CN106755315B - Tobacco glandular hairs secretion characteristic related gene Nt-te Molecular mapping and preparation method and application - Google Patents
Tobacco glandular hairs secretion characteristic related gene Nt-te Molecular mapping and preparation method and application Download PDFInfo
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Abstract
The invention discloses tobacco glandular hairs secretion characteristic related gene Nt-te Molecular mapping and preparation method and applications.The present invention constructs K326 × TI1406F using glandular hairs secreting type flue-cured tobacco K326 and burley tobaccos mutant glandular hairs nonsecreting type TI14062Hybrid Population, F2:3Family and BC1F1Backcross population finds and located a gene relevant to glandular hairs secretion characteristic on No. 14 chromosome of tobacco for the first time, and develops the molecular labeling with the non-secreting gene close linkage of glandular hairs.The molecular marker SNP 930887 cannot be only used for identifying or assist to identify tobacco glandular hairs secretion phenotype to be measured, it can also be used in molecular marker assisted selection breeding (MAS), it is not only to clone the gene and sequence analysis and carry out finely positioning to the gene to lay a good foundation, also the cultivation for many plant glandular hairs secretory products, flavouring essence quality and the excellent new varieties of insect resistace is provided fundamental basis.
Description
Technical field
The invention belongs to agricultural biotechnology engineering fields, and in particular to tobacco glandular hairs secretion characteristic related gene Nt-te
Molecular mapping and preparation method and application.
Background technique
Plant epidermal hair (trichomes) is a kind of specialization organ for being widely present in higher plant surface, although form
It is different, but usually may be logically divided into glandular hairs (glandular trichomes) and nonglandular hair (non-glandular trichomes)
2 major class.Plant glandular hairs be made of 3 base cells, shank cell and apical cell parts (Maffei et al, 2010；
, including secreting type glandular hairs and nonsecreting type glandular hairs (Wagner et al, 1991) McDowell et al, 2011).Secreting type gland
The ability that hair has very strong synthesis, stores or secrete a large amount of secondary metabolites, secretion is as natural insecticide, food
Additive plays an important role (McDowell et al, 2011) in pharmaceuticals industry.For example, Lamiaceae plant peppermint, white flower
The Trichome exudates such as sweet basil, lavender, thyme are rich in a variety of aromatic oil, wherein many fragrance oil components are for medicinal
(Schilmiller et al,2008)；The secreting type glandular hairs of sweet wormwood have the energy of the multiple compounds such as synthesis Sesquiterpene
Power is the important place (Weathers et al, 2011) of important anti-malaria medicaments artemisinin synthesis and secretion；Cotton glandular hairs point
The gossypol and other related compounds secreted have very strong antifungic action, and are natural insecticide (Dayan et
al,2003)；Western three enediol of cypress of Tobacco Glandular Trichome Major Secretory object has important role (Johnson to the fragrance of cured tobacco leaf
Et al., 1985), and there are the bioactivity such as stronger anti-tumor activity, antibacterial and coordinate plant growth, on nicotine
Addiction also has certain inhibiting effect (Han Jinfeng etc., 2013).
Research thinks that glandular hairs difference secretion synthesizing site is different.The study found that golgiosome is the conjunction of polysaccharose substance
At place (Amelunxen et al, 1968；Danilova et al,1987；Schnepf et al,1968).Close lipid
The place that matter synthesizes in secretory cell always exists dispute.Werker etc. (1981) thinks Inula (Inula) plant most
It just by the lipid material that cell head is secreted is generated by tubulose smooth surfaced endoplasmic reticulum.Hammond etc. (1987) studies hemp gland
The ultra microstructure discovery of hair, plastid number sharply increases with secretion process, therefore, it is considered that the conjunction of plastid and lipid secretion object
At related.Plastid be the synthesis of glandular hairs terpenes secretion place (Turner et al, 2000；Gersbach et al,
2002).The secretion activity for opening the magnificent presence for waiting (2008) to demonstrate chloroplaset and tobacco glandular hairs is closely related.Nielsen etc.
