CN102492774A - Primers and method for quickly distinguishing orange varieties - Google Patents
Primers and method for quickly distinguishing orange varieties Download PDFInfo
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Abstract
The invention discloses primers and a method for quickly distinguishing orange varieties, and belongs to the field of molecular marking in molecular biology. The method comprises the steps of: designing RAPD (Random Amplified Polymorphic Deoxyribonucleic Acid) random primers according to the gene sequences of the orange, and screening to obtain 16 random primers; extracting DNA (Deoxyribonucleic Acid) of a plurality of orange varieties, and diluting for later use; and amplifying the DNA of the unknown orange varieties by utilizing the random primers one by one, distinguishing out the variety with unique characteristic band, and building a tree-shaped identification figure. According to the invention, 48 orange varieties can be distinguished on a molecular level through a plurality of PCR (Polymerase Chain Reaction), the practicability of RAPD molecular marking utilizing 11 base primers in plant variety identification is embodied to a certain extent, and the obtained tree-shaped identification figure is more illustrative than the clustering tree, namely the primers for distinguishing any two varieties can be found out according to the variety identification figure; and the method can realize early identification of orange seedlings, and has wide universality in other species.
Description
Technical field
The present invention relates to molecular biology molecule marker field, relate in particular to a kind of primer and method of quick differentiation oranges and tangerines kind.
Background technology
Oranges and tangerines belong to the Rutaceae fruit tree, comprise tangerine, mandarin orange, shaddock, orange, lemon etc., mainly are distributed in the zone on the south 35 ° of the north latitude in the world.Citrus plant is one of main fruit tree species of planting of the China and the world always.The Citrus variety source is very abundant.Therefore, set up Citrus kind rapid and reliable authenticate technology system for nursery stock identify in early days, the kind rights protection, produce in sibship and the genetic diversity etc. of differentiation and research Citrus germ plasm resource of kind have important and practical meanings.Up to the present, traditional single morphology cultivar identification method is owing to receive the influence of environment to be difficult to effectively distinguish or differentiate numerous kinds; On the other hand, many Citrus breeding parents more and more concentrate on the minority improved seeds or article are fastened, and make that the new variety of institute's seed selection are more similar on more proterties, and are difficult to distinguish.How effectively to identify kind in the research and production, just become one and usually run into, and very important problem.Still do not utilize dna marker to carry out the quick and measure efficiently of cultivar identification so far, can't form foundation and reference when carrying out cultivar identification yet.Therefore, cultivar identification just lacks purpose, and non-directly perceived, randomness is too strong simultaneously, inefficiency.For this reason, it is significant that the new measure of cultivar identification is carried out in exploitation fast.
Molecule marker is the one type of genetic marker that produces along with the development of molecular cloning and recombinant DNA technology.In recent years, molecular marking technique has been widely used in Idioplasm identification and the fingerprinting of vegetable crop.Because the DNA analysis technology does not receive the influence of environment, etap, sampling point, the polymorphic site that detects is unlimited, and the result has the safety of height, and resolving ability is strong, and repeatability is high.In RAPD commonly used, RFLP, SSR, AFLP equimolecular labeling technique; RAPD (Random Amplified Polymorphic DNA; Randomly amplified polymorphic DNA) developing history is long; Be to use one of dna marker technology the earliest; It with quick, easy, be difficult for the characteristic and advantage that is affected by the external environment and need do not foresee genome sequence, be widely used in the aspect such as Study on Genetic Basis, gene linkage mark, construction of genetic atlas of sort research, the germ plasm resource of kind.
Existing RAPD adopts computer software to draw the digitizing finger printing in the Idioplasm identification of vegetable crop more, and combines statistics software to carry out cluster analysis, differentiates but the result of cluster analysis often can't be used for kind.Chinese patent " based on the cowpea variety molecular assay method of Genome RAPD analysis " (patent No. ZL200510018593.5) discloses the method for distinguishing and judge the cowpea different varieties based on RAPD; Obtain 23 primers through screening, adopt the 0-1 fingerprint and construct clustering tree.This patent adopts primer more, and operational ton is very big when quality is identified, do not possess ageing, through the primer of the bad judgement differentiation of clustering tree kind, and directly perceived inadequately.
Summary of the invention
Goal of the invention: the purpose of this invention is to provide a kind of primer and method of quick differentiation oranges and tangerines kind, be used for quick differentiation, identify the oranges and tangerines kind.
Technical scheme: to achieve these goals, the primer of a kind of quick differentiation oranges and tangerines kind of the present invention comprises:
Y17：5’-AGGGGTCTTGG-3’，
Y23：5’-GGACCCAACCG-3’，
Y28：5’-GTGTGCCCCAT-3’，
Y40：5’-AGCGTCCTCCT-3’，
Y41：5’-AGCGTCCTCCG-3’，
Y47：5’-ACGACCGACAG-3’，
Y54：5’-TGGTGGCGTTC-3’，
Y55：5’-ACCCCCGACTT-3’，
Y59：5’-ACCCCCGACTG-3’，
Y60：5’-ACCCCCGACTC-3’，
1S2：5’-GGGTAACGCCA-3’，
1S3：5’-GGGTAACGCCT-3’，
1S4：5’-GGGTAACGCCT-3’，
5S2：5’-CCGCTACCGAA-3’，
5S5：5’-CCGCTACCGAG-3’，
A5：5’-GTCCACACGGT-3’；
Because RAPD amplification self, the design of random primer is seemed particularly important.The primer of general RAPD is 9～10 bases, and in theory, primer is short more, and template sequence two ends and primer complementary site quantity are many more, the amplification PCR rich polymorphism; But primer is shorter, also can cause the low problem of template complementary pairing stability.The present invention adopts the primer of 11 bases, can improve the stability of RAPD reaction.
In addition, the annealing temperature of primer is also extremely important, so the present invention will carry out strict screening to primer behind the some random primers of design.Said primer is to filter out the primer that band is clear, the PCR product repeats to occur through the random primer that designs through 3 subgradient PCR.Primer will be selected the random primer that amplification property is strong, stability is high, polymorphum is good, satisfies the primer requirement that is used for analyzing gene group polymorphum.
