CN107881255B

CN107881255B - Double-significance marker for accurately identifying onion S/N cytoplasm genotype and application thereof

Info

Publication number: CN107881255B
Application number: CN201711424260.1A
Authority: CN
Inventors: 杨妍妍; 吴雄; 刘冰江; 霍雨猛; 曹德高
Original assignee: Vegetable Research Institute of Shandong Academy of Agricultural Sciences
Current assignee: Vegetable Research Institute of Shandong Academy of Agricultural Sciences
Priority date: 2017-12-25
Filing date: 2017-12-25
Publication date: 2020-03-13
Anticipated expiration: 2037-12-25
Also published as: CN107881255A

Abstract

The invention discloses a double-significance marker for accurately identifying the S/N cytoplasm genotype of an onion and application thereof. The length of the specific fragment of the S-type cytoplasm is 232bp, and the length of the specific fragment of the N-type cytoplasm is 196bp in the double-display marker, and the invention also discloses the SCAR marker primers SEQ ID NO.5 and SEQ ID NO. 6. The obtained SCAR molecular marker is used for quickly and accurately judging the cytoplasm type of the onion material, so that the wrong classification of the cytoplasm genotype caused by experimental operation errors and invalid PCR amplification is avoided, and when a maintainer line is selected in a large group, a group sample can be mixed to extract DNA for preliminary screening of individual plants with or without the maintainer line, so that the selection efficiency is greatly improved, the time and the cost are saved, and the method has important significance for onion breeding sterile line and maintainer line matching and onion hybrid breeding.

Description

Double-significance marker for accurately identifying onion S/N cytoplasm genotype and application thereof

Technical Field

The invention belongs to the technical field of plant breeding, relates to a molecular marker for onion breeding and application thereof, and particularly relates to a double-display molecular marker for identifying the S/N cytoplasmic genotype of an onion and application thereof, which can be applied to molecular marker-assisted breeding to accurately select an onion male sterile line and a matched maintainer line.

Background

Onion (Allium cepa.L), namely onion bulb, onion, scallion and scallion, and the like, are diploid plants growing for two years, belong to crops of Allium in the family of liliaceae, have recorded that the cultivation history is more than 5000 years, the recent generations of the onions are introduced into China, the cultivation in China has the history of more than 100 years, but the onion is strong in adaptability and resistant to storage and transportation, the cultivation history in China is short, but the development speed is very high, and the onions are widely cultivated all over the country. The producing areas mainly include Yunnan, Sichuan, Jiangsu, Shandong, Gansu, inner Mongolia and Xinjiang. At present, our country has the largest onion production area and yield in the world, and the total yield and area accounts for nearly 30% of the total yield and area in the world, and is one of 4 countries (china, india, usa and japan) with large onion production. The onion can be eaten fresh, is also used for processing products in a large quantity, is rich in various sulfides and flavonoid substances, has the health-care effects of reducing and preventing thrombosis, reducing blood fat, reducing blood sugar, resisting oxidation, resisting aging, diminishing inflammation, preventing cancer and the like, is praised as a vegetable queen and is deeply loved by consumers.

Male sterile plants were first discovered in "Italian Red" (Italian Red) variety by Jones and Emsweller in the United states in 1936, the sterility of which was commonly controlled by a single nuclear gene and cytoplasm [ Jones HA. and Emsweller SL. American terriere ion. Proc. Amer. Soc. Hort. Sci.1936,34: 582-. In 1943 Jones and Clarke discovered a male sterile line [ Jones HAand Clarke A. history of male sterility in the ketone and the production of hybrid seed, Proc Amer. Soc. Hort.Sci.1943,43:189-194], and subsequently onion became one of the earliest vegetable crops in the world that utilized heterosis. The cytoplasmic-nuclear interaction controls the fertility of the onion, and has great advantages when being used for producing onion hybrid seeds. However, due to the high frequency of the Ms gene or the S-type cytoplasm, it is difficult to successfully obtain maintainers from several cultivars, such as: "Texas 1015Y" (United States), "SappoKi" (Japan), "Pukekohe Longkeeper" (New Zealand). Therefore, the breeding of the onion male sterility maintainer line is a very time-consuming and labor-consuming work.

