CN111500766A

CN111500766A - Molecular marker for detecting dwarf gene ndf1 in brassica napus and application thereof

Info

Publication number: CN111500766A
Application number: CN202010550874.XA
Authority: CN
Inventors: 王茂林; 魏涛; 赵云; 王睿
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-06-16
Filing date: 2020-06-16
Publication date: 2020-08-07

Abstract

The invention belongs to the technical field of crop molecular marker screening and application. In particular to a molecular marker for detecting dwarf gene ndf1 in cabbage type rape and application thereof. The sequences of the molecular markers are shown in SEQ ID NO. 1 and SEQ ID NO. 1. The molecular marker can be used for detecting whether the brassica napus carries the dwarf gene ndf1 or not, and can perform molecular identification on a single plant in progeny of a segregation population obtained after a ndf1 gene-carrying rape variety or a common variety is hybridized with a high-stalk rape variety. The molecular marker primer of the invention can carry out specific PCR according to alleles, can extract genome DNA at any growth stage of the cabbage type rape, directly electrophoreses the obtained product after PCR amplification, and can detect whether a certain individual carries the dwarf allele ndf1 according to the existence of banding patterns. The marker primer can be applied to the auxiliary selection of the cabbage type rape plant height and plant type.

Description

Molecular marker for detecting dwarf gene ndf1 in brassica napus and application thereof

Technical Field

The invention belongs to the technical field of crop molecular marker screening and application, and particularly relates to a molecular marker for detecting dwarf gene ndf1 in brassica napus and application thereof. The molecular marker can be used for detecting whether a cabbage type rape variety carries the dwarf gene or not or identifying whether a single plant of a progeny of a segregation population obtained after the dwarf rape variety is combined with a tall rape variety carries the dwarf gene or not, is named as ndf1-M1024 and is a pair of marker primers, PCR can be carried out on the genome of the single plant of the rape by utilizing the pair of the marker primers, and whether the material to be detected carries the dwarf gene ndf1 or not can be judged through an electrophoresis band of a product. The molecular marker of the invention can be used for auxiliary selection of cabbage type rape molecular breeding.

Background

Rape is an oil crop widely planted in the world, rape also occupies a relatively high proportion in oil crop planting, rape seed meal is rich in protein which can be used for preparing high-quality feed, rape is also applied to biodiesel, environmental control and other aspects, rape Plant height is easy to lodging, harvesting difficulty is unfavorable for mechanical operation and the like, selection of proper Plant height is one of important subjects of rape Breeding, reports of rape dwarf positioning and cloning are less (Wang Y, He J, Yang L, Wang Y, Chen W, Wan S, Chup, Guan R.Fine mapping of a dwarf Plant and transforming Plant height using high-density-nuclear gene map in BMC application, 2016, 9-91, Korean strain, transgenic Plant, WO 26, WO 7, strain No. 7, No..

The traditional method for breeding different plant heights is to identify the phenotype of the plant height of the rape plants after the rape plants grow to a certain stage. The invention can select the rape plant height related gene at any growth stage through the molecular marker, and further analyze and determine the plant height and the phenotype of the plant to be detected. The invention designs a molecular marker ndf1-M1024, ndf1-M1024 sequence for assisting in selecting dwarf gene based on the sequence mutation of the dwarf gene ndf1 as an upstream sequence: 5'-TGTTGTATGTAGAGCCCAAGTGGTCGA-3', and the downstream sequence: 5'-TTGAAAGATCCTCTTGTCTCTTGCCA-3' are provided. Compared with the traditional identification method, the method can greatly improve the breeding efficiency. For example, the traditional method is to perform plant height phenotypic identification at the flowering or mature period of rape, determine candidate single plants, and then use the candidate single plants for hybridization or other purposes in the next generation; the method provided by the invention can determine whether a single plant carries the dwarf gene ndf1 in the whole growth period of the rape, and can perform hybridization or other purposes in the current generation, thereby greatly improving the breeding efficiency.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a molecular marker of a dwarf gene ndf1 located on a cabbage type rape A3 chromosome for rape auxiliary selection, the molecular marker is named as ndf1-M1024, and the upstream primer of the marker is as follows: 5'-TGTTGTATGTAGAGCCCAAGTGGTCGA-3', respectively; the downstream primer is: 5'-TTGAAAGATCCTCTTGTCTCTTGCCA-3' are provided. The molecular marker ndf1-M1024 can be applied to detecting single plants of a segregation population Fn (n is more than or equal to 2) obtained by combining (hybridizing) a brassica napus dwarf variety carrying ndf1 gene with other high-stalk rape varieties, the ndf1-M1024 marker is completely linked with the dwarf trait based on ndf1 gene sequence mutation design, the plant height trait identification does not need to be carried out until the flowering period or the mature period of rape, and whether a single plant carries the dwarf gene ndf1 can be detected in the full growth period of the segregation population. Therefore, the molecular marker of the invention can greatly improve the breeding auxiliary selection efficiency of rape.

