CN105506148B - Tobacco black shank resistant Ph gene linked molecular marker and application thereof - Google Patents
Tobacco black shank resistant Ph gene linked molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a tobacco black shank resistant foodPhGene-linked molecular marker and application thereof, and tobacco black shank resistancePhThe serial numbers of the gene-linked molecular markers are Scf _30K, TM62 and ST141, the InDel marker is InDel0617, and the nucleotide sequences of the amplification products are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4. Tobacco black shank resistancePhThe gene linked molecular marker is applied to the hybridization 1 generation, the backcross 2 generation and the subsequent backcross and selfing improved line (population) and the black shank resistance improvement. The invention provides a medicine for resisting tobacco black shankPhGene-linked molecular marker and application thereof in improving resistance of existing excellent varieties to black shankPhThe method lays a foundation for fine positioning of genes and gene cloning, can be used for resistance screening of tobacco resistance improvement candidate materials in hybridization, backcross and subsequent generations thereof, has the advantages of convenience, accuracy, high efficiency and co-dominance, and provides a convenient, practical, scientific and efficient way for improving the resistance of tobacco varieties by using molecular marker-assisted selection (MAS).
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a tobacco black shank resistance agentPhA gene-linked molecular marker and application thereof.
Background
Honghuadajinyuan is a good flue-cured tobacco variety bred from Dajinyuan variety variant introduced in the United states and is named after deep red flower color. The Honghuadajinyuan tobacco leaf has unique aroma characteristics and excellent quality and style, is one of the most distinctive high-quality tobacco varieties in China all the time, and is favored by cigarette industry enterprises. But due to the fact that the variety is used for treating the black shank: (Phytophthoranicotianae) The resistance is poor, and the production scale is severely limited. Therefore, the improvement of the resistance breeding of the Honghuadajinyuan black shank has important practical significance.
The resistance improvement of the tobacco black shank naturally relates to the resistance breeding of the tobacco black shank. The tobacco black shank resistance breeding research started in the United states, Tisdale found that big copa and small copa are resistant to black shank in Florida in 1922, and through hybridization and selection, some cigar wrapper tobacco lines with high black shank resistance were bred in 1931, wherein Florida 301 is the best resistant and is one of various types of resistance. And breeding the black shank resistant variety Rg in the same year. In 1943, the first black shank resistant flue-cured tobacco variety Oxford is bred, and then a series of black shank resistant flue-cured tobacco varieties such as DB101, DB102 and the like are bred. In addition, Johnson E S, et al (Jonhson E S, Wolff M F, Wernsman E A,et al. Origin of the black shankresistance gene，Phin tobaco cubvar Coker 371 gold Plant Disease, 2002, 86(10):1080-N.plumbaginifoliaAndN.longiflorathe resistance gene of the phytophthora parasitica is allele, and is found out by a BSA methodSeed Coker 371-Gold for resisting black shankPhA gene-linked RAPD marker. The inventor also developed a method for resisting black shankPhGene-assisted selection of molecular markers TM29 and TM50, located at positions corresponding to the markersPhThe molecular markers TM29 and closely linked to the two sides of the genePhThe genetic distance of the gene is 0.091cM, and the molecular markers TM50 andPhthe genetic distance of the gene was 0.148 cM. But the requirement of precisely and directionally improving the black shank resistance of the high-quality tobacco variety is still far from being met, so that deep development and research are necessary.
Disclosure of Invention
The invention aims to provide a tobacco black shank resistant agentPhA gene-linked molecular marker; the second purpose is to provide the anti-tobacco black shankPhThe application of gene-linked molecular markers; the third purpose is to provide the anti-tobacco black shankPhTobacco varieties obtained by applying the gene-linked molecular markers, and seeds and asexual propagules thereof; the fourth purpose is to provide the tobacco black shank resistancePhAn expression cassette for a gene-linked molecular marker; the fifth purpose is to provide the anti-tobacco black shankPhThe gene linked molecular marker expression box is applied to the hybridization 1 generation, the backcross 2 generation and the subsequent backcross and selfing improved line (population); the sixth purpose is to provide the anti-tobacco black shankPhA transgenic cell line of gene-linked molecular markers; the seventh purpose is to provide the anti-tobacco black shankPhThe gene-linked molecular marker transgenic cell line is applied to the 1-generation hybridization, the 2-generation backcross and subsequent backcross and selfing improved lines (groups); the eighth purpose is to provide the anti-tobacco black shankPhRecombinant bacteria of gene-linked molecular markers; the ninth purpose is to provide the anti-tobacco black shankPhThe recombinant bacteria of the gene linked molecular marker is applied to the hybridization 1 generation, the backcross 2 generation and the subsequent backcross and selfing improved line (colony).
