CN107541517A - A kind of external source radish fragment specific mark and its preparation method and application - Google Patents

Abstract

The present invention relates to the preparation method and application of a kind of cabbage type rape external source radish fragment specific mark and external source radish fragment specific mark, compared by cabbage type rape radish cytoplasmic sterility restorer CLR650 full-length genomes weight sequencing data and radish gene group Sketchy data, develop 1~CLR9 of specific molecular marker CLR9 20 of external source radish fragment.By the target individual plant of cytoplasmic sterility restorer CLR650 backcross populations, BC is obtained₄Segregating population；By BC₄Fertile plant in colony, more mixing from generation to generation are carried out using mixing method and selected, until obtaining hybrid separation colony of the fertile plant proportion more than 75%；Homozygous stable, acquisition cabbage type rape radish cytoplasmic sterility restorer is carried out finally by inbreeding of more generation.Instant invention overcomes the serious relatively low, character of restoring gene genetic force in existing method to be difficult to homozygous stable obstacle.

Description

A kind of external source radish fragment specific mark and its preparation method and application

Technical field

The invention belongs to rapeseed breeding technical field and biology field, and in particular to a kind of external source radish fragment is special The preparation method of heterolabeling and external source radish fragment specific mark, further relate to external source radish fragment specific mark and educated in cabbage type rape Application in kind.

Background technology

Rape has obvious hybrid vigour, is domestic and international reply edible vegetable oil shortage, developing regenerated biological energy Most potential oil crops.Male sterile with radish cytoplasm (Ogu CMS) be cabbage type rape heterosis utilization it is preferable not Type is educated, is the sterile source that the small storehouse of Japan is found in nineteen sixty-eight in radish and the Cyto-plasmtc male sterile system established.Due to this Sterile system sterility is highly stable, thorough, has very important value in crop in cruciferae heterosis utilization.With Deepening continuously for rape molecular biology research, genomic sequencing technique is able to perfect, and sequencing cost also further declines, point The exploitation of son mark and assisted Selection technology are using increasingly mature and practical, and this is studies cabbage type rape radish cytoplasm not Educate restorer and provide technical support.

Heyn in 1976 by generic cross successfully by the restoring gene of radish imported into cabbage type rape (Heyn, 1976).Then, French Pelletier et al. is also led the restoring gene in parsnip by Protoplast Fusion Technique Enter into cabbage type rape.But due to restoring gene from radish be transferred to cabbage type rape when, the radish fragment of redundancy also penetrates into Rapeseed gene group, restorer is caused recovery capability to be present undesirable, part female sterile and compared with high-sulfur glucoside and restoring gene The problems such as chain.

China introduces cabbage type rape ogura-CMS systems the eighties in last century, and each rapeseed breeding unit in the whole nation is subsequent Numerous studies are carried out.Restoring gene is imported into rape from radish mutation Oil radish as Li Xufeng was once reported, but obtains the extensive of material Multiple genes is on episome, but can not stablize heredity；2015, magnificent Jinsui River etc. utilized the Wild cabbage type oil purchased from French INRA After dish restorer R2000 is hybridized with sterile line, to F₁The recovery individual plant to be changed for radioinduction selection markers, so as to Genetic improvement is carried out to R2000, but its recovery resource used and molecular labeling derive from external studied.China is still without certainly The molecular labeling available for ogura-CMS restorer breedings of main exploitation.

The content of the invention

The technical problems to be solved by the invention are to overcome the shortcomings of to mention in background above technology and defect, there is provided one Target gene specific mark and preparation method thereof kind based on genome weight sequencing technologies and comparative genomics technological development and Application in cabbage type rape breeding.The specific molecular marker of the present invention derives from external source radish fragment, and preparation method is simply easy OK, accurate and effective, it is combined with conventional hybridization, backcrossing, mixing selection, selfing breeding technique, it is sweet can effectively solves current China Distant hybridization external source target gene genetic force existing for blue type rape radish cytoplasm sterile restoring system Breeding Process it is serious it is relatively low, Character is difficult to the problems such as homozygous stable.

Our the Ogu CMS restorers of the novel cabbage type rape to the autonomous cultivation of Hu'nan Prov. Crops Research Inst. CLR650 enter A large amount of molecular biology and CYTOGENETIC ANALYSIS OF ONE are gone, the results showed that the restorer genome has tended towards stability, and it is educated Property restoring gene is Rfo genes.The problem of serious, character is difficult to stablize is separated based on CLR650 filial generations fertility partially, we Sequence and reference gene group sequence alignment the independent development specific molecular marker of external source radish fragment are resurveyed by genome.It is described Radish fragment specific mark include CLR9-1, CLR9-2, CLR9-3, CLR9-4, CLR9-5, CLR9-6, CLR9-7, CLR9-8, CLR9-9、CLR9-10、CLR9-11、CLR9-12、CLR9-13、CLR9-14、CLR9-15、CLR9-16、CLR9-17、CLR9- 18、CLR9-19、CLR9-20.The external source radish fragment specific mark CLR9-1, it has SEQ ID No：Nucleosides shown in 1 Acid sequence.

The external source radish fragment specific mark CLR9-2, it has SEQ ID No：Nucleotide sequence shown in 2.

The external source radish fragment specific mark CLR9-3, it has SEQ ID No：Nucleotide sequence shown in 3.

The external source radish fragment specific mark CLR9-4, it has SEQ ID No：Nucleotide sequence shown in 4.

The external source radish fragment specific mark CLR9-5, it has SEQ ID No：Nucleotide sequence shown in 5.

The external source radish fragment specific mark CLR9-6, it has SEQ ID No：Nucleotide sequence shown in 6.

The external source radish fragment specific mark CLR9-7, it has SEQ ID No：Nucleotide sequence shown in 7.

The external source radish fragment specific mark CLR9-8, it has SEQ ID No：Nucleotide sequence shown in 8.

