LU504641B1 - Development and application of kasp molecular marker for capsicum milky white fruit color - Google Patents
Development and application of kasp molecular marker for capsicum milky white fruit color Download PDFInfo
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- LU504641B1 LU504641B1 LU504641A LU504641A LU504641B1 LU 504641 B1 LU504641 B1 LU 504641B1 LU 504641 A LU504641 A LU 504641A LU 504641 A LU504641 A LU 504641A LU 504641 B1 LU504641 B1 LU 504641B1
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- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 65
- 235000002566 Capsicum Nutrition 0.000 title claims abstract description 56
- 239000001390 capsicum minimum Substances 0.000 title claims abstract description 56
- 239000003147 molecular marker Substances 0.000 title claims abstract description 29
- 238000011161 development Methods 0.000 title description 6
- 240000008574 Capsicum frutescens Species 0.000 title 1
- 241000208293 Capsicum Species 0.000 claims abstract description 59
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000009395 breeding Methods 0.000 claims abstract description 8
- 230000001488 breeding effect Effects 0.000 claims abstract description 8
- 239000003550 marker Substances 0.000 claims abstract description 6
- 241000196324 Embryophyta Species 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 4
- 238000001506 fluorescence spectroscopy Methods 0.000 claims description 4
- 238000003205 genotyping method Methods 0.000 claims description 3
- 230000003247 decreasing effect Effects 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 7
- 238000007844 allele-specific PCR Methods 0.000 abstract description 2
- 230000035772 mutation Effects 0.000 abstract 1
- 238000012408 PCR amplification Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 210000000349 chromosome Anatomy 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000003086 colorant Substances 0.000 description 3
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 238000012257 pre-denaturation Methods 0.000 description 2
- KEQXNNJHMWSZHK-UHFFFAOYSA-L 1,3,2,4$l^{2}-dioxathiaplumbetane 2,2-dioxide Chemical compound [Pb+2].[O-]S([O-])(=O)=O KEQXNNJHMWSZHK-UHFFFAOYSA-L 0.000 description 1
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 description 1
- 240000008384 Capsicum annuum var. annuum Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
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- 101150042675 glk gene Proteins 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
- A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/08—Fruits
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
- A01H6/822—Capsicum sp. [pepper]
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses a KASP molecular marker of capsicum milky white fruit color and its application. According to the invention, a capsicum material B1-2 with milky white fruit color and a capsicum material D50 with dark green fruit color are hybridized to construct a F1 generation, the F1 generation is inbred to obtain a F2 generation, and the locus for controlling the milky white fruit color of capsicum is obtained by applying a BSA-Seq (Bulked Segregant Analysis-Seq) method. Using this mutation site, the marker KASP (Kompetive Allele Specific PCR) is developed. This marker can genotype F2 population, improve the efficiency of breeding selection, and has important application value in capsicum milky white fruit color breeding.
Description
DESCRIPTION LU504641
DEVELOPMENT AND APPLICATION OF KASP MOLECULAR MARKER FOR
CAPSICUM MILKY WHITE FRUIT COLOR
The invention relates to a molecular marker detection technology, and in particular to a KASP molecular marker for identifying capsicum milky white fruits.
Capsicum spp. is not only an important vegetable, but also a popular condiment. widely planted in more than 140 countries all over the world, the sown area in China exceeds 2 million hm? every year. The fruit color of capsicum is an important commodity character, and excellent fruit color appearance will prompt consumers to buy it. As an important breeding goal, fruit color has attracted more and more breeders’ attention.
Capsicum has rich fruit color variation, mostly green, yellow-green, yellow, red, purple, a small amount of milky white and white. Different colors are due to different kinds and quantities of pigment substances contained in capsicum fruits, and different relative contents will show different colors, among which milky white fruit color is due to less or no chlorophyll. According to existing research reports, green fruit color is dominant to white fruit color traits, so varieties and materials with white fruit color traits are rare.