For utilizing above-mentioned primer Rapid identification to distinguish the method for oranges and tangerines kind, comprise the steps:
(1) extraction need be identified the oranges and tangerines DNA of differentiation, diluted for use;
(2) utilize primer 1S3 that oranges and tangerines DNA is carried out the RAPD amplification,
Then be classified as the A group as if characteristic spectrum belt and 550bp atypism bands of a spectrum occur at 480bp, 600bp, get into step (3);
Then be classified as the B group if characteristic spectrum belt all occurs, get into step (4) at 480bp, 550bp, 600bp;
Then be classified as the C group as if characteristic spectrum belt and 600bp atypism bands of a spectrum occur at 480bp, 550bp, get into step (5);
(3) utilize primer Y40 that the DNA of A group is carried out the RAPD amplification, then be classified as the A1 group, get into step (31) as if characteristic spectrum belt and 650bp atypism bands of a spectrum occur at 550bp; If 550bp and 650bp all the atypism bands of a spectrum then be classified as the A2 group, entering step (32); If at 1400bp atypism bands of a spectrum, oranges and tangerines kind ' Xie Cheng ' is distinguished; If characteristic spectrum belt occurs at 650bp, the oranges and tangerines kind ' is not known fire ' and is distinguished;
(31) utilize primer 5S2 that the DNA of A1 group is carried out the RAPD amplification, if at 600bp atypism bands of a spectrum, oranges and tangerines kind ' navel is orange in vain ' is distinguished; If characteristic spectrum belt occurs at 600bp, oranges and tangerines kind ' fragrant and sweet orange ' is distinguished;
(32) utilize primer Y41 that the DNA of A2 group is carried out the RAPD amplification, then be classified as the A2-1 group, get into step (321) if characteristic spectrum belt occurs at 1400bp; If at 1400bp atypism bands of a spectrum, oranges and tangerines kind ' companion is wild ' is distinguished;
(321) utilize primer A5 that the DNA of A2-1 group is carried out the RAPD amplification, if at 1000bp atypism bands of a spectrum, oranges and tangerines kind ' between upright ' is distinguished; If characteristic spectrum belt occurs at 1000bp, oranges and tangerines kind ' fragrant citrus ' is distinguished;
(4) utilize primer Y40 that the DNA of B group is carried out the RAPD amplification, if characteristic spectrum belt all occurs and 950bp atypism bands of a spectrum then are classified as the B1 group, entering step (41) at 550bp and 600bp; If 550bp and 600bp all the atypism bands of a spectrum then be classified as the B2 group, entering step (42); If characteristic spectrum belt occurs and 550bp atypism bands of a spectrum then are classified as the B3 group, entering step (43) at 600bp and 950bp; Then be classified as the B4 group if characteristic spectrum belt all occurs, get into step (44) at 550bp, 600bp, 950bp; As if characteristic spectrum belt and 550bp, 950bp atypism bands of a spectrum occur at 600bp, oranges and tangerines kind ' early red ' is distinguished; If characteristic spectrum belt occurs at 600bp, 1100bp, oranges and tangerines kind ' u'eno ' is distinguished; As if characteristic spectrum belt and 600bp, 950bp atypism bands of a spectrum occur at 550bp, oranges and tangerines kind ' from generation to generation ' is distinguished;
(41) utilize primer Y59 that the DNA of B1 group is carried out the RAPD amplification, then be classified as the B1-1 group, get into step (411) if characteristic spectrum belt occurs at 1800bp; If at 1800bp atypism bands of a spectrum, oranges and tangerines kind ' bridge originally ' is distinguished;
(411) utilize primer Y17 that the DNA of B1-1 group is carried out the RAPD amplification, if characteristic spectrum belt occurs at 1000bp, oranges and tangerines kind ' various household supplies ' is distinguished; If at 1000bp atypism bands of a spectrum, oranges and tangerines kind ' between upright ' is distinguished;
(42) utilize primer Y55 that the DNA of B2 group is carried out the RAPD amplification, if characteristic spectrum belt all occurs at 300bp, 350bp, 1200bp, oranges and tangerines kind ' tertia ' is distinguished; If characteristic spectrum belt all occurs at 450bp, 750bp, oranges and tangerines kind ' southern mandarin orange ' is distinguished; If characteristic spectrum belt and 750bp atypism bands of a spectrum occur at 450bp, oranges and tangerines kind ' Nanfeng orange ' is distinguished; If characteristic spectrum belt occurs at 300bp, characteristic spectrum belt does not appear simultaneously at 350bp, 1200bp, be classified as the B2-1 group, get into step (421); If 450bp, 1200bp all the atypism bands of a spectrum then be classified as B2-2 group, get into step (422);
(421) utilize primer Y28 that the DNA of B2-1 group is carried out the RAPD amplification, then be classified as the B2-11 group, get into step (4211) if characteristic spectrum belt occurs at 2600bp; If then be classified as the B2-12 group at 2600bp atypism bands of a spectrum, get into step (4212);
(4211) using the primers 5S5 B2-11 group on the DNA RAPD amplification, if the characteristic bands appear at 850bp and 1200bp in 750bp or no characteristic bands, variety 'HB grapefruit' is distinguished; if 850bp , 1200bp characteristic bands appeared, species 'wilderness' is distinguished; if 750bp, 850bp characteristic bands appeared, species 'long can' be distinguished; if 850bp, 1200bp are no characteristic bands, variety 'Great Fen' is distinguished; if 850bp, 1800bp characteristic bands appeared, re-use for the RAPD primers 1S2, if the characteristic bands appear at 1300bp compared to 'feed the red'; if no characteristic 1300bp band was 'up and down the red';
(4212) using the primers 1S2 B2-12 group on the DNA RAPD amplification, if the characteristic bands appear at 1300bp, variety 'citrus 1' be distinguished; if the characteristic bands appear at 900bp, species' Youth Work 'be distinguished; if 1300bp and 900bp are no characteristic bands, variety' City People 'be distinguished;
(422) utilize primer 1S4 that the DNA of B2-2 group is carried out the RAPD amplification, if characteristic spectrum belt occurs at 750bp, kind ' this morning No. 6 ' is distinguished; If at 750bp atypism bands of a spectrum, kind ' orange mandarin orange No. 3 ' is distinguished;
(43) utilize primer Y59 that the DNA of B3 group is carried out the RAPD amplification,, get into step (431) if having characteristic spectrum belt then to be classified as the B3-1 group at 1800bp; If then be classified as the B3-2 group at 1800bp atypism bands of a spectrum, get into step (432);
(431) utilize primer Y17 that the DNA of B3-1 group is carried out the RAPD amplification, if at 1000bp, 450bp characteristic spectrum belt is arranged all, kind ' flat red lemon ' is distinguished; If all do not have characteristic spectrum belt at 250bp, 600bp, kind ' Calusena lansium ' is distinguished; If at 600bp characteristic spectrum belt is arranged, kind ' Gan Xia ' is distinguished; If all do not have characteristic spectrum belt at 1800bp, 450bp, kind ' granulated sugar tangerine ' is distinguished; If have characteristic spectrum belt and 1000bp atypism bands of a spectrum then to be classified as one type, increase with primer Y47 again, if characteristic spectrum belt is arranged then ' Ying Liman I ' is if at 1800bp atypism bands of a spectrum then be kind ' between standing ' for kind at 1800bp at 450bp;
