CN117512203A - Molecular marker related to papaya fruit width, method and application - Google Patents
Molecular marker related to papaya fruit width, method and application Download PDFInfo
- Publication number
- CN117512203A CN117512203A CN202410008699.XA CN202410008699A CN117512203A CN 117512203 A CN117512203 A CN 117512203A CN 202410008699 A CN202410008699 A CN 202410008699A CN 117512203 A CN117512203 A CN 117512203A
- Authority
- CN
- China
- Prior art keywords
- molecular marker
- papaya
- cpa05g
- width
- genotype
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 235000009467 Carica papaya Nutrition 0.000 title claims abstract description 95
- 241000219173 Carica Species 0.000 title claims abstract description 92
- 239000003147 molecular marker Substances 0.000 title claims abstract description 81
- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000009395 breeding Methods 0.000 claims abstract description 21
- 230000001488 breeding effect Effects 0.000 claims abstract description 21
- 210000000349 chromosome Anatomy 0.000 claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims description 30
- 230000003321 amplification Effects 0.000 claims description 21
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 21
- 239000002773 nucleotide Substances 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- 108700039691 Genetic Promoter Regions Proteins 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 abstract description 12
- 238000001514 detection method Methods 0.000 abstract description 11
- 238000009396 hybridization Methods 0.000 abstract description 5
- 230000002349 favourable effect Effects 0.000 abstract description 2
- 239000003550 marker Substances 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 238000011161 development Methods 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 8
- 108700028369 Alleles Proteins 0.000 description 7
- 240000006432 Carica papaya Species 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 102000054766 genetic haplotypes Human genes 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000007119 Ananas comosus Nutrition 0.000 description 1
- 244000099147 Ananas comosus Species 0.000 description 1
- 235000014649 Carica monoica Nutrition 0.000 description 1
- 241000219172 Caricaceae Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 241000948223 Octomys Species 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000012098 association analyses Methods 0.000 description 1
- 235000021015 bananas Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000013574 canned fruits Nutrition 0.000 description 1
- PHIQHXFUZVPYII-UHFFFAOYSA-N carnitine Chemical compound C[N+](C)(C)CC(O)CC([O-])=O PHIQHXFUZVPYII-UHFFFAOYSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 235000011869 dried fruits Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- 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)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and discloses a SNP molecular marker related to papaya fruit width, a method and application thereof, wherein the molecular marker is Cpa05g 006390-1724, the molecular marker Cpa05g 006390-1724 is positioned at 8116762 th basic group of a Chr05 chromosome, and the fruit width of a CC genotype is smaller than that of a TT genotype. The molecular marker can be applied to detecting the breeding materials with high fruit width of papaya, is favorable for rapidly detecting the breeding materials related to fruit width, assists in hybridization breeding, shortens the breeding period of new varieties, has lower detection cost, is not limited by environment, and has high accuracy and easy repeatability of detection results.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to SNP molecular markers related to papaya fruit width traits, a method and application thereof.
Background
Papaya (Carica papaya L.) is a evergreen fruit tree of the genus Carica of the family Caricaceae, and has soft and juicy pulp, fragrant and sweet smell, and full year of flower and fruit period. Papaya originally produces tropical america, which is transmitted into china in the clear period, and is now widely planted in tropical and warmer subtropical regions of the world. China is mainly distributed in Guangdong, xianhu vegetable garden, hainan, guangxi, yunnan and Fujian. Papaya, bananas and pineapples are called as a tropical three-major herbaceous fruit tree, and the fruits are rich in nutrition and amino acids, vitamins and trace elements; the meat quality is fragrant, sweet and juicy; papaya can also be processed, and the products comprise canned fruits, preserved fruits, dried fruits and the like; the seeds can squeeze oil, and the olive milk is also processed into papain, so that the papain can be widely applied to industries such as food, cosmetics, medicines, leather making and the like, and has very broad industrial development prospect.
