CN109355419B - Group of molecular markers for breaking N downstream (3' end) linkage drag of tobacco TMV resistance gene and application thereof - Google Patents
Group of molecular markers for breaking N downstream (3' end) linkage drag of tobacco TMV resistance gene and application thereof Download PDFInfo
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Abstract
The invention discloses a group of molecular markers for breaking N downstream (3' end) linkage drag of tobacco TMV resistance genes and application thereof. The molecular markers for breaking the linkage drag of the downstream (3' end) of the tobacco TMV resistance gene are numbered as TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn383, and the nucleotide sequences of the PCR amplification products are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, SEQ ID No.9 and SEQ ID No. 10. The molecular markers have the characteristics of simplicity, high efficiency and high flux, and can be used for accurately breaking the linkage drag at different positions of the downstream (3' end) of the N gene of the tobacco and cultivating a new tobacco TMV-resistant variety which contains the N gene and can completely or partially break the influence of the adverse linkage drag.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a group of molecular markers for breaking N downstream (3' end) linkage drag of a tobacco TMV resistance gene and application thereof.
Background
Tobacco Mosaic disease caused by Tobacco Mosaic Virus (TMV) is one of important Tobacco diseases in China and even in global Tobacco producing areas. In order to reduce and avoid huge economic loss to the tobacco industry caused by tobacco mosaic disease, measures such as medicament prevention and artificial destruction of field disease residues are mainly adopted in production for prevention, and although a certain effect is achieved in controlling the occurrence prevalence of TMV, the situation that TMV is exploded in local fields still happens occasionally. Therefore, breeding disease-resistant varieties and improving the disease resistance of tobacco are fundamental ways for preventing and treating TMV.
Currently, The TMV antigens widely used in production are mainly derived from wild tobacco-leaf tobacco (Nicotiana luteosa), The resistance of which is controlled by a dominant single gene N (Gerstel D U, Inheritance in Nicotiana tabacum. XIX. Identification of The tabacum chromosom replantation by one of N. glutinosa in molar-resistant sugar save. tomasoco, Genetics, 1945, 30: 448-454), and The N gene has been successfully cloned in 1994 (Whitham S, Dinne-mar S P, Choi D, Hehl R, Corr C and Baker 1101, The product of The tobacco genetic mineral resistance N: Similarity toxin Receptor and 1, Reyna Cell 78). Subsequently, The work of improving TMV resistance in common tobacco (cultivated tobacco) using The N gene has been greatly advanced (T Ternovsky M F, Methods of cutting tobaco variations resistance to tobaco mosaic and powdery minute, The A.I. Mikoyan san Soviet Sci.Res. Inst. Tob. & Indian Tob. Ind. Krasnodar Pub, 1945, 143: 126-.
In tobacco varieties with TMV resistance containing N genes obtained by traditional crossbreeding methods, the N genes are carried with encumbrance fragments (derived from wild tobacco-heart tobacco chromosome fragments) which are closely linked on the downstream (3 'end) of the N genes (5' end) in addition to the target gene N. Due to the existence of linkage drag, the tobacco has the adverse effects of lower yield, slower upper leaf yellowing, difficult baking of later-stage mature leaves and the like while obtaining the TMV resistance, and the large-area popularization and application of the tobacco containing the TMV resistance gene N in production is seriously hindered. It has been shown that The N gene itself, in addition to providing TMV resistance, has no adverse effect on yield and production value, whereas The adverse trait or effect caused by linkage drag is derived from other genes in a tightly linked segment downstream (3' end) of The N gene (Lewis RS., Linger LR., Wolff MF., Wernsman EA., The novel infection of N-mediated TMV resistance on yield in tobacaco: linkage drag expression of The gene expression application, 2007, 115: 169-.
