CN104726564A

CN104726564A - Primer set, kit and method for identifying different tobacco varieties

Info

Publication number: CN104726564A
Application number: CN201510079604.4A
Authority: CN
Inventors: 张建奎; 陈婷婷; 戴秀梅; 曲存民; 尹国英; 袁洪
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2015-02-15
Filing date: 2015-02-15
Publication date: 2015-06-24
Anticipated expiration: 2035-02-15
Also published as: CN104726564B

Abstract

The invention discloses a primer set, an application of the primer set in identification of tobacco varieties and/or determination of genetic relationships of different tobacco varieties, a kit, an application of the kit in identification of the tobacco varieties and/or determination of the genetic relationships of the different tobacco varieties, a method for identifying the tobacco varieties and a method for determining the genetic relationships of the different tobacco varieties. The primer set comprises a primer 1, a primer 2, a primer 3 and a primer 4. By using the primer set to perform multiple PCR (polymerase chain reaction) and selectively perform single PCR, the different tobacco varieties can be quickly and efficiently identified and/or the genetic relationships and genetic diversities of the different tobacco varieties can be identified.

Description

A kind of primer sets, test kit and method thereof identifying different tobacco bred

Technical field

The present invention relates to agricultural biological technical field, especially, the present invention relates to biological tobacco technical field, more particularly, the present invention relates to one group of primer sets, a kind of test kit, primer sets or test kit differentiating tobacco bred and/or determining purposes in different tobacco bred sibship, a kind ofly differentiate the method for tobacco bred and a kind of method determining different tobacco bred sibship.

Background technology

In leaf tobacco production and cigarette blending process, tobacco bred is discerned between right and wrong often important, only in this way the accuracy of guarantee raw material of cigarette kind.And the tobacco bred discrimination method Main Basis Agronomic characteristic, junior tobacco leaf outward appearance etc. of current use, it is more difficult and inaccurate to differentiate, particularly less to outward appearance Traits change kind, is difficult to differentiate according to morphological specificity and economical character.Therefore, utilize molecular marking technique develop accurate, simple, quick and efficient Variety identification technology to promotion tobacco leaf and production of cigarettes significant.Malins etc. [differentiate by the SCAR mark of tobacco bred, " Chinese tobacco journal " 2012,5th phase, 79-84 page] utilize the genomic dna of 6 pairs of SCAR primer pairs, 12 tobacco bred Redried laminas to carry out SCAR-PCR Amplification Analysis, according to the amplification difference of 6 SCAR mark in different tobacco bred, establish the molecule I D of 12 tobacco breds and differentiate route, although 12 kinds can be differentiated to come completely, but differentiate that route is very loaded down with trivial details, cause identification efficiency low.Therefore, it is very necessary for developing a kind of efficient and cost-saving discrimination method.

Secondly, tobacco germplasm is the basic substance of tobacco bred improvement, is related to the success or failure of tobacco breeding.Sibship and analysis of genetic diversity are carried out to tobacco germplasm, can be the utilization of variety source in tobacco breeding and important biology and genetics information are provided.5210 parts of resources are collected in current China's tobacco germplasm storehouse altogether, be that tobacco germplasm preserves the maximum country of quantity in the world, since the nineties in 20th century, the research of variety source has also developed into from aspects such as morphology, cytology, Physiology and biochemistries with molecular marking technique is gradually the molecular level of core.At present, tobacco genetic diversity and Genetic relationship study in molecule marker mainly based on ISSR, SRAP, RAPD, AFLP, what all adopt is substance round pcr, and amplification efficiency needs to be improved further.

Multiplex PCR amplification technique (be called for short " multiplex PCR ") refers to and to be improved on the basis of regular-PCR, add in a PCR reaction system two to and above primer, the different zones for multiple DNA profiling or same template increases the special round pcr of a class of multiple object fragment.This technology can adapt to the needs that high-throughput DNA fingerprint is analyzed, and save DNA profiling and test consumable, simplify the operation step, accelerated test process.Current tobacco Multiplex PCR technological development application only has report in tobacco virus detection, Transgenic Tobacco detection etc., and have not been reported in tobacco bred discriminating, tobacco sibship and analysis of genetic diversity.

Summary of the invention

The object of the present invention is to provide a kind of method rapidly and efficiently being differentiated different tobacco bred or qualification tobacco germplasm sibship and genetic diversity by DNA molecular marker.

According to an aspect of of the present present invention, the invention provides one group of primer sets, it is characterized in that, described primer sets comprises, primer one, primer two, the combination of primer three and primer four; And/or the combination of primer five and primer six.

Described primer one is made up of SEQ ID NO:1 and SEQ ID NO:2, described primer two is made up of SEQ ID NO:3 and SEQ IDNO:4, described primer three is made up of SEQ ID NO:5 and SEQ ID NO:6, and described primer four SEQ ID NO:7 and SEQ ID NO:8 forms.Any two in primer one, primer two, primer three and primer four, any more than three or whole four different zones that can both be specifically bound to template under same PCR reaction system, same reaction conditions separately carry out amplified reaction simultaneously, and amplified production is easy to distinguish, these four primers and combination thereof be contriver based on different tobacco gene group DNA sequence dna, obtain through long-time Design and optimization and a large amount of composite test screening verification.

According to one embodiment of present invention, this primer sets also comprises primer five and primer six, and described primer five is made up of SEQ ID NO:9 and SEQ ID NO:10, and described primer six is made up of SEQ ID NO:11 and SEQ ID NO:12.The different zones that primer five and primer six can be specifically bound to template separately under same PCR reaction system, same reaction conditions carries out amplified reaction simultaneously, and amplified production is easy to distinguish, these two primers and combination thereof be contriver based on different tobacco gene group DNA sequence dna, through long-time Design and optimization and lot of experiments screening and checking obtain.

According to one embodiment of present invention, described primer sets comprises primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, at least one in primer 14 and primer 15, described primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, primer 14 and primer 15 are respectively by SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ IDNO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ IDNO:30 forms.These primers are the difference of contriver based on different tobacco gene group DNA sequence dna, through long-time Design and optimization and lot of experiments screening and checking obtain.Following primer 1 to primer 15 is equal to primer one to primer 15 respectively.

According to an aspect of of the present present invention, the primer sets that the invention provides in any embodiment of aforementioned one aspect of the present invention is being differentiated tobacco bred and/or is being identified the purposes in the sibship of different tobacco bred.

According to another aspect of the present invention, the invention provides a kind of test kit, it comprises the primer sets in aforementioned one aspect of the present invention or any embodiment.

According to another aspect of the present invention, the test kit that the invention provides one aspect of the present invention is being differentiated tobacco bred and/or is being identified the purposes in the sibship of different tobacco bred.The advantage of the aforementioned primer sets to one aspect of the present invention and the description of technical characteristic, be suitable for the purposes of the purposes of the primer sets of this one side of the present invention, test kit or test kit equally, do not repeat them here.

According to another aspect of the invention, the invention provides a kind of method differentiating tobacco bred, it is characterized in that, employ described primer sets or described test kit, concrete, the method comprises: the genomic dna extracting tobacco to be checked; Primer one, primer two, primer three and primer four is utilized to carry out Quadruple-PCR to described genomic dna, obtain the first amplified production, described primer one is made up of SEQ ID NO:1 and SEQ ID NO:2, described primer two is made up of SEQ ID NO:3 and SEQ ID NO:4, described primer three is made up of SEQ ID NO:5 and SEQ ID NO:6, and described primer four SEQ ID NO:7 and SEQID NO:8 forms; Utilize described first amplified production to differentiate the kind of described tobacco to be checked, obtain the first identification result.

Described tobacco to be checked can be selected from cloud and mist 97, cloud and mist 87, Yun yan85, cloud and mist 99, cloud and mist 105, No. 1, Bi Na, No. 1, gold sea, peace No. 2, cigarette, No. 11, Henan cigarette, blue or green cigarette 201, middle cigarette 103, middle cigarette 104, CF223, PVH2254, NC196, coker176, coker206, coker254, coker258, coker298, coker319, CZ-1, G23, HB030, K326, K346, K358, M373, MC944, MSK326, MS cloud and mist 87, NC13, NC27NF, NC567, NC628, NC82, NC89, NC95, RG11, RG8, RG97, Samsun, SC71, TW90, Viginia, Bath horse, reach white No. 1, reach white No. 2, E'yan 1, reform No. 3, No. 1, your cigarette, No. 2, your cigarette, No. 3, your cigarette, Guiyan 4, rustica, No. 2, leek level ground, No. 1, Lou Shan, Maryland, Qin's cigarette 201, Qin's cigarette 96, fine large black smoke falls, prestige rather spends cigarette in vain, No. one, cloud, at least one in Yunyan201 S and cloud and mist 317, but be not limited to these tobacco breds.

The genomic dna of tobacco can from the blade of tobacco seedling, leaf bud and tobacco leaf one of at least, in one embodiment of the invention, the genomic dna of tobacco is the mixture of the DNA of the blade of this seedling that grows tobacco, leaf bud and tobacco leaf three part.

