CN105420368A - Method for constructing fingerprint of phaseolus vulgaris according to SSR (simple sequence repeat) molecular marker and application - Google Patents
Method for constructing fingerprint of phaseolus vulgaris according to SSR (simple sequence repeat) molecular marker and application Download PDFInfo
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Abstract
The invention discloses a method for constructing a fingerprint of phaseolus vulgaris according to an SSR (simple sequence repeat) molecular marker and applications, and belongs to the technical field of finger print construction. The method comprises the following steps of extracting phaseolus vulgaris DNA (deoxyribonucleic acid), performing PCR (polymerase chain reaction) amplified reaction by utilizing the phaseolus vulgaris DNA and SSR core primers, and after electrophoresis detection, constructing the characteristic fingerprint of the phaseolus vulgaris according to an electrophoresis result; the nucleotide sequences of the SSR core primers are as shown by SEQIDNO.1-16. Compared with conventional morphological identification, the method disclosed by the invention is accurate and reliable in identification result and short in consumed time, is not influenced by environments, is simple in operation, can be used for identifying multiple varieties simultaneously, and is convenient and efficient.
Description
Technical field
The present invention relates to a kind of method and the application that build Kidney bean finger printing based on SSR molecular marker, belong to fingerprint map construction technical field.
Background technology
Kidney bean (Phaseolusvulgaris) is one of most important Food Legume, accounts for 50% of global Food Legume output, and China's germplasm resources on Phaseolus vulgaris is abundant, various in style, and cultivation of choosing seeds through for a long time, creates dissimilar variety source.The taxonomy and nomenclature standard disunity of current Bean Varieties, single is difficult to different Bean Varieties to distinguish from view of morphology.
DNA fingerprinting refers to the DNA electrophoretogram can differentiating difference between biont, and it is a kind of authenticate technology be based upon on DNA molecular marker basis.The structure of DNA fingerprinting to be without prejudice very important effect for the rights and interests of protection breeder, and SSR molecular marker has been proved to be a kind of cultivar identification method efficiently.SSR (Simplesequencerepeat), also known as microsatellite DNA, is made up of short tandem repetitive sequence, and be distributed in eukaryotic gene group, the difference due to multiplicity makes site present polymorphism.The single-copy sequence that one section relatively conservative is had in both sides, SSR seat, the SSR sequence of the different multiplicity that increases according to this sequences Design a pair Auele Specific Primer, after poly-electrophoresis, compare the migration distance of amplified band, just can judge the length polymorphism of Different Individual on certain SSR seat.SSR is codominant marker, and be that one of the most frequently used mark studied by current genetic diversity and genetic construction, SSR marker has stable, polymorphism advantages of higher.Also utilize molecular marking technique to conduct a research in Kidney bean, but mostly lay particular emphasis on the sibship of certain class Kidney bean resource material is studied.Also do not set up the report of Bean Varieties DNA fingerprinting thus differential variety at present.
Summary of the invention
For solving the deficiencies in the prior art, the invention provides a kind of method building Kidney bean finger printing based on SSR molecular marker, the technical scheme of employing is as follows:
The object of the present invention is to provide a kind of method building Kidney bean finger printing based on SSR molecular marker, the method extracts Kidney bean DNA, utilize Kidney bean DNA and SSR core primers to carry out pcr amplification reaction, after electrophoresis detection, build Kidney bean characteristic fingerprint pattern according to electrophoresis result; The nucleotide sequence of described SSR core primers is as shown in SEQIDNO.1-16.
Described method steps is as follows:
1) extract Kidney bean DNA, obtain Kidney bean DNA;
2) SSR core primers is obtained by screening; Described SSR core primers, often pair of SSR core primers comprises upstream primer and downstream primer, and nucleotide sequence is as shown in SEQIDNO.1-16;
3) step 1 is utilized) gained Kidney bean DNA and step 2) gained SSR core primers carries out pcr amplification reaction, then carry out 8% native polyacrylamide gel electrophoresis to PCR primer to detect, the colour developing of silver dye, builds Kidney bean characteristic fingerprint pattern according to electrophoresis result.