Based on the sequence of TMV resistance gene N of tobacco, researchers at home and abroad successfully developed a plurality of Molecular markers for detecting TMV resistance containing N gene (Lewis R S, Milla S R, and Levin J S, Molecular and genetic characterization of Nicotiana glutamosa L. chromosom segments in tomato viral resistance nutrient tobacco access, Crop Sci, 2005, 45(6): 2355-2362; Qinghua, Jun, Lishui, Marzhuwen, Qiu Tao, Zhang Shachen, N gene fragment sequence research of anti-TMV germplasm resources, Guangdong agricultural science, 2011, 1(29): 96-99; Chenshuang, Prain, Yulan, tobacco anti-TMV N gene and its application in Molecular marker assisted breeding, university, 34(4) 355-361; zhangyu, Rouginggang, Yiying, Huwavelet, Dyeberg, Zhang Bo, tobacco N gene and its application in flue-cured tobacco genetic breeding, Chinese agronomy bulletin, 2013, 29(19): 89-92), and simultaneously establishes a corresponding detection system (Liu Yi, Guo Mega Quiqing, Wanxiuqing, Yanpeqiang, Chong, Liudan, the establishment of the N gene marker gene PCR detection method and its application in genetic breeding, molecular plant breeding, 2010, 8(1): 167-; CN 102140546B; CN 103866038B). However, the above markers and methods are only used to detect TMV resistant tobacco varieties having the N gene, and do not have the function of breaking the cumbersome segments of the N gene that are closely linked. Although there are related documents (Lewis RS, Milla SR, Levin JS, Molecular and genetic characterization of Nicotiana glutamosa L. chromosome segments in nanobucic virus-resistant bacteria of crack Sci., 2005, 45:2355 2362) and patents (CN 105200052B; CN 105200149B) that use Molecular markers to measure and estimate the length of linked fragments carried by the introduction of N gene, these studies disclose Molecular markers based on tomato genome data rather than on sequence information of cumber fragments closely linked to N gene, and therefore, these Molecular markers can only be used to roughly estimate the length of cumber fragments linked downstream (3' end) of N gene in tobacco without precise detection, judgment and judgment of cumber fragments linked to N gene in tobacco and specific position information, and thus, the breeding of such markers is limited, and the linked cumber fragments and specific position information of N gene in tobacco is also limited Application in tobacco anti-TMV varieties.
Disclosure of Invention
The first purpose of the invention is to provide a molecular marker for breaking the N downstream (3' end) linkage drag of tobacco TMV resistance gene; the second purpose is to provide the application of the molecular marker for breaking the N downstream (3' end) linkage drag of the tobacco TMV resistance gene.
The first purpose of the invention is realized by that the molecular markers for breaking the N downstream (3' end) linkage drag of the tobacco TMV resistance gene are numbered as TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn383, and the nucleotide sequences of the PCR amplification products are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, SEQ ID No.9 and SEQ ID No. 10.
The second purpose of the invention is realized by using the group of molecular markers to accurately detect and judge the breaking degree of the N gene downstream (3' end) linkage drag and the specific position information in the tobacco to be detected.
Another objective of the invention is to provide a group of primer pairs for PCR amplification to break the N downstream (3' end) linkage drag of tobacco TMV resistance gene.
Still another object of the present invention is to provide a method for accurately detecting and determining whether or not a linkage drag downstream (3' end) of the N gene in tobacco to be detected is broken and positional information of the broken linkage drag.
The invention further aims to provide application of the molecular marker, the primer pair and the application method in breaking adverse linkage drag influence of N gene downstream (3' end) in breeding of tobacco anti-TMV varieties.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention discloses a group of markers for breaking the linkage drag of the N upstream (5' end) of a tobacco TMV resistance gene, and the molecular markers are SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 and SEQ ID No. 10.
