CN114150084B - Primer pair for identifying carrot petaloid cytoplasmic male sterility and application thereof - Google Patents

Abstract

The invention discloses a primer pair for identifying carrot petal-type cytoplasmic male sterility and its application. The primer pair is designed according to the Indel molecular markers screened by transcriptome analysis. The application is to use the primer pair to identify carrot petal Cytoplasmic male sterility. In this application, the total RNA of a group of stable carrot petal-type cytoplasmic male sterile lines and their maintainers was analyzed by transcriptome sequencing, compared with the published reference mitochondrial genome of carrots, and the male sterile lines and maintainers were screened out. Differential Indel sites between lines were identified and primers were designed. Through specific amplification verification between fertile and sterile carrots of different varieties, the results show that the primer pair of the present application can completely distinguish the fertility of the varieties to be tested, and can quickly screen out cytoplasmic male sterile carrots at the seedling stage The variety has a high accuracy rate and has important practical application value and market prospect for removing miscellaneous plants in the breeding process.

Description

A pair of primers for identifying carrot petal-type cytoplasmic male sterility and its application

technical field

The invention belongs to the field of biotechnology breeding, and in particular relates to a pair of primers for identifying carrot petal-type cytoplasmic male sterility and an application thereof.

Background technique

Carrot (Daucus carota L.) belongs to a class of important root vegetables belonging to the genus Carota of Umbelliferae. Its fleshy root is rich in carotenoids and has high nutritional value. It is widely planted all over the world and is a global One of the top ten vegetable crops. The rapid increase of carrot planting area not only greatly increases the demand for seeds, but also requires higher seed quality and production efficiency.

Carrot is a biennial crop with high degree of cross-pollination, and the flower organs are extremely small, so it is difficult to artificially detassell, and it is difficult to carry out conventional artificial cross-breeding. Due to the difficulty in carrying out conventional artificial cross breeding, the utilization of heterosis in carrots is relatively backward. Therefore, the early breeding methods mainly used group selection and pedigree selection, but the consistency and stability of the varieties bred by this method are relatively poor, the main reason is that the heterozygosity of the conventional variety itself and the self-crossing within the group cause serious species characteristics. decline.

With the discovery of carrot cytoplasmic male sterile material, the selection and breeding of carrot hybrids became a reality. At present, there are mainly two types of carrot cytoplasmic male sterility: petalized type and brown drug type. With the in-depth study of the genetic mechanism of carrot male sterility, carrot male sterile materials are widely used in actual breeding work, so as to ensure that the variety has high consistency and stability. In the 1960s, the United States pioneered the release of the first generation of carrot hybrids. At present, most of the carrot varieties produced and used in European and American countries are first-generation hybrids, but different countries or seed companies use different sources of male sterility. More use of flap type.

The traditional conventional method of cultivating male sterile lines of carrots is to use the method of backcrossing and transferring, using the backcrossing of high-quality fertile single plants to carry out fertility identification on the backcross offspring, focusing on examining the method of fertility identification with maintainer lines and no restorer genes. However, due to the complex genetic background of carrot male sterility, fertile plants often appear in pre-selected sterile lines after several generations in breeding.

Therefore, the development of cytoplasmic gene molecular markers to carry out fertility identification research on backcross materials can distinguish the fertility of carrots at the seedling stage and remove fertile plants that may be adulterated in advance, which can greatly save manpower, material resources and breeding space, and lay a solid foundation for subsequent flowering. Post-back crossing provides convenience.

Contents of the invention

In order to solve the above problems, the object of the present invention is to provide a primer pair designed using Indel molecular markers and the method and application of using the primer pair to identify carrot petal-type cytoplasmic male sterile plants. The primer pair and The method has the advantages of simplicity, strong specificity and high stability.

Technical content of the present invention is as follows:

In a first aspect, the present invention provides a pair of primers for identifying carrot petal-type cytoplasmic male sterility,

The forward primer sequence of the primer pair is SEQ ID NO.1:

5'-TTGTGCGGTTAAGGTTGTTGT-3';

The reverse primer sequence of the primer pair is SEQ ID NO.2:

5'-CCTGTTCTTGTGGATCAATGGA-3'.

The primer pair is composed of two upstream and downstream primers for amplifying a specific DNA fragment, i.e. a forward primer and a reverse primer; The target sequence was added and also located in the carrot reference mitochondrial genome, the URL of the reference mitochondrial genome is as follows:

http://ftp.ensemblgenomes.org/pub/plants/release-51/fasta/daucus_carota/dna/.