In recent years, the development of molecular biology provides a new means for plant genetic markers based on DNA variation, namely molecular marker technology. It is directly appeared in DNA form, can be detected in every tissue and every development period of plant body, and is not limited by season and environment. Havey et al found that there was a region of high variation between trnT and trnL in onion chloroplast DNA, i.e., there was an insertion of 100bp between 2 genes in the N-type cytoplasm compared with the S-type cytoplasm, and designed primers to obtain characteristic bands that can distinguish between S-type and N-type cytoplasm in onions [ Havey MJ. identification of cytoplasms using the polymerase reaction to aid in the extraction of main amino acids from modified proteins of ligands of the onion genes.1995, 90:263-268 ]. Later, SCAR markers (MS7/MS8) were developed based on this insertion sequence, and a specific fragment of 850bp was amplified in the S-type cytoplasm and a specific fragment of 950bp was amplified in the N-type cytoplasm, thereby efficiently identifying onion N-type and S-type cytoplasm [ Cho KS, Yang TJ, Kwon YS, Wo JG. development and application of SCAR marker related to cytoplasma of large steric in noise, journal of Koreasobiology for Hot tissue sciences.2001,42 (5): 527-532.]. Sato finds that a specific chloroplast gene insertion sequence is arranged at the upstream of the mitochondrial cob gene of the S-type cytoplasm of the onion, utilizes the specific sequence to design a primer for PCR amplification, amplifies 1 specific 414bp fragment in the S-type cytoplasm, amplifies 1 specific 180bp fragment in the N-type cytoplasm, and utilizes the marker to quickly and accurately identify the S-type and N-type cytoplasm of the onion [ Sato Y.PCRamplification of CMS-specific mitogenic nucleotide sequences to important genes of onions (Allium cepa L.). 1998,96:367-370 ]. Engelke et al found that the mitochondrial gene orfA501 used in chives to distinguish cytoplasmic fertility was also present in onion mitochondria, primer orfA501 was designed with a specific sequence of the atp9 gene of chives male sterile cytoplasmic mitochondrial DNA, onion mitochondrial DNA was amplified, and specific PCR markers were successfully developed that can distinguish fertile N-type from sterile (S-type or T-type) cytoplasm [ Engelke T, Terefe D, Taloglu T.A PCR-based marker systematic CMS- (S), CMS- (T) and (N) -toplasms in the cytoplasm (Allium cepa L.) Theotetic and Applied genetics.2003,107(1): 162-. Kim ET al found a chimeric open reading frame orf725 in S-type and T-type cytoplasm and developed a molecular marker that can distinguish three onion cytoplasm types by a single simple PCR [ Kim S, Lee ET, Cho DY, Han T, Bang H, Patil BS, Ahn YK, Yoon NK. identification of a novel polymeric gene, orf725, and its use in a later description of a molecular marker for distinguishing between the same and the same cytoplasma type in on (Allium cepa L.) the application gene 2009,118:433-441 ]. Plum Yongbo et al obtained the flanking sequence of chive orfA501 by chromosome walking, developed the SCAR marker for identifying onion cytoplasmic genotype, which amplified a specific 2175bp band and a 1053bp band in the onion sterile line, while only amplified the 1053bp band in the maintainer line [ plum Yongbo, Liu Bing Jiang, Huo Ramson, Miao army, Yang Jianping, Wu Xiong, Yan.