The invention also discloses a group of primer pairs for PCR amplification for breaking the downstream (3' end) linkage drag of the tobacco TMV resistance gene N, wherein the primer pairs are TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn383, and the group of primer pairs respectively comprise:
the sequence of TMn324 is TMn 324F: 5'-CGACTTCATGAAAGCACCAG-3' the flow of the air in the air conditioner,
TMn324R：5’- CTACTGGACTCCACCCACGA -3’；
the TMn330 sequence is TMn 330F: 5'-TGGTTAAAACTCCCATCCCTA-3' the flow of the air in the air conditioner,
TMn330R：5’- TGGAAAATGAATTTTTCGAATG -3’；
the sequence of TMn332 is TMn 332F: 5'-TTTGTGCCAAAAGAAAAGGAA-3' the flow of the air in the air conditioner,
TMn332R：5’- TGGAGAGATCGTGAAGGTCA -3’；
the sequence of TMn349 is TMn 349F: 5'-TGCACGTCTGATACTTTTTGA-3' the flow of the air in the air conditioner,
TMn349R：5’- CAACTTTGCACCACCATCAC -3’；
TMn359 sequence TMn 359F: 5'-CAACACGCACCATCACAAGT-3' the flow of the air in the air conditioner,
TMn359R：5’- CTATGCTGCCCCTTCTTCAC -3’；
TMn360 sequence is TMn 360F: 5'-TGGTTAAAGCATGCGACTTG-3' the flow of the air in the air conditioner,
TMn360R：5’- AGGAGTTTTTCGGCGATTG -3’；
the TMn383 sequence is TMn 383F: 5'-TTTACGCCCTCACAAAAACC-3' the flow of the air in the air conditioner,
TMn383R：5’- GATTTCACCGTCGGGACTAA -3’。
the invention also discloses a method for accurately detecting and judging whether the linkage drag of the downstream (3' end) of the N gene in the tobacco to be detected is broken and the position information of the broken linkage drag. Amplifying the genomic DNA of the tobacco to be detected respectively by 7 pairs of primers including TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn383, detecting PCR amplification products, and judging whether the linkage drag of the downstream (3' end) of the N gene in the tobacco to be detected is broken and the accurate position information of the linkage drag broken according to the existence of product sequences or different combination modes of the product sequences, wherein the specific information is shown in Table 1:
nucleotide sequence information of PCR amplification product of 7 pairs of primers disclosed in Table 1 and position information thereof in the downstream (3' end) of tobacco N gene for breaking linkage drag
The primer sequence (Seq ID No. 11-Seq ID No. 24) and the molecular marker sequence (Seq ID No. 1-Seq ID No. 10) disclosed by the invention are obtained by the following technical scheme. Firstly, performing 15 times of re-sequencing on wild tobacco-heart tobacco (Nicotiana luteinosa) containing dominant single gene N and anti-TMV flue-cured tobacco variety Coker176 containing N genes and linkage burdensome fragments thereof cultivated through artificial hybridization by using a genome re-sequencing technology (mainly using a second-generation sequencing technology and simultaneously using a third-generation sequencing technology as an auxiliary technology), and filtering, splicing and assembling obtained re-sequencing data by using a bioinformatics method to finally form high-quality Scaffold data; secondly, comparing The obtained Scaffold data of The heart-leaf tobacco and The flue-cured tobacco Coker176 with The published full-genome data information of The flue-cured tobacco K326 (Sierro N., Battiey JN., Ouadi S., Bakaher N., Bovet L., Willig A., Goepfert S., Peitsch M C and Ivanov NV. The tobacaco genome sequence and its complex with The depth of The tomato and lattice 2014, Nat. Commun, 5, doi:10.1038/ncomms 4833) respectively to obtain The complete sequence information of The N-gene lower (3' end) linked neoplastic fragments; and finally, developing a group of molecular markers with accurate position information based on the obtained sequence information of the N gene downstream (3' end) linked redundant fragments and carrying out experimental verification.
The invention also discloses application of the group of molecular markers for breaking the downstream (3' end) linkage drag of the tobacco TMV resistance gene N in cultivating tobacco anti-TMV varieties with adverse linkage drag influence broken. The molecular marker of the invention can be used for cultivating tobacco anti-TMV varieties which break the adverse linkage drag influence of the N gene, and the technical personnel in the field can understand that whether the linkage drag of the downstream (3' end) of the N gene in the tobacco to be detected is broken and the specific position information of linkage drag breaking are accurately judged according to the existence of the amplification product sequence or different combination modes among sequences by detecting whether the genome DNA of the tobacco containing the N gene contains the nucleic acid sequence of the PCR amplification product corresponding to the primer pair.
The detection may be a method of PCR detection, in particular, PCR amplification may be performed using primer pairs TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn 383. The detection can also be performed by sequencing methods. The auxiliary selection method for tobacco breeding has the advantages of simplicity, rapidness and high sensitivity.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a group of molecular markers for breaking N downstream (3 'end) linkage drag of tobacco TMV resistance genes, which are developed and obtained based on complete sequence information of N downstream (3' end) linkage drag fragments and have accurate position information. Compared with the molecular marker related to N gene resistance or the marker for estimating the length of the N introduced segment, which is reported in the literature or patents, the molecular marker is used for accurately detecting, judging and breaking the linked redundant segments at different positions downstream (3' end) of the N gene, and is not used for evaluating the resistance of the N gene and roughly estimating the length of the linked redundant segment of the N gene introduction. According to the group of molecular markers disclosed above, linked drag fragments at different positions downstream (3' end) of the N gene can be accurately detected, judged and broken, so that the TMV-resistant flue-cured tobacco variety with the effect of completely breaking or partially breaking adverse linked drag at different positions can be obtained.