The function of the primer pair is as follows: after using the primer pair to perform PCR amplification on the total DNA of the carrot sample to be tested, when the size of the amplified fragment is 264bp, it is determined that the carrot variety is sterile; when the size of the amplified fragment is 272bp, it is determined that This carrot variety is fertile.

During the long-term scientific research experiment, the applicant used the technical idea of transcriptome analysis to screen Indel molecular markers, and performed transcriptome sequencing by extracting total RNA from the inflorescences of a group of stable carrot petal-type cytoplasmic male sterile lines and their maintainers Analysis, compared with the published reference mitochondrial genome of carrots, screened out dozens of Indel sites with differences between the sterile line and the maintainer line, designed upstream and downstream primers containing Indel sites, and tested them in different species Carrot specific amplification verification between fertile and sterile carrot plants, using capillary electrophoresis to separate fragments, and read the analysis results to identify carrot cytoplasmic male sterility. Finally, after repeated experimental screening and verification, the applicant obtained a pair of specific primers, which can completely distinguish the fertility of the varieties to be tested. When the size of the amplified fragment was 264bp, it was determined that the carrot variety was sterile; when the size of the amplified fragment was 272bp, it was determined that the carrot variety was fertile. By using the pair of specific primers provided by the invention, cytoplasmic male sterile carrot varieties can be quickly screened out at the seedling stage, with high accuracy, and have important practical application value for removing miscellaneous plants in the breeding process.

In a second aspect, the present invention provides a method for identifying carrot petal-type cytoplasmic male sterility, comprising the following steps:

(1) extracting the total DNA of the carrot sample to be tested;

(2) using the primers described in the first aspect to perform PCR amplification on the extracted total DNA to obtain a product;

(3) The product is subjected to electrophoresis to separate bands, and the fertility of carrot varieties is judged according to the size of the bands.

Preferably, the criterion for judging the fertility of a carrot variety is: when the size of the amplified fragment is 264bp, it is judged that the carrot variety is sterile; when the size of the amplified fragment is 272bp, it is judged that the carrot variety is fertile.

Preferably, said carrot sample includes leaves and inflorescences.

Preferably, the leaves are young leaves.

Preferably, the electrophoresis method includes capillary electrophoresis or polyacrylamide gel electrophoresis.

Preferably, the PCR amplification reaction system is: a total volume of 10 μL, including 5 μL of 2×Taq Master Mix forPAGE, 0.5 μL of 10 μmol/L forward primer and 0.5 μL of reverse primer, 1 μL of 25 ng/μL DNA template, and Make up to 10 μL with sterile water.

Preferably, the reaction program of the PCR amplification is: pre-denaturation at 95°C for 5 min, 1 cycle; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 20 s, 35 cycles, final extension at 72°C for 5 min, 1 cycle cycle.

Preferably, the electrophoresis is capillary electrophoresis, and the capillary electrophoresis step: after the PCR is completed, 15 μL of TE is added to each sample well to be tested, 25 μL of Ladder is added to the Marker well, and then the sample plate is placed in the capillary electrophoresis instrument, After the electrophoresis was completed, the analysis software was used to analyze the results.

In a third aspect, the present invention provides an application of the primer pair described in the first aspect in identifying carotidized male sterility.

In a fourth aspect, the present invention provides an application of the primer pair as described in the first aspect in the preparation of reagents and/or kits for identifying carrot petal male sterility.

In the fifth aspect, the present invention provides a reagent and/or kit for identifying carrot petalized male sterility, comprising the primer pair described in the first aspect.

Specifically, the PCR reaction system of this application: a total volume of 10 μL, including 5 μL of 2×Taq Master Mix (for PAGE), 0.5 μL of upstream and downstream primers (10 μmol/L), and 1 μL of DNA template (25ng/μL). Make up to 10 μL with water.

Specifically, the PCR reaction amplification procedure of this application: 95°C pre-denaturation for 5 minutes, 1 cycle; 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 20 s, 35 cycles, 72°C final extension for 5 min, 1 cycle .

Specifically, capillary electrophoresis in this application: after PCR, add 15 μL of TE to the sample well, add 25 μL of Ladder to the marker well, and then place the sample plate in the capillary electrophoresis instrument. After the electrophoresis is completed, use the corresponding analysis software PROSize 3.0 analysis results. Experiments have proved that the carrot samples carrying the 264bp fragment are sterile; the carrot samples carrying the 272bp fragment are fertile.