Preferably, step 1) described Bean Varieties is assorted flower No. 1, beans, short rice bean, rabbit leg, flower waist beans, nest youth beans, spend waist beans in vain, purple spends beans in vain, coloured kidney bean, the large horse's hoe, Phaseolus vulgarisl, red coloured kidney bean, little black kidney bean, duricrust beans, broadside beans, meat angle beans, long white Nanjing, 40 smallpox beans, early red bean, Caulis et folium clerodendri bungei kidney beans, Big White Flower river beans, local river beans, duricrust flower river beans, small ocean beans, great Hua ocean beans, Mei Dou, orange plum beans, powder yellow Fen beans, the red golden flower of large grain, red sword bean, right beans, black frame beans, flower plum beans, black little red bean, red cow gram, white grain red bean or cow gram.
Preferably, step 1) described extraction Kidney bean DNA, method, for getting Kidney bean spire, adds liquid nitrogen grinding powdered, adopts RNA isolation kit to extract DNA, detects the quality of STb gene, then STb gene is diluted to 100ng/ μ L, be stored in-20 DEG C for subsequent use.
Preferably, step 2) described screening, method is:
Step one, primary dcreening operation primer: filter out that to have polymorphism, banding pattern between different Bean Varieties clear and can stablize the SSR primary dcreening operation primer of repetition;
Step 2, sieve primer again: in the SSR primary dcreening operation primer that step one obtains, filter out the core primers that the highest and SSR primer that loci number is maximum of polymorphism information content values between Bean Varieties builds as Bean Varieties fingerprint databases, loci number according to core primers divides monoid, and the kind being in same site is divided into same monoid;
Other primers are divided subclass group by step 3 in the monoid divided in step 2, by that analogy, only have a kind until each monoid is assigned to.
Preferably, step 3) described PCR reaction system comprises DNA profiling 0.8 μ L, 10pmol/L upstream primer 0.2 μ L, 10pmol/L downstream primer 0.2 μ L, 10 × PCRbuffer2 μ L, 25mmol/LMg
2+1.2 μ L, 10mmol/LdNTP0.5 μ L, 5U/ μ LTaq enzyme 0.2 μ L, ddH
2o14.4 μ L; Described PCRbuffer is not containing Mg
2+.
Preferably, step 3) described PCR response procedures is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 15s, annealing 15s, 72 DEG C extend 30s, totally 35 circulations; 72 DEG C extend 10min; 4 DEG C of preservations; The annealing temperature of described each SSR core primers is: the annealing temperature of CD11 and CD15 is 50 DEG C, and CD16, CD17 and CD18 annealing temperature is 52 DEG C, and the annealing temperature of CD12 is 53 DEG C, and the annealing temperature of CD13 and CD14 is 55 DEG C.
Preferably, step 3) described structure Kidney bean characteristic fingerprint pattern, be the mobility record electrophoresis result according to stranded DNA molecule amount and primer, the maximum band of the often pair of primer mobility is designated as 1, and what mobility was taken second place is designated as 2, the like, be designated as 0 without band, separate with '-' between different primers, set up SSR genotype information data, encode according to primer order, the finger-print code obtained is the characteristic fingerprint pattern of this kind.