The method comprises the following specific steps:

extracting genomic DNA (i.e. total DNA) of a material to be identified by a conventional CTAB method, or extracting genomic DNA of a material to be identified by using a commercial kit, which is now described by taking a sample of 0.2g leaves of Brassica napus into a 1.5ml EP tube, adding an appropriate amount of liquid nitrogen, sufficiently grinding with a homogenizer, adding 0.6m L extraction buffer (100mM Tris-HCl pH8.0,10mM EDTA pH8.0,100mM KCl) in a 65 ℃ water bath for 10min, centrifuging at 12000rpm for 3min, taking supernatant, adding a double volume of precooled absolute ethanol, immediately centrifuging at 4000rpm for 5min when a flocculent precipitate appears, dissolving the precipitate with 50. mu.l TE-RNase (5.0mM Tris-HCl pH8.0, 1.0mM EDTA pH8.0, RNase A30. mu.g/m L), incubating at 37 ℃ for 15min, adding a double volume of precooled absolute ethanol, precipitating at-20 ℃ for 10min, centrifuging at 4000rpm for 3min, adding an appropriate amount of Tris-70% EDTA, drying at 37 ℃ or dry L. mu.g/m₂Dissolving O for later use. If the commercial kit is used, the instructions of the commercial kit are referred to.

The extracted DNA is further amplified by PCR, the PCR amplification system and conditions are that the 20L system contains 1 × PCRbuffer, 2.5 mmol/L MgCl₂0.15 mmol/L dNTPs, 1.0U Taq enzyme, 0.5 mol/L primer (such as ndf1-M1024 molecular marker primer of the invention), 50ng template DNA, and ddH₂O to a final volume of 20. mu. L PCR was performed by a procedure of pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 20sec, renaturation at 63.5 ℃ for 20sec, extension at 72 ℃ for 30sec for 35 cycles, and finallyExtension to 72 ℃ for 5min, the PCR product was stored at 4 ℃.

DNA amplification products were separated on a 1.5% agarose gel under 1 × TAE buffer, constant pressure 130V, ethidium bromide staining, D L2000 Marker as standard molecular weight and analyzed by photography using a conventional Bio-Rad gel imaging system.

The invention has the advantages that:

(1) the molecular marker screened by the invention can identify whether a sample for extracting DNA carries the dwarf gene ndf1 at any stage of the growth and development period of the rape, and the molecular marker is completely linked with the dwarf gene ndf1 and is not easy to generate false positive.

(2) The identification process of the invention is simpler and more convenient than other molecular marking methods. The obtained PCR product can be directly subjected to agarose gel electrophoresis without the steps of enzyme digestion and the like, and can be judged by judging whether a band exists or not without electrophoresis with high resolution such as PAGE (polyacrylamide gel electrophoresis).

The molecular marker can be applied to the detection of the cabbage type rape plant height property, and is suitable for large-batch screening and identification.

