LU504454B1 - Molecular markers for distinguishing dafang line peppers and dafang wrinkled peppers and application thereof - Google Patents

Molecular markers for distinguishing dafang line peppers and dafang wrinkled peppers and application thereof Download PDF

Info

Publication number
LU504454B1
LU504454B1 LU504454A LU504454A LU504454B1 LU 504454 B1 LU504454 B1 LU 504454B1 LU 504454 A LU504454 A LU 504454A LU 504454 A LU504454 A LU 504454A LU 504454 B1 LU504454 B1 LU 504454B1
Authority
LU
Luxembourg
Prior art keywords
dafang
indel
seq
pepper
insdseq
Prior art date
Application number
LU504454A
Other languages
German (de)
Inventor
Dongfu Huang
Original Assignee
Guizhou Pepper Res Institute
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Guizhou Pepper Res Institute filed Critical Guizhou Pepper Res Institute
Priority to LU504454A priority Critical patent/LU504454B1/en
Application granted granted Critical
Publication of LU504454B1 publication Critical patent/LU504454B1/en

Links

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
    • 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
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Botany (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the field of biotechnology, in particular to molecular markers for distinguishing Dafang line peppers and Dafang wrinkled peppers and application thereof, and molecular marker combination comprises molecular markers chr4-InDel-8, chr5-InDel-22, chr6-InDel-8, chr7-InDel-39, chr10-InDel-39 and chr11-indel-38. According to the invention, six pairs of InDel molecular marker primers evenly distributed on six chromosomes are screened out, and after PCR amplification is respectively carried out on the Dafang line pepper and the Dafang wrinkled pepper by using the six pairs of InDel molecular marker primers, the amplified PCR products are subjected to 3% agarose gel electrophoresis to obtain an identification gel map, and then the identification gel map is converted into a 0,1 data matrix, and the Dafang line pepper and the Dafang wrinkled pepper can be identified according to the 0,1 data matrix.