Described method concrete steps are:
1) get Kidney bean spire, add liquid nitrogen grinding powdered, adopt RNA isolation kit to extract DNA, detect the quality of STb gene, then STb gene is diluted to 100ng/ μ L, be stored in-20 DEG C for subsequent use, obtain Kidney bean DNA;
2) SSR core primers is obtained by screening; Described SSR core primers, often pair comprises upstream primer and downstream primer, and nucleotide sequence is as shown in SEQIDNO.1-16; Described screening, method is: step one, primary dcreening operation primer: filter out that between different Bean Varieties, to have polymorphism, banding pattern clear and can stablize the SSR primary dcreening operation primer of repetition; Step 2, sieve primer again: in the SSR primary dcreening operation primer that step one obtains, filter out the core primers that the highest and SSR primer that loci number is maximum of polymorphism information content values between Bean Varieties builds as Bean Varieties fingerprint databases, loci number according to core primers divides monoid, and the kind being in same site is divided into same monoid; Other primers are divided subclass group by step 3 in the monoid divided in step 2, by that analogy, only have a kind until each monoid is assigned to;
3) step 1 is utilized) gained Kidney bean DNA and step 2) gained SSR core primers carries out pcr amplification reaction; Then carry out 8% native polyacrylamide gel electrophoresis to PCR primer to detect, the colour developing of silver dye, according to the mobility record electrophoresis result of stranded DNA molecule amount and primer, the maximum band of the often pair of primer mobility is designated as 1, and what mobility was taken second place is designated as 2, the like, be designated as 0 without band, separate with '-' between different primers, set up SSR genotype information data, encode according to primer order, the finger-print code obtained is the characteristic fingerprint pattern of this kind;
Described PCR reaction system comprises DNA profiling 0.8 μ L, 10pmol/L upstream primer 0.2 μ L, 10pmol/L downstream primer 0.2 μ L, 10 × PCRbuffer2 μ L, 25mmol/LMg
2+1.2 μ L, 10mmol/LdNTP0.5 μ L, 5U/ μ LTaq enzyme 0.2 μ L, ddH
2o14.4 μ L; Described PCRbuffer is not containing Mg
2+; Described PCR response procedures is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 15s, annealing 15s, and 72 DEG C extend 30s, totally 35 circulations; 72 DEG C extend 10min; 4 DEG C of preservations;
The annealing temperature of described each SSR core primers is: the annealing temperature of CD11 and CD15 is 50 DEG C, and CD16, CD17 and CD18 annealing temperature is 52 DEG C, and the annealing temperature of CD12 is 53 DEG C, and the annealing temperature of CD13 and CD14 is 55 DEG C
The above either method is building Kidney bean finger printing and is differentiating the application in Bean Varieties.
Beneficial effect of the present invention:
1, the construction process of Kidney bean DNA fingerprinting of the present invention, make use of a certain Bean Varieties DNA of SSR primer pair to increase, obtain a specific fingerprint, and one group of this specific kind of SSR primer pair is carried out DNA cloning this kind will be made may to obtain one group of distinctive DNA fingerprint, so the inventive method adopts combination of primers mirror method for distinguishing again analyze primer and screen, each kind is made to have found its distinctive DNA fingerprint.
2, compare with conventional identification of morphology, the inventive method qualification result accurately and reliably, consuming time short, not affected by environment, simple to operate, multiple kind can be identified simultaneously, convenient and swift.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not by the restriction of embodiment.
Embodiment 1:
The present embodiment, using 36 parts of Bean Varieties as sample material, utilizes the SSR core primers filtered out to build the finger print data of Bean Varieties, to reach the object of cultivar identification.
36 parts as shown in table 1 below for examination Bean Varieties information.Plant matter numbering in table 1 and derive from Crops In China kind matter Information Network.
Table 136 part supplies examination Bean Varieties information
The structure of Bean Varieties standard DNA fingerprint databases, carries out as follows:
1. each kind total DNA extraction of Kidney bean
Kidney bean is planted in soil, incubated at room temperature, get Kidney bean spire after growing 12 days and save backup in-80 DEG C after liquid nitrogen freezing.Get the tender blade 0.2g of each kind Kidney bean children in 1.5mL centrifuge tube, add liquid nitrogen grinding powdered.Adopt RNA isolation kit to extract DNA, operation experiments step is carried out according to test kit specification sheets.Detect the quality of STb gene with agarose gel electrophoresis and spectrophotometer, then STb gene be diluted to 100ng/ μ L, be stored in-20 DEG C for subsequent use.
