CN110578013B

CN110578013B - Identification method for orientation of two pepper fruit stalks and application thereof

Info

Publication number: CN110578013B
Application number: CN201810582011.3A
Authority: CN
Inventors: 肖晗; 江铸颜; 刘新华
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2018-06-07
Filing date: 2018-06-07
Publication date: 2023-03-28
Anticipated expiration: 2038-06-07
Also published as: CN110578013A

Abstract

The invention relates to two methods for identifying the orientation of pepper fruit stalks and application thereof. The invention discloses a molecular marker capable of specifically identifying the orientation of pepper carpopodium for the first time. The molecular marker can be well applied to identifying the orientation of the pepper fruit stalks and has good specificity and accuracy.

Description

Identification method for orientation of two pepper fruit stalks and application thereof

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to two methods for identifying orientations of pepper fruit stalks and application thereof.

Background

Capsicum (Capsicum) is an annual or perennial plant of the genus Capsicum of the family Solanaceae (Solanaceae). Cultivated in all parts of China, is one of the main vegetable crops.

Pod pepper (Capsicum annuum var frutescens L) is a variety of Capsicum, has the advantages of small fruit, high pungency, easy drying and the like, and has extremely high edible and medicinal values. Capsaicin was first isolated from capsicum by Thres in 1876, and capsaicin, mainly composed of capsaicin and dihydrocapsaicin, is the main source of pungent taste. The pod pepper is rich in capsaicin and capsorubin. Meanwhile, the capsaicinoids also have the effects of antibiosis, tumor resistance, pain relief and the like, so the capsaicinoids have higher application value in medical health care. The peppery taste in pepper fruits is determined by the capsaicin content, and varies greatly from species to species. An important difference in the pepper and non-pepper phenotype is the orientation of the fruit stalks, with an upward orientation indicating a pepper that is not a pepper. This phenotype plays an important role in the Construction of genetic linkage maps in Capsicum (Lefebvre, V.et al (1995); construction of an intraspecific integrated linkage map of pepper using molecular markers and double-halo generations. Genome 38, 112-121). The method can identify the orientation of the pepper stalks and has important significance for pepper breeding.

In order to better develop the research and utilization of the germplasm resources of the pod pepper and the development and breeding of the excellent variety of the pod pepper, the development of a simple, convenient, rapid and effective molecular marker for distinguishing the pod pepper from the non-pod pepper is particularly important, particularly the pod pepper and the non-pod pepper can be distinguished in the early planting stage, a large amount of planting space, manpower, material resources and other resources can be saved, and the method has important value in the subsequent breeding process for screening the breeding population of the pod pepper and the non-pod pepper.

Disclosure of Invention

The invention aims to provide two methods for identifying the orientation of pepper fruit stalks and application thereof.

In a first aspect of the invention, there is provided a method of specifically identifying a pepper stem orientation phenotype, the method selected from the group consisting of: (a) Identifying the nucleic acid sequence of the pepper to be detected, wherein if double copies of the nucleotide sequence of SEQ ID NO. 9 exist (preferably, two copies of the sequence are connected in front of and behind), the pepper to be detected is of a carpopodium-up phenotype, and if only a single copy of the nucleotide sequence of SEQ ID NO. 9 exists, the pepper to be detected is of a carpopodium-down phenotype; or

(b) Identifying the nucleic acid sequence of the pepper to be detected, if the 27 th position of the sequence region shown in SEQ ID NO. 3 is a base G, indicating that the pepper to be detected is of a phenotype with an upward fruit stem, and if the 27 th position is a base C, indicating that the pepper to be detected is of a phenotype with a downward fruit stem.

In a preferred embodiment, the identification of the nucleic acid sequence is carried out by sequencing, PCR amplification, probe, hybridization, chip, restriction analysis, allelic polymorphism analysis (e.g., melting curve).

In another preferred embodiment, the single-copy or double-copy form of the nucleotide sequence of SEQ ID NO. 9 is identified by PCR amplification and the 27 th base of the sequence region indicated by SEQ ID NO. 3 is identified.