In one embodiment of the invention, for obtaining good multiplex PCR result, contriver obtains reaction conditions and the reaction system of Quadruple-PCR through plenty of time optimization Test, the response procedures of the said Quadruple-PCR through optimizing comprises: { 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, each cycle down 0.5 DEG C of 10 circulations, 72 DEG C of 30s}, { 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s} and 72 DEG C 10min of 25 circulations; And/or the reaction system of the Quadruple-PCR optimized comprises: the Easy Taq archaeal dna polymerase 0.25 μ L of 5U/ μ L, the dNTPs 0.5 μ L of 10mmol/L, containing MgCL ₂10 × Easy Taq solution 2 μ L, the MgCl of 20mmol/L ₂the genomic dna 2 μ L of each 0.25 μ L, the 20ng/ μ L of positive anti-primer of the corresponding primer of 1 μ L, 50ng/ μ L, ddH ₂o adds to 25 μ L.In one embodiment of the invention, the described kind utilizing the first amplified production to differentiate described tobacco to be checked, comprise: carry out electrophoresis detection to described first amplified production, obtain the first electrophoresis result, the specific band comprised based on described first electrophoresis result carries out described discriminating.Said electrophoresis result comprises electrophorogram, naked eyes on dyeing rear electrophoresis figure the band of clear resolution all can be used as specific band, first electrophorogram is contrasted the DNA fingerprinting of the tobacco of known kind, comprise the number of the electrophoretic band of both contrasts, relative position etc. between the absolute location of band and band, when the electrophorogram of the amplified production of tobacco to be checked is consistent with the DNA fingerprinting of certain known tobacco or basically identical, differentiate as said known kind by this tobacco to be checked.Said DNA fingerprinting can be an electrophorogram, the DNA fingerprinting storehouse of tobacco comprises the DNA fingerprinting of multiple known kind, the DNA fingerprinting storehouse of tobacco can be passed through the tobacco of each known kind, obtain with the genomic dna amount same with tobacco to be checked, same multi-PRC reaction system condition, same electrophoresis applied sample amount, same deposition condition and same dyeing condition etc., can obtaining in advance and save backup, also can obtain when differentiating tobacco to be checked simultaneously.

According to one embodiment of present invention, differentiating the kind of multiple tobacco, such as 20 when growing tobacco, through above-mentioned Quadruple-PCR, may not distinguish completely and identify all kinds, when the kind distinguished is failed in existence in the first identification result, the method can also comprise the following steps: utilize primer five and primer six to carry out double PCR to the genomic dna of U/I tobacco in described first identification result, obtain the second amplified production, described primer five is made up of SEQ ID NO:9 and SEQID NO:10, described primer six is made up of SEQ ID NO:11 and SEQ ID NO:12, the second amplified production is utilized to differentiate the kind of U/I tobacco in described first identification result, obtain the second identification result, make to differentiate the kind failing to distinguish in the first identification result further.In one embodiment of the invention, for obtaining good multiplex PCR result, contriver obtains reaction conditions and the reaction system of this double PCR through plenty of time optimization Test, the response procedures of the said double PCR through optimizing comprises: { 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, each cycle down 0.5 DEG C of 10 circulations, 72 DEG C of 30s}, { 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s} and 72 DEG C 10min of 25 circulations; And/or the reaction system of the double PCR optimized comprises: the Easy Taq archaeal dna polymerase 0.25 μ L of 5U/ μ L, the dNTPs 0.5 μ L of 10mmol/L, containing MgCL ₂10 × Easy Taq solution 2 μ L, the MgCl of 20mmol/L ₂the genomic dna 2 μ L of each 0.25 μ L, the 20ng/ μ L of positive anti-primer of the corresponding primer of 1 μ L, 50ng/ μ L, ddH ₂o adds to 25 μ L.In one embodiment of the invention, the described kind utilizing the second amplified production to differentiate U/I tobacco in the first identification result, comprise: electrophoresis detection is carried out to described second amplified production, obtain the second electrophoresis result, the specific band comprised based on described second electrophoresis result carries out described discriminating.Said electrophoresis result comprises electrophorogram, naked eyes on dyeing rear electrophoresis figure the band of clear resolution all can be used as specific band, first electrophorogram is contrasted the DNA fingerprinting of the tobacco of known kind, comprise the number of the electrophoretic band of both contrasts, relative position etc. between the absolute location of band and band, when the electrophorogram of the amplified production of tobacco to be checked is consistent with the DNA fingerprinting of certain known tobacco or basically identical, differentiate as said known kind by this tobacco to be checked.Said electrophoresis result comprises electrophorogram, naked eyes on dyeing rear electrophoresis figure the band of clear resolution all can be used as specific band, by the second electrophorogram, the DNA fingerprinting storehouse optionally contrasting the tobacco of known kind in conjunction with its corresponding first electrophorogram, comprise the contrast number of electrophoretic band, relative position etc. between the absolute location of band and band, when the electrophorogram of the amplified production of tobacco to be checked is consistent with the DNA fingerprinting of certain known tobacco or basically identical, differentiate as said known kind by this tobacco to be checked.Said DNA fingerprinting can be an electrophorogram, preferably, the DNA fingerprinting of known kind can pass through the tobacco of each known kind, obtain with the genomic dna amount same with tobacco to be checked, same multi-PRC reaction system condition, same electrophoresis applied sample amount, same deposition condition and same dyeing condition etc., can obtaining in advance and save backup, also can obtain when differentiating tobacco to be checked simultaneously.

According to one embodiment of present invention, primer seven is utilized, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, one in primer 14 and primer 15 is carried out substance PCR to the genomic dna of U/I tobacco in described second identification result, obtains the 3rd amplified production, described primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, primer 14 and primer 15 are respectively by SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ IDNO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30 forms, and utilizes described 3rd amplified production to differentiate the kind of U/I tobacco in described second identification result, obtains the 3rd identification result.In one embodiment of the invention, for obtaining preferably PCR primer, contriver spends plenty of time energy optimization Test to obtain reaction conditions and the reaction system of this substance PCR, the response procedures of the substance PCR of described optimization comprises: { 94 DEG C of 5min of 35 circulations, 94 DEG C of 30s, 55 DEG C ~ 60 DEG C 30s, 72 DEG C of 30s} and 72 DEG C 10min; And/or the amplification system of the substance PCR of described optimization comprises: containing MgCL ₂10 × Easy Taq solution 1.67 μ L, the Easy Taq archaeal dna polymerase 0.125 μ L of the dNTPs 0.25 μ L of 10mmol/L, 5U/ μ L, the genomic dna 1 μ L of each 0.365 μ L, the 20ng/ μ L of positive anti-primer of the corresponding primer of 50ng/ μ L, ddH ₂o adds to 10 μ L.In one embodiment of the invention, the described kind utilizing the 3rd amplified production to differentiate U/I tobacco in the second identification result, comprise: electrophoresis detection is carried out to described 3rd amplified production, obtain the 3rd electrophoresis result, the specific band comprised based on described 3rd electrophoresis result carries out described discriminating.Said electrophoresis result comprises electrophorogram, naked eyes on dyeing rear electrophoresis figure the band of clear resolution all can be used as specific band, by the 3rd electrophoresis result, the optional DNA fingerprinting that must contrast the tobacco of known kind in conjunction with the first electrophoresis result of its correspondence and the second electrophoresis result, comprise the number of the electrophoretic band of both contrasts, relative position etc. between the absolute location of band and band, when the electrophorogram of the amplified production of tobacco to be checked is consistent with the DNA fingerprinting of certain known tobacco or basically identical, differentiate as said known kind by this tobacco to be checked.Said DNA fingerprinting can be an electrophorogram, the DNA fingerprinting storehouse of tobacco comprises the DNA fingerprinting of multiple known kind, the DNA fingerprinting storehouse of tobacco can be passed through the tobacco of each known kind, obtain with the genomic dna amount same with tobacco to be checked, same multi-PRC reaction system condition, same electrophoresis applied sample amount, same deposition condition and same dyeing condition etc., can obtaining in advance and save backup, also can obtain when differentiating tobacco to be checked simultaneously.

The DNA molecular marker method of the employing multiplexed PCR amplification that the invention described above provides and various embodiment thereof, differentiate for different tobacco bred, greatly can improve the efficiency that tobacco bred is differentiated.

According to another aspect of the present invention, the invention provides a kind of method for the identification of different tobacco bred sibship, this that employ described primer sets or described test kit.Concrete, the method comprises: the genomic dna extracting multiple tobacco respectively; Primer one, primer two, primer three and primer four is utilized to carry out Quadruple-PCR to the genomic dna respectively grown tobacco respectively, obtain the first amplified production respectively grown tobacco, described primer one is made up of SEQ ID NO:1 and SEQ ID NO:2, described primer two is made up of SEQ ID NO:3 and SEQ ID NO:4, described primer three is made up of SEQ ID NO:5 and SEQID NO:6, and described primer four SEQ ID NO:7 and SEQ ID NO:8 forms; Utilize described the first amplified production respectively grown tobacco to determine the sibship of the tobacco at least partially in described multiple tobacco, obtain the first sibship.