The first purpose of the invention is realized by that the tobacco black shank resistancePhThe numbers of the gene-linked molecular markers are Scf _30K, TM62, ST141 and InDel0617, the nucleotide sequences of the amplification products are respectively shown as SEQ ID No.1 (196 bp), SEQ ID No.2 (490 bp), SEQ ID No.3 (294 bp) and SEQ ID No.4 (298 bp).
The second purpose of the invention is realized by that the tobacco black shank resistancePhThe gene linked molecular marker is applied in the improved line (colony) of hybridization 1 generation, backcross 2 generation and subsequent backcross and selfing.
The tobacco black shank resistancePhThe application of the gene-linked molecular marker is that the gene-linked molecular marker resists the tobacco black shankPhThe application of the gene-linked molecular marker in the improvement of the black shank resistance comprises the following specific steps:
(1) respectively carrying out PCR amplification on the genomic DNA of the improved tobacco variety by adopting the primers of the molecular markers;
(2) carrying out gel electrophoresis detection on the amplification result;
(3) and screening and amplifying the material containing the nucleotide sequence fragment of the amplification product shown as SEQ ID number 1-4 from the detection result.
The third purpose of the invention is realized by that the tobacco black shank resistancePhTobacco variety obtained by applying gene-linked molecular marker, and seed and asexual propagule thereof.
The fourth object of the present invention is achieved by the fact that said tobacco black shank resistancePhAn expression cassette for a gene-linked molecular marker.
The fifth object of the present invention is achieved by the fact that said tobacco black shank resistancePhThe gene linked molecular marker expression cassette is used in the hybridization of 1 generation, backcross 2 generation and subsequent backcross and selfing improved line.
The sixth object of the present invention is achieved by the fact that said tobacco black shank resistancePhA transgenic cell line of a gene-linked molecular marker.
The seventh object of the present invention is achieved by the fact that said tobacco black shank resistancePhThe transgenic cell line of the gene linked molecular marker assists in selecting improved lines of 1 generation of hybridization, 2 generations of backcross and subsequent backcross and selfing (Population).
The eighth object of the present invention is achieved by the fact that said tobacco black shank resistancePhRecombinant bacteria of gene-linked molecular markers.
The ninth object of the present invention is achieved by the fact that said tobacco black shank resistancePhThe recombinant bacteria of the gene linked molecular marker is applied to the hybridization 1 generation, the backcross 2 generation and the subsequent backcross and selfing improved line (colony).
The invention constructs F by taking the Honghuadajinyuan of a flue-cured tobacco variety with excellent susceptibility as the male parent of the recurrent parent and the disease-resistant tobacco breeding material RBST with the introduced segment of the wild tobacco chromosome as the non-recurrent parent1Backcrossing and selfing to resist black shank by the phenotypic identification of 6 generation populationPhAnd (5) analyzing the genetic rule of the gene. At BC1F1The population is a mapping population and is realized by using a BSA method and a chromosome walking technology based on molecular markersPhThe fine positioning of the chromosome segment of the wild tobacco in which the gene is positioned and the acquisition of the closely linked marker are not only for resisting black shankPhLays a foundation for fine positioning of genes and gene cloning, and provides a theoretical basis for improving the resistance of the existing excellent varieties by using molecular Marker Assisted Selection (MAS).
The invention provides a medicine for resisting tobacco black shankPhThe gene linked molecular marker provides the application of the gene linked molecular marker in improving the resistance of the existing excellent variety to black shank resistancePhThe method lays a foundation for fine positioning of genes and gene cloning, can be used for screening resistance of tobacco resistance improvement candidate materials in hybridization, backcross and subsequent generations thereof, has the advantages of convenience, accuracy, high efficiency and co-dominance, and provides a convenient, practical, scientific and efficient way for improving the resistance of tobacco varieties by using molecular marker-assisted selection (MAS).
