CN117512202B - SNP molecular marker remarkably related to papaya fruit chamber diameter, method and application - Google Patents
SNP molecular marker remarkably related to papaya fruit chamber diameter, method and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a SNP molecular marker, a method and application thereof, wherein the SNP molecular marker is Cpa03G 018680-337, the molecular marker Cpa03G 018680-337 is positioned at 234 th site of a nucleotide sequence shown in SEQ ID No.1, and the 234 th site base is G or T, wherein the diameter of a fruit chamber of a GG genotype is smaller than that of a TT genotype. The G allele of the-337 (G/T) locus of CPA03G018680 is extremely obviously related to the diameter of the papaya fruit chamber, and the detection of the carrying allele at the-337 (G/T) locus of CPA03G018680 is beneficial to the screening of the advance selection of the diameter of the papaya fruit chamber.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an SNP molecular marker Cpa03g 018680-337 which is obviously related to the diameter of a papaya fruit chamber, a method and application thereof.
Background
Papaya is a tropical evergreen fruit tree, which is a perennial fleshy herb plant of papaya family, also known as fructus Benincasae, dan Gua, papaw, fructus Citri Reticulatae viride, fructus Benincasae, fructus Chaenomelis, and semen Nelumbinis. It is native to mexico and central america, widely cultivated in tropical and subtropical regions of the world, transferred from the 17 th century into our country, mainly produced in guangdong, southwest, guangxi, yunnan, fujian, etc. The fresh papaya has beautiful appearance, thin skin, thick meat, sweet juice, fragrant and sweet smell and rich nutrition. The mature papaya pulp is yellow or red, has rich carotene and lycopene content, and has excellent health care effect, important edible value and industrial value. The development of papaya production greatly stimulates the development of food processing, medicine and health, beauty and health care, aquaculture and other related industries, and has important significance.
The papaya breeding fully utilizes natural variation varieties, screens and cultures excellent papaya varieties by using modern biotechnology means (genetic engineering technology, distant hybrid embryo in vitro culture technology and functional genomics), and researchers in the related field complete sequencing of the whole genome of papaya, so that the possibility is provided for developing high-density specific markers by better utilizing a bioinformatics technology method, related researches become more accurate, convenient and rapid, and good preconditions are provided for cloning and application of further genes; the continuous development of clone culture engineering technology, embryo culture and the like can enable distant hybridization breeding to be possible; the QTL positioning analysis can be used when the papaya is subjected to molecular marker analysis, so that related genes can be accurately positioned and expressed; by genetic engineering technology, disease-resistant genes and other useful genes are introduced, so as to achieve the aim of improving papaya varieties.
Whole genome association analysis (GWAS) is a low-cost and high-precision genotyping method based on linkage disequilibrium theory, and is used for locating genes or genome regions affecting complex traits. Along with the subsequent publication of the genome of various trees of fruit trees, the GWAS is widely applied to fruit tree researches in recent years. Molecular markers associated with specific traits can be identified, and molecular marker-assisted breeding (MAB) can be used for molecular marker assisted breeding, so that the breeding of new varieties is quickened.
Papaya can be divided into papaya for fruits and papaya for vegetables according to eating purposes at present, the wild papaya fruits are larger, and for the papaya for eating purposes, the large fruit type is not popular in the market which is oriented by consumers at present, but the small fruit type which can be eaten by one person is favored by consumers, so that the selection of the fruit size in papaya breeding is important. The diameter of the papaya fruit chamber is of great importance in the selection of the complex trait of fruit size.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide SNP molecular markers Cpa03g 018680-337 which are obviously related to the diameter of a papaya fruit chamber, a method and application.
The technical scheme adopted for solving the technical problems is as follows:
SNP molecular markers which are remarkably related to the diameter of papaya fruit chambers are Cpa03G 018680-337, wherein the molecular markers Cpa03G 018680-337 are positioned at the 234 th position of the nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, and the diameter of the fruit chambers of GG genotypes is smaller than that of TT genotypes.
Further, the molecular marker is very significantly associated with the papaya fruit cavity size gene CPA03G018680 with P <0.01.
The SNP molecular marker is applied to papaya breeding.
The SNP molecular marker is applied to the screening of papaya fruit size.