Molecular markers for detecting differences between biological individuals based on molecular mutation follow morphological markers, cell markers and biochemical markers, solve the defects of the previous generation genetic marker method, and the molecules are in an ideal genetic marker form. DNA molecular markers have relatively unique advantages: the environment can influence the expression of the gene without changing the original structure of the gene, and the DNA molecular marker is not influenced by the environment; part of the molecular markers are co-dominant, so that whether the genotype is homozygous or not can be identified, and complete genetic information can be provided; the number of marks is rich; is not affected by the development stage, and improves the analysis accuracy. The completion and disclosure of the sequencing of the papaya genome provide possibility for developing high-density specific markers by better utilizing a bioinformatic technology method, so that related researches become more accurate, convenient and rapid, and a good precondition is 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.
In traditional molecular breeding, breeders mainly select plant individuals with excellent characters and fix the excellent characters through hybridization or backcrossing. Molecular Marker-assisted selection (MAS) is an effective tool for genetic improvement of crops. Along with the development and application of high-throughput low-cost SNP markers and the rapid development of bioinformatics, the application of MAS is expanded to whole genome selection (Genomic Selection, GS), and the selection efficiency and accuracy are greatly improved. The sequencing of the papaya genome is completed and published, so that the development and application of molecular markers of important papaya fruit characters are greatly promoted, and convenience is provided for molecular marker assisted breeding.
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. Papaya fruit width is of great importance in the selection of complex traits such as fruit size.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide an SNP molecular marker related to papaya fruit width characters, a method and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
a SNP molecular marker related to papaya fruit width property is characterized in that the molecular marker is Cpa05g 006390-1724, the marker is positioned at 8116762 th base of a Chr05 chromosome, the marker is positioned in a promoter region of a gene, a C/T polymorphism exists at 358 th site of a sequence shown in amplification products with 527bp length, namely SEQ ID No.1, the molecular marker Cpa05g 006390-1724 is positioned at 358 th site of a nucleotide sequence shown in SEQ ID No.1, the 358 th base is C or T, and the fruit width of a CC genotype is smaller than that of a TT genotype.
Further, the molecular marker is very significantly related to gene Cpa05g006390 related to papaya fruit width, P < 0.01.
The SNP molecular marker is applied to papaya breeding.
The SNP molecular marker is applied to the screening of the width and the size of papaya.
The application of the SNP molecular marker in the reagent for detecting the SNP molecular marker related to the width of papaya is characterized in that: the application is to predict the width of papaya fruits, wherein the molecular marker is Cpa05g 006390-1724, the molecular marker Cpa05g 006390-1724 is located at 8116762 th base of a chromosome of Chr05 and is located in a region of a gene promoter, a C/T polymorphism exists at 358 th base of a sequence shown as an amplification product with 527bp length, the molecular marker Cpa05g 006390-1724 is located at 358 th base of a nucleotide sequence shown as SEQ ID No.1, the 358 th base is C or T, and the fruit width of a CC 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 width and the size of the papaya is the selection of the variety with high width and the size of the papaya, wherein the molecular marker is Cpa05g 006390-1724,
the molecular marker Cpa05g 006390-1724 is positioned at 8116762 th base of the chromosome of the Chur 05, the gene is in the promoter region, a C/T polymorphism exists at 358 th base of a sequence shown by an amplification product with 527bp length, the molecular marker Cpa05g 006390-1724 is positioned at 358 th base of a nucleotide sequence shown by SEQ ID No.1, the 358 th base is C or T, and the fruit width of a CC 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 width and the size of papaya is assisted breeding by the molecular marker related to the width and the size of papaya, wherein the molecular marker is Cpa05g 006390-1724, the molecular marker Cpa05g 006390-1724 is positioned at 8116762 th base of a chromosome of Chr05, the gene has C/T polymorphism at 358 th position of a sequence shown as an amplification product with 527bp length in a promoter region, the molecular marker Cpa05g 006390-1724 is positioned at 358 th position of a nucleotide sequence shown as SEQ ID No.1, the 358 th base is C or T, and the fruit width of a CC genotype is smaller than a TT genotype.