In another preferred embodiment, (a) the capsicum to be detected is subjected to PCR amplification, and if the nucleotide sequence shown as SEQ ID NO. 1 exists, the capsicum is shown to be of a fruit stem upward phenotype; if the nucleotide sequence shown in SEQ ID NO. 2 exists, the phenotype is that the carpopodium is downward.

In another preferred example, primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 are used for PCR amplification, and if the amplification product is 332bp, the phenotype that the fruit stem is upward is indicated; if only 295bp amplification products appear or two amplification products of 332bp and 295bp appear simultaneously, the phenotype is that the carpopodium faces downwards.

In another preferred embodiment, the size of the amplification product is determined by electrophoresis.

In another preferred embodiment, (b) the capsicum to be detected is subjected to PCR amplification, and if the nucleotide sequence shown as SEQ ID NO. 3 exists, the capsicum is shown to be of a fruit stem-up phenotype; if the nucleotide sequence shown in SEQ ID NO. 4 exists, the phenotype is that the fruit stem faces downwards.

In another preferred example, primers shown in SEQ ID NO 7 and SEQ ID NO 8 are used for PCR amplification, and the amplified product is subjected to enzyme digestion by restriction enzyme DpnII, if the amplified product cannot be subjected to enzyme digestion, the amplified product is shown to be of a phenotype with an upward fruit stalk; if the enzyme can be cut, the fruit stem is shown to be of a downward-facing phenotype.

In another preferred embodiment, the determination of the cleavage and the size of the cleavage product is performed by electrophoresis: if the enzyme can not be cut by enzyme, a 191bp electrophoresis band exists, which indicates that the fruit stem is of an upward phenotype; if the enzyme can be cut to generate two electrophoresis bands of 23bp and 168bp or three electrophoresis bands of 23bp, 168bp and 191bp, the phenotype of the fruit stem is shown as the downward fruit stem.

In another aspect of the invention, the application of the polynucleotide or the polymorphic locus as a molecular marker for specifically identifying the pepper carpopodium orienting phenotype is provided, wherein the nucleotide sequence of the polynucleotide is shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and/or SEQ ID NO. 9; or the polymorphic sites are: base 27 of the sequence region shown in SEQ ID NO. 3.

In another aspect of the present invention, there is provided the use of a primer specifically amplifying a nucleic acid fragment comprising the region of SEQ ID NO. 9 as an agent for specifically identifying the phenotype of pepper carpopodium orientation; preferably, the nucleotide sequence of the primer is shown as SEQ ID NO. 5 and SEQ ID NO. 6; or the primer specifically amplifies a fragment containing the polymorphic sites: base 27 of the sequence region represented by SEQ ID NO. 3; preferably, the nucleotide sequence of the primer is shown as SEQ ID NO. 7 and SEQ ID NO. 8.

In a preferred embodiment, said molecular marker or primer is further used for: identifying pepper germplasm resources; and/or breeding and breeding the pepper.

In another aspect of the present invention, there is provided a primer for specifically identifying the pepper carpopodium orientation phenotype, which specifically amplifies a nucleic acid fragment comprising the region of SEQ ID NO. 9; preferably, the primer sequence is shown as SEQ ID NO. 5 and SEQ ID NO. 6.

In another aspect of the present invention, there is provided a primer for specifically identifying the pepper carpopodium orientation phenotype, the primer being a primer that specifically amplifies a fragment containing a polymorphic site: base 27 of the sequence region represented by SEQ ID NO. 3; preferably, the primer sequence is shown as SEQ ID NO. 7 and SEQ ID NO. 8.

In another aspect of the present invention, there is provided a kit for specifically identifying a pepper stalk orientation phenotype, comprising: a primer for specifically amplifying a nucleic acid fragment containing a region of SEQ ID NO. 9; preferably, the primer sequence is shown as SEQ ID NO. 5 and SEQ ID NO. 6; or a primer for specifically amplifying a fragment containing a polymorphic site: base 27 of the sequence region represented by SEQ ID NO. 3; preferably, the primer sequences are shown as SEQ ID NO. 7 and SEQ ID NO. 8.

In a preferred embodiment, when the kit comprises a primer for specifically amplifying a fragment containing a polymorphic site, the kit further comprises: restriction enzyme DpnII.