Said tobacco can be selected from cloud and mist 97, cloud and mist 87, Yun yan85, cloud and mist 99, cloud and mist 105, No. 1, Bi Na, No. 1, gold sea, peace No. 2, cigarette, No. 11, Henan cigarette, blue or green cigarette 201, middle cigarette 103, middle cigarette 104, CF223, PVH2254, NC196, coker176, coker206, coker254, coker258, coker298, coker319, CZ-1, G23, HB030, K326, K346, K358, M373, MC944, MSK326, MS cloud and mist 87, NC13, NC27NF, NC567, NC628, NC82, NC89, NC95, RG11, RG8, RG97, Samsun, SC71, TW90, Viginia, Bath horse, reach white No. 1, reach white No. 2, E'yan 1, reform No. 3, No. 1, your cigarette, No. 2, your cigarette, No. 3, your cigarette, Guiyan 4, rustica, No. 2, leek level ground, No. 1, Lou Shan, Maryland, Qin's cigarette 201, Qin's cigarette 96, fine large black smoke falls, prestige rather spends cigarette in vain, No. one, cloud, at least two kinds in Yunyan201 S and cloud and mist 317, but be not limited to these tobacco breds.

The genomic dna of tobacco can from the blade of tobacco seedling, leaf bud and tobacco leaf one of at least, in one embodiment of the invention, the genomic dna of tobacco is the mixture of the genomic dna of the blade of this seedling that grows tobacco, leaf bud and tobacco leaf three part.In one embodiment of the invention, for obtaining good multiplex PCR result, contriver obtains reaction conditions and the reaction system of Quadruple-PCR through plenty of time optimization Test, the response procedures of the said Quadruple-PCR through optimizing comprises: { 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, each cycle down 0.5 DEG C of 10 circulations, 72 DEG C of 30s}, { 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s} and 72 DEG C 10min of 25 circulations; And/or the reaction system of the Quadruple-PCR optimized comprises: the Easy Taq archaeal dna polymerase 0.25 μ L of 5U/ μ L, the dNTPs 0.5 μ L of 10mmol/L, containing MgCL ₂10 × Easy Taq solution 2 μ L, the MgCl of 20mmol/L ₂the genomic dna 2 μ L of each 0.25 μ L, the 20ng/ μ L of positive anti-primer of the corresponding primer of 1 μ L, 50ng/ μ L, ddH ₂o adds to 25 μ L.In one embodiment of the invention, the described sibship utilizing the first amplified production to determine the tobacco at least partially in described multiple tobacco, comprise: electrophoresis detection is carried out to described the first amplified production respectively grown tobacco, obtain the first electrophoresis result, the number of the specific band comprised based on described first electrophoresis result carries out the determination of described sibship.Said electrophoresis result comprises electrophorogram, and because used multi-primers is the specific regions sequences Design based on different tobacco, the naked eyes on dyeing rear electrophoresis figure the band of clear resolution can all to can be used as the specific band of this tobacco.The kind General Sequences similarity nearer due to sibship is higher, and according to this, in one embodiment of the invention, assert that the tobacco sibship jointly with more specific bands is nearer, namely genetic distance is nearer.Obtain in some embodiments in the present invention, by amplified production certain position on electrophorogram have band with regard to assignment be 1, this position without band assignment be 0 building database, STATISTIC analysis software NTSYSpc1.0.0.0 is adopted to calculate genetic distance (GD according to default parameters, genetic distance), and by Ni-like ions method (unweighted pair-group method with arithmetic means, UPGMA) according to default parameters constructing system clustering tree.

According to one embodiment of present invention, when determining that the tobacco bred of sibship is more or wherein comprise a part of kind that to show sibship after Quadruple-PCR qualification above very near, the method can also comprise: utilize primer five and the genomic dna of primer six to the tobacco at least partially in described first sibship to carry out double PCR, obtain corresponding second amplified production, described primer five is made up of SEQ ID NO:9 and SEQ ID NO:10, described primer six is made up of SEQ ID NO:11 and SEQ ID NO:12, the second amplified production is utilized to determine the sibship of the tobacco at least partially in described first sibship, obtain the second sibship, far and near to determine each interracial sibship further.In one embodiment of the invention, for obtaining good multiplex PCR result, contriver obtains reaction conditions and the reaction system of this double PCR through plenty of time optimization Test, the response procedures of the said double PCR through optimizing comprises: { 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, each cycle down 0.5 DEG C of 10 circulations, 72 DEG C of 30s}, { 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s} and 72 DEG C 10min of 25 circulations; And/or the reaction system of the double PCR optimized comprises: the Easy Taq archaeal dna polymerase 0.25 μ L of 5U/ μ L, the dNTPs 0.5 μ L of 10mmol/L, containing MgCL ₂10 × Easy Taq solution 2 μ L, the MgCl of 20mmol/L ₂the genomic dna 2 μ L of each 0.25 μ L, the 20ng/ μ L of positive anti-primer of the corresponding primer of 1 μ L, 50ng/ μ L, ddH ₂o adds to 25 μ L.In one embodiment of the invention, the first amplified production that described utilization respectively grows tobacco determines the sibship of the tobacco at least partially in multiple tobacco, comprise: electrophoresis detection is carried out to described the second amplified production respectively grown tobacco, obtain the second electrophoresis result, the number of the specific band comprised based on described second electrophoresis result carries out the determination of described sibship.Said electrophoresis result comprises electrophorogram, and because used multi-primers is the specific regions sequences Design based on different tobacco, the naked eyes on dyeing rear electrophoresis figure the band of clear resolution can all to can be used as the specific band of this tobacco.The kind General Sequences similarity nearer due to sibship is higher, and according to this, in one embodiment of the invention, assert that the tobacco sibship jointly with more specific bands is nearer, namely genetic distance is nearer.Obtain in some embodiments in the present invention, by amplified production certain position on electrophorogram have band with regard to assignment be 1, this position without band assignment be 0 building database, STATISTIC analysis software NTSYSpc1.0.0.0 is adopted to calculate genetic distance (GD according to default parameters, genetic distance), and by Ni-like ions method (unweighted pair-group methodwith arithmetic means, UPGMA) according to default parameters constructing system clustering tree.

According to one embodiment of present invention, the method also comprises, and utilizes primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, one in primer 14 and primer 15 is carried out substance PCR to the genomic dna of the tobacco at least partially in described second sibship, obtains corresponding 3rd amplified production, described primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, primer 14 and primer 15 are respectively by SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ IDNO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ IDNO:29 and SEQ ID NO:30 forms, and utilizes described 3rd amplified production to determine the sibship of the tobacco at least partially in described second sibship, obtains the 3rd sibship.In one embodiment of the invention, for obtaining preferably PCR primer, contriver spends plenty of time energy optimization Test to obtain reaction conditions and the reaction system of this substance PCR, the response procedures of the substance PCR of described optimization comprises: { 94 DEG C of 5min of 35 circulations, 94 DEG C of 30s, 55 DEG C ~ 60 DEG C 30s, 72 DEG C of 30s} and 72 DEG C 10min; And/or the amplification system of the substance PCR of described optimization comprises: containing MgCL ₂10 × Easy Taq solution 1.67 μ L, the Easy Taq archaeal dna polymerase 0.125 μ L of the dNTPs 0.25 μ L of 10mmol/L, 5U/ μ L, the genomic dna 1 μ L of each 0.365 μ L, the 20ng/ μ L of positive anti-primer of the corresponding primer of 50ng/ μ L, ddH ₂o adds to 10 μ L.In one embodiment of the invention, the described sibship utilizing the second amplified production to determine the tobacco at least partially in the first sibship, comprise: the described sibship utilizing the 3rd amplified production to determine the tobacco at least partially in the second sibship, comprise: electrophoresis detection is carried out to described 3rd amplified production, obtain the 3rd electrophoresis result, the number of the specific band comprised based on described 3rd electrophoresis result carries out the determination of described sibship.Said electrophoresis result comprises electrophorogram, and because used multi-primers is the specific regions sequences Design based on different tobacco, the naked eyes on dyeing rear electrophoresis figure the band of clear resolution can all to can be used as the specific band of this tobacco.The kind General Sequences similarity nearer due to sibship is higher, and according to this, in one embodiment of the invention, assert that the tobacco sibship jointly with more specific bands is nearer, namely genetic distance is nearer.Obtain in some embodiments in the present invention, by amplified production certain position on electrophorogram have band with regard to assignment be 1, this position without band assignment be 0 building database, STATISTIC analysis software NTSYSpc1.0.0.0 is adopted to calculate genetic distance (GD according to default parameters, genetic distance), and by Ni-like ions method (unweighted pair-group method with arithmetic means, UPGMA) according to default parameters constructing system clustering tree.

Method in this method on the one hand of the invention described above or arbitrary embodiment, can the edge relation of the different tobacco bred of determination rapidly and efficiently, the genetic diversity of research tobacco.