The external source radish fragment specific mark CLR9-9, it has SEQ ID No：Nucleotide sequence shown in 9.

The external source radish fragment specific mark CLR9-10, it has SEQ ID No：Nucleotide sequence shown in 10.

The external source radish fragment specific mark CLR9-11, it has SEQ ID No：Nucleotide sequence shown in 11.

The external source radish fragment specific mark CLR9-12, it has SEQ ID No：Nucleotide sequence shown in 12.

The external source radish fragment specific mark CLR9-13, it has SEQ ID No：Nucleotide sequence shown in 13.

The external source radish fragment specific mark CLR9-14, it has SEQ ID No：Nucleotide sequence shown in 14.

The external source radish fragment specific mark CLR9-15, it has SEQ ID No：Nucleotide sequence shown in 15.

The external source radish fragment specific mark CLR9-16, it has SEQ ID No：Nucleotide sequence shown in 16.

The external source radish fragment specific mark CLR9-17, it has SEQ ID No：Nucleotide sequence shown in 17.

The external source radish fragment specific mark CLR9-18, it has SEQ ID No：Nucleotide sequence shown in 18.

The external source radish fragment specific mark CLR9-19, it has SEQ ID No：Nucleotide sequence shown in 19.

The external source radish fragment specific mark CLR9-20, it has SEQ ID No：Nucleotide sequence shown in 20.

The technical concept total as one, present invention also offers a kind of preparation method of external source radish fragment specific mark, Comprise the following steps：

(1) it is maternal and cabbage type rape routine product using cabbage type rape radish cytoplasmic sterility restorer CLR650 It is that A is hybridized, obtains F₁Colony, select more plants of F₁Colony plant carries out bagging isolation selfing, obtains F₂Fertility segregation colony is single Strain, and extract the genome DNA of each individual plant；F is investigated in full-bloom stage₂Colony's individual plant fertility, establishes fertile plant and infertility respectively Pnca gene mixing pit；

(2) full-length genome is carried out to cabbage type rape radish cytoplasmic sterility restorer CLR650 and resurveys sequence, sequencing is tied Fruit is compared with radish gene group sketch and radish cytoplasm sterile restoring gene sequence, obtain CLR650 restoring gene and its Side external source radish sequence information, special primer is designed according to target area；

(3) special primer of acquisition is screened using gene mixing pit, retains fertile gene mixing pit PCR amplifications in sun Property, the primer that is negative of sterile gene mixing pit amplification, and by the primer screened in F₂Carried out on Fertility segregation colony individual plant Checking is isolated, the primer that PCR amplification positive performances isolate with fertility performance is as external source radish fragment specific mark.

Preferably, cabbage type rape routine strain A is 20B in the step (1).The conventional strain genome is relatively pure The state of conjunction, filial generation are not in massive separation phenomenon, reduce to the full extent characters of progenies separate it is pure to restorer Close, adverse effect caused by stabilization.In addition, conventional strain is free of other kinds of restoring gene, at the same it is also bad without other Cytoplasmic effect, be more beneficial for the acquisition of progeny population molecular marker screening and target strain.

Preferably, more plants of F of selection in the step (1)₁Colony plant carries out bagging isolation selfing and refers to select 6-8 strains F₁ Colony plant carries out bagging isolation selfing.

The present invention relates to molecular labeling be by genome weight sequencing result be compared with radish gene group and, From radish gene group information, restoring gene region is not limited only to, also relates to the sequence faced by restoring gene, can With the more effective change for detecting the external source radish fragment for penetrating into rapeseed gene group during transformation.

The technical concept total as one, present invention also offers a kind of above-mentioned external source radish fragment specific mark CLR9-1 Any of~20 application in cabbage type rape Breeding for restoration lines, the application comprise the following steps：

1) it is maternal and cabbage type rape routine strain using cabbage type rape radish cytoplasmic sterility restorer CLR650 A is hybridized, and obtains F₁Colony；Select more plants of F₁Returned for plant and cabbage type rape routine strain A as recurrent parent Hand over, build BC₁Segregating population；

2) in Seedling Stage, using the above-mentioned external source radish fragment specific mark of at least one to BC₁Segregating population carries out molecule Marker assisted selection, pull out the individual plant that labeled analysis result is negative；More plants of BC are selected at the florescence₁Phenotype approaches in segregating population (i.e. character is similar) cabbage type rape routine strain A individual plant continues to be returned with cabbage type rape routine strain A as female parent Hand over；Repeat the above steps until obtaining cabbage type rape C LR650 BC₄Fertility segregation colony；

3) BC is pulled out₄Sterile individual plant in Fertility segregation colony, the fertile individual plant of reservation is all mixed in isolated area Pollination and harvest are closed, and in the fertility performance of full-bloom stage investigation progeny population, until obtaining fertile plant proportion more than 75% Colony；

4) by fertile plant in segregating population of the fertile plant ratio more than 75%, all isolation is selfed, and continues to select in self progeny Fertile plant all isolation selfings are selected, are recovered until obtaining the cabbage type rape radish cytoplasmic sterility that fertility does not separate, phenotype is consistent It is CLR095.

Compared with prior art, beneficial effects of the present invention are：

1. molecular labeling of the present invention is the external source radish according to the weight sequencing result independent development of CLR650 genomes Fragment specific mark, there is initiative meaning, can be provided for China's cabbage type rape radish cytoplasmic sterility recovery resource transformation newly Molecular marking technique supports；

2. the present invention's carries out seed selection using the method for conventional breeding means and molecular marker assisted selection, can be in seedling stage Carry out initial option, on the one hand, non-targeted individual plant can be pulled out ahead of time, reduce the work of practice ground and later stage phenotypic evaluation Amount；On the other hand, efficiency of selection and accuracy are greatly improved, excludes mechanical admixture and biology occur in Breeding Process in time The influence mixed；

3. the novel cabbage type rape radish cytoplasm sterile restoring system inheritance stability obtained using the present invention, in certain journey The unfavorable character of distant hybridization of CLR650 carryings is eliminated on degree, the cabbage type rape with initiative meaning can be provided for China Radish cytoplasm sterile restoring source；

4. the present invention is selected using first backcrossing orientation transformation, then by mixing, it is homozygous at utmost to increase target gene Probability, finally by homozygous, the stable Breeding Strategies of selfing, the restoring gene genetic force that distant hybridization can be overcome to bring is seriously inclined Low, character is difficult to stable etc. obstacle, and and can avoids inbreeding depression phenomenon caused by continuous multi-generation selfing, and facts have proved this Strategy is easier to obtain homozygous stable radish cytoplasm sterile restoring system.