In another preferred embodiment, the kit further comprises: DNA extraction reagents, taq enzyme, PCR buffer, DNA polymerase, and/or instructions for use that instruct a method of identifying the orientation of the pepper stem.

In another aspect of the present invention, there is provided an isolated polynucleotide for specifically identifying the pepper stalk orientation phenotype comprising the nucleic acid segment set forth in SEQ ID NO. 9.

In another aspect of the present invention, there is provided an isolated polynucleotide specifically identifying the carpopodium capsulatum phenotype orientation comprising at least the 27 th base of the sequence region represented by SEQ ID NO. 3 and the bases adjacent thereto; the length of the polynucleotide is 15-5000 bp; e.g., 20, 30, 50, 80, 100, 200, 300, 500, 1000, 2000, 3000bp; preferably, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO. 3 or SEQ ID NO. 4.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 shows the comparison result of the sequence difference sites of the molecular marker 1 for identifying pod pepper and non-pod pepper in pepper;

FIG. 2 is the comparison result of the sequence difference sites of the molecular marker 2 for identifying pod pepper and non-pod pepper in pepper;

FIG. 3 shows PCR amplification of genomic DNA of Capsicum annuum using 30ul Easy Taq DNA Polymerase PCR amplification system using Primer1/Primer2 of molecular marker 1;

FIG. 4 shows PCR amplification of genomic DNA of Capsicum annuum using 30ul Easy Taq DNA Polymerase PCR amplification system using molecular marker 2 Primer3/Primer 4; the obtained PCR product was digested with DpnII and subjected to electrophoretic analysis: wherein the non-pod pepper with the downward fruit stem is 1,8,9, 12, 15 and 17; the pepper with the fruit stem facing upwards is 5,6,7, 10, 13, 14 and 16; the remaining 2,3,4, 11 was heterozygous, with the stalk phenotype also facing downward. Remarking: during electrophoresis, the 23bp fragment is too weak, and the gel is low in visualization degree after imaging.

Detailed Description

The inventor of the invention, after extensive and intensive research and experiment, firstly reveals a molecular marker which can specifically identify the orientation of the pepper carpopodium. The molecular marker can be well applied to identifying the orientation of the pepper fruit stalks and has good specificity and accuracy.

As used herein, the "stem-up phenotype of a pepper" is "pod pepper," which refers to a single or cluster pepper that grows with its stem up or obliquely up after flowering pollination.

As used herein, the "stemmed downward phenotype" of a pepper is "non-pod pepper" and refers to a pepper that grows stemmed downward or obliquely downward after flowering pollination.

The gene or genes by which the carpopodium orientation of a pepper is controlled in the pepper genome sequence has not been known to those skilled in the art, and thus there is no molecular marker in the prior art to identify the carpopodium orientation of a pepper. The genome of the pepper is very complex, various genes with functions or functional positions exist, a large number of SNP (single nucleotide polymorphism) sites, insertion deletion sites and the like exist, and the specific molecular marker for identifying the orientation of the pepper carpopodium is very difficult to locate from the middle. Therefore, the inventor has conducted long-term and extensive research and screening, and through repeated experimental verification on various samples, finally determined a specific molecular marker suitable for identifying the orientation of the pepper carpopodium, a primer for identifying the molecular marker, and an identification strategy.

Accordingly, the present invention provides a method for specifically identifying a pepper stalk phenotype, comprising: (a) Identifying the nucleic acid sequence of the pepper to be detected, wherein if double copies of the nucleotide sequence of SEQ ID NO. 9 exist (preferably, two copies of the sequence are connected in front of and behind), the pepper to be detected is of a carpopodium-up phenotype, and if only a single copy of the nucleotide sequence of SEQ ID NO. 9 exists, the pepper to be detected is of a carpopodium-down phenotype; or (b) identifying the nucleic acid sequence of the pepper to be detected, wherein if the 27 th position of the sequence region shown in SEQ ID NO. 3 is a base G, the pepper to be detected is of a phenotype that the fruit stem faces upwards, and if the 27 th position is a base C, the pepper to be detected is of a phenotype that the fruit stem faces downwards.