2. the screening of primer
A.SSR primer synthesizes: the purity requirement of primer synthesis is PAGE purifying, and is distributed into 1OD/ pipe, and the concentration of upstream and downstream primer is all diluted to 10pmolL
-1.
The screening method of b.SSR primer pair sequence:
Step one, primary dcreening operation primer: filter out that to have polymorphism, banding pattern between different Bean Varieties clear and can stablize the SSR primary dcreening operation primer of repetition; Step 2, sieve primer again: in the SSR primary dcreening operation primer that step one obtains, find out the core primers that a pair the highest the and SSR primer that loci number is maximum of polymorphism information content values builds as Bean Varieties fingerprint databases between Bean Varieties, divide monoid according to its loci number, the kind being namely in same site divides monoid; Step 3, with the differentiation result of core primers for benchmark, then detects at each monoid with another pair of primers, the same division subclass group in same site in each monoid, by that analogy, be divided into more monoid like this, only had a kind until each monoid is assigned to.
Obtain following 8 pairs of SSR core primers by screening, Primer is as shown in table 2.
Table 2 Primer
The screening step of c.SSR primer pair sequence:
With described Bean Varieties for material, utilize the SSR primer of described synthesis to carry out pcr amplification to the DNA of each Bean Varieties respectively, the PCR reaction system adopted in PCR reaction test is 20 μ L systems: DNA profiling 0.8 μ L, F-primer (10pmolL
-1) 0.2 μ L, R-primer (10pmolL
-1) 0.2 μ L, 10 × PCRbuffer be (containing Mg
2+) 2 μ L, Mg
2+(25mmolL
-1) 1.2 μ L, dNTP (10mmolL
-1) 0.5 μ L, Taq enzyme (5U μ L
-1) 0.2 μ L, ddH
2o14.4 μ L, amounts to 20.0 μ L.
PCR response procedures is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 15s, annealing temperature (different primers annealing temperature is in table 2) annealing 15s, and 72 DEG C extend 30s, totally 35 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.
D. gel electrophoresis and the colour developing of silver dye
Amplified production adopts 8% native polyacrylamide gel electrophoresis to detect, and electrophoresis carries out in Sequi-GenGT nucleic acid electrophoresis system (Bio-Rad, USA).
The colour developing of silver dye: utilize stationary liquid (containing 50% ethanol and 2% acetic acid) fixing 3min; Outwell stationary liquid, distillation washing 1 time, time 30s, dye 5min to add staining fluid (Silver Nitrate of 0.2%); Outwell staining fluid, distillation washing 2 times, each time 30s, adds nitrite ion (3%NaOH and 0.5% formaldehyde), to bands of a spectrum are clear; Photographic recording.
3. the structure of Kidney bean DNA characteristics finger printing
The SSR primer pair sequence utilizing described screening to obtain, respectively pcr amplification is carried out to described each Bean Varieties DNA, band is read with GeneMapper4.0 software and the mode manually combined, according to the mobility record result of DNA standard molecular weight and primer, ignore assorted band weak band, the maximum band of the often pair of primer mobility is designated as 1, what mobility was taken second place is designated as 2, the like, 0 is designated as without band, separate with '-' between different primers, set up for examination material SSR genotype information data, encode according to primer order, namely according to CD11, CD12, CD13, CD14, CD15, CD16, CD17, the order of CD18 primer is carried out, the finger-print code obtained is the finger printing (table 3) of this kind.