(1991) cultivar TI1068 of the year to tobacco glandular hairs with secretion capacity and the mutant material without secretion capacity
TI1406 is studied, and discovery secreting type glandular hairs have complete chloroplaset and cytoplasmic domains, and nonsecreting type Tobacco Glandular Trichome
Chloroplast structure is lacked, disclosing tobacco glandular hairs chloroplaset is indispensable to secretory substance synthesis.
The functional gene that some participation glandular hairs secondary metabolites that reflect out at present synthesize.The synthesis of peppermint monoterpene terpane
Gene GPPS and monooxygenase gene P450 participates in the synthesis of glandular hairs terpene substances.The Amorpha-4,11-diene synthase ADS of sweet wormwood is catalyzed
Amorphadiene generates Arteannuic acid.Bleeker etc. (2011) has found to participate in the cis- different of short terpene synthesis in the glandular hairs of tomato
Amylene based transferase NDPS1 and participation monoterpene α-phellandrene synthesis terpene synzyme PHS1.Isopentene group in hops glandular hairs
Transferase HlPT1L and HlPT2 participate in picric acid synthesis (Xu et al, 2013；Li etal,2015).Schilmiller etc.
(2012) find that an acyltransferase SlAT2 participates in the conjunction of four acyl sucroses in the apical cell of IV type glandular hairs of cultivated tomato
At.Arabidopsis AtMYB103 gene controls the generation (Higginson et al, 2003) of glandular hairs branch.In cotton epidermis glandular hairs
Specific expression gene GaMYB2 and cotton fiber are originated and are developed closely related (Shangguan et al, 2008).In tobacco
The type and quantity (Wang et al, 2002) of CYP71D16 gene controllable tobacco leaf leaf surface secretion metabolin.
Summary of the invention
The first purpose of the invention is to provide a kind of methods that tobacco glandular hairs secretion phenotype to be measured is identified in identification or auxiliary.
The method that identification provided by the invention or auxiliary identify tobacco glandular hairs secretion phenotype to be measured is detection tobacco individual
Genotype is AA genotype or GG genotype, determines that the tobacco individual glandular hairs are secreted according to the genotype of the tobacco individual
Phenotype:
If the genotype of the tobacco individual is GG genotype, the tobacco is or candidate is glandular hairs secreting type；
If the genotype of the tobacco individual is AA genotype, the tobacco is or candidate is glandular hairs nonsecreting type；
The GG genotype is the homozygote that the 331st deoxyribonucleotide of DNA fragmentation first is G；
The AA genotype is the homozygote that the 331st deoxyribonucleotide of DNA fragmentation first is A；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
In the above method,
The genotype of the detection tobacco individual is AA genotype or GG Genotypic methods are following A) or B):
A) direct Sequencing；
B the pcr amplification product containing the 331st deoxyribonucleotide of DNA fragmentation first) is sequenced；
1) or 2) primer used in the pcr amplification product is:
1) single strand dna group shown in sequence 2 in the single strand dna as shown in sequence 1 in sequence table and sequence table
At primer pair A；
2) the primer pair B that single strand dna shown in the single strand dna shown in sequence A and sequence B forms；
The sequence A is sequence 1 to be deleted to or increase or change one or several nucleotide, and have with sequence 1 identical
The nucleotide of function；
The sequence B is sequence 2 to be deleted to or increase or change one or several nucleotide, and have with sequence 2 identical
The nucleotide of function.
A second object of the present invention is to provide the genotype of detection the 331st deoxyribonucleotide of DNA fragmentation first
The new application of substance.
The present invention provides detection the 331st deoxyribonucleotide of DNA fragmentation first genotype substance it is following 1)-
6) in it is any in application:
1) it identifies or assists to identify that tobacco glandular hairs to be measured secrete phenotype；
2) preparation identification or auxiliary identify the product of tobacco glandular hairs secretion phenotype to be measured；
3) it identifies or assists to identify that tobacco to be measured is glandular hairs secreting type or glandular hairs nonsecreting type；
4) preparation is identified or assists to identify the product that tobacco to be measured is glandular hairs secreting type or glandular hairs nonsecreting type；
5) breeding glandular hairs secreting type tobacco or glandular hairs nonsecreting type tobacco；
6) product of breeding glandular hairs secreting type tobacco or glandular hairs nonsecreting type tobacco is prepared；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
Third object of the present invention is to provide the products that tobacco glandular hairs secretion phenotype to be measured is identified in a kind of identification or auxiliary.