Based on the novel findings of the present invention, one skilled in the art can perform the analysis of nucleic acid sequences using any of a variety of techniques known in the art or being developed, and such techniques can be included in the present invention. Such methods include, for example, but are not limited to: sequencing, PCR amplification, probe, hybridization, restriction analysis, allelic polymorphism analysis (e.g., melting curve), and the like.

In a preferred embodiment, the pepper to be detected is subjected to PCR amplification, and if the nucleotide sequence shown as SEQ ID NO. 1 exists, the pepper is shown to have a phenotype that the fruit stem faces upwards; if the nucleotide sequence shown in SEQ ID NO. 2 exists, the phenotype is that the carpopodium is downward. In a more preferred mode, primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 are used for PCR amplification, and if the amplification product is 332bp, the phenotype is that the fruit stem faces upwards; if only one 295bp amplification product appears or two amplification products of 332bp and 295bp appear simultaneously, the phenotype is that the carpopodium is downward.

The invention also provides another method for specifically identifying the pepper stem orientation phenotype, which comprises the following steps: identifying the nucleic acid sequence of the pepper to be detected, if the 27 th position of the sequence region shown in SEQ ID NO. 3 is a base G, indicating that the pepper to be detected is of a phenotype with an upward fruit stem, and if the 27 th position is a base C, indicating that the pepper to be detected is of a phenotype with a downward fruit stem. In a preferred mode, the pepper to be detected is subjected to PCR amplification, and if the nucleotide sequence shown as SEQ ID NO. 3 exists, the pepper is shown to be of a phenotype with an upward fruit stem; if the nucleotide sequence shown in SEQ ID NO. 4 exists, the phenotype is that the fruit stem is downward. In a more preferable mode, primers shown in SEQ ID NO. 7 and SEQ ID NO. 8 are used for PCR amplification, and the amplified product is cut by restriction enzyme DpnII, if the amplified product cannot be cut by enzyme, the amplified product is shown to be of a phenotype that the fruit stem faces upwards; if the enzyme can be cut, the fruit stem is shown to be of a downward-facing phenotype. In a further preferred mode, whether the enzyme is cleaved and the size of the cleavage product is determined by electrophoresis: if the enzyme can not be cut by enzyme, a 191bp electrophoresis band exists, which indicates that the fruit stem is of an upward phenotype; if the enzyme can be cut to generate two electrophoresis bands of 23bp and 168bp or three electrophoresis bands of 23bp, 168bp and 191bp, the phenotype of the fruit stem is shown as the downward fruit stem.

Aiming at the molecular marker of the invention, the orientation of the capsicum frutescens fruit stem of the sample to be detected can be accurately and rapidly judged only by carrying out PCR reaction and/or agarose gel electrophoresis and judging whether the corresponding PCR product exists or not, the cost is low, the method is suitable for large-scale identification, and the required sample amount is small.

Methods for obtaining DNA from a sample to be tested are well known to those skilled in the art, and may be, for example, the conventional phenol/chloroform/isoamyl alcohol method, or may be some commercially available DNA extraction kits, which are well known to those skilled in the art.

The Polymerase Chain Reaction (PCR) technique is well known to those skilled in the art and its basic principle is the in vitro enzymatic synthesis of specific DNA fragments. The method of the present invention can be carried out using conventional PCR techniques.

The invention also relates to a kit for identifying the orientation of the pepper carpopodium, which contains primers shown by SEQ ID NO. 5 and SEQ ID NO. 6; or the primers shown in SEQ ID NO. 7 and SEQ ID NO. 8. In a preferred mode, when the kit comprises the primers shown in SEQ ID NO. 7 and SEQ ID NO. 8, the kit further comprises: restriction enzyme DpnII.

In addition, the kit may also contain other agents for identifying the orientation of the capsicum fructicola handle, such as (but not limited to):

(A) Various reagents for PCR reactions, such as, but not limited to: taq enzyme, PCR buffer solution, dNTP, DNA polymerase and the like; or

(B) Various reagents required for DNA extraction (i.e. preparation of PCR reaction templates) such as, but not limited to: phenol, chloroform, isoamyl alcohol, naCl, etc.; or

(C) Kit for extracting DNA.