Table 336 part material fingerprint code
| Variety name | Finger-print code | Variety name | Finger-print code |
| No. 1, hydridization beans | 2-2-5-2-3-5-3-4 | Caulis et folium clerodendri bungei kidney beans | 2-1-5-3-2-5-14-7 |
| Short rice bean | 3-4-5-5-3-5-3-7 | Big White Flower river beans | 3-2-3-4-4-4-2-6 |
| Rabbit leg | 1-2-4-5-1-3-4-7 | Local river beans | 1-3-5-4-2-2-3-6 |
| Flower waist beans | 2-1-4-1-3-5-2-3 | Duricrust flower river beans | 1-4-3-3-3-3-2-2 |
| Nest youth beans | 1-2-1-1-1-1-1-1 | Small ocean beans | 1-2-2-2-1-5-2-2 |
| Spend waist beans in vain | 1-4-5-1-4-1-5-4 | Great Hua ocean beans | 0-1-2-5-1-2-2-2 |
| Purple spends beans in vain | 3-2-5-4-3-5-4-7 | Mei Dou | 1-3-2-4-1-2-2-2 |
| Coloured kidney bean | 1-3-2-2-2-5-3-5 | Orange plum beans | 0-3-2-2-1-1-1-1 |
| The large horse's hoe | 2-2-2-4-2-5-2-2 | Powder yellow Fen beans | 4-3-2-4-1-2-2-7 |
| Phaseolus vulgarisl | 1-1-2-3-1-5-1-4 | The red golden flower of large grain | 1-4-2-2-1-1-1-0 |
| Red coloured kidney bean | 4-4-1-5-2-5-0-4 | Red sword bean | 1-3-2-2-1-1-2-2 |
| Little black kidney bean | 1-4-0-1-2-3-2-3 | Right beans | 1-4-3-1-1-2-2-3 |
| Duricrust beans | 1-4-2-3-1-4-2-3 | Black frame beans | 4-2-3-4-3-5-4-6 |
| Broadside beans | 0-4-3-5-3-3-2-2 | Flower plum beans | 1-4-3-3-2-3-4-2 |
| Meat angle beans | 2-4-5-3-1-5-14-3 | Black little red bean | 1-4-3-3-2-2-4-3 |
| Long white Nanjing | 2-2-1-1-2-1-2-1 | Red cow gram | 1-5-4-0-2-3-4-2 |
| 40 smallpox beans | 1-4-3-3-1-3-2-2 | White grain red bean | 2-4-3-0-1-2-4-2 |
| Early red bean | 1-4-3-3-3-2-2-2 | Cow gram | 1-4-3-2-2-4-3-3 |
Can be reached a conclusion by table 3, the DNA fingerprinting of any two kinds is not identical, and differential primer number, all more than 1, can judge that 36 parts supply examination Bean Varieties to be different kinds, and corresponding finger-print code is fingerprint specific to this kind.4. data statistics and analysis
Utilize the number of alleles (Na) of Popgene3.2 computed in software primer sites, Shannon diversity index, polymorphism information amount (PIC).In 36 parts of materials, 8 SSR marker detect 40 allelotrope altogether, and the number of alleles that each site detects is 4-7, average 5, each site; Primer polymorphism information amount (PIC) variation range is 0.5350 ~ 0.7737, average out to 0.6690, and all primer PIC values, all more than 0.5, show that these primers can detect abundant heritable variation (table 4).
Table 4 genetic diversity index
| Primer | Number of alleles | Effective number of allele | Shannon index | Polymorphism information content |
| CD11 | 4 | 2.3810 | 1.0889 | 0.535 |
| CD12 | 5 | 3.1984 | 1.3216 | 0.6385 |
| CD13 | 5 | 3.9690 | 1.462 | 0.7013 |
| CD14 | 4 | 3.6636 | 1.3377 | 0.6764 |
| CD15 | 4 | 3.4286 | 1.2861 | 0.6519 |
| CD16 | 6 | 4.6957 | 1.6261 | 0.7538 |
| CD17 | 5 | 2.9673 | 1.2993 | 0.6217 |
| CD18 | 7 | 4.9846 | 1.7537 | 0.7737 |
Result shows, 8 pairs of combination of primers can be used among the qualification of plant genotype, carries out genetic typing to make the material to those do not have accurate pedigree to record.The hereditary feature of qualification Bean Varieties can be applied to, protect these kinds to weigh from infringement.