Onion originates from the middle Asia region, and the history of onion transmitted to China is short. Because the onion is a two-year plant, the breeding age is 2-4 times of that of vegetables such as Chinese cabbage, radish and the like, moreover, the germplasm resources of the onion in China are relatively deficient, the utilization of the male sterility technology on the vegetables is far behind that of other developed countries, and the market of hybrid is almost monopolized by foreign varieties. With the adjustment of agricultural industrial structure, the demand for good varieties of onions is increasing day by day, the production mainly takes variety introduction and conventional variety breeding as main parts, a first filial generation variety with the proprietary intellectual property right of China is lacked, a large amount of foreign exchange imported seeds from foreign countries is needed, and the production cost of the onions is high. Therefore, the lagging situation of China in the field of onion breeding is changed, and the key point is to widely collect variety resources as soon as possible and to take the way of combining modern molecular biology technology and conventional breeding. The research of molecular marker-assisted selection of onion maintainer lines is accelerated, so that the breeding period is shortened, and onion hybrids which have the independent intellectual property rights of China and meet the market demand are bred as soon as possible. Therefore, the breeding of the excellent male sterile line and the maintainer line is a key link to be solved urgently in onion breeding and is also the source work of onion industrialization.

The purpose of gene tagging is to achieve molecular Marker Assisted breeding (MAS). The auxiliary selection of the marker linked with the cytoplasmic locus and the nuclear gene can reduce the workload and improve the breeding efficiency. The cytoplasm type of a single plant can be identified by using the marker linked with the cytoplasm locus, S-type cytoplasm in fertile materials is eliminated, and maintainers are selected from the single plant of the N-type cytoplasm type, so that the number of test cross combinations and selfing plants is reduced, the workload is reduced, blindness is avoided, and the selection effect is improved. In the actual breeding work of onion, the selection of N-type cytoplasmic single plants from a population with unknown cytoplasmic types is the most critical core work. The group of the previous inventors have developed an onion cytoplasmic male sterility SCAR marker (patent No.: ZL201410153951.2) which can amplify a specific band of 2175bp and a band of 1053bp in S-type cells, and only a band of 1053bp in N-type cytoplasm. Although the marker can effectively avoid the situation of wrong classification of cytoplasm types caused by experimental operation errors and ineffective PCR amplification, due to the lack of a stable N-type cytoplasm specific marker, when a maintainer line is selected in a large population, DNA cannot be extracted from a mixed population sample to carry out primary screening on the maintainer line individual plant, only individual plant verification can be carried out, and the selection efficiency is influenced. Therefore, the development of a new N-type cytoplasm specific marker, especially an S/N specific double-significance marker, has important significance for breeding onion hybrids by matching the onion breeding sterile line with the maintainer line.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a double-significance marker for accurately identifying the S/N cytoplasmic genotype of onion. The invention uses onion (Allium cepa L.) male sterile line and maintainer line as test material, and carries out differential expression analysis on 58 ORFs in onion mitochondrion whole genome, and discovers an expression differential fragment orf 393. The genome DNA of the onion S-type cytoplasmic male sterile line S118 and the genome DNA of the maintainer line N218 are subjected to PCR amplification by using the full-length primer of orf393, and the sizes of amplified fragments of the orf393 in the sterile line and the maintainer line are slightly different. Through recovery, cloning, sequencing and DNA sequence comparison, the amplified fragment of orf393 in the onion maintainer line is deleted 36 bases compared with the sterile line, and the double-dominant SCAR marker for identifying the onion cytoplasm genotype is developed according to the difference sequence of orf393 in the sterile line and the maintainer line. The molecular marker obtained is used for quickly and accurately judging the cytoplasm type of the onion material, so that the wrong classification of the cytoplasm genotype caused by experimental operation errors and invalid PCR amplification is avoided, and when a maintainer line is selected from a large number of groups, the group samples can be mixed to extract DNA for preliminary screening of individual plants with or without the maintainer line, so that the selection efficiency is greatly improved, the time and the cost are saved, the breeding of the onion maintainer line is accelerated, and a novel onion hybrid parent matching system is established.