Use of a SNP molecular marker as described above in a reagent for detecting a SNP molecular marker associated with papaya fruit size, said use being for predicting papaya fruit size, wherein said SNP molecular marker is located at position 234 of the nucleotide sequence shown in SEQ ID No.1, said base at position 234 being G or T, wherein the fruit chamber diameter of the GG genotype is smaller than the TT genotype.
The nucleotide sequence shown in SEQ ID No. 1:
ACAACTTCCCAAATAATGTGAGTCTTTTATTATCGTAAATTTGAAAATTTTAACTTTTTT
ATCTTTTAGTAACTTCAGCATTCAATATTTTTTTAATTTTTTTCATTTATTTATTCTTAAT
TAACATTATTTAGCAGCAAATTATTAGAAAAATTAATTCTTAATTATAAAATAAAATTA
AGAATCATTAATTCACGAGTTTCTTTGTCTCTTATAATAATTTTAACCATGAAGAAATT
GAAAGCAAACAATATGTATTAAATTTATATATGTTGGTTTTTTTTTTTAATATTATCAAA
CAATAAGCATATGTTAAAGTGAGACTATCCATCAAGTCCAATACATATATTGTTATTTA
ATTTTTAATTTGAATCATAATTTAAAGTCTTCATAAATTATAATAAATATTAACAATTA
CATAAATCTCACTCCGAAACAGTATAAAAAAATATAACTTCAATTGAAATCTTATTAA
AATGAAGAAACTCAAAAATCTTATGTAACAGCCCCGAAAATTCATAATGGAATGTATATTAAAAATATCAAATGCGAAAGCGAAGTCA。
the application of the SNP molecular marker in detecting the reagent of the SNP molecular marker related to the size of papaya fruits, wherein the application is the selection of a variety with high papaya fruit size, the SNP molecular marker is positioned at 234 th position of a nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, and the diameter of a fruit cavity of a GG genotype is smaller than that of a TT genotype.
The application of the SNP molecular marker in the reagent for detecting the SNP molecular marker related to the papaya fruit size, wherein the application is auxiliary breeding of the molecular marker related to the papaya fruit size, the SNP molecular marker is positioned at the 234 th position of a nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, and the diameter of a fruit cavity of a GG genotype is smaller than that of a TT genotype.
A method for detecting SNP molecular markers related to the size of papaya fruits, wherein the SNP molecular markers are positioned at the 234 th position of a nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, a primer pair is designed by taking the nucleotide sequence containing the SNP molecular markers as a base sequence, PCR amplification is carried out by taking papaya genome DNA as a template, and the genotype of amplified products is detected, wherein the diameter of a fruit cavity of a GG genotype is smaller than that of a TT genotype, and the upstream primer of the primer pair is: SEQ ID No.2, the downstream primer is: SEQ ID No.3.
The beneficial effects obtained by the invention are as follows:
1. the G allele of the-337 (G/T) locus of CPA03G018680 is extremely obviously related to the diameter of the papaya fruit chamber, and the detection of the carrying allele at the-337 (G/T) locus of CPA03G018680 is beneficial to the screening of the advance selection of the diameter of the papaya fruit chamber.
2. The invention obtains a SNP molecular marker related to the cavity diameter of papaya fruit based on GWAS analysis screening, the fruit cavity diameter is the diameter of the largest position of the central cavity of the measured fruit, and the average value is calculated. The unit is cm, the accuracy is 0.1cm, the SNP molecular marker can be applied to detecting the papaya fruit chamber diameter breeding material, is favorable for rapidly detecting the breeding material, assists in hybridization breeding, shortens the new variety cultivation period, has lower detection cost, is not limited by environment, has high detection result accuracy and is easy to repeat. At present, no one uses the detection method to detect SNP molecular markers related to fruit cavity diameters in papaya.
Drawings
FIG. 1 is a Manhattan diagram of SNP markers highly correlated with the diameter of papaya fruit cavities in the present invention; wherein the point of the SNP marker exceeds a threshold value;
FIG. 2 is a Manhattan diagram and a linkage disequilibrium diagram of a local region of chromosome 3 where SNP markers extremely significantly related to the cavity diameter of papaya fruit are located in the invention; the arrow pointing sites in the interval are molecular markers screened by the invention, and the screened markers belong to the same linkage disequilibrium block;
FIG. 3 is a graph showing the comparison of fruit cavity diameter differences between different genotype subgroups of SNP markers which are extremely significantly related to the fruit cavity diameter of papaya fruit in the present invention; TT alleles are significantly correlated with fruit cavity diameter.
