CN1702175A - Cowpea variety molecular identification method based on genome RAPD analysis - Google Patents
Cowpea variety molecular identification method based on genome RAPD analysis Download PDFInfo
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Abstract
The invention discloses a method for molecular inspection of cowpea variety based on analysis of genome RAPD. Extracting DNA of the available genome cowpea varieties, and generating separately polyase chain reaction to prepare the random augmentative polymorphic DNA product, then processing electrophoretic separation to obtain random augmentative polymorphic DNA standard atlas; analyzing the inspection-required cowpea variety following said steps to get the random augmentative polymorphic DNA atlas, comparing and deciding it with standard atlas, the inspection result is obtained. With the invention, it can definitely decide the different varieties of cowpea, and realize precise and rapid inspection of variety truth and variety purity, providing reliable technique of variety inspection, market control of seed, variety protection, quality control of pre-sowing seed, stock breeding and so on.
Description
Technical field
The present invention relates to the method for a kind of plant genetic mark and kind Molecular Identification, specifically a kind of cowpea variety molecular assay method of analyzing based on Genome RAPD.
Background technology
Vigna annual herb plant.Originate from African torrid areas, be distributed widely in the torrid zone and subtropical zone.Common cowpea (Vigna unguiculata ssp.unguiculata), long pod cowpea (Vigna unguiculata ssp.sesquipedalis), three cultivations of short pod cowpea (Vigna unguiculata ssp.cylindrica) subspecies are arranged.China is the secondary origin center of asparagus bean (Vigna unguiculata cv-gr.sesquipedalis E.Westphal), and long cultivation history and abundant variety source are arranged.In recent years, though genetic breeding work has obtained very big achievement, but still can not satisfy an urgent demand of market to new variety, by genetic improvement seed selection cowpea new variety, particularly realize high-quality, ripe property is supporting and the various breeding objective of proterties such as pod color is a significant job.The variety authentication of seed and purity are one of most important indexs of estimating seed quality; it is identified with check is the key and the difficult point in kind protection, seed quality control and standard seed market, also is performance improved seeds genetic potential and the important measures of guaranteeing producer's increasing both production and income and market supply.The tradition authentication method mainly is to plant by the sub-district, field to carry out, it is not only time-consuming, effort, cost height, and it is bigger influenced by environment, climatic factor, postpone as a result, can not prevent effectively that defective seed from coming into the market, can not provide valuable to the peasant with kind of an information.Therefore, accurately, Rapid identification variety authentication and variety are the directions of seed quality detection technique development, also are to guarantee agriculture production safety, the key of protection peasant benefit.
In the process of kind genetic improvement, correctly divide the product population, the sibship between distinct crop varieties has directive significance to parent's selection and apolegamy, also is one of breeding work key of success.About the classification of asparagus bean kind, mainly to divide in the past according to qualitative character such as shape of the seed, color, also useful quantitative character such as florescence, single pod is heavily waited and carries out the report that cluster analysis and employing isozyme feature are carried out assortment.With these technological methods assortment is identified that significant limitation is all arranged, the interracial genetic affinity of discrimination is difficult to instruct breeding work well.Specifically, morphological markers is accredited as feature with the field, has advantages such as directly perceived, easy, but it is easily affected by environment that this phenotype is identified, poor accuracy, and the proterties polymorphism is also not enough, length consuming time and identify afterwards is difficult to take precautions against production loss, is the starting stage of genetic marker.Cytological marker is to check that kind chromosome number and structure variation are feature, it plays an important role to kind and ploidy inspection, but division seems that then quantity of information is little to kind, equally the nearer material of some relation is difficult to accurately be distinguished, and operation is very complicated.Along with people to the deepening constantly and development of molecular biology of variety source hereditary property understanding, molecular marking technique is widely used in the genetic analysis to species.
The RAPD mark is that (Random Amplified PolymorphicDNA, RAPD) technology is to be set up on the basis of PCR (polymerase chain reaction) by people (1990) such as U.S. scientist Williams and Welsh to randomly amplified polymorphic DNA.The RAPD reaction is by high-temperature denatured (92～94 ℃), and low-temperature annealing (35～55 ℃) and middle temperature are extended (68～72 ℃) three step one-period and formed.(general 25～40 circulations) are carried out in circulation.RAPD is the detection of species polymorphism, genetic map construction, assortment, sibship is identified and the Perfected process of viral detection, good character and resistance screening.The RAPD technology is being used for showing remarkable advantages when kind (being) is identified, Williams (1990), and Welsh (1990) has carried out the RAPD fingerprint map construction on corn, soybean and paddy rice, and kind and strain have been carried out evaluation and pedigree analysis.Studies show that in a large number; its technical system of different plant species there is very big-difference; therefore; research optimization is suitable for the RAPD analytical technology system of cowpea genomic dna; the technology of a renewal both can be provided for the evaluation of cowpea variety sibship, cultivar identification, kind protection and stock breeding etc.; can deepen understanding again, for genetic improvement provides foundation to the cowpea variety genetic affinity.
To the molecule marking research of asparagus bean kind genetic polymorphism, do not launch so far both at home and abroad.That has reported studies show that, the degree of different plant species explanations of genomic DNA polymorphism be because of species different.For example eggplant is 20.15%, and root of Szemao crotalaria are 39.43%; The DNA band number of average each primer amplification is also inequality: eggplant is 5.9,6.1 of mango.The asparagus bean genomic DNA polymorphism is higher than these species significantly, although on average the DNA band number (6.13) of each primer amplification is little with above-mentioned species difference.But on the dna molecular level, asparagus bean exists abundant heritable variation.