In addition, the kit can also contain an instruction book and/or standard operating procedures for identifying the orientation of the capsicum frutescens stems.

The kit provided by the invention can realize the purposes of rapid detection and batch detection of the orientation of the pepper fruit stalks.

Compared with the prior art, the invention has the following advantages and effects:

the invention overcomes the technical defects that the prior technology can not identify the pod pepper and the non-pod pepper, and provides two simple and effective molecular markers for identifying the pod pepper and the non-pod pepper and the optimal primers thereof, thereby providing a feasible method for quickly and effectively identifying the pod pepper and the non-pod pepper and providing a powerful tool for screening breeding groups of the pod pepper and the non-pod pepper.

By using the molecular marker or the primer thereof, the orientation of the capsicum frutescens can be rapidly detected in large batch, pod pepper and non-pod pepper can be rapidly and accurately distinguished from the sample to be detected, the amount of the required sample is small, and the operation is simple.

The pod pepper and the non-pod pepper can be identified in the early planting stage, great convenience is brought to the breeding work of the pod pepper and the non-pod pepper, and the development of pepper breeding can be greatly promoted.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBrook et al, molecular cloning, A laboratory Manual, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 establishment of two molecular markers for identifying orientation of capsicum frutescens stalks

1. Establishment of molecular marker 1 for identifying orientation of pepper carpopodium

Through a large number of screening, verification and phenotype observation, the inventor finds that a section of InDel (Insertion/Deletion) in a pepper genome is closely related to the fruit stalk orientation of pepper, and if only 1 36bp nucleotide sequence exists at the position, the phenotype that the fruit stalk is downward is generated; the presence of two 36bp repeats of the nucleotide sequence is of the carpopodium-up phenotype.

(1) Designing a primer: 332bp sequences are selected before and after 36bp InDel for comparison (figure 1), and primers are designed according to sequence difference, wherein the primer sequences are as follows:

upstream Primer1:5'-TGGCAGACAAATGGGTGTGTT-3' (SEQ ID NO: 5);

downstream Primer2:5'-GTTACATGGTCAGGAGGT-3' (SEQ ID NO: 6).

(2) Analysis of amplified fragments:

the pod pepper genome DNA can amplify a segment (SEQ ID NO: 1) of 332 bp; the non-pod pepper genome DNA can amplify a 295bp segment (SEQ ID NO: 2); the samples were separated by 3% agarose gel electrophoresis.

SEQ ID NO:1：

/>

SEQ ID NO:2：

TGGCAGACAAATGGGTGTGTTACTACCATAAAAGAAGGTAGAGCACTAGCATTGATATTGATGCGACAAGTTTCGCATTTTGCTCCGTTATTGAACCGTCTACTTGTACAATTTAATTTAAATTAACCAAATAAAGTAATTTTTTTTTTTTTAAAAAGTCAGCTCACTATACTAAAGTTTTCGTTATGCATGGGGTTCGAGAAATGATCAAACCACACTGATTTCTTATATGCAGTCTTACCTTGTATTTCTGCCAAAGACTATTTCTGCACAAGTGACCTCCTGACCATGTAAC(295bp)

The 36bp sequence is represented by SEQ ID NO: 9: TAAAGTTTTCGTTATGCATGGGGTTCGAGAAATGAT.

2. Establishment of molecular marker 2 for identifying orientation of pepper carpopodium

(1) Designing a primer: 191bp sequences are selected before and after 1bp SNP (single nucleotide polymorphism) for comparison (figure 2), and primers are designed according to sequence differences, wherein the primer sequences are as follows:

upstream Primer3:5'-TTACAAATTTCACTGATTTTTTAGAT-3' (SEQ ID NO: 7);

downstream Primer4:5'-CAAGTACTAGCATAATATCG-3' (SEQ ID NO: 8).