(432) utilize primer Y60 that the DNA of B3-2 group is carried out the RAPD amplification, if at 750bp characteristic spectrum belt is arranged, kind ' Wanbai pummelo ' is distinguished; If at 800bp characteristic spectrum belt is arranged, kind ' orange mandarin orange No. 3 ' is distinguished; If all do not have characteristic spectrum belt at 750bp, 850bp, kind ' the big tangerine of Western Hills ' is distinguished;
(44) utilize primer Y59 that the DNA of B4 group is carried out the RAPD amplification,, get into step (441) if all have characteristic spectrum belt then to be classified as the B4-1 group at 1200bp, 1800bp; Then be classified as the B4-2 group as if characteristic spectrum belt and 1200bp atypism bands of a spectrum occur at 1800bp, get into step (442); If at 1200bp and 1800bp atypism bands of a spectrum all, kind ' good fortune tangerine ' quilt is distinguished;
(441) utilize primer Y17 that the DNA of B4-1 group is carried out the RAPD amplification; If characteristic spectrum belt all occurs at 200bp and 1000bp; Utilize primer Y54 to increase again; If characteristic spectrum belt occurs then for kind ' sweet orange shaddock ', if then be kind ' 4 days National Day ' at 1600bp atypism bands of a spectrum at 1600bp; If characteristic spectrum belt occurs and at 1000bp atypism bands of a spectrum at 200bp; Utilize primer Y23 to increase again; If characteristic spectrum belt occurring at 350bp then is kind ' Mi Jin '; If characteristic spectrum belt occurs then for kind ' Qingniao Co. ' at 650bp, if 350bp, 650bp all the atypism bands of a spectrum then be kind ' three guarantors '; If characteristic spectrum belt and 200bp atypism bands of a spectrum occur at 1000bp, kind ' No. 3, emerging Tianjin ' is distinguished; If at 1000bp atypism bands of a spectrum, kind ' Bei Kou ' is distinguished;
(442) utilize primer Y23 that the DNA of B4-2 group is carried out the RAPD amplification, if characteristic spectrum belt occurs at 250bp, kind ' 5 days National Day ' is distinguished; If at 250bp atypism bands of a spectrum, kind ' warm mandarin orange No. 3 ' is distinguished;
(5) utilize primer Y40 that the DNA of C group is carried out the RAPD amplification, if characteristic spectrum belt occurs at 650bp, kind ' little pomelo ' is distinguished; If at 650bp atypism bands of a spectrum, kind ' shaddock recklessly ' is distinguished.
The RAPD amplification of this method is the PCR reaction system of 30 μ l, comprises 10 * PCR Buffer, 3 μ l, 2.5mmol/LdNTPs 2.4 μ l, 25mmol/LMgCl
21.8 μ l, 1.25UTaq enzyme, random primer 10pmol/ μ l 1.2 μ l, template DNA 40～50ng; React on 95 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, renaturation 1min, 72 ℃ are extended 2min, 42 circulations; 72 ℃ are extended 10min.
The annealing temperature of each primer is as shown in the table in the said RAPD amplified reaction:
Notice, the annealing temperature when the annealing temperature here refers to renaturation.
The present invention just can distinguish and identifies through 16 primers 48 oranges and tangerines kinds; For primer clear, intactly expression evaluation employing and the kind that identifies; Other people distinguish, identify the oranges and tangerines kind for ease simultaneously, and the present invention has also set up " the tree type is differentiated figure ".Said tree type discriminating figure is based on the result that the bands of a spectrum form behind the pcr amplification counts; The tree type is differentiated and is schemed with primer as node; Behind the node differentiation result of this primer to different varieties; Comprise the classification distinguished or the kind that directly distinguishes, the branch behind node also mark the foundation distinguished each other of Different Results, i.e. difference between the bands of a spectrum.For example: 650bp (+) is illustrated in 650bp has characteristic spectrum belt; 1200bp (-) is illustrated in 1200bp atypism bands of a spectrum.The tree type is differentiated the use of figure for ease, and the oranges and tangerines of 48 kinds are as shown in table 1 with the numbering mark, and " the tree type is differentiated figure " please refer to the Fig. 1 in " Figure of description ".
Table 148 oranges and tangerines variety name and reference numeral
Utilize method of the present invention; And " tree type differentiate figure " can greatly make things convenient for evaluation, the differentiation of said 48 oranges and tangerines kinds, and, under the known situation that needs the differentiation kind; Can on " the tree type is differentiated figure ", find the primer of distinguishing these kinds, convenient and swift.
Beneficial effect: the primer that the primer of a kind of quick differentiation oranges and tangerines kind of the present invention adopts 11 bases and obtains through strict screening; Improved the stability of RAPD reaction effectively; Can be easily the oranges and tangerines of 48 kinds be made a distinction; Easy to operate, quick and precisely, embodied the primer that utilizes 11 bases to a certain extent and carried out the RAPD molecule marker and on plant variety is identified, have practicality; The tree type discriminating figure of gained of the present invention has more intuitive than clustering tree, and figure just can find out the primer that can distinguish any two kinds according to the evaluation of tree type, and this method can realize the early stage evaluation of oranges and tangerines nursery stock, on other species, also has versatility widely.
Description of drawings
Fig. 1 utilizes 16 primers the oranges and tangerines of 48 kinds to be distinguished the tree type discriminating figure of evaluation;
Fig. 2 is the RAPD collection of illustrative plates that utilizes 48 oranges and tangerines kind DNA of primer 1S3 amplification, and line is a specific spectruming belt, and the file numeral is corresponding with table 1; M:DNA marker;
Fig. 3 is the RAPD collection of illustrative plates that utilizes primer Y59 that the B3 group is identified, line is a specific spectruming belt, and the file numeral is corresponding with table 1; M:DNA marker;
Fig. 4 is the RAPD collection of illustrative plates that utilizes primer Y60 that 21,25, No. 26 kind DNA cloning are obtained, and line is a specific spectruming belt, and the file numeral is corresponding with table 1; M:DNA marker;
Fig. 5 is that line is a specific spectruming belt to kind 8,12,15,17,20,22, and the file numeral is corresponding with table 1; M:DNA marker;
Fig. 6 adopts the primer 1S2 RAPD collection of illustrative plates that 17,20 amplifications obtain to kind, and line is a specific spectruming belt, and the file numeral is corresponding with table 1; M:DNA marker.