Specifically, the invention relates to the following technical scheme:

firstly, the invention discloses a double-significance marker for identifying onion S/N cytoplasm genotypes, wherein the specific fragment lengths of the molecular markers are 232bp and 196bp respectively, wherein the marker with the specific fragment length of 232bp is specific to S-type cytoplasm, and the nucleotide sequence of the marker is shown as SEQ ID NO. 1; the marker with the specific fragment length of 196bp is unique to N-type cytoplasm, and the nucleotide sequence of the marker is shown as SEQ ID NO. 2.

Secondly, the invention discloses a double-significance SCAR marker primer for identifying onion cytoplasm genotype, which comprises the following components in percentage by weight:

a forward primer: GXX-F1: 5'-CCCGAAGGAGTGACTTTTTCTGATTT-3' (SEQ ID NO. 5);

reverse primer: GXX-R1: 5'-TCTCTGGAGCACCGACTGAAGGG-3' (SEQ ID NO. 6).

Based on the double-dominant SCAR marker primer, the invention also discloses a kit for identifying the onion cytoplasm genotype, and the kit comprises the double-dominant SCAR marker primer and a conventional PCR amplification reagent.

In addition, the invention discloses application of the double-dominant marker, the mitochondrial orf393 or the double-dominant SCAR marker primer in onion breeding, in particular to breeding of onion maintainer lines.

In the application, the amplified fragment of orf393 in the onion sterile line has the size of 1179bp, and the nucleotide sequence of the amplified fragment is shown as SEQID No. 3; the amplified fragment of orf393 in the onion maintainer line was 1143bp in size, with the nucleotide sequence shown in SEQ id No. 4.

In a specific embodiment, the double-significance marker, mitochondrial orf393 or double-significance SCAR marker primer described above is used to identify the onion S/N cytoplasmic genotype.

The double-significance marker is used as a molecular marker to be applied to the auxiliary breeding of the onion male sterile line and the matched maintainer line, and the specific application mode is as follows: and carrying out PCR amplification on the genome DNA of the individual to be detected by using the double-dominant labeled primer, wherein if the individual to be detected only amplifies a 232bp fragment, the cytoplasm type of the individual to be detected is S-type cytoplasm, and if the individual to be detected amplifies a 196bp fragment, the cytoplasm type of the individual to be detected is N-type cytoplasm. The onion male sterile line and the onion maintainer line can be directly screened by combining the technology (granted patent No. ZL201310125445.8) which is previously developed by the applicant and used for judging the types of cell nuclei.

Based on the application, the invention particularly discloses a method for screening a maintainer line from an onion mixed population sample, which comprises the processes of designing and amplifying primers aiming at the double-obvious marker and the mitochondrial orf 393; or the process of amplification by using the double dominant SCAR marker primer.

Specifically, when selecting maintainers in a large population, DNA can be extracted from mixed population samples to primarily screen the maintainer individual, and if a specific band of 196bp is amplified in the DNA of the mixed population, which indicates that N-type cytoplasm exists in the population, the individual screening is necessary.