Summary of the invention
Technical problem to be solved by this invention is: optimize the technical system that the cowpea Genome RAPD is analyzed, distinct cowpea variety genetic affinity, a kind of cowpea variety molecular assay method of analyzing based on Genome RAPD is provided, and it has overcome identifies the not scientific of cowpea variety molecule kind existence according to the similarities and differences of kind mode of appearance now.
Technical scheme of the present invention is achieved in that it comprises the steps,
(1), extracts the DNA of existing each cowpea variety;
(2), carry out PCR (polymerase chain) reaction respectively, obtain random amplified polymorphic DNA product with extracted cowpea variety genomic dna;
(3), respectively random amplified polymorphic DNA product is carried out electrophoretic separation with 1.0% agarose, EB directly is added in the gel, and total applied sample amount is 8 μ l, wherein 4 μ l amplified productions, other adds 40% sucrose solution, 2 μ l and 0.1% tetrabromophenol sulfonphthalein, 2 μ l, constant voltage electrophoresis 1.5h under 80V;
(4), electrophoresis finishes the back and takes a picture on the UV-light detector, obtains the randomly amplified polymorphic DNA standard diagram respectively;
(5) select clear randomly amplified polymorphic DNA collection of illustrative plates to adopt " 0-1 " system log (SYSLOG) bands of a spectrum, the assignment that band will be arranged is 1, and the assignment of not having band is 0; Read the data of each material, set up database at different primer extension product electrophoretic bands;
(6), by the arithmetical av method being carried out cluster analysis not to be weighted to, obtain system's clustering tree.
(7), the cowpea variety of required evaluation is analyzed by above-mentioned steps, obtain the randomly amplified polymorphic DNA collection of illustrative plates of required evaluation cowpea variety, this randomly amplified polymorphic DNA collection of illustrative plates and standard sample spectrum are compared judgement, obtain the Analysis and Identification result.
Wherein the reaction conditions of polymerase chain reaction is as follows: primer (25pmol/L) 1.0 μ l, Taq archaeal dna polymerase (1u/ μ l) 1 μ l, dNTPs (10mmol/L) 0.5 μ l, MgCl
2(25mmol/L) 1.5 μ l, 10 * buffer, 2.5 μ l, template DNA (50ng/ μ l) 1 μ l, ultrapure water 17.5 μ l.
The described primer in polymerase chain reaction is:
OPA03?TTAGCGCCCC、OPA07?CTACGCTCAC、OPC01?TCCCAGCAGA、
OPC02?GTGAGGCGTC、OPC06?CCAGAACGGA、OPC07?GTCCCGACGA、
OPC08?TTTGGGTGCC、OPC09?CTCACCGTCC、OPC11?AAAGCTGCGG、
OPI02?AGCCGTTCAG、OPJ03?AGCACCTCGT、OPJ09?ACGGCACGCA、
OPP17?AAGGCACGAG、OPS07?TCCGATGCTG、OPS10?ACCGTTCCAG、
OPS18?CTGGCGAACT、OPS19?GAGTCAGCAG、OPS20?TCTGGACGGA、
OPU10?ACCTCGGCAC、OPU11?AGACCCAGAG、OPU17?ACCTGGGGAG、
OPU18?GAGGTCCACA、OPU20?ACAGCCCCCA。
Mg in the polymerase chain reaction of described asparagus bean genomic dna
2+Concentration is 1.5mmol/L.
The consumption of Taq enzyme is 1.0 ~ 1.5u in the polymerase chain reaction of described asparagus bean genomic dna.
The concentration of oligonucleotide is 0.025 μ mol/L in the randomly amplified polymorphic DNA analytical procedure of described asparagus bean genomic dna.
Tm is 38.0 ℃ in the randomly amplified polymorphic DNA analytical procedure of described asparagus bean genomic dna.
The present invention's asparagus bean variety genome DNA template that can effectively increase, its amplified production polymorphism ratio is reached more than 95%, the kind number of times that average every band occurs is 18.39 (luffing is 1～39), and frequency is 0.46 (luffing is 0.30～0.98), and its variation coefficient is 71.45%.
The main factor that influences the randomly amplified polymorphic DNA reaction is Mg
2+Parameters such as concentration, Taq enzyme, random primer, dNTP, template, annealing temperature.
Mg
2+Be the activator of Taq polysaccharase, influence outside the enzymic activity, also influence the melting temperature(Tm) of annealing, template and the PCR product of primer, the specificity of product, the formation of primer dimer etc.Mg
2+Concentration can adopt typical two step optimizing processs.
The consumption of Taq enzyme is generally 0.5 ~ 2.0u in RAPD, and the too high meeting of the vigor of Taq enzyme non-specific amplification occurs and produces fuzzy small segment, a little less than mistake is hanged down and then caused amplified band too, even does not have amplified band.
The primer of RAPD is followed the design of primers principle of round pcr, and for reaching ideal specific amplification and amplification efficiency, the concentration of Oligonucleolide primers is important, specifically is controlled to be 0.025 μ mol/L.
The concentration that dNTP is commonly used is 20 ~ 200 μ mol/L, and the final concentration of 4 kinds of dNTP equates.The dNTP of high density can fast reaction speed, but the mistake that also can increase base participates in rate, and suitable lower concentration can improve the tolerance range of reaction.Template DNA can add the PCR mixed solution with strand or double-stranded form.Usually the PCR efficient of the template DNA of small segment will be higher than macromole DNA, to improve output.