Embodiment 2 primer polymorphism is verified
Pair primer pair 36 parts of Kidney beans in except core primers 40 are carried out pcr amplification for examination material, builds Kidney bean finger printing.Statistics primer polymorphism information, below list wherein 8 to the non-core primer pair wherein result that builds of 6 Variety fingerprinting, 8 pairs of non-core primer information are as shown in table 6,5 experimental cultivar finger-print codes are as shown in table 7, finger-print code primer order CD98, CD1, CD67, CD85, CD97, CD20, CD5,
cG1
The non-core primer sequence of table 5 part
The non-core primer information of table 6
| Primer | Number of alleles | PIC |
| CD98 | 1 | 0 |
| CD1 | 2 | 0.0912 |
| CD67 | 2 | 0.121 |
| CD85 | 1 | 0 |
| CD97 | 1 | 0 |
| CD20 | 1 | 0 |
| CD5 | 1 | 0 |
| CG1 | 2 | 0.151 |
A table 75 varieties systematics code
| Variety name | Finger-print code |
| Assorted flower No. 1, beans | 1-1-2-1-1-1-1-2 |
| Short rice bean | 1-1-1-1-1-1-1-2 |
| Rabbit leg | 1-2-2-1-1-1-1-1 |
| Flower waist beans | 1-1-2-1-1-1-1-2 |
| Nest youth beans | 1-2-2-1-1-1-1-1 |
Be 1 by the number of alleles of table 5 and the known primer CD98 of table 6, primer CD67, primer CD20 and primer CD5, there is no polymorphism, kind can not be distinguished; Primer CD1, primer CD67, primer CG1 polymorphism content value are low, can only by part variety plot separately, and flower waist beans are identical with assorted colored beans No. 1 fingerprint, can not be separated.Through overtesting, utilize other non-core primers can not identification of species, therefore, the primer that only embodiment 1 is screened has good identification result, and other primers all can not be identified.
Embodiment 3
Get 10 Bean Varieties (different from the place of production in embodiment 1) as verifying that material is as shown in table 8, the core primers of screening is utilized to increase to 10 Kidney beans, adopt the method for embodiment 1 to build finger printing to anonymous sample, finger-print code is as shown in table 8.
A table 810 checking Bean Varieties
A table 910 varieties systematics code
| Numbering | Variety name | Finger-print code |
| 1 | Yellow kidney bean | 4-3-3-2-2-2-3-1 |
| 2 | Flower waist beans | 2-1-4-1-3-5-2-3 |
| 3 | Coloured kidney bean | 2-3-4-2-3-2-2-1 |
| 4 | Coloured kidney bean | 1-4-1-2-3-2-3-1 |
| 5 | Mei Dou | 1-4-3-2-2-4-3-3 |
| 6 | The large horse's hoe | 2-2-2-4-2-5-2-2 |
| 7 | Duricrust beans | 2-3-1-1-2-4-1-1 |
| 8 | Paint face's beans | 1-2-5-4-3-4-2-4 |
| 9 | Phaseolus vulgarisl | 1-1-2-3-1-5-1-4 |
| 10 | Paint face's beans | 1-4-4-3-1-1-1-1 |
From table 8 and table 3, plum beans are all not identical with cow gram place of production different names in embodiment 1, but fingerprint is identical, and both explanations are same breed; Same Phaseolus vulgarisl is different from the large horse's hoe place of production in the Phaseolus vulgarisl in embodiment 1, the large horse's hoe and embodiment 1 and fingerprint is all identical, illustrates it is identical kind each other.Coloured kidney bean (comprising embodiment 1) from Different sources finds that after comparison fingerprint is each other not identical, illustrates that three is not identical kind; The yellow kidney bean (comprising embodiment 1) of same Different sources, the fingerprint of paint face's beans are also different separately, although of the same name, but not identical kind.
Illustrate thus, 36 parts of Kidney bean finger printings that 8 pairs of core primers through screening construct can be used in cultivar identification, and the method for embodiment 1 is also applicable to the qualification to other Bean Varieties.