Embodiment
Below in conjunction with accompanying drawing the present invention is done explanation further.
16 primers of the present invention are to screen and get through strictness at original 60 random primers that design.To 60 random primers of genome design of existing oranges and tangerines kind, therefrom choose random primer 11 bases, that amplification property is strong, stability is high, polymorphum is good earlier, filter out the primer that band is clear, the PCR product repeats to occur through 3 subgradient PCR.Specifically, the reaction system of grads PCR is 15 μ l, comprising ddH
2O 8.3 μ l, 10 * Buffer, 1.5 μ l, the Mg2 of 25mmol/L
+1.2 μ l, the dNTPs 1.8 μ l of 2.5mmol/L, the primer 1.2 μ l of 10pmol/ μ L, the template DNA 1 μ l of 30ng/ μ l, the DNApolymerase 0.08 μ l of 5U/ μ l.Response procedures is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30sec, 35～45 ℃ of renaturation 1min, 72 ℃ are extended 2min, and totally 40 circulations extend below 10min at 72 ℃ of temperature condition at last, 4 ℃ of preservations.The annealing temperature of selecting bands of a spectrum are clear, the PCR product repeats to occur primer, this primer according to continuous three subgradient PCR with higher for good, for the bigger then selection medium temperature of optimal temperature scope.
Primer and corresponding annealing temperature that screening obtains are as shown in table 1:
The gene order and the annealing temperature of table 116 primer
The oranges and tangerines sample all picks up from Dongshan, Dongting Lake, Jiangsu Province, the DNA extraction method with reference to the room through your method (Fang Jinggui, chapter town. a kind of short-cut method [J] biotechnology of extracting fruit tree leaf DNA, 1999,9 (2): 31-32.).The DNA that extracts is diluted to 30～50ng/ μ l.For ease the oranges and tangerines kind is described, the oranges and tangerines of different varieties are numbered, as shown in table 2.
Table 248 oranges and tangerines variety name and reference numeral
Utilize primer that oranges and tangerines DNA is carried out the RAPD amplification as required, set up the RAPD amplification reaction system: the PCR reaction system of 30 μ l comprises 10 * PCR Buffer, 3 μ l, 2.5mmol/L dNTPs 2.4 μ l, 25mmol/L MgCl
21.8 μ l, the 1.25UTaq enzyme, random primer 10pmol/ μ l 1.2 μ l, template DNA 40～50ng adds ddH
2O polishing to 30 μ l.The amplified reaction program is: 95 ℃ of preparatory sex change 3min, and 94 ℃ of sex change 30s, renaturation 1min (annealing temperature of different primers is as shown in table 1), 72 ℃ are extended 2min, 42 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations.Be reflected on the eppendorf amplification appearance and carry out, amplified production is electrophoresis 30～60min in 1.3% sepharose, and ethidium bromide staining is observed on the ultraviolet projectoscope and taken pictures.
" the tree type is differentiated figure " also drawn in discriminating, statistical work for ease simultaneously." the tree type is differentiated figure " is based on the result that the bands of a spectrum form behind the pcr amplification counts; The tree type is differentiated and is schemed with primer as node; Behind the node differentiation result of this primer to different varieties; Comprise the classification distinguished or the kind that directly distinguishes, the branch behind node also mark the foundation distinguished each other of Different Results, i.e. difference between the bands of a spectrum.Utilize the oranges and tangerines of 48 kinds to extract DNA after the RAPD amplification of 16 primers can be drawn out complete tree type discriminating figure, as shown in Figure 1.
Utilize 16 primers specific as follows to evaluation, the differentiation of above-mentioned 48 oranges and tangerines kinds:
(1) utilizes primer 1S3 that oranges and tangerines DNA is carried out RAPD amplification (please refer to Fig. 2), then be classified as the A group, get into step (2) as if characteristic spectrum belt and 550bp atypism bands of a spectrum occur at 480bp, 600bp; Then be classified as the B group if characteristic spectrum belt all occurs, get into step (3) at 480bp, 550bp, 600bp; Then be classified as the C group as if characteristic spectrum belt and 600bp atypism bands of a spectrum occur at 480bp, 550bp, get into step (4).
(2) utilize primer Y40 that the DNA of A group is carried out the RAPD amplification, then be classified as the A1 group, get into step (21) as if characteristic spectrum belt and 650bp atypism bands of a spectrum occur at 550bp; If 550bp and 650bp all the atypism bands of a spectrum then be classified as the A2 group, entering step (22); If at 1400bp atypism bands of a spectrum, oranges and tangerines kind ' Xie Cheng ' is distinguished; If characteristic spectrum belt occurs at 650bp, the oranges and tangerines kind ' is not known fire ' and is distinguished.
(21) utilize primer 5S2 that the DNA of A1 group is carried out the RAPD amplification, if at 600bp atypism bands of a spectrum, oranges and tangerines kind ' navel is orange in vain ' is distinguished; If characteristic spectrum belt occurs at 600bp, oranges and tangerines kind ' fragrant and sweet orange ' is distinguished.
(22) utilize primer Y41 that the DNA of A2 group is carried out the RAPD amplification, then be classified as the A2-1 group, get into step (221) if characteristic spectrum belt occurs at 1400bp; If at 1400bp atypism bands of a spectrum, oranges and tangerines kind ' companion is wild ' is distinguished.
(221) utilize primer A5 that the DNA of A2-1 group is carried out the RAPD amplification, if at 1000bp atypism bands of a spectrum, oranges and tangerines kind ' between upright ' is distinguished; If characteristic spectrum belt occurs at 1000bp, oranges and tangerines kind ' fragrant citrus ' is distinguished.