Description

DESCRIPTION LU504454
MOLECULAR MARKERS FOR DISTINGUISHING DAFANG LINE PEPPERS AND
DAFANG WRINKLED PEPPERS AND APPLICATION THEREOF
TECHNICAL FIELD
The invention relates to the field of biotechnology, in particular to molecular markers for distinguishing Dafang line peppers and Dafang wrinkled peppers and application thereof.
BACKGROUND
Dafang peppers are mainly "wrinkled peppers", "chicken feet peppers" and "line peppers", which have enjoyed the reputation of "Guizhou wrinkled peppers", "tribute peppers" and "pride among peppers" in history. They are characterized by fleshy meat, bright red color, moderate spicy taste, rich fragrance, rich in various amino acids, minerals and vitamins, and the content of red pigment is 2.5%-3.0%.
At present, the identification of Dafang line pepper and Dafang wrinkled pepper still relies on traditional evaluation methods, such as physical and chemical index detection and artificial sensory evaluation. Both of these methods have some problems, such as complex sample pretreatment and chemical reagents, which pose a threat to operators and ecological environment; the artificial sensory evaluation method has the disadvantages of strong subjectivity, poor repeatability, complex influencing factors and long time consumption. These shortcomings lead to the two methods cannot be widely promoted in the market; therefore, it is urgent to develop a new detection technology.
SUMMARY LU504454
The purpose of the present invention is to provide molecular markers for distinguishing Dafang line peppers and Dafang wrinkled peppers and application thereof, so as to solve the problems existing in the prior art. The molecular markers provided by the present invention can accurately and efficiently identify Dafang line pepper and
Dafang wrinkled pepper.
In order to achieve the above objectives, the present invention provides the following scheme.
The invention provides molecular markers for distinguishing Dafang line peppers and Dafang wrinkled peppers, wherein molecular marker combination comprise molecular markers chr4-InDel-8, chr5-InDel-22, chr6-InDel-8, chr7-InDel-39, chr10-InDel-39 and chr11-indel-38; the chr4-InDel-8 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.1-2; the chr5-InDel-22 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.3-4; the chr6-InDel-8 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.5-6; the chr7-InDel-39 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.7-8; the chr10-InDel-39 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.9-10; the chr11-InDel-38 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.11-12.
The invention also provides primer pair combination for distinguishing Dafang line peppers and Dafang wrinkled peppers, comprising primer pairs as shown in SEQ ID
NO.1-2, primer pairs as shown in SEQ ID NO.3-4, primer pairs as shown in SEQ ID
NO.5-6, primer pairs as shown in SEQ ID NO.7-8, primer pairs as shown in SEQ ID
NO.9-10 and primer pairs as shown in SEQ ID NO.11-12.
The invention also provides an application of the primer pair combination 1504454 preparing a Kit for identifying Dafang line pepper and Dafang wrinkled pepper.
The invention also provides a kit for identifying Dafang line pepper from Dafang wrinkled pepper, comprising the primer pair combination.
The invention also provides a method for distinguishing Dafang line pepper from
Dafang wrinkled pepper, comprising: (1) obtaining genomic DNA of samples to be detected; (2) using the genomic DNA as a template, using the primer pairs in the primer pair combination according to claim 2 to perform PCR amplification respectively; after detecting the amplified products by electrophoresis, obtaining an identification gel map, and then converting into a 0,1 data matrix, wherein the band is marked as 1 and the band is marked as 0: (3) according to the 0,1 data matrix, judging whether the sample to be detected is
Dafang line pepper or Dafang wrinkled pepper: when the primer pairs shown in SEQ ID
NO.1-2, the primer pairs shown in SEQ ID NO.3-4, the primer pairs shown in SEQ ID
NO.5-6, the primer pairs shown in SEQ ID NO.7-8, the primer pairs shown in SEQ ID
NO.9-10 and the primer pairs shown in SEQ ID NO.11-12 have the 0, 1 data matrices of the amplified products as follows: (0, 1), (1, 0), (1, 0), (0, 1), (1, 0), (1, 0), (0, 1), (1, 0), (0, 1), and (0, 1), the sample to be tested is Dafang line pepper; when the primer pairs shown in SEQ ID NO.1-2, the primer pairs shown in SEQ ID NO.3-4, the primer pairs shown in SEQ ID NO.5-6, the primer pairs shown in SEQ ID NO.7-8, the primer pairs shown in SEQ ID NO.9-10 and the primer pairs shown in SEQ ID NO.11-12 have a 0-1 data matrix of (1, 0), (0, 1), (0, 1), (1, 0), (0, 0), (0, 1), (0, 1), (1, 0), (1, 0), and (1, 0) for the amplified products, the sample to be tested is Dafang wrinkled pepper.
Further, in step (2), the reaction system of PCR amplification includes 1 uL of DNA, 1 HL of 10 umol/L InDel forward primer, 1 UL of 10 umol/L InDel reverse primer, 12.5 uL of 2xPCR reaction mixture, 0.3 uL of DNA polymerase and 9.2 pL of ultrapure water.
Further, in step (2), the reaction procedure of PCR amplification comprises: 94°C for min; 35 cycles at 94°C for 30 s, 65°C for 30 s and 72°C for 30s; 72°C for 5 min.
The invention also provides an application of molecular marker combination, primét)504454 pair combination or kit in the identification of Dafang line pepper and Dafang wrinkled pepper.
The invention discloses the following technical effects:
According to the invention, six pairs of InDel molecular marker primers evenly distributed on six chromosomes are screened. The six pairs of InDel molecular markers were used for PCR amplification of pepper and pepper, respectively. The amplified PCR products are subjected to 3% agarose gel electrophoresis to obtain the identification gel map, and then the identification gel map is converted into a 0,1 data matrix, according to which the pepper and pepper could be identified.
The invention realizes the rapid identification of the Dafang line pepper and the
Dafang wrinkled pepper, is convenient and quick to operate, uses less samples and does not need complicated sample pretreatment, and has high application and popularization values.
DESCRIPTION OF THE INVENTION
A number of exemplary embodiments of the present invention will now be described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.
It should be understood that the terminology described in the present invention is only for describing specific embodiments and is not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Intermediate values within any stated value or stated range, as well as each smaller range between any other stated value or intermediate values within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
Unless otherwise specified, all technical and scientific terms used herein have tH&J504454 same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated document, the contents of this specification shall prevail.
It is obvious to those skilled in the art that many improvements and changes can be made to the specific embodiments of the present invention without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the invention. The description and example of that present invention are exemplary only.
The terms "comprising", "including", "having" and "containing" used in this article are all open terms, which means including but not limited to.
Example 1 (1) The whole genome of "Niuchang Pepper" was re-sequenced, and compared with the reference genome, a large number of InDel markers were preliminarily screened out. (2) From the InDel markers obtained by preliminary screening, the InDel markers with difference <4bp and heterozygosity were removed, and the rest were used as InDel marker library. (3) In the InDel marker library, according to the size of each chromosome, 21 InDel markers are evenly selected on each chromosome. Pepper has 12 chromosomes, and 252 InDel markers were selected. (4) For 252 InDel markers, design InDel primers according to the principle that the primer length is 18-24bp, the GC content is 40%-60%, and the product length is about 300bp; two pairs of InDel primers were designed for each InDel marker, and a total of 504 pairs of InDel primers were designed. (5) Validation of 504 pairs of InDel primers:
S1: the verification sample is Zunla No.1, Ping Huang pepper; LU504454
S2, extracting the DNA of the verification sample by using a plant DNA extraction kit;
S3, using 504 pairs of InDel primers to carry out PCR amplification on the extracted verification sample DNA;
S4, the total reaction system of PCR amplification is 25uL, including 1uL of DNA, 1uL of 10pmol/L InDel forward primer, 1 UL of 10umol/L InDel reverse primer, 12.5uL of 2xPCR reaction mixture, 0.3uL of DNA polymerase and 9.2 uL of ultrapure water.
S5, the procedure of PCR amplification is: 94°C for 5min; 35 cycles at 94°C for 30s, 65°C for 30s and 72°C for 30s; 5 min at 72°C and stored at 4°C;
S6, the amplified PCR products were subjected to 3% agarose gel electrophoresis, and the obtained data were analyzed. InDel labeled primer pairs without amplification bands and polymorphism were removed, and 47 pairs of InDel primers with high stability and good polymorphism were obtained. (6) 47 pairs of InDel primers were used to identify Dafang line pepper and Dafang wrinkled pepper;
S1, using a plant DNA extraction kit to extract the DNA of the big pepper and the big wrinkled pepper;
S2, using 47 pairs of InDel primers to amplify the DNA of pepper and pepper by
PCR;
S3, the total reaction system of PCR amplification is 25uL, including 1uL of DNA, 1uL of 10pmol/L InDel forward primer, 1 y | of 10pmol/L InDel reverse primer, 12.5uL of 2xPCR reaction mixture, 0.3uL of DNA polymerase and 9.2 yu | of ultrapure water.
S4, the procedure of PCR amplification is: 94°C for 5min; 35 cycles at 94°C for 30s,
TM°C for 30s and 72°C for 30s; 5 min at 72°C and stored at 4°C;
S5, the amplified PCR products were subjected to 3% agarose gel electrophoresis, and the identification gel map was obtained. The obtained data were analyzed, and
InDel labeled primer pairs without amplification bands and polymorphism were removed to verify whether 47 pairs of InDel primers had fragment polymorphism. Results Six pairs of InDel molecular marker primers were screened out, which were evenly distributed on six chromosomes. 6 pairs of InDel molecular marker primers are shown in Table 1.
SE: transforming the identification gel diagram into a 0,1 data matrix. According tdJ504454 the characteristic bands on the rubber map, the bands with clear, no drag and easy to distinguish in the same position of Dafang line pepper and Dafang wrinkled pepper are recorded as 1 and no band as 0, and the 0,1 data matrix is established. The results are shown in Table 2.
The results showed that six pairs of InDel molecular marker primers could be used to identify Dafang line pepper and Dafang wrinkled pepper, and the identification criteria were as follows:
If the 0,1 data matrix of chr4-InDel-8, chr5-InDel-22, chr6-InDel-8, chr7-InDel-39, chr10-InDel-39, chr11-InDel-38 are in the order of (0,1), (1,0), (1,0), (0,1), (1,0), (0,0), (0,1), (0,1 ), then the detected pepper sample is Dafang line pepper; if the 0,1 data matrix of chr4-InDel-8, chr5-InDel-22, chr6-InDel-8, chr7-InDel-39, chr10-InDel-39, and chr11-InDel-38 are (1, 0), (0, 1), (0, 1), (1, 0), (0, 1), (0, 1), (0, 1), and (1, 0) in that order, the detected pepper sample is a Dafang wrinkled pepper.
Table 1 Six pairs of InDel molecular marker primers for identifying Dafang lingJ504454 pepper and Dafang wrinkled pepper
Molecular Serial
Primer | Nucleotide sequence (5'-3') Chromosome marker number
Forward SEQ ID
GGCTCCACTCTGTTGGTTAG primer NO.1 chr4-InDel-8 4
Reverse SEQ ID
GGGGTAAGGCGAGACATTTT primer NO.2
Forward SEQ ID
ACATTGGTGTTAGTCTTGGCATTA primer NO.3 chr5-InDel-22 5
Reverse SEQ ID
AGGGCTTAGTCGTGTTCGTTC primer NO.4
Forward SEQ ID
TATTCTGTGGACTTGACGCA primer NO.5 chr6-InDel-8
Reverse SEQ ID
TTTACCGAGGGTTCATCTGA primer NO.6
Forward SEQ ID
GTTCGCACCATCTTCCTTTT primer NO.7 chr7-InDel-39 7
Reverse SEQ ID
TACTACCCATCACATTACAC primer NO.8
Forward SEQ ID
TTGGCATATAACAAATCCCT primer NO.9 chr10-InDel-39 10
Reverse SEQ ID
CTAGTTTTTCTGGTTTGCGT primer NO.10
Forward SEQ ID
AGCATGATTTCTTTTTCTCA primer NO. 11 chr11-InDel-38 11
Reverse SEQ ID
ACTACTTTTGAGGATCCGAC primer NO.12
Table 2 0,1 data matrix of 6 pairs of InDel primers for detecting Dafang line peppét)504454 and Dafang wrinkle pepper chr4-InDel- | chr5-InDel- | chr6-InDel- | chr7-InDel- | chr10-InDel | chr11-InDel e ee ee ban |ban |ban |ban |ban |ban ‘ban |ban |ban |ban |ban | ban
RSS
Dafa ng line 1 1 1 1 1 1 pepp er
Dafa ng wrink 1 1 1 1 1 1 le pepp er
SEQUENCE LISTING LU504454 <ST26SequenceListing dtdVersion="V1_3" fleName="sequence.xml" softwareName="
WIPO Sequence" softwareVersion="2.3.0" productionDate="2023-06-08"> <ApplicantFileReference> Guizhou Pepper Research
Institute </ApplicantFileReference> <ApplicantName languageCode="en"> Guizhou Pepper Research
Institute </ApplicantName> <InventorName languageCode="en">Dongfu Huang </InventorName> <InventionTitle languageCode="en">MOLECULAR MARKERS FOR DISTINGUISHING
DAFANG LINE PEPPERS AND DAFANG WRINKLED PEPPERS AND APPLICATION
THEREOF </InventionTitle> <SequenceTotalQuantity>12</SequenceTotalQuantity> <SequenceData sequencelDNumber="1"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q2"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier>
</INSDFeature_quals> LU504454 </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>ggctccactctgttggttag</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="2"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q4"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>ggggtaaggcgagacatttt</INSDSeq_sequence> </INSDSeq> </SequenceData>
<SequenceData sequencelDNumber="3"> LU504454 <INSDSeq> <INSDSeq_length>24</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..24</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q6"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>acattggtattagtettggcatta</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="4"> <INSDSeq> <INSDSeq_length>21</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table>
<INSDFeature> LU504454 <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..21</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q8"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>agggcttagtcgtgttcgttc</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="5"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value> LU504454 </INSDQualifier> <INSDQualifier id="q10"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>tattctgtggacttgacgca</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="6"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q12"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier>
</INSDFeature_quals> LU504454 </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>tttaccgagggttcatctga</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="7"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q14"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>gttcgcaccatcttcctttt</INSDSeq_sequence> </INSDSeq> </SequenceData>
<SequenceData sequencelDNumber="8"> LU504454 <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q16"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>tactacccatcacattacac</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="9"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table>
<INSDFeature> LU504454 <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q18"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>ttggcatataacaaatccct</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="10"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSD Qualifier_value> LU504454 </INSDQualifier> <INSDQualifier id="q20"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>ctagtttttctgatttgcat</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="11"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q22"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier>
</INSDFeature_quals> LU504454 </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>agcatgatttctttttctca</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="12"> <INSDSeq> <INSDSeq_length>20</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..20</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q24"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>synthetic construct</INSD Qualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>actacttttgaggatccgac</INSDSeq_sequence> </INSDSeq> </SequenceData>
</ST26SequenceListing> LU504454

