CN117248052B - Molecular marker and primer group related to ornamental collard color and application - Google Patents

Molecular marker and primer group related to ornamental collard color and application Download PDF

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CN117248052B
CN117248052B CN202311113706.4A CN202311113706A CN117248052B CN 117248052 B CN117248052 B CN 117248052B CN 202311113706 A CN202311113706 A CN 202311113706A CN 117248052 B CN117248052 B CN 117248052B
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ornamental
collard
molecular marker
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CN117248052A (en
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程锋
张扬勇
张磊
李幸
张亢
吕红豪
王勇
王雪霖
季家磊
韩风庆
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/13—Plant traits
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    • 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

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Abstract

The invention discloses a molecular marker related to ornamental collard color, a primer group and application thereof. The invention develops a molecular marker of a structural variation type based on structural variation sites on MYB2 gene promoters of ornamental collard and other cabbage vegetables, which is a specific structural variation in the ornamental collard, and whether the structural variation exists in the cabbage vegetables or not is detected by the molecular marker, so that whether a detection material is the ornamental collard can be rapidly identified, and the molecular marker can be further used for biotechnology assisted breeding.

Description

Molecular marker and primer group related to ornamental collard color and application
Technical Field
The invention relates to the field of molecular breeding, in particular to a molecular marker related to ornamental collard color, a primer group and application thereof.
Background
Ornamental collardBrassica oleracea var.acephala) Is a horticultural variety of edible cabbage belonging to Brassica genus of BrassicaceaeBrassicaL.) for two yearsThe raw plants have ornamental and edible values. The ornamental collard has bright leaf color, is an important leaf-observing plant in winter and early spring, and is mostly used for arranging flower beds and flower scenes in Yangtze river basin and the south area thereof or used as a pot plant. The ornamental collard has high nutritive value, contains rich substances such as vitamin A, vitamin B, vitamin C, potassium, iron, calcium and the like, and tender leaves of the ornamental collard can be fried, cold-mixed and made into soup, and are commonly used for being matched with vegetables with various colors to make salad.
The ornamental collard has rich plant type, leaf shape and leaf color, high diversity and important significance for resource evaluation and creation and new variety cultivation by researching the genetic relationship and genetic diversity of the ornamental collard and related species. The molecular marker typing technology has the advantages of no environmental influence, short test period, more selectable markers and capability of performing high-throughput sequencing analysis, and is widely used for research and application of genetic diversity and kindred relation analysis, new variety identification, seed purity and variety authenticity detection, molecular marker assisted breeding and the like. However, there is still a lack of accurate, stable and reliable molecular markers for ornamental collards.
Disclosure of Invention
In order to solve at least part of the technical problems in the prior art, the invention provides an ornamental collardBoMYB2Specific primers and application of molecular markers of Structural Variation (SV) of a gene promoter, thereby providing theoretical basis for molecular assisted breeding of ornamental collard. Specifically, the present invention includes the following.
In a first aspect of the invention there is provided a molecular marker associated with the colour of ornamental collard which can be used to identify ornamental collard, said molecular marker being a structural variant molecular marker located atBoMYB2The upper reaches of the gene promoter are 293bp-580bp.
In certain embodiments, the molecular markers associated with ornamental collard color according to the present invention have the sequence set forth in SEQ ID NO. 1.
In a second aspect of the invention, there is provided a primer combination for amplifying the molecular marker of the first aspect.
In certain embodiments, a primer combination according to the present invention comprises a forward primer having the sequence set forth in SEQ ID NO.2 and a reverse primer.
In certain embodiments, the primer combination according to the invention, wherein the reverse primer has the sequence shown in SEQ ID No. 3.
In a third aspect of the invention, there is provided a kit for identifying ornamental collard comprising the primer combination described above.