Molecular markers based on the traits of capsicum milky white fruit color can be used to screen and identify capsicum seedling materials, which can improve the selection efficiency and shorten the breeding cycle without waiting for the development stage of capsicum fruit. Compared with the first generation markers based on molecular hybridization technology and molecular markers based on PCR technology, KASP (Kompetive Allele-specific PCR) is a high-throughput genotyping technology based on
SNP, and KASP molecular markers have the advantages of accuracy, short time and strong operability. At present, some sites or genes related to capsicum milky white fruit color have also been reported before, but they have not been developed into effective 59 4641 molecular markers.
In order to solve the problems existing in the prior art, the invention aims to provide a KASP molecular marker for capsicum milky white fruit color. The marker is located in the genome of Capsicum.annuum.L Zunla, at the 8345286th position on chromosome 10, with a nucleotide sequence with a polymorphism of G/A, or contains a nucleotide sequence as shown in SEQ ID NO.1, with a polymorphism of G/A at the 2440th position.
According to the invention, the BSA-seq technology is utilized to find that the last base of the second intron of a CaGLK gene in the mutant of capsicum milky white fruit color is mutated, and the base of the SNP site is mutated from G to A, and the SNP site is located at chromosome 10, position 8345286, and position 2440 of the nucleotide sequence described in SEQ ID NO.1 which has been sequenced, and the position is A.
The color of young capsicum fruit is milky white, and the locus is G, and the young 5is dark green.
The KASP molecular marker of capsicum milky white fruit color can be amplified by the following primers:
Primer_AlleleFAM: (SEQ ID NO.2:
GAAGGTGACCAAGTTCATGCTTTCGATGAGCTCGATATTTT)
Primer_AlleleHEX: (SEQ ID NO.3:
GAAGGTCGGAGTCAACGGATTTTCGATGAGCTCGATATTTC)
Primer_Common: (SEQ ID NO.4: TGCATGTAACTCAAATTCTTAT)
The amplification primer of KASP molecular marker of capsicum milky white fruit color of the invention has the following nucleotide sequence:
Primer_AlleleFAM: (SEQ ID NO.2:
GAAGGTGACCAAGTTCATGCTTTCGATGAGCTCGATATTTT)
Primer_AlleleHEX: (SEQ ID NO.3:
GAAGGTCGGAGTCAACGGATTTTCGATGAGCTCGATATTTC)
Primer_Common: (SEQ ID NO.4: TGCATGTAACTCAAATTCTTAT)
By using the molecular marker provided by the invention, the rapid and accurate identification of the capsicum milky white fruit color can be realized.
Therefore, the invention also provides an application of the molecular marker Plis04641 capsicum milky white fruit color or the primer thereof in identifying the traits of capsicum milky white fruit color.
Specifically, the application comprises the following steps: (1) extracting genomic DNA of capsicum to be detected (2) using the genomic DNA of capsicum to be detected as a template, and amplifying PCR reaction by using the primer labeled by KASP molecule of capsicum milky white fruit color. (3) analyzing the SNP locus of the amplified product, where G corresponds to the dark green young capsicum fruit and A corresponds to the milky white young capsicum fruit.
In the specific embodiment provided by the invention, the system and procedure of the PCR amplification reaction in the step (2) are as follows:
The total volume of PCR amplification reaction system is 1.6L, in which the DNA to be detected is 0.8uL, 2x KASP Master mix + Assay: 0.8 uL. The PCR reaction procedure is as follows: pre-denaturing at 94°C for 15min, 10 cycles (denaturing at 94°C for 20s, extending at 55-60°C for 60s, and decreasing by 0.6°C once per cycle) and 26 cycles (denaturing at 94°C for 20s and extending for 60s).
The analysis of SNP sites in the step (3) refers to reading and analyzing fluorescence data by using IntelliQube machine, where fluorescence FAM excites 485nm to emit 520nm, fluorescence HEX excites 535nm to emit 556nm and fluorescence ROX excites 575nm to emit 610nm. The fluorescent signal is read at the end point (SNPLine/Araya).
Those skilled in the art should know that the PCR amplification system and the reaction procedure are different according to the actual use, or the volume of other reagent components needs to be adjusted, and the reaction temperature and time are different. Therefore, the PCR amplification system, the reaction procedure and the KASP molecular marker data setting and data analysis described in the present invention include but are not limited to the above contents.
