNPs and InDels in papaya genotypes and its potential for marker assisted selection of fruit quality traits;Dieimes Bohry 等;《Scientific Reports》;第11卷;摘要 * |
| 番木瓜干旱胁迫相关CpDHN1基因的克隆与分析;郭静远 等;《热带作物学报》;第42卷(第12期);摘要 * |
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