The present invention utilizes above-mentioned technical system and 8 pairs of Kidney bean SSR polymorphism primers filtering out, constructs 36 Kidney bean DNA fingerprintings, 36 Bean Varieties can be distinguished, and provides technical support by for the qualification of Kidney bean and variety right.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention; any person skilled in the art; not departing from spirit and scope of the invention; various changes and modification can be done; therefore, what protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. one kind builds Kidney bean fingerprint based on SSR molecular marker
figurethe method of spectrum, is characterized in that, extracts Kidney bean DNA, utilizes Kidney bean DNA and SSR core primers to carry out pcr amplification reaction, builds Kidney bean characteristic fingerprint after electrophoresis detection according to electrophoresis result
figurespectrum; The nucleotide sequence of described SSR core primers is as shown in SEQIDNO.1-16.
2.
according to claimmethod described in 1, is characterized in that, step is as follows:
1) extract Kidney bean DNA, obtain Kidney bean DNA;
2) SSR core primers is obtained by screening; Described SSR core primers, often pair of SSR core primers comprises upstream primer and downstream primer, and nucleotide sequence is as shown in SEQIDNO.1-16;
3) step 1 is utilized) gained Kidney bean DNA and step 2) gained SSR core primers carries out pcr amplification reaction, then carry out 8% native polyacrylamide gel electrophoresis to PCR primer to detect, the colour developing of silver dye, builds Kidney bean characteristic fingerprint according to electrophoresis result
figurespectrum.
3.
according to claimmethod described in 2, it is characterized in that, step 1) described Bean Varieties is assorted flower No. 1, beans, short rice bean, rabbit leg, flower waist beans, nest youth beans, spend waist beans in vain, purple spends beans in vain, coloured kidney bean, the large horse's hoe, Phaseolus vulgarisl, red coloured kidney bean, little black kidney bean, duricrust beans, broadside beans, meat angle beans, long white Nanjing, 40 smallpox beans, early red bean, Caulis et folium clerodendri bungei kidney beans, Big White Flower river beans, local river beans, duricrust flower river beans, small ocean beans, great Hua ocean beans, Mei Dou, orange plum beans, powder yellow Fen beans, the red golden flower of large grain, red sword bean, right beans, black frame beans, flower plum beans, black little red bean, red cow gram, white grain red bean or cow gram.
4.
according to claimmethod described in 2, is characterized in that, step 1) described extraction Kidney bean DNA, method, for getting Kidney bean spire, adds liquid nitrogen grinding powdered, adopts RNA isolation kit to extract DNA, detect the quality of STb gene, then STb gene be diluted to 100ng/ μ L, be stored in-20 DEG C for subsequent use.
5.
according to claimmethod described in 2, is characterized in that, step 2) described screening, method is:
Step one, primary dcreening operation primer: filter out that to have polymorphism, banding pattern between different Bean Varieties clear and can stablize the SSR primary dcreening operation primer of repetition;
Step 2, sieves primer: in the SSR primary dcreening operation primer that step one obtains, filter out the highest and SSR primer that loci number is maximum of polymorphism information content values between Bean Varieties as Bean Varieties fingerprint again
figurethe core primers that spectrum storehouse builds, the loci number according to core primers divides monoid, and the kind being in same site is divided into same monoid;
Other primers are divided subclass group by step 3 in the monoid divided in step 2, by that analogy, only have a kind until each monoid is assigned to.
6.
according to claimmethod described in 2, is characterized in that, step 3) described PCR reaction system comprises DNA profiling 0.8 μ L, 10pmol/L upstream primer 0.2 μ L, 10pmol/L downstream primer 0.2 μ L, 10 × PCRbuffer2 μ L, 25mmol/LMg
2+1.2 μ L, 10mmol/LdNTP0.5 μ L, 5U/ μ LTaq enzyme 0.2 μ L, ddH
2o14.4 μ L; Described PCRbuffer is not containing Mg
2+.