The fruit color of capsicum in green maturity is an important commodity character of capsicum. Excellent fruit color can attract consumers to buy, and milky white fruit color is a scarce capsicum germplasm resource. The invention provides an application of the 504641 molecular marker and the primer thereof in molecular marker-assisted breeding for identifying and cultivating creamy white capsicum young fruits.
Finally, the invention provides a detection reagent or a kit containing the molecular marker primer.
The kit also contains dNTP, DNA polymerase, PCR reaction buffer, PCR amplification primers and experimental consumables required by PCR experiments.
The invention has the beneficial effects that: 1. The molecular marker provided by the invention can accurately, rapidly and specifically identify the traits of young capsicum milk white fruit, and realize the seedling identification of capsicum fruit color. 2. The data can be read by amplification with the KASP molecular marker primer provided by the invention, without polyacrylamide gel electrophoresis operation, and the result is reliable and stable, with the advantages of rapidness and convenient detection.
FIG. 1 shows the results of ASNP-index of the test material distributed on 12 chromosomes in Embodiment 1 of the present invention.
FIG. 2 shows the genotyping results of F2 population by KASP markers in
Embodiment 2; where the X-axis is FAM ray, and the Y-axis is HEX-ray. AA and aa are homozygous genotypes, and Aa is heterozygous genotype.
DESCRIPTION OF THE INVENTION LU504641
Embodiment 1 Development and application of KASP molecular marker for capsicum milky white fruit color 1. Capsicum materials and construction of recombinant inbred lines
The high-generation inbred line capsicum B1-2 with milky white fruit color mutant is used as the female parent, and the dark green capsicum D50 is used as the male parent, and F4 is obtained and represented as green. F4 is inbred to obtain F2 generation with milky white, yellow-green and dark green fruit colors, and the phenotypic traits are separated. 2. Preliminary mapping of related loci in milky white young fruits of capsicum
Selecting 30 capsicum plants with extreme phenotypes of milky white fruit color and capsicum plants with extreme phenotypes of dark green in Fa population, extracting
DNA from each plant by CTAB method, adjusting the DNA concentration to the same, and mixing the DNA of 30 young white fruit plants and 30 young dark green fruit plants to construct a mixed pool, named W-pool and G-pool. Samples of B1-2, D50, W-pool and
G-pool are sent to Huada Gene Co., Ltd. for the second generation sequencing of molecules. The depth is 50x.
Taking Capsicum.annuum.L var. Zunla as the reference genome, after splicing and filtering the sequencing data, the correct double-end matching degree is more than 89% and the sequencing depth is more than 47.15. The SNP of two parents, W-pool and
G-pool, was detected by GATK (GATK3.4) software. The SNP-index parameters are calculated by sliding window, and the results are analyzed by correlation. The results show that the SNP locus related to milky white fruit is controlled at 39.54M on chromosome 10. As shown in FIG. 1. 3. Development of molecular markers
There is a reported GLK gene in the localization interval, which is involved in the regulation of tomato fruit color formation, so it is inferred that the gene CaGLK may be involved in the formation of capsicum milky white young fruit. Primers CaGLK-F: (SEQ
ID No.5: ATGATGCTTGTTGTATCTACACCATTGAG) and CaGLK-R: (SEQ ID No.6:
TCAAGTTGGAGGTATTTTTGTAATCCCTTGA) are designed, and the CaGLK genes in parents B1-2 and D50 are amplified and sequenced. It is found that in parents B1-2, The 504641 base of the 2440 site of CaGLK gene is A, and the base of D50 is G with that of the reference genome Capsicum.annuum.L Zunla. The KASP molecular marker primers are designed through the variation of this SNP site.
