CN101880658A - Molecular marker of line with genic sterile recessive epistatic interaction in brassica napus and application - Google Patents

Abstract

The invention belongs to a preparation field of a cole molecular marker, in particular to a molecular marker linked to recessive genic sterile gene in brassica napus and breeding of a temporary maintainer line homologous with a genic sterile line. The molecular maker is characterized by comprising the following steps of: establishing near-isogenic lines 7-7365AB, 7-736512AB and 7-7365AC of three genes BnMs3, BnMs4 and BnRf by using two types of male sterile lines 7-7365AB in the brassica napus, brassica napus 7-749 and temporary maintainer line materials 7-7365C as raw materials, and then sieving SSR (Simple Tandem Repeat) primers and AFLP (Amplified Fragment Length Polymorphism) primers and transforming a SCAR marker to obtain a codominant SSR molecular marker SR12384 linked to the BnMs3, dominant SCAR markers SM129, SM98, SM113 and codominant SCAR markers SM23 and H3 which are linked to the BnMs4, and a codominant SSR marker D1 linked to the BnRf. The invention can be used for auxiliary selection and breeding of recessive genic sterile markers and can also be used for fine mapping and cloning of sterile genes.

Description

The molecule marker of line with genic sterile recessive epistatic interaction in brassica napus and application

Technical field

The invention belongs to the molecule marker preparation field of swede type rape recessive cytoblast sterile gene, be specifically related to the preparation of the chain molecule marker of fertility-related gene and in swede type rape with the application of the aspects such as marking supplementary breeding of sterile line homologous temporary maintainer line.

Background technology

Male sterile is the effective way that rape heterosis utilizes.Up to the present, three the being hybrid seedings that utilize cytoplasmic male sterility to carry out still are the main approach of swede type rape heterosis utilization.With pol CMS and Shan 2A CMS is that the cytoplasmic male sterility of representative is used for cross-fertilize seed in a large number and produces.Nuclear male sterility is that the important channel, particularly recessive karyon male sterile that rape heterosis utilizes shows the characteristic that some are better than cytoplasmic male sterility: sterility is thorough, stable; Have and recover the source widely; The risk that does not have sterile cytoplasm negative effect and tenuigenin simplification.Therefore, nuclear male sterility demonstrates huge application potential in the utilization of rape heterosis, has become the research focus that rape heterosis utilizes.Yet, nuclear male sterility is in the process of hybrid seeding, can not produce 100% complete sterility colony, must pull out in the maternal row 50% educated strain, bring very big inconvenience to production of hybrid seeds production, so the nuclear male sterility that develops into of complete sterility colony is widely used in the key that the rape hybrid produces.

7-7365AB is the novel recessive karyon male sterile material of a class, and its fertility is subjected to 1 pair of recessive sterile gene (Bnms3) and 1 pair of multiple equipotential epistatic gene (BnMs4/Bnrf) control.When the Bnrf gene is that recessiveness is isozygotied or BnMs4 gene dominance is isozygotied or can suppresses the normal expression of sterile gene (Bnms3) that isozygotys during heterozygosis, thereby fertility is restored.Therefore, genotype is that the individual plant performance of Bnms3ms3Ms4_ and Bnms3ms3rfrf can be educated, and genotype is that the individual plant of Bnms3ms3RfRf and Bnms3ms3Rfrf then shows sterile.Therefore, this material can the male sterile of analog cell matter be realized three the being production of hybrid seeds (Fig. 1): homozygous two-type line (AB system, genotype: Bnms3ms3RfRf and BnMs3ms3RfRf), temporary maintainer line (C system, genotype: Bnms3ms3rfrf), recover system (R system, genotype: BnMs3Ms3_ and Bn_Ms4Ms4).Promptly utilize the hybridization of sterile line and temporary maintainer line to produce 100% sterile population, utilize this complete sterility colony to produce cross-fertilize seed again, in hybrid seeding, must pull out the difficult problem that maternal row 50% can be educated strain thereby solved this recessive cytoblast sterile with recovering system.This hereditary pattern has not yet to see report.

Owing to relate to 1 pair of recessive sterile gene and 1 pair of multiple equipotential epistatic gene, so the seed selection difficulty of nuclear male sterility temporary maintainer line is bigger.Therefore, the molecule marker of development and BnMs3, BnMs4 and three gene linkages of BnRf is for homozygous two-type line, and is especially particularly important with the seed selection of homozygous two-type line homologous temporary maintainer line.AFLP technology (Vos et al, 1995) is widely used in (Jorge et al, 1999 in the structure and analysis of genetic diversity of location, genetic linkage map of the Main Agronomic Characters gene of various crop because of its plurality of advantages; Tang et al, 2006; Zhang et al, 2006; Price et al, 2000; Guo et al, 2003).AFLP more can be directly at target gene place section in conjunction with the BSA analytical method, and the acquisition that makes mark is more fast with accurate.Therefore, this method is widely used in Position Research (Yi et al, 2006 of range gene; Lei et al, 2007; Ke et al, 2005).(Hong et al such as Hong, 2006) utilize this method in dominant genic male sterile material Rs1046AB, to obtain 6 AFLP marks chain with dominance suppressor gene Rf, (Huang et al such as Huang, 2007) by 1024 pairs of combination of primers of screening, obtain 15 AFLP marks chain with BnMs3; Xiao etc. (Xiao et al, 2008) screening obtains 7 AFLP marks chain with BnRf, and BnRf is navigated to N7 linkage group upper end.The present invention utilizes similar method to obtain 8 important molecule markers, for the application of molecular marker assisted selection (MAS) in swede type rape provides a great convenience, for the seed selection of recessive epistatic interaction in brassica napus genie male sterile line temporary maintainer line provides approach fast and effectively.

Summary of the invention

The purpose of this invention is to provide a kind of and the chain molecule marker of swede type rape recessive cytoblast sterile fertility-related gene, and the application in the swede type rape molecular marker assisted selection, comprising with the aspects such as marking supplementary breeding of swede type rape sterile line homologous temporary maintainer line.