By carrying out large-scale screening verification test to PCR primer, the primer sequence finally determined is specific as follows:

SEQ ID NO:1TGTGGAGCTCCTTTCTTTGC；

SEQ ID NO:2TCAAATCAACAACAAATCCAAT；

SEQ ID NO:3AAACTTGAAGCAGAGACGGC；

SEQ ID NO:4GCACATGCGGATCTTGATTT；

SEQ ID NO:5TGTGTGCACCCTCCAATTTA；

SEQ ID NO:6TGATCTCTAGAGTGGTGGCATC；

SEQ ID NO:7AATGTCTGCCCAATCGAAAG；

SEQ ID NO:8CGAATAACGACACTCGAACG；

SEQ ID NO:9CCAACTCTACCGCTAACTTCAAA；

SEQ ID NO:10CACGACTGACGAGACATGGT；

SEQ ID NO:11TCACCAATTATGCCGATTCA；

SEQ ID NO:12CACACACTTGTTTGGAATTGAGA。

SEQ ID NO:13TTTAAGCTCATTTGAGCCCG；

SEQ ID NO:14CGTATTTGAGATTTATATGCCTTCG；

SEQ ID NO:15CATTTGAACATGGTTGGCTG；

SEQ ID NO:16CTCAACTCTCGTCGCTCTTG；

SEQ ID NO:17GCATGCATATGAACATGGGA；

SEQ ID NO:18TTTGACATCTCTACTCTTCCGTTT；

SEQ ID NO:19CTTTGCAGCAACAATCTCAA；

SEQ ID NO:20CACTTGGCTAGGCTAAATAAGCA；

SEQ ID NO:21CAGCCAAGAGAACCCTTCAG；

SEQ ID NO:22GATTACCCTTCAAATGCCGA；

SEQ ID NO:23AACGAGTGTAAACATGCTCCC；

SEQ ID NO:24CCACCAGCACAATAATGAACA；

SEQ ID NO:25TTTGGAATCAATAAGACGACAA；

SEQ ID NO:26TTGACCAGTAGGCTTATCACACA；

SEQ ID NO:27TCAAATCAAATCAACCCTCTCC；

SEQ ID NO:28TGGTTGGAGCTTCTCTCGTT；

SEQ ID NO:29CGTACCCTGAATGCCATCTT；

SEQ ID NO:30ATGCAGCGTTTCAGGAATTT。

Carried out a large amount of shaker tests to the combination of primers of multiplex PCR, the multiple PCR primer finally determined combination is by primer 1, primer 2, primer 3, the Quadruple-PCR primer that primer 4 forms, the double PCR primer be made up of primer 5 and primer 6.

Final selected, when differentiating different tobacco bred, application primer 1, primer 2, primer 3, primer 4 carries out Quadruple-PCR amplification, and or application primer 5 and primer 6 carry out double PCR amplification and different tobacco breds can be distinguished or identify and be divided into a few class.To not by the kind distinguished completely, can also according to different practical situation continuation primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14, any one or more pairs of primer in primer 15 carries out the qualification of substance pcr amplification, until finally distinguish all tobacco breds completely.

When analyzing the sibship of different tobacco bred and genetic diversity, application primer 1, primer 2, primer 3, primer 4 carries out Quadruple-PCR amplification, with or application primer 5, primer 6 carries out double PCR amplification, application primer 7 can also be continued according to different practical situation, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14, any one or more pairs of primer in primer 15 proceeds substance pcr amplification, then just can according to the result of pcr amplification electrophoretic band, generally acknowledge that known method calculates genetic distance and carries out cluster analysis with STATISTIC analysis software etc.

The PCR program of described quadruple, double PCR amplification is 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, each cycle down 0.5 DEG C, 72 DEG C extend 30s, after 10 circulations, and 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 45s, 25 circulations, and last 72 DEG C extend 10min.PCR amplification system is the Easy Taq archaeal dna polymerase 0.25 μ L of 5U/ μ L, the dNTPs 0.5 μ L of 10mmol/L; Containing MgCL ₂10 × Easy Taqbuffer 2 μ L, the MgCl of 20mmol/L ₂the tobacco template DNA 2 μ L of each 0.25 μ L, the 20ng/ μ L of positive anti-primer of 1 μ L, 50ng/ μ L, adds ddH ₂o to 25 μ L;

The PCR program of described substance pcr amplification is 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 55 DEG C ~ 60 DEG C annealing 30s, and 72 DEG C extend 30s, 35 circulations, and last 72 DEG C extend 10min.PCR amplification system is for containing MgCL ₂10 × Easy Taq buffer 1.67 μ L, the template DNA 1 μ L of each 0.365 μ L, the 20ng/ μ L of positive anti-primer of the Easy Taq DNA polymerase 0.125 μ L of the dNTPs 0.25 μ L of 10mmol/L, 5U/ μ L, 50ng/ μ L, adds ddH ₂o to 10 μ L.

The present invention has creatively carried out a large amount of tests to the combination of primers of Multiplex PCR, the amplification system of Multiplex PCR, the amplification program of Multiplex PCR.Main experimental process is as follows:

The candidate drugs of multiplex PCR is to screening:

First this experiment uses general PCR program, increase with 486 pairs of primer pair tobacco germplasms, preliminary screening goes out rich polymorphism, banding pattern 15 pairs of primers clearly, i.e. primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14, primer 15.Then, with above-mentioned 15 pairs of primer amplifications from the very near tobacco material of 9 sibships in Yunnan and Guizhou, comprise cloud and mist 97 and variation choosing be QY97M, cloud and mist 87 and two variation choosing are T24, recessed leaf cigarette, K326 and variation choosing thereof are that blade is yellow, and other two kind T09-1 and objective field cigarette.Amplified band is shown by polyacrylamide gel electrophoresis, verify that above-mentioned 15 pairs of primer single amplification are to the resolving ability of different varieties, the stability that increases and band sharpness, result shows that above-mentioned 15 pairs of primer PCRs amplification is stable, electrophoretic band is clear, rich polymorphism, Variety identification power strong, meets requirement of experiment.3rd step, selects design multiple PCR primer according to following principle: try not between primer to form stable aggressiveness, especially 3' head, as having aggressiveness at head, free energy preferably controls below 2, and the free energy of other aggressiveness is no more than 4; Annealing temperature is close; Difference will be had between the amplification object fragment length size of each pair of primer.Screen through test of many times, preliminary screening goes out SEQID NO:1-2 (primer 1), SEQ ID NO:3-4 (primer 2), SEQ ID NO:5-6 (primer 3), SEQ ID NO:7-8 (primer 4), SEQ ID NO:9-10 (primer 5), SEQ ID NO:11-12 (primer 6), these 7 pairs of primers of SEQ ID NO:19-20 (primer 10), as the candidate drugs of multiplex PCR.4th step, the primer 1 previous step filtered out, primer 2, primer 3, primer 4, primer 5, primer 6, primer 10, carry out combination of two according to multiplex PCR rule, have larger difference between screening amplified fragments size and the less primer of Tm value difference for combining, determine that combination of two is to being primer 1+2,1+3,1+4,2+3,2+4,2+15,4+10,3+4,5+6.These 9 groups of double combination of primers are used for increasing and verify from the tobacco material that the sibship in Yunnan and Guizhou is very near by the 5th step.In order to reach best expanding effect, then multiplexed PCR amplification system and amplification program are optimized.

The optimization experiment of multiplex PCR procedure:

In order to optimize the amplification condition of multiplex PCR, choose tobacco material K326, T24, blade is yellow, pcr amplification is carried out by above-mentioned 9 groups of double combination of primers, by screening the comparison of different amplification system and amplification program, best to reach amplification effect, namely specific band is obviously clear, and non-specific band is few, and the object stripe size of multiplex amplification is consistent with the stripe size of corresponding primer substance pcr amplification.Through many experiments, finally determine reaction system and the amplification program of multiplex PCR.Determine multiplexed PCR amplification reaction in each reagent dosage be: Easy Taq archaeal dna polymerase (5U/ μ L) 0.25 μ L, dNTPs (10mmol/L) 0.5 μ L 10 × Easy Taq buffer (containing MgCl ₂) 2 μ L, MgCl ₂(20mmol/L) 1 μ L, each 0.25 μ L of positive anti-primer (50ng/ μ L), tobacco template DNA (20ng/ μ L) 2 μ L, add ddH ₂o to 25 μ L.Determine that multiplexed PCR amplification program takes touch down program, 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s (each cycle down 0.5 DEG C), 72 DEG C extend 30s, and after 10 circulations, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 45s, 25 circulations, and last 72 DEG C extend 10min.This response procedures can the product that correctly matches of enriching primer and template, reduces the appearance of non-specific band.

The amplification experiment screening of double PCR primer pair:

The screening of double PCR primer pair is determined: by the tobacco material in 9 Yunnan and Guizhou, with above-mentioned 9 groups of 1+2, 1+3, 1+4, 2+3, 2+4, 2+15, 10+4, 3+4, the double combination of primers primer of 5+6, increase according to the PCR system after optimization and program, analysis contrast is carried out according to electrophorogram, screen satisfactory combination of primers pair, result shows, combination of primers 1+2, 1+3, 1+4, 2+3, 5+6 effect is best, amplified band is clear, polymorphic bands quantity is moderate, the stripe size that multiplexed PCR amplification goes out and the stripe size that corresponding primer substance pcr amplification goes out basically identical, meet requirement of experiment, primer 2+15,10+4 does not meet requirement of experiment, is eliminated, though the expanding effect of primer 2+4 and primer 3+4 meets the requirements reluctantly, its effect is inferior to combination of primers 1+2, and therefore 1+3,1+4,2+3,5+6 are also eliminated.