Drawings

FIG. 1: the invention relates to pictures of representative variety materials of brassica napus with three different plant heights and (phenotype) plant types. Description of reference numerals: FIG. 1A is a diagram showing the plant height and phenotype of a maturity dwarf brassica napus mutant containing ndf1 gene; FIG. 1, panel B, is the plant height and phenotype of F1 generation dwarf Brassica napus containing ndf1 gene crossed with wild Brassica napus 156B; panel C of FIG. 1 is the plant height and phenotype of wild type tall Brassica oleracea variety 156B.

FIG. 2: the molecular marker ndf1-M1024 of the invention is applied to the gum running chart for detecting the parents of the cabbage type rape, the F1 generation, the F2 generation and the other 2 conventional tall rape varieties.

Description of reference numerals: samples for each lane are as follows:

lane 3 is the brassica napus parent (containing ndf1 gene);

lanes

2, 8, 11 and 24 are tall stalk brassica napus cultivar 156B;

lane 15 is first cross (F1) of brassica napus;

lanes

4, 5, 6, 10, 11, 12, 13, 18, 22 are the second filial generation of brassica napus carrying ndf1 gene (F2);

lanes

1, 7, 14, 17, 19, 23, 24 are the second filial generation of brassica napus carrying no ndf1 gene (F2);

lane 21 is brassica canada variety Westar;

lane 9 is cole variety cole colchicle oil.

Detailed Description

Description of sequence listing:

sequence listing SEQ ID NO:1 is the upstream primer sequence of the molecular marker primer of the invention.

Sequence listing SEQ ID NO: 2 is a downstream primer sequence of the molecular marker primer of the invention.

Example 1

Obtaining cabbage type rape mutant material NDF1 through artificial mutagenesis

After the stem mutation is carried out, the dry seeds of the Brassica napus homozygous inbred line 3529 (a high-stalk rape variety, a literature source: Wang F L, Zhao Y, Chen F, et al. Inheritance and potentials of a mutted dwarfing and fi in Brassica napus [ J ]. Plant Breeding,2008,23(5): 449) 453, Wanhualin, rape (Brassica napus) petential petal-free mutant creating, heredity and high-quality hybrid rape research [ D ], Sichuan university, 2005) are artificially mutagenized by a fast neutron generator, the processing parameters and steps are as follows, the seeds of the Brassica napus homozygous inbred line 3529 are placed at a distance of 1lcm from the target center, the seeds are irradiated by fast neutrons with average energy of 14Mev, the temperature is 25-26 ℃, the relative humidity is 20%, the temperature is interrupted by irradiation for 14 hours every day, the daily irradiation is about 14 hours, the total throughput is about 1010 hours, the total throughput of the stem mutation is observed by a constant temperature, the strain growing speed after the stem mutation is carried out, the stem mutation is carried out by a high-speed seedling growing, the strain transplanting is carried out by a fast neutron generator, the high-speed seedling transplanting is carried out, the high-speed mutation is carried out by a high-speed seedling transplanting, the high-speed seedling transplanting test of the strain growing test of the strain transplanting test of the strain No-speed rape (D rape transplanting, the strain transplanting test of the strain No. 16. the strain No. 16, the strain of the.

Example 2

This example relates to the use of molecular markers in brassica napus variety NDF1 (from example 1), brassica napus variety 156B (i.e., fertile from the "hybrid" of the "Sichuan" variety of crops committee, the "variety approval code" of "Sichuan" variety of oils 29) and progeny of the two crosses (including the two parents, the "combined" F1 "and" F2 "segregating populations), as well as in two other conventional high-stalk brassica napus varieties. However, the practice of the present invention is not limited to the above varieties.

The method comprises the following specific steps:

(1) a cabbage type rape variety (containing ndf1 genes) is taken as a male parent and hybridized with a cabbage type rape variety 156B as a female parent to obtain an F1 generation, and an F2 generation segregation population is constructed. Other conventional tall rape varieties include Westar (a double low cabbage type rape variety from Canada, a commonly used material in the art), the cabbage type rape variety Kele oil (national academy of agriculture agency Admission (2011, No. 017), the applicant planted the above materials in the test base of Deyang City, Sichuan province.