Brief description of the drawings

In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are the present invention Some embodiments, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis These accompanying drawings obtain other accompanying drawings.

Fig. 1 is the transformation schematic flow sheet of cabbage type rape Ogu CMS restorers CLR095 in the embodiment of the present invention.

Fig. 2 is F of the present invention in cabbage type rape radish matter sterile restoring material C LR650₂Fertility segregation colony gene mixes Close the PCR amplifications between pond.

Fig. 3 is F of the present invention in cabbage type rape radish matter sterile restoring material C LR650₂Segregating population individual plant PCR is expanded As a result, F represents fertile plant in figure, and S represents sterile plant.

Fig. 4 is the cabbage type rape radish cytoplasmic sterility restorer CLR095 and CLR650 that are obtained in the present invention contrast, In Fig. 4：A：Seedling leaf contrasts；B：Floral organ contrasts；C：Silique contrasts.Fig. 4 is using CLR650 as comparison other, by Fig. 4 It can be seen that CLR095 blade, flower and Pod length occur substantially to change.

Fig. 5 is the cabbage type rape radish cytoplasmic sterility restorer CLR095 and CLR650 that are obtained in the present invention contrast. Fig. 5 is using CLR650 as comparison other, and CLR095 blade, flower and Pod length occur substantially to change as seen from Figure 5.

Embodiment

For the ease of understanding the present invention, the present invention is done below in conjunction with Figure of description and preferred embodiment more complete Face, meticulously describe, but protection scope of the present invention is not limited to specific examples below.

Unless otherwise defined, all technical terms used hereinafter are generally understood that implication phase with those skilled in the art Together.Technical term used herein is intended merely to describe the purpose of specific embodiment, is not intended to the limitation present invention's Protection domain.

Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention can pass through city Field is commercially available or can be prepared by existing method.

Embodiment 1：

The cabbage type rape radish cytoplasmic sterility restorer CLR095 of the embodiment of the present invention selection such as institute of accompanying drawing 1 Show, specifically include following steps.

1. male sterile with radish cytoplasm restoring gene specific molecular marker and linked marker exploitation

1.1 implement material

(for radish male sterile cytoplasm, leaf margin is zigzag to cabbage type rape radish matter sterile restoring material C LR650, leaf Point is sharper, dark green leaf color, silique tubbiness, sees document：Chen Weijiang etc., cabbage type rape radish cytoplasmic male sterility recover material The initiative of material, Scientia Agricultura Sinica, 2012,45 (8)：1465-1474), the material is by trailing plants-indigo plant (Raphanobrassica) (AACCRR, 2n=56) is come with cabbage type rape by graft technology selection cross, and chromosome number is between 38-40.Its Cytoplasm is radish male sterile cytoplasm, and nucleus contains CLR650 radish cytoplasm restoring gene Rfo, therefore shows as just Chang Keyu；(for conventional cabbage type rape cytoplasm, for leaf margin to incise, blade tip is medium, leaf to cabbage type rape routine strain 20B Color is light green, and silique is elongated, sees document：Lee's certain kind of berries etc., rich oily 730 seed selection of cabbage type rape hybrid new varieties, Agriculture in Hunan science, 2008, (6)：19-20), it is the low conventional strain of semi-winterness Brassica napus with double.

1.2 male sterile with radish cytoplasm restoring gene F₂The structure of segregating population

Spring in 2011, when CLR650 enters initial bloom stage, after still unopened bud extracts stamen on CLR650 plant Hybridized with 20B, obtain F₁Seed；In mid-May, 2011 is by the F of acquisition₁Seed sowing is in climatic chamber nutritive cube, acquisition 20 Individual F₁Plant, and 8 healthy and strong individual plants of growing way are selected in initial bloom stage, bagging isolation selfing is carried out with parchment sack, obtains F₂Point Peel off body seed；In October, 2011 selects 1 part of above-mentioned F₂Rape experimental plot of the seed after the rice field-upland field rotation of Changsha is sowed, Obtain F₂Fertility segregation colony plant, divide individual plant to carry out drop in the 3-5 leaf phases, and 2-3cm is chosen from each individual plant²Size Young leaflet tablet carries out Genome DNA extraction, and extracting method is with reference to (Li Jia etc., a kind of side of effectively extraction rape leaf STb gene such as Li Jia Method, Hua Zhong Agriculture University's journal, 1994,13 (5)：521-523) method of report is carried out, using 0.8% Ago-Gel electricity Swimming detection DNA mass, DNA concentration is detected with ultraviolet specrophotometer (Pharmacia Biotech, GeneQuant II). When next year rape enters full-bloom stage, investigation, record F₂The fertility performance of each individual plant in colony.The F2 separation groups that the present invention obtains Body shares 210 individual plants, wherein fertile individual plant is 74 plants, sterile individual plant is 136 plants.According to fertility investigation result from F₂Separate group In body, the STb gene for choosing 10 fertile individual plants and 10 sterile individual plants respectively carries out mixed in equal amounts, obtains 1 fertile plant respectively Mixing pit and 1 sterile plant mixing pit, it is that target fragment molecular markers development is standby.