Drawings
FIG. 1 shows the specific positional information of 7 pairs of primers on the downstream (3' end) linked drag fragment of the N gene. In the figure, the left side is a specific physical distance (unit is Mb), the right side is a primer name, wherein N _ gene _ start and N _ gene _ start are position information of the N gene, a marker TMn323_ N is a specific primer for detecting the N gene, and Scf _ End is an ending End of a downstream (3' End) linkage drag fragment of the N gene;
FIG. 2 is an electrophoretogram of PCR amplification products of primers TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn383 in three materials of Coker176 (N-gene-containing flue-cured tobacco, genotype NN), K326 (N-gene-free flue-cured tobacco, genotype NN) and its next generation (F1, Coker176 XK 326, genotype NN). Wherein the three parts of tobacco material of each pair of primers are respectively as follows from left to right: coker176, K326 and F1. The rightmost lane of the electrophoretogram is 100 bp DNA Ladder, which is 500bp, 400bp, 300bp and 200bp from top to bottom.
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 examples of the present invention do not include specific techniques or conditions, and are performed according to techniques or conditions described in the literature in the art or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
The invention discloses a group of primer pairs and a molecular marker for breaking the linkage drag of the N downstream (3' end) of a tobacco TMV resistance gene. By utilizing the primer pair disclosed by the invention and taking the tobacco genome DNA as a template to carry out PCR, the linked redundant fragments at different positions of the downstream (3' end) of the N gene of the tobacco can be accurately detected, judged and broken. It is to be noted that, as will be understood by those skilled in the art, the molecular marker of the present invention can be obtained by chemical synthesis in addition to the above-mentioned PCR amplification.
The serial numbers of the molecular markers for breaking the N downstream (3' end) linkage drag of the tobacco TMV resistance gene are TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn383, and the nucleotide sequences of the amplification products are shown in SEQ ID Nos. 1-10.
The primer sequences of 7 sites corresponding to the molecular markers are respectively as follows:
the sequence of TMn324 is TMn 324F: 5'-CGACTTCATGAAAGCACCAG-3' the flow of the air in the air conditioner,
TMn324R：5’- CTACTGGACTCCACCCACGA -3’；
the TMn330 sequence is TMn 330F: 5'-TGGTTAAAACTCCCATCCCTA-3' the flow of the air in the air conditioner,
TMn330R：5’- TGGAAAATGAATTTTTCGAATG -3’；
the sequence of TMn332 is TMn 332F: 5'-TTTGTGCCAAAAGAAAAGGAA-3' the flow of the air in the air conditioner,
TMn332R：5’- TGGAGAGATCGTGAAGGTCA -3’；
the sequence of TMn349 is TMn 349F: 5'-TGCACGTCTGATACTTTTTGA-3' the flow of the air in the air conditioner,
TMn349R：5’- CAACTTTGCACCACCATCAC -3’；
TMn359 sequence TMn 359F: 5'-CAACACGCACCATCACAAGT-3' the flow of the air in the air conditioner,
TMn359R：5’- CTATGCTGCCCCTTCTTCAC -3’；
TMn360 sequence is TMn 360F: 5'-TGGTTAAAGCATGCGACTTG-3' the flow of the air in the air conditioner,
TMn360R：5’- AGGAGTTTTTCGGCGATTG -3’；
the TMn383 sequence is TMn 383F: 5'-TTTACGCCCTCACAAAAACC-3' the flow of the air in the air conditioner,
TMn383R：5’- GATTTCACCGTCGGGACTAA -3’。
the group of molecular markers for breaking the downstream (3' end) linkage drag of tobacco TMV resistance gene N and the application thereof are as follows: respectively amplifying the genomic DNA of the tobacco to be detected by 7 pairs of primers including TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn383, detecting the nucleic acid sequence of a PCR amplification product, and accurately judging whether the linkage drag at the downstream (3' end) of the N gene in the tobacco to be detected is broken and the specific position information of the linkage drag broken according to the existence of the amplification product sequence or different combination modes among sequences.