The product provided by the invention for identifying or assisting to identify tobacco glandular hairs secretion phenotype to be measured is detection DNA fragmentation first the
The substance of the genotype of 331 deoxyribonucleotides；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
In above-mentioned application or product,
The substance of the genotype of detection the 331st deoxyribonucleotide of DNA fragmentation first be it is following 1) or 2) or 3)
Or 4):
1) single strand dna group shown in sequence 2 in the single strand dna as shown in sequence 1 in sequence table and sequence table
At primer pair A；
2) the primer pair B that single strand dna shown in the single strand dna shown in sequence A and sequence B forms；
The sequence A is sequence 1 to be deleted to or increase or change one or several nucleotide, and have with sequence 1 identical
The nucleotide of function；
The sequence B is sequence 2 to be deleted to or increase or change one or several nucleotide, and have with sequence 2 identical
The nucleotide of function；
3) contain 1) the primer pair A or 2) PCR reagent of the primer pair B；
4) contain 1) the primer pair A or 2) the primer pair B or 3) kit of the PCR reagent.
In above-mentioned application or product,
3) final concentration of each primer in primer pair A or primer pair B in the PCR reagent described in is 10M.
Fourth object of the present invention is to provide a kind of method of breeding glandular hairs secreting type tobacco.
The method of breeding glandular hairs secreting type tobacco provided by the invention includes that the tobacco of GG genotype is selected to carry out breeding；
The GG genotype is the homozygote that the 331st deoxyribonucleotide of DNA fragmentation first is G；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
Fifth object of the present invention is to provide a kind of methods of breeding glandular hairs nonsecreting type tobacco.
The method of breeding glandular hairs nonsecreting type tobacco provided by the invention includes that the tobacco of AA genotype is selected to carry out breeding；
The AA genotype is the homozygote that the 331st deoxyribonucleotide of DNA fragmentation first is A；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
In the above method or above-mentioned application or the said goods,
The glandular hairs secreting type tobacco is the tobacco that glandular hairs have secretion；
The glandular hairs nonsecreting type tobacco is the tobacco that glandular hairs do not have secretion.
Effective effect of the invention is as follows:
1, the present invention located a tobacco glandular hairs secretion characteristic related gene Nt- on No. 14 chromosome of tobacco for the first time
te.The gene is obtained using the non-secreting mutant TI1406 of tobacco.The gene pairs fully understands the machine of plant glandular hairs secretion
It manages and promotes its utility value in the cultivation of plant anti-insect, flavouring and metabolite new varieties that all there is important guidance
Meaning.
2, the present invention uses molecule labelling method, determines a tobacco glandular hairs secretion characteristic related gene positioning, and develop
The SNP marker of 1 close linkage.
3, the molecular labeling of the invention with Nt-te gene close linkage is in the stable filial generation family of phenotype
The new label obtained, can be used for molecular marker assisted selection breeding.
The present invention constructs K326 using glandular hairs secreting type flue-cured tobacco K326 and burley tobaccos mutant glandular hairs nonsecreting type TI1406
×TI1406F2Hybrid Population, F2:3Family and BC1F1Backcross population finds and located on No. 14 chromosome of tobacco for the first time
One gene relevant to glandular hairs secretion characteristic, and develop the molecular labeling with the non-secreting gene close linkage of glandular hairs.Pass through
Genetic analysis shows that the gene by the regulation of Recessive genes, is temporarily named as Nt-te by Tobacco Glandular Trichome non-secretory.It is marked with SNP
Note analysis shows, Nt-te is positioned on No. 14 chromosome, and molecular marker SNP 930887 and the gene close linkage.
The molecular marker SNP 930887 cannot be only used for identifying or assist to identify tobacco glandular hairs secretion phenotype to be measured, it may also be used for molecule
Marker assisted selection breeding (MAS) is not only to clone the gene and sequence analysis and carry out finely positioning to the gene to establish
Basis, also the cultivation for many plant glandular hairs secretory products, flavouring essence quality and the excellent new varieties of insect resistace is provided fundamental basis.