1.3 resurvey the radish cytoplasm sterile restoring gene special primer design of sequence based on CLR650 genomes

When CLR650 seed being seeded in into nutritive cube, but growing to 3-4 piece true leaves, 3-4cm is taken²Young leaflet tablet, send by Shanghai Ou Yi biomedicines Science and Technology Ltd. carries out weight sequencing analysis using Illumina MiSeq platforms, obtains unidirectional The sequencing data of the segments of 67.169G alkali cardinal sum 447,797,399.Wherein, clean reads ratios reach 96.09%, height The ratio of quality base reaches 99.21%,>71% genome reaches the sequencing depth of 50 × above.Then by these sequences The region of radish gene group and radish cytoplasm sterile restoring gene Rfo the place 127Kb with having been announced on NCBI are compared respectively It is right, radish gene group Scaffold 131 sequence information where obtaining OguCMS restoring gene, and it is inferred to CLR650 Middle external source radish matter sterile restoring gene is located at radish gene group R₉On chromosome.

1.4 male sterile with radish cytoplasm restoring gene specific molecular markers screen and checking

For radish R₉127Kb regional sequence where the sequences of Scaffold 131 and Rfo that chromosome has been announced, is used Primer3 design of primers instrument devises 80 pairs of radish special primers altogether.It is first that 80 pairs of primers are fertile in step 1.2 Strain mixing pit and sterile plant mixing pit enter performing PCR amplification, and the mixing pit performance of screening fertile plant is positive, the performance of sterile plant mixing pit is cloudy The primer of property, then by the primer of acquisition in F₂Carry out isolating checking (referring to accompanying drawing 2,3) in Fertility segregation colony, by testing Card determines specific mark of 20 marks for radish fragment：CLR9-1、CLR9-2、CLR9-3、CLR9-4、CLR9-5、 CLR9-6、CLR9-7、CLR9-8、CLR9-9、CL R9-10、CLR9-11、CLR9-12、CLR9-13、CLR9-14、CLR9-15、 CLR9-16, CLR9-17, CLR9-18, CLR9-19, CLR9-20 (primer sequence and Product Sequence difference of each specific mark Such as table 1 below, shown in table 2).

Table 1：The primer sequence of radish specific mark and the size of product fragment of the present invention

The PCR primer sequence of the radish specific mark of the present invention of table 2

2. the seed selection of male sterile with radish cytoplasm restorer

2.1 molecular marker assisted selection backcross breedings

Spring in 2011, the F that will be obtained in above-mentioned steps 1.2₁Plant is returned with 20B, obtains BC₁Seed；2011 10 Month is by the BC of acquisition₁Seed after planting, obtains CLR650/20B BC in Changsha rape experimental plot₁Segregating population plant.Due to There is serious inclined segregation phenomenon in radish matter sterile restoring gene, the present invention utilizes to be obtained in a step 1.4 in CLR650 offsprings External source radish fragment specific mark CLR9-3, CLR9-12, CLR9-18 combination, to BC₁The 3-5 leaf phases in segregating population just open Begin to select, pull out the individual plant that labeled analysis result is negative.Spring rape full-bloom stage in 2012, to the fertile individual plant of above-mentioned reservation Fertility investigation is carried out, the fertile plant main inflorescence by phenotypic character close to 20B, bagging isolation selfing is carried out with parchment sack, And unopened bud on branch is removed and is returned with 20B after stamen, obtain BC₂The seed of segregating population.Maturity period adjusts Silique shape, Pod length and every seed number per pod of each backcrossing individual plant under the conditions of open-pollinated are looked into, and it is natural to divide individual plant to harvest its The seed of pollination, and three oil content, content of erucic acid and glucosinolate content Quality Traits Analysis are carried out using near-infrared analyzer, protect Phenotype and quality trait are stayed closest to 20B individual plant, continues to be returned by the use of 20B as recurrent parent, until obtaining phenotype and 20B mono- Cause, the backcross population that quality trait is improved.

The present invention is from BC₁In segregating population in 163 individual plants, what 31 molecular marker analysis results of acquisition were positive can Individual plant is educated, wherein 10 phenotypic characters are close to 20B.Using near-infrared analyzer (Matrix-1, Bruker, Germany, OPUS/QUANT5.5software) to 10 BC of acquisition₁The self-fertility seed of fertile individual plant carries out oil content, erucic acid contains Amount and three Quality Traits Analysis (the results are shown in Table 3) of glucosinolate content, reservation glucosinolate content is minimum, oil content is more than 35%, mustard Acid content is less than the BC of 5% backcrossing individual plant₂Segregating population seed.The first tenday period of a month in May, 2012, by above-mentioned BC₂Segregating population seed, broadcasts Kind obtains BC in Xining, Qinghai rape experimental plot₂Segregating population plant, and continue according to BC₁Transfer method, further carry out Backcrossing, until obtaining the consistent BC of phenotype₄Segregating population plant.It is relatively uniform that the present invention in Qinghai obtains phenotype in 2014 BC₄Segregating population plant, the segregating population totally 191 individual plants, fertile and 139 individual plants performances wherein 52 individual plants are acted normally For male sterile with radish cytoplasm.

The cabbage type rape radish matter sterile restoring material BC that the present invention of table 3 obtains₁-BC₄It is elected to fertile individual plant product in colony Matter analysis result

2.2 cabbage type rape radish cytoplasmic sterility restorers are homozygous, stably

In full-bloom stage, according to initial bloom stage fertility investigation result, the BC that will be obtained in step 2.1₄Infertility in segregating population Individual plant is pulled out, and the fertile plant of reservation is all carried out into isolation mixed pollination, carries out mixing harvest after ripe, the next generation continues to retain Fertile plant carries out isolation mixed pollination in segregating population, until obtaining segregating population of the fertile plant ratio more than 75%.Then will All fertile individual plants in the segregating population of acquisition are selfed with parchment bagging isolation, until obtaining the selfing unseparated strain of fertility System, i.e., new cabbage type rape radish cytoplasmic sterility restorer.