A method for detecting SNP molecular markers related to the width of papaya comprises the steps of locating a 8116762 th base of a chromosome of Chr05, locating a gene in a promoter region, locating a C/T polymorphism at a 358 th position of a sequence shown as an amplification product with 527bp length, locating a molecular marker Cpa05g006390: -1724 at a 358 th base of a nucleotide sequence shown as SEQ ID No.1, locating the 358 th base as C or T, locating the nucleotide sequence containing the SNP molecular marker as a base sequence, designing a primer pair, carrying out PCR amplification by using a papaya genome DNA as a template, and detecting the genotype of an amplification product, wherein the diameter of a fruit chamber of the GG genotype is smaller than that of a TT genotype, and the upstream primer of the primer pair is as follows: SEQ ID No.2, the downstream primer is: SEQ ID No.3.
The beneficial effects obtained by the invention are as follows:
1. the SNP molecular marker can be applied to detecting the breeding materials with high fruit width of papaya, is favorable for rapidly detecting the breeding materials related to the fruit width, assists in hybridization breeding, shortens the breeding period of new varieties, has lower detection cost, is not limited by environment, has high accuracy of detection results and is easy to repeat.
2. The SNP molecular marker related to the papaya fruit width is obtained based on GWAS analysis and screening, and the molecular marker (Cpa 05g006390: -1724) on the Cpa05g00639 gene can be applied to detecting the papaya fruit width, thereby being beneficial to rapid detection of breeding materials, assisting in hybridization breeding, shortening the cultivation period of new varieties, and being lower in detection cost, free from environmental limitation, high in detection result accuracy and easy to repeat.
3. The SNP molecular marker is developed based on 358 papaya germplasm resources collected in mexico, south Africa, guangxi of China, hainan of China and the like, and the genetic diversity of the material is rich, and the material has wide representativeness in the papaya domestication process.
4. The invention obtains a papaya fruit width related SNP molecular marker Cpa05g 006390-1724 based on GWAS analysis screening, which is positioned at 8116762 th base of a Chr05 chromosome, a gene is positioned in a promoter region, and a C/T polymorphism exists at 358 th site of a sequence shown as an amplification product with 527bp length, wherein a C allele is extremely obviously related to the papaya fruit width.
Drawings
FIG. 1 is a Manhattan diagram of SNP markers highly correlated with the width of papaya fruits in the present invention; wherein, the SNP marker locus-log 10 (P-value) > 6, the SNP marker locus and the papaya fruit width are extremely obvious;
FIG. 2 is a Manhattan diagram and a linkage disequilibrium haplotype block diagram of a local region of chromosome 5 in which SNP markers which are extremely significantly related to the fruit width of papaya are located; wherein the arrow indicates the molecular marker locus;
FIG. 3 is a graph showing comparison of fruit width differences between different genotype subgroups of SNP markers which are extremely significantly correlated with fruit width of papaya in the present invention; wherein the T allele is significantly associated with fruit width;
FIG. 4 is a gel electrophoresis chart of PCR amplified products of primers for detecting SNP molecular marker Cpa05g006390: -1724 genotype in papaya genome provided in the present invention.
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.
A haplotype SNP molecular marker related to papaya fruit width character is Cpa05g 006390-1724, wherein the marker is positioned at 8116762 th base of a chromosome Chr05, a gene is in a promoter region, and a C/T polymorphism exists at 358 th site of a sequence shown as an amplification product with 527bp length, namely SEQ ID No.1, wherein the fruit width of a CC genotype is smaller than that of a TT genotype.