(3) utilize primer Y40 that the DNA of B group is carried out the RAPD amplification, if characteristic spectrum belt all occurs and 950bp atypism bands of a spectrum then are classified as the B1 group, entering step (31) at 550bp and 600bp; If 550bp and 600bp all the atypism bands of a spectrum then be classified as the B2 group, entering step (32); If characteristic spectrum belt occurs and 550bp atypism bands of a spectrum then are classified as the B3 group, entering step (33) at 600bp and 950bp; Then be classified as the B4 group if characteristic spectrum belt all occurs, get into step (34) at 550bp, 600bp, 950bp; As if characteristic spectrum belt and 550bp, 950bp atypism bands of a spectrum occur at 600bp, oranges and tangerines kind ' early red ' is distinguished; If characteristic spectrum belt occurs at 600bp, 1100bp, oranges and tangerines kind ' u'eno ' is distinguished; As if characteristic spectrum belt and 600bp, 950bp atypism bands of a spectrum occur at 550bp, oranges and tangerines kind ' from generation to generation ' is distinguished.
(31) utilize primer Y59 that the DNA of B1 group is carried out the RAPD amplification, then be classified as the B1-1 group, get into step (311) if characteristic spectrum belt occurs at 1800bp; If at 1800bp atypism bands of a spectrum, oranges and tangerines kind ' bridge originally ' is distinguished.
(311) utilize primer Y17 that the DNA of B1-1 group is carried out the RAPD amplification, if characteristic spectrum belt occurs at 1000bp, oranges and tangerines kind ' various household supplies ' is distinguished; If at 1000bp atypism bands of a spectrum, oranges and tangerines kind ' between upright ' is distinguished.
(32) utilize primer Y55 that the DNA of B2 group is carried out the RAPD amplification, if characteristic spectrum belt all occurs at 300bp, 350bp, 1200bp, oranges and tangerines kind ' tertia ' is distinguished; If characteristic spectrum belt all occurs at 450bp, 750bp, oranges and tangerines kind ' southern mandarin orange ' is distinguished; If characteristic spectrum belt and 750bp atypism bands of a spectrum occur at 450bp, oranges and tangerines kind ' Nanfeng orange ' is distinguished; If characteristic spectrum belt occurs at 300bp, characteristic spectrum belt does not appear simultaneously at 350bp, 1200bp, be classified as the B2-1 group, get into step (321); If 450bp, 1200bp all the atypism bands of a spectrum then be classified as B2-2 group, get into step (322).
(321) utilize primer Y28 that the DNA of B2-1 group is carried out the RAPD amplification, then be classified as the B2-11 group, get into step (3211) if characteristic spectrum belt occurs at 2600bp; If then be classified as the B2-12 group at 2600bp atypism bands of a spectrum, get into step (3212).
(3211) using the primers 5S5 B2-11 group on the DNA RAPD amplification, if the characteristic bands appear at 850bp and 1200bp in 750bp or no characteristic bands, variety 'HB grapefruit' is distinguished; if 850bp , 1200bp characteristic bands appeared, species 'wilderness' is distinguished; if 750bp, 850bp characteristic bands appeared, species 'long can' be distinguished; if 850bp, 1200bp are no characteristic bands, variety 'Great Fen' is distinguished; if 850bp, 1800bp characteristic bands appeared, re-use for the RAPD primers 1S2, if the characteristic bands appear at 1300bp compared to 'feed the red'; if no characteristic 1300bp band was 'up and down the red'.
(3212) using the primers 1S2 B2-12 group on the DNA RAPD amplification, if the characteristic bands appear at 1300bp, variety 'citrus 1' be distinguished; if the characteristic bands appear at 900bp, species' Youth Work 'be distinguished; if 1300bp and 900bp are no characteristic bands, variety' City People 'be distinguished.
(322) utilize primer 1S4 that the DNA of B2-2 group is carried out the RAPD amplification, if characteristic spectrum belt occurs at 750bp, kind ' this morning No. 6 ' is distinguished; If at 750bp atypism bands of a spectrum, kind ' orange mandarin orange No. 3 ' is distinguished.
(33) utilize primer Y59 that the DNA of B3 group is carried out the RAPD amplification,, get into step (331) if having characteristic spectrum belt then to be classified as the B3-1 group at 1800bp; If then be classified as the B3-2 group at 1800bp atypism bands of a spectrum, get into step (332).
(331) utilize primer Y17 that the DNA of B3-1 group is carried out the RAPD amplification, if at 1000bp, 450bp characteristic spectrum belt is arranged all, kind ' flat red lemon ' is distinguished; If all do not have characteristic spectrum belt at 250bp, 600bp, kind ' Calusena lansium ' is distinguished; If at 600bp characteristic spectrum belt is arranged, kind ' Gan Xia ' is distinguished; If all do not have characteristic spectrum belt at 1800bp, 450bp, kind ' granulated sugar tangerine ' is distinguished; If have characteristic spectrum belt and 1000bp atypism bands of a spectrum then to be classified as one type, increase with primer Y47 again, if characteristic spectrum belt is arranged then ' Ying Liman I ' is if at 1800bp atypism bands of a spectrum then be kind ' between standing ' for kind at 1800bp at 450bp.
(332) utilize primer Y60 that the DNA of B3-2 group is carried out RAPD amplification (please refer to Fig. 3), if at 750bp characteristic spectrum belt is arranged, kind ' Wanbai pummelo ' is distinguished; If at 800bp characteristic spectrum belt is arranged, kind ' orange mandarin orange No. 3 ' is distinguished; If all do not have characteristic spectrum belt at 750bp, 850bp, kind ' the big tangerine of Western Hills ' is distinguished.
(34) utilize primer Y59 that the DNA of B4 group is carried out the RAPD amplification, (please refer to Fig. 4) gets into step (341) if all have characteristic spectrum belt then to be classified as the B4-1 group at 1200bp, 1800bp; Then be classified as the B4-2 group as if characteristic spectrum belt and 1200bp atypism bands of a spectrum occur at 1800bp, get into step (342); If at 1200bp and 1800bp atypism bands of a spectrum all, kind ' good fortune tangerine ' quilt is distinguished.
(341) utilize primer Y17 that the DNA of B4-1 group is carried out the RAPD amplification; If characteristic spectrum belt all occurs at 200bp and 1000bp; Utilize primer Y54 to increase again; If characteristic spectrum belt occurs then for kind ' sweet orange shaddock ', if then be kind ' 4 days National Day ' at 1600bp atypism bands of a spectrum at 1600bp; If characteristic spectrum belt occurs and at 1000bp atypism bands of a spectrum at 200bp; Utilize primer Y23 to increase again; If characteristic spectrum belt occurring at 350bp then is kind ' Mi Jin '; If characteristic spectrum belt occurs then for kind ' Qingniao Co. ' at 650bp, if 350bp, 650bp all the atypism bands of a spectrum then be kind ' three guarantors '; If characteristic spectrum belt and 200bp atypism bands of a spectrum occur at 1000bp, kind ' No. 3, emerging Tianjin ' is distinguished; If at 1000bp atypism bands of a spectrum, kind ' Bei Kou ' is distinguished.