54 fertile plants of reservation were all carried out isolation mixed pollination by the present invention in the spring in 2014, were mixed after ripe Harvest.The seed for mixing harvest is seeded in Changsha rape experimental plot by October, 2014, obtains the Fertility segregation of 155 individual plants Colony, full-bloom stage in 2015, colony's individual plant fertility investigation show that fertile plant ratio (is compiled in field in 30% or so, 1 segregating population Number 14Q017) fertile plant ratio is 50% or so；5 fertile plants continue paired brother and sister's friendship in selection segregating population 14Q14, Obtain 10 brother and sister and hand over segregating population.10 segregating populations are seeded in Changsha rape experimental plot by October, 2014, secondary Year full-bloom stage investigates the fertility performance of each colony, have 8 segregating populations (field numbering 14C041,14C043,14C044, 14C045,14C046,14C047,14C048,14C050) fertile plant ratio 30% or so, 1 segregating population (field number 14Q042) fertile plant ratio is 50% or so；1 segregating population (field numbering 14C049) fertile plant ratio is 70% or so； All fertile individual plants in segregating population 14Q049 are isolated with parchment sack and are selfed, obtain the seed of 32 selfing strains. The seed of above-mentioned 32 selfing strains is seeded in Xining, Qinghai rape experimental plot by May, 2015, during full-bloom stage to it is each from Hand over strain to carry out fertility investigation, obtain selfing strain (field numbering 15Q028) of the fertile plant ratio 90% or so.Will Strain 15Q028 fertile individual plant is isolated with parchment sack to be selfed, and obtains the seed of 28 selfing strains.2015 10 parts, will The seed of above-mentioned 28 selfings strain is seeded in Changsha rape experimental plot.During next year full-bloom stage, each selfing strain is entered Row fertility is investigated, and is obtained a complete fertile selfing strain (field numbering 15C048), is utilized near-infrared analyzer (Matrix- 1, Bruker, Germany, OPUS/QUANT5.5software) oil-containing is carried out to the solid seed of strain 15Q028 individual plant selfings Three amount, content of erucic acid and glucosinolate content Quality Traits Analysis show that its oil content is 38.90%, and sulphur resources are 68.65 μ Mol/g, content of erucic acid 0.7%.

10 parts of selfed seeds by strain 15C048 are seeded in Changsha rape experimental plot within 2016, and next year full-bloom stage is educated The investigation of sex investigation fertility shows that the strain individual plant fertility is all normal (field numbering 16C095), the entitled Wild cabbage type oil of the strain Dish radish matter sterile restoring system CLR095.

The character investigation of 2.3 cabbage type rape radish cytoplasmic sterility restorers

The restorer CLR095 that step 2.2 is obtained under field conditions (factors) sow by single file area, 2 repetitions, and line-spacing is 0.27m, spacing in the rows are 0.15m (often 10 plants of row), and respectively using CLR650 and 20B as control.Seedling stage investigates leaf color, leaf margin shape, Florescence free pollination, and investigate petal shape and raw state, maturity period and investigate plant height, primary branch number, primary branch silique Number, Pod length, Seed number per pod, and three oil content, content of erucic acid and glucosinolate content quality traits are analyzed, concrete outcome is shown in Table 4.The restorer CLR095 that the present invention obtains largely eliminates the unfavorable of external source redundancy radish fragment in genome Influence, original cabbage type rape radish matter is clearly distinguishable from quality, blade profile, leaf color and silique shape isophenous economical character Sterile restoring material C LR650 (referring to accompanying drawing 4, Fig. 5 and table 4), illustrate the breeding method of the present invention in seed selection cabbage type rape trailing plants It is effective to foretell in matter restorer.

The cabbage type rape radish matter sterile restoring system CLR095 of table 4 trait expression

Relational language is explained：

Cytoplasmic male sterility：Technical term, it is a kind of natural phenomena being widely present in higher plant, shows as mother Body heredity, pollen abortion and gynoecium are normal.

Strain：Technical term, the previous materials in breeding process, has a number of similar individual, in variety protection General designation before experiment.

PCR：Technical term, it is a kind of to be used to amplify the Protocols in Molecular Biology for expanding specific DNA fragmentation.

Low sulfatide：Technical term, the standard of low sulfatide material is used as using 30 μm of ol/g of glucosinolate content ＜.

It is double low:Technical term, i.e., technical term of the present invention are the abbreviation of low erucic acid, low sulfatide.

Sisters hand over：Technical term, it is mutual between the shape cultivated by same original parent similar Sister Lines or individual plant Hybridization.