SEQ ID No.1：
GACCCGGTTCGTCCACTATCTCCCAGGATATCTAGTTAGGCTGTCCAAATTATCTATTTATCAATTCACATAACATTTTTTCCATTTTCTTTGATCTCAATCATCCAATCATACACAAACACGTAAACTAATAATGAACACCAAACAAAATAACACAACAATGGGTTCACCATAATTCAAACCATAATAAAGGAAATCATGATTCATGCATACATAACCATAAAATAAACACATCAGATACCATACTGTATTTAAAACTGTAAAATAACTAAAACAGCAACTGACCAAAAATATATCAAACTCGAGCTCCATTAGCAACCTTAATTATTTATATATATCTATTAATTCACGAAATGCTGTTTTATTATTTATTGAGCTTTAATACCGTGATAGGAAATAGGGTGAGAAATCCTTACTTGTTTTCGGGGTTTGAAATCGGCTCTACAATGTACTTACGGGCTGTCCCTAAATTCCCTGAATTTTCTCTGGGAAACTGAAAGGCACTACTGGTACCTGTACGTGTCCCA。
Preferably, the molecular marker is very significantly related to the papaya fruit width related gene Cpa05g006390 with P < 0.01.
The SNP molecular marker is applied to papaya breeding.
The SNP molecular marker is applied to the screening of the width and the size of papaya.
The application of the SNP molecular marker in the reagent for detecting the SNP molecular marker related to the width of papaya is characterized in that: the application is to predict the width of papaya fruits, wherein the molecular marker is Cpa05g 006390-1724, the marker is positioned at 8116762 th base of a Chr05 chromosome, a gene is in a promoter region, and a C/T polymorphism exists at 358 th site of a sequence shown as an amplification product with 527bp length, wherein the fruit width of a CC genotype is smaller than that of a TT genotype;
the application of the SNP molecular marker in a reagent for detecting the SNP molecular marker related to the width and the size of papaya, wherein the application is the selection of a variety with high width and size of papaya, wherein the molecular marker is Cpa05g 006390-1724, the marker is positioned at 8116762 th base of a chromosome of Chr05, the gene is in a promoter region, and a C/T polymorphism exists at 358 th site of a sequence shown as an amplification product with 527bp length, wherein the fruit width of a CC genotype is smaller than that of a TT genotype;
the application of the SNP molecular marker in a reagent for detecting the SNP molecular marker related to the width and the size of papaya, wherein the application is assisted breeding of the molecular marker related to the width and the size of papaya, wherein the molecular marker is Cpa05g 006390-1724, the marker is positioned at 8116762 th base of a chromosome of Chr05, the gene is in the promoter region, and a C/T polymorphism exists at 358 th site of a sequence shown by an amplification product with 527bp length, wherein the fruit width of a CC genotype is smaller than that of a TT genotype;
a method for detecting SNP molecular markers related to the width of papaya, wherein the SNP molecular markers are positioned at 8116762 base of a chromosome Chr05, a gene is in a master region, a C/T polymorphism exists at 358 th position of a sequence shown as an amplification product with 527bp length, a primer pair is designed by taking a nucleotide sequence containing the SNP molecular markers as a base sequence, PCR amplification is carried out by taking papaya genome DNA as a template, the genotype of the amplification product is detected, wherein the fruit width of a CC genotype is smaller than that of a TT genotype, and upstream primers of the primer pair are as follows: 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 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 (3) taking 1-2 g of fresh and tender leaves from papaya plants, 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 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 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. The reference genome was selected from the newly assembled papaya fruit material "kamiya" genome of the subject group, clear reads were aligned with the reference genome sequence using BWA-mem software, the results were sequenced using samtools software alignment, mutation detection was performed after PCR repeat removal by GATK4.0 software, and mutation site datasets were retained with statistical significance using hard standard filter mutation sets 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.
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 width of papaya fruits is detected, so that candidate genes Cpa05g006390 related to the width of papaya fruits are determined.
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 loci associated with papaya fruit width were selected in the Cpa05g006390 gene promoter region (Cpa 05g006390: -1724 (C/T).
4. Marker development and detection
The sequence containing SNP sites (Cpa 05g006390: -1724 (C/T)) which are obviously related to the width of papaya fruits obtained by the previous screening is taken as a template SEQ ID NO.1, and a pair of primers are designed by using Primer 5.0 software, wherein the sequences of the primers are as follows:
forward primer: GACCCGGTTCGTCCACTATC;
reverse primer: TGGGACACGTACAGGTACCA.