The screening experiment of triple PCR and Quadruple-PCR combination of primers:

According to the dual combination of primers filtered out, attempt to set up combination of primers 1+2+3; 1+3+4; The triple PCR of 2+3+4.In like manner, use it for 9 tobacco materials of amplification from Guizhou, Yunnan, electrophoretic band shows, the triple PCR of combination of primers 2+3+4, polymorphic bands comparatively small amt, the bad identification of band; The electrophoretic band of combination of primers 1+2+3 and combination of primers 1+3+4 triple PCR is clear easily to be distinguished, polymorphism is good, therefore attempts a more step and sets up Quadruple-PCR, be i.e. the Quadruple-PCR of combination of primers 1+2+3+4.Quadruple combination of primers 1+2+3+4 is used for 9 tobacco materials increased from Guizhou, Yunnan, can be found out by electrophoresis result Fig. 1, the specific band of the Quadruple-PCR amplification of combination of primers 1+2+3+4 is obvious, present rich polymorphism, illustrate that the Quadruple-PCR amplifier molecule mark of combination of primers 1+2+3+4 can be used for the discriminating of tobacco bred.

Multiplex PCR amplifier molecule mark is used for the experimental verification of different tobacco bred:

In order to verify the practical application effect that above-mentioned multiplexed PCR amplification molecule marker is identified for different tobacco bred, by Chongqing cigarette section produce in carrying out Chongqing City high-quality characteristic new flue-cured tobacco varieties 2014 years the cloud and mist 97 shown, cloud and mist 87, Yun yan85, cloud and mist 99, cloud and mist 105, No. 1, your cigarette, No. 2, your cigarette, No. 1, Bi Na, No. 1, gold sea, peace No. 2, cigarette, No. 11, Henan cigarette, Qin's cigarette 201, middle cigarette 104, middle cigarette 203, CF223, K326, Coker206, PVH1452, PVH2254, NC196 etc. 20 produce main cultivation flue-cured tobacco cultivars as material of participating in the experiment, carry out the discriminating checking of kind.The first step, 1+2+3+4 quadruple combination of primers is utilized to carry out the Quadruple-PCR amplification of 20 flue-cured tobacco cultivars, as can be seen from electrophorogram (accompanying drawing 2), amplified band is high-visible, polymorphism is obvious, known through result comparison (accompanying drawing 2, accompanying drawing 8, accompanying drawing 9), 9 kinds such as cloud and mist 97, cloud and mist 99, No. 1, your cigarette, No. 1, Bi Na, Qin's cigarette 201, CF223, PVH1452, NC196, middle cigarette 203 can directly be differentiated to make a distinction by a Quadruple-PCR amplification from other kind, and all the other 11 kinds are divided into 4 groups.Second step, in order to all the other 11 kinds are distinguished completely, double PCR amplification is carried out to these 11 material combination of primers 5+6, as can be seen from electrophorogram (accompanying drawing 3), expanding effect is good, through result comparison (accompanying drawing 3, accompanying drawing 8, accompanying drawing 9) known, this time double PCR amplification can divide open No. 1, gold sea well with PVH2254, No. 11, Henan cigarette, middle cigarette 104, Coker206 is separated from each other, cloud and mist 87 and Yun yan85 are separated, cloud and mist 105, No. 2, your cigarette, K326 and the cloud and mist 105 of pacifying in cigarette No. 2 these groups with No. 2, your cigarette distinguish, but K326 is still consistent with the amplified band size of peace No. 2, cigarette in this group, do not make a distinction.3rd step, carries out substance pcr amplification with primer SEQ ID NO:13-14 (primer 7), and as can be seen from electrophorogram Fig. 4 and Fig. 8 and Fig. 9, this substance pcr amplification can make a distinction K326 and No. 2 discriminatings of peace cigarette.Therefore, these 20 kinds can be differentiated to make a distinction completely by three pcr amplifications of the present invention.Malins etc. [SCAR mark of tobacco bred is differentiated, " Chinese tobacco journal " 2012, the 5th phase, 79-84 page] utilize 6 pairs of SCAR primer pairs, 12 tobacco breds to differentiate, need to carry out 11 pcr amplifications, 12 Variety identification could be made a distinction.To sum up prove, the present invention is differentiated for different tobacco bred in conjunction with substance pcr amplification by multiplexed PCR amplification, has high efficiency obviously and practicality.

Multiplex PCR molecule marker is used for the Relationship iden-tification of different tobacco bred and the experimental verification of cluster analysis

The present invention not only can be used for the discriminating of different tobacco bred, can also be used for Relationship iden-tification and the cluster analysis of different tobacco bred.The same, above-mentioned 20 tobacco breds from Chongqing, wherein 7 pairs of primers of the present invention are used to carry out 3 pcr amplification (Quadruple-PCR amplifications altogether, a double PCR amplification, a substance pcr amplification), coamplification goes out 22 bands, and wherein polymorphic bands has 19, accounts for 86.37% of total band number.Band assignment is had to be 1, without being with assignment to be 0 building database by amplified production, STATISTIC analysis software NTSYSpc1.0.0.0 is adopted to calculate genetic distance (GD, geneticdistance), and with Ni-like ions method (unweighted pair-group method with arithmetic means, UPGMA) constructing system clustering tree.The genetic cluster figure of 20 tobacco breds is shown in accompanying drawing 5.The genetic distance of 20 flue-cured tobacco cultivars is between 0.0667 ~ 1.0000.When similarity factor is 0.69,20 tobacco breds are divided into three major types, monoid I comprises 10 kinds such as cloud and mist 97, PVH1452, PVH2254, No. 2, your cigarette, K326, cloud and mist 105, peace No. 2, cigarette, cloud and mist 99, Qin's cigarette 201, middle cigarette 104, monoid II comprises No. 1, your cigarette, No. 11, Henan cigarette, No. 1, gold sea, Coker206, middle cigarette 203,7 kinds such as CF223, NC196, and monoid III comprises 3 kinds such as No. 1, cloud and mist 87, Yun yan85 and Bi Na.Between the kind of realm genetic distance and sibship nearer, as in monoid III between cloud and mist 87 and Yun yan85 genetic distance nearest, be 0.0667, both explanations sibship is nearer, and middle cigarette in monoid I 104 and No. 1, Bi Na have genetic distance farthest, both explanations sibship is far away.

As can be seen from above, contriver just draws combination of primers of the present invention and amplification system and program through a large amount of performing creative labour and a large amount of tests.The invention provides a kind of method rapidly and efficiently differentiating different tobacco bred or qualification tobacco germplasm sibship and genetic diversity.

Accompanying drawing explanation

Fig. 1 be in one embodiment of the present of invention utilize primer 1+2+3+4 to from Guizhou, Yunnan 9 tobacco breds with carry out Quadruple-PCR amplification qualification electrophorogram.

Fig. 2 is the electrophoretogram utilizing primer 1+2+3+4 to increase to 20 tobacco bred Quadruple-PCRs from Chongqing in one embodiment of the present of invention.

Fig. 3 is the electrophoretogram utilizing primer 5+6 to increase to 11 tobacco bred double PCRs from Chongqing in one embodiment of the present of invention.

Fig. 4 utilizes primer 7 to the electrophoretogram of 11 tobacco bred pcr amplifications from Chongqing in one embodiment of the present of invention.

Fig. 5 is the sibship genetic cluster figure of the utilization in one embodiment of the present of invention from 20 tobacco breds in Chongqing.

Fig. 6 is that the primer 1+2+3+4 that utilizes in one embodiment of the present of invention carries out the DNA fingerprinting of quadruple primer PCR amplification to 9 tobacco breds from Guizhou, Yunnan.

Fig. 7 is the discriminating route map of 9 tobacco breds from Guizhou, Yunnan in one embodiment of the present of invention.

Fig. 8 is the DNA fingerprinting of 20 tobacco breds from Chongqing in one embodiment of the present of invention.

Fig. 9 is the discriminating route map of 20 tobacco breds from Chongqing in one embodiment of the present of invention.

Figure 10 is the sibship genetic cluster figure of 7 tobacco breds from Guizhou in one embodiment of the present of invention.

Embodiment

Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Be exemplary below by the embodiment be described with reference to the drawings, be intended to for explaining the present invention, and can not limitation of the present invention be interpreted as.In description of the invention, " first ", " second ", " the 3rd " etc. are for referring to or describe conveniently, ordinal relation can not be interpreted as or have relative importance to indicate, except as otherwise noted, the implication of " multiple ", " many groups ", " multiple " is more than two (groups or heavy) or two (group or heavy).Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.

Below in test, the primer synthesizes by prompt base (Shanghai) trade Co., Ltd in the English Weihe River.