Sequence table

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<210> 5

<211> 367

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

tcggaaaggg caagaagcaa gttgctgttt aattccggat cctttttgct tcgtcatttt 60

atattgattt acaaaatttc tttaaatgtc tactttaatt gggctccctg aggttttccc 120

agttagggcc cttactggca aacacatcca gagcccaatt ccagtttacc cttaatttaa 180

ctttttcaaa tcaatttaaa aagaagatta tcaaaggggt ccggcgtgaa atcagccccg 240

gaaaaaaaaa aacaaaagaa aaaaaaatct ggaacagcgc tgccccacga ggattatcgg 300

ggtgccatga cttcacataa aaacatgaac taggtaatac cccggccaaa atggaaaaaa 360

aaatgat 367

<210> 6

<211> 349

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

tcgcgaggtg caagagggag gcaaggacgc gtaaaaagta ctggtatggt attaattcgc 60

gtagaataag tgacaaaata tttagtttta atatggcaca caagattaga caagaagacg 120

ttgacgaata aaaaaaaaaa aaaaaaaact ttaacggggt cgttgggggt ttaaattttg 180

gtttttaccc tttccccgga aaaccctttt taaaaaaaaa aaaaagatcc atcatgtagg 240

actttgacct ggccacccag acaatgcaac gactggtttc cttttttact atgtattaat 300

gcaatcatat tattaccatt aactaagtaa gaccctgcca aaatggaat 349

<210> 7

<211> 364

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

ccgaagggaa caaaagaagg aaaacgaaaa ataaaaaaaa gaagttaaga catctttttc 60

ttttgttaat ataaataaac aaccagacca acattgatct gccttttcct ttgtggatta 120

aaatcaattg ggccaccaaa atcaaataat ccccaatcat gggtattaat cccttctgga 180

ggccccactc aattttgccc aacaaaaaaa tcgctttact ttttttgctt tgtttatttt 240

tttatattac caaaaaaaaa aaattttttc cccattttaa ttaaaaggga aaagcgggaa 300

aataaaaatt tctttttttt ttattatggg gtctaaccct tttcctttgg tttaaattaa 360

aaaa 364

<210> 8

<211> 374

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

ccgatcggaa aaagctgaag gacgttcaat ccttattccc tttttgattt gtaaactatg 60

gcatctattt tttttattaa tattaacaaa caaacaaaaa aaaattgatc ttgcttttcc 120

attgatgatc aaaatcgatt ggacgaccaa gaccagatta atacccgaca tctgcgatta 180

tccatgaagg aggcacaact caaattatca caacaagaaa aatgctttac tttttgtgct 240

ctgcttattt atttatataa ccaaaaaaaa aaaatttttt tccctattta attaaaatgg 300

aaaaaaagga aaaacaaaaa ttccttgtat tttattattg tgtcttacct tttttccttg 360

ttttccaata aaaa 374

<210> 9

<211> 392

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

gcgccgcgcg aagagagaaa gcgcagtgta tgttagtgat gatataaact tctatttgag 60

gctgatgcct gaaaaaaatt ggatattttc ttatactttc gatattttgg ataaaacata 120

ttaggaaaat tttggatgat caaatatttg attctacttt aatttttcaa atatgttata 180

tcttttaaat acattttttt tttttatatc tattttttgt tatgccacta cctcatatgt 240

atgtttatat cttctctttc tacattccca ttgaggggat gtgcgaggcg gggattgtgt 300

aagacgtaca actctcctgt ttcgctcttt gattttgggc gggggggtca acccgttttt 360

ctggggatca aaaaaaaaaa aagggggggg gg 392

<210> 10

<211> 348

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

taggagacgg gagaaccaaa ctgtgagtaa tactgcgtga gagaaatggt cttcctgctc 60

agatctctcc ctttcactaa agataaataa accctaaaac tttcccgcag tttctttctc 120

cttccgagtt tccctaaaag tacgaactac tcctcttcaa agacccatct gcttgaatta 180

ccaaaatcat cagcaaaaat acccaggtaa aaattactgc ttttacttct tcatctttat 240

aagggccttt cacttttttt tttttttttt cccgtttttg gttttttacc cggggggggg 300

gggttttccc tttttaactc tttcccttaa aagggggggt cgaaaaaa 348

<210> 11

<211> 369

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

tccgagccga gagaaagaag agtgcaacga atttcatcga actggtcgtc ctcctttggg 60

cggcatgact cctcctcttt cttcatccat cttgtcttac gtcgcttcga ctctcgcggg 120

gggttcttct tcctgaacct ctcacccatt catctctcct tccttttttc ccttactctc 180

tctctctctc tctctatctc ttcctccccc atagacgtgc ttcacgttgt taggttcttt 240

cattttttcc tggaacataa agaatacacg tgtcttcttc ctcacccctc ccgttattta 300

taaaaaaaga tttctttgta ttttataggc gggggggccc cctttccttt tcgagtgcaa 360

aaaaaaaaa 369

<210> 12

<211> 360

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

tcggtgctgg taggcgggaa ctttacctcg atcctaaggg tatatttcaa gattgctgta 60

agttcggtga catatatatg ataatatatt gttccaattt tttaatatat atatatatat 120

atatatatat agagagtgta tttaaattat ttttttactg tatacccatc tgaaaaaaat 180

gtgtacttga ttttcgtaca aaaattactc tccctcccgt ttctccgggg gggtatataa 240

aaaaatctat cctgggatgg cccccgccct ctaaatatag ggtttaaagg aaatttcaaa 300

accgttacgt tttttacgcc ccccgccccc cccggtttcc ggggagtaaa ataaaaaaag 360

<210> 13

<211> 385

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 13

tacaatataa atgaaatttt atttgtgatc ttgccgctcc caactcgagc gaaaccaggt 60

acgcaaaacg ttacatatat gttaggttgt tgccctttgg cccaattttt tctaatttgc 120

ccctcccttt aaaaaaaaac caaattaaaa ccgcctttaa aaaaaatttt cccccaagac 180

gggcaaaaaa aaaaaggggt ttttaatttt ttttcaaaca aaaaaaattt gccaaaattt 240

ttctttgttt ctttccaaaa aaaaaccata ataattttga attttttttg acccggtttc 300

cccccgaggg ggggccccac aacccgcccc cccagggggg ggaaaattcc cccccccctt 360

tttgaaccga cataagggaa aattt 385

<210> 14

<211> 367

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 14

taactataaa aaattttttc cgcgttacca ctcttatact accagttgct tatggtaaac 60

tactattgga tagtaatcta gtggatttat gctattctgc ctactggtgg ttttccttgg 120

ctgttgaaaa aaaaccccga ggggacgaag aaaaaacatg tatctatata tatatatata 180

tatatatata tatacatata tctatatacc catatatcca aaacccgggt tgtctttttt 240

ctttctcaca atacattatt acaacaggga cactttttcc tctcgctccc aatcaaacgg 300

gggagaaggc accaacaaaa aaaaaaaaaa aggagcctgg tctcaagccg attaaaaagg 360

ggccaat 367

<210> 15

<211> 566

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 15

gaaggttaaa gataagacga gcttgctttg caggagaatc tatggaattc cttaagcttg 60

gccgacgaac ccgaagcctg aggacacgcc gctttctcct ccggcgactt cgcctccgcc 120

gtcaaccact tcaccgaagc gatcaacctc gccccgacca accacgtcct ctactccaac 180

cgctccgccg cccacgcctc cctcctccgc tacgaggaag ctctctccga cgccaagaag 240

accgtcgagc tcaaacccga ctgggccaag ggctacagcc gcctcggcgc cgctcacctc 300

ggcctcaacc aatccgacga agccgtcgag gcctgctcca agggtctcga gatcgatcca 360

agcaacgacg cgctttaatc gggttctgca gacggcttcg agggtccgcg ccgcccctcc 420

ccccccgaat ccgtttgggg acgcgttcaa ggggccagag catgtgggcg aaggtgaggg 480

cggatccgtc gacgaggggg ttcttgtgtg agcctgactt cgtcaacatg atgcaggaga 540