The present invention is achieved through the following technical solutions:

A kind ofly examine sterile relevant BnMs3 with recessive epistatic interaction in brassica napus, BnMs4 and the closely linked molecule marker of BnRf gene, described molecule marker is used to identify the genotype of swede type rape at described three gene locuss, be used to utilize this recessive karyon male sterile to carry out the assisted Selection of molecule marker simultaneously, it is characterized in that it is one of following molecule marker:

(1) be used to distinguish BnMs3ms3RfRf, BnMs3Ms3RfRf and three kinds of genotypic codominance SSR molecule marker SR12384 of Bnms3ms3RfRf with the BnMs3 gene is closely linked, its nucleotide sequence is shown in SEQ ID NO:11-12;

(2) be used to distinguish Bnms3ms3rfrf, Bnms3ms3Rfrf and three kinds of genotypic codominance SSR molecule marker D1 of Bnms3ms3RfRf with the BnRf gene is closely linked, its nucleotide sequence is shown in SEQ ID NO:13-14;

(3) with the closely linked dominant SCAR molecule mark SM129 of BnMs4 gene, SM98 and SM113, its nucleotide sequence is shown in SEQ ID NO:1-6, and with the three kinds of genotype codominance molecule marker SM25 and the H3 that are used to distinguish Bnms3ms3rfrf, Bnms3ms3Rfrf and Bnms3ms3RfRf of BnMs4 gene linkage, its nucleotide sequence is shown in SEQ ID NO:7-10.

Molecule marker of the present invention can be examined in the sterile marker assisted selection at recessive epistatic interaction in brassica napus and be applied.

Molecule marker of the present invention can also be applied in BnMs4 and BnRf Fine Mapping and the map based cloning at recessive epistatic interaction in brassica napus genic male sterile gene BnMs3.

The applicant provides a kind of and the preparation method closely linked molecule marker of swede type rape recessive karyon male sterility gene, and this method is according to following steps:

(1) with the preparation of the codominance SSR molecule marker SR12384 of BnMs3 gene linkage:

A) utilizing the male sterile amphitypy of swede type rape recessive karyon is that the brother and sister that genotype carried out at least 10 generations for educated strain and the genotype of BnMs3ms3RfRf for the sterile strain of Bnms3ms3RfRf among the 7-7365AB hand over the near isogenic line that obtains the BnMs3 site, with its called after 7-7365AB;

B) adopt CTAB in a small amount method extract previous step rapid a) in sterile strain and can educate strain DNA, use the primer PCR amplification shown in sequence table SEQ ID NO:11-12 can educate the DNA of individual plant and sterile individual plant;

C) utilize 6% polyacrylamide gel electrophoresis to detect amplification, obtain distinguishing the codominance SSR molecule marker SR12384 of three kinds of genotype: BnMs3ms3RfRf, BnMs3Ms3RfRf and Bnms3ms3RfRf, its sequence is shown in SEQ ID NO:11-12.

(2) with the preparation of the codominance SSR molecule marker D1 of BnRf gene linkage:

A) utilize the brother and sister that genotype carried out at least 10 generations for educated strain and the genotype of Bnms3ms3rfrf for the sterile strain of Bnms3ms3Rfrf among the swede type rape recessive karyon male sterile line 7-7365AC to hand over the near isogenic line that obtains the BnRf site, with its called after 7-7365AC;

B) adopt CTAB in a small amount method extract previous step rapid a) in sterile strain and can educate strain DNA, adopt group's analytical method, use the primer PCR amplification shown in sequence table SEQ ID NO:13-14 can educate the DNA of individual plant and sterile individual plant;

C) utilize 6% polyacrylamide gel electrophoresis to detect amplification, obtain distinguishing the codominance SSR molecule marker D1 of three kinds of genotype Bnms3ms3rfrf, Bnms3ms3Rfrf and Bnms3ms3RfRf, its sequence is shown in SEQ ID NO:13-14.

(3) with the preparation of SCAR molecule marker SM129, SM98, SM113, SM25 and the H3 of BnMs4 gene linkage:

A) utilize the brother and sister that genotype carried out at least 10 generations for educated strain and the genotype of Bnms3ms3Ms4Rf for the sterile strain of Bnms3ms3RfRf among the swede type rape recessive karyon male sterile line 7-736512AB to hand over the near isogenic line that obtains the BnMs4 site, with its called after 7-736512AB;

B) adopt CTAB in a small amount method extract previous step rapid a) in sterile strain and can educate strain DNA, adopt group's analytical method, use the primer PCR amplification shown in sequence table SEQ ID NO:1-10 can educate the DNA of individual plant and sterile individual plant;

C) utilize 6% polyacrylamide gel electrophoresis to detect amplification, obtain dominance molecule marker SM129, SM98 and the SM113 chain with BnMs4, its sequence is shown in SEQID NO:1-6; And the codominance molecule marker SM25 and the H3 that can distinguish three kinds of genotype Bnms3ms3rfrf, Bnms3ms3Rfrf and Bnms3ms3RfRf, its sequence is shown in SEQ ID NO:11-14.