The necessary strict regulation of annealing temperature, by formula Tm=4 (G+C)+2 (A+T) calculates.In fact, the Tm value also is subjected to the influence of other heterogeneities in damping fluid, so the Tm value of calculating all should be regarded about value as.The strict template DNA purity and the mensuration of concentration have been carried out before the PCR reaction;
The present invention catches the cowpea genetic affinity to establish and variety is checked key issue in these two research and production, has founded the technological method of effective solution.Identification of species method of the present invention directly detects the dna sequence dna difference of kind; both had genetic stability; has information diversity again; can clearly distinguish and differentiate the different varieties of cowpea; realize accurately, Rapid identification variety authentication and variety, thereby for cultivar identification, seed market management, kind protection, broadcast before seed quality control and stock breeding etc. robust techniques is provided.The present invention optimizes the RAPD technical system of cowpea genomic dna, makes that cowpea variety Molecular Identification speed is fast, the time is short, polymorphism is many, can be solve problem such as true and false Seed Inspection in advance, to avoid causing the loss of producer's interests.All asparagus bean kinds can be distinguished and identify to 1 of present method utilization and above effective primer, realizes that the dna fingerprint of asparagus bean kind is identified.
Description of drawings
Fig. 1 is the RAPD amplified production electrophorogram of primer OPJ03
Fig. 2 is the RAPD amplified production electrophorogram of primer OPU17
Fig. 3 is the RAPD amplified production electrophorogram of primer OPA03
Fig. 4 is the RAPD amplified production electrophorogram of primer OPA07
Fig. 5 is the RAPD amplified production electrophorogram of primer OPC07
Fig. 6 is the RAPD amplified production electrophorogram of primer OPC09
Fig. 7 is the RAPD amplified production electrophorogram of primer OPJ09
Fig. 8 is the RAPD amplified production electrophorogram of primer OPS10
Fig. 9 is the RAPD amplified production electrophorogram of primer OPJ20
Figure 10 is the RAPD amplified production electrophorogram of primer OPU11
The dendrogram that Figure 11 obtains for cluster analysis
Table 1 is 40 asparagus bean kind code names and source and RAPD amplification
Table 2 is 30 10bp random primer sequences and effective primer sequence amplification situation
Table 3 is band, kind distribution number of times and the frequency statistics table of effective primer amplification
Table 4 is result's statistics of 23 effective primer amplifications in each product population
Embodiment
Below the invention will be further described, the present invention includes following steps:
(1), extracts the DNA of existing 40 asparagus bean kind blade genome cowpea variety molecules;
(2), carry out the polymerase chain reaction respectively, obtain random amplified polymorphic DNA product with extracted cowpea variety genomic dna;
(3), respectively random amplified polymorphic DNA product is carried out electrophoretic separation with 1.0% agarose, EB directly is added in the gel, and total applied sample amount is 8 μ l, wherein 4 μ l amplified productions, other adds 40% sucrose solution, 2 μ l and 0.1% tetrabromophenol sulfonphthalein, 2 μ l, constant voltage electrophoresis 1.5h under 80V;
(4), electrophoresis finishes the back and takes a picture on the UV-light detector, obtains the randomly amplified polymorphic DNA standard diagram respectively;
(5) select clear randomly amplified polymorphic DNA collection of illustrative plates to adopt " 0-1 " system log (SYSLOG) bands of a spectrum, the assignment that band will be arranged is 1, and the assignment of not having band is 0; Read the data of each material, set up database at different primer extension product electrophoretic bands;
(6), by the arithmetical av method being carried out cluster analysis not to be weighted to, obtain system's clustering tree.
(7), the cowpea variety of required evaluation is analyzed by above-mentioned steps, obtain the randomly amplified polymorphic DNA collection of illustrative plates of required evaluation cowpea variety, this randomly amplified polymorphic DNA collection of illustrative plates and standard sample spectrum are compared judgement, obtain the Analysis and Identification result.
Wherein the reaction conditions of polymerase chain reaction is as follows: primer (25pmol/L) 1.0 μ l, Taq archaeal dna polymerase (1u/ μ l) 1 μ l, dNTPs (10mmol/L) 0.5 μ l, MgCl
2(25mmol/L) 1.5 μ l, 10 * buffer, 2.5 μ l, template DNA (50ng/ μ l) 1 μ l, ultrapure water 17.5 μ l.
Primer described in the polymerase chain reaction is at least a of table 2.
Mg in the polymerase chain reaction of described asparagus bean genomic dna
2+Concentration is 1.5mmol/L.
The consumption of Taq enzyme is 1.0 ~ 1.5u in the polymerase chain reaction of described asparagus bean genomic dna.
The concentration of oligonucleotide is 0.025 μ mol/L in the randomly amplified polymorphic DNA analytical procedure of described asparagus bean genomic dna.
Tm is 38.0 ℃ in the randomly amplified polymorphic DNA analytical procedure of described asparagus bean genomic dna.
40 asparagus bean kind blade genomic dnas of the concrete extraction of the present invention, utilization RAPD technology is carried out cowpea variety genetic affinity and Molecular Identification research.It is as shown in table 2 to filter out 23 effective primers from 30 random primers, obtains 141 amplified bands altogether, and wherein 140 is the polymorphism band, polymorphism ratio 99.29%, and average each primer amplification 6.13 band, the polymorphic bands number is 6.09.Average every kind shows 64.35 of amplified bands of polymorphism (luffing is 26～82), and its variation coefficient is 15.62%; The kind number of times that average every band occurs is 18.39 (luffing is 1～39), and frequency is 0.46 (luffing is 0.30～0.98), and its variation coefficient is 71.45%.As seen the number of RAPD amplified band is different between kind, its site variation is bigger, there is difference in various degree in this explanation for dna sequence dna between the kind of examination, and the dna fingerprint that forms according to each kind can determine interracial hereditary difference effectively, technology is provided for the Molecular Identification of kind.Experiment detects 18 characteristic strips altogether, article 15, the performance of do as one likes shape has with the Semen Phaseoli kind of drying that other kinds differ greatly, be distributed in 10 primers, demonstrate 1 characteristic strip from Filipine luxuriant and rich with fragrance 7 kinds in the amplified production of OPC01 primer, the silver-colored wild goose kind of our new seed selection demonstrates OPS20-1 and two amplified bands of OPS20-4 in the amplified production of OPS20 primer.