In a fourth aspect of the invention, there is provided the use of a molecular marker of the invention, a primer combination of the invention, or a kit of the invention in the genetic analysis of ornamental collard.
In certain embodiments, the use according to the invention, wherein the genetic analysis comprises genetic diversity analysis, germplasm identification or assisted breeding.
In a fifth aspect of the invention, there is provided a method for identifying whether a plant comprises ornamental collard, comprising detecting the presence or absence of a marker located in the DNA of the plant to be identifiedBoMYB2A step of molecular marking of 293bp-580bp upstream of the gene promoter.
In certain embodiments, a method for identifying whether a plant comprises ornamental collard according to the present invention comprises the steps of:
(1) Extracting sample DNA from a plant sample to be identified;
(2) Amplifying nucleic acid containing the molecular marker from the sample DNA by PCR using a forward primer having a sequence shown as SEQ ID NO.2 and a reverse primer having a sequence shown as SEQ ID NO. 3; and
(3) The plants were identified by detecting the length of the sample PCR products by electrophoresis.
The invention is based on ornamental collard and other cabbage vegetablesBoMYB2An SV molecular marker developed at the structural variation site of gene promoter is a specific structural variation in ornamental collard by detecting the vegetables of the cabbage familyWhether the SV molecular marker exists in vegetables can be used for rapidly identifying whether the detection material is ornamental collard.
Drawings
FIG. 1 is a schematic view of ornamental collard and other brassica oleracea vegetables.
FIG. 2 shows a cabbage vegetableBoMYB2PCR amplification electrophoretogram of promoter SV molecular marker primer.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
Unless otherwise indicated, the gene position in the present invention refers to a position relative to cabbage T10.
The ornamental collard of the invention refers to the cruciferae family of the genus BrassicaceaeCruciferae) Brassica genusBrassicaL.) a variant. The term "variant" refers to an academic speciesThe journals are initially published without a grade of the seed (i.e., variety, subspecies, variants) and later as one gets a full and deep understanding of the seed, it is found that there are individuals or groups of plants within the seed that have a different variation from the characteristics of the seed that were recognized when the seed was initially published, and that the variation is obvious and stable, and it is worth separating them individually to show the differences, and the plant scientists would name the type of variation and publish it as a variant within the seed according to the circumstances. In contrast, a type that has the original characteristics and does not undergo significant variation is called the original variant of this variant.
Molecular markers
In a first aspect, the present invention provides a specific molecular marker having a characteristic sequence for distinguishing ornamental collard from other collard species, and the present invention verifies that the molecular marker is closely related to the color of ornamental collard, thereby enabling distinguishing of ornamental collard, the molecular marker being a structural variation of 288bp in length, which is located in the ornamental collardBoMYB2The upper reaches 293bp-580bp of the gene promoter. In a specific embodiment, the sequence has the nucleotide sequence shown in SEQ ID NO. 1.
The characteristic sequence in the present invention refers to a gene fragment which is present only in ornamental collard vegetables among all subspecies and varieties of the brassica vegetables, for example. In the present invention, other brassica species are intended to include any cruciferous species other than ornamental collard, preferably any brassica species, examples of brassica species including, but not limited to, common head cabbage, broccoli, cauliflower, cabbage mustard, kohlrabi, edible collard, brussels sprouts, and the like.
Primer combination
In a second aspect of the invention, there is provided a specific primer combination for amplifying a molecular marker of the invention, comprising a forward primer and a reverse primer. In a specific embodiment, the forward primer has the sequence shown in SEQ ID NO.2 and the reverse primer has the sequence shown in SEQ ID NO. 3.
Kit for detecting a substance in a sample
The invention also provides a kit for identifying ornamental collard comprising the primer combination of the invention and optionally reagents for a polymerase chain reaction. Reagents for the polymerase chain reaction include any of those used in conventional PCR, such as a polymerase, a buffer, and the like.