(342) utilize primer Y23 that the DNA of B4-2 group is carried out the RAPD amplification, if characteristic spectrum belt occurs at 250bp, kind ' 5 days National Day ' is distinguished; If at 250bp atypism bands of a spectrum, kind ' warm mandarin orange No. 3 ' is distinguished.
(4) utilize primer Y40 that the DNA of C group is carried out the RAPD amplification, if characteristic spectrum belt occurs at 650bp, kind ' little pomelo ' is distinguished; If at 650bp atypism bands of a spectrum, kind ' shaddock recklessly ' is distinguished.
In sum, in every case relate in above-mentioned 48 kinds the oranges and tangerines of kind arbitrarily or its combination, can accomplish kind according to above-mentioned steps (1)～(4) and differentiate and distinguish.
" the tree type is differentiated figure " of utilizing method statistic of the present invention to go out not only can be accomplished the evaluation to unknown kind, also can inquire to identify the needed primer of different varieties and differentiate foundation.Please refer to Fig. 1 below, can find out, when needs are inquired about discrimination method and the required primer of any oranges and tangerines kind, only need find corresponding kind numbering, recall forward and get final product in tree type discriminating figure branches end.For example; Need interrogation zone branch kind ' flat red lemon ', ' Calusena lansium ', ' Ying Liman I ', ' sweet orange shaddock '; According to the method for the invention, can ' the sweet orange shaddock ' that have 600bp, 550bp, 950bp characteristic spectrum belt simultaneously be distinguished through primer Y40, and all the other kinds do not have characteristic spectrum belt at 550bp; 3 remaining kinds are through primer Y17; Separablely go out ' the flat red lemon ' that has characteristic spectrum belt at 450bp, 1000bp, and all do not have ' Calusena lansium ' of characteristic spectrum belt at 450bp, 600bp, remaining can confirm as kind ' Ying Liman I '.
Give a further embodiment, the method of the present invention is distinguished HB grapefruit (type number 8), fields (type number 12), a long time to (type number 15), the material of red (type number 17), a large Fen (type number 20), up and down the red (type number 22) six varieties of citrus.Differentiate figure through using for reference the tree type, as shown in Figure 1, select primer 5S5 for use; Gel electrophoresis spectrum through amplification obtains is as shown in Figure 5, can find out at about 850bp place 8,15; 17; 22 four strains have characteristic spectrum belt, reduce one group, No. 12 with No. 20 kinds the atypism bands of a spectrum then are divided into another group at the 850bp place.Simultaneously No. 12 kinds have characteristic spectrum belt at about 1200bp place, and No. 20 kinds do not have, and just can make a distinction with No. 20 kinds for such No. 12; In addition, can be divided into two groups to No. 8, No. 15, No. 17, No. 22 kinds again at the 1800bp place, No. 8, No. 15 kinds are the atypism bands of a spectrum at the 1800bp place, and No. 17, No. 22 kinds have; Further; Can make a distinction No. 15 with No. 8 kinds again at the 750bp place, 17 and No. 22 kind can't be distinguished with primer 5S5, continues to select for use primer 1S2; Electrophoresis result please refer to shown in Figure 6; No. 17 characteristic spectrum belt is arranged at the 1300bp place, and do not have for No. 22, so just can make a distinction No. 17 with No. 22 two kinds.
SEQUENCE?LISTING
< 110>Agricultural University Of Nanjing
< 120>a kind of primer and method of quick differentiation oranges and tangerines kind
<130> NJAU120901
<160> 16
<170> PatentIn?version?3.3
<210> 1
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> Y17
<400> 1
aggggtcttg?g 11
<210> 2
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> Y23
<400> 2
ggacccaacc?g 11
<210> 3
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> Y28
<400> 3
gtgtgcccca?t 11
<210> 4
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> Y40
<400> 4
agcgtcctcc?t 11
<210> 5
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> Y41
<400> 5
agcgtcctcc?g 11
<210> 6
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> Y47
<400> 6
acgaccgaca?g 11
<210> 7
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> Y54
<400> 7
tggtggcgtt?c 11
<210> 8
<211> 10
<212> DNA
< 213>synthetic
<220>
<221> Y55
<400> 8
acccccgact?t 11
<210> 9
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> Y59
<400> 9
acccccgact?g 11
<210> 10
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> Y60
<400> 10
acccccgact?c 11
<210> 11
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> 1S2
<400> 11
gggtaacgcc?a 11
<210> 12
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> 1S3
<400> 12
gggtaacgcc?t 11
<210> 13
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> 1S4
<400> 13
gggtaacgcc?t 11
<210> 14
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> 5S2
<400> 14
ccgctaccga?a 11
<210> 15
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> 5S5
<400> 15
ccgctaccga?g 11
<210> 16
<211> 11
<212> DNA
< 213>synthetic
<220>
<221> A5
<400> 16
gtccacacgg?t 11
Claims (5)
1. primer of distinguishing fast the oranges and tangerines kind is characterized in that comprising:
Y17：5’-AGGGGTCTTGG-3’，
Y23：5’-GGACCCAACCG-3’，
Y28：5’-GTGTGCCCCAT-3’，
Y40：5’-AGCGTCCTCCT-3’，
Y41：5’-AGCGTCCTCCG-3’，
Y47：5’-ACGACCGACAG-3’，
Y54：5’-TGGTGGCGTTC-3’，
Y55：5’-ACCCCCGACTT-3’，
Y59：5’-ACCCCCGACTG-3’，
Y60：5’-ACCCCCGACTC-3’，
1S2：5’-GGGTAACGCCA-3’，
1S3：5’-GGGTAACGCCT-3’，
1S4：5’-GGGTAACGCCT-3’，
5S2：5’-CCGCTACCGAA-3’，
5S5：5’-CCGCTACCGAG-3’，
A5：5’-GTCCACACGGT-3’；
2. the primer of a kind of quick differentiation oranges and tangerines kind according to claim 1 is characterized in that, said primer is to filter out the primer that band is clear, the PCR product repeats to occur through the random primer to the design of oranges and tangerines genome through 3 subgradient PCR.