Experimental technique

1. tobacco gene group DNA extraction method

Tobacco seed is sent out seedling and is got the similar a few strain seedling mixing of proterties 1 week rear (tri-leaf period), CTAB method is utilized to extract tobacco gene group DNA, and detect purity and concentration with Beckman DU600 ultraviolet spectrophotometer, be diluted to 100ng/ μ L according to concentration ,-20 DEG C save backup.

2. substance pcr amplification

Pcr amplification reaction is totally 10 μ L, and reactant comprises 10 × Easy Taq buffer (containing MgCl ₂) 1.67 μ L, dNTPs (10mmol/L) 0.25 μ L, Easy Taq archaeal dna polymerase (5U/ μ L) 0.125 μ L, each 0.365 μ L of positive anti-primer (50ng/ μ L), template DNA (20ng/ μ L) 1 μ L, adds ddH ₂o to 10 μ L.PCR program is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 55 DEG C-60 DEG C annealing 30s, and 72 DEG C extend 30s, 35 circulations, and last 72 DEG C extend 10min, 4 DEG C of preservations.PCR reaction is carried out on System-9700 type amplification instrument.The detection of amplified production is undertaken by argentation.

3. multiplexed PCR amplification

In multiplexed PCR amplification reaction, each reagent dosage is as follows: be totally 25 μ L, reactant comprises Easy Taq archaeal dna polymerase (5U/ μ L) 0.25 μ L, dNTPs (10mmol/L) 0.5 μ L, 10 × Easy Taq buffer is (containing MgCl ₂) 2 μ L, MgCl ₂(20mmol/L) 1 μ L, each 0.25 μ L of positive anti-primer (50ng/ μ L), tobacco template DNA (20ng/ μ L) 2 μ L, add ddH ₂o to 25 μ L.

Amplification program Touchdown program, is specially 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s (each cycle down 0.5 DEG C), 72 DEG C extend 30s, and after 10 circulations, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 45s, 25 circulations, and last 72 DEG C extend 10min.

PCR reaction is still carried out on System-9700 type amplification instrument, and the detection of amplified production is undertaken by argentation.

4. data analysis

Get banding pattern clear, press out with or no record, amplified production with O, 1 statistics building database, on identical migration position (same molecular amount fragment) have band assignment be 1, without band note assignment be 0.STATISTIC analysis software NTSYSpc1.0.0.0 is adopted to calculate genetic distance (GD, genetic distance), and with Ni-like ions method (unweightedpair-group method with arithmetic means, UPGMA) constructing system clustering tree.

Embodiment 1: the DNA fingerprinting from 9 tobacco breds in Guizhou, Yunnan is identified

9 certified tobacco materials are respectively: QY97M, cloud and mist 97, K326, cloud and mist 87, T24, blade Huang, recessed leaf cigarette, T09-1, objective field cigarette, wherein QY97M is the variation lines selected through Systematic selection from cloud and mist 97 colony, T24 and recessed leaf cigarette are the variant strains selected through Systematic selection from cloud and mist 87 colony, blade Huang is through variation lines that Systematic selection selects from K326, their sibship is very near, Agronomic trait and economical character close, with ordinary method be difficult to differentiate.

Tobacco gene group DNA extraction: with reference to " tobacco gene group DNA extraction method " operation above.

Pcr amplification and result judge:

Multiplexed PCR amplification reaction in each reagent dosage as follows: Easy Taq archaeal dna polymerase (5U/ μ L) 0.25 μ L, dNTPs (10mmol/L) 0.5 μ L 10 × Easy Taq buffer (containing MgCl ₂) 2 μ L, MgCl ₂(20mmol/L) 1 μ L, each 0.25 μ L of positive anti-primer (50ng/ μ L), tobacco template DNA (20ng/ μ L) 2 μ L, add ddH ₂o to 25 μ L.

Primer is the quadruple primer of primer 1+ primer 2+primer 3+ primer 4.Concrete sequence is as follows:

Primer 1:

SEQ ID NO:1(5’→3’)TGTGGAGCTCCTTTCTTTGC

SEQ ID NO:2(5’→3’)TCAAATCAACAACAAATCCAAT

Primer 2:

SEQ ID NO:3(5’→3’)AAACTTGAAGCAGAGACGGC

SEQ ID NO:4(5’→3’)GCACATGCGGATCTTGATTT

Primer 3:

SEQ ID NO:5(5’→3’)TGTGTGCACCCTCCAATTTA

SEQ ID NO:6(5’→3’)TGATCTCTAGAGTGGTGGCATC

Primer 4:

SEQ ID NO:7(5’→3’)AATGTCTGCCCAATCGAAAG

SEQ ID NO:8(5’→3’)CGAATAACGACACTCGAACG

Response procedures is touch down program, is specially 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s (each cycle down 0.5 DEG C), 72 DEG C extend 30s, and after 10 circulations, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 45s, 25 circulations, and last 72 DEG C extend 10min.

PCR reaction is carried out on System-9700 type amplification instrument, the detection of amplified production is undertaken by argentation, electrophorogram is shown in accompanying drawing 1 (wherein swimming lane 1 is respectively QY97M, cloud and mist 97, K326, cloud and mist 87, T24, blade Huang, recessed leaf cigarette, T09-1, objective field cigarette for DNA molecular amount Marker, swimming lane 2-12).As can be seen from accompanying drawing 1, variant banding pattern between the QY97M that systematic breeding goes out from cloud and mist 97 and cloud and mist 97, the T24 that systematic breeding goes out from cloud and mist 87 and mutual variant banding pattern between recessed leaf cigarette and cloud and mist 87 three, variant banding pattern between the blade Huang that systematic breeding goes out from K326 and K326, also all mutual variant banding pattern between cloud and mist 87, cloud and mist 97, K326, T09-1, objective field cigarette etc., can differentiate by the quadruple primer PCR method of this invention.

Contriver, according to the presence or absence of these 9 kind pcr amplification characteristic bands, obtains the DNA fingerprinting (accompanying drawing 6) of 9 kinds, can as the appraisal basis of these 9 kinds.The discriminating route map of these 9 kinds is shown in accompanying drawing 7.In Fig. 6, black table is shown with onesize PCR band appearance, and such as K326 has 110bp band, and cloud and mist 87 does not have 110bp band.

Embodiment 2: the DNA fingerprinting from 20 tobacco breds in Chongqing is identified

20 tobacco breds from Chongqing are respectively: cloud and mist 97, cloud and mist 87, Yun yan85, cloud and mist 99, cloud and mist 105, No. 1, your cigarette, No. 2, your cigarette, No. 1, Bi Na, No. 1, gold sea, peace No. 2, cigarette, No. 11, Henan cigarette, Qin's cigarette 201, middle cigarette 104, middle cigarette 203, CF223, K326, Coker206, PVH1452, PVH2254, NC196.

1, Quadruple-PCR amplification:

Pcr amplification and result judge:

Primer is four pairs of primers of sequence SEQ ID NO:1-8.Concrete sequence is shown in embodiment 1.

PCR reaction is carried out on System-9700 type amplification instrument, and the detection of amplified production is undertaken by argentation.From accompanying drawing 2, (wherein the left side first swimming lane is that DNA molecular amount Marker, 1-20 represent 20 tobacco breds respectively, and variety name is respectively 1 cloud and mist 97, 2 clouds and mists 87, 3 Yun yan85, 4 clouds and mists 99, 5 clouds and mists 105, 6 No. 1, your cigarette, 7 No. 2, your cigarette, 8 No. 1, Bi Na, 9 No. 1, gold medal seas, 10 peace No. 2, cigarettes, 11 No. 11, Henan cigarettes, 12 Qin's cigarettes 201, cigarette 104 in 13, cigarette 203 in 14, 15CF223, 16K326, 17Coker206, 18PVH1452, 19PVH2254, 20NC196), accompanying drawing 8 and accompanying drawing 9 can be found out, can directly by cloud and mist 97 by a Quadruple-PCR amplification, cloud and mist 99, No. 1, your cigarette, No. 1, Bi Na, Qin's cigarette 201, CF223, PVH1452, NC196, 9 kinds such as middle cigarette 203 grade are differentiated to make a distinction from other kind, all the other 11 kinds are divided into 4 groups, but in group not separately, wherein, cloud and mist 105, No. 2, your cigarette, four materials such as peace No. 2, cigarette and K326 are one group, No. 1, gold sea and PVH2254 two materials are one group, No. 11, Henan cigarette, middle cigarette 104 and Coker206 tri-materials are one group, cloud and mist 87 and Yun yan85 two materials are one group.In Fig. 8, black table is shown with onesize PCR band appearance, and such as cloud and mist 97 has 110bp band, and cloud and mist 87 does not have 110bp band.

In order to distinguish above-mentioned 4 groups of totally 11 tobacco breds do not separated separately, carry out double PCR amplification.

2, double PCR amplification:

Pcr amplification and result judge:

The total system of pcr amplification reaction is totally 25 μ L, wherein Easy Taq archaeal dna polymerase (5U/ μ L) 0.25 μ L, dNTPs (10mmol/L) 0.5 μ L 10 × Easy Taq buffer (containing MgCl ₂) 2 μ L, MgCl ₂(20mmol/L) 1 μ L, each 0.25 μ L of positive anti-primer (50ng/ μ L), tobacco template DNA (20ng/ μ L) 2 μ L, add ddH ₂o to 25 μ L.