Above-mentioned steps a) described in the CTAB small-sample method as follows: the tender plant of children or blade are put into mortar add 0.75-1.0ml CTAB extraction buffer, smash into it to pieces homogenate with grinding rod immediately, move into again in the 2.0mL centrifuge tube, then at 55 ℃ of-60 ℃ of water-bath 45-60min, fluctuate lightly therebetween, take out centrifuge tube and be cooled to room temperature, add isopyknic volume ratio again and be 24: 1 chloroform: the abundant mixing 10-20min of primary isoamyl alcohol, the centrifugal 12-15min of 12000r/min, shift supernatant liquor in another 2.0mL centrifuge tube, in the centrifuge tube of supernatant liquor is housed, add 60ul5mmol/LNH ₄AC, and then add the freezing in advance dehydrated alcohol or the isopropanol precipitating DNA of 2 times of volumes places 1-2hrs or spends the night at-20 ℃, goes out or the centrifugal 3min deposit D of 10000r/min NA with the rifle choicest, again with containing 10mM NH ₄Ac 70% ethanol ammonia soaks DNA 5hrs, removes ethanolic soln, and dry DNA at room temperature or in the stink cupboard adds 300 μ l TE buffered soln or 300 μ l ddH again ₂The O dissolving DNA ,-20 ℃ of preservations are standby;

Wherein:

CTAB extraction buffer composition is (per-cent by volume): 2%CTAB, 1.4M NaCl, and 20mM EDTA, 0.2% β mercaptoethanol, 100mM Tris-HCl, pH 8.0, add water and are settled to 1L;

TE buffer preparation method is as follows: contain 10mM Tris-HCl and 1mM EDTA-Na2 in the 500ml TE damping fluid, room temperature preservation after 8.0,121 ℃ of sterilizations of pH.

Table 1. primer sequence of the present invention

Positively effect of the present invention

1. separate and obtained and BnMs3, BnMs4 and the closely linked molecule marker of BnRf gene, made up the highdensity genetic linkage maps in BnMs3 site and BnMs4/BnRf site, for the further Fine Mapping and the clone of above 3 genes haves laid a good foundation.

2. obtained a large amount of molecule markers, especially based on codominance SCAR mark and the SSR mark of PCR, examine the assist-breeding of sterile homozygous two-type line and temporary maintainer line thereof mutually necessary condition is provided for utilizing molecule marker to carry out recessive epistasis, made things convenient for the heterosis utilization of recessive cytoblast sterile material, for the production of cross-fertilize seed provides good condition.

More detailed technical scheme is as described in " embodiment ".

Description of drawings

Fig. 1. recessive epistatic interaction in brassica napus is examined sterile 7-7365AB " three being " production of hybrid seeds mode chart.

Fig. 2. the molecule marker genetic linkage map of swede type rape recessive karyon male sterility gene BnMs3.

Fig. 3. with the amplification figure of codominant marker sR12384 in microcommunity of BnMs3 gene linkage.

Fig. 4. with the mark linkage map of BnRf gene linkage.

Fig. 5. with the amplification figure of codominance SSR mark D1 in microcommunity of BnRf gene linkage

Fig. 6. be marked at amplification in the microcommunity with the chain SCAR of BnMs4.Among the figure:

A：SM129，B：SM98，C：SM113；

Fig. 7. (a) the N7 linkage map that is made up by Tapidor * Ningyou7: diagram EC09MG12 is positioned between XM1 and the CNU063, and XM1 is and the AFLP mark of BnRf gene linkage in the square frame, is positioned in the N7 upper end.(b) genetic linkage map of the BnMs4 gene that makes up by near isogenic line 7-736512AB: diagram and 13 chain AFLP marks of BnMs4,4 SCAR marks, 5 with BnRf gene common molecule marker, dotted line is illustrated common molecule marker in two linkage maps.(c) genetic linkage map of the BnRf gene that is made up by near isogenic line 7-7365AC: diagram BnRf gene is between common tags ENA06 and SSR1.(d) from the N7 genetic linkage map of Quantum * No2127-17: diagram P16MC08 is positioned between ME7EM12E and the sR4047.

Fig. 8. (a) the vertical electrophoresis amplification figure of SCAR mark SM25 in the different genotype individual plant.(b) the vertical electrophoresis amplification figure of SSR mark D1 in the different genotype individual plant.(c) the vertical electrophoresis amplification figure of SCAR mark H3 in the different genotype individual plant.

1: genotype is the educated individual plant of Bnms3ms3Ms4Rf among the near isogenic line 7-736512AB

2: genotype is the educated individual plant of Bnms3ms3Ms4Ms4 among the near isogenic line 7-736512AB

3: genotype is the sterile individual plant of Bnms3ms3RfRf among the near isogenic line 7-736512AB

4: genotype is the sterile individual plant of Bnms3ms3Rfrf among the near isogenic line 7-7365AC

5: genotype is the sterile individual plant of Bnms3ms3RfRf among the near isogenic line 7-736512AB

6: genotype is the educated individual plant of Bnms3ms3rfrf among the near isogenic line 7-7365AC

Fig. 9. the application of molecular marker assisted selection in seed selection swede type rape recessive nuclear sterile temporary guarantor system

Embodiment

Embodiment 1: the swede type rape recessive epistasis is examined sterile related gene B nMs3, the 7-7365AB of 1. 3 near isogenic line colonies of acquisition of BnMs4 and BnRf specific molecular marker, and the structure of 7-736512AB and 7-7365AC:

Used basic material is swede type rape recessive karyon male sterile homozygous two-type line 7-7365AB (Bnms3ms3RfRf/BnMs3ms3RfRf) (Huang et al, 2007), novel swede type rape 7-749 (Bnms3ms3Ms4Ms4) (yellow town, Ph D dissertation, 2008, http://www.cnki.net/) and temporary maintainer line 7-7365C (Bnms3ms3rfrf) (Xiao et al, 2008), obtain three near isogenic line 7-7365AB (Huang et al by these three materials, 2007), 7-736512AB (yellow town, Ph D dissertation, 2008, middle National IP Network, http://www.cnki.net/) and 7-7365AC) (Xiao et al, 2008), be respectively applied for and BnMs3 the screening of these three closely linked molecule markers of gene of BnMs4 and BnRf.

The structure of near isogenic line 7-7365AB: with the sterile strain (Bnms3ms3RfRf) among the homozygous two-type line 7-7365AB with can educate strain (BnMs3ms3RfRf) and carry out the above hybridization of 10 generations and preserve, the final difference that obtains between the individual plant exists only in the near isogenic line 7-7365AB (Huang et al, 2007) of Bnms3 site and near zone.7-7365AB comprises 50% sterile individual plant (Bnms3ms3RfRf) and 50% educated individual plant (BnMs3ms3RfRf).Sow this 7-7365AB, the professional and technical personnel utilizes naked eyes just can differentiate sterile strain and can educate strain.