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 12.5.5. 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 was made to the synthesis of Beijing Octomy Dingsheng Biotech Co., ltd.) (Cpa 05g006390: -1724 (C/T) PCR amplification was performed at 95℃for 5min, 95℃for 30s, 62℃for 30s,72℃for 30s,35 cycles, and 72℃for 5min, giving a 527bp length of amplification product, i.e., the genotype carried by the SNP site at 368 th of the sequence shown in SEQ ID No. 1.
Therefore, the method can detect or predict the fruit width of the papaya variety, effectively select the fruit width of the papaya variety and accelerate the breeding process of the papaya variety.
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 to 2.
Table 1 50 papaya strains Cpa05g006390: -1724 different genotypes and fruit widths (mm)
Table 2 50 papaya strains average fruit width (mm) at Cpa05g006390: -1724 different genotypes
As can be seen from tables 1 and 2, the detection of the carrying allele at the Cpa05g 006390:1724 locus is helpful for screening papaya fruit width. The GWAS peak on chromosome 5 correlates with papaya fruit width traits in a radial scan between papaya with vegetables and fruits. Papaya material carrying the TT allele at this site has a greater fruit width than papaya material carrying the CC allele.
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 (6)
1. An application of SNP molecular markers related to papaya fruit width traits in papaya breeding, which is characterized in that: the molecular marker is Cpa05g 006390-1724, the molecular marker Cpa05g 006390-1724 is positioned at 8116762 th base of chromosome Chr05, the gene is in the promter region, the C/T polymorphism exists at 358 th site of the amplified product sequence with 527bp length, namely SEQ ID No.1, the molecular marker Cpa05g 006390-1724 is positioned at 358 th site of the nucleotide sequence shown in SEQ ID No.1, the 358 th base is C or T, the fruit width of CC genotype is smaller than TT genotype, and the gene Cpa05g006390 related to the fruit width of papaya is extremely obviously related to P < 0.01.
2. The use of the SNP molecular marker of claim 1 for papaya width size screening, characterized in that: the molecular marker is Cpa05g 006390-1724, the molecular marker Cpa05g 006390-1724 is positioned at 8116762 th base of chromosome Chr05, the gene is in the promter region, the C/T polymorphism exists at 358 th site of the amplified product sequence with 527bp length, namely SEQ ID No.1, the molecular marker Cpa05g 006390-1724 is positioned at 358 th site of the nucleotide sequence shown in SEQ ID No.1, the 358 th base is C or T, the fruit width of CC genotype is smaller than TT genotype, and the gene Cpa05g006390 related to the fruit width of papaya is extremely obviously related to P < 0.01.
3. The use of the SNP molecular marker according to claim 1 in a reagent for detecting SNP molecular marker related to papaya width size, characterized in that: the application is to predict the width of papaya fruits, wherein the molecular marker is Cpa05g 006390-1724, the molecular marker is Cpa05g 006390-1724, the molecular marker Cpa05g 006390-1724 is located at 8116762 base of a chromosome of Chr05, the gene is in a promter region, a C/T polymorphism exists at 358 th site of a sequence shown as an amplification product with 527bp length, the molecular marker Cpa05g 006390-1724 is located at 358 th site of a nucleotide sequence shown as SEQ ID No.1, the 358 th base is C or T, the fruit width of CC genotype is smaller than that of TT genotype, and the gene Cpa05g006390 related to the papaya fruit width of the molecular marker is extremely obviously related to P < 0.01.
4. The use of the SNP molecular marker according to claim 1 in a reagent for detecting SNP molecular marker related to papaya width size, characterized in that: the application is the selection of a variety with high papaya width, wherein the molecular marker is Cpa05g 006390-1724, the molecular marker Cpa05g 006390-1724, the molecular marker Cpa05g 006390-1724 is positioned at 8116762 base of a chromosome of Chr05, the gene is in a promter region, a C/T polymorphism exists at 358 th position of a sequence shown by an amplification product with 527bp length, the molecular marker Cpa05g 006390-1724 is positioned at 358 th position of a nucleotide sequence shown in SEQ ID No.1, the 358 th base is C or T, the fruit width of a CC genotype is smaller than that of a TT genotype, and the gene Cpa05g006390 related to the papaya fruit width of the molecular marker is extremely obviously related to P < 0.01.