Primer is the double primer of primer 5+ primer 6, and sequence is as follows:

Primer 5:

SEQ ID NO:9(5’→3’)CCAACTCTACCGCTAACTTCAAA

SEQ ID NO:10(5’→3’)CACGACTGACGAGACATGGT

Primer 6:

SEQ ID NO:11(5’→3’)TCACCAATTATGCCGATTCA

SEQ ID NO:12(5’→3’)CACACACTTGTTTGGAATTGAGA

PCR program is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s (each cycle down 0.5 DEG C), 72 DEG C extend 30s, and after 10 circulations, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 45s, 25 circulations, and last 72 DEG C extend 10min.

PCR reaction is carried out on System-9700 type amplification instrument, and the detection of amplified production is undertaken by argentation.As can be seen from accompanying drawing 3, accompanying drawing 8 and accompanying drawing 9, increased by this double PCR, last quadruple primer PCR No. 1, the gold sea do not distinguished of increasing is distinguished with PVH2254, No. 11, Henan cigarette, middle cigarette 104, Coker206 are distinguished, cloud and mist 87, Yun yan85 are distinguished, and cloud and mist 105, No. 2, your cigarette are distinguished from " cloud and mist 105, No. 2, your cigarette; peace No. 2, cigarette and K326 " group, and only remaining K326 and peace No. 2, cigarette do not make a distinction.

In order to No. 2, the peace cigarette do not made a distinction and K326 two material sections are separated, proceed pcr amplification.

3, substance pcr amplification:

Pcr amplification and result judge:

Substance pcr amplification reaction is totally 10 μ L, and reactant comprises 10 × Easy Taq buffer (containing MgCl ₂) 1.67 μ L, dNTPs (10mmol/L) 0.25 μ L, Easy Taq archaeal dna polymerase (5U/ μ L) 0.125 μ L, each 0.365 μ L of positive anti-primer (50ng/ μ L), template DNA (20ng/ μ L) 1 μ L, adds ddH ₂o to 10 μ L.

Primer is primer 7.Sequence is as follows:

Primer 7:

SEQ ID NO:13(5’→3’)TTTAAGCTCATTTGAGCCCG

SEQ ID NO:14(5’→3’)CGTATTTGAGATTTATATGCCTTCG

PCR program is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 55 DEG C ~ 60 DEG C annealing 30s, and 72 DEG C extend 30s, 35 circulations, and last 72 DEG C extend 10min, 4 DEG C of preservations.

PCR reaction is carried out on System-9700 type amplification instrument.The detection of amplified production is undertaken by argentation.As can be seen from accompanying drawing 4, accompanying drawing 8 and accompanying drawing 9, by this substance pcr amplification, peace No. 2, cigarette and K326 two material sections can be separated.

So far, through three pcr amplifications, all 20 materials can be distinguished completely.

Contriver, according to the presence or absence of these 20 kind pcr amplification characteristic bands, obtains the DNA fingerprinting (see accompanying drawing 8) of 20 kinds, can as the appraisal basis of these 20 kinds.Discriminating route map from these 20 kinds in Chongqing is shown in accompanying drawing 9.

Embodiment 3: from Genetic relationship and the genetic cluster of 7 tobacco breds in Guizhou

Experimental cultivar is: " K326 ", " cloud and mist 87 ", " T24 ", " blade is yellow ", " recessed leaf cigarette ", " T09-1 ", " objective field cigarette " totally 7 kinds.

Pcr amplification and result judge:

Primer 1:

SEQ ID NO:1(5’→3’)TGTGGAGCTCCTTTCTTTGC

SEQ ID NO:2(5’→3’)TCAAATCAACAACAAATCCAAT

Primer 2:

SEQ ID NO:3(5’→3’)AAACTTGAAGCAGAGACGGC

SEQ ID NO:4(5’→3’)GCACATGCGGATCTTGATTT

Primer 3:

SEQ ID NO:5(5’→3’)TGTGTGCACCCTCCAATTTA

SEQ ID NO:6(5’→3’)TGATCTCTAGAGTGGTGGCATC

Primer 4:

SEQ ID NO:7(5’→3’)AATGTCTGCCCAATCGAAAG

SEQ ID NO:8(5’→3’)CGAATAACGACACTCGAACG

PCR reaction is carried out on System-9700 type amplification instrument, and the detection of amplified production is undertaken by argentation, and electrophorogram is shown in accompanying drawing 1.Get banding pattern clear, press out with or no record, amplified production with O, 1 statistics building database, on identical migration position (same molecular amount fragment) have band assignment be 1, without band note assignment be 0.STATISTIC analysis software NTSYSpc1.0.0.0 is adopted to calculate genetic distance (GD, genetic distance), and with Ni-like ions method (unweightedpair-group method with arithmetic means, UPGMA) constructing system clustering tree.The genetic cluster figure of 7 tobacco breds is shown in accompanying drawing 10.

As can be seen from accompanying drawing 10,7 experimental cultivars can be divided three classes when genetic distance is 0.27, " K326 ", " blade is yellow ", " T09-1 " they are a class, and " cloud and mist 87 ", " T24 ", " recessed leaf cigarette " they are a class, objective field cigarette " for another kind of.From cultivar origin, " T24 " and " recessed leaf cigarette " is the strain selected through Systematic selection from " cloud and mist 87 ", applies method of the present invention, and " cloud and mist 87 ", " T24 ", " recessed leaf cigarette " in order to a class, are originated very identical with its pedigree by poly-; " blade yellow " be from " K326 " through the strain that Systematic selection selects, apply method of the present invention, " K326 ", " blade Huang " also gathered in a class, originates also very identical with its pedigree.Therefore prove, method of the present invention is used for tobacco bred Relationship iden-tification and genetic cluster analysis is feasible, reliable.

Embodiment 4: from Genetic relationship and the genetic cluster of 20 tobacco breds in Chongqing

20 tobacco breds are respectively: cloud and mist 97, cloud and mist 87, Yun yan85, cloud and mist 99, cloud and mist 105, No. 1, your cigarette, No. 2, your cigarette, No. 1, Bi Na, No. 1, gold sea, peace No. 2, cigarette, No. 11, Henan cigarette, Qin's cigarette 201, middle cigarette 104, middle cigarette 203, CF223, K326, Coker206, PVH1452, PVH2254, NC196.

Pcr amplification and result judge:

Primer is the quadruple primer of primer 1+ primer 2+primer 3+ primer 4.Concrete sequence is with above embodiment.

PCR reaction is carried out on System-9700 type amplification instrument, and the detection of amplified production is undertaken by argentation.Electrophorogram is shown in accompanying drawing 2.Get banding pattern clear, press out with or no record, amplified production with O, 1 statistics building database, on identical migration position (same molecular amount fragment) have band assignment be 1, without band note assignment be 0.STATISTIC analysis software NTSYSpc1.0.0.0 is adopted to calculate genetic distance (GD, genetic distance), and with Ni-like ions method (unweightedpair-group method with arithmetic means, UPGMA) constructing system clustering tree.

The genetic cluster figure obtaining 20 tobacco breds is shown in accompanying drawing 5.20 flue-cured tobacco cultivars genetic distance is between any two between 0.0667 ~ 1.0000.When genetic similarity is 0.69,20 tobacco breds are divided into three major types, monoid I comprises 10 kinds such as cloud and mist 97, PVH1452, PVH2254, No. 2, your cigarette, K326, cloud and mist 105, peace No. 2, cigarette, cloud and mist 99, Qin's cigarette 201, middle cigarette 104, monoid II comprises No. 1, your cigarette, No. 11, Henan cigarette, No. 1, gold sea, Coker206, middle cigarette 203,7 kinds such as CF223, NC196, and monoid III comprises 3 kinds such as No. 1, cloud and mist 87, Yun yan85 and Bi Na.Between the kind of realm genetic distance and sibship nearer, as in monoid III between cloud and mist 87 and Yun yan85 genetic distance nearest, be 0.0667, both explanations sibship is nearer, and middle cigarette in monoid I 104 and No. 1, Bi Na have genetic distance farthest, both explanations sibship is far away.

Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention when not departing from principle of the present invention and aim, revising, replacing and modification.

Claims

1. one group of primer sets, is characterized in that, described primer sets comprises, primer one, primer two, the combination of primer three and primer four; And/or the combination of primer five and primer six;

Described primer one is made up of SEQ ID NO:1 and SEQ ID NO:2,

Described primer two is made up of SEQ ID NO:3 and SEQ ID NO:4,

Described primer three is made up of SEQ ID NO:5 and SEQ ID NO:6,

Described primer four is made up of SEQ ID NO:7 and SEQ ID NO:8;

Described primer five is made up of SEQ ID NO:9 and SEQ ID NO:10,

Described primer six is made up of SEQ ID NO:11 and SEQ ID NO:12;

Optional, described primer sets comprises primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, at least one in primer 14 and primer 15;

Described primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, primer 14 and primer 15 are made up of SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ IDNO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ IDNO:28, SEQ ID NO:29 and SEQ ID NO:30 respectively.