Drawings
FIG. 1 shows that 4 molecular markers of Scf _30K, TM62, ST141 and InDel0617 of the present invention are applied to recurrent parent materials of Honghuadajinyuan, non-recurrent parent materials of RBST and BC3F1And BC3F2Detecting results of the selected individual plants in generation;
wherein: left side 8 selected BC3F1Single plant and recurrent parent material safflower large golden element, non-recurrent parent material RBST, F1Genotype of a total of 11 samples; the right side is 5 selected BC3F2Single plant and recurrent parent material safflower large golden element, non-recurrent parent material RBST, F1Genotypes of a total of 8 samples; the rightmost lane in the figure is Marker, and the 3 lanes adjacent to the Marker are respectively: (from left to right) recurrent parent material safflower big gold dollar, non-recurrent parent material RBST, F1。
Detailed Description
The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The invention relates to a tobacco black shank resistance agentPhThe serial numbers of the gene-linked molecular markers are Scf _30K, TM62, ST141 and InDel0617, and the nucleotide sequences of the amplification products are respectively shown as SEQ ID No.1 (196 bp), SEQ ID No.2 (490 bp), SEQ ID No.3 (294 bp) and SEQ ID No.4 (298 bp).
The primer sequences of the 4 sites corresponding to the molecular markers are respectively as follows:
the Scf _30K sequence is Scf _30 KF: 5'-GAGAAGCCCATCACCTTTTG-3' the flow of the air in the air conditioner,
Scf_30KR：5’-TTCGAAATAAAGGCTCCCTCT-3’；
TM62 sequence is TM 62F: 5'-AG ACGGGGC TAAATTTGACA-3' the flow of the air in the air conditioner,
T M62R：5’-AGCGGAAGAGTT GAGGA CA A-3’；
ST141 sequence ST 141F: 5'-CCATTCTAGCAAAGCCCATAA-3' the flow of the air in the air conditioner,
ST141R：5’-CAGA GGCAACAATGCATACG-3’；
the sequence of InDel0617 is InDel 0617F: 5'-TGGCTTTGCGGTCCTATTAC-3' the flow of the air in the air conditioner,
InDel0617R：5’- GCTCTGCAGATGCAAAGGTT-3’。
said composition containsPhThe tobacco breeding material of the gene is the black shank resistant bacterium No. 0The physiological race.
The invention relates to a tobacco black shank resistance agentPhThe application of the gene-linked molecular marker is that the gene-linked molecular marker resists the tobacco black shankPhThe gene linked molecular marker is applied in the improved line (colony) of hybridization 1 generation, backcross 2 generation and subsequent backcross and selfing.
The invention relates to a tobacco black shank resistance agentPhThe application of the gene-linked molecular marker is that the gene-linked molecular marker resists the tobacco black shankPhThe application of the gene-linked molecular marker in the improvement of the black shank resistance comprises the following specific steps:
(1) respectively carrying out PCR amplification on the genomic DNA of the improved tobacco variety by adopting the primers of the molecular markers;
(2) carrying out gel electrophoresis detection on the amplification result;
(3) and screening and amplifying the material containing the nucleotide sequence fragment of the amplification product shown as SEQ ID number 1-4 from the detection result.
The recurrent parent female parent of the improved tobacco variety is safflower Honghuadajinyuan, and the non-recurrent parent male parent is RBST.
The PCR reaction system is as follows: 30-50 ng/muL DNA, 1.0 mumol/L forward and reverse primers, 1.5mmol/LdNTPs, 2 muL 10 × PCR Buffer (Mg)2+plus），0.75~1.0 UTaqPolymerase (r)Taq，TaKaRa Ltd.), and double distilled water is added to 20 μ L.
The PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30sec, renaturation at 60 ℃ for 30sec, extension at 72 ℃ for 30sec, and after 30 cycles, extension at 72 ℃ for 5 min.
The gel electrophoresis detection refers to adopting 6% non-denatured polyacrylamide gel and 1 xTBE electrophoresis buffer solution, carrying out separation at 500V constant pressure electrophoresis for 1.5 hours, and finally carrying out silver staining and color development.
The tobacco black shank resistancePhTobacco variety obtained by applying gene-linked molecular marker, and seed and asexual propagule thereof.
Contains the said tobacco black shank resisting agentPhAn expression cassette for a gene-linked molecular marker.
The tobacco black shank resistancePhThe gene linked molecular marker expression cassette is used in the hybridization of 1 generation, backcross 2 generation and subsequent backcross and selfing improved line.
Contains the said tobacco black shank resisting agentPhA transgenic cell line of a gene-linked molecular marker.
The tobacco black shank resistancePhThe gene linked molecular marker transgenic cell line is applied in the 1 st filial generation, the 2 nd backcross and subsequent backcross and selfing improved line (colony).
Contains the said tobacco black shank resisting agentPhRecombinant bacteria of gene-linked molecular markers.
