CN114150084B

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

Info

Publication number: CN114150084B
Application number: CN202111542676.XA
Authority: CN
Inventors: 孙海波; 王一衡; 付任胜; 岳东霞; 陈磊; 杨迎霞
Original assignee: Tianjin Academy of Agricultural Sciences
Current assignee: Tianjin Academy of Agricultural Sciences
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2023-04-18
Anticipated expiration: 2041-12-16
Also published as: CN114150084A

Abstract

The invention discloses a primer pair for identifying carrot petalled cytoplasmic male sterility and application thereof. The application screens Indel sites with difference between the sterile line and the maintainer line and designs a primer by carrying out transcriptome sequencing analysis on total RNA of a group of stable carrot petaloid cytoplasmic male sterile lines and the maintainer line and comparing the transcriptome sequencing analysis with published carrot reference mitochondrial genomes. The results show that the primer pair of the application can completely distinguish fertility of the variety to be detected, can quickly screen out cytoplasmic male sterile carrot varieties in the seedling stage, has high accuracy, and has important practical application value and market prospect for removing hybrid plants in the breeding process.

Description

Primer pair for identifying carrot petaloid cytoplasmic male sterility and application thereof

Technical Field

The invention belongs to the field of biotechnology breeding, and particularly relates to a primer pair for identifying carrot petaloid cytoplasmic male sterility and application thereof.

Background

Carrot (Daucus carota L.) belongs to an important root vegetable of carrot genus of Umbelliferae, and its fleshy root is rich in carotenoid, has high nutritive value, is widely planted all over the world, and is one of ten vegetable crops all over the world. The rapid increase of the planting area of the carrots not only greatly increases the demand of seeds, but also has higher requirements on the quality and the production efficiency of the seeds.

Carrot is a biennial high-degree cross pollination crop, the floral organs are extremely small, the artificial emasculation is difficult, and the conventional artificial hybridization breeding is difficult to develop. Due to the difficulty in developing conventional artificial crossbreeding, the utilization of the heterosis of the carrot is laggard. Therefore, the early breeding method mainly adopts population selection and pedigree selection, but the variety produced by the method has poor consistency and stability, mainly because of the heterozygosity of the conventional variety and serious sexual decline caused by self-crossing in the population.

With the discovery of the cytoplasmic male sterile material of the carrot, the breeding of the first generation hybrid of the carrot becomes practical. At present, the cytoplasmic male sterility types of carrots mainly comprise a petal type and a brown drug type. With the deep research on the genetic mechanism of carrot male sterility, the carrot male sterility material is widely applied to the actual breeding work, thereby ensuring that the variety has higher consistency and stability. In the 60's of the 20 th century, the first carrot generation hybrid was first introduced in the united states. At present, carrot varieties produced and used in Europe and America countries are mostly first generation hybrids, but male sterility sources used in different countries or seed companies are different, for example, the U.S. mainly adopts a petaloid type, most seed companies in Europe use a brown medicine type, and most of Asia use the petaloid type.

The conventional method for cultivating the carrot male sterile line is to apply a backcross transformation method, backcross excellent fertile single plants, and perform fertility identification on backcross progeny, wherein the method focuses on checking that a maintainer line has no restoring gene, and the fertility identification method needs to be performed in the plant flowering period.

Therefore, the development of cytoplasmic gene molecular markers for fertility identification research on backcross materials can distinguish carrot fertility in the seedling stage, remove possibly-doped fertile plants in advance, greatly save manpower and material resources and breeding space, and provide convenience for backcross after subsequent flowering.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a primer pair designed by Indel molecular markers, a method for identifying carrot petaloid cytoplasmic male sterile plants by using the primer pair and application of the method.

The technical content of the invention is as follows:

in a first aspect, the invention provides a primer pair for identifying carrot petaloid 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 consists of an upstream primer and a downstream primer for amplifying a specific DNA fragment, namely a forward primer and a reverse primer; the specific DNA fragment has a target sequence which can be amplified by a primer pair consisting of a forward primer and a reverse primer and is simultaneously positioned in a carrot reference mitochondrial genome, and the website of the reference mitochondrial genome is as follows:

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

the primer pair has the following functions: after PCR amplification is carried out on the total DNA of the carrot sample to be detected by adopting a primer pair, when the size of an amplified fragment is 264bp, the carrot variety is judged to be sterile; when the size of the amplified fragment is 272bp, the carrot variety is judged to be fertile.

In the long-term scientific research experiment process, the applicant utilizes the technical thought of screening Indel molecular markers by transcriptome analysis, extracts total RNA from a group of stable carrot petaloid cytoplasmic male sterile lines and inflorescences of maintainer lines thereof, performs transcriptome sequencing analysis, compares the total RNA with published carrot reference mitochondrial genomes, screens out tens of Indel sites which are different between the sterile lines and the maintainer lines, respectively designs upstream and downstream primers containing the Indel sites, performs specific amplification verification between different types of carrot fertile plants and sterile plants, separates fragments by capillary electrophoresis, reads analysis results, and thereby identifies the cytoplasmic male sterility of the carrots. Finally, through screening and verification of a heavy experiment, the applicant obtains a pair of specific primers which can completely distinguish the fertility of the variety to be detected. 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. By utilizing the specific primer pair provided by the invention, a carrot variety with cytoplasmic male sterility can be quickly screened out in the seedling stage, the accuracy is high, and the method has important practical application value for removing hybrid plants in the breeding process.

In a second aspect, the present invention provides a method of identifying carrot petalled cytoplasmic male sterility comprising the steps of:

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

(2) Carrying out PCR amplification on the extracted total DNA by adopting the primer pair of the first aspect 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.

Preferably, the standard for judging the fertility of the carrot variety is as follows: when the amplified fragment size is 264bp, the carrot variety is judged to be sterile; when the size of the amplified fragment is 272bp, the carrot variety is judged to be fertile.

Preferably, the carrot sample comprises leaves and inflorescences.

Preferably, the leaves are young leaves.

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

Preferably, the reaction system of the 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.

Preferably, 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,35 cycles, final extension at 72 ℃ for 5min,1 cycle.

Preferably, the electrophoresis is capillary electrophoresis, the capillary electrophoresis step: after 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 a Marker hole, then the sample plate is placed in a capillary electrophoresis instrument, and after electrophoresis is finished, analysis software is adopted to analyze results.

In a third aspect, the present invention provides the use of a primer pair as described in the first aspect for identifying carrot petaline type male sterility.

In a fourth aspect, the invention provides an application of the primer pair in the first aspect in preparing a reagent and/or a kit for identifying carrot petaline type male sterility.

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

Specifically, the PCR reaction system of the present application: 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 the upstream and downstream primers (10. Mu. Mol/L), 1. Mu.L of DNA template (25 ng/. Mu.L), was supplemented to 10. Mu.L with sterile water.

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

Specifically, the capillary electrophoresis of the present application: after the PCR is finished, 15 mu L of TE is added into the sample hole, 25 mu L of Ladder is added into the Marker hole, the sample plate is placed in a capillary electrophoresis apparatus, and after the electrophoresis is finished, the result is analyzed by adopting corresponding analysis software PROSize 3.0. Experiments prove that the carrot sample carrying the 264bp fragment is sterile; carrot samples carrying the 272bp fragment were fertile.

Specifically, the method comprises the following steps:

extracting total DNA of carrot leaves to be detected, carrying out PCR amplification on the extracted total DNA by using the primer pair, separating fragments of a product through capillary electrophoresis, and judging the fertility of the carrot variety to be detected according to the size of a strip;

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 comprises: a total volume of 10. Mu.L, including 5. Mu.L of 2 XTAQQ Master Mix (for PAGE), 0.5. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), 1. Mu.L of DNA template (25 ng/. Mu.L), was supplemented to 10. Mu.L with sterile water.

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

Capillary electrophoresis step: after 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 a Marker hole, then the sample plate is placed in a capillary electrophoresis instrument, and after electrophoresis is finished, the result is analyzed by adopting corresponding analysis software.

Has the advantages that:

the invention mainly researches the identification problem of carrot petaline cytoplasmic sterile plants, solves the problem of early impurity removal in the backcross transformation process, and mainly solves the identification problem of screening proper molecular markers for different types of carrot petaline male sterile varieties.

The invention provides a method for identifying cytoplasmic male sterile plants of carrots by using a primer pair designed by Indel molecular markers. The molecular marker and the primer provided by the invention can be used for quickly screening cytoplasmic male sterile carrot varieties in the seedling stage, and the method has the advantages of good specificity, good stability, high accuracy and high screening speed, provides technical support for removing hybrid plants in the backcross transformation process and accelerating the quick breeding of carrot strains, and has important practical application value and wide market prospect.

Description of the drawings:

FIG. 1: the nine pairs of primers present a polymorphic electropherogram between fertile and sterile samples.

FIG. 2 is a schematic diagram: identifying a carrot cytoplasmic male sterility electrophoresis pattern by a pair of Indel molecular marked specific primers Mt-1; and if the icons are respectively 1: a sterile line; 2: a holding system; 3: a sterile line; 4: a holding system; 5: a sterile line; 6: a holding system; 7: a sterile line; 8: a holding system.

FIG. 3: identifying a carrot cytoplasmic male sterility electrophoresis pattern by a pair of Indel molecular marked specific primers Mt-1; and if the icons are respectively 1: mendelian No. 3; 2: SK316;3: chun ao 709;4: nobel II; 5: rose bengal; 6: wax twisting; 7: chun ao 705;8: hongbao Liucun ginseng.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.

Example 1

Through respectively extracting a group of carrot petal type cytoplasmic male sterile lines of Hongye strains selected by vegetable research institute of Tianjin city agricultural academy of sciences and inflorescence RNAs of maintainer lines (HYs 698 and HYd 698), establishing a cDNA library, sequencing a transcriptome, and comparing and analyzing with carrot reference mitochondrial genome, the websites of the reference mitochondrial genome are as follows:http://ftp.ensemblgenomes.org/pub/plants/release-51/fasta/daucus_carota/dna/(ii) a 32 positions with base number difference (Indel is more than or equal to 4 bp) between fertile and sterile are screened out, and the schematic example is shown in Table 1.

TABLE 1 9 differential positions screened by transcriptome analysis

On the basis of containing the differential sites, the sequences are vertically extended by about 100bp, 9 pairs of primer amplification bands are sequentially designed according to the sequence and respectively correspond to the differential positions 1-9, and the table 2 shows.

TABLE 2 primer pair sequences

Carrot petiolated cytoplasmic male sterile lines and maintenance lines thereof (HYs 698 and HYd 698) are detected through 9 primer pairs Mt-1 to Mt-9 designed by us, PCR amplification, electrophoretic separation and capillary electrophoresis verification results are shown in figure 1. Wherein, the amplification bands of the primer pairs Mt-2, mt-3, mt-4, mt-7 and Mt-8 have almost no difference between fertile and sterile, and fertility can not be distinguished. The amplified fragments of the primer pairs Mt-5, mt-6 and Mt-9 have small difference, which is not beneficial to observation and application. The difference of the amplified bands of the primer pair Mt-1 is obvious, and the amplified bands are compared and analyzed with the reference mitochondrial genome, the fertile band is consistent with the reference mitochondrial genome sequence, the size is 272bp, and the sterile band is 264bp. Therefore, the specific primer pair Mt-1 and the method can clearly distinguish fertility from sterility (HYs 698 and HYd 698) according to the bands, and further verify other fertility varieties by using the primer pair.

Example 2

(1) Sample set 1: WMA-477 and WMB-477 are derived from the Japanese black field type carrot petal type cytoplasmic male sterile line and the maintainer line thereof; LSsA-128 and LSsB-128 are derived from American cut carrot petaloid cytoplasmic male sterile line and maintainer line thereof; SKs35-027 and SKd-027 are derived from carrot petaloid cytoplasmic male sterile line and maintainer line thereof with southward and black field combined type; HYs698 and HYd698 are derived from the carrot petal type cytoplasmic male sterile line and its maintainer line of the Hongye line bred at vegetable research institute of agricultural and scientific institute of Tianjin.

(2) Reagent: 2 XTaq Master Mix (for PAGE); jin Weizhi.

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

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

(3) And (3) polymorphism detection: extracting the genome DNA of the young leaf of the carrot to be detected, and carrying out PCR amplification by using the genome DNA as a template and the specific primer pair provided by the invention.

(4) And (3) PCR reaction system: 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 the upstream and downstream primers (10. Mu. Mol/L), 1. Mu.L of DNA template (25 ng/. Mu.L), was supplemented to 10. Mu.L with sterile water.

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

(6) Capillary electrophoresis: after the PCR is finished, 15 mu L of TE is added into a sample hole, 25 mu L of Ladder is added into an H12 hole, then the sample plate is placed in a capillary electrophoresis apparatus, and after the electrophoresis is finished, the result is analyzed by adopting corresponding analysis software PROSize3.0.

(7) And (5) judging a result: if the PCR amplification product is a banding pattern A (showing a band, 264 bp), the carrot to be detected is a sterile line; if the PCR amplification product is banding pattern B (showing a band, 272 bp), the carrot to be tested is the maintainer line.

(8) The carrot petal type male sterile line and the maintenance line thereof are taken as test samples, and the size of the amplified DNA fragment of the primer pair of the universal molecular marker is tested. The results are shown in FIG. 2 and Table 3, and the DNA fragment of the carrot petaloid male sterile line is 264bp; the carrot petal type male sterility maintainer line DNA fragment is 272bp.

Experimental results show that the universal molecular marker and the specific primer pair screened by the invention have good application in identification of cytoplasmic male sterile plants of carrots. By applying the Indel molecular marker and the primer pair, carrot petalinized male sterile lines and carrot petalinized male sterile lines of different types of resources can be accurately distinguished.

TABLE 3 identification of cytoplasmic male sterility in carrot

Example 3

(1) Sample set 2: commercial varieties 8 (Mendelian No. 3, SK316, chunrao 709, nobel No. two, rose bengal, rad twist, chunrao 705, hongbao six cun ginseng) are commercially available.

(2) Reagent: 2 × Taq Master Mix (for PAGE); jin Weizhi.

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

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

(4) And (3) PCR reaction system: a total volume of 10. Mu.L, including 5. Mu.L of 2 XTAQQ Master Mix (for PAGE), 0.5. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), 1. Mu.L of DNA template (25 ng/. Mu.L), was supplemented to 10. Mu.L with sterile water.

(6) Capillary electrophoresis: after the PCR is finished, 15 mu L of TE is added into a sample hole, 25 mu L of Ladder is added into an H12 hole, then the sample plate is placed in a capillary electrophoresis apparatus, and after the electrophoresis is finished, the result is analyzed by adopting corresponding analysis software.

(7) And (5) judging a result: if the PCR amplification product is a banding pattern A (showing a band, 264 bp), the carrot to be detected is a sterile variety; if the PCR amplification product is the banding pattern B (showing a band, 272 bp), the carrot to be detected is a fertile variety.

(8) The results are shown in Table 4 and FIG. 3, and it can be seen from Table 4 and FIG. 3 that the carrot petaloid male sterile and fertile lines can be accurately distinguished by using the Indel molecular marker and the primer pair, and the marker is suitable for wide application.

TABLE 4 identification of cytoplasmic male sterility in carrot

In conclusion, the Indel molecular markers are screened through transcriptome analysis and a pair of specific primers for identifying carrot petaline cytoplasmic male sterility is obtained through design, and the identification method adopting the primer pair has the advantages of good specificity, high stability and high screening speed, is suitable for being applied to identification of carrot cytoplasmic male sterility plants, and provides technical support for accelerating rapid breeding of carrot strains.

It will be apparent to those skilled in the art that various changes and modifications can be made in the above embodiments without departing from the scope and spirit of the invention, and it is intended that all such changes and modifications as fall within the true spirit and scope of the invention be interpreted in accordance with the principles of the invention. And the invention is not limited to the example embodiments set forth in the description.

Sequence listing

<110> Tianjin City academy of agricultural sciences

<120> primer pair for identifying carrot petaline cytoplasmic male sterility and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> carrot (Daucus carota)

<400> 1

ttgtgcggtt aaggttgttg t 21

<210> 2

<211> 22

<212> DNA

<213> carrot (Daucus carota)

<400> 2

cctgttcttg tggatcaatg ga 22

<210> 3

<211> 22

<212> DNA

<213> carrot (Daucus carota)

<400> 3

ggcttcttgg ctggaatcat aa 22

<210> 4

<211> 20

<212> DNA

<213> carrot (Daucus carota)

<400> 4

ggtggtgaga cgaggtgtag 20

<210> 5

<211> 20

<212> DNA

<213> carrot (Daucus carota)

<400> 5

caatcagacg gagcggaatc 20

<210> 6

<211> 21

<212> DNA

<213> carrot (Daucus carota)

<400> 6

tgttagcagc cagagatgag t 21

<210> 7

<211> 20

<212> DNA

<213> carrot (Daucus carota)

<400> 7

gctgttcggt tacgcaatct 20

<210> 8

<211> 21

<212> DNA

<213> carrot (Daucus carota)

<400> 8

cttacttctc ggaggcagga t 21

<210> 9

<211> 23

<212> DNA

<213> carrot (Daucus carota)

<400> 9

ggaactcgtg tcttatgctt ctt 23

<210> 10

<211> 24

<212> DNA

<213> carrot (Daucus carota)

<400> 10

gatagtgcgg tattgttgtt atgg 24

<210> 11

<211> 21

<212> DNA

<213> carrot (Daucus carota)

<400> 11

atcgtagcgt ggtagcatac a 21

<210> 12

<211> 20

<212> DNA

<213> carrot (Daucus carota)

<400> 12

acatctcggc ttggttggaa 20

<210> 13

<211> 21

<212> DNA

<213> carrot (Daucus carota)

<400> 13

ctcgctcaac actcgttctt c 21

<210> 14

<211> 24

<212> DNA

<213> carrot (Daucus carota)

<400> 14

gcaatggtaa tctcgtcctt agtt 24

<210> 15

<211> 25

<212> DNA

<213> carrot (Daucus carota)

<400> 15

ctcaagtata ggctcttctt ctgtt 25

<210> 16

<211> 25

<212> DNA

<213> carrot (Daucus carota)

<400> 16

gtatagtata gtgcggctta tggtt 25

<210> 17

<211> 23

<212> DNA

<213> carrot (Daucus carota)

<400> 17

aactagcaga atcgtggaac atc 23

<210> 18

<211> 21

<212> DNA

<213> carrot (Daucus carota)

<400> 18

cgcaggcact gaatgaatga a 21

Claims

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. 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.