Primer_AlleleFAM: (SEQ ID NO.2:
GAAGGTGACCAAGTTCATGCTTTCGATGAGCTCGATATTTT)
Primer_AlleleHEX: (SEQ ID NO.3:
GAAGGTCGGAGTCAACGGATTTTCGATGAGCTCGATATTTC)
Primer_Common: (SEQ ID NO.4: TGCATGTAACTCAAATTCTTAT)
The specific PCR reaction system and procedures are as follows:
The total volume of PCR amplification reaction system is 1.6L, in which the DNA to be detected is 0.8L, 2x KASP Master mix + Assay: 0.8 uL. The PCR reaction procedure is pre-denaturation at 94°C for 15min, 10 cycles (denaturation at 94°C for 20s, extension at 55-60°C for 60s, with each cycle reduced by 0.6°C) and 26 cycles (denaturation at 94°C for 20s and extension for 60s). 4. Data reading
Using IntelliQube machine to read and analyze the fluorescence data; where fluorescence FAM excites 485nm to emit 520nm, fluorescence HEX excites 535nm to emit 556nm and fluorescence ROX excites 575nm to emit 610nm. The fluorescent signal is read at the end point (SNPLine/Araya).
According to the invention, the KASP molecular marker is used for identifying the young capsicum milky white fruits, so that the color of the young capsicum fruits can be accurately, rapidly and massively predicted, and the breeding selection efficiency of the young capsicum milky white fruit is improved.
Embodiment 2 Identification of KASP molecular markers of capsicum milky white fruit color in F2 population
F1 generation is obtained by crossing milky white young fruit B1-2 with dark green young fruit D50, and F2 population is inbred by F4. In 2015, 3000 F2 plants are planted in
Quzhou Academy of Agricultural Sciences, and DNA is extracted from each plant by
CTAB method at seedling stage for KASP marker detection. The reaction system is as follows: DNA to be detected is 0.8uL, 2x KASP Master mix + Assay: 0.8 pL. The reaction procedure is pre-denaturation at 94°C for 15min, 10 cycles (denaturation at 94°C for 208 504641 extension at 55-60°C for 60s, with each cycle reduced by 0.6°C) and 26 cycles (denaturation at 94°C for 20s and extension for 60s). The IntelliQube machine reads and analyzes the fluorescence data, and the fluorescence FAM excites 485nm to emit 520nm, the fluorescence HEX excites 535nm to emit 556nm, and the fluorescence ROX excites 575nm to emit 610nm. The fluorescent signal is read at the end point (SNPLine/Araya), as shown in FIG. 2.
When the capsicum plants grow to green maturity, 2264 capsicum plants are counted, where 555 plants are milky white, 637 plants are dark green and 1072 plants are yellow-green, which are between the male parent and the female parent, and are completely consistent with the KASP tag.
The KASP molecular marker can accurately predict the color of young fruits in different capsicum materials, and has important application value in breeding work.
The general description and implementation used in this paper describe the present invention in detail, but some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art. Therefore, modifications or improvements made on the basis of not deviating from the core content of the present invention belong to the scope of the present invention.
SEQUENCE LISTING LU504641
Quzhou Academy of Agricultural and Forestry Sciences Quzhou Academy of