The tobacco black shank resistancePhThe recombinant bacteria of the gene linked molecular marker is applied to the hybridization 1 generation, the backcross 2 generation and the subsequent backcross and selfing improved line (colony).
The following is further illustrated with reference to the examples:
materials and methods
Parent selection: a breeding material RBST (tobacco wild species-containing RBST) with high resistance to tobacco black shank No. 0 physiological race and tobacco mosaic disease is obtained by taking a high-quality flue-cured tobacco variety Honghuadajinyuan as a recurrent parent in a tobacco industry biotechnological key laboratory of Yunnan institute of tobacco agricultural science and technology and cultivating the breeding material by utilizing a distant hybridization technology and combining a tobacco haploid breeding technology originated from a female parentN.plumbaginifoliaThe chromosome segment of (1) has extremely high resistance of tobacco black shank No. 0 physiological race) (Chinese patent application No. 201410819251.2) is a non-recurrent parent, and the target resistance in the segment is introduced by hybridization, backcross andPhthe gene molecular marker positioning and molecular marker assisted selection technology carries out continuous 4-year resistance detection on the black shank No. 0 physiological race of the strain containing the introduced segment of the wild tobacco chromosome, and an introduced line W67-23-115 with high resistance to the tobacco black shank No. 0 physiological race is screened out. The resistance of the black shank No. 0 physiological race of the introgression line is consistent with that of a donor parent, and the other characters are equivalent to those of a receptor parent, namely the introgression line has the genetic background of the receptor parent-safflower large gold. The parent and introduction are preserved in laboratory and guaranteed to be self-containedThe public was issued for validation experiments within twenty years from the filing date.
Molecular primer: consisting of two parts, one of which is 5119 pairs of PT sequence primers published by Binder et al, and detailed results can be found in article 123 curly Table of the book of Binder et al, the name of the same applied Gene 2011 (A high diversity genetic map of tobacaco)NicotianatabacumL.) an object from large scale scientific marker definition; the other part is 13648 pairs of TM sequence primers (the data of the part are not published at present) which are self-developed by the Chinese Tobacco Genome Database (CTGDB). PCR experiments were carried out using Takara LtdTaqPolymerase (r)Taq，TaKaRa Ltd.) and dNTPs (2.5 mM each，TaKaRaLtd.); the electrophoresis gel was diluted to 6% using 30% non-denatured polyacrylamide (taiwan institute of technology, inc.).
Example 1 resistance to blacklegPhScreening of Gene-Linked molecular markers
Step 1, identifying the tobacco black shank phenotype
In 2010, in the field of the test base of tobacco agricultural science research institute in Yunnan province, Honghuadajinyuan is used as female parent and RBST is used as male parent to prepare hybrid F1Planting two rows of Honghuadajinyuan and F in summer in base field in 2011 summer1Using Honghuadajinyuan as male parent, F1Hybridization for female parent to generate BC1F1After the seeds are ripe, all the seeds are mixed and harvested; carrying out F simultaneously1Selfing to obtain F2. Planting two rows of Honghua Dajinyuan and BC in the sunshine greenhouse of the base from winter of the same year to spring of the next year1F1Using Honghuadajinyuan as male parent, BC1F1Hybridization for female parent to generate BC2F1And all the ingredients are mixed and harvested after being matured. Obtaining F through hybridization, selfing and backcrossing1、F2And BC1F1、BC2F1And (4) a group.
Adopting 162-hole floating seedling-raising tray to carry out treatment on Honghua Dajinyuan, RBST and F in base experimental greenhouse in summer of 20121、F2、BC1F1、BC2F1Float onAnd (5) seedling cultivation, wherein 10 trays are used for each group, and the seedling is managed to be mature conventionally. 1080 seedlings with uniform size are screened from each group and transplanted into a small flowerpot, and the resistance of the black shank at the seedling stage is determined by a hypha block micro-wound grafting stem base method (Chinese patent application No. 201510617233.0).
In the four generation population grown, the black shank resistance phenotype was investigated, segregation ratios calculated, statistical data analysis was performed using microsoft excel 2003 software, and results were chi-square tested using SAS 8.0.
The results show that: filial generation F1All show resistance; f2The separation ratio of the anti-influenza is in accordance with 3:1 by a chi-square test; in the backcross population, the ratio of the resistance to the disease is 1: 1. From this, it was confirmed that the tobacco black shank resistance was controlled by 1 pair of nuclear genes and was a dominant gene.
Step 2, DNA extraction and molecular marker analysis
Taking young leaves of tobacco plants, and extracting genome DNA of each individual plant of parents and groups by using an improved CTAB (cetyl trimethyl ammonium bromide) method.