Detailed description of the invention
Fig. 1 is that mutant TI1406 and K326 Tobacco Glandular Trichome secretes situation.Figure 1A is the gland of glandular hairs secreting type flue-cured tobacco K326
Hair secretion situation, the glandular hairs that Figure 1B is burley tobaccos glandular hairs nonsecreting type mutant TI1406 secrete situation.
Fig. 2 is the peak value map marked after SNP930887 sequencing.
Fig. 3 is that tobacco glandular hairs secrete related gene Nt-te positioning map.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
Parent material glandular hairs secreting type flue-cured tobacco K326 and burley tobaccos glandular hairs nonsecreting type mutant in following embodiments
TI1406 is provided by Tobacco Institute, Chinese Academy of Agricultural Science's germplasm resource bank, and the public can grind from Chinese Academy of Agricultural Sciences tobacco
Study carefully institute's germplasm resource bank (network address is http://www.ycsjk.com.cn/) to obtain.
The SNP marker exploitation of embodiment 1, the positioning of tobacco glandular hairs secretion characteristic related gene and its close linkage
One, informative population
Using glandular hairs secreting type flue-cured tobacco K326 and burley tobaccos glandular hairs nonsecreting type mutant TI1406, hybridization obtains F1Generation kind
Son, by hybridizing F1F is obtained for self-pollination2In generation, obtains F by single seed descent2:3Seed, F1It is returned and obtains with mutant TI1406
BC1F1Group.By parent TI1406 and K326, F1、F2、F2:3And BC1F1Group's tobacco seed is seeded in seedlings nursing plate.After 30 days,
Choose 100 plants of F1 plant, 705 plants of F2Single plant, 300 plants of BC1F1Group's single plant.The each family of 153 F2:3 familys sows 21 plants,
50 plant mutant body TI1406,50 plants of K326 seedling temporary plantings are transplanted after about 60 days to crop field into hole tray.Utilize 153 F2:3Family
Carry out phenotypic genetic analysis；Selectivity utilizes recessive homozygous single plant (F2115 plants in group and BC1F1114 plants of generation) it is used as gland
Hair secretor target group, reduces test error, accelerates positioning process.
Two, glandular hairs phenotypic genetic is analyzed
After planting, tobacco leaf it is long to about 15cm when, take TI1406 and parent K326, F1, F2 and F respectively2:3Family group
The fresh blade of single plant, and whether there is secretion using stereoscopic microscope observing glandular hairs, and then judge that glandular hairs secrete phenotype.Tool
Body detecting step reference literature " Nielsen et al, Crop Science, 1982,22 (5): the method in 1051-1053 ".
The result shows that tobacco bred K326 glandular hairs head surface includes a large amount of secretion (Figure 1A), and mutant TI1406
Surface folding, without or only a small amount of secretion (Figure 1B).Each F2:3Family is chosen 21 plants of single plants and is observed.21 plants of cigarettes
Leaf glandular hairs show as secreting type, corresponding F entirely2Single plant is homozygous dominant genotype TeTe；21 plants of Tobacco Glandular Trichomes are shown as presumptuously entirely
Secrete type, corresponding F2Single plant is homozygous recessive genotype tete；21 plants of Tobacco Glandular Trichomes show as secreting type and nonsecreting type, corresponding F2
Single plant is heterozygous dominant genotype Tete.The separation of filial generation phenotype is obvious, and classical genetics analyze F2For the non-of segregating population
Secreting type and secreting type segregation ratio are close to 1:3；By F2:3Family phenotype, which speculates, finds corresponding F2For genotype (homozygous dominant gene:
Heterozygous dominant gene: homozygous recessive gene) segregation ratio meets 1:2:1, Tobacco Glandular Trichome secretory is specified by the tune of Recessive genes
Control, is temporarily named as Nt-te (table 1) for the gene.