Agricultural and Forestry Sciences Development and application of KASP molecular marker for capsicum milky white fruit color 6 6066 DNA PAT source 1..6066 mol_type other DNA organism synthetic construct atgatgcttgttgtatctacaccattgagctacaaaaatgaaaggggaaattatgatttatt tcaagattttcctgatgggaatttaatcgacaccattgattttgatgacttttttgagggaatcaacgatggagatttge tgcaaaatttgaaaatccttgatgaatttgatattagcaagaataatacaactactaatctaaatgtgaaaacaaagtca aaggaaaatgataaatccaagaaatcgtcaagccaaatcaagaatcctgaagggaagaaaaaagtgaaggtaatttit tt tttgttatattattattatitttgaaaaaaaaaaagataaticaaaaatatcatctccatgcticggaatttaggtttit ctgtaagagtttaagttatatacattatagcgtaatgaaattttgcatcattatataacctttgtatgttttagcaatta gtagtttgtctcacttitaagattacaaatgtcacgtgttaaagaggttagattatattacttgttgatcgatatctttt gactgacttgattatctgtigtagcaaagaataatgatttacatgatgtcgatattctttgaatgacctgatagtataaa aaaaaacttttatcatgcatgtaatccatgaaactaaagatcactagctagttatagtcaatttctatgaagaatttaag ttatttatatggataatgtaataaatttttaataaaaatattgtgggtaattgacctttittcccaaatgaaagtttaat ttcttgaagtatgttgttgttgttcatattgtctctcticccaattttgttcaacatggctgagacattacattattata ttttccatacttgatttattatgctttatitaaticgagaatctatcaaaaatagatttacgtatatattttcatatatt ttatttatgagattactctgaataagacgttgtigtigttaaggttgattggactccagagctgcacaggagatttgtaa aagcagtagagaaattaggtgtggataaagcagttccatcaagacttttagagcttatggctactgatggtctcactaga cataacattgccagccatcttcaagtaaattttcttcgaaaaaaactcagtcaticttaattagagattttgagttcgag ttttgaatgtgaaattagtatcttattaggaagtgctctcctagatgtgaaagtttccgattagcgtgaaacaggtaccg cacatcagacagagaaccaaaaagtaatgatgtgaagaaatatttatataatcatccttaatcagaaatcttgagtttaa atcttgtatatggagttatcttatagcgagcattctctccgatataatgtgagaatticgatiggaagaatccaattttt tcccacttttttcattaaaatgaaaaaagaaaatcagataaaagtgatgaaaticicatagtcaaatgcttatttagtct ccaatacaactatcgaacatcgagcgaagaaataaaaaaaaattatatttaaaaaataatacttatatcgagggaagaa
C taaaaaaatattaaacaattatacgtattttcttgagtttcaataccttttaggttgtcattttccatatatgagaaagg caagaatactaatttagtatttictitaaagagaaaagataattticttgcctgaaaatttgctcataatagaaacatgtg tgaacgaaaaaatgaacaattaggacctagttcaaatttgcatcttctgaaggaaactcttatgaatcaatgagagattt cgaatttgaatatcttaaaatgaaaaaaataaattattaggaccactgccttttaactagaaatagacactacaatgcta gcatgtcacagtattgttttttcaaaagtattctagtticaattaaatttagtttaatttatttttgatgtctatgtcgaa caccagatgaaaaaaagagaattatgaaactaataagaaaactatctccaaaaatagatcatacaatgctagaatgtta | 504641
C agtattatttttttgtaattactctagttcaattagatttgattaatataataactctcttcatttaaaattttatattt tagttttaatctgttttaaaaaaaaaaattagctttatctaattaaacctagaaagttttcatacttacattcttttgat tatattgaaaggagggagtatctttttaaatcttattgttatatgagataagtgtttttttatatatatatattattaga ttctgttcacatataataataagtaaatttcctcaataagcatgatatagtaacttgaaaaaagttatgatgatattaaa tttgtttatattttttatgcatgtaactcaaattcttattattattacacatatgcaaaaatatcgagctcatcgaaaac atttattagcaagggaagctgaggcagcaagctggacccaaaggaagcaaatgtatgatggagccattgcgatcggag gc ggagggaagagagttataatgaacccatggtctgcaccaccaaccatgggttttccacccatggctcatcatattagacc cttacatgtttgggggcatccatatgtaaataattctttttggcatccacattatcaaggagtgagttccttttatatat atattctttattattttgttttcttcttatcttatcgtatgactggacacaaagttttaagttaaaaaggagaaaaaagt ttgaaaataaaatttgttataacacgcttaaattttgcgggaattctatgattcacctcgactatttattatcgtacttc tgtgtcatatatttgcctaactggaccactatgtgaatactcatacactgagcatgtgagggtttgaagtggtccagttg gacaggtatatgccacaaaaatacgtaataaatagtcgagggggtcataggattcccgtaaagttgaggcegtgttacaat aaatttcgtcaaaatttaggtatatttctgacccttticccttaaaaaaaatgaaaatcttigaaaatttatgattatata acatatttaacatttatacgattatattattttgaatttcttatgatctaaatatgatataatatttgtgtggtggttaa gttagtgtaacaaatgattttccctgtgagtggtactcaaggattgattctctigttaatcaatcgagttcgtcacatca agcttctcttagggticcaaacaaatatatttatatcgttaatatatatatatatatatatatatatatgtacttgtata aatcataaataacatcaataagcatgaaagtgagtacattatgtttcccaaattacacctcccaaaaagtataaagtcgt