The PCR reaction system is as follows: 20 muL of total reaction system, 30-50 ng/muL of DNA, 1.0 mumol/L of forward primer and reverse primer, 1.5mmol/L of dNTPs and 2 muL of 10 XPCR Buffer (Mg)2+plus），0.75~1.0 UTaqPolymerase (r)Taq，TaKaRa Ltd.), and double distilled water is added to 20 μ L. The molecular primers used were 13648 pairs of TM sequence primers (the data of which are not published at present) developed by the Chinese Tobacco Genome Database (CTGDB) and 5119 pairs of primers developed by Binder et al. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30sec, renaturation at 60 ℃ for 30sec, extension at 72 ℃ for 30sec, 30 cycles, re-extension at 72 ℃ for 5min, and re-extension at 16 ℃ for ever. Separating the amplified product by 6.0% non-denatured polyacrylamide gel, electrophoresis buffer solution of 1.0 xTBE, performing 500V constant-pressure electrophoresis separation for 1.5h, performing silver staining and developing after electrophoresis, and counting banding patterns.
Step 3, molecular marker screening, data statistics and linkage map construction
Mainly comprises 4 steps: (1) screening showed polymorphism between parents and in the first filial generation (F)1) A co-dominant molecular marker of (1); (2) at BC1F1Respectively selecting 15 parts of DNA of anti-and susceptible individual plants in the population, constructing anti-and susceptible 2 near-isogenic pools according to a BSA method, and further screening target genes ((Ph) Closely linked markers; (3) analysis of BC Using the obtained polymorphic primers1F1The genotype of each individual plant of the population; (4) linkage maps were generated using the Joinmap 4.0 software.
Statistical methods for molecular labeling: the band pattern corresponding to the female parent (Honghuadajinyuan) is designated as a, the band pattern corresponding to the male parent (RBST) is designated as b, the heterozygous band pattern is designated as h, and the unamplified or unclear band pattern is designated as u.
The results show that: the molecular marker primer pair P1 (Honghuadajinyuan) and P2 (RBST) were screened with 18767 (13648 TM +5119 PT), with 1501 pairs of primers showing polymorphism between the two parents, with 8% polymorphism.
Combining BSA method to construct pool, further screening to obtain 32 target genes: (Ph) Linked molecular markers.
BC combining safflower Honghua Dajinyuan RBST by utilizing screened polymorphic markers1F1The population was genotyped and a linkage map was constructed using Joinmap 4.0 in conjunction with resistance survey data. The results show that: a total of 84 molecular markers and target genes: (Ph) Located on the same linkage group (LOD = 10), and finally obtainedPh4 molecular markers Scf _30K, TM62, ST141 and InDel0617 with closely linked genes, the genetic distance is 0.089 cM. The 4 marks are respectively located atPhOn both sides of the gene, the molecular markers InDel0617 andPhthe genetic distance of the gene was 0.018cM (i.e., on the same side as the previously reported marker TM29, but from a distancePhCloser; chinese patent application No. 201410250226.7), the molecular markers Scf _30K, TM62 and ST141 are in coseparation state (i.e. the genetic distance between the 3 markers is 0) and are in co-segregation withPhThe genetic distance of the gene was 0.071cM (i.e.on the same side as the previously reported marker TM 50; Chinese patent application No. 201410250226.7). The linkage map obtained in this experiment was further combined with a published tobacco genetic map (Bindler)et al2011) found a total of 23 markers (PT lines) shared by this linkage group and chromosome 20A sequence of columns) according to whichPhThe gene is located on chromosome 20.
Step 4, recovery, purification and sequencing of PCR amplification difference bands marked by Scf _30K, TM62, ST141 and InDel0617
(1) Recovery of the fragment of interest
The boiling method is adopted. The specific operation is as follows: firstly, respectively digging down differential bands of PCR amplification of markers Scf _30K, TM62, ST141 and InDel0617 from glue, putting the bands into a 1.5mL Eppendorf tube, and adding 100 mu L of ultrapure water into the tube, wherein the water addition amount is determined according to the color depth of the adhesive tape; soaking at normal temperature for 24h, boiling in 95 deg.C water bath (or PCR instrument) for 30min, and centrifuging at 5000r/m for 3 min. The product is PCR amplified with supernatant 3 μ L as template, and the rest product is stored at-20 deg.c for further use.