Information by the RAPD amplified band is carried out the UPMGA cluster analysis to asparagus bean kind sibship, obtains its dna molecular dendrogram.The asparagus bean kind can be divided into 5 product populations, that is: product population A (containing 12 kinds); Product population B (containing 18 kinds); Product population C (containing 4 kinds); Product population D (containing 4 kinds) and product population E (containing 2 kinds).Product population B can be divided into 2 kind subgroups again.
Optimize the RAPD reaction system of asparagus bean, realized the kind Molecular Identification.As identify that cowpea No. two kinds in Hubei Province can utilize primer OPC11 earlier, the asparagus bean variety genome DNA has 3 amplified bands under this primer guiding, Hubei Province cowpea is arranged No. two (early emerald green) and line fresh kidney beans and three kinds of No. 5 cowpeas of 110 banding patterns wherein, can get rid of other kinds, utilize a primer just passable again, the three is distinguished, as use primer OPS10, three's banding pattern is respectively: 00010000,00100110 and 00100010.Choose arrow cowpea four strains as duricrust beans, white skin incense coil cowpea, short tiger and Kaifeng, they all have identical amplified band number between any two, the primer number of apparent band too, but the above two just can be distinguished with primer OPJ03, their banding pattern is respectively 10011 and 11011, both just can distinguish the back with OPC11, and their banding pattern is respectively 111 and 000.In a word, use 1 and above effective primer can distinguish and identify all asparagus bean kinds, realize the dna molecular fingerprint identification of asparagus bean kind.
The random primer of 30 10bp that the present invention adopts, therefrom having filtered out 23 effective primers (promptly can amplify the primer of dna fragmentation, obtain the different primer guiding electrophoretogram of 40 asparagus bean kind blade genomic dnas of amplification down respectively in the genomic dna of 40 asparagus bean kinds.Detect 18 characteristic strips altogether, removing No. 12 kind phenanthrene 7 has 1 (OPC01-6) and No. 26 kind silver wild geese that 2 (OPS20-1 are arranged in the amplified production of No. 18 primer OPS20 in the amplified production of No. 3 primer OPC01, OPS20-4) outside the characteristic strip, all the other 15 characteristic strips are No. 27 kinds Semen Phaseoli that dries and produce, its primer distributes: No. 4 primer OPC02, No. 7 primer OPC08, No. 8 primer OPC09, No. 13 primer OPP17, each 1 of No. 19 primer OPU10 and No. 20 primer OPU11, No. 2 primer OPA07, each 2 of No. 12 primer OPJ09 and No. 14 primer OPS07, No. 23 primer OPU20 are totally 3 (table 1, table 3).Fig. 1 to Figure 10 is respectively the RAPD amplified production electrophorogram of 10 primers enumerating.
Based on of the cluster analysis of RAPD technology to the asparagus bean kind:
140 RAPD that read 23 40 variety genome DNAs that primer produced analyze the polymorphism amplified band, set up database.Neighbor program with the PHYLIP3.6 software package is carried out cluster analysis by not being weighted to arithmetical av (UPGMA, Unweighted pair groupmethod with arithmetic mean) method.What at first obtain is 40 interracial genetic distance matrixes of asparagus bean.Genetic distance is big more, and interracial sibship is also just far away more; Carry out interracial cluster analysis again and obtain dendrogram (Figure 11).
According to cluster result, it is more reasonable that these domestic representational 40 asparagus bean kinds are divided into 5 product populations, that is:
Product population A:11,19,25,32,33,34,35,36,37,38,39,40
Product population B:04,05,07,08,09,14,15,16,17,18,21,22,23,24,28,29,30,31
Product population C:02,03,06,26
Product population D:10,12,20,27
Product population E:01,13
Wherein, product population B can be divided into 2 kind subgroups again, is called B-1 and B-2.
Product population A mainly is made of 12 short living kinds and half sprawling species etc.Have in the product population to show altogether and be with 16, the percentage of polymorphism bands of a spectrum is 85.19% (table 4).Asparagus bean be by the common cowpea that originates from Africa in the result of ground long-term evolution such as China, the most approaching common cowpea of this product population proterties is to carry out the transition to that to overgrow be the intermediate type of the asparagus bean kind led, is than primary asparagus bean product population.Its properties and characteristics is that short life, pod are short, pod color is darker, and seed is arranged closely etc.Varietal character is that historical the evolution forms, and by natural selection and artificial selection, the transgenation accumulation causes dna sequence dna difference, thus decision proterties difference.Illustrate that also habit and pod color isophenous proterties difference reflect the more different of hereditary basis.
Product population B mainly is made of 18 cowpea varieties of overgrowing, and has in the product population to show altogether to be with 3, and the percentage of polymorphism bands of a spectrum is 97.81% (table 4).It is overgrow, and pod is long, and much more shallow pod color is, the seed proterties such as sparse of arranging belongs to the characteristic feature of China's asparagus bean local variety, is the variety type that the shorter life and the type kind of partly overgrowing are evolved, and is the long-term result who selects of China working people, the interior heritable variation of group is bigger, and varietal character is various.The B-1 subgroup mostly is the northern China kind, and these kinds show as pod simultaneously on proterties more elongated.Kailu line beans (31) as the Inner Mongol.U.S. ground beans also gather in this subgroup, but itself and other kind genetic distance farthest.The B-2 subgroup mostly is China's south kind, also has the cowpea variety green for a long time of Bangladesh.As if its cluster is similar and gather earlier in cluster according to pod shape etc. again, and as Du's cowpea (15), fast (29) the three kind pods of red Ricefield eel bone (22) and yellow are thicker, press careless beans (18), cowpea (24) and skin incense coil cowpea (23) three kind pods weak point and crooked slightly month in and month out in vain.Product population C mainly is made of 4 kinds of seed selection, has in the group to show altogether to be with 33, and the percentage of polymorphism bands of a spectrum is 65.26% (table 4).As wherein short brave kind (02) be us with cowpea 14 (07, belong to the B-2 subgroup) do maternal and U.S.'s ground beans (08, genus B-1 subgroup) are made male parent, the new variety that select in the filial generation.Silver wild goose (26) is that we are maternal with Du's cowpea (15, belong to the B-2 subgroup), with the new variety that select in No. four (14, belong to the B-2 subgroup) filial generations of high yield.Because hybridization causes gene recombination, make the dna sequence dna difference increase of seed selection kind and China's local variety, so cluster is together thereafter.