The invention is further illustrated by the following specific examples:
example 1
First, experimental material
63 parts of a tobacco material containing an N gene and having TMV resistance, wherein 20 parts of burley tobacco, 40 parts of flue-cured tobacco, 1 part of aromatic tobacco, sun tobacco and wild tobacco are respectively, and the N gene in the 63 parts of the tobacco material is derived from wild tobacco-heart tobacco (N.glutinosa), so that linked burdensome segments are more or less present at the downstream (3' end) of the N gene. In addition, 1 part of flue-cured tobacco (K326) which does not contain N genes and does not have TMV resistance is used as a control, 64 parts of tobacco materials in total are common tobacco germplasm resources, and the public can obtain tobacco germplasm resource storage units or tobacco agriculture science research institutes in Yunnan province. Specific tobacco material information is shown in table 2:
TABLE 264 parts of tobacco materials
| Numbering | Name (R) | Type (B) | Source | Numbering | Name (R) | Type (B) | Source |
| T01 | Burley21 | Burley tobacco | USA | T33 | Mu Dan 80-7 | Flue-cured tobacco | China |
| T02 | Burley22 | Burley tobacco | USA | T34 | Mu Dan 81-56 | Flue-cured tobacco | China |
| T03 | Burley49 | Burley tobacco | USA | T35 | Cigarette No.5 | Flue-cured tobacco | China |
| T04 | Burley64 | Burley tobacco | USA | T36 | Tai Yan No.6 | Flue-cured tobacco | China |
| T05 | BanketA-1 | Burley tobacco | USA | T37 | Zhongwei No.1 | Flue-cured tobacco | China |
| T06 | K10 | Burley tobacco | USA | T38 | RBST | Flue-cured tobacco | China |
| T07 | K14(ergo) | Burley tobacco | USA | T39 | Coker86 | Flue-cured tobacco | USA |
| T08 | Kentucky9 | Burley tobacco | USA | T40 | Coker176 | Flue-cured tobacco | USA |
| T09 | Kentucky10 | Burley tobacco | USA | T41 | H423 | Flue-cured tobacco | USA |
| T10 | Kentucky12 | Burley tobacco | USA | T42 | NC567 | Flue-cured tobacco | USA |
| T11 | Kentucky14 | Burley tobacco | USA | T43 | NC628 | Flue-cured tobacco | USA |
| T12 | Kentucky15 | Burley tobacco | USA | T44 | NC7 | Flue-cured tobacco | USA |
| T13 | Kentucky17 | Burley tobacco | USA | T45 | NC86 | Flue-cured tobacco | USA |
| T14 | Kentucky908 | Burley tobacco | USA | T46 | SC71 | Flue-cured tobacco | USA |
| T15 | Kentucky8959 | Burley tobacco | USA | T47 | SC72 | Flue-cured tobacco | USA |
| T16 | Virginia1048 | Burley tobacco | USA | T48 | Vamorr48 | Flue-cured tobacco | USA |
| T17 | NC3 | Burley tobacco | USA | T49 | Virginia3160 | Flue-cured tobacco | USA |
| T18 | TN90 | Burley tobacco | USA | T50 | Virginia645 | Flue-cured tobacco | USA |
| T19 | Primer No.1 | Burley tobacco | Brazil rubber | T51 | Virginia770 | Flue-cured tobacco | USA |
| T20 | BYS | Burley tobacco | Korea | T52 | Virginia80 | Flue-cured tobacco | USA |
| T21 | 78-3013 | Flue-cured tobacco | China | T53 | WE-12 | Flue-cured tobacco | USA |
| T22 | 7915 | Flue-cured tobacco | China | T54 | K326 | Flue-cured tobacco | USA |
| T23 | 8211 | Flue-cured tobacco | China | T55 | PVH01 | Flue-cured tobacco | Brazil rubber |
| T24 | 8212 | Flue-cured tobacco | China | T56 | PVH02 | Flue-cured tobacco | Brazil rubber |
| T25 | 9501 | Flue-cured tobacco | China | T57 | RGH04 | Flue-cured tobacco | Brazil rubber |
| T26 | B09 | Flue-cured tobacco | China | T58 | PVH06 | Flue-cured tobacco | Brazil rubber |
| T27 | CV85 | Flue-cured tobacco | China | T59 | PVH07 | Flue-cured tobacco | Brazil rubber |
| T28 | CV87 | Flue-cured tobacco | China | T60 | Korean anti-red blood | Flue-cured tobacco | Korea (Korea) |
| T29 | Big white rib 2518 | Flue-cured tobacco | China | T61 | AK6 | Flue-cured tobacco | Is not detailed in |
| T30 | Liao cigarette No.9 | Flue-cured tobacco | China | T62 | Virginia878 | Sun-cured tobacco | USA |
| T31 | Liaoyan No.13 cigarette | Flue-cured tobacco | China | T63 | Samsun-NN | Aromatic cigarette | Japanese |
| T32 | Liaoyan No.14 cigarette | Flue-cured tobacco | China | T64 | N .glutinosa | Wild cigarette | South America |
Second, extracting tobacco genome DNA
The conventional CTAB method or plant tissue DNA extraction kit can be adopted, and the method can refer to the existing literature report or the instruction in the kit.