FIG. 4 is a gel electrophoresis of the products of PCR amplification in the papaya genome using primer pairs according to the present invention.
The GWAS peak on chromosome 3 correlates with fruit cavity diameter traits in the radial scan between fruit papaya and vegetable papaya. Papaya material carrying the TT allele has a higher fruit cavity diameter than papaya material carrying the GG allele. The diameter of the fruit cavity of the papaya for vegetables is higher than that of the papaya for fruits, and TT alleles are almost absent in the papaya for fruits, which indicates that the allele differences influence the diameter of the fruit cavity.
Detailed Description
The present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are conventional commercial products unless specified otherwise, the methods used in the invention are conventional methods in the art unless specified otherwise, and the mass of each substance used in the invention is conventional.
SNP molecular markers which are remarkably related to the diameter of papaya fruit chambers are Cpa03G 018680-337, wherein the molecular markers Cpa03G 018680-337 are positioned at the 234 th position of the nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, and the diameter of the fruit chambers of GG genotypes is smaller than that of TT genotypes.
Preferably, the molecular marker is very significantly associated with the papaya fruit cavity size gene CPA03G018680 by P <0.01.
The SNP molecular marker is applied to papaya breeding.
The SNP molecular marker is applied to the screening of papaya fruit size.
Use of a SNP molecular marker as described above in a reagent for detecting a SNP molecular marker associated with papaya fruit size, said use being for predicting papaya fruit size, wherein said SNP molecular marker is located at position 234 of the nucleotide sequence shown in SEQ ID No.1, said base at position 234 being G or T, wherein the fruit chamber diameter of the GG genotype is smaller than the TT genotype.
The application of the SNP molecular marker in detecting the reagent of the SNP molecular marker related to the size of papaya fruits, wherein the application is the selection of a variety with high papaya fruit size, the SNP molecular marker is positioned at 234 th position of a nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, and the diameter of a fruit cavity of a GG genotype is smaller than that of a TT genotype.
The application of the SNP molecular marker in the reagent for detecting the SNP molecular marker related to the papaya fruit size, wherein the application is auxiliary breeding of the molecular marker related to the papaya fruit size, the SNP molecular marker is positioned at the 234 th position of a nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, and the diameter of a fruit cavity of a GG genotype is smaller than that of a TT genotype.
A method for detecting SNP molecular markers related to the size of papaya fruits, wherein the SNP molecular markers are positioned at the 234 th position of a nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, a primer pair is designed by taking the nucleotide sequence containing the SNP molecular markers as a base sequence, PCR amplification is carried out by taking papaya genome DNA as a template, and the genotype of amplified products is detected, wherein the diameter of a fruit cavity of a GG genotype is smaller than that of a TT genotype, and the upstream primer of the primer pair is: SEQ ID No.2, the downstream primer is: SEQ ID No.3.
Specifically, the related preparation and detection are as follows:
1. papaya genome-wide SNP marker development
340 papaya (the batch of materials has rich genetic diversity and wide representativeness in the domestication process of papaya) collected from mexico, south africa, guangxi of China, hainan of China and the like are sown in Wenchang bases of the national academy of tropical agriculture, the soil fertility is moderate, diseases and insect pests are avoided, phenotypic character data are planted and collected for many years, and the phenotypic character data are used for subsequent analysis after being processed by EXCE L2016.
1-2 g of fresh and tender leaves are taken from papaya plants, and DNA of papaya materials is extracted according to a selected root plant genome DNA extraction kit (DP 305) after liquid nitrogen grinding. Detecting the quality and concentration of the DNA sample by using an ultra-micro spectrophotometer and 1% agarose gel electrophoresis, selecting the DNA sample with clear electrophoresis strip and no obvious protein residue in a gel hole, wherein OD260/OD280 is between 1.8 and 1.9, and the concentration is more than 100 NG/mL, and carrying out library construction and sequencing.