The structure of near isogenic line 7-736512AB: by 7-7365A (Bnms3ms3RfRf) and novel swede type rape 7-749 (Bnms3ms3Ms4Ms4) hybridization, backcross with 7-7365A then, sterile and separating of can educating appear in its offspring, utilize with the closely linked molecule marker sR12384 scanning of BnMs3 and can educate the individual plant offspring of individual plant acquisition genotype for Bnms3ms3Ms4Rf, this can be educated individual plant and sterile individual plant and carries out continuous hybrid and build up the 7-736512AB of near isogenic line colony (the yellow town that difference between the individual plant exists only in BnMs4 site and near zone then, Ph D dissertation, 2008, http://www.cnki.net/).Comprise 50% sterile individual plant (Bnms3ms3RfRf) and 50% educated individual plant (Bnms3ms3Ms4Rf) among the 7-736512AB.Sow this 7-736512AB, the professional and technical personnel utilizes naked eyes just can differentiate sterile strain and can educate strain.

The structure of near isogenic line 7-7365AC: the F that utilizes 7-7365A and 7-7365C hybridization to obtain ₁Backcross with 7-7365A again and obtain BC ₁Segregating population (Bnms3ms3Rfrf/Bnms3ms3rfrf).Use this BC ₁The educated strain of colony inside and sterile strain were hybridized more than 10 generations, and the difference between the acquisition individual plant exists only in the near isogenic line 7-7365AC (Xiao et al, 2008) of BnRf site and near zone.Comprise 50% sterile individual plant (Bnms3ms3Rfrf) and 50% educated individual plant (Bnms3ms3rfrf) among the 7-7365AC.Sow this 7-7365AC, the professional and technical personnel utilizes naked eyes just can differentiate sterile strain and can educate strain.

2.DNA the structure of extraction and sample pool

To in three near isogenic line colonies separately sterile individual plant and can educate individual plant and list, choose the fresh and tender blade of 0.5g seedling stage, be loaded on the 2.0ml centrifuge tube, store for future use in-20 ℃ or-70 ℃.Extracting genome DNA adopts CTAB method (Lu et al, 2004).Concrete steps are: near isogenic line colony swede type rape blade is fallen in mortar, add the CTAB damping fluid (2%CTAB of 700-800 μ l2%; EDTA20mmol/L; PH=8.0Tris-HCl100mmol/L; NaCl1.4mol/L; PVP1%), wear into homogenate.65 ℃ of water-bath 60min, every the 15min jog once.Room temperature is placed 10min, adds isopyknic chloroform: primary isoamyl alcohol (24: 1) jog, mixing.12, the centrifugal 15min of 000r/min takes out gently, draws supernatant to the centrifuge tube of new 2ml, adds the 3M NaAC (PH=5.2) of 1/10 volume, adds the freezing dehydrated alcohol of two volumes again, spins upside down mixing for several times, ice bath 30min.10, the centrifugal 2min of 000r/min, the careful supernatant that goes adds 1,000 μ l rinsing liquid (76% ethanol, 10mM ammonium acetate), twice of rinsing.The careful rinsing liquid of outwelling dries to clear, colorless under room temperature.Look the DNA amount, take the circumstances into consideration to add TE damping fluid (containing 0.1% RNase) dissolving DNA ,-20 ℃ of preservations are standby.Total DNA quality detects with 0.8% agarose gel electrophoresis.Total DNA concentration is measured with ultraviolet spectrophotometer (Pharmacia Biotech, Gene QuantII), according to detected result, with the DNA concentration ddH of each sample ₂O adjusts to 50ng/ μ l, and is standby in-20 ℃ of preservations.

Result according to florescence fertility investigation chooses 16 separately and can educate individual plant and 16 sterile individual plants from three near isogenic lines, 8 individual plant DNA balanced mix make up 2 respectively and can educate pond (BF) and 2 sterile ponds (BS).

3.AFLP analyze with BSA

3.1 the enzyme of total DNA is cut and is connected

Analyze two simultaneously and can educate pond and two sterile ponds, to improve screening efficiency.Reaction system is: total DNA 75ng, and 2.5U EcoR I (or PstI, Sac I), (MBI Fermentas, Lithuania), 1 * buffer Tango, cumulative volume are 12.5 μ l to 1.5U Mse I.Earlier bathe 5.5-6h 37 ℃ of temperature, after change 65 ℃ of water-bath 1h over to, enzyme cuts the back to add isopyknic connection mixed solution (0.75U T4 dna ligase, 25pmol EcoRI or Pst I or Sac I joint, 25pmol Mse I joint), in 4 ℃ of refrigerator overnight.After connection is finished, use ddH ₂10 times of templates of O dilution as the pre-amplification of PCR.Adopted 3 kinds of enzymes to cut combination EcoR I/Mse I, Pst I/Mse I and Sac I/Mse I in the aflp analysis of this research altogether.AFLP joint and design of primers are all undertaken by (1995) reported method such as Vos, give birth to worker's biotechnology company limited by Shanghai and synthesize, and sequence information is as shown in the table:

Table 2.AFLP joint and pre-amplification primer

3.2 pre-amplification

10 kinds of pre-amplification combination of primers: EA/MC, EA/MG, EC/MG, EC/MC, EC/P0, EC/TG, P0/MC, P0/MG, SA/MC, SA/MG are adopted in this research altogether.

The PCR reaction system: 5 μ l enzymes are cut the connection product, 1 times of (*) PCR damping fluid, 2.0mmol/L MgCl ₂, 0.16mmol/L dNTPs, 1U Taq enzyme 50ng EA (or P0, SA, EC) primer, 50ng MC (or MG, TG) primer, the reaction cumulative volume is 25 μ l.PCR reaction cycle parameter: 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min carry out 20 circulations altogether.Get 5 μ l PCR products and detect in 1.0% sepharose, (Smear 50bp-1000bp), shows that pre-expansion synergy fruit is then good if be the disperse shape.15-30 times of resultant product dilution is as the template of selective amplification.

