CN117210602A - Molecular marker of common head cabbage leaf bulb development gene BoACS4 and application - Google Patents

Abstract

The application discloses a molecular marker of a common head cabbage leaf bulb development gene BoACS4 and application thereof. The molecular marker of the structural variation type is developed based on structural variation sites of the upstream of the BoACS4 genes of the common head cabbage and other cabbage vegetables, is specific structural variation in the common head cabbage, and can be used for rapidly identifying whether a detection material is the common head cabbage or not by detecting whether the structural variation exists in the cabbage vegetables through the molecular marker, so that the molecular marker can be used for biotechnology-assisted breeding.

Description

Molecular marker of common head cabbage leaf bulb development gene BoACS4 and application

Technical Field

The application relates to the technical field of molecular breeding, in particular to a molecular marker for identifying a common head cabbage leaf bulb development gene BoACS4 and application thereof.

Background

Common head cabbage (Brassica oleracea l.var.capitata l.), abbreviated cabbage, also known as: cabbage, lotus white, cabbage, are an important cruciferous vegetable crop. Cabbage is rich in high quality proteins, cellulose, minerals, vitamins, etc., and is cultivated universally throughout the world. The planting area of the cabbage in China is about 90 ten thousand hm2 (Yang Limei, fang Zhiyuan, zhang Yangyong, etc. Chinese common head cabbage disease-resistant and stress-resistant genetic breeding has been studied and developed in recent years [ J ]. Gardening school report, 2020,47 (9): 11.DOI: 10.16420/j.issn.0513-353x.2020-0494), and the cabbage has very important position in annual supply and export trade of vegetables in the whole world.

The leaf head is an important agronomic character of the cabbage, the quality of the leaf head directly influences the economic value of the cabbage, and research on the genetic relationship and genetic diversity of the leaf head and related species has important significance for resource evaluation and creation and new variety cultivation. 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.

For example, chinese patent application CN113528698A discloses an InDel molecular marker for identifying and/or differentiating brassica oleracea vegetables and uses thereof. The application screens an InDel locus on the second exon of the WUSCHEL (WUS) gene of the cabbage C09 chromosome. In the InDel locus, the sequences of the common head cabbage, the corm cabbage and the cabbage mustard are consistent with those of the wild cabbage, and 12bp deletion exists in the broccoli and the cauliflower, so that an InDel molecular marker is developed.

There is still a need to develop an accurate, stable and reliable molecular marker for common head cabbage.

Disclosure of Invention

In order to solve at least part of the technical problems in the prior art, the application provides a molecular marker, a primer group, a method and application for identifying the common head cabbage, thereby providing a theoretical basis for auxiliary breeding of common head cabbage molecules. Specifically, the present application includes the following.

In a first aspect of the application, there is provided a molecular marker associated with head of common head cabbage development, which can be used to identify the head cabbage, the molecular marker is a structural variation molecular marker located 2186bp upstream (or at a position on the genome) of the boncs 4 gene.

In certain embodiments, the molecular marker related to development of common head cabbage according to the present application has a sequence shown in SEQ ID NO. 1.

In a second aspect of the application, 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 application 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 application, wherein the reverse primer has the sequence shown in SEQ ID No. 3.

In a third aspect of the application, there is provided a kit for identifying common head cabbage comprising the above primer combination.

In a fourth aspect of the application, there is provided the use of a molecular marker of the application, a primer combination of the application, or a kit of the application in genetic analysis of common head cabbage.

In certain embodiments, the use according to the application, wherein the genetic analysis comprises genetic diversity analysis, germplasm identification or assisted breeding.

In a fifth aspect of the application, there is provided a method for identifying whether a plant comprises a common head cabbage, comprising the step of detecting in the DNA of the plant to be identified the presence of a molecular marker located 2186bp upstream of the BoACS4 gene.

In certain embodiments, a method for identifying whether a plant comprises broccoli according to the present application 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 SV molecular marker is developed based on structural variation sites on the BoACS4 genes of the common head cabbage and other cabbage vegetables, is specific structural variation in the common head cabbage, and can rapidly identify whether the detection material is the common head cabbage by detecting whether the SV molecular marker exists in the cabbage vegetables. In addition, the application also verifies that the SV molecular marker has correlation with the leaf head development of the common head cabbage.

Drawings

Fig. 1 is a schematic view of a common head cabbage and other cabbage-type vegetables.

Fig. 2 shows the distinguishing effect of the head cabbage with SV and other materials without SV.

Detailed Description

Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.

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 application. In addition, for numerical ranges in the present application, 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 application. 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 application 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 application. 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 application refers to a position relative to cabbage T10.

"head cabbage" according to the application refers to a variant of the Brassica genus (Brassica L.) of the Cruciferae family. The term "variant" means that a species is initially published on an academic journal without a grade under the species (i.e., variant, subspecies, variant), and later as one gets knowledge of the species, it is found that there are individuals or groups of plants within the species that have a variation from the characteristics of the species that were recognized when the species was initially published, and that the variation is obvious and stable, and it is worth separating them individually to show the difference, the plant scientist would name the type with the variation and publish it as a variant within the species according to 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 of the application, a specific molecular marker is provided, the molecular marker has a characteristic sequence for distinguishing common head cabbage from other cabbages, and meanwhile, the application verifies that the molecular marker is closely related to the development of the leaf head of the common head cabbage, so that the common head cabbage can be distinguished, and the molecular marker is a structural variation with the length of 1671bp and is positioned at 2186bp upstream of the BoACS4 gene of the common head cabbage. In a specific embodiment, the sequence has the nucleotide sequence shown in SEQ ID NO. 1.