The invention uses onion (Allium cepa L.) male sterile line and maintainer line as test material, and carries out differential expression analysis on 58 ORFs in onion mitochondrion whole genome, and discovers an expression differential fragment orf 393. The genomic DNA of the onion S-type cytoplasmic male sterile line S118 and the genomic DNA of the onion maintainer line N218 are subjected to PCR amplification by using the full-length primers of orf393, and as a result, the sizes of amplified fragments in the sterile line and the maintainer line are slightly different. The inventor carries out recovery sequencing and DNA sequence comparison analysis on the differential fragment, the size of the amplified fragment of orf393 in the onion sterile line is 1179bp, and the nucleotide sequence of the amplified fragment is shown as SEQID No. 3; and the amplified fragment of orf393 in the onion maintainer line is 1143bp in size, the nucleotide sequence of the fragment is shown in SEQ ID No.4, and 36 bases are deleted compared with the sterile line. Based on the differential sequence of orf393 in the sterile and maintainer lines, the inventors designed a pair of molecular tagged primers, forward Primer: GXX-F1: 5'-CCCGAAGGAGTGACTTTTTCTGATTT-3', respectively; reverse primer: GXX-R1: 5'-TCTCTGGAGCACCGACTGAAGGG-3' are provided. The forward primer GXX-F1 and the reverse primer GXX-R1 are used for carrying out PCR amplification on the total DNA of the onion cytoplasmic male sterile line S118 and the corresponding maintainer line N218, wherein a specific strip of 232bp is amplified in each sterile line S118, and a specific strip of 196bp is amplified in the corresponding maintainer line N218. PCR verification is carried out on 10 combined materials with known cytoplasm genotypes with different genetic backgrounds (see table 1), a 232bp specific strip is amplified in all sterile lines, a 196bp specific strip is amplified in all maintainer lines, and the PCR result is matched with the field judgment result. The primers can be used for completely and accurately identifying the S-type cytoplasm and the N-type cytoplasm of the onion, so that the misclassification of the cytoplasm genotype caused by experimental operation errors and invalid PCR amplification is avoided, and when a maintainer line is selected from a large number of groups, the DNA can be extracted from a group sample to primarily screen the maintainer line single plant, so that the selection efficiency is greatly improved.

The screening process of the molecular marker primer is as follows:

(1) selecting an onion cytoplasmic male sterile line 118 and a corresponding maintainer line N218;

(2) extracting total DNA and RNA of onion genome respectively by using a rapid plant genome extraction kit and an RNAprep Pure plant total RNA extraction kit produced by Beijing Tiangen Biochemical technology limited;

(3) differential expression analysis of 58 ORFs in the whole genome of onion mitochondria shows that an expression differential fragment orf393 is obtained.

(4) Amplifying the total DNA of the onion cytoplasmic male sterile line and the onion cytoplasmic male sterile line by using a full-length primer expressing a differential fragment orf 393;

(5) recovering fragments from the sterile line and the maintainer line, and obtaining a DNA difference sequence between the sterile line and the maintainer line after connection, transformation, cloning and sequencing;

(6) designing primers by using the DNA difference sequence, and verifying the sterile line S118 and the maintainer line N218;

(7) the feasibility of this marker was verified using 200 additional individual materials of known genotype, resulting in a double-dominant marker identifying onion S/N cytoplasmic types.

The invention achieves the following beneficial effects:

the invention selects the total DNA of the onion cytoplasmic male sterile line S118 and the corresponding maintainer line N218 for PCR amplification to obtain the stable double-significance marker for distinguishing the S/N cytoplasm, the marker is stable and reliable, and the invention lays a foundation for the onion molecular marker-assisted selection breeding system. Compared with the prior art, the method has the advantages that: the molecular marker for identifying onion cytoplasm genotype has a 232bp specific band in S-type cytoplasm and a 196bp band in N-type cytoplasm, and is a double-significance marker. The amplification result not only avoids the wrong classification of cytoplasm genotypes caused by experimental operation errors and ineffective PCR amplification, but also can extract DNA from mixed population samples to carry out preliminary screening on individual strains of maintainer lines when the maintainer lines are selected in a large population, and if a specific band of 196bp is amplified in the DNA of the mixed population, the individual screening is necessary. If only a 232bp specific band is amplified in the DNA of the mixed population, which indicates that no N-type cytoplasm single plant exists in the population, the population can be directly eliminated, and the selection efficiency is greatly improved. Not only ensures the accuracy and reliability of the identification result, but also saves time and cost.

Drawings

FIG. 1 shows the DNA electrophoretogram of sterile line and maintainer line. Wherein: m is Marker, 1-8 are sterile single plants, and 9-16 are fertile single plants.

FIG. 2 is the RNA electrophoresis pattern of sterile line and its maintainer line. Wherein: m is Marker, 1-5 are sterile single plants, 6-10 are fertile single plants.

FIG. 358 ORFs partial PCR amplification results. Wherein: wherein: m is Marker, S is sterile individual plant of S118, and N is fertile individual plant of N218.