The method of ultrasonic breaking (or enzyme cutting) is adopted to break DNA randomly into fragments of about 300bp, and the construction of a sequencing library is completed by end repair, addition of A at the 3' end, addition of sequencing adapter pair, purification and PCR amplification of the DNA fragments. And sequencing the library through an ILLUMINA 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 (RA W READS) is required according to a certain standard, and the filtering standard is as follows: (1) removing the sequence with linker (ADAPTER), (2) removing a pair of sequences with a single-ended sequence nitrogen content >10%, and (3) removing a pair of sequences with a low quality base number exceeding 50%. CLEAN READS after removing the low-quality sequence, the linker sequence and the inaccurate sequence is subjected to next sequence alignment. The reference genome was selected from the newly assembled papaya fruit material "KAMIYA" genome of the subject group, CLEAN READS was aligned with the reference genome sequence using BWA-MEM software, the results were sequenced using samtoils software alignment, mutation detection was performed after removal of the PCR repeat by GATK4.0 software, the mutation set was filtered with QD >2.0, qual >30.0, fs <60.0, mq >40.0 hard criteria, and the statistically significant mutation site dataset was retained. And filtering the mutation sites again according to MAF (minor allele frequency) > = 0.05 and MISS (deletion rate) <= 0.2 as a standard to obtain a high-quality mutation site collection.
2. Analysis of papaya fruit width-related SNP loci by GWAS
Annotating the mutation sites by utilizing the genome DNA interval characteristic description file, and respectively counting the number of the mutation sites falling in a gene coding region, a non-coding region, an inter-gene region, a non-synonymous mutation and the like. After a population genetic relationship matrix is obtained through population structure analysis, full genome association analysis is carried out by combining phenotypic character data, and a linkage disequilibrium region (-LOG 10 (P-VALUE) > 6) with the size of 20KB related to the cavity diameter of papaya fruits is detected, so that candidate genes CPA03G018680 related to the cavity size of papaya fruits are determined.
FIG. 1 is a Manhattan diagram obtained by analyzing the above loci by GWAS, wherein the X-axis represents the position of the SNP marker on the chromosome, and the Y-axis represents the-LOG 10 (P) value, and the larger the value, the higher the correlation between the SNP locus and the trait. The SNP markers screened at this time are located at the arrows in the figure;
FIG. 2 is a Manhattan diagram and a linkage disequilibrium diagram of a local region of chromosome 3 where SNP markers extremely significantly related to the cavity diameter of papaya fruit are located in the invention; the arrow pointing sites in the interval are molecular markers screened by the invention, and the screened markers belong to the same linkage disequilibrium block;
3. screening candidate markers
The allele frequency, the trait differences, and the gene expression level differences were compared based on the SNP markers in the region associated with the GWAS analysis, and the results are shown in FIG. 3. SNP sites related to the chamber diameter of papaya fruits were selected in the CPA03G018680 gene PROMOTER region (CPA 03G018680: -337 (G/T)).
4. Marker development and detection
The sequence containing SNP sites (CPA 03G018680: -337 (G/T)) which are obviously related to the chamber diameter of papaya fruits obtained by the previous screening is taken as a template SEQ ID NO.1, and a pair of PRIMERs is designed by using PRIMER 5.0 software, wherein the sequences of the PRIMERs are as follows:
forward primer: ACAACTTCCCAAATAATGTGAGTCT (SEQ ID NO. 2);
reverse primer: TGACTTCGCTTTCGCATTTGA (SEQ ID NO. 3).
The primers are used for carrying out common PCR amplification on the genomic DNA of the materials to be screened, and the amplification system is as follows: 2X RAPID TAQ MASTER MIX 12.5 MmL, 10 MmM forward primer 1 MmL, 10 MmM reverse primer 1 MmL, DN A template 1 Mm L, DDH2O 9.5 MmL. (MIX (amplification buffer) purchased from Novamat biosciences Inc., primer commission Beijing Oriental biosciences Inc..) PCR amplification was performed at 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 to completion of China large gene company.) to obtain 819bp DNA fragment, sequencing, comparing the sequencing result with the related gene fragment SEQ ID NO.1 of the Chinese papaya, analyzing, and detecting the genotype carried by SNP locus at 234 th position of the sequence. Therefore, the diameter of the papaya variety fruit chamber can be detected or predicted, the diameter of the papaya variety fruit chamber can be effectively selected, and the process of papaya variety breeding is accelerated.