In addition to the components described above, the kits of the present invention may also include precautions related to regulating manufacturing, use, or marketing of the diagnostic kit in a form prescribed by a government agency. In addition, the kits of the invention may also be provided with detailed instructions for use, storage and troubleshooting. The kit may also optionally be provided in a suitable device, preferably for robotic operation in a high throughput setting.
In certain embodiments, the components of the kits of the invention may be provided as dry powders. When the reagents and/or components are provided as dry powders, the powders may be restored by the addition of a suitable solvent. It is contemplated that the solvent may also be disposed in another container. The container will typically include at least one vial, test tube, flask, bottle, syringe, and/or other container means, with the solvent optionally being placed in aliquots. The kit may further comprise means for a second container comprising a sterile, pharmaceutically acceptable buffer and/or other solvent.
In certain embodiments, the components of the kits of the invention may be provided in solution, e.g., in aqueous solution. Where present in aqueous solution, the concentration or amount of these ingredients can be readily determined by one skilled in the art according to various needs. For example, for storage purposes, the concentration of the components may be present in a higher form, and the concentration may be reduced to the working concentration by, for example, diluting the higher concentration solution when in the working state or in use.
Where more than one component is present in a kit, the kit will also typically contain a second, third or other additional container in which additional components may be placed separately. In addition, combinations of various components may be included in the container. Any combination or reagent described herein may be a component in a kit.
Use of the same
The invention further provides molecular markers, substances capable of detecting the molecular markers, primer combinations, and the use of kits in ornamental collard genetic analysis including, but not limited to, genetic diversity analysis, germplasm identification, assisted breeding, and the like.
Method for identifying whether plants contain ornamental collard
The present invention provides a method for identifying whether a plant contains ornamental collard. Preferably, the plant is from the crucifer familyCruciferae) More preferably, the plant described herein is from Brassica speciesBrassica) Further preferred, the plant described herein is a plant from the brassica oleracea class of vegetables. In certain embodiments, a "plant" herein is a type of plant, where the methods of the invention refer to methods for identifying whether the plant is an ornamental collard. In certain embodiments, a "plant" herein is a mixture of multiple types of plants or plant components thereof, where the methods of the invention refer to methods for identifying whether ornamental collard is included in a plant.
The method of the invention comprises detecting the presence or absence of a DNA sequence from a plant to be identified in the presence of a DNA sequence from an ornamental collardBoMYB2A step of gene characteristic sequence. The feature sequences have already been described and will not be described in detail here.
In the invention, whether the ornamental collard exists or not is detectedBoMYB2The method of characterizing the gene may employ any method known in the art, examples of which include a method of amplifying a nucleic acid comprising the characterizing sequence, a method of directly sequencing a plant genome, and the like.
In certain embodiments, the methods of the invention for identifying whether a plant comprises ornamental collard comprise the step of amplifying a gene sequence comprising a signature sequence from DNA of a plant sample to be identified by PCR using forward and reverse primers.
In an exemplary embodiment, a method for identifying whether a plant contains ornamental collard comprises the steps of:
(1) Extracting sample DNA from a plant sample to be identified;
(2) Amplifying nucleic acid containing the molecular marker from the sample DNA by PCR using forward primer and reverse primer;
(3) The length of the sample PCR product was checked by electrophoresis to identify whether the plant was ornamental collard.
In step (1), the "plant sample to be identified" refers to the whole plant or a part of the plant, e.g., the plant root, leaf or stem, etc.
In step (2), the ratio of the forward primer to the reverse primer is 1:1.PCR reaction procedure: 85-98deg.C for 1-5min, 85-98deg.C for 10-30s, 40-70deg.C for 10-30s, 50-80deg.C for 10-30s, and 10-40 cycles for 1-10 min. Also preferably, the PCR reaction procedure: 90-96 ℃ for 1-3min, 90-96 ℃,12-20s, 50-65 ℃,15-20s, 70-75 ℃ for 10-20s, 10-30 cycles, 65-75 ℃ for 1-5 min.