3. a method of utilizing the said primer Rapid identification of claim 1 to distinguish the oranges and tangerines kind is characterized in that this method comprises the steps:
(1) extraction need be identified the oranges and tangerines DNA of differentiation, diluted for use;
(2) utilize primer 1S3 that oranges and tangerines DNA is carried out the RAPD amplification,
Then be classified as the A group as if characteristic spectrum belt and 550bp atypism bands of a spectrum occur at 480bp, 600bp, get into step (3);
Then be classified as the B group if characteristic spectrum belt all occurs, get into step (4) at 480bp, 550bp, 600bp;
Then be classified as the C group as if characteristic spectrum belt and 600bp atypism bands of a spectrum occur at 480bp, 550bp, get into step (5);
(3) utilize primer Y40 that the DNA of A group is carried out the RAPD amplification,
Then be classified as the A1 group as if characteristic spectrum belt and 650bp atypism bands of a spectrum occur at 550bp, get into step (31);
If 550bp and 650bp all the atypism bands of a spectrum then be classified as the A2 group, entering step (32);
If at 1400bp atypism bands of a spectrum, oranges and tangerines kind ' Xie Cheng ' is distinguished;
If characteristic spectrum belt occurs at 650bp, the oranges and tangerines kind ' is not known fire ' and is distinguished;
(31) utilize primer 5S2 that the DNA of A1 group is carried out the RAPD amplification,
If at 600bp atypism bands of a spectrum, oranges and tangerines kind ' navel is orange in vain ' is distinguished;
If characteristic spectrum belt occurs at 600bp, oranges and tangerines kind ' fragrant and sweet orange ' is distinguished;
(32) utilize primer Y41 that the DNA of A2 group is carried out the RAPD amplification,
Then be classified as the A2-1 group if characteristic spectrum belt occurs, get into step (321) at 1400bp;
If at 1400bp atypism bands of a spectrum, oranges and tangerines kind ' companion is wild ' is distinguished;
(321) utilize primer A5 that the DNA of A2-1 group is carried out the RAPD amplification,
If at 1000bp atypism bands of a spectrum, oranges and tangerines kind ' between upright ' is distinguished;
If characteristic spectrum belt occurs at 1000bp, oranges and tangerines kind ' fragrant citrus ' is distinguished;
(4) utilize primer Y40 that the DNA of B group is carried out the RAPD amplification,
If characteristic spectrum belt all occurs and 950bp atypism bands of a spectrum then are classified as the B1 group, entering step (41) at 550bp and 600bp;
If 550bp and 600bp all the atypism bands of a spectrum then be classified as the B2 group, entering step (42);
If characteristic spectrum belt occurs and 550bp atypism bands of a spectrum then are classified as the B3 group, entering step (43) at 600bp and 950bp;
Then be classified as the B4 group if characteristic spectrum belt all occurs, get into step (44) at 550bp, 600bp, 950bp;
As if characteristic spectrum belt and 550bp, 950bp atypism bands of a spectrum occur at 600bp, oranges and tangerines kind ' early red ' is distinguished;
If characteristic spectrum belt occurs at 600bp, 1100bp, oranges and tangerines kind ' u'eno ' is distinguished;
As if characteristic spectrum belt and 600bp, 950bp atypism bands of a spectrum occur at 550bp, oranges and tangerines kind ' from generation to generation ' is distinguished;
(41) utilize primer Y59 that the DNA of B1 group is carried out the RAPD amplification,
Then be classified as the B1-1 group if characteristic spectrum belt occurs, get into step (411) at 1800bp;
If at 1800bp atypism bands of a spectrum, oranges and tangerines kind ' bridge originally ' is distinguished;
(411) utilize primer Y17 that the DNA of B1-1 group is carried out the RAPD amplification,
If characteristic spectrum belt occurs at 1000bp, oranges and tangerines kind ' various household supplies ' is distinguished;
If at 1000bp atypism bands of a spectrum, oranges and tangerines kind ' between upright ' is distinguished;
(42) utilize primer Y55 that the DNA of B2 group is carried out the RAPD amplification,
If characteristic spectrum belt all occurs at 300bp, 350bp, 1200bp, oranges and tangerines kind ' tertia ' is distinguished;
If characteristic spectrum belt all occurs at 450bp, 750bp, oranges and tangerines kind ' southern mandarin orange ' is distinguished;
If characteristic spectrum belt and 750bp atypism bands of a spectrum occur at 450bp, oranges and tangerines kind ' Nanfeng orange ' is distinguished;
If characteristic spectrum belt occurs at 300bp, characteristic spectrum belt does not appear simultaneously at 350bp, 1200bp, be classified as the B2-1 group, get into step (421);
If 450bp, 1200bp all the atypism bands of a spectrum then be classified as B2-2 group, get into step (422);
(421) utilize primer Y28 that the DNA of B2-1 group is carried out the RAPD amplification,
Then be classified as the B2-11 group if characteristic spectrum belt occurs, get into step (4211) at 2600bp;
If then be classified as the B2-12 group at 2600bp atypism bands of a spectrum, get into step (4212);
(4211) utilize primer 5S5 that the DNA of B2-11 group is carried out the RAPD amplification,
If characteristic spectrum belt occurs at 850bp, and at 750bp or 1200bp atypism bands of a spectrum, kind ' HB shaddock ' is distinguished;
If characteristic spectrum belt all occurs at 850bp, 1200bp, kind ' open country ' is distinguished;
If characteristic spectrum belt all occurs at 750bp, 850bp, kind ' ability for a long time ' is distinguished;
If 850bp, 1200bp are no characteristic bands, variety 'big Fen' be distinguished;
If characteristic spectrum belt all occurs at 850bp, 1800bp, utilize primer 1S2 to carry out the RAPD amplification again, then be ' expecting red ' if characteristic spectrum belt occurs at 1300bp; If at 1300bp atypism bands of a spectrum then is ' red up and down ';
(4212) utilize primer 1S2 that the DNA of B2-12 group is carried out the RAPD amplification,
If the characteristic bands appear at 1300bp, variety 'citrus 1' be distinguished;
If characteristic spectrum belt occurs at 900bp, kind ' green grass or young crops sees ' is distinguished;
If at 1300bp and 900bp atypism bands of a spectrum all, kind ' city people ' quilt is distinguished;
(422) utilize primer 1S4 that the DNA of B2-2 group is carried out the RAPD amplification,
If characteristic spectrum belt occurs at 750bp, kind ' this morning No. 6 ' is distinguished;