4 kinds that the main origin of product population D comes from South East Asia constitute, and have in the product population to show altogether to be with 39, and the percentage of polymorphism bands of a spectrum is 61.39% (table 4).Cowpea is the self-pollinated plant of comparison strictness, limited gene exchange, this product population long-term with China in kind under different envrionment conditionss, develop, this geographical isolation, cause it obviously to be divided into the different varieties group, hereditary difference is big even surpassed the dna sequence dna difference that hybridization back genetic recombination is brought between the kind in China same product population.The also mixing breed in the explanation product population, the degree of genetic improvement is limited.
Product population E is made up of new seed selection kind, has in the product population to show altogether to be with 51, and the percentage of polymorphism bands of a spectrum is 35.44% (table 4).Wherein Hubei Province cowpea No. two (01) is that we are to belong to different varieties group's No. 5 cowpeas (06, genus product population C) and cowpea 19 (05, belong to B-1 kind subgroup) be the new variety of orthoselection among the offspring of parent's hybridization, it has tangible heterobeltiosis at aspects such as precocity, pod commodity property and high yields.The gene recombination that this different varieties group's interracial hybridization causes, make the kind of selection cross in dna sequence dna and China's local variety and the product population, kind as selection cross in the above-mentioned kind group, accelerated evolutionary more, aggravated dna sequence dna difference, 23 effective primers to the amplification of cowpea variety Genome RAPD, it has 6 not have amplified production, has two primers (OPC07 and OPS10) only to have a kind identical with its banding pattern.Illustrate also that the kind sibship is determined and the parent selects to match extreme importance in successfully improving the breed.
| The kind code name | Variety name | The source | The primer number of tool amplified production | Occupy the ratio of imitating primer | The total band of kind amplification number | Account for the ratio of all band numbers | Characteristic strip |
| ?01 | No. two, Hubei Province cowpea | Jianghan University | ?17 | ?73.91％ | ?55 | ?39.00％ | ?0 |
| ?02 | Short tiger | Jianghan University | ?21 | ?91.30％ | ?72 | ?51.06％ | ?0 |
| ?03 | The line fresh kidney beans | Guangxi | ?20 | ?86.96％ | ?66 | ?46.81％ | ?0 |
| ?04 | Ground, Yinchuan fresh kidney beans | Ningxia | ?18 | ?78.26％ | ?62 | ?43.97％ | ?0 |
| ?05 | Cowpea 19 | Zhejiang | ?18 | ?78.26％ | ?55 | ?39.00％ | ?0 |
| ?06 | No. 5 cowpeas | Hubei | ?20 | ?86.96％ | ?62 | ?43.97％ | ?0 |
| ?07 | Cowpea 14 | Zhejiang | ?11 | ?47.83％ | ?27 | ?19.15％ | ?0 |
| ?08 | U.S.'s ground beans | Draw from the U.S. | ?22 | ?95.65％ | ?67 | ?47.52％ | ?0 |
| ?09 | Cowpea green for a long time | Broad-mouthed receptacle for holding liquid Jia La state | ?18 | ?78.26％ | ?55 | ?39.00％ | ?0 |
| ?10 | Hong Kong powder beans | Draw from Britain | ?22 | ?95.65％ | ?66 | ?46.81％ | ?0 |
| ?11 | Red kind three chi Changhong beans | Japan | ?22 | ?95.65％ | ?69 | ?48.94％ | ?0 |
| ?12 | Luxuriant and rich with fragrance 7 | Philippines | ?23 | ?100％ | ?77 | ?54.61％ | ?OPC01-6 |
| ?13 | Hand over the black and white beans | Taiwan | ?20 | ?86.96％ | ?75 | ?53.19％ | ?0 |
| ?14 | No. four, high yield | Guangdong | ?17 | ?73.91％ | ?60 | ?42.55％ | ?0 |
| ?15 | Du's cowpea | Hubei | ?20 | ?86.96％ | ?70 | ?49.65％ | ?0 |
| ?16 | Purple chief | Jianghan University | ?21 | ?91.30％ | ?66 | ?46.81％ | ?0 |