Third, PCR amplification and electrophoresis detection
PCR amplification systems are conventional and reference is made to published literature; in the PCR amplification program, the annealing temperature of the 7 pairs of primers is 60 ℃, and the specific PCR amplification program information can refer to relevant documents; the electrophoresis detection is also carried out by conventional methods, and reference is made to published relevant documents.
Linkage drag breaking information of downstream (3' end) of four, N genes
And respectively amplifying 64 parts of tobacco genome DNA to be detected by utilizing the 7 pairs of primers, detecting a PCR amplification product sequence, and judging the breaking degree of the downstream (3' end) linkage drag of the N gene in 64 parts of tobacco to be detected and the accurate position information of the breaking of the linkage drag according to the existence of the product sequence or different combination modes. Detailed results table 3:
table 3 analysis of downstream (3' end) linkage drag disruption of N gene in 64 parts of tobacco material using 7 primer pairs
Note: the number in parentheses behind the primer indicates the position of the marker from the N gene; +: the primer has a PCR amplification product and the linkage drag at the position is not broken; -: the primer has no PCR amplification product and the linkage drag at that position is completely broken; +/+: the primer has a PCR amplification product (genotype NN) and the linkage drag at that location has not been broken; +/-: the primer has two PCR amplification products (genotype is Nn) and the linkage drag at the position is partially broken; -/-: the primer has a PCR amplification product (genotype nn) and the linkage drag at that location is completely broken; the sequence information of the PCR amplification product of each primer is detailed in Table 1.
SEQUENCE LISTING
<110> research institute of tobacco agricultural science in Yunnan province
<120> a group of molecular markers for breaking N downstream (3' end) linkage drag of tobacco TMV resistance gene and application thereof
<130> 2018
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 195
<212> DNA
<213> TMn324 amplification product nucleotide sequence
<400> 1
cctaccctgc cctaatccac tatcttttta aaaaaaattt aatcctgccc tgcctcattt 60
agcgtttgcc ctgccctgcc ctgccccatt tagcgtttgc tctgccttgc cccattctaa 120
ttgtcatccc tatttttgtt tcctatttat aacttaatca aaaatataat cagttgtctt 180
actgcaacga ccaca 195
<210> 2
<211> 287
<212> DNA
<213> TMn330 amplification product nucleotide sequence
<400> 2
tctcttgcca caaccacaag tttttcacat acagttttct tacgcttttc agagaattaa 60
taccgagtcc atggcatcat cttcttcttc ttctgctaga tggagctatg atgtttttct 120
aagttttaga ggtgaagata ctcggaaaac atttacaagt cacttgtacg aagtcttgaa 180
taataaggga ataaaaacct ttcaagatga aaaaaggcta gagtacggtt caactatccc 240
agaagaactc tgtaaagcta tagaagagtc tcaatttgcc atcatcg 287
<210> 3
<211> 241
<212> DNA
<213> TMn332 amplification product nucleotide sequence
<400> 3
caccctcctc ttttccgatt tatatcaaaa tttggaaacc tatttcattt taactcaaca 60
aaaaaaaaaa agtaaaaatg gcacggggcg ccctatttgg tcgcccccat ttaacatata 120
ctcatttttt taaaaagttt taacttgtac tcacttttta aacaacttca gccccctttc 180
tcctcctcct tctcctcctt tgttttcttc ttcttctttc ttctgctact gttggtgctg 240
c 241
<210> 4
<211> 225
<212> DNA
<213> TMn349
<400> 4
cgattaagtt gcataccttc caccccctcc cccctccaaa ccaaaaaaat caaaatcaaa 60
atcgacattc taactcgagg gtatggcata atggtcaatg aagtgggatg aaaacatgag 120