3.3 selective amplification

PCR reaction system: the 3 μ l cut back that increases in advance, 1 times of (*) PCR damping fluid, 2.0mmol/L MgCl ₂, 0.2mmol/L dNTPs, 1U Taq enzyme, 50ng E+3 primer (P0+3 primer, M+3 primer, T+3 primer or S+3 primer) (above-mentioned combination of primers see Table 3 and remarks), the reaction cumulative volume is 15 μ l.The PCR loop parameter is: 94 ℃ (30s), 65 ℃ (0.7 ℃/circulation) (30s), 72 ℃ (1min), 14 circulations; 94 ℃ (30s), 56 ℃ (30s), 72 ℃ (1min) 25 circulations.Selective amplification primer sequence and numbering see the following form:

Table 3.AFLP selective amplification primer

Remarks: E+3: wherein E represents: 5 '-GACTGCGTACCAATTC-3 ' adds three bases of respective column in the table 3

P+3: wherein P represents: 5 '-GACTGCGTACATGCAG-3 ' adds three bases of respective column in the table 3

S+3: wherein S represents: 5 '-GACTGCGTACAAGCTC-3 ' adds three bases of respective column in the table 3

M+3: wherein M represents: 5 '-GATGAGTCCTGAGTAA-3 ' adds three bases of respective column in the table 3

T+3: wherein T represents: 5 '-GATGAGTCTGTCACGA-3 ' adds three bases of respective column in the table 3

3.4 the electrophoresis detection of amplified production

In the selective amplification product, add isopyknic sample-loading buffer (98% deionized formamide, 10mM/L EDTA, the blue or green FF of 0.005% dimethylbenzene, 0.005% tetrabromophenol sulfonphthalein), ice bath cooling immediately behind 95 ℃ of sex change 5min, last sample 2 μ l, the 80W electrophoresis stopped electrophoresis when the blue or green FF of dimethylbenzene ran 2/3 offset plate.Cma staining develops behind the electrophoresis.

3.5 electrophoresis detection result

By screening AFLP primer, obtained 17 the AFLP marks chain with BnMs3, the 13AFLP mark chain with BnMs4, as shown in the table with the sequence information of chain 12 these marks of AFLP mark of BnMs4

The AFLP mark of table 4.nMs3 gene linkage

The AFLP mark of table 5.BnMs4 gene linkage

The AFLP mark of table 6. and BnRf gene linkage

4.SCAR mark transforms

4.1 the recovery of differential fragment and purifying

At 6% denaturing polyacrylamide gel offset plate (argentation) as yet not before the drying, maybe with dried glue ddH ₂O is moistening, the characteristic fragment that occurs in the sterile individual plant is dug down gently and changes over to blade be added with 20 μ l ddH ₂In the 0.5ml centrifuge tube of O, smash to pieces with disposable sterilized rifle head, in 95 ℃ of insulations be put in behind the 10min 4 ℃ standby, with preceding centrifugal a little.Get 2 μ l supernatant liquors and do template, the reaction conditions of Kuo Zeng primer and reaction conditions and AFLP selective amplification is identical again.After finishing, PCR place 72 ℃ to be incubated 10min product.Get 20 μ l reaction product and on 1.0% sepharose, detect, if amplified production size and the original consistent and assorted band of nothing then use test kit (worker's biotechnology company limited is given birth in Shanghai) to reclaim master tape.Detailed process is as follows: accurately cut out the blob of viscose that includes target fragment with blade under ultraviolet lamp, put in the 1.5ml centrifuge tube, add 500 μ l Binding Buffer, 50 ℃～60 ℃ insulation 10min, spun upside down mixing every two minutes, glue is thoroughly melted; Then the sol solution of fusing is transferred to cover and be put in the UNIQ-10 post of 2ml collection tube, room temperature leaves standstill 2min; The centrifugal 1min of 8000r/min removes the waste liquid in the collection tube, adds 500 μ l Washing Solution in the UNIX-10 post, and the centrifugal 1min of room temperature 8000r/min adds fresh Washing Solution and repeats once, removes the waste liquid in the collection tube; The centrifugal 15s of room temperature 12000r/min.Add ElutionBuffer 30 μ l (accurately dripping on the filtering membrane) in the UNIQ-10 post, room temperature is placed 2min, be inserted in the new 1.5ml centrifuge tube, and the centrifugal 1min of 12000r/min, collected solution can be used for ligation.

4.2 ligation

Adopt pMD18-T vector test kit (TaKaRra) to carry out the clone of target fragment.Linked system is: 2 μ l DNA, 0.5 μ l pMD18-Tvector, 2.5 μ l solution I.Of short duration centrifugal collection mixture spends the night in 4 ℃ of connections.

4.3 the preparation of intestinal bacteria (Ecoli) competent cell

The coli strain DH-5 α bacterium liquid of going bail for and being stored in-70 ℃ at first separates mono-clonal on the LB solid medium, choose a mono-clonal then and carry out liquid culture and spend the night.Therefrom getting 0.5ml bacterium liquid transfers in the triangular flask that 45ml LB liquid nutrient medium is housed, cultivate (37 ℃ of 2.5～3h in shaking table, 240r/min), after be transferred in the 50ml centrifuge tube of precooling, ice bath 10min, low-temperature centrifugation 10min (4 ℃, 4000r/min) collect thalline, add the 0.1mol/L CaCl of 2ml precooling ₂Resuspended culture, ice bath 15min, 4 ℃ of centrifugal 10min of 4000r/min remove supernatant liquor, and handstand is dried, and adds the 0.1mol/L CaCl of 2ml precooling again ₂Re-suspended cell is sub-packed in the aseptic Eppendorf tube of ice bath, puts into-70 ℃ of refrigerators behind the glycerol adding mixing and preserves standby.