FIG. 4 shows the PCR amplification results of primers GXX-F1 and GXX-R1 on the onion sterile line and the onion maintainer line. Wherein: m is Marker, S is sterile individual plant of S118, and N is fertile individual plant of N218.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.

Example 1

Materials and methods

1. Extraction and detection of DNA and RNA

The extraction method of the onion cytoplasmic male sterile line and the corresponding maintainer line genome DNA refers to the specification of a rapid plant genome extraction kit produced by Beijing Tiangen Biochemical technology Co., Ltd, and the extraction of the total RNA refers to the specification of an RNAprep Pure plant total RNA extraction kit produced by Beijing Tiangen Biochemical technology Co., Ltd, and the quality of the DNA and the RNA is detected by agarose gel electrophoresis and a spectrophotometer.

2. Obtaining expression differential fragments

In the published onion CMS-S mitochondria whole genome sequence (GenBank access: KU318712), 58 new ORFs were found, and the inventors performed differential expression analysis on 58 ORFs by using cDNAs of the onion sterile line S118 and the maintainer line N218 as templates to obtain an expression differential fragment orf393 (FIG. 3).

3 specific band recovery, purification, cloning and sequencing

The genomic DNA of the onion S-type cytoplasmic male sterile line S118 and the genomic DNA of the onion maintainer line N218 are subjected to PCR amplification by using the full-length primers of orf393, and as a result, the sizes of amplified fragments in the sterile line and the maintainer line are slightly different. The inventor carries out recovery sequencing and DNA sequence comparison analysis on the differential fragment, the size of the amplified fragment of orf393 in the onion sterile line is 1179bp, and the nucleotide sequence of the amplified fragment is shown as SEQ ID No. 3; and the amplified fragment of orf393 in the onion maintainer line is 1143bp in size, the nucleotide sequence of the fragment is shown in SEQ ID No.4, and 36 bases are deleted compared with the sterile line.

The recovery of specific bands was performed according to the Biospin Gel Extraction Kit instructions; molecular cloning of specific bands method of molecular cloning II is adopted

4 design and Synthesis of primers

Based on the difference in the DNA sequences of orf393 in the onion sterile and maintainer lines, the inventors designed a pair of molecular marker primers, forward primers: GXX-F1: 5'-CCCGAAGGAGTGACTTTTTCTGATTT-3', respectively; reverse primer: GXX-R1: 5'-TCTCTGGAGCACCGACTGAAGGG-3' are provided. Synthesized by Beijing Boshang Biotechnology Co., Ltd, and purified by PAGE.

DNA sequencing was performed by Beijing Boshang Biotechnology Co.

5. PCR amplification and detection

PCR amplification was performed on a TC-XP-D Gene Amplifier from Burley, USA, in a 25. mu.L system, 2 XTAQA PCRMaster Mix 12.5. mu.L, primers 1, 2 (0.5. mu. mol/L) each 1.0. mu.L, DNA 1.0. mu.L, ddH₂O9.5. mu.L, with a reaction program of 94 ℃ pre-denaturation for 5min, [94 ℃ denaturation for 30sec, [ 60 ℃ annealing for 30sec, [ 72 ℃ extension for 30 sec%]35 cycles, extension for 10min at 72 ℃, and storage at 4 ℃; the PCR product was separated and analyzed by electrophoresis on 2.0% agarose gel, stained with ethidium bromide and imaged automatically on a gel imaging system.

6. Individual plant verification

Judging the fertility of each strain through field observation to obtain 10 combinations of a sterile line and a maintainer line, randomly extracting 10 plants of materials from the sterile line and the maintainer line of each combination, extracting total DNA of a genome, and performing field test on the total DNA of the genome by using a forward primer GXX-F1: 5'-CCCGAAGGAGTGACTTTTTCTGATTT-3' and reverse primer: GXX-R1: 5'-TCTCTGGAGCACCGACTGAAGGG-3', PCR amplification was performed on 200 parts of these materials with known fertility, and the reaction system and procedure were the same as those of FIG. 5.