In the step (3), the length of the PCR product of the sample is detected by electrophoresis, and the ornamental collard is 495bp.
Examples
The following shows the relationship with the color of the ornamental collardBoMYB2Development of molecular markers for Structural Variation (SV) of gene promoters and identification of ornamental collard.
The SV-based patterned flood genome was constructed using high quality genomes of 27 different cabbage varieties, including 2 wild cabbage, 7 common head cabbage, 7 collard, 3 broccoli, 2 kohlrabi, 2 cabbage mustard, 1 brussels sprout, 1 ornamental cabbage, as follows.
With wild cabbage T10 as the reference genome, other genomes were aligned to T10 using NUCmer (v4.0.0) (Mar ç ais et al, 2018) with parameters set as: -maxmtch-c 100-l50, filtering the comparison results, using delta-filters with parameters: -m-i90-l100, using show-records for the filtered result conversion format, the parameters being: -T-H-r-d.
SV was identified from the alignment using SyRI (Goel et al, 2019) and pooled for redundancy between different genomes.
The non-redundant SV was combined with the T10 reference genome using VG ToolKit (v1.33.0) (Garrison et al 2018) to construct a patterned flood genome based on SV.
Resequencing 704 parts of a different variety of cabbage material, comprising: 36 parts of wild cabbage, 310 parts of common head cabbage, 153 parts of broccoli, 63 parts of broccoli, 46 parts of kohlrabi, 34 parts of kale, 24 parts of cabbage mustard, 20 parts of brussels sprouts and 18 parts of ornamental cabbage, and the sequencing platform is Illumina HiSeq 2000, and the original data is filtered by using Trimmomatic v0.39 to remove joints, poly-N and low quality fragments, so that clean data is obtained. The VG Index is utilized to establish indexes in xg and gcsa formats for the SV-based graphical pan genome, then the VG Map is utilized to compare clean data to the graphical pan genome, and the VG Pack is utilized to filter the low-quality comparison result, wherein the parameters are as follows: -Q5. The SV genotype of each sample was obtained using VG Call with the parameters: -a-s.
The SV is related to cabbage materials of different variety types, and the special SV of one ornamental collard is found, wherein the SV is 288bp in length, and is not found in 18 ornamental collards and is not found in other cabbage materials. The SV is located atBoMYB2A promoter region of 289bp upstream of the gene.
Extracting genome DNA from young seedlings of ornamental collard and common head cabbage, and synthesizing SV molecular marker primers with the following sequences: boMYB2SV-F: GCCACGTGTCGAGATATTATTGG (SEQ ID NO. 2); boMYB2SV-R: CTCTGCAATTGGGATGTCTTTTGTC (SEQ ID NO. 3).
And carrying out PCR reaction by taking the DNA of different types of cabbage materials as templates, and carrying out agarose gel electrophoresis detection on the obtained products, wherein the electrophoresis result is shown in figure 2, the obtained product of the ornamental collard material is 495bp, and the obtained product of the common head cabbage material is 207bp. Meanwhile, the product is subjected to sanger sequencing, and the sequencing result shows that the SV region sequence is shown as SEQ ID NO.1, and 288bp is obtained.
By using the molecular marker primer, 48 parts of cabbage materials are identified, wherein the molecular marker primer comprises 13 parts of ornamental collard materials, 35 parts of other cabbage materials, and the other cabbage materials comprise 15 parts of common head cabbage, 5 parts of broccoli, 5 parts of cauliflower, 3 parts of cabbage mustard, 3 parts of kohlrabi, 2 parts of edible collard and 2 parts of brussels sprouts. Extracting genome DNA of all materials, and performing PCR reaction identification.
The results show that: 13 ornamental collard materials are 495bp, and the identification accuracy is 100%; of 35 parts of other cabbage materials, 34 parts are 207bp,1 part of undetected bands, and the accuracy is 97%.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.

Claims (1)

1. A method for identifying whether a plant is an ornamental collard, characterized in that the presence or absence of a nucleotide sequence located in the DNA of the plant to be identified is detectedBoMYB2The sequence of the molecular marker is shown as SEQ ID NO.1, and the molecular marker comprises the following steps:
(1) Extracting sample DNA from a plant sample to be identified;
(2) Amplifying nucleic acid containing the molecular marker from the sample DNA by PCR using a forward primer and a reverse primer, wherein the sequence of the forward primer is shown as SEQ ID NO.2, and the sequence of the reverse primer is shown as SEQ ID NO. 3; and
(3) Identifying the plant by detecting the length of the sample PCR product by electrophoresis, wherein the plant is ornamental collard when the length of the obtained PCR product is 495bp.
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Title
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