4.4 the conversion of connexon

Get 2 μ l ligation things and change in the 1.5ml centrifuge tube, ice bath is stand-by.Take out competent cell and place on ice from-70 ℃ of refrigerators, (about 5min) carefully draws 50 μ l cells and is transferred in the top centrifuge tube when treating that it just melts, and leaves standstill 20min on ice.Accurate heat shock 90s (not shaking) in 42 ℃ of water-baths.Put back to 2min on ice rapidly, add LB substratum (room temperature) 400 μ l then, 37 ℃ of shaking tables are cultivated 1.5h (150r/min).Therefrom get conversion nutrient solution 100 μ l and be tiled in LB flat board (Tryptone 10g/L, Yeast extract 5g/L NaCl 10g/L, Agar 15g/L, Amp50 μ g/mL) on, positive horizontalization plate to liquid all is absorbed, and is inverted flat board then and in 37 ℃ of cultivation 16h～20h indigo plant, hickie is fully presented.

4.5 the screening of recon and evaluation

Each target cloned sequence is respectively selected 6 white mono-clonal bacterium colonies and is carried out liquid culture.(derive from PMD-18 carrier sequence with universal sequencing primer thing M13, M13-47:5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ' RV-M:5 '-GAGCGGATAACAATTTCACACAGG-3 ') bacterium liquid being carried out PCR detects, reaction system is: 2 μ l bacterium liquid, 1 times of (*) PCR damping fluid, 1.5mmol/L MgCl ₂, 0.2mmol/L dNTPs, 1.0U Taq enzyme (MBI Fermentas), each 35ng of universal sequencing primer thing, the reaction cumulative volume is 20 μ l.The PCR loop parameter is: 94 ℃ (2min); 94 ℃ (30s), 55 ℃ (30s), 72 ℃ (60s), 30 circulations; 4 ℃ of preservations.Product detects in 1.2% agargel electrophoresis, if amplified fragments size and original purpose clip size suitable (or big slightly) then are illustrated as positive monoclonal.Each specific fragment send 2 to be cloned into the order-checking of Beijing AudioCodes biotechnology limited liability company.

4.6SCAR design of primers

According to sequencing result, utilize primer-design software Primer 3 design primers, synthetic by Beijing AudioCodes biotechnology limited liability company.

4.7SCAR labeled analysis

The reaction system of specific PCR amplification is: 50ng DNA, 1 times of (*) PCR buffer, 2.0mmol/L Mg ²⁺, 0.15mmol/L dNTPs, 1.0U Taq polymerase, each 50ng of forward and reverse special primer, the reaction cumulative volume is 20 μ l.The PCR loop parameter is: 94 ℃ (2min); 94 ℃ (30s), optimum annealing temperature (1min), 72 ℃ (1min), 35 circulations.Do the thermograde test with parents earlier, determine suitable annealing region, annealing and the time of extending adjust in addition according to fragment length.Amplified production is electrophoresis detection on 1.2% agar gel.

If between the parent, detect polymorphism, then each individual plant in the segregating population is carried out pcr amplification and electrophoresis detection.As do not have polymorphism can consider to redesign primer, and still then do not implement the PCR walking, the flanking sequence of marker site is cloned out.Finally obtained with 5 SCAR marks (table 7) of BnMs3 gene linkage and with chain 3 dominant SCAR mark: SM129, the SM98 of BnMs4, SM113 (Fig. 7) and two codominance SCAR mark SM25 and H3 (Fig. 8 A, Fig. 8 C), their sequence is shown in sequence table SEQ ID NO:1-10.

The SCAR mark of table 7. and BnMs3 gene linkage

5.BnMs3 gene locus and BnMs4/BnRf gene locus construction of genetic atlas and collection of illustrative plates location thereof

Will with the statistic data input computer of closely linked molecule marker title of each gene and morphological characters, the mapping of operation MAPMAKER/EXPVersion 3.0 software analysis, obtain 3 genes genetic linkage maps (Fig. 2, Fig. 4, Fig. 5).

Utilize 2 double haploids (DH) Tapidor * Ningyou7 of colony (Qiu et al, 2006) and Quantum * No2127-17 (Chen etal, 2007) on the N19 linkage group of the swede type rape that will be positioned to have delivered with the chain AFLP mark P06MG04 of BnMs3, and obtained 1 and can distinguish three kinds of genotypic codominance SSR molecule marker sR12384 of BnMs3ms3RfRf/BnMs3Ms3RfRf/Bnms3ms3RfRf (Fig. 3) on N19, the sequence of this SSR mark is shown in sequence table SEQ ID NO:11-12.Also be decided to be upper end with the chain AFLP mark P01MC07 of BnRf in the N7 linkage group, screening obtains 1 can distinguish three kinds of genotypic codominance SSR molecule marker D1 of Bnms3ms3Rfrf/Bnms3ms3RfRf/Bnms3ms3rfrf, the sequence of this SSR mark is shown in sequence table SEQ ID NO:13-14, the upper end that utilizes these 2 double haploid colonies to be positioned at the N7 linkage group with chain AFLP mark P19MC08 of BnMs4 and EC09MG12 equally, and on N7, obtained and BnMs4 and all chain common molecule marker ENA06 of BnRf, CNU063 and sR4047, and find that SM25 and D1 also are common molecule marker (Fig. 8 A, Fig. 8 B).

6.SSR analysis system

PCR reaction system: 50ng DNA, 1 * PCR damping fluid, 2.0mmol/L MgCl ₂, 0.2mmol/L dNTPs, 0.5U Taq enzyme, forward and reverse each 12.5ng of SSR primer, the reaction cumulative volume is 10 μ l.The PCR loop parameter is: 94 ℃ (2min); 94 ℃ (30s), 60 ℃ (0.5 ℃/circulation) (30s), 72 ℃ (45s), 14 circulations; 94 ℃ (30s), 55 ℃ (30s), 72 ℃ (1min), 30 circulations; 4 ℃ of preservations.Amplified production is electrophoretic separation on 6% sex change PAGE gel, uses 0.5 * TBE usually, 80W voltage.After electrophoresis was finished, argentation showed band.