agaataaggt tcagttacaa gtggcccgca aagtcgctag ggcaccgttc tttagaagat 180
tctggcaagc cccatttact tctagaagat tcaagaacat gcccc 225
<210> 5
<211> 217
<212> DNA
<213> TMn359 amplification product nucleotide sequence
<400> 5
cggggaaaac agcaatagag tgaagttaaa gggaatgttt ggtgaccact tctattcagt 60
attattttat gcatgaacca tcgtgttatt cgtttcaaat gattgaatct atttctttaa 120
caatttaact ttatacatta atttatattc acacaatata tatatatata tatatacttc 180
attttacact acgagttcaa aagttttctt tcttttt 217
<210> 6
<211> 221
<212> DNA
<213> TMn359 amplification product nucleotide sequence
<400> 6
cggggaaaac agcaatagag tgaagttaaa gggaatgttt ggtgaccact tctattcagt 60
attattttat gcatgaacca tcgtgttatt cgtttcaaat gattgaatct atttctttaa 120
caatttaact ttatacatta atttatattc acacaatata tatatatata tatatatata 180
cttcatttta cactacgagt tcaaaagttt tctttctttt t 221
<210> 7
<211> 225
<212> DNA
<213> TMn360 amplification product nucleotide sequence
<400> 7
ggaattacta ccgtcccaac aaaaatatac tactccttca acttaaattt caattgaaat 60
gatggtaaaa taaaaaattt aacgttaaat tatatatata tatatatata tatatattag 120
aacatactaa taaaaaaaat atgtaatatt ataattaaaa tagagaaaag taaaaaccag 180
attagcacca aaattttgcc acgcatttgg acacgtggct tacaa 225
<210> 8
<211> 233
<212> DNA
<213> TMn360 amplification product nucleotide sequence
<400> 8
ggaattacta ccgtcccaac aaaaatatac tactccttca acttaaattt caattgaaat 60
gatggtaaaa taaaaaattt aacgttaaat tatatatata tatatatata tatatatata 120
tatattagaa catactaata aaaaaaatat gtaatattat aattaaaata gagaaaagta 180
aaaaccagat tagcaccaaa attttgccac gcatttggac acgtggctta caa 233
<210> 9
<211> 221
<212> DNA
<213> TMn383 amplification product nucleotide sequence
<400> 9
ctggcaattt gtccccttta gcacctacta ctactactac tactactact actactacta 60
ctactactac tactcctgct actcctgcta ctactactac tactgctgct actactgctg 120
ctgctgctac tgctgctgct gccgccgctg ccgctgctgc tgctactgcc gctgctacca 180
ctgctaccac tgccgccact gccactactg ccgccactac t 221
<210> 10
<211> 209
<212> DNA
<213> TMn383 amplification product nucleotide sequence
<400> 10
ctggcaattt gtccccttta gcacctacta ctactactac tactactact actactacta 60
ctcctgctac tcctgctact actactacta ctgctgctac tactgctgct gctgctactg 120
ctgctgctgc cgccgctgcc gctgctgctg ctactgccgc tgctaccact gctaccactg 180
ccgccactgc cactactgcc gccactact 209
<210> 11
<211> 20
<212> DNA
<213> TMn324F
<400> 11
cgacttcatg aaagcaccag 20
<210> 12
<211> 20
<212> DNA
<213> TMn324R
<400> 12
ctactggact ccacccacga 20
<210> 13
<211> 21
<212> DNA
<213> TMn330F
<400> 13
tggttaaaac tcccatccct a 21
<210> 14
<211> 22
<212> DNA
<213> TMn330R
<400> 14
tggaaaatga atttttcgaa tg 22
<210> 15
<211> 21
<212> DNA
<213> TMn332F
<400> 15
tttgtgccaa aagaaaagga a 21
<210> 16
<211> 20
<212> DNA
<213> TMn332R
<400> 16
tggagagatc gtgaaggtca 20
<210> 17
<211> 21
<212> DNA
<213> TMn349F
<400> 17
tgcacgtctg atactttttg a 21
<210> 18
<211> 20
<212> DNA
<213> TMn349R
<400> 18
caactttgca ccaccatcac 20
<210> 19
<211> 20
<212> DNA
<213> TMn359F
<400> 19
caacacgcac catcacaagt 20
<210> 20
<211> 20
<212> DNA
<213> TMn359R
<400> 20
ctatgctgcc ccttcttcac 20
<210> 21
<211> 20
<212> DNA
<213> TMn360F
<400> 21
tggttaaagc atgcgacttg 20
<210> 22
<211> 19
<212> DNA
<213> TMn360R
<400> 22
aggagttttt cggcgattg 19
<210> 23
<211> 20
<212> DNA
<213> TMn383F
<400> 23
tttacgccct cacaaaaacc 20
<210> 24
<211> 20
<212> DNA
<213> TMn383R
<400> 24
gatttcaccg tcgggactaa 20
Claims (2)