(2) Purification of the fragment of interest
Direct purification method using PCR product. 2 times of absolute ethyl alcohol is added into the PCR product, the mixture is placed at the temperature of minus 20 ℃ overnight, and the mixture is centrifuged at 1,2000r/m for 5min to obtain a purified product.
(3) Ligation of the fragment of interest to the vector
The reaction system is 10 μ L: PMD18-T vector 1.0. mu.L; ligation buffer I5.0 μ L; the target fragment was 4.0. mu.L.
Sample is added on a clean bench, the reactants are mixed evenly, the mixture is centrifuged for a short time, and the mixture is connected for about 1h at the temperature of 16 ℃, so that the connection efficiency is not influenced after the mixture is kept overnight.
(4) Conversion of ligation products
1) Taking out competent cells, Solution A and Solution B, and melting on ice;
2) competence (50. mu.L) + 5. mu.L Solution A + 4. mu.L Solution B + 46. mu.L of pre-chilled deionized water;
3) subpackaging the suspension into 1.5mL centrifuge tubes by using a cooled sterile gun head, adding 105 mu L of the suspension into each tube, adding 5 mu L of target DNA, and slightly rotating and uniformly mixing;
4) performing water bath heat shock for 90s at 42 ℃, and paying attention not to shake the centrifuge tube;
5) quickly transferring a 1.5mL centrifuge tube into an ice bath to cool the cells for 3-5 min;
6) 500. mu.L of LB liquid medium was added. Pre-culturing for 1h on a shaking table of 150r/m at 37 ℃;
7) coating the bacterial liquid on an LB solid culture medium containing 100 mu g/mL Amp, 25 mu g/mL IPTG and 40 mu g/mL X-GAL, evenly spreading the bacterial liquid by using a sterile elbow glass rod, and placing the liquid at room temperature until the liquid is absorbed;
8) inverting the plate, and culturing at 37 ℃ for 12-16 h.
(5) Blue-white screening of recombinant plasmids
After incubation at 37 ℃ a few blue colonies and more white colonies appear on the X-Gal/IPTG-coated LB plates, the white colonies being recombinant clones. White single colonies were picked and smeared in a checkered LB liquid medium, and cultured overnight at 37 ℃ at 150 rpm.
(6) Detection of colony PCR
1 mul of bacterial liquid is taken as a template for PCR amplification. Taking 4 microliter of PCR product, detecting by 1.5% agarose gel electrophoresis, comparing with DNA Marker standard molecular weight, detecting the size of the inserted fragment, and obtaining the clone with the same size as the inserted target fragment as the positive clone.
(7) Sequencing and analysis of Positive clones
Each marker was prepared by storing 3 positive clones in glycerol (330. mu.L glycerol plus 1000. mu.L bacterial suspension) in duplicate, one copy was stored at-20 ℃ and the other copy was sent for sequencing. The sequencing result is shown in SEQ ID No. 1-4.
Example 2PhApplication of gene close linkage marker
Variety breeding: in the experimental base of tobacco agricultural science research institute in Yunnan province from winter to spring of the next year in 2009, a flue-cured tobacco variety Honghuadajinyuan popularized in Yunnan tobacco district is taken as a female parent, RBST is taken as a male parent to prepare hybrid F1. 2010 respectively planting two rows of Honghuadajinyuan and F in the base in summer1Using Honghuadajinyuan as male parent, F1Hybridization for female parent to generate BC1F1And all the ingredients are mixed and harvested after being matured. Planting two rows of Honghua Dajinyuan and BC in the sunshine greenhouse of the base from winter of the same year to spring of the next year1F1Using Honghuadajinyuan as male parent, BC1F1Preparing the male and female parentsGeneration of BC by crossing2F1And all the ingredients are mixed and harvested after being matured. The field management is carried out according to a conventional method.
Molecular marker assisted selection: 2011 summer three-row BC planted in base2F115 lines, 1-2 fresh leaves per line were harvested into an ultra-low temperature freezer at-80 ℃ and then ground using liquid nitrogen and applied to the CTAB method (Paterson AH, Brubaker CL, Wendel JF. A Rapid method for extraction of cottons: (Paterson A, Bio-Rad laboratories, Inc.)Gossypiumspp.） genomic DNA suitable for RFLP or PCR analysis [J]Plant MolBiol Rep,1993, 11, 2: 122) 127) and the resistance to the black shank No. 0 physiological race on the chromosome-introduced fragment of wild tobacco in RBSTPhPerforming PCR amplification on a molecular marker (table 1) with genes closely linked, wherein the amplification system is 20uL, the DNA template is 1.5uL (30-50 ng/uL), the pre-denaturation is performed at 95 ℃ for 5min, the denaturation is performed at 95 ℃ for 30sec, the renaturation is performed at 60 ℃ for 30sec, the extension is performed at 72 ℃ for 30sec, after 30 cycles, the re-extension is performed at 72 ℃ for 5min, and the amplification product is subjected to non-denaturing polyacrylamide gel electrophoresis: the gel concentration was 6%, the electrophoresis buffer was 1 XTBE, and electrophoresis was carried out at 500V for 1.5 hours at constant pressure. 8 individuals containing 4 molecular marker target bands are selected to be continuously backcrossed with the safflower gold to obtain BC3F1And harvesting seeds according to single plants.