Embodiment 2: utilize the upper sterile codominance molecular selection good temporary maintainer line and the technological line following (Fig. 9) of improveing back sterile line homologous temporary maintainer line seed selection chain with BnMs3/BnMs4/BnRf of the recessive nuclear of swede type rape:

1. the sterile individual plant 7-7365A (Bnms3ms3RfRf) that utilizes recessive epistasis to do mutually in the nuclear male sterility homozygous two-type line is hybridized with temporary maintainer line 7-7365C (Bnms3ms3rfrf), obtains a complete sterility colony, with its called after S ₁(Bnms3ms3Rfrf) generation.

2. utilize the S that obtains ₁Hybridize for the educated individual plant 7-7365B (BnMs3ms3RfRf) in individual plant and the homozygous two-type line, obtain a segregating population, we are with its called after S ₂Generation.

3.S ₂Comprise 4 kinds of genotype (BnMs3ms3Rfrf, BnMs3ms3RfRf, Bnms3ms3Rfrf and Bnms3ms3RfRf) in generation, closely linked dominance of utilization and BnMs3 and BnRf and codominant marker screen the target gene type and (are defined as: candidate's individual plant) for the individuality of BnMs3ms3Rfrf.The individual plant that obtains for screening utilizes the AFLP technology to carry out background and selects selection and the most approaching individual plant of 7-7365A background simultaneously.

4. the candidate's individual plant and the 7-7365A that obtain in the previous step are hybridized, obtain a segregating population, its called after S ₃Generation.At S ₃In generation, same utilization and BnMs3 and the closely linked dominance of BnRf and codominant marker screen the individuality of target gene type for BnMs3ms3Rfrf.The individual plant that obtains for screening simultaneously utilizes the AFLP technology to carry out context analyzer, selects and the most approaching individual plant of 7-7365A background.

5. the candidate's individual plant that obtains in the previous step is carried out selfing respectively, the segregating population called after S that obtains ₄Generation.At S ₄In generation, our utilization and the closely linked dominance of BnMs3, BnRf and codominant marker screen the individuality of target gene type for Bnms3ms3rfrf.

6. in each segregating generation, carry out prospect and background and select.Finally select the good and quality of economical character temporary maintainer line preferably

Reference:

[1]VosP，Hogers?R，Bleeker?M，Reijans?M，van?de?Lee?T，Hornes?M，Freijters?A，Pot?J，Peleman?J，Kuiper?M，Zabeau?M.AFLP：A?new?technique?for?DNA?fingerprinting.Nucleic?Acids?Research，1995，23：4407-4414.

[2]Jorge?L?F，Fabio?E，Alba?A，Geraldo?G，Miriam?C?D，Mirle?F，Juan?E?D，Joe?M?T.Analyses?of?geneticdiversity?in?Cuban?rice?varieties?using?isozyme，RAPD?and?AFLP?markers.Euphytica，1999，109：107-115.

[3]Tang?S?Q，Bin?X?Y，Wang?L，Zhong?Y.Genetic?diversity?and?population?structure?of?yellow?camellia(Camellia?nitidissima)in?China?as?revealed?by?RAPD?and?AFLP?markers.Biochemical?Genetics，2006，9：444-456.Zhang?Z?F，Wang?Y，Zheng?Y?L.AFLP?and?PCR-based?markers?linked?to?Rf3，a?fertility?restorergene?for?S?cytoplasmic?male?sterility?in?maize.Molecular?Genetics?and?Genomics，2006，2：162-169.

[4]Price?A?H，Steele?K?A，Moore?B?J，Barraclough?P?P，Clark?L?J.A?combined?RFLP?and?AFLP?linkage?map?ofupland?rice(Oryza?sativa?L.)used?to?identify?QTLs?for?root-penetration?ability.Theoretical?and?AppliedGenetics，2000，1：49-56.

[5]Guo?P?G，Bai?G?H，Shaner?G?E.AFLP?and?STS?tagging?of?a?major?QTL?for?Fusarium?head?blight?resistancein?wheat.Theoretical?and?Applied?Genetics，2003，106：101l-1017.

[6]Yi?B，Chen?Y?N，Lei?S?L，Tu?J?X，Fu?T?D.Fine?mapping?of?the?recessive?genic?male-sterile?gene(Bnms1)inBrassica?napus?L.Theoretical?and?Applied?Genetics，2006，113：643-650.

[7]Lei?S?L，Yao?X?Q，Yi?B，Chen?W，Ma?C?Z，Tu?J?X，Fu?T?D.Towards?map-based?cloning：fine?mapping?of?arecessive?genic?male-sterile?gene(BnMs2)in?Brassica?napus?L.and?syntenic?region?identification?based?onthe?Arabidopsis?thaliana?genome?sequences.Theoretical?and?Applied?Genetics，2007，115：643-651.

[8]Ke?L?P，Sun?Y?Q，Hong?DF，Liu?P?W，Yang?G?S.Identification?of?AFLP?markers?linked?to?one?recessivegenic?male?sterility?gene?in?oilseed?rape，(Brassica?napus?L).Plant?Breeding，2005，124：367-370.

[9]Hong?D?F，Wan?L?L，Liu?P?W，Yang?G?S，He?Q?B.AFLP?and?SCAR?markers?linked?to?the?suppressorgene(Rf)of?a?dominant?genetic?male?sterility?in?rapeseed(Brassica?napus?L).Euphytica，2006，151：401-409.

[10]Huang?Z，Chen?Y?F，Yi?B，Xiao?L，Ma?C?Z，Tu?J?X，Fu?T?D.Fine?mapping?of?the?recessive?genic?malesterility?gene(Bnms3)in?Brassica?napus?L.Theoretical?and?Applied?Genetics，2007，115：113-118.