| ?17 | Thailand's cowpea | Thailand | ?20 | ?86.96％ | ?74 | ?52.48％ | ?0 |
| ?18 | Press careless beans | Hainan | ?23 | ?100％ | ?75 | ?53.19％ | ?0 |
| ?19 | Collude red | Heilungkiang | ?23 | ?100％ | ?81 | ?57.45％ | ?0 |
| ?20 | The duricrust beans | Guizhou | ?21 | ?91.30％ | ?67 | ?47.52％ | ?0 |
| ?21 | Long white cowpea | Sichuan | ?21 | ?91.30％ | ?70 | ?49.65％ | ?0 |
| ?22 | Red Ricefield eel bone | Hubei | ?21 | ?91.30％ | ?55 | ?39.00％ | ?0 |
| ?23 | White skin incense coil cowpea | Jiangsu | ?21 | ?91.30％ | ?67 | ?47.52％ | ?0 |
| ?24 | Cowpea month in and month out | Anhui | ?23 | ?100％ | ?65 | ?46.10％ | ?0 |
| ?25 | Climb beans in vain | Hebei | ?22 | ?95.65％ | ?72 | ?51.06％ | ?0 |
| ?26 | The silver wild goose | Jianghan University | ?20 | ?86.96％ | ?63 | ?44.68％ | ?OPS20-1， ?OPS20-4 |
| ?27 | Semen Phaseoli dries | Guangdong | ?22 | ?95.65％ | ?6O | ?42.55％ | ?15 |
| ?28 | Chrysanthemum green grass or young crops | Beijing | ?22 | ?95.65％ | ?65 | ?46.10％ | ?0 |
| ?29 | Chrysanthemum is fast | Tianjin | ?21 | ?91.30％ | ?62 | ?43.97％ | ?0 |
| ?30 | Ivory white | Hebei | ?17 | ?73.91％ | ?45 | ?31.91％ | ?0 |
| ?31 | Kailu line beans | The Inner Mongol | ?23 | ?100％ | ?83 | ?58.87％ | ?0 |
| ?32 | The quarter butt cowpea | Jilin | ?22 | ?95.65％ | ?76 | ?53.90％ | ?0 |
| ?33 | Changde rascal cowpea | The Hunan | ?21 | ?91.30％ | ?76 | ?53.90％ | ?0 |
| ?34 | The arrow cowpea is chosen in Kaifeng | Henan | ?21 | ?91.30％ | ?72 | ?51.06％ | ?0 |
| ?35 | An IOU issued by a post office cowpea | Gansu | ?21 | ?91.30％ | ?73 | ?51.77％ | ?0 |
| ?36 | Hot-tempered cowpea | Anhui | ?20 | ?86.96％ | ?58 | ?41.13％ | ?0 |
| ?37 | Ground, Baoji fresh kidney beans | Shaanxi | ?22 | ?95.65％ | ?64 | ?45.39％ | ?0 |
| ?38 | Tea sieve rice one | Japan | ?17 | ?73.91％ | ?54 | ?38.30％ | ?0 |
| ?39 | The blue or green cowpea of high yield | Jianghan University | ?19 | ?82.61％ | ?68 | ?48.23％ | ?0 |
| ?40 | White cowpea | Guizhou | ?19 | ?82.61％ | ?65 | ?46.10％ | ?0 |
Table 1
| Sequence number | Primer | Sequence 5 ' → 3 ' | ?GC％ | Total DNA band number increases | Polymorphic dna band number | The percentage of polymorphism bands of a spectrum |
| ?1 | ?OPA03 | ?TTAGCGCCCC | ?70 | ?5 | ?4 | ?80％ |
| ?2 | ?OPA07 | ?CTACGCTCAC | ?60 | ?6 | ?6 | ?100％ |
| ?3 | ?OPC01 | ?TCCCAGCAGA | ?60 | ?7 | ?7 | ?100％ |
| ?4 | ?OPC02 | ?GTGAGGCGTC | ?70 | ?11 | ?11 | ?100％ |
| ?5 | ?OPC06 | ?CCAGAACGGA | ?60 | ?4 | ?4 | ?100％ |
| ?6 | ?OPC07 | ?GTCCCGACGA | ?70 | ?6 | ?6 | ?100％ |
| ?7 | ?OPC08 | ?TTTGGGTGCC | ?60 | ?6 | ?6 | ?100％ |
| ?8 | ?OPC09 | ?CTCACCGTCC | ?70 | ?7 | ?7 | ?100％ |
| ?9 | ?OPC11 | ?AAAGCTGCGG | ?60 | ?3 | ?3 | ?100％ |
| ?10 | ?OPI02 | ?AGCCGTTCAG | ?60 | ?5 | ?5 | ?100％ |
| ?11 | ?OPJ03 | ?AGCACCTCGT | ?60 | ?5 | ?5 | ?100％ |
| ?12 | ?OPJ09 | ?ACGGCACGCA | ?70 | ?7 | ?7 | ?100％ |
| ?13 | ?OPP17 | ?AAGGCACGAG | ?60 | ?4 | ?4 | ?100％ |
| ?14 | ?OPS07 | ?TCCGATGCTG | ?60 | ?7 | ?7 | ?100％ |
| ?15 | ?OPS10 | ?ACCGTTCCAG | ?60 | ?8 | ?8 | ?100％ |
| ?16 | ?OPS18 | ?CTGGCGAACT | ?60 | ?8 | ?8 | ?100％ |
| ?17 | ?OPS19 | ?GAGTCAGCAG | ?60 | ?4 | ?4 | ?100％ |
| ?18 | ?OPS20 | ?TCTGGACGGA | ?60 | ?4 | ?4 | ?100％ |
| ?19 | ?OPU10 | ?ACCTCGGCAC | ?70 | ?7 | ?7 | ?100％ |
| ?20 | ?OPU11 | ?AGACCCAGAG | ?60 | ?8 | ?8 | ?100％ |
| ?21 | ?OPU17 | ?ACCTGGGGAG | ?70 | ?6 | ?6 | ?100％ |
| ?22 | ?OPU18 | ?GAGGTCCACA | ?60 | ?6 | ?6 | ?100％ |
| ?23 | ?OPU20 | ?ACAGCCCCCA | ?70 | ?7 | ?7 | ?100％ |
| Amount to | ?141 | ?140 | ||||
| On average | ?6.13 | ?6.09 |
Table 2
| Primer | The band sequence number | Number of times | Frequency | Primer | The band sequence number | Number of times | Frequency | Primer | The band sequence number | Number of times | Frequency | Primer | The band sequence number | Number of times | Frequency |