2011 each BC in the sunshine greenhouse of the base from winter to spring of the next year3F1Three rows of 15 plants were planted for a total of 360 plants, each leaf was harvested and DNA was extracted, and BC was determined using the molecular markers provided in Table 13F1Genotype of the individual plant, selection of the individual plant with 4 markers all present for selfing to obtain 5 BC3F2。
Each BC in base in summer 20123F2Planting three rows of 15 plants in each row, totaling 15 rows of 225 plants, collecting fresh leaves of each plant, extracting DNA, and determining BC by using the molecular markers provided in Table 13F2Genotype of single plant, selecting 4 markers which are simultaneously existed and are homozygous, selfing to obtain 5 BC3F3。
And (3) field resistance identification: in summer of 2013, each BC selected3F3Respectively in Yuxi Yunnan province, Dali Yunnan province,Two lines of 30 plants are planted in four tobacco black shank No. 0 physiological race disease gardens at four points of Yunnan Emei mountain and Yunnan Shilin, and the two lines are repeated twice to detect the resistance of the black shank No. 0 physiological race disease. BC of the improved Honghuadajinyuan3F3And selecting a family of a physiological race with high tobacco black shank resistance No. 0 (all individuals in disease gardens with four points have no susceptible disease), namely the bred strain is improved red. The resistance of the black shank No. 0 physiological race of the strain is consistent with that of a non-recurrent parent RBST, and the quality, economic character and field agronomic character of the strain are equivalent to those of a recurrent parent safflower Hongjinyuan.
TABLE 1 resistance of wild tobacco introgression in RBST to black shank # 0 physiological racePhMolecular marker with closely linked genes
SEQUENCE LISTING
<110> research institute of tobacco agricultural science in Yunnan province
<120> molecular marker closely linked with tobacco black shank resistant Ph gene and application thereof
<130>2015
<160>12
<170>PatentIn version 3.3
<210>1
<211>196
<212>DNA
<213>Scf_30K
<400>1
gagaagccca tcaccttttg attctattaa gcttataaac ctgactgttt caccgcgctt 60
aagctatata tatatatata tatatatata tatatctgta tatgacatca atttacataa 120
ccccttctca actgaaaaat catgtaaaca tataacaaca actgtaaaaa agagaagagg 180
gagcctttat ttcgaa 196
<210>2
<211>490
<212>DNA
<213>TM62
<400>2
agacggggct aaatttgaca aacaaaaact caactcccta agcttataga tgatttaagg 60
agtaccaaaa agccaagaga accttaattt ccatatattg taaaaatgga aagcttctta 120
tcagacaaag agcgcatgca acctcatccc agcaacccag aaaaatacta gatgatgttt 180
gacacacaca cacacacaca cacacacaca cacacacaca cacacagtta agattaaaca 240
aaagccttgt tggtatttca cctgctcctg caacaaagga cttagaaaaa aagaggcaaa 300
aatcaggata actcatagcc caaaagatgt tcaatcattg aaactgcaat ataatgaagc 360
catgaagcaa catttaccat gtcatttaag tggaatgcta gacgcaatga gtcaaggtac 420
tctagtgagg attatagcag aaaaggactc aaaaaactcg gcaatattac ttgtcctcaa 480
ctcttccgct 490
<210>3
<211>294
<212>DNA
<213>ST141
<400>3
ccattctagc aaagcccata acttctctct taatttactc tttctctctc tagtagaata 60
atcagtatct tcagctcact tcaaatagct cgttgctgtt tcatcaggta aaaactctta 120
attccgttaa tcttcaatta aaatacttca aattcgttga atttctgaat atatgaatgt180
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgg cattggcatt atggtaccgt 240
caagtttaac agtcagtatt aacatgctcg attacgtatg cattgttgcc tctg 294
<210>4
<211>298
<212>DNA
<213>Tab052
<400>4
atcccccatt ctagcaaagc ccataacttc tctcttaatt tactctttct ctctctagta 60
gaataatcag tatcttcagc tcacttcaaa tagctcgttg ctgtttcatc aggtaaaaac 120
tcttaattcc gttaatcttc aattaaaata cttcaaattc gttgaatttc tgaatatata 180