[11] yellow town. molecular marker assisted selection is cultivated novel swede type rape recessive karyon male sterile line .[doctorate paper]. Wuhan: Hua Zhong Agriculture University Library, 2008

[12]Xiao?L，YiB，Chen?Y?F，Huang?Z，Chen?W，Ma?C?Z，Tu?J?X，Fu?T?D.Molecular?markers?linked?toBn；rf：Arecessive?epistatic?inhibitor?gene?of?recessive?genic?male?sterility?in?Brassica?napus?L..Euphytica，2008，164：377-384.

[13]Lu?G?Y，Yang?G?S，Fu?T?D.Molecular?mapping?of?a?dominant?genic?male?sterility?gene?Ms?in?rapeseed(Brassica?napusL).Plant?Breeding，2004，123：262-265.

[14]Vos?P，Hogers?R，Bleeker?M，Reijans?M，van?de?Lee?T，Hornes?M，Freijters?A，Pot?J，Peleman?J，Kuiper?M，Zabeau?M.AFLP：A?new?technique?for?DNA?fingerprinting.Nucleic?Acids?Research，1995，23：4407-4414.

[15]Qiu?D，Morgan?C，Shi?J，Long?Y，Liu?J，Li?R，Zhuang?X，Wang?Y，Tan?X，Dietrich?E，Weihmann?T，Everett?C，Vanstraelen?S，Beckett?P，Fraser?F，Trick?M，Barnes?S，Wilmer?J，Schmidt?R，Li?J，Li?D，Meng?J?L，Bancroft?I.A?comparative?linkage?map?of?oilseed?rape?and?its?use?for?QTL?analysis?of?seed?oil?and?erucic?acid?content.Theoretical?and?Applied?Genetics，2006，114：67-80.

[16]Chen?W，Zhang?Y，Liu?X?P，Chen?B?Y，Tu?J?X，Fu?T?D.Detection?of?QTL?for?six?yield-related?traits?in?oilseedrape(Brassica?napus?L)using?DH?and?immortalized?F ₂?populations.Theoretical?and?Applied?Genetics，2007，115：849-858.

Sequence table

＜110〉Hua Zhong Agriculture University

＜120〉molecule marker of line with genic sterile recessive epistatic interaction in brassica napus and application

<130>

<141>2010-04-14

<160>14

<170>PatentIn?version?3.1

 

<210>1

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

 

<400>1

ccaacacact?acatttgttc 20

 

<210>2

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>2

tcccggagat?cagaagccct 20

 

<210>3

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>3

acaccatgaa?acgccacaag 20

 

<210>4

<211>22

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(22)

<223>

<220>

<221>primer_bind

<222>(1)..(22)

<223>

<400>4

gagttctctg?cttctcagcc?gt 22

 

<210>5

<211>22

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(22)

<223>

<220>

<221>primer_bind

<222>(1)..(22)

<223>

<400>5

ccaactgagc?gtcttcattg?at 22

 

<210>6

<211>22

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(22)

<223>

<220>

<221>primer_bind

<222>(1)..(22)

<223>

<400>6

ggtggttagc?cttagatcgt?gg 22

 

<210>7

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>7

catccgagag?aagagtagag 20

 

<210>8

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>8

tacacaggag?gtgatctgac 20

 

<210>9

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>9

ctgcagactg?gccactcctc 20

 

<210>10

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>10

aacacaagag?tttaaggagc 20

 

<210>11

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>11

gtctcgccgt?ggaatgttat 20

 

<210>12

<211>22

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(22)

<223>

<220>

<221>primer_bind

<222>(1)..(22)

<223>

<400>12

ggtttcagct?ttctcaaaat?ca 22

 

<210>13

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>13

ctcgatacgg?ataaggttcg 20

 

<210>14

<211>20

<212>DNA

＜213〉swede type rape (Brassica napus)

<220>

<221>gene

<222>(1)..(20)

<223>

<220>

<221>primer_bind

<222>(1)..(20)

<223>

<400>14

ttttgcttcg?gttgtgtatg 20

Claims (3)

1. examine sterile relevant BnMs3 with recessive epistatic interaction in brassica napus for one kind, BnMs4 and the closely linked molecule marker of BnRf gene, described molecule marker is used to identify the genotype of swede type rape at described three gene locuss, be used to utilize this recessive karyon male sterile to carry out the assisted Selection of molecule marker simultaneously, it is characterized in that it is one of following molecule marker:

(1) be used to distinguish BnMs3ms3RfRf, BnMs3Ms3RfRf and three kinds of genotypic codominance SSR molecule marker SR12384 of Bnms3ms3RfRf with the BnMs3 gene is closely linked, its nucleotide sequence is shown in SEQ ID NO:11-12;

(2) be used to distinguish Bnms3ms3rfrf, Bnms3ms3Rfrf and three kinds of genotypic codominance SSR molecule marker D1 of Bnms3ms3RfRf with the BnRf gene is closely linked, its nucleotide sequence is shown in SEQ ID NO:13-14;

(3) with the closely linked dominant SCAR molecule mark SM129 of BnMs4 gene, SM98 and SM113, its nucleotide sequence is shown in SEQ ID NO:1-6, and with the three kinds of genotype codominance molecule marker SM25 and the H3 that are used to distinguish Bnms3ms3rfrf, Bnms3ms3Rfrf and Bnms3ms3RfRf of BnMs4 gene linkage, its nucleotide sequence is shown in SEQ ID NO:7-10.

2. the described molecule marker of claim 1 is examined application in the sterile marker assisted selection at recessive epistatic interaction in brassica napus.

3. the described molecule marker of claim 1 is at recessive epistatic interaction in brassica napus genic male sterile gene BnMs3, the application in BnMs4 and BnRf Fine Mapping and the map based cloning.

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