7.
according to claimmethod described in 2, is characterized in that, step 3) described PCR response procedures is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 15s, annealing 15s, 72 DEG C extend 30s, totally 35 circulations; 72 DEG C extend 10min; 4 DEG C of preservations; The annealing temperature of described each SSR core primers is: the annealing temperature of CD11 and CD15 is 50 DEG C, and CD16, CD17 and CD18 annealing temperature is 52 DEG C, and the annealing temperature of CD12 is 53 DEG C, and the annealing temperature of CD13 and CD14 is 55 DEG C.
8.
according to claimmethod described in 2, is characterized in that, step 3) described structure Kidney bean characteristic fingerprint
figurespectrum, it is the mobility record electrophoresis result according to stranded DNA molecule amount and primer, the maximum band of the often pair of primer mobility is designated as 1, what mobility was taken second place is designated as 2, the like, be designated as 0 without band, separate with '-' between different primers, set up SSR genotype information data, encode according to primer order, the finger-print code obtained is the characteristic fingerprint of this kind
figurespectrum.
9.
according to claimmethod described in 2, is characterized in that, concrete steps are:
1) get Kidney bean spire, add liquid nitrogen grinding powdered, adopt RNA isolation kit to extract DNA, detect the quality of STb gene, then STb gene is diluted to 100ng/ μ L, be stored in-20 DEG C for subsequent use, obtain Kidney bean DNA;
2) SSR core primers is obtained by screening; Described SSR core primers, often pair comprises upstream primer and downstream primer, and nucleotide sequence is as shown in SEQIDNO.1-16; Described screening, method is: step one, primary dcreening operation primer: filter out that between different Bean Varieties, to have polymorphism, banding pattern clear and can stablize the SSR primary dcreening operation primer of repetition; Step 2, sieves primer: in the SSR primary dcreening operation primer that step one obtains, filter out the highest and SSR primer that loci number is maximum of polymorphism information content values between Bean Varieties as Bean Varieties fingerprint again
figurethe core primers that spectrum storehouse builds, the loci number according to core primers divides monoid, and the kind being in same site is divided into same monoid; Other primers are divided subclass group by step 3 in the monoid divided in step 2, by that analogy, only have a kind until each monoid is assigned to;
3) step 1 is utilized) gained Kidney bean DNA and step 2) gained SSR core primers carries out pcr amplification reaction; Then carry out 8% native polyacrylamide gel electrophoresis to PCR primer to detect, the colour developing of silver dye, according to the mobility record electrophoresis result of stranded DNA molecule amount and primer, the maximum band of the often pair of primer mobility is designated as 1, and what mobility was taken second place is designated as 2, the like, be designated as 0 without band, separate with '-' between different primers, set up SSR genotype information data, encode according to primer order, the finger-print code obtained is the characteristic fingerprint of this kind
figurespectrum;
Described PCR reaction system comprises DNA profiling 0.8 μ L, 10pmol/L upstream primer 0.2 μ L, 10pmol/L downstream primer 0.2 μ L, 10 × PCRbuffer2 μ L, 25mmol/LMg
2+1.2 μ L, 10mmol/LdNTP0.5 μ L, 5U/ μ LTaq enzyme 0.2 μ L, ddH
2o14.4 μ L; Described PCRbuffer is not containing Mg
2+; Described PCR response procedures is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 15s, annealing 15s, and 72 DEG C extend 30s, totally 35 circulations; 72 DEG C extend 10min; 4 DEG C of preservations; The annealing temperature of described each SSR core primers is: the annealing temperature of CD11 and CD15 is 50 DEG C, and CD16, CD17 and CD18 annealing temperature is 52 DEG C, and the annealing temperature of CD12 is 53 DEG C, and the annealing temperature of CD13 and CD14 is 55 DEG C.
10.
according to claimeither method described in 1-9 is at structure Kidney bean fingerprint
figurecompose and differentiate the application in Bean Varieties.
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