(2) The individual plant genome DNA of the rape material is respectively extracted, and PCR detection (the PCR detection method is a common method) is carried out on each individual plant one by utilizing the molecular marker ndf1-M1024 of the invention. ndf1-M1024 molecular marker is designed according to the SNP of ndf1 gene and high-stalk rape allele (allele-specific PCR molecular marker), so whether a single plant carries ndf1 dwarf gene can be shown by the band type of the amplification product (tests show that all the single plant carrying ndf1 gene can amplify a band, but the high-stalk rape not carrying ndf1 gene can not amplify a corresponding band)

(3) The rape individuals with different plant height phenotypes were subjected to PCR detection using molecular markers ndf1-M1024, followed by electrophoresis and analysis of the banding pattern, and the results are shown in FIG. 2.

(4) Whether the dwarf gene ndf1 is carried or not can be determined according to the band types of different alleles, and if the single plant carries the dwarf gene ndf1, the single plant is indicated to carry the dwarf gene. Meanwhile, the plant heights of single plants in field segregation populations and different parents in a mature period are measured in a matched mode, and the results prove that the plant height of the single plant carrying the dwarf gene is short stalk or semi-short stalk, and the plant height of the single plant not carrying the dwarf gene ndf1 is long stalk (Table 1).

TABLE 1 average plant height of Brassica napus parents carrying different alleles, F1 generation, F2 generation and another 2 conventional tall stalked Brassica napus varieties

The above examples all employ conventional CTAB methods to extract brassica napus fresh leaf genomic DNA for PCR amplification. The PCR amplification procedure was: 3 minutes at 95 ℃; 20 seconds at 95 ℃,20 seconds at 63.5 ℃, 30 seconds at 72 ℃,35 cycles of reaction, and finally 5 minutes of extension at 72 ℃. The PCR amplification products were detected by electrophoresis on a 1.5% agarose gel. A single sample can complete the entire assay within hours.

It should be noted that the present invention is not limited to the above embodiments, and all relevant cases directly derived from the disclosure of the present invention by related researchers are within the scope of the present invention. For example, the ndf1-M1024 molecular marker is not only applied to the separation population Fn (n is more than or equal to 2) of the filial generation containing ndf1 gene and 156B or the aforementioned rape varieties, but also applied to the identification of any rape varieties carrying ndf1 dwarf genes and not carrying ndf1 dwarf genes.

Appendix: description of noun terms:

in the present invention: the dwarf gene is represented by ndf 1; the mutant strain is represented by NDF 1; the molecular marker of the dwarf gene is represented by ndf 1-M1024. The molecular marker and the molecular marker primer are synonymous.

Sequence listing

<110> Sichuan university

<120> molecular marker for detecting dwarf gene ndf1 in brassica napus and application thereof

<141>2020-06-16

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<170>SIPOSequenceListing 1.0

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<211>27

<212>DNA

<213> Brassica napus (Brassica napus)

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tgttgtatgt agagcccaag tggtcga 27

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<212>DNA

<213> Brassica napus (Brassica napus)

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<221>primer_bind

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ttgaaagatc ctcttgtctc ttgcca 26

Claims

1. A molecular marker primer for detecting whether a cabbage type rape variety carries dwarf genes or whether a segregation population progeny single plant obtained by combining the cabbage type rape variety with a tall stalk rape variety carries the dwarf genes is separated and cloned, and is characterized in that the nucleotide sequence of the molecular marker primer is as follows:

an upstream primer: 5'-TGTTGTATGTAGAGCCCAAGTGGTCGA-3' the flow of the air in the air conditioner,

a downstream primer: 5'-TTGAAAGATCCTCTTGTCTCTTGCCA-3' are provided.

2. The application of the molecular marker primer in detecting whether a cabbage type rape variety carries dwarf genes or identifying a segregation population progeny single plant obtained after the rape variety is combined with a tall-stalk rape variety is characterized in that the nucleotide sequence of the molecular marker primer is as follows:

a downstream primer: 5'-TTGAAAGATCCTCTTGTCTCTTGCCA-3' are provided.

3. The application of the molecular marker primer of claim 1 in rape plant height auxiliary selection.