tgtgaatcaaatatagaacccaacaaggttggagtcaaacacacagagaataggacaaaatttgatcaagtattigtcta ggtaactggtatatatatattttagaatacaagttttaaaaagagttatttattaaaatttatgatgctcaatcaaatat cgttacataaattgaaatatttgttgcaggtaccaaattctcttgcaccaggcactccttgctttccttcaccaacggta gctactttttctaagttttattgtttcagtctcgtactagtatttagcagtgttttaaagagcgggggcegtaagttaaag cgttttatttaacataggtttcaaagcaatgaggcgtaagcecttatgaaacttatttittaatttataatgtagtaaaat aacatcataaatatagacacctattttticctaagttttgttagcgagccccaagceattgacggtttggegtgcettagggt gtagaatcagacgcttaaggtataagtctcataaaattaagccttttacctatgcctcgaggtgttitgccagtgctttg cttccaaagcgagcctcaaaactaccttttaaaacactgatatttaggaggtgtttgaccaagtaatttagagctcaagt ttaaaattttaatctattagcgataagcaaagaaattaagaaaatcaaatttgagtaaagcatttagtacccctttgaac tatgaccaaatttgttatgacacactttaactttacggggatcctattacccctaaactaaattttagegtatttitatc atcctttagttgacgtgatactttttgtcaacctttttaactgacatgacaactttaatgtggactccattitatataag aaaaatatcacgtctgcacaaaagagtgacagaaatacgttaaaattgagtttaagagagtaataggaccctctaaagtt gaagtgtgtcatatcaactttgatcataattcaaaaaattattgaatacttatctcatcaaattttattatatataaata LU50464- aaataataagtttaattttaaatatttgatactataatattttgctaaatatgaaccccttacaaccacgtgactcgect ttgcagagatttgcagcagctcttatggtccctggegttccaccaccctttgcatcaggacaaactccacatttgecatcece totgagecttttttgtctctcaacaattataattatttcccctacctacattgacttgaaacaaaatttaagaaaataaa gaatatttttaaattttatgaccttaaattaaagttacattaaatatatcaaaatactctttaatcttatgatcctaaat atgccatgtgaaaaatagaaactaaagtattotcaaaaaaaaaaaaagttattttttgaaacgaactaaaaataaaagca gatcattctttttgaaactgagagagtattagtatatgatttattgctataagaatatctatatttaaatatagagttag gattttaaattttaaaattaaatgacgattttaagtcttaataattaagattgttiggtttaaaagaacatgttagtttg aaaaatattttctactaacataagtgaattttacttattticttatgttigttatgtaagcaaaaatatttatcataaag aatatacaatgtcacttggaaaacttgtttttcatacttttactagagaagttatttttticttttttaaataacttatt ttttcagagaaagtgtctticaaaaattttgacaaatcaaacatgaaaatttttaacaaattgaatacaccctacgttct aaatttaatatttgtgtgcatttaatgaattttttittgatacaaatacattatttaagcaaatgctattaaatttgatc gaacaattatcttcagacttctagetetgctectgatatgattttttttttttaatatttattataagttttaaaagtct tteccttattttctaaatttatcgacctagtggettaagccaaattttacaccaacagagttcatcatctactatataaac atttaaaaaaaaaaaaagacaaataatccagtactccgtcgaactatgaccaaagttgctacgacacactccaacatca
C aaggatcctattaccccctgagctcaaatttaacatatttttgtcacccttttgtgctgatgtggtatctttattacata aaatgaggctcacatcaaagatgtcacatcagctaaagaggttgataaaaggtatcacgtcagctaaaaagattgataa a aatatgctaaaatttagtttggaggggggaggggttaataggaccgtgaagtiggaatgtatcatagcaaatttgatcat aattcagagatgtactagatgctttactcaaacaaaaaaaatgaaccttatatataggatgaaattttactgacatatta tcttgatatttgttacacagacaaaagagagcatagatgcagcaattgaagatgttttatcaaagccacaaacgcecacitt cctataggattgaaacctccatcaattgatagtgtgttgaatgaattacaatgtcaagggattacaaaaatacctccaac ttga 41 DNA PAT source 1..41 mol_type other DNA organism synthetic construct gaaggtgaccaagttcatgctttcgatgagctcgatatttt 41 DNA PAT source 1..41 mol_type other
DNA organism synthetic construct gaaggtcggagtcaacggattttcgatgagctcgatatttc 22 DNA
PAT source 1.22 mol_type other DNA organism synthetic construct tgcatgtaactcaaattcttat 29 DNA PAT source 1..29 mol_type other DNA organism synthetic construct atgatgcttgttgtatctacaccattgag 31 DNA PAT source 1..31 mol_type other DNA organism synthetic construct tcaagttggaggtatttttgtaatcccttga
Claims (10)
1. A KASP molecular marker for identifying the color of capsicum milky white fruit, characterized in that the nucleotide sequence of the molecular marker primer is shown in SEQ ID NO 1.