2. a test kit, is characterized in that, comprises primer sets according to claim 1.

3. the test kit of primer sets according to claim 1 or claim 2 is being differentiated tobacco bred and/or is being identified the purposes in the sibship of different tobacco bred.

4. differentiate a method for tobacco bred, it is characterized in that, employ primer sets according to claim 1 or test kit according to claim 2.

5. the method for discriminating tobacco bred according to claim 4, is characterized in that, comprise step,

Extract the genomic dna of tobacco to be checked;

Primer one, primer two, primer three and primer four is utilized to carry out Quadruple-PCR to described genomic dna, obtain the first amplified production, described primer one is made up of SEQ ID NO:1 and SEQ ID NO:2, described primer two is made up of SEQ ID NO:3 and SEQ ID NO:4, described primer three is made up of SEQ ID NO:5 and SEQ ID NO:6, and described primer four SEQ ID NO:7 and SEQ ID NO:8 forms;

Utilize described first amplified production to differentiate the kind of described tobacco to be checked, obtain the first identification result;

Optional, also comprise,

Primer five and primer six is utilized to carry out double PCR to the genomic dna of U/I tobacco in described first identification result, obtain the second amplified production, described primer five is made up of SEQ ID NO:9 and SEQ ID NO:10, and described primer six is made up of SEQ ID NO:11 and SEQ ID NO:12

Utilize the second amplified production to differentiate the kind of U/I tobacco in described first identification result, obtain the second identification result;

Optional,

Utilize primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, one in primer 14 and primer 15 is carried out substance PCR to the genomic dna of U/I tobacco in described second identification result, obtain the 3rd amplified production, described primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, primer 14 and primer 15 are respectively by SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30 forms,

Utilize described 3rd amplified production to differentiate the kind of U/I tobacco in described second identification result, obtain the 3rd identification result.

6. the method for discriminating tobacco bred according to claim 5, is characterized in that, described genomic dna from the blade of tobacco seedling, leaf bud and tobacco leaf one of at least;

Optional, the response procedures of described Quadruple-PCR and described double PCR is { 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, each cycle down 0.5 DEG C, 72 DEG C of 30s}, { 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 45s} and 72 DEG C 10min of 25 circulations of 10 circulations;

Optional, the reaction system of described Quadruple-PCR and double PCR is the Easy Taq archaeal dna polymerase 0.25 μ L of 5U/ μ L, and the dNTPs 0.5 μ L of 10mmol/L, containing MgCL ₂10 × Easy Taq solution 2 μ L, the MgCl of 20mmol/L ₂the genomic dna 2 μ L of each 0.25 μ L, the 20ng/ μ L of positive anti-primer of the corresponding primer of 1 μ L, 50ng/ μ L, ddH ₂o adds to 25 μ L;

Optional, the response procedures of described substance PCR comprises: { 94 DEG C of 5min, 94 DEG C of 30s, 55 DEG C ~ 60 DEG C 30s, 72 DEG C of 30s} and 72 DEG C 10min of 35 circulations;

Optional, the amplification system of described substance PCR comprises: containing MgCL ₂10 × Easy Taq solution 1.67 μ L, the Easy Taq archaeal dna polymerase 0.125 μ L of the dNTPs 0.25 μ L of 10mmol/L, 5U/ μ L, the genomic dna 1 μ L of each 0.365 μ L, the 20ng/ μ L of positive anti-primer of the corresponding primer of 50ng/ μ L, ddH ₂o adds to 10 μ L;

Optional, the described kind utilizing the first amplified production to differentiate described tobacco to be checked, comprises,

Electrophoresis detection is carried out to described first amplified production, obtains the first electrophoresis result,

The specific band comprised based on described first electrophoresis result carries out described discriminating;

Optional, the described kind utilizing the second amplified production to differentiate U/I tobacco in the first identification result, comprises,

Electrophoresis detection is carried out to described second amplified production, obtains the second electrophoresis result,

The specific band comprised based on described second electrophoresis result carries out described discriminating;

Optional, the described kind utilizing the 3rd amplified production to differentiate U/I tobacco in the second identification result, comprises,

Electrophoresis detection is carried out to described 3rd amplified production, obtains the 3rd electrophoresis result,

The specific band comprised based on described 3rd electrophoresis result carries out described discriminating.

7. identify a method for different tobacco bred sibship, it is characterized in that, employ primer sets according to claim 1 or test kit according to claim 2.

8. identify the method for different tobacco bred sibship described in claim 7, it is characterized in that, comprise step,

Extract the genomic dna of multiple tobacco respectively;

Primer one, primer two, primer three and primer four is utilized to carry out Quadruple-PCR to the genomic dna respectively grown tobacco respectively, obtain the first amplified production respectively grown tobacco, described primer one is made up of SEQ ID NO:1 and SEQ ID NO:2, described primer two is made up of SEQ ID NO:3 and SEQ ID NO:4, described primer three is made up of SEQ ID NO:5 and SEQ ID NO:6, and described primer four SEQ ID NO:7 and SEQ ID NO:8 forms;

Utilize described the first amplified production respectively grown tobacco to determine the sibship of the tobacco at least partially in described multiple tobacco, obtain the first sibship;

Optional, also comprise,

Primer five and the genomic dna of primer six to the tobacco at least partially in described first sibship is utilized to carry out double PCR, obtain corresponding second amplified production, described primer five is made up of SEQ ID NO:9 and SEQ ID NO:10, described primer six is made up of SEQ ID NO:11 and SEQ ID NO:12

Utilize the second amplified production to determine the sibship of the tobacco at least partially in described first sibship, obtain the second sibship;

Optional,

Utilize primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, one in primer 14 and primer 15 is carried out substance PCR to the genomic dna of the tobacco at least partially in described second sibship, obtain corresponding 3rd amplified production, described primer seven, primer eight, primer nine, primer ten, primer 11, primer 12, primer 13, primer 14 and primer 15 are respectively by SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ IDNO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30 forms,

Utilize described 3rd amplified production to determine the sibship of the tobacco at least partially in described second sibship, obtain the 3rd sibship.

9. identify the method for different tobacco bred sibship described in claim 8, it is characterized in that, described genomic dna from the blade of tobacco seedling, leaf bud and tobacco leaf one of at least;

Optional, the response procedures of described substance PCR is { 94 DEG C of 5min, 94 DEG C of 30s, 55 DEG C ~ 60 DEG C 30s, 72 DEG C of 30s} and 72 DEG C 10min of 35 circulations;

Optional, the amplification system of described substance PCR is for containing MgCL ₂10 × Easy Taq solution 1.67 μ L, the Easy Taq archaeal dna polymerase 0.125 μ L of the dNTPs 0.25 μ L of 10mmol/L, 5U/ μ L, the genomic dna 1 μ L of each 0.365 μ L, the 20ng/ μ L of positive anti-primer of the corresponding primer of 50ng/ μ L, ddH ₂o adds to 10 μ L;

Optional, the first amplified production that described utilization respectively grows tobacco determines the sibship of the tobacco at least partially in multiple tobacco, comprises,

Electrophoresis detection is carried out to described the first amplified production respectively grown tobacco, obtains the first electrophoresis result,

The number of the specific band comprised based on described first electrophoresis result carries out the determination of described sibship;

Optional, the described sibship utilizing the second amplified production to determine the tobacco at least partially in the first sibship, comprises,

The number of the specific band comprised based on described second electrophoresis result carries out the determination of described sibship;

Optional, the described sibship utilizing the 3rd amplified production to determine the tobacco at least partially in the second sibship, comprises,

The number of the specific band comprised based on described 3rd electrophoresis result carries out the determination of described sibship.

10. the purposes of claim 3 or the method for the different tobacco bred of the arbitrary described discriminating of claim 4-6 or the arbitrary described method determining different tobacco bred sibship of claim 7-9, it is characterized in that, described tobacco bred is cloud and mist 97, cloud and mist 87, Yun yan85, cloud and mist 99, cloud and mist 105, No. 1, Bi Na, No. 1, gold sea, peace No. 2, cigarette, No. 11, Henan cigarette, blue or green cigarette 201, middle cigarette 103, middle cigarette 104, CF223, PVH2254, NC196, coker176, coker206, coker254, coker258, coker298, coker319, CZ-1, G23, HB030, K326, K346, K358, M373, MC944, MSK326, MS cloud and mist 87, NC13, NC27NF, NC567, NC628, NC82, NC89, NC95, RG11, RG8, RG97, Samsun, SC71, TW90, Viginia, Bath horse, reach white No. 1, reach white No. 2, E'yan 1, reform No. 3, No. 1, your cigarette, No. 2, your cigarette, No. 3, your cigarette, Guiyan 4, rustica, No. 2, leek level ground, No. 1, Lou Shan, Maryland, Qin's cigarette 201, Qin's cigarette 96, fine large black smoke falls, prestige rather spends cigarette in vain, No. one, cloud, Yunyan201 S, more than one in cloud and mist 317.