The characteristic sequence in the present application refers to a gene fragment which is not present in the common head cabbage vegetable, for example, in all subspecies and varieties of the cabbage vegetable. In the present application, other brassica is intended to include any cruciferous species other than common head cabbage, preferably any brassica species, examples of which include, but are not limited to, ornamental collard, broccoli, cauliflower, cabbage mustard, kohlrabi, edible collard, brussels sprouts, and the like.

Primer combination

In a second aspect of the application, there is provided a specific primer combination for amplifying a molecular marker of the application, 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 application also provides a kit for identifying common head cabbage, which comprises the primer combination and optional reagents for 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 application 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 application 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 application 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 application 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 application further provides molecular markers, substances capable of detecting the molecular markers, primer combinations, and the use of the kit in genetic analysis of common head cabbage, including but not limited to genetic diversity analysis, germplasm identification, assisted breeding, and the like. Substances capable of detecting the molecular markers include those polynucleotide molecules capable of complementary pairing with the target molecule, and the like.

Method for identifying whether plants contain common head cabbage

The present application provides a method for identifying whether a plant comprises a common head cabbage. Preferably, the plant described herein refers to a plant from the Cruciferae family (Cruciferae), more preferably the plant described herein is a plant from the Brassica genus (Brassica), further preferably the plant described herein is a plant from the Brassica class of vegetables. In certain embodiments, a "plant" herein is a type of plant, where the methods of the application refer to methods for identifying whether the plant is a head cabbage. In certain embodiments, a "plant" herein is a mixture of multiple types of plants or plant components thereof, where the methods of the application refer to methods for identifying whether or not a common head cabbage is included in a plant.

The method of the application comprises the step of detecting in the DNA of the plant to be identified the presence or absence of a characteristic sequence of the BoACS4 gene located in the head cabbage. The feature sequences have already been described and will not be described in detail here.

In the present application, the method of detecting the presence or absence of the characteristic sequence of the BoACS4 gene located in the common head cabbage may employ any method known in the art, examples of which include a method of amplifying a nucleic acid comprising the characteristic sequence, a method of directly sequencing a plant genome, and the like.

In certain embodiments, the methods of the application for identifying whether a plant comprises common head cabbage 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 comprises a common head cabbage includes 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 common head cabbage.

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 1-10min. 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 1-5min.

In the step (3), the length of the PCR product of the sample is detected by electrophoresis, the common head cabbage is 572bp, and the non-common head cabbage is 2243bp.

Examples

The following shows structural variations associated with head of common head cabbage development development of molecular markers of (SV) and identification of common head cabbage.

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.

Wild cabbage T10 is used as a reference genome, and NUCmer (v4.0.0) is utilizedet al, 2018) to compare other genomes to T10, set parameters: -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.

Correlation analysis of SV with cabbage materials of different variety types revealed a SV of 1671bp in length that was specifically negative selected in the head cabbage, was absent in 289 parts of cabbage, was present in 3 parts of cabbage only, was present in the other 232 parts of non-head cabbage material, and was absent in 82 parts of non-head cabbage material. The SV is located 2186bp upstream of the BoACS4 gene.

Extracting genome DNA from common head cabbage and young seedlings of common head cabbage, and synthesizing SV molecular marker primers with the following sequences: boACS4SV-F: GGTAATAAGCGGTCACTCATTTCAAC (SEQ ID NO. 2); boACS4SV-R: GGAGGTTTCAGAGTCATCGAAGAAGG (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 products of the common head cabbage materials are 572bp, and the obtained products of the non-common head cabbage materials are 2243bp. 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 the total is 1671bp.

By using the molecular marker primer, 50 parts of cabbage materials are identified, wherein the molecular marker primer comprises 25 parts of common head cabbage materials, 25 parts of other cabbage materials, and the other cabbage materials comprise 5 parts of broccoli, 5 parts of cauliflower, 3 parts of cabbage mustard, 3 parts of kohlrabi, 3 parts of edible collard, 3 parts of ornamental collard and 3 parts of brussels sprouts. Extracting genome DNA of all materials, and performing PCR reaction identification.

The results show that: 25 parts of common head cabbage materials are 572bp, and the identification accuracy is 100%; in 25 parts of other cabbage materials, 20 parts of the cabbage material are 2243bp,5 parts of the cabbage material are 572bp, and the accuracy is 80%.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.

Claims (10)

1. A molecular marker associated with head cabbage leaf development, wherein said molecular marker is a structural variant molecular marker located upstream of the BoACS4 gene.

2. The molecular marker related to the development of the head of common head cabbage of claim 1, wherein the molecular marker has a sequence shown in SEQ ID NO. 1.

3. A primer combination for amplifying the molecular marker according to claim 1 or 2.

4. The primer combination of claim 3, comprising a forward primer and a reverse primer.

5. The primer combination of claim 4, wherein 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.

6. A kit for identifying common head cabbage, comprising a primer combination according to any one of claims 3 to 5.

7. Use of a molecular marker according to claim 1 or 2, a primer combination according to any one of claims 3-5, or a kit according to claim 6 in a genetic analysis of common head cabbage.

8. The use according to claim 7, wherein the genetic analysis comprises genetic diversity analysis, germplasm identification or assisted breeding.

9. A method for identifying whether a plant comprises broccoli, comprising the step of detecting in the DNA of the plant to be identified the presence of a molecular marker located upstream structural variation of the boncs 4 gene.

10. The method for identifying whether a plant contains a common head according to claim 9, comprising 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.