Claims (8)

CLAIMS LU504454
1. Molecular markers for distinguishing Dafang line peppers and Dafang wrinkled peppers, wherein molecular marker combination comprises molecular markers chr4-InDel-8, chr5-InDel-22, chr6-InDel-8, chr7-InDel-39, chr10-InDel-39 and chr11-indel-38; chr4-InDel-8 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.1-2; chr5-InDel-22 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.3-4; chr6-InDel-8 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.5-6; chr7-InDel-39 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.7-8; chr10-InDel-39 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.9-10; chr11-InDel-38 is obtained by using the capsicum genome as a template and amplifying the primer pair shown in SEQ ID NO.11-12.
2. A primer pair combination for distinguishing Dafang line peppers and Dafang wrinkled peppers, comprising primer pairs as shown in SEQ ID NO.1-2, primer pairs as shown in SEQ ID NO.3-4, primer pairs as shown in SEQ ID NO.5-6, primer pairs as shown in SEQ ID NO.7-8, primer pairs as shown in SEQ ID NO.9-10 and primer pairs as shown in SEQ ID NO.11-12.
3. An application of the primer pair combination according to claim 2 in preparing a kit for identifying Dafang line pepper and Dafang wrinkled pepper.
4. A kit for identifying Dafang line pepper and Dafang wrinkled pepper, comprising the primer pair combination according to claim 2.
5. A method for distinguishing Dafang line pepper and Dafang wrinkled pepper/504454 comprising: (1) obtaining genomic DNA of samples to be detected; (2) using the genomic DNA as a template, using the primer pairs in the primer pair combination according to claim 2 to perform PCR amplification respectively; after detecting the amplified products by electrophoresis, obtaining an identification gel map, and then converting into a data matrix 0 or 1, wherein the data matrix with the band is marked as 1 and the data matrix without the band is marked as O; (3) according to the data matrix 0 or 1, judging whether the samples to be detected are Dafang line pepper or Dafang wrinkled pepper: when the primer pairs shown in SEQ ID NO.1-2, the primer pairs shown in SEQ ID NO.3-4, the primer pairs shown in SEQ ID
NO.5-6, the primer pairs shown in SEQ ID NO.7-8, the primer pairs shown in SEQ ID
NO.9-10 and the primer pairs shown in SEQ ID NO.11-12 have the 0, 1 data matrices of the amplified products as follows: (0, 1), (1, 0), (1, 0), (0, 1), (1, 0), (1, 0), (0, 1), (1, 0), (0, 1), and (0, 1), the sample to be tested is Dafang line pepper; when the primer pairs shown in SEQ ID NO.1-2, the primer pairs shown in SEQ ID NO.3-4, the primer pairs shown in SEQ ID NO.5-6, the primer pairs shown in SEQ ID NO.7-8, the primer pairs shown in SEQ ID NO.9-10 and the primer pairs shown in SEQ ID NO.11-12 have a 0-1 data matrix of (1, 0), (0, 1), (0, 1), (1, 0), (0, 0), (0, 1), (0, 1), (1, 0), (1, 0), and (1, 0) for the amplified products, the sample to be tested is Dafang wrinkled pepper.
6. The method according to claim 5, wherein in step (2), the reaction system of PCR amplification includes 1 uL of DNA, 1 uL of 10 umol/L InDel forward primer, 1 uL of 10 pmol/L InDel reverse primer, 12.5 pL of 2xPCR reaction mixture, 0.3 pL of DNA polymerase and 9.2 uL of ultrapure water.
7. The method according to claim 5, wherein in step (2), the reaction procedure of PCR amplification comprises: 94°C for 5 min; 35 cycles at 94°C for 30 s, 65°C for 30 s and 72°C for 30s; 72°C for 5 min.
8. An application of molecular marker combination according to claim 1, primer paitJ504454 combination according to claim 2 or kit according to claim 4 in the identification of Dafang line pepper and Dafang wrinkled pepper.
LU504454A 2023-06-08 2023-06-08 Molecular markers for distinguishing dafang line peppers and dafang wrinkled peppers and application thereof LU504454B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
LU504454A LU504454B1 (en) 2023-06-08 2023-06-08 Molecular markers for distinguishing dafang line peppers and dafang wrinkled peppers and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
LU504454A LU504454B1 (en) 2023-06-08 2023-06-08 Molecular markers for distinguishing dafang line peppers and dafang wrinkled peppers and application thereof