If at 750bp atypism bands of a spectrum, kind ' orange mandarin orange No. 3 ' is distinguished;
(43) utilize primer Y59 that the DNA of B3 group is carried out the RAPD amplification,
If have characteristic spectrum belt then to be classified as the B3-1 group at 1800bp, get into step (431);
If then be classified as the B3-2 group at 1800bp atypism bands of a spectrum, get into step (432);
(431) utilize primer Y17 that the DNA of B3-1 group is carried out the RAPD amplification,
If at 1000bp, 450bp characteristic spectrum belt is arranged all, kind ' flat red lemon ' is distinguished;
If all do not have characteristic spectrum belt at 250bp, 600bp, kind ' Calusena lansium ' is distinguished;
If at 600bp characteristic spectrum belt is arranged, kind ' Gan Xia ' is distinguished;
If all do not have characteristic spectrum belt at 1800bp, 450bp, kind ' granulated sugar tangerine ' is distinguished;
If have characteristic spectrum belt and 1000bp atypism bands of a spectrum then to be classified as one type, increase with primer Y47 again, if characteristic spectrum belt is arranged then ' Ying Liman I ' is if at 1800bp atypism bands of a spectrum then be kind ' between standing ' for kind at 1800bp at 450bp;
(432) utilize primer Y60 that the DNA of B3-2 group is carried out the RAPD amplification,
If at 750bp characteristic spectrum belt is arranged, kind ' Wanbai pummelo ' is distinguished;
If at 800bp characteristic spectrum belt is arranged, kind ' orange mandarin orange No. 3 ' is distinguished;
If all do not have characteristic spectrum belt at 750bp, 850bp, kind ' the big tangerine of Western Hills ' is distinguished;
(44) utilize primer Y59 that the DNA of B4 group is carried out the RAPD amplification,
If all have characteristic spectrum belt then to be classified as the B4-1 group at 1200bp, 1800bp, get into step (441);
Then be classified as the B4-2 group as if characteristic spectrum belt and 1200bp atypism bands of a spectrum occur at 1800bp, get into step (442);
If at 1200bp and 1800bp atypism bands of a spectrum all, kind ' good fortune tangerine ' quilt is distinguished;
(441) utilize primer Y17 that the DNA of B4-1 group is carried out the RAPD amplification,
If characteristic spectrum belt all occurs at 200bp and 1000bp, utilize primer Y54 to increase again, then be kind ' sweet orange shaddock ' if characteristic spectrum belt occurs, as if then being kind ' 4 days National Day ' at 1600bp atypism bands of a spectrum at 1600bp;
If characteristic spectrum belt occurs and at 1000bp atypism bands of a spectrum at 200bp; Utilize primer Y23 to increase again; If characteristic spectrum belt occurring at 350bp then is kind ' Mi Jin '; If characteristic spectrum belt occurs then for kind ' Qingniao Co. ' at 650bp, if 350bp, 650bp all the atypism bands of a spectrum then be kind ' three guarantors ';
If characteristic spectrum belt and 200bp atypism bands of a spectrum occur at 1000bp, kind ' No. 3, emerging Tianjin ' is distinguished;
If at 1000bp atypism bands of a spectrum, kind ' Bei Kou ' is distinguished;
(442) utilize primer Y23 that the DNA of B4-2 group is carried out the RAPD amplification,
If characteristic spectrum belt occurs at 250bp, kind ' 5 days National Day ' is distinguished;
If at 250bp atypism bands of a spectrum, kind ' warm mandarin orange No. 3 ' is distinguished;
(5) utilize primer Y40 that the DNA of C group is carried out the RAPD amplification, if characteristic spectrum belt occurs at 650bp, kind ' little pomelo ' is distinguished; If at 650bp atypism bands of a spectrum, kind ' shaddock recklessly ' is distinguished.
4. a kind of Rapid identification according to claim 3 is distinguished the method for oranges and tangerines kind, it is characterized in that: the RAPD amplification of this method is the PCR reaction system of 30 μ l, comprises 10 * PCR Buffer, 3 μ l, 2.5mmol/L dNTPs2.4 μ l, 25mmol/LMgCl
21.8 μ l, 1.25UTaq enzyme, random primer 10pmol/ μ l 1.2 μ l, template DNA 40～50ng; React on 95 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, renaturation 1min, 72 ℃ are extended 2min, 42 circulations; 72 ℃ are extended 10min.
5. a kind of Rapid identification according to claim 4 is distinguished the method for oranges and tangerines kind, and it is characterized in that: the annealing temperature of each primer is as shown in the table in the said RAPD amplified reaction:
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103146687A (en) * | 2013-03-27 | 2013-06-12 | 吉林烟草工业有限责任公司 | Random amplified polymorphic deoxyribonucleic acid-polymerase chain reaction (RAPD-PCR) kit as well as amplification method and application thereof |
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| CN111593049A (en) * | 2020-06-18 | 2020-08-28 | 西南大学 | Application of short DNA sequence in identification of citrus group and citrus seedling |
| CN114438249A (en) * | 2022-02-22 | 2022-05-06 | 江西省农业科学院园艺研究所 | Primer group, kit and identification method for identifying cultivation types of valium odoratum honey pomelos |
| CN116042905A (en) * | 2023-03-10 | 2023-05-02 | 湖北省农业科学院果树茶叶研究所 | SV (space velocity) marker for identifying citrus varieties and application thereof |
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2011
- 2011-12-09 CN CN2011104077244A patent/CN102492774A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146687A (en) * | 2013-03-27 | 2013-06-12 | 吉林烟草工业有限责任公司 | Random amplified polymorphic deoxyribonucleic acid-polymerase chain reaction (RAPD-PCR) kit as well as amplification method and application thereof |
| CN103146687B (en) * | 2013-03-27 | 2015-04-22 | 吉林烟草工业有限责任公司 | Random amplified polymorphic deoxyribonucleic acid-polymerase chain reaction (RAPD-PCR) kit as well as amplification method and application thereof |
| CN109266776A (en) * | 2018-10-24 | 2019-01-25 | 华中农业大学 | Utilize the kit and method of InDel Marker Identification citrus shaddock hybrid generation |
| CN111593049A (en) * | 2020-06-18 | 2020-08-28 | 西南大学 | Application of short DNA sequence in identification of citrus group and citrus seedling |
| CN114438249A (en) * | 2022-02-22 | 2022-05-06 | 江西省农业科学院园艺研究所 | Primer group, kit and identification method for identifying cultivation types of valium odoratum honey pomelos |
| CN116042905A (en) * | 2023-03-10 | 2023-05-02 | 湖北省农业科学院果树茶叶研究所 | SV (space velocity) marker for identifying citrus varieties and application thereof |
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