2. The molecular marker according to claim 1, characterized in that the KASP molecular marker is used to genotype individual plants with milky white fruit color and green fruit color; the genotypes of milky white fruit, dark green fruit color and yellow-green fruit color heterozygosity between milky white and dark green are screened out.
3. For detecting primers for the molecular marker according to claim 1 or 2, characterized by comprising Primer_AlleleFAM: (SEQ ID NO.2: GAAGGTGACCAAGTTCATGCTTTCGATGAGCTCGATATTTT) Primer_AlleleHEX: (SEQ ID NO.3: GAAGGTCGGAGTCAACGGATTTTCGATGAGCTCGATATTTC) Primer_Common: (SEQ ID NO.4: TGCATGTAACTCAAATTCTTAT).
4. An application of the molecular marker of claim 1 or 2 or the primer of claim 3 in identifying the fruit color of capsicum.
5. The application according to claim 4, characterized by comprising the following steps: (1) extracting genomic DNA of capsicum to be detected; (2) carrying out genotyping by using the genomic DNA of the capsicum to be tested as a template and using the primer of claim 3.
6. The application according to claim 5, characterized in that the reaction system in the step (2) is 1.6uL, comprising DNA: 0.8 pL, 2x KASP Master mix + Assay: 0.8 uL.
7. The application according to claim 5, characterized in that the PCR reaction 504641 procedure in the step (2) is pre-denaturing at 95°C for 15min, 10 cycles (denaturing at 94°C for 20s, extending at 50-60°C for 60s, and decreasing by 0.6°C once per cycle) and 26 cycles (denaturing at 94°C for 20s and extending at 55°C for 60s).
8. The application according to claim 5, characterized in that in the step (2), the fluorescence data are read and analyzed by using an IntelliQube machine, and fluorescence FAM excites 485nm to emit 520nm, fluorescence HEX excites 535nm to emit 556nm and fluorescence ROX excites 575nm to emit 610nm; The fluorescent signal is read at the end point (SNPLine/Araya).
9. The application according to claim 5, characterized in that the marker test results show that the KASP marker is capable of stably distinguishing the capsicum with white fruit color and the capsicum with green fruit color.
10. An application of the molecular marker of claim 1 or 2 or the primer of claim 3 in molecular marker-assisted breeding of capsicum milky white fruit color.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU504641A LU504641B1 (en) | 2023-06-30 | 2023-06-30 | Development and application of kasp molecular marker for capsicum milky white fruit color |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU504641A LU504641B1 (en) | 2023-06-30 | 2023-06-30 | Development and application of kasp molecular marker for capsicum milky white fruit color |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| LU504641B1 true LU504641B1 (en) | 2024-02-01 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| LU504641A LU504641B1 (en) | 2023-06-30 | 2023-06-30 | Development and application of kasp molecular marker for capsicum milky white fruit color |
Country Status (1)
| Country | Link |
|---|---|
| LU (1) | LU504641B1 (en) |
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2023
- 2023-06-30 LU LU504641A patent/LU504641B1/en active IP Right Grant
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Effective date: 20240201 |