tgcagaggaa tcctataacg ggaaaa 566

<210> 16

<211> 311

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 16

taaggcatag gcagaagagc aactgctcaa gatttctact taaactgaaa cattttctgt 60

gacacactca ttgtagccgg aagaaaagag attgtcgtat ctattcactc ctcacagcag 120

ctctctcgat catgaacatg accacagtga gagagagaga gagagagaga tatccaaatt 180

tactttcact atctctcctt caagtttttc agatgcttca tttgcttcct cttgacttta 240

aaaacaactt cggcgctata tcaacctcct accttctttg gtggctgcac aaacatgatc 300

ctctctacga a 311

<210> 17

<211> 346

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 17

taaatatttt tcttttctcc ggatagatac tccagggagt cgtgcaagtg gccgactctg 60

tgcgcccact cgggcagaga aggcgggcct caattggtgg ttttcgtggc tgtgcttgta 120

tgattgttga ctttcggttg tctccatgtc tctttgtgat atgttcttcc tccaggaatt 180

tcccccagct attgaccaag atatccccgg cctggaggtt cttttttctt tttcacgata 240

cggtactaca acaggcagat aggattccag tgtctccctc acaacgaaga agcttattcc 300

gttgctgagt ttgctcagga gcctggtcac aagcgggatg agaagg 346

<210> 18

<211> 346

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 18

tacggaaaca agggacgaag agcaatggag tcaacagatg acttatctgt tgcttttttt 60

tgaaatcctc acaaaaatcc gaagaacaac cgaactgaaa acatatgagt tttgtagttt 120

ccataattga tttcatcaat cgaacctaaa atataatttt ttcttggtct gctgatgaaa 180

tccaatatta gaatcacaaa atctctatat ctctcatttc gtttttcttt ttatttatga 240

taaattatta tttttaaaaa ttattgataa ctttttcatg aataacttgt tttttttttg 300

gaacgtcttt gagaaaaata atgtgccata tgttgtaccg aaaaaa 346

<210> 19

<211> 343

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 19

tccgggaagg aaagcgaaca tgaaccgttg taaacgatat cttatctgtt gctttttttt 60

gaaatcttca caataatccg aacaacaacc gaacagaaaa catatgagta tcgtaatatc 120

cataattgat ttcatcaacc gaacctaaaa tataatttta tcttggtctg ctgatgaaat 180

ccaatattgg aataacaaaa tatatatatc ttttatttcg ttgttctttt tatttatgat 240

aaattattat ttttaaaaat tattgataac tttttcatga ataacttgtt ttttttttgg 300

aacgtctttg agaaaaataa tgtgccatag gttgtaccga gga 343

<210> 20

<211> 535

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 20

tacacaagaa aagcgagaca gagagttctt ttagtatttt gacgggtact tagactttca 60

tacttctatt acctaacgtt ttggaagaat aacaatctct actccatgtg aggagctagt 120

attaagtgta aggaaaaatt tcttccgaaa gcgctctgtc tctcactcta tatagataaa 180

tatatagata tttaaatatt tatatataaa tatccagatt tgtaaaaaat taaaaacctt 240

cccattttca atttcggaaa atcaaaaaaa tttttttttt tttatatata tatatatata 300

tataaaaaaa taaaaaaata tatatatata tatatataga gagagacgtg ttccactctc 360

tcaaaaaaat atctatatat atatagagag acaaaagaga gagagagagt gtgtgtcact 420

cccacacaca ctcgaaaaag agagagagtg tgtgtgtgtc tctgtgacaa aaaaaaattt 480

tgtgagacac tgtgtgtatt tttatcaaca ccccctctct gtgtgtatag agaaa 535

<210> 21

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 21

gaacttatgg cactccgatc tc 22

<210> 22

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 22

ggtcattttc ttccttgata gc 22

<210> 23

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 23

atatcgcacg ggttccttac 20

<210> 24

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 24

tgttaaaacc gagggaaaag ag 22

<210> 25

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 25

cgtagggcag ctttgatttt ag 22

<210> 26

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 26

aaagaagtct cgcctgaaca ag 22

<210> 27

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 27

aagagaaaac cagagcgaca ag 22

<210> 28

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 28

gcagcgatag gaaattggat aa 22

<210> 29

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 29

cgaacagaat tgaaaccgaa c 21

<210> 30

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 30

cagaagcaag tcgagagaga ca 22

<210> 31

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 31

tataaaacct ggggattgtt gc 22

<210> 32

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 32

aaatgccttc cttgataact gg 22

<210> 33

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 33

tggtggtgtc tcaaaatggt a 21

<210> 34

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 34

tgctttgtat tcatctctcc cc 22

<210> 35

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 35

tgctagggtt cctctggatc t 21

<210> 36

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 36

ctgagaggat catgttttgt gc 22

<210> 37

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 37

ctttatctgc ttctgctgtt gc 22

<210> 38

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 38

aatagcttcc tcacctgtca cc 22

<210> 39

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 39

tgcatacaaa ccgagaatca 20

<210> 40

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 40

ggacaaacaa ggatggagtt tc 22

<210> 41

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 41

cgaagcaagt aagaaacaca cg 22

<210> 42

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 42

atataactag gtgttttgcc cg 22

<210> 43

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 43

catccatcag ttcaatcggt ta 22

<210> 44

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 44

tctgcacttg ggtcactaca at 22

<210> 45

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 45

gcttgtacgc ttcttccaga tt 22

<210> 46

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 46

atgagaatgg ctagtccggt ta 22

<210> 47

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 47

gcagcgatag gaaattggat aa 22

<210> 48

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 48

aagagaaaac cagagcgaca ag 22

<210> 49

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 49

gttgtacgtc ttccactttc cc 22

<210> 50

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 50

aggaaccgac attcagagag ag 22

<210> 51

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 51

aattaacctt gtcgggtgaa ga 22

<210> 52

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 52

cgaagatttc attgctgata cg 22

<210> 53

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 53

catggtactc ctgagcttat ttg 23

<210> 54

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 54