| ?OPA0 ?3 | ?1 | ?12 | ?0.30 | ?OPC0 ?7 | ?36 | ?28 | ?0.70 | ?OPJ0 ?9 | ?71 | ?1 | ?0.03 | ?OPS2 ?0 | ?106 | ?1 | ?0.03 |
| ?2 | ?39 | ?0.98 | ?37 | ?17 | ?0.43 | ?OPP1 ?7 | ?72 | ?1 | ?0.03 | ?OPU1 ?0 | ?107 | ?32 | ?0.80 | ||
| ?3 | ?36 | ?0.90 | ?38 | ?15 | ?0.38 | ?73 | ?37 | ?0.93 | ?108 | ?28 | ?0.70 | ||||
| ?4 | ?38 | ?0.95 | ?OPC0 ?8 | ?39 | ?1 | ?0.03 | ?74 | ?39 | ?0.98 | ?109 | ?1 | ?0.03 | |||
| ?OPA0 ?7 | ?5 | ?1 | ?0.03 | ?40 | ?11 | ?0.28 | ?75 | ?2 | ?0.05 | ?110 | ?31 | ?0.78 | |||
| ?6 | ?1 | ?0.03 | ?41 | ?34 | ?0.85 | ?OPS0 ?7 | ?76 | ?11 | ?0.28 | ?111 | ?7 | ?0.18 | |||
| ?7 | ?28 | ?0.70 | ?42 | ?34 | ?0.85 | ?77 | ?3 | ?0.08 | ?112 | ?6 | ?0.15 | ||||
| ?8 | ?38 | ?0.95 | ?43 | ?34 | ?0.85 | ?78 | ?35 | ?0.88 | ?113 | ?4 | ?0.10 | ||||
| ?9 | ?9 | ?0.23 | ?44 | ?24 | ?0.60 | ?79 | ?15 | ?0.38 | ?OPU1 ?1 | ?114 | ?35 | ?0.88 | |||
| ?10 | ?34 | ?0.85 | ?OPC0 ?9 | ?45 | ?34 | ?0.85 | ?80 | ?3 | ?0.08 | ?115 | ?25 | ?0.63 | |||
| ?OPC0 ?1 | ?11 | ?7 | ?0.18 | ?46 | ?31 | ?0.78 | ?81 | ?1 | ?0.03 | ?116 | ?36 | ?0.90 | |||
| ?12 | ?33 | ?0.83 | ?47 | ?20 | ?0.50 | ?82 | ?1 | ?0.03 | ?117 | ?32 | ?0.80 | ||||
| ?13 | ?6 | ?0.15 | ?48 | ?23 | ?0.58 | ?OPS1 ?0 | ?83 | ?2 | ?0.05 | ?118 | ?34 | ?0.85 | |||
| ?14 | ?12 | ?0.30 | ?49 | ?1 | ?0.03 | ?84 | ?2 | ?0.05 | ?119 | ?28 | ?0.70 | ||||
| ?15 | ?15 | ?0.38 | ?50 | ?21 | ?0.53 | ?85 | ?24 | ?0.60 | ?120 | ?1 | ?0.03 | ||||
| ?16 | ?1 | ?0.03 | ?51 | ?10 | ?0.25 | ?86 | ?21 | ?0.53 | ?121 | ?29 | ?0.73 | ||||
| ?17 | ?28 | ?0.70 | ?OPC1 ?1 | ?52 | ?37 | ?0.93 | ?87 | ?5 | ?0.13 | ?OPU1 ?7 | ?122 | ?29 | ?0.73 | ||
| ?OPC0 ?2 | ?18 | ?1 | ?0.03 | ?53 | ?5 | ?0.13 | ?88 | ?32 | ?0.80 | ?123 | ?8 | ?0.20 | |||
| ?19 | ?6 | ?0.15 | ?54 | ?2 | ?0.05 | ?89 | ?31 | ?0.78 | ?124 | ?12 | ?0.30 | ||||
| ?20 | ?36 | ?0.90 | ?OPI0 ?2 | ?55 | ?31 | ?0.78 | ?90 | ?7 | ?0.18 | ?125 | ?28 | ?0.70 | |||
| ?21 | ?8 | ?0.20 | ?56 | ?26 | ?0.65 | ?OPS1 ?8 | ?91 | ?6 | ?0.15 | ?126 | ?9 | ?0.23 | |||
| ?22 | ?24 | ?0.60 | ?57 | ?27 | ?0.68 | ?92 | ?14 | ?0.35 | ?127 | ?33 | ?0.83 | ||||
| ?23 | ?6 | ?0.15 | ?58 | ?36 | ?0.90 | ?93 | ?2 | ?0.05 | ?OPU1 | ?128 | ?28 | ?0.70 |
| ?8 | |||||||||||||||
| ?24 | ?16 | ?0.40 | ?59 | ?30 | ?0.75 | ?94 | ?7 | ?0.18 | ?129 | ?32 | ?0.80 | ||||
| ?25 | ?3 | ?0.08 | ?OPJ0 ?3 | ?60 | ?34 | ?0.85 | ?95 | ?2l | ?0.53 | ?130 | ?32 | ?0.80 | |||
| ?26 | ?28 | ?0.70 | ?61 | ?15 | ?0.38 | ?96 | ?26 | ?0.65 | ?131 | ?10 | ?0.25 | ||||
| ?27 | ?3 | ?0.08 | ?62 | ?2 | ?0.05 | ?97 | ?6 | ?0.15 | ?132 | ?27 | ?0.68 | ||||
| ?28 | ?10 | ?0.25 | ?63 | ?39 | ?0.98 | ?98 | ?26 | ?0.65 | ?133 | ?3 | ?0.08 | ||||
| ?OPC0 ?6 | ?29 | ?3 | ?0.08 | ?64 | ?39 | ?0.98 | ?OPS1 ?9 | ?99 | ?29 | ?0.73 | ?OPU2 ?0 | ?134 | ?1 | ?0.03 | |
| ?30 | ?18 | ?0.45 | ?OPJ0 ?9 | ?65 | ?30 | ?0.75 | ?100 | ?26 | ?0.65 | ?135 | ?30 | ?0.75 | |||
| ?31 | ?25 | ?0.63 | ?66 | ?1 | ?0.03 | ?101 | ?27 | ?0.68 | ?136 | ?1 | ?0.03 | ||||
| ?32 | ?25 | ?0.63 | ?67 | ?30 | ?0.75 | ?102 | ?25 | ?0.63 | ?137 | ?20 | ?0.50 | ||||
| ?OPC0 ?7 | ?33 | ?34 | ?0.85 | ?68 | ?27 | ?0.68 | ?OPS2 ?0 | ?103 | ?1 | ?0.03 | ?138 | ?13 | ?0.33 | ||
| ?34 | ?3 | ?0.08 | ?69 | ?2 | ?0.05 | ?l04 | ?29 | ?0.73 | ?139 | ?1 | ?0.03 | ||||
| ?35 | ?5 | ?0.13 | ??70 | ?34 | ?0.85 | ?105 | ?29 | ?0.73 | ?140 | ?3 | ??0.08 |
Table 3
| Product population A | Product population B | Product population C | Product population D | Product population E | |
| Total DNA band number increases | ?108 | ?137 | ?95 | ?101 | ?79 |
| Polymorphic dna band number | ?92 | ?134 | ?62 | ?62 | ?28 |
| The percentage of polymorphism bands of a spectrum | ?85.19％ | ?97.81％ | ?65.26％ | ?61.39％ | ?35.44％ |
Table 4
Claims (7)