Table 1, parent and group's Tobacco Glandular Trichome secretion characteristic genetic analysis
| Parent or filial generation | Secreting type single plant number | Nonsecreting type single plant number | Total strain number | P value |
| Mutant Te | 0 | 30 | 30 | |
| K326 | 30 | 0 | 30 | |
| F2Group | 535 | 170 | 705 | 0.59 |
| F2:3Family | 38(TeTe)81(Tete) | 34(tete) | 153 | 0.74 |
Three, the positioning of glandular hairs secretor and the exploitation of molecular labeling
1, the extraction of gene DNA and the building of anti-sense pond
Take parent TI1406 and K326,115 F2The homozygous single plant of recessiveness and 114 BC in group1F1In generation, is recessive homozygous
Single plant tobacco leaf extracts DNA using CTAB method.Fresh blade is placed in liquid nitrogen and is ground into powder, to centrifuge tube
The middle CTAB Extraction buffer that 65 DEG C of 800 μ L preheatings are added, keeps the temperature 30-50min in 65 DEG C of water-baths after mixing, shakes frequently
In order to avoid agglomerating；Centrifuge tube is taken out, is cooled to room temperature, 800 μ L phenol: chloroform: isoamyl alcohol (25:24:1) are added, extracts 10min, phase
Between gently overturn at regular intervals, mix well it, 12000rpm be centrifuged 10min；Supernatant is taken to be centrifuged to another 1.5mL
Isometric chloroform: isoamyl alcohol (24:1) is added in Guan Zhong, and after mixing well 10min, 12000rpm is centrifuged 10min；Supernatant is taken to arrive
In another 1.5mL centrifuge tube, the isopropanol of 0.8 times of volume pre-cooling is added, slowly mixes, places 30-60min at -20 DEG C, make
DNA is with Precipitation；Choose DNA precipitating to be transferred in 1.5mL centrifuge tube, with 75% ethanol wash 2 times, finally uses dehydrated alcohol
Dehydration, drying are added the ultrapure water that sterilizes in right amount, 37 DEG C of dissolutions, the integrality of 1% agarose gel electrophoresis detection DNA and
Ultraviolet spectrophotometry detects the concentration of DNA sample, and DNA concentration is adjusted to 25ng/ μ L.
Choose 10 homozygous glandular hairs nonsecreting type single plants, 10 homozygous glands respectively according to the methods of Michelmore (1991)
Hair secreting type single plant building is used for the separate tank of segregating population fractional analysis (Bulk Segregating Analysis, BSA).
2, RAD sequencing positioning is carried out based on BSA mixing pit
Each 50 parts of mixing of sample genes of individuals group DNA for extracting two kinds of phenotypes, using interrupting after corresponding enzyme digestion and at random,
The DNA fragmentation of length needed for electrophoresis recycles, and cluster preparation is carried out plus connector, finally upper machine sequencing.Total amount, which is sequenced, is more than
80G, each 10G of two of them parent, each 30G of mixing pit.By the data obtained after filtering BWA software and refer to genome sequence
It is compared, comparison result is merged using Picart-tools, uses SOAPsnp software detection SNP (table 2).
Table 2, SNP detection and annotation result statistics
It is that genotype frequency is carried out to wild type filial generation and saltant type filial generation respectively with reference to genotype with wild-type parent K326
Rate analysis；The reads depth for supporting the reads depth of saltant type total divided by the site in each site obtains all sites
The frequency index of mutant-type genotype.On chromosome to the frequency index of site mutation type genotype in two mixing pits
Distribution mapping finds that the second half section part of No. 14 chromosomes is whole higher, thus it is speculated that the section should be with mutant phenotype close linkage.
For the SNP site of the section, software Premier 5.0 (Premier BiosoftInternational, Palo are utilized
Alto, CA) devise 30 pairs of SNP primers.
3, the exploitation of SNP930887 molecular labeling
30 pairs of SNP primer pair phenotypes using BSA mixing pit RAD sequencing exploitation are 115 F of nonsecreting type2Single plant and 114
A BC1F1Single plant is expanded.PCR reaction system is 20 μ l, contains 2 × DreamTaqTM Green DNA Polymerase
10 μ l, 10M upstream and downstream primers are respectively 1 μ l, 25ng/ μ L DNA, 3 μ L.PCR reacts amplification program, 95 DEG C of initial denaturation 5min, and 95
DEG C denaturation 30s, 53-58 DEG C of annealing 45s, 72 DEG C of extensions 45s-55s, 35 recycle, last 72 DEG C of extensions 10min, and 4 DEG C save.
Sequencing analysis is carried out after the detection of SNP amplified production.