(2) After amplification of the fragment, it was digested with DpnII:

both pod pepper and non-pod pepper can amplify a 191bp DNA fragment, SEQ ID NO 3 and SEQ ID NO 4, respectively, after enzyme digestion with DpnII, the DNA fragments are subjected to 4% agarose gel electrophoresis, and can be distinguished: if the PCR product can not be cut by the enzyme DpnII, a 191bp DNA fragment is electrophoresed, and the PCR product is pod pepper; if the PCR product can be cut by DpnII enzyme, the cut product is two DNA fragments of 23bp and 168bp, the PCR product is the non-pod pepper; if the PCR product can be cut by DpnII enzyme, the cut product is three DNA fragments of 23bp, 168bp and 191bp, and is the non-pod pepper.

SEQ ID NO:3：

SEQ ID NO:4：

Example 2 application of molecular marker 1 of pod pepper and non-pod pepper

1. Extraction of genomic DNA from pepper leaves

The test material included 24 materials from a heterozygous population of pod peppers and non-pod peppers.

Selecting young leaves of a single pepper plant, and extracting the genome DNA of the pepper by adopting a CTAB method, wherein the method comprises the following specific steps:

(1) Placing about 0.2g of plant material in a 2ml centrifuge tube, adding 400uL of DNA extract, adding a steel ball, shaking for 1min with a tissue disruption grinder, and fully grinding the material;

(2) Standing at 65 deg.C for 20min, and shaking for full cracking;

(3) After the material is cooled to room temperature, adding 400 mu l of chloroform, and violently and uniformly mixing;

(4) Centrifuging at room temperature at 12000rpm for 10min, and sucking supernatant into a new centrifuge tube;

(5) Adding isopropanol with the same volume, reversing, uniformly mixing, centrifuging at room temperature, and centrifuging at 12000rpm for 10min;

(6) Discarding the supernatant, adding 1ml of 75% ethanol, and centrifuging at 12000rpm for 5min at room temperature;

(7) Discarding the supernatant, adding 1ml of 75% ethanol, and centrifuging at 12000rpm for 5min at room temperature;

(8) The supernatant solution is discarded, the mixture is dried at room temperature, then 30-50uL of 1x TE solution is added, and the mixture is placed at 65 ℃ for 10min to help dissolution.

(9) After the extracted DNA is detected for quality, the DNA can be stored temporarily at the temperature of minus 20 ℃ or stored for a long time at the temperature of minus 80 ℃.

2. Analyzing the carpopodium orientation of 24 samples of pepper heterozygous population by using a molecular marker 1 for identifying the carpopodium orientation of pepper

PCR amplification is carried out by using an identifying Primer1 (SEQ ID NO: 5)/Primer 2 (SEQ ID NO: 6), and the reaction system is a 30uL Easy Taq DNA Polymerase system and comprises the following components: 100ng/uL of genomic DNA (1 uL); 10uM upstream Primer 1.6 uL;10uM downstream Primer 2.6 uL;10XEasy Taq buffer 3uL;2.5mM dNTPs 2.4uL; easy Taq DNA Polymerase 0.3uL; ddH ₂ O makes up 30uL. The PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 5min; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 35 cycles; final extension at 72 ℃ for 10min.

3. Agarose gel electrophoresis detection of amplification products and carpopodium orientation determination

The PCR amplification products were electrophoresed through a 3% agarose gel (0.5XTBE, 120V, about hour) and visualized using a gel imaging system. The results of the electrophoretic analysis of the obtained PCR products using agarose gel are shown in FIG. 3:

lanes

1,3,4,7, 14, 19, 22 are non pod pepper with its stalk facing down (a 295bp band is present); 2,9, 10, 17, 20, 21 lanes (band 332 bp) are pod pepper with its stem facing up; the remaining 5,6,8, 11, 12, 13, 15, 16, 18, 23, 24 are heterozygous (with both 295bp and 332bp bands present), with the carpopodium phenotype facing downward.

The above results indicate that genomic DNA of both pod pepper and non-pod pepper amplified a single band and were clearly distinguishable in size.

And comparing the identification result with the fruit stalk phenotype observed in the late planting stage, wherein the result is completely consistent. Therefore, the molecular marker 1 can effectively distinguish pod pepper from non-pod pepper.

Example 3 use of molecular markers 2 for pod pepper and non-pod pepper

(1) The extraction of genomic DNA from pepper leaves was performed as described in example 2.