1, a kind of cowpea variety molecular assay method of analyzing based on Genome RAPD, it comprises the steps:
(1), extracts the existing genomic DNA of each cowpea variety;
(2), institute extracted the genomic DNA of cowpea variety carry out the polymerase chain reaction respectively, obtain random amplified polymorphic DNA product;
(3), respectively random amplified polymorphic DNA product is carried out electrophoretic separation with 1.0% agarose, EB directly is added in the gel, and total applied sample amount is 8 μ l, wherein 4 μ l amplified productions, other adds 40% sucrose solution, 2 μ l and 0.1% tetrabromophenol sulfonphthalein, 2 μ l, constant voltage electrophoresis 1.5h under 80V;
(4), electrophoresis finishes the back and takes a picture on the UV-light detector, obtains the randomly amplified polymorphic DNA standard diagram respectively;
(5) select clear randomly amplified polymorphic DNA collection of illustrative plates to adopt " 0-1 " system log (SYSLOG) bands of a spectrum, the assignment that band will be arranged is 1, and the assignment of not having band is 0; Read the data of each material, set up database at different primer extension product electrophoretic bands;
(6), by the arithmetical av method being carried out cluster analysis not to be weighted to, obtain system's clustering tree;
(7), the cowpea variety of required evaluation is analyzed by above-mentioned steps, obtain the randomly amplified polymorphic DNA collection of illustrative plates of required evaluation cowpea variety, this randomly amplified polymorphic DNA collection of illustrative plates and standard sample spectrum are compared judgement, obtain the Analysis and Identification result.
2, according to a kind of cowpea variety molecular assay method of analyzing based on Genome RAPD of claim 1, wherein the reaction conditions of polymerase chain reaction is as follows: primer (25pmol/L) 1.0 μ l, Taq archaeal dna polymerase (1u/ μ l) 1 μ l, dNTPs (10mmol/L) 0.5 μ l, MgCl
2(25mmol/L) 1.5 μ l, 10 * buffer, 2.5 μ l, template DNA (50ng/ μ l) 1 μ l, ultrapure water 17.5 μ l.
3, according to a kind of cowpea variety molecular assay method of analyzing based on Genome RAPD of claim 2, wherein: the described primer in polymerase chain reaction is OPA03 TTAGCGCCCC, OPA07 CTACGCTCAC, OPC01 TCCCAGCAGA, OPC02 GTGAGGCGTC, OPC06 CCAGAACGGA, OPC07 GTCCCGACGA, OPC08 TTTGGGTGCC, OPC09 CTCACCGTCC, OPC11 AAAGCTGCGG, OPI02 AGCCGTTCAG, OPJ03 AGCACCTCGT, OPJ09 ACGGCACGCA, OPP17 AAGGCACGAG, OPS07 TCCGATGCTG, OPS10 ACCGTTCCAG, OPS18 CTGGCGAACT, OPS19 GAGTCAGCAG, OPS20 TCTGGACGGA, OPU10 ACCTCGGCAC, OPU11 AGACCCAGAG, OPU17 ACCTGGGGAG, OPU18 GAGGTCCACA, OPU20 ACAGCCCCCA.
4, according to a kind of cowpea variety molecular assay method of analyzing based on Genome RAPD of claim 1, wherein: Mg in the polymerase chain reaction of described asparagus bean genomic dna
2+Concentration is 1.5mmol/L.
5, according to a kind of cowpea variety molecular assay method of analyzing based on Genome RAPD of claim 1, wherein: the consumption of Taq enzyme is 1.0 ~ 1.5u in the polymerase chain reaction of described asparagus bean genomic dna.
6, according to a kind of cowpea variety molecular assay method of analyzing based on Genome RAPD of claim 1, wherein: the concentration of oligonucleotide is 0.025 μ mol/L in the randomly amplified polymorphic DNA analytical procedure of described asparagus bean genomic dna.
7, according to a kind of cowpea variety molecular assay method of analyzing based on Genome RAPD of claim 1, wherein: Tm is 38.0 ℃ in the randomly amplified polymorphic DNA analytical procedure of described asparagus bean genomic dna.
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