5. The use of the SNP molecular marker according to claim 1 in a reagent for detecting SNP molecular marker related to papaya width size, characterized in that: the application is auxiliary breeding of papaya width-size related molecular markers, wherein the molecular markers are Cpa05g 006390-1724, the molecular markers are Cpa05g 006390-1724, the molecular markers Cpa05g 006390-1724 are positioned at 8116762 th base of a chromosome of Chr05, the gene is in a promoter region, a C/T polymorphism exists at 358 th site of a sequence shown by amplification products with 527bp length, the molecular markers Cpa05g 006390-1724 are positioned at 358 th site of a nucleotide sequence shown in SEQ ID No.1, the 358 th base is C or T, the fruit width of a CC genotype is smaller than that of a TT genotype, and the molecular markers are very obviously related to the gene Cpa05g006390 related to the papaya fruit width, and P is less than 0.01.
6. A method for detecting SNP molecular markers associated with papaya width size, characterized by: the molecular marker is Cpa05g 006390-1724, the SNP molecular marker is positioned at 8116762 th base of a Chr05 chromosome, a gene is in a promter region, a C/T polymorphism exists at 358 th base of a sequence shown as an amplification product with 527bp length, the Cpa05g 006390-1724 is positioned at 358 th base of a nucleotide sequence shown as SEQ ID No.1, the 358 th base is C or T, a nucleotide sequence containing the SNP molecular marker is used as a base sequence, a primer pair is designed, PCR amplification is carried out by using papaya genome DNA as a template, the genotype of an amplification product is detected, the fruit width of the GG genotype is smaller than that of a TT genotype, the P < 0.01 is extremely obviously related to the gene Cpa05g006390 related to the fruit width of papaya, and the upstream primer of the primer pair is: SEQ ID No.2, the downstream primer is: SEQ ID No.3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410008699.XA CN117512203B (en) | 2024-01-04 | 2024-01-04 | Molecular marker related to papaya fruit width, method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410008699.XA CN117512203B (en) | 2024-01-04 | 2024-01-04 | Molecular marker related to papaya fruit width, method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117512203A true CN117512203A (en) | 2024-02-06 |
| CN117512203B CN117512203B (en) | 2024-03-22 |
Family
ID=89745942
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410008699.XA Active CN117512203B (en) | 2024-01-04 | 2024-01-04 | Molecular marker related to papaya fruit width, method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117512203B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180146633A1 (en) * | 2014-06-06 | 2018-05-31 | Nunhems B.V. | MELON PLANTS WITH A DOMINANT MELON YELLOWING ASSOCIATED VIRUS (MYaV) RESISTANCE GENE |
| CN114574629A (en) * | 2022-05-06 | 2022-06-03 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to papaya fruit weight and application thereof |
| CN114622034A (en) * | 2022-04-26 | 2022-06-14 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to papaya fruit cavity diameter and application |
| CN114672588A (en) * | 2022-05-26 | 2022-06-28 | 中国热带农业科学院三亚研究院 | An SNP molecular marker Cpa03g017250:8, amplification primer, detection kit and application thereof |
| CN114672586A (en) * | 2022-05-26 | 2022-06-28 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to width character of papaya fruit, amplification primer, detection kit and application thereof |
| CN114672587A (en) * | 2022-05-26 | 2022-06-28 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to fructose content of papaya fruit, amplification primer, detection kit and application thereof |
-
2024
- 2024-01-04 CN CN202410008699.XA patent/CN117512203B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180146633A1 (en) * | 2014-06-06 | 2018-05-31 | Nunhems B.V. | MELON PLANTS WITH A DOMINANT MELON YELLOWING ASSOCIATED VIRUS (MYaV) RESISTANCE GENE |
| CN114622034A (en) * | 2022-04-26 | 2022-06-14 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to papaya fruit cavity diameter and application |