(II) results and analysis

1. Detection and analysis of sterile line and corresponding maintainer line genome DNA and RNA

Referring to the specification of a rapid plant genome extraction kit produced by Beijing Tiangen Biotechnology Limited, total DNA of onion genome is extracted, and the result of 0.8 percent agarose gel electrophoresis (figure 1) shows that the extracted DNA has no obvious residue at a sample application hole, which indicates that the contents of polysaccharide and protein are not high; the DNA bands are positioned at the same position, and the band shapes are clear, free of dispersion and degradation. As can be seen from FIG. 2, the extracted total RNA band pattern is clear, both 28S and 18S bands are present, and no DNA and protein residues are left. Therefore, the extracted DNA and RNA can meet the requirements of subsequent experiments.

2. PCR amplification results

In the published onion CMS-S mitochondria whole genome sequence (GenBank access: KU318712), 58 new ORFs were found, and the inventors performed differential expression analysis on 58 ORFs by using cDNAs of the onion sterile line S118 and the maintainer line N218 as templates to obtain an expression differential fragment orf393 (FIG. 3). The genomic DNA of the onion S-type cytoplasmic male sterile line S118 and the genomic DNA of the onion maintainer line N218 are subjected to PCR amplification by using the full-length primers of orf393, and as a result, the sizes of amplified fragments in the sterile line and the maintainer line are slightly different. The inventor carries out recovery sequencing and DNA sequence comparison analysis on the differential fragment, the size of the amplified fragment of orf393 in the onion sterile line is 1179bp, and the nucleotide sequence of the amplified fragment is shown as SEQ ID No. 3; and the amplified fragment of orf393 in the onion maintainer line is 1143bp in size, the nucleotide sequence of the fragment is shown in SEQ ID No.4, and 36 bases are deleted compared with the sterile line.

Based on the difference in the DNA sequences of orf393 in the onion sterile and maintainer lines, the inventors designed a pair of molecular marker primers, forward primers: GXX-F1: 5'-CCCGAAGGAGTGACTTTTTCTGATTT-3', respectively; reverse primer: GXX-R1: 5'-TCTCTGGAGCACCGACTGAAGGG-3' are provided. The forward primer GXX-F1 and the reverse primer GXX-R1 are used for carrying out PCR amplification on the total DNA of the onion cytoplasmic male sterile line S118 and the corresponding maintainer line N218, and the result is shown in FIG. 4, wherein it can be clearly seen that a specific strip of 232bp is amplified from the sterile line S118 and a specific strip of 196bp is amplified from the corresponding maintainer line N218.

3. Individual authentication

In order to verify the feasibility of the double-significance marker in onion molecular marker assisted breeding application, 10 combined materials with different genetic backgrounds and known cytoplasm genotypes are utilized to carry out PCR verification (table 1), 2% agarose gel electrophoresis results show that all sterile lines amplify a 232bp strip, a corresponding maintainer line amplifies a 196bp fragment, and the PCR amplification results are completely consistent with field judgment results. A232 bp specific band exists in a sterile line single plant, and a 196bp specific band is amplified in a maintainer line single plant, which indicates that the primer pair can be completely applied to identifying the onion cytoplasmic genotype, so that the relationship between orf393 and onion cytoplasmic male sterility can be known.

TABLE 1 Single plant validation results of double-phenotypic markers in onion Male sterile and maintainer lines

Note: s: a male sterile line; n: a holding system; +: with amplified fragments, -: no fragments were amplified.

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ttagcggctt tatgtggtaa tcaattcaat tcatagtata ttaatttagc atctaatgtt 300

ctaagacagg agctacctat tctttctatg caaggtattt cggcacagaa tcagagttct 360

agtgcggata ctcagtctgt cctttcttcc attgcccgct cacttggtct tactactaat 420

tctactgcac atccacttat cacagctttt gttgttcaaa ttctcaaaga gatggttaga 480

agaggtcctc ccgaaggagt gactttttct gattttaatg aatctaagtt ccgtaattct 540

attagtacca ttttacagtc tgtgtttact ggacatcatg aagacccttc atctcctcag 600

tcggtgttta ctggacctca tacagactct tcatcccctc agtctactgg acctcatgaa 660

gacccttcag tcggtgctcc agagagctcc ttttagtccc atgttccaga gatcaccacc 720

ctcaatcaat cagctctaga ccctcagtct tcttctgact cgcctacgtc cacttcctct 780

tcctttcact ctgtttcatc tggggagatt tctgttgtgc gtgaattgtt gtctttacct 840

gatttcattt ctttgtgtat aatggttatt ttgactatta taaatatata tgctttgcca 900

gaccctttgc cagagccagc agacgcttta cctcctgctc tctctaatag tatagtgaca 960

gggcctagtg aagtggctgg gtctcaagct gatgatcaca ttgcgaatga gacttatgtt 1020

ggttatagta ctaagtctat tatcttaagt ggtattgtaa tattaacaat tatatggtta 1080

gggtttccag ctgatggatt aagcccaatc gatgttgatc ctcaattggc atttccaaca 1140

tga 1143

<210>5

<211>26

<212>DNA

<213> Forward primer: GXX-F1

<400>5

cccgaaggag tgactttttc tgattt 26

<210>6

<211>23

<212>DNA

<213> reverse primer: GXX-R1

<400>6

tctctggagc accgactgaa ggg 23

Claims

1. A double-dominant marker for identifying onion S/N cytoplasm genotypes is characterized in that the lengths of specific fragments of the double-dominant marker are 232bp and 196bp respectively, wherein the marker with the specific fragment length of 232bp is unique to an S-type cytoplasm, and the nucleotide sequence of the marker is shown as SEQ ID No. 1; the marker with the specific fragment length of 196bp is unique to N-type cytoplasm, and the nucleotide sequence of the marker is shown as SEQ ID NO. 2.

2. A double-significance SCAR marker primer for identifying onion cytoplasm genotype is characterized in that the primers are respectively as follows:

a forward primer: GXX-F1: 5'-CCCGAAGGAGTGACTTTTTCTGATTT-3', respectively;

reverse primer: GXX-R1: 5'-TCTCTGGAGCACCGACTGAAGGG-3' are provided.

3. A kit for identifying the cytoplasmic genotype of onion, comprising the dual dominant scarr marker primer of claim 2, and further comprising PCR amplification reagents.

4. Use of the double dominant marker of claim 1 or the double dominant SCAR marker primer of claim 2 in onion breeding.

5. The application of the onion mitochondria orf393 in onion breeding is characterized in that the amplification fragment size of the orf393 in the onion sterile line is 1179bp, and the nucleotide sequence is shown as SEQ ID No. 3; the amplified fragment of orf393 in the onion maintainer line was 1143bp in size and its nucleotide sequence is shown in SEQ ID No. 4.

6. The use of claim 4 or 5, wherein the use is in the breeding of onion maintainers.

7. The use of claim 4 or 5, wherein said use is for identifying onion S/N cytoplasmic genotype.

8. A method for breeding onion male sterile line and matched maintainer line in an auxiliary way, which is characterized in that the double dominant SCAR marker primer of claim 2 is used for carrying out PCR amplification on the genome DNA of an individual to be detected, if the individual to be detected only amplifies 232bp fragments, the cytoplasm type of the individual to be detected is S-type cytoplasm, and if the individual to be detected amplifies 196bp fragments, the cytoplasm type of the individual to be detected is N-type cytoplasm.

9. A method of screening a maintainer line from a sample of a mixed population of onions, comprising amplification using the dual dominant scarr marker primers of claim 3.

10. The method of claim 9, wherein the double dominant SCAR marker primers of claim 3 are used for amplification, and if a specific band of 196bp is amplified in DNA of a mixed population, the single-plant screening is further performed.