It is as shown in Figure 3 that analysis result is compared after product sequencing.It was found that SNP930887 and target gene Nt-Te close linkage,
And target gene Nt-Te is locked in No. 14 chromosomes about 5.4Mb section.The primer sequence of SNP930887:
SNP930887F:5'-TCTAAACGACAGCGGTAAT-3'(sequence 1) and SNP930887R5'-TGGATACGAACCTGGAAA-
3'(sequence 2).Genetic distance with 4.0 software evaluation of markers of Mapmanger and gene is respectively 2.6cM.
Four, the verifying of molecular marker SNP 930887
Respectively with parent's glandular hairs secreting type flue-cured tobacco K326, burley tobaccos glandular hairs nonsecreting type mutant TI1406, F2:3 family
Phenotypic evaluation is the single plant of glandular hairs secreting type (genotype is homozygous dominant) and phenotypic evaluation is that (genotype is glandular hairs nonsecreting type
Homozygous recessive) single plant genomic DNA be template, using molecular marker SNP 930887 carry out PCR amplification, respectively obtain PCR
Amplified production, and pcr amplification product is sequenced.PCR reaction system is 20 μ l, contains 2 × DreamTaqTM Green DNA
10 μ l, 10M upstream and downstream primer of Polymerase is respectively 1 μ l, 25ng/ μ L DNA, 3 μ L.PCR reacts amplification program, and 95 DEG C pre-
It is denaturalized 5min, 95 DEG C of denaturation 30s, 53-58 DEG C of annealing 45s, 72 DEG C of extension 45s-55s, 35 circulations, last 72 DEG C extend
10min, 4 DEG C of preservations.
Sequencing result shows: phenotypic evaluation is the pcr amplification product of the single plant of glandular hairs secreting type and phenotypic evaluation is glandular hairs
The size of the pcr amplification product of the single plant of nonsecreting type is 485bp, wherein phenotypic evaluation is the single plant of glandular hairs secreting type
331st deoxyribonucleotide of pcr amplification product is that (phenotypic evaluation is that the PCR amplification of the single plant of glandular hairs secreting type produces to G
The nucleotides sequence of object is classified as sequence 3), the genotype that the 331st deoxyribonucleotide of pcr amplification product is G is named
For GG genotype；And phenotypic evaluation is the 331st deoxyribonucleotide of the pcr amplification product of the single plant of glandular hairs nonsecreting type
It is A (phenotypic evaluation is that the nucleotides sequence of the pcr amplification product of the single plant of glandular hairs nonsecreting type is classified as sequence 4), by PCR amplification
331st deoxyribonucleotide of product is that the genotype of A is named as AA genotype.Therefore can sentence according to the following method
Tobacco to be measured of breaking is glandular hairs secreting type or glandular hairs nonsecreting type:
PCR amplification is carried out to tobacco to be measured with molecular marker SNP 930887, obtains pcr amplification product；Detect PCR amplification
Product determines that the genotype of tobacco individual is AA genotype or GG genotype according to pcr amplification product:
If the genotype of tobacco individual is GG genotype, tobacco is or candidate is glandular hairs secreting type；
If the genotype of tobacco individual is AA genotype, tobacco is or candidate is glandular hairs nonsecreting type；
The GG genotype is the homozygote that the 331st deoxyribonucleotide of pcr amplification product is G；
The AA genotype is the homozygote that the 331st deoxyribonucleotide of pcr amplification product is A.