cgactcttca atgtgcatct ct 22

<210> 55

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 55

tctccttcaa agcaatctct cc 22

<210> 56

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 56

gcagagactt cttcaccgtc tt 22

<210> 57

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 57

tttctccctg atgacctttt gt 22

<210> 58

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 58

ggtttagacg gcacctagtc ag 22

<210> 59

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 59

cggtctaaca tattgcacat tc 22

<210> 60

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 60

ccaaatctga atgcgagaga at 22

Claims (15)

1. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-1, and it has SEQ ID No：Nucleotide sequence shown in 1.

2. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-4, and it has SEQ ID No：Nucleotide sequence shown in 4.

3. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-8, and it has SEQ ID No：Nucleotide sequence shown in 8.

4. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-10, and it has SEQ ID No：Nucleotide sequence shown in 10.

5. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-11, and it has SEQ ID No：Nucleotide sequence shown in 11.

6. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-13, and it has SEQ ID No：Nucleotide sequence shown in 13.

7. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-14, and it has SEQ ID No：Nucleotide sequence shown in 14.

8. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-15, and it has SEQ ID No：Nucleotide sequence shown in 15.

9. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-19, and it has SEQ ID No：Nucleotide sequence shown in 19.

10. a kind of external source radish fragment specific mark, it is characterised in that the specific mark is CLR9-20, and it has SEQ ID No：Nucleotide sequence shown in 20.

11. a kind of preparation method of external source radish fragment specific mark, comprises the following steps：

(1) entered using cabbage type rape radish cytoplasmic sterility restorer CLR650 for female parent with cabbage type rape routine strain A Row hybridization, obtains F₁Colony, select more plants of F₁Colony plant carries out bagging isolation selfing, obtains F₂Fertility segregation colony individual plant, and Extract the genome DNA of each individual plant；F is investigated in full-bloom stage₂Colony's individual plant fertility, establishes fertile plant and sterile pnca gene respectively Mixing pit；

(2) to cabbage type rape radish cytoplasmic sterility restorer CLR650 carry out full-length genome resurvey sequence, by sequencing result with Radish gene group sketch and radish cytoplasm sterile restoring gene sequence are compared, and obtain CLR650 restoring gene and its side External source radish sequence information, special primer is designed according to target area；

(3) special primer of acquisition is screened using gene mixing pit, retains fertile gene mixing pit PCR amplifications and be positive, no The primer that the amplification of gene mixing pit is negative is educated, and by the primer screened in F₂Isolated on Fertility segregation colony individual plant Checking, the primer that PCR amplification positive performances are isolated with fertility performance is as external source radish fragment specific mark.

12. the preparation method of external source radish fragment specific mark according to claim 11, it is characterised in that the step (1) cabbage type rape routine strain A is 20B in.

13. the preparation method of external source radish fragment specific mark according to claim 11, it is characterised in that the step (1) more plants of F of selection in₁Colony plant carries out bagging isolation selfing and refers to select 6-8 strains F₁Colony plant carries out bagging isolation certainly Hand over.

14. it is a kind of as any one of claim 1-10 or as any one of claim 11-13 preparation side Application of the external source radish fragment specific mark in cabbage type rape Breeding for restoration lines made from method, it is characterised in that the application bag Include following steps：

1) entered using cabbage type rape radish cytoplasmic sterility restorer CLR650 for female parent with cabbage type rape routine strain A Row hybridization, obtains F₁Colony；Select more plants of F₁It is returned for plant with cabbage type rape routine strain A as recurrent parent, structure Build BC1 segregating populations；

2) in Seedling Stage, using it is any one of at least one claim 1-10 or as described in claim 11-13 system External source radish fragment specific mark is to BC made from Preparation Method₁Segregating population carries out molecular marker assisted selection, pulls out labeled analysis As a result the individual plant being negative；More plants of BC are selected at the florescence₁Individual plant of the phenotype close to cabbage type rape routine strain A in segregating population As female parent, continue to be returned with cabbage type rape routine strain A；Repeat the above steps until obtaining cabbage type rape CLR650 BC₄Fertility segregation colony；

3) BC is pulled out₄Sterile individual plant in Fertility segregation colony, the fertile individual plant of reservation is all subjected to mixed pollination in isolated area And harvest, and in the fertility performance of full-bloom stage investigation progeny population, until obtaining colony of the fertile plant proportion more than 75%；

4) by fertile plant in segregating population of the fertile plant ratio more than 75%, all isolation is selfed, and continuing selection in self progeny can Strain all isolation selfings are educated, until obtaining the cabbage type rape radish cytoplasmic sterility restorer that fertility does not separate, phenotype is consistent CLR095。

15. application according to claim 14, it is characterised in that cabbage type rape routine strain A is in the step (1) 20B。