To verify the practicality of the SNP markers in the invention, 50 papaya plants (excluding 340 papaya plants for SNP marker development) were randomly selected from the papaya planting area of Wenchang base, the biological technology of national academy of tropical agricultural sciences, and were subjected to genotyping and papaya fruit width trait investigation after sequencing. The results are shown in tables 1 and 2.
TABLE 150 papaya in CPA03G018680: -337 different genotypes and fruit chamber diameter (mm)
TABLE 2 50 papaya at Cpa03g018680: -337 different genotypes and fruit chamber diameter (mm)
The diameter of the largest part of the fruit's central cavity was measured and the average value calculated. The unit is cm, and the accuracy is 0.1cm.
As can be seen from tables 1 and 2, the G allele at the-337 (G/T) locus of CPA03G018680 is extremely obviously related to the diameter of the papaya fruit chamber, and the detection of the genotype carried by the CPA03G018680 (G/T) locus of CPA03G018680 enables rapid genotype detection so as to quickly screen materials carrying GG genes, thereby being beneficial to screening the diameter of the papaya fruit chamber in advance.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (5)
1. Use of a reagent for detecting SNP molecular markers significantly associated with papaya fruit chamber diameter in papaya fruit size screening, characterized in that: the SNP molecular marker is Cpa03G 018680-337, the molecular marker Cpa03G 018680-337 is positioned at the 234 th position of the nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, wherein the diameter of a fruit cavity of the GG genotype is smaller than that of the TT genotype, and the SNP molecular marker is obviously related to the papaya fruit cavity size gene Cpa03G 018680P <0.01.
2. The application of the reagent for detecting SNP molecular markers in preparing the reagent for detecting SNP molecular markers related to the size of papaya fruits is characterized in that: the application is to predict the size of papaya fruits, wherein the SNP molecular marker is Cpa03G 018680-337, the SNP molecular marker is positioned at the 234 th position of the nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, the diameter of a fruit cavity of a GG genotype is smaller than that of a TT genotype, and the SNP molecular marker is remarkably related to the Cpa03G018680 gene P <0.01.
3. The application of the reagent for detecting SNP molecular markers in preparing the reagent for detecting SNP molecular markers related to the size of papaya fruits is characterized in that: the application is the selection of papaya fruit size varieties, wherein the SNP molecular marker is Cpa03G 018680-337, the SNP molecular marker is positioned at the 234 th position of the nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, the diameter of a fruit cavity of the GG genotype is smaller than that of the TT genotype, and the SNP molecular marker is very obviously related to the papaya fruit cavity size gene Cpa03G018680 and P <0.01.
4. The application of the reagent for detecting SNP molecular markers in preparing the reagent for detecting SNP molecular markers related to the size of papaya fruits is characterized in that: the application is auxiliary breeding of papaya fruit size related molecular markers, wherein the SNP molecular markers are Cpa03G 018680-337, the SNP molecular markers are positioned at 234 th position of a nucleotide sequence shown in SEQ ID No.1, the 234 th position base is G or T, the diameter of a fruit cavity of a GG genotype is smaller than that of a TT genotype, and the P <0.01 is extremely obviously related to the papaya fruit cavity size gene Cpa03G018680.
5. A method for detecting SNP molecular markers associated with papaya fruit size, characterized by: the SNP molecular marker is Cpa03G 018680-337, the SNP molecular marker is positioned at the 234 th position of a nucleotide sequence shown in SEQ ID No.1, the 234 th base is G or T, a primer pair is designed by taking the nucleotide sequence containing the SNP molecular marker as a basic sequence, PCR amplification is carried out by taking papaya genome DNA as a template, the genotype of an amplified product is detected, wherein the diameter of a fruit cavity of the GG genotype is smaller than that of the TT genotype, the SNP molecular marker and the Cpa03G018680 gene of the papaya cavity size are extremely obviously related to P <0.01, and the upstream primer of the primer pair is: SEQ ID No.2, the downstream primer is: SEQ ID No.3.
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