(2) Analyzing the carpopodium orientation of 17 samples of pepper heterozygous population by utilizing molecular marker 2 for identifying pepper carpopodium orientation

PCR amplification was performed using the identifying primers Primer3 (SEQ ID NO: 7)/Primer 4 (SEQ ID NO: 8), and the PCR reaction system and the PCR reaction procedure were the same as those shown in example 2.

After amplification of the fragment, it was digested with DpnII:

both pod pepper and non-pod pepper amplified a 191bp DNA fragment, using 30uL DpnII enzyme digestion system (NEB company), comprising the following components: 10X NE Buffer 3.1 3uL; 10ul of PCR product; 1ul of DpnII enzyme; ddH ₂ O makes up 30uL. The enzyme was cleaved at 37 ℃ for 1 hour.

(3) Agarose gel electrophoresis detection of enzyme digestion products and determination of carpopodium orientation

4% agarose gel electrophoresis (0.5 XTBE,120V, about 2 hour) is carried out on the PCR amplification product, a gel imaging system is used for observation, the result shows that the sizes of the fragments of the pod pepper and the non-pod pepper after enzyme digestion can be obviously distinguished, if the PCR product can not be digested by DpnII, a 191bp DNA fragment is electrophoresed, and the pod pepper is obtained; if the PCR product can be cut by DpnII enzyme, the cut product is two DNA fragments of 23bp and 168bp, the PCR product is the non-pod pepper; if the PCR product can be cut by DpnII enzyme, the cut product is three DNA fragments of 23bp, 168bp and 191bp, and is the non-pod pepper. Consistent with the phenotype of the fruit stalks observed at the later stage of planting. It was demonstrated that the developed molecular marker 2 was able to effectively distinguish pod pepper from non-pod pepper (as shown in fig. 4).

Because of the diversity of pepper varieties, the

molecular markers

1 and 2 have a slight difference from the sequences and sizes disclosed by the invention, but the primers using the

molecular markers

1 and 2 disclosed by the invention can be effectively distinguished.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, simplifications, etc. which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes and modifications are intended to be included in the scope of the present invention. Also, all references mentioned in this application are incorporated by reference as if each reference were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

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<120> identification method of orientation of two pepper fruit stalks and application thereof

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Claims

1. A method for specifically identifying a pepper stalk orientation phenotype, said method selected from the group consisting of:

(a) Identifying the nucleic acid sequence of the hot pepper to be detected, if double copies of the nucleotide sequence of SEQ ID NO. 9 exist, indicating that the hot pepper to be detected is of a carpopodium-upward phenotype, and if only single copies of the nucleotide sequence of SEQ ID NO. 9 exist, indicating that the hot pepper to be detected is of a carpopodium-downward phenotype; or

2. The method of claim 1, wherein the nucleic acid sequence is identified by sequencing, PCR amplification, probe, hybridization, microarray, restriction analysis, or allelic polymorphism analysis.

3. The method as claimed in claim 1, characterized in that the single-copy or double-copy form of the nucleotide sequence of SEQ ID No. 9 and the base 27 of the sequence region indicated by SEQ ID No. 3 are identified by means of PCR amplification.

4. The method according to claim 1 or 3, wherein in (a), the pepper to be tested is subjected to PCR amplification, and if the nucleotide sequence shown as SEQ ID NO. 1 exists, the pepper is shown to have a stem-up phenotype; if the nucleotide sequence shown in SEQ ID NO. 2 exists, the phenotype is that the fruit stem faces downwards.

5. The method of claim 4, wherein the primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 are used for PCR amplification, and if the amplification product is 332bp, the phenotype is that the fruit stem is upward; if only 295bp amplification products appear or two amplification products of 332bp and 295bp appear simultaneously, the phenotype is that the carpopodium faces downwards.

6. The method of claim 5, wherein the size of the amplification product is determined by electrophoresis.

7. The method according to claim 1 or 3, wherein in (b), the pepper to be tested is subjected to PCR amplification, and if the nucleotide sequence shown in SEQ ID NO. 3 exists, the pepper is shown to have a stem-up phenotype; if the nucleotide sequence shown in SEQ ID NO. 4 exists, the phenotype is that the fruit stem faces downwards.