Sequence table
<110>Tobacco Institute, Chinese Academy of Agricultural Science
<120>tobacco glandular hairs secretion characteristic related gene Nt-te Molecular mapping and preparation method and application
<160>4
<210>1
<211>19bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>1
tctaaacgac agcggtaat 19
<210>2
<211>18bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>2
tggatacgaa cctggaaa 18
<210>3
<211>485bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>3
tctaaacgac agcggtaata aaatcatgta ataaaaaacg tatttagtaa aattaagata 60
attaagccaa aaaaataaaa aaacagttaa gcgaccgtgc tagaaccacg aaattcggaa 120
atgcctaaca ccttcttctg aattaacaga attccttact caggatttct ggttcgcaga 180
ataacaaaca gagtcatatt ctcctcgatt cagggattaa aacctgtgac ttgggacgcc 240
ttaaaattcc caggtggcga ctctgaaaca aataaacaag tcccgtttcg actgtccttc 300
aattggagaa aactccctac gcgccccgcg ggtgcggaaa aaggaggtgc gacagagacc 360
agtgattgaa ctcctcccaa gtgcactcat accccaaacc aaatgtagta ccatgtttct 420
tcagcttgat gggtttggca attccctgga ggtttttgcc gagtcccttt ccaggttcgt 480
atcca 485
<210>4
<211>485bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>4
tctaaacgac agcggtaata aaatcatgta ataaaaaacg tatttagtaa aattaagata 60
attaagccaa aaaaataaaa aaacagttaa gcgaccgtgc tagaaccacg aaattcggaa 120
atgcctaaca ccttcttctg aattaacaga attccttact caggatttct ggttcgcaga 180
ataacaaaca gagtcatatt ctcctcgatt cagggattaa aacctgtgac ttgggacgcc 240
ttaaaattcc caggtggcga ctctgaaaca aataaacaag tcccgtttcg actgtccttc 300
aattggagaa aactccctac gcgccccgcg agtgcggaaa aaggaggtgc gacagagacc 360
agtgattgaa ctcctcccaa gtgcactcat accccaaacc aaatgtagta ccatgtttct 420
tcagcttgat gggtttggca attccctgga ggtttttgcc gagtcccttt ccaggttcgt 480
atcca 485
Claims (10)
1. it is AA that a kind of method that identification or auxiliary identify tobacco glandular hairs secretion phenotype to be measured, which is the genotype of detection tobacco individual,
Genotype or GG genotype determine that the tobacco individual glandular hairs secrete phenotype according to the genotype of the tobacco individual:
If the genotype of the tobacco individual is GG genotype, the tobacco is or candidate is glandular hairs secreting type；
If the genotype of the tobacco individual is AA genotype, the tobacco is or candidate is glandular hairs nonsecreting type；
The GG genotype is the homozygote that the 331st deoxyribonucleotide of DNA fragmentation first is G；
The AA genotype is the homozygote that the 331st deoxyribonucleotide of DNA fragmentation first is A；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
2. according to the method described in claim 1, it is characterized by: it is described detection tobacco individual genotype be AA genotype also
Be GG Genotypic methods it is following A) or B):
A) direct Sequencing；
B the pcr amplification product containing the 331st deoxyribonucleotide of DNA fragmentation first) is sequenced；
1) or 2) primer used in the pcr amplification product is:
1) single strand dna shown in sequence 2 forms in the single strand dna shown in sequence 1 in sequence table and sequence table
Primer pair A；
2) the primer pair B that single strand dna shown in the single strand dna shown in sequence A and sequence B forms；
The sequence A is sequence 1 to be deleted to or increases or change one or several nucleotide, and have identical function with sequence 1
Nucleotide；
The sequence B is sequence 2 to be deleted to or increases or change one or several nucleotide, and have identical function with sequence 2
Nucleotide.
3. method according to claim 1 or 2, it is characterised in that:
The glandular hairs secreting type tobacco is the tobacco that glandular hairs have secretion；
The glandular hairs nonsecreting type tobacco is the tobacco that glandular hairs do not have secretion.
4. detect the substance of the genotype of the 331st deoxyribonucleotide of DNA fragmentation first following 1) -6) in it is any in
Using:
1) it identifies or assists to identify that tobacco glandular hairs to be measured secrete phenotype；
2) preparation identification or auxiliary identify the product of tobacco glandular hairs secretion phenotype to be measured；
3) it identifies or assists to identify that tobacco to be measured is glandular hairs secreting type or glandular hairs nonsecreting type；
4) preparation is identified or assists to identify the product that tobacco to be measured is glandular hairs secreting type or glandular hairs nonsecreting type；
5) breeding glandular hairs secreting type tobacco or glandular hairs nonsecreting type tobacco；
6) product of breeding glandular hairs secreting type tobacco or glandular hairs nonsecreting type tobacco is prepared；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
5. application according to claim 4, it is characterised in that: the 331st dezyribonucleoside of the detection DNA fragmentation first
The substance of the genotype of acid be it is following 1) or 2) or 3):
1) single strand dna shown in sequence 2 forms in the single strand dna shown in sequence 1 in sequence table and sequence table
Primer pair A；
2) contain the PCR reagent of 1) the primer pair A；
3) contain 1) the primer pair A or 2) kit of the PCR reagent；
The glandular hairs secreting type tobacco is the tobacco that glandular hairs have secretion；
The glandular hairs nonsecreting type tobacco is the tobacco that glandular hairs do not have secretion.