Specifically, the method of this application is:

Extracting the total DNA of carrot leaves to be tested, using the primers to perform PCR amplification on the extracted total DNA, separating the fragments of the product by capillary electrophoresis, and judging the fertility of the carrot variety to be tested by the size of the band;

The forward primer sequence of the primer pair is SEQ ID NO.1:

5'-TTGTGCGGTTAAGGTTGTTGT-3';

The reverse primer sequence of the primer pair is SEQ ID NO.2:

5'-CCTGTTCTTGTGGATCAATGGA-3'.

The PCR reaction system: a total volume of 10 μL, including 5 μL of 2×Taq Master Mix (for PAGE), 0.5 μL of upstream and downstream primers (10 μmol/L), 1 μL of DNA template (25ng/μL), supplemented with sterile water to 10 μL.

PCR reaction amplification program: pre-denaturation at 95°C for 5 min, 1 cycle; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 20 s, 35 cycles, final extension at 72°C for 5 min, 1 cycle.

Capillary electrophoresis step: After PCR, add 15 μL of TE to each sample well to be tested, add 25 μL Ladder to the Marker well, and then place the sample plate in the capillary electrophoresis instrument. After the electrophoresis is completed, use the corresponding analysis software Analyze the results.

Beneficial effect:

The present invention mainly studies the identification of carrot petal-type cytoplasmic sterile plants, solves the problem of early removal of impurities in the process of backcross transfer, and focuses on solving the problem of screening suitable molecular markers for different types of carrot petal-type male sterility For the identification of varieties, the difficulty of the invention is that a pair of molecular markers can be used to completely distinguish most of the carrot petal-type male sterile and fertile varieties. The innovation of the present invention is to use the results of transcriptome sequencing to compare the results of the reference mitochondrial genome. Obtaining more accurate screening of petalized male sterility and fertility molecular markers provides a reliable technical means for the identification of carrot cytoplasmic male sterility and is conducive to accelerating the progress of carrot breeding.

The invention provides a method for identifying carrot cytoplasmic male sterile plants using primer pairs designed by Indel molecular markers. The molecular markers and primer pairs provided by the present invention can quickly screen out cytoplasmic male sterile carrot varieties at the seedling stage. The method has good specificity, good stability, high accuracy, fast screening speed, and is suitable for backcross breeding. The removal of miscellaneous plants in the process provides technical support for accelerating the rapid breeding of carrot strains, and has important practical application value and broad market prospects.

Description of the drawings:

Figure 1: Electropherograms of polymorphisms presented by nine pairs of primers between fertile and sterile samples.

Figure 2: A pair of Indel molecular marker specific primer pair Mt-1 identification of carrot cytoplasmic male sterility electrophoresis; the figure is marked as 1: sterile line; 2: maintainer line; 3: sterile line; 4: Maintainer line; 5: sterile line; 6: maintainer line; 7: sterile line; 8: maintainer line.

Figure 3: A pair of Indel molecular marker specific primer pair Mt-1 identification of carrot cytoplasmic male sterility electrophoresis; the figure is marked as 1: Mendel 3; 2: SK316; 3: Chunao 709; 4: Nobel No. 2; 5: rose red; 6: wax twist; 7: Chunao 705; 8: Hongbao six-inch ginseng.

Detailed ways

The present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be considered as illustrative rather than limiting the scope of the invention, the spirit and scope of which is defined only by the claims. For those skilled in the art, on the premise of not departing from the spirit and scope of the present invention, various changes or modifications to the material components and dosage in these embodiments also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are commercially available.

Example 1

A group of inflorescence RNAs from carrot petal cytoplasmic male sterile lines and their maintainers (HYs698 and HYd698) of the Hongyue strain bred by the Vegetable Research Institute of Tianjin Academy of Agricultural Sciences were extracted, a cDNA library was established, and the transcriptome was sequenced. , compared with carrot reference mitochondrial genome, the website of the reference mitochondrial genome is as follows: http://ftp.ensemblgenomes.org/pub/plants/release-51/fasta/daucus_carota/dna/ ; Table 1 shows a schematic example of the position where there is a difference in the number of bases (Indel≥4bp) between fertile and sterile.

Table 1 Nine differential positions screened by transcriptome analysis

On the basis of including this difference site, the sequence was extended up and down by about 100 bp each, and 9 pairs of primers were designed in order to amplify the bands, corresponding to the difference positions 1-9 respectively, as shown in Table 2.