tatatatata tatatatata tatatatata tatatatata tatatatata tatatatata 240
tatatagcat tggcattatg tattaacatg ctcgattagc aatgcattgt tgcctgac 298
<210>5
<211>20
<212>DNA
<213>Scf_30KF
<400>5
gagaagccca tcaccttttg 20
<210>6
<211>21
<212>DNA
<213>Scf_30KR
<400>6
ttcgaaataa aggctccctc t 21
<210>7
<211>20
<212>DNA
<213>TM62F
<400>7
agacggggct aaatttgaca 20
<210>8
<211>20
<212>DNA
<213>T M62R
<400>8
agcggaagag ttgaggacaa 20
<210>9
<211>21
<212>DNA
<213>ST141F
<400>9
ccattctagc aaagcccata a 21
<210>10
<211>20
<212>DNA
<213>ST141R
<400>10
cagaggcaac aatgcatacg 20
<210>11
<211>20
<212>DNA
<213>Tab052F
<400>11
atcccccatt ctagcaaagc 20
<210>12
<211>20
<212>DNA
<213>Tab052R
<400>12
gtcaggcaac aatgcattgc 20
Claims (6)
1. Tobacco black shank resistancePhGene-linked molecular markers characterized by resistance to tobacco black shankPhThe serial numbers of the gene-linked molecular markers are Scf _30K, TM62, ST141 and InDel0617, and the nucleotide sequences of the amplification products are respectively shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4; the primer sequences of the 4 sites corresponding to the molecular markers are respectively as follows:
the Scf _30K sequence is Scf _30 KF: 5'-GAGAAGCCCATCACCTTTTG-3' the flow of the air in the air conditioner,
Scf_30KR：5’-TTCGAAATAAAGGCTCCCTCT-3’；
TM62 sequence is TM 62F: 5'-AG ACGGGGC TAAATTTGACA-3' the flow of the air in the air conditioner,
TM62R：5’-AGCGGAAGAGTT GAGGA CA A-3’；
ST141 sequence ST 141F: 5'-CCATTCTAGCAAAGCCCATAA-3' the flow of the air in the air conditioner,
ST141R：5’-CAGA GGCAACAATGCATACG-3’；
the sequence of InDel0617 is InDel 0617F: 5'-TGGCTTTGCGGTCCTATTAC-3' the flow of the air in the air conditioner,
InDel0617R：5’-GCTCTGCAGATGCAAAGGTT-3’。
2. the molecular marker of claim 1, characterized by comprisingPhThe tobacco breeding material of the gene is a black shank resistant bacterium No. 0 physiological race.
3. The use of the molecular marker of claim 1 or 2 for the improvement of blackleg resistance, comprising the following specific steps:
(1) the male parent of the recurrent parent of the improved tobacco variety is safflower large golden dollar, the male parent of the non-recurrent parent is RBST, and the primers of the molecular markers are adopted to carry out PCR amplification on the genomic DNA of the improved tobacco variety respectively;
(2) carrying out gel electrophoresis detection on the amplification result;
(3) and screening and amplifying the material containing the fragment of the nucleotide sequence of the amplification product shown as SEQ ID number 1-4 from the detection result.
4. Use according to claim 3, characterized in that the PCR reaction system is: 30-50 ng/muL DNA, 1.0 mumol/L forward and reverse primers respectively, 1.5mmol/L dNTPs, 2 muL 10 × PCR Buffer, and the Buffer is Mg2+Buffer of plus, 0.75-1.0UTaqThe polymerase is a polymerase, and the polymerase is a polymerase,Taqthe polymerase is r of TaKaRa LtdTaqAnd adding double-distilled water to 20 mu L.
5. The use according to claim 3, characterized in that the PCR amplification procedure is: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30sec, renaturation at 60 ℃ for 30sec, extension at 72 ℃ for 30sec, and after 30 cycles, extension at 72 ℃ for 5 min.
6. The use of claim 3, wherein the gel electrophoresis detection is performed by using 6% non-denaturing polyacrylamide gel, 1 XTBE electrophoresis buffer, separation at 500V constant pressure electrophoresis for 1.5 hours, and finally silver staining for color development.
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