Publications (1)

Publication Number Publication Date
LU504454B1 true LU504454B1 (en) 2023-12-11

Family

ID=89123326

Family Applications (1)

Application Number Title Priority Date Filing Date
LU504454A LU504454B1 (en) 2023-06-08 2023-06-08 Molecular markers for distinguishing dafang line peppers and dafang wrinkled peppers and application thereof

Country Status (1)

Country Link
LU (1) LU504454B1 (en)

Similar Documents

Publication Publication Date Title
KR102049399B1 (en) CAPS marker for discriminating Solenopsis invicta and Solenopsis geminata and uses thereof
LU504454B1 (en) Molecular markers for distinguishing dafang line peppers and dafang wrinkled peppers and application thereof
CN110184387B (en) RT-LAMP detection primer for detecting ANSVV, application thereof, detection reagent and detection method
KR102327643B1 (en) Molecular markers for identification of antlers and their use
CN101824468B (en) Primer group for detecting Yersinia pestis, rapid diagnosis kit and detection method
EP0851941B1 (en) Control for pcr
Faggi et al. Use of magnetic beads to extract fungal DNA
CN110982909B (en) Primer, kit and detection method for detecting Taihe black-bone chicken eggs
CN101712992B (en) Chlamys nobilis molecular marker with orange shell color and identification method thereof and kit
CN105132542B (en) Method, the primer and probe of broad-leaved yellow wingceltis in redwood are identified using round pcr
CN112795663B (en) Primer pair composition, kit and method for distinguishing cyprinus carpio from non-cyprinus carpio
CN112680531B (en) Primer pair, kit and method for quickly detecting horse-derived components in horse skins and mule skins
CN110894532A (en) RAA constant temperature fluorescence detection method and reagent for bacterial septicemia (FBS)
CN109136390A (en) It is a kind of at the same identify chicken, duck, sheep Species origin PCR-STR method
JP6853523B2 (en) PCR using helicase
CN107805664B (en) Composition, kit and method for identifying deer species by GeXP multiple PCR
KR102301392B1 (en) Primer set for distinguishing of Bacillus thuringiensis and Bacillus cereus, and using thereof
KR102563613B1 (en) Development of SNP markers and use thereof for discrimination of Pyogo mushroom cultivar Chamaram
CN118147343B (en) Method for high-throughput identification of tulip tree varieties and special KASP primer combination thereof
KR102571496B1 (en) Multiplex primer sets for identification between Chionoecetes japonicus and Chionoecetes opilio, and uses thereof
KR102373255B1 (en) Genetic markers and methods for identifying species and origin of family Pleuronectidae
KR102550866B1 (en) Species-specific multiplex PCR primer sets for identification of six species of Bivalvia, and uses thereof
CN113005216B (en) Specific genetic marker composition for identifying poppy and 3 allied species thereof
CN108796088A (en) The kit and detection method of chicken derived components in a kind of detection food
KR102254330B1 (en) Primer set for determining identity of Miichthys miiuy and Sciaenops ocellatus, method of determining identity of Miichthys miiuy and Sciaenops ocellatus using the same and kit comprising the same

Legal Events

Date Code Title Description
FG Patent granted

Effective date: 20231211