Table 2 Primer pair sequence

Carotidized cytoplasmic male sterile line and its maintainer line (HYs698 and HYd698) were detected by 9 primer pairs Mt-1 to Mt-9 designed by us. After PCR amplification, electrophoresis separation, and capillary electrophoresis verification, the results were as follows: Figure 1 shows. Among them, the primer pairs Mt-2, Mt-3, Mt-4, Mt-7 and Mt-8 had almost no difference in amplified bands between fertile and sterile, and could not distinguish fertility. The fragments amplified by the primer pair Mt-5, Mt-6 and Mt-9 had little difference, which was not conducive to observation and application. The difference in the bands amplified by the primer pair Mt-1 was obvious. Compared with the reference mitochondrial genome, the fertile band was consistent with the reference mitochondrial genome sequence, with a size of 272bp, while the sterile band was 264bp in size. Therefore, the specific primer pair Mt-1 and the method can clearly distinguish fertile and sterile (HYs698 and HYd698) according to the bands, and then use this primer pair to verify other fertile varieties.

Example 2

(1) Sample group 1: WMA-477 and WMB-477 are derived from Japanese Kuroda-type carrot petal-type cytoplasmic male sterile lines and their maintainers; LSsA-128 and LSsB-128 are derived from American cut-type carrots The petalized cytoplasmic male sterile line and its maintainer line; SKs35-027 and SKd35-027 are derived from the carrot petalized cytoplasmic male sterile line and its maintainer line with the combination of Nantes and Kuroda; HYs698 and HYd698 are derived from Carrot petal-type cytoplasmic male sterile line and its maintainer line of Hongyue strain bred by Vegetable Research Institute of Tianjin Academy of Agricultural Sciences.

(2) Reagents: 2×Taq Master Mix (for PAGE); Specific amplification primers synthesized by Jinweizhi.

The forward primer sequence is SEQ ID NO.1: 5'-TTGTGCGGTTAAGGTTGTTGT-3'

The reverse primer sequence is SEQ ID NO.2: 5'-CCTGTTCTTGTGGATCAATGGA-3'.

(3) Polymorphism detection: extract the genomic DNA of young carrot leaves to be tested, and use it as a template to perform PCR amplification using the specific primer pair provided by the present invention.

(4) PCR reaction system: a total volume of 10 μL, including 5 μL of 2×Taq Master Mix (for PAGE), 0.5 μL of upstream and downstream primers (10 μmol/L), 1 μL of DNA template (25ng/μL), supplemented with sterile water to 10 μL.

(5) PCR amplification program: pre-denaturation at 95°C for 5 min, 1 cycle; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 20 s, 35 cycles, final extension at 72°C for 5 min, 1 cycle.

(6) Capillary electrophoresis: After PCR, add 15 μL of TE to the sample well, add 25 μL of Ladder to the H12 well, and then place the sample plate in the capillary electrophoresis instrument. After the electrophoresis is completed, use the corresponding analysis software PROSize3.0 Analyze the results.

(7) Result judgment: if the PCR amplification product is band type A (displays a band, is 264bp), then the carrot to be tested is a sterile line; if the PCR amplification product is band type B (displays a band, is 272bp) , the carrot to be tested is the maintainer line.

(8) Taking the carrot petal-type male sterile line and its maintainer line as test samples, the size of the DNA fragment amplified by the primer pair of the universal molecular marker was tested. The results are shown in Figure 2 and Table 3. The DNA fragment of the carrot petal-type male sterile line is 264bp; the DNA fragment of the carrot petal-type male sterile maintainer line is 272bp.

The experimental results show that the universal molecular marker and the specific primer pair screened by the present invention have a good application in the identification of carrot cytoplasmic male sterile plants. The application of the Indel molecular marker and the primer pair can accurately distinguish the carrot petalized male sterile line and its maintainer line of different types of resources.

Table 3 Identification results of carrot cytoplasmic male sterility

Example 3

(1) Sample group 2: 8 commercial varieties available on the market (Mendel No. 3, SK316, Chunao 709, Nobel No. 2, Rose Red, Laxian, Chunao 705, Hongbao Six-inch Ginseng).

(2) Reagents: 2×Taq Master Mix (for PAGE); Specific amplification primers synthesized by Jinweizhi.

The forward primer sequence is SEQ ID NO.1: 5'-TTGTGCGGTTAAGGTTGTTGT-3'

The reverse primer sequence is SEQ ID NO.2: 5'-CCTGTTCTTGTGGATCAATGGA-3'.

(3) Polymorphism detection: extract the genomic DNA of young carrot leaves to be tested, and use it as a template to perform PCR amplification using the specific primer pair provided by the present invention.

(4) PCR reaction system: a total volume of 10 μL, including 5 μL of 2×Taq Master Mix (for PAGE), 0.5 μL of upstream and downstream primers (10 μmol/L), 1 μL of DNA template (25ng/μL), supplemented with sterile water to 10 μL.

(5) PCR amplification program: pre-denaturation at 95°C for 5 min, 1 cycle; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 20 s, 35 cycles, final extension at 72°C for 5 min, 1 cycle.

(6) Capillary electrophoresis: After PCR, add 15 μL of TE to the sample well, add 25 μL of Ladder to the H12 well, and then place the sample plate in the capillary electrophoresis instrument. After the electrophoresis is completed, use the corresponding analysis software to analyze the results.

(7) Result judgment: if the PCR amplification product is band type A (displays a band, is 264bp), then the carrot to be tested is a sterile variety; if the PCR amplification product is band type B (displays a band, is 272bp) , the carrot to be tested is a fertile variety.

(8) The results are shown in Table 4 and Figure 3. From Table 4 and Figure 3, it can be seen that the Indel molecular marker and primer pair can be used to accurately distinguish carrot petal male sterile and fertile lines, and this marker is suitable for wide application.

Table 4 Identification results of carrot cytoplasmic male sterility

In summary, the present invention screens Indel molecular markers through transcriptome analysis and designs a pair of specific primers for identifying carrot petal-type cytoplasmic male sterility. The identification method using the primer pair has good specificity and stability The advantages of high and fast screening speed are suitable for the identification of carrot cytoplasmic male sterile plants, and provide technical support for accelerating the rapid breeding of carrot lines.

After the preferred embodiment described in detail, those skilled in the art can clearly understand that various changes and modifications can be carried out without departing from the scope and spirit of the above-mentioned patent application. Any simple modifications, equivalent changes and modifications all belong to the scope of the technical solutions of the present invention. And the present invention is not limited by the example implementations in the specification.

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Claims (10)

1. A primer pair for identifying carrot petaloid cytoplasm male sterility is characterized in that,

the forward primer sequences of the primer pairs are as follows: 5'-TTGTGCGGTTAAGGTTGTTGT-3';

the reverse primer sequences of the primer pairs are as follows: 5'-CCTGTTCTTGTGGATCAATGGA-3'.

2. A method for identifying carrot petaloid cytoplasmic male sterility, comprising the steps of:

(1) Extracting total DNA of a carrot sample to be detected;

(2) Performing PCR amplification on the extracted total DNA by using the primer pair of claim 1 to obtain a product;

(3) And (4) carrying out electrophoresis separation on the product to obtain strips, and judging the fertility of the carrot variety according to the size of the strips.

3. The method according to claim 2, wherein the criterion for judging the fertility of the carrot variety is: when the amplified fragment size is 264bp, the carrot variety is judged to be sterile; when the amplified fragment size is 272bp, the carrot variety is judged to be fertile.

4. A method according to claim 2 or 3, characterized in that: the carrot samples included leaves and inflorescences.

5. A method according to claim 2 or 3, wherein the method of electrophoresis comprises capillary electrophoresis or polyacrylamide gel electrophoresis.

6. The method of claim 2 or 3, wherein the reaction system for PCR amplification is: a total volume of 10. Mu.L, including 5. Mu.L of 2 XTaq Master Mix for PAGE, 0.5. Mu.L of each of 10. Mu. Mol/L of forward and reverse primers, 1. Mu.L of 25 ng/. Mu.L of DNA template, was supplemented to 10. Mu.L with sterile water.

7. The method of claim 2 or 3, wherein the reaction procedure of the PCR amplification is: pre-denaturation at 95 ℃ for 5min,1 cycle; denaturation at 95 ℃ for 15s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 20s, and 35 cycles; final extension at 72 ℃ for 5min,1 cycle.

8. The method according to claim 2 or 3, wherein the electrophoresis is capillary electrophoresis, and the capillary electrophoresis step is: after the PCR is finished, 15 mu L of TE is added into each sample hole to be detected, 25 mu L of Ladder is added into the Marker hole, then the sample plate is placed in a capillary electrophoresis instrument, and after the electrophoresis is finished, the result is analyzed by adopting analysis software.

9. Use of the primer pair of claim 1 in the preparation of a kit for identifying carrot petalinized male sterility.

10. A kit for identifying carrot petaline type male sterility, comprising the primer of claim 1.

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