1. The molecular markers are numbered as TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn383, and the nucleotide sequences of PCR amplification products are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, SEQ ID No.9 and SEQ ID No. 10; the primer sequences of 7 sites corresponding to the molecular markers are respectively as follows:
the sequence of TMn324 is TMn 324F: 5'-CGACTTCATGAAAGCACCAG-3', as shown in SEQ ID No.11, TMn 324R: 5'-CTACTGGACTCCACCCACGA-3', as shown in SEQ ID No. 12;
the TMn330 sequence is TMn 330F: 5'-TGGTTAAAACTCCCATCCCTA-3', as shown in SEQ ID No.13, TMn 330R: 5'-TGGAAAATGAATTTTTCGAATG-3', as shown in SEQ ID No. 14;
the sequence of TMn332 is TMn 332F: 5'-TTTGTGCCAAAAGAAAAGGAA-3', as shown in SEQ ID No.15, TMn 332R: 5'-TGGAGAGATCGTGAAGGTCA-3', as shown in SEQ ID No. 16;
the sequence of TMn349 is TMn 349F: 5'-TGCACGTCTGATACTTTTTGA-3', as shown in SEQ ID No.17, TMn 349R: 5'-CAACTTTGCACCACCATCAC-3', as shown in SEQ ID No. 18;
TMn359 sequence TMn 359F: 5'-CAACACGCACCATCACAAGT-3', as shown in SEQ ID No.19, TMn 359R: 5'-CTATGCTGCCCCTTCTTCAC-3', as shown in SEQ ID No. 20;
TMn360 sequence is TMn 360F: 5'-TGGTTAAAGCATGCGACTTG-3', as shown in SEQ ID No.21, TMn 360R: 5'-AGGAGTTTTTCGGCGATTG-3', as shown in SEQ ID No. 22;
the TMn383 sequence is TMn 383F: 5'-TTTACGCCCTCACAAAAACC-3', as shown in SEQ ID No.23, TMn 383R: 5'-GATTTCACCGTCGGGACTAA-3', as shown in SEQ ID No. 24.
2. The application of the molecular marker as claimed in claim 1, characterized in that the molecular marker is used for accurately detecting and judging the breaking degree and specific position information of the downstream 3' end linkage drag of the N gene in the tobacco to be detected; respectively amplifying the genomic DNA of the tobacco to be detected by using 7 primers including TMn324, TMn330, TMn332, TMn349, TMn359, TMn360 and TMn383, detecting the nucleic acid sequence of a PCR amplification product, wherein if the PCR amplification product contains all 7 sequences shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7 and SEQ ID No.9, namely that the completely linked burdensome exists at the downstream 3' end of the N gene; if none of the 7 PCR amplification product sequences exists, the linkage drag at the downstream 3' end of the N gene is completely broken; if only part of the 7 PCR amplification product sequences exists, the linkage drag part at the downstream 3' end of the N gene is broken, namely, the linkage drag is not broken at the position where the PCR amplification product sequences exist, and the linkage drag is completely broken at the position where the PCR amplification product sequences do not exist; in addition, if only three sequences of SEQ ID No.6, SEQ ID No.8 and SEQ ID No.10 exist and the rest sequences do not exist, the linkage drag at the downstream 3' end of the N gene is completely broken; if any combination of two sequence combinations shown as SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10 exists in the PCR amplification product, the linkage drag of the part existing at the downstream 3' end of the N gene is indicated, namely, the linkage drag part is broken at the position where the two PCR amplification product sequences exist in a combined form, and the specific information is shown in Table 1:
table 1 discloses nucleotide sequence information of PCR amplification products of 7 pairs of primers and position information of PCR amplification products of 7 pairs of primers on downstream 3' end broken linkage drag of N gene of tobacco
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