6. a kind of identification or auxiliary identify the product of tobacco glandular hairs secretion phenotype to be measured, to detect the 331st deoxidation of DNA fragmentation first
The substance of the genotype of ribonucleotide；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
7. product according to claim 6, it is characterised in that: the 331st dezyribonucleoside of the detection DNA fragmentation first
The substance of the genotype of acid be it is following 1) or 2) or 3):
1) single strand dna shown in sequence 2 forms in the single strand dna shown in sequence 1 in sequence table and sequence table
Primer pair A；
2) contain the PCR reagent of 1) the primer pair A；
3) contain 1) the primer pair A or 2) kit of the PCR reagent；
The tobacco glandular hairs secretion phenotype is glandular hairs secreting type tobacco glandular hairs secreting type tobacco or glandular hairs nonsecreting type tobacco；
The glandular hairs secreting type tobacco is the tobacco that glandular hairs have secretion；
The glandular hairs nonsecreting type tobacco is the tobacco that glandular hairs do not have secretion
8. a kind of method of breeding glandular hairs secreting type tobacco, including selecting the tobacco of GG genotype to carry out breeding；
The GG genotype is the homozygote that the 331st deoxyribonucleotide of DNA fragmentation first is G；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
9. a kind of method of breeding glandular hairs nonsecreting type tobacco, including selecting the tobacco of AA genotype to carry out breeding；
The AA genotype is the homozygote that the 331st deoxyribonucleotide of DNA fragmentation first is A；
The nucleotide sequence of the DNA fragmentation first is as shown in sequence 3.
10. according to method described in power claim 8 or 9, it is characterised in that:
The glandular hairs secreting type tobacco is the tobacco that glandular hairs have secretion；
The glandular hairs nonsecreting type tobacco is the tobacco that glandular hairs do not have secretion.
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| CN108823329B (en) * | 2018-06-28 | 2022-03-22 | 中国农业科学院烟草研究所 | Molecular marker for identifying cytoplasmic male sterile line of tobacco sua-CMS |
| CN109943642B (en) * | 2019-02-28 | 2022-05-06 | 中国科学院海洋研究所 | Method for identifying Suhong No.1 molecule of Clariaceae Exopalaemon carinicauda |
| CN110387383B (en) * | 2019-07-30 | 2022-12-06 | 贵州省烟草科学研究院 | Method for regulating expression of NtCBT gene in tobacco tissue and application thereof |
| CN112680464B (en) * | 2020-12-10 | 2022-04-26 | 南京农业大学 | Monomethylarsenic and trivalent antimony oxidase gene arsV, and protein coded by same and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2813141B1 (en) * | 2013-06-14 | 2015-08-05 | Keygene N.V. | Directed strategies for improving phenotypic traits |
| CN104946781A (en) * | 2015-07-16 | 2015-09-30 | 湖北省烟草科学研究院 | Method for early-stage screening of high-nicotine and low-nicotine tobacco converter plants |
| CN105624277A (en) * | 2014-11-28 | 2016-06-01 | 中国农业科学院烟草研究所 | Method for obtaining molecular marker closely linked to plant height growth property of tobacco |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2813141B1 (en) * | 2013-06-14 | 2015-08-05 | Keygene N.V. | Directed strategies for improving phenotypic traits |
| EP2949204A1 (en) * | 2013-06-14 | 2015-12-02 | Keygene N.V. | Directed strategies for improving phenotypic traits |
| CN105624277A (en) * | 2014-11-28 | 2016-06-01 | 中国农业科学院烟草研究所 | Method for obtaining molecular marker closely linked to plant height growth property of tobacco |
| CN104946781A (en) * | 2015-07-16 | 2015-09-30 | 湖北省烟草科学研究院 | Method for early-stage screening of high-nicotine and low-nicotine tobacco converter plants |
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