8. The method of claim 7, wherein the primers shown in SEQ ID NO. 7 and SEQ ID NO. 8 are used for PCR amplification, and the amplification product is digested by restriction enzyme DpnII, and if the amplification product cannot be digested, the phenotype that the carpopodium faces upwards is indicated; if the enzyme can be cut, the fruit stem is shown to be of a downward-facing phenotype.

9. The method of claim 8, wherein the determination of the cleavage and the size of the cleavage product is performed by electrophoresis: if the enzyme can not be cut by enzyme, a 191bp electrophoresis band exists, which indicates that the fruit stem is of an upward phenotype; if the enzyme can be cut to generate two electrophoretic bands of 23bp and 168bp or three electrophoretic bands of 23bp, 168bp and 191bp, the phenotype of the fruit stem is shown as the downward fruit stem.

10. Application of polynucleotide or polymorphic locus as a molecular marker for specifically identifying pepper carpopodium orientation phenotype, wherein the nucleotide sequence of the polynucleotide is shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and/or SEQ ID NO. 9; or the polymorphic sites are: base 27 of the sequence region shown in SEQ ID NO. 3.

11. Use of a primer as a reagent for specifically identifying the phenotype of pepper carpopodium orientation, said primer specifically amplifying a nucleic acid fragment comprising the region of SEQ ID No. 9; or

The primer specifically amplifies a fragment containing a polymorphic site, wherein the polymorphic site is as follows: base 27 of the sequence region shown in SEQ ID NO. 3.

12. The use of claim 11, wherein the primer has the nucleotide sequence shown in SEQ ID NO 5 and SEQ ID NO 6, and specifically amplifies a nucleic acid fragment comprising the region of SEQ ID NO 9.

13. The use of claim 11, wherein the primer has the nucleotide sequence shown in SEQ ID nos. 7 and 8, and specifically amplifies a fragment containing a polymorphic site: base 27 of the sequence region shown in SEQ ID NO. 3.

14. The use of any one of claims 10 to 13, wherein the molecular marker or primer is further used to:

identifying pepper germplasm resources; and/or

And (4) breeding and selecting the peppers.

15. The primer for specifically identifying the pepper carpopodium orientation phenotype is characterized by specifically amplifying a nucleic acid fragment containing a region of SEQ ID NO. 9, and the sequence of the primer is shown as SEQ ID NO. 5 and SEQ ID NO. 6.

16. The primer for specifically identifying the pepper carpopodium orientation phenotype is characterized in that the primer is used for specifically amplifying a segment containing a polymorphic site, and the polymorphic site is as follows: base 27 of the sequence region represented by SEQ ID NO. 3; the primer sequences are shown as SEQ ID NO. 7 and SEQ ID NO. 8.

17. A kit for specifically identifying a pepper carpopodium orientation phenotype, comprising:

a primer for specifically amplifying a nucleic acid fragment containing a region of SEQ ID NO. 9; the primer sequences are shown as SEQ ID NO. 5 and SEQ ID NO. 6; or

A primer for specifically amplifying a fragment containing a polymorphic site: base 27 of the sequence region represented by SEQ ID NO. 3; the primer sequences are shown as SEQ ID NO. 7 and SEQ ID NO. 8.

18. The kit of claim 17, wherein the primer that specifically amplifies the fragment containing the polymorphic site is further comprised of: restriction enzyme DpnII.

19. The kit of claim 17, further comprising: DNA extraction reagents, taq enzyme, PCR buffer, DNA polymerase, and/or instructions for use that instruct a method of identifying the orientation of the pepper stem.

20. An isolated polynucleotide for specifically identifying the pepper carpopodium orientation phenotype comprising the nucleic acid fragment set forth in SEQ ID NO 9.

21. An isolated polynucleotide which specifically identifies a pepper carpopodium-oriented phenotype and comprises at least the 27 th base of the sequence region shown in SEQ ID NO. 3 and bases adjacent thereto; the length of the polynucleotide is 15-5000 bp, and the nucleotide sequence of the polynucleotide is shown as SEQ ID NO. 3 or SEQ ID NO. 4.