| CN114574629A (en) * | 2022-05-06 | 2022-06-03 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to papaya fruit weight and application thereof |
| CN114672588A (en) * | 2022-05-26 | 2022-06-28 | 中国热带农业科学院三亚研究院 | An SNP molecular marker Cpa03g017250:8, amplification primer, detection kit and application thereof |
| CN114672586A (en) * | 2022-05-26 | 2022-06-28 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to width character of papaya fruit, amplification primer, detection kit and application thereof |
| CN114672587A (en) * | 2022-05-26 | 2022-06-28 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to fructose content of papaya fruit, amplification primer, detection kit and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| P. K. ANISH KUMAR等: "Home Genetics and Genomics of Papaya Chapter Allele Discovery Platform (ADP) in Papaya (Carica papaya L.)", GENETICS AND GENOMICS OF PAPAYA, 1 January 2013 (2013-01-01), pages 409 - 421 * |
| 郭霞;张艳;刘志勇;: "番木瓜性别连锁的AFLP及其SCAR标记的建立", 中国农业科学, no. 03, 10 March 2009 (2009-03-10), pages 967 - 973 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117512203B (en) | 2024-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114672587B (en) | SNP molecular marker related to fructose content of papaya fruit, amplification primer, detection kit and application thereof | |
| CN114672586B (en) | SNP molecular marker related to width character of papaya fruit, amplification primer, detection kit and application thereof | |
| CN114622034B (en) | SNP molecular marker related to papaya fruit chamber diameter and application thereof | |
| CN116426678B (en) | SNP molecular marker related to fructose content of papaya pulp and application thereof | |
| CN114672588B (en) | A SNP molecular marker Cpa03g017250:8, amplification primer, detection kit and application thereof | |
| CN114574629B (en) | SNP molecular marker related to papaya fruit weight and application thereof | |
| CN110607386A (en) | KASP primer combination suitable for construction of tomato DNA fingerprint database and application thereof | |
| CN117144055B (en) | Application of haplotype molecular marker related to regulation and control of papaya fruit length | |
| KR20180077873A (en) | SNP markers for selection of marker-assisted backcross in watermelon | |
| CN111719012A (en) | dCAPS molecular marker primer pair for identifying dehydration rate genotype of corn kernel and application | |
| CN116622876B (en) | Haplotype molecular marker related to vitamin C content of papaya pulp and application thereof | |
| CN114592086B (en) | SNP molecular marker related to resistance of ring spot virus PRSV and application | |
| CN110616277A (en) | Rice grain length gene function marker and application thereof | |
| CN117512203B (en) | Molecular marker related to papaya fruit width, method and application | |
| CN107354150B (en) | Differential sequence for identifying tomato fruit dry juice character, molecular marker and identification method thereof | |
| CN107338246B (en) | Specific sequence of tomato fruit dry juice character and its molecular marker and identification method | |
| CN117512202B (en) | SNP molecular marker remarkably related to papaya fruit chamber diameter, method and application | |
| CN108424975B (en) | Pueraria SSR marker primer pair developed based on transcriptome sequence and application thereof | |
| CN111363844A (en) | Water chestnut SSR primer group and application thereof | |
| CN109868330A (en) | It is a kind of based on RNA-Seq exploitation lens EST-SSR label and application | |
| CN117144056B (en) | Application of haplotype molecular marker related to papaya fruit fructose accumulation | |
| CN115838790B (en) | Molecular marker for sex identification of actinidia arguta and application of specific primer pair M4 | |
| CN116334269B (en) | Molecular marker for sex identification of actinidia arguta and specific primer pair M3 thereof | |
| CN117165718B (en) | Application of SNP molecular marker related to fructose content of papaya pulp | |
| CN117604155A (en) | SNP molecular marker for screening oil palm germplasm resources with high callus induction rate and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |