CN101748195A - Creation method of sweet pepper SSR finger-print and application thereof - Google Patents
Creation method of sweet pepper SSR finger-print and application thereof Download PDFInfo
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- CN101748195A CN101748195A CN200810204361A CN200810204361A CN101748195A CN 101748195 A CN101748195 A CN 101748195A CN 200810204361 A CN200810204361 A CN 200810204361A CN 200810204361 A CN200810204361 A CN 200810204361A CN 101748195 A CN101748195 A CN 101748195A
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Abstract
The invention discloses a creation method of sweet pepper SSR finger-print and an application thereof. The method is as follows: screening SSR primer for different sweet pepper varieties and inbred line total DNA to obtain 12 pairs of SSR polymorphism molecular markers, and performing electrophoretic separation to obtain corresponding SSR molecular marker polymorphism gram through the 12 pairs of SSR polymorphism molecular markers. The technology can make definite conclusion on the sweet pepper genetype, performs genetic classification to sweet pepper breeding material which is not recorded on an exact pedigree, makes up the deficiency of the pedigree record, also can be used for identifing the hereditary feature of the inbred line of sweet pepper, and protects the variety of the inbred line against invasion.
Description
Technical field
The present invention relates to a kind of Idioplasm identification technology, concrete relate to a kind of creation method of sweet pepper SSR finger-print and application thereof.
Background technology
Refer to reflect between biont or population the DNA fragment specific of certain species diversity in the genome on the dna molecular marker technological essence.(simple sequence repeat, SSR), the series connection that also claims microsatellite DNA (microsatellite DNA) to be meant 1～5 Nucleotide in the dna molecular repeats simple repeated sequence.SSR in animal-plant gene group stochastic distribution, high information quantity and advantages such as polymorphism, codominance and Mendelian inheritance, has generally acknowledged superiority and application prospect at aspects such as construction of genetic atlas, analysis of genetic diversity, sibship evaluation, dna fingerprinting structure, functional gene marks with it.Dna molecular marker is also referred to as the finger printing of DNA usually mostly with DNA difference between the form performance biont of electrophoretic band.
SSR fingerprint good reproducibility, simple easy handling, and be the codominant marker, can be used to identification of species and seed purity analysis.In recent years, some scholars have done a large amount of research work aspect the utilizing of SSR finger printing.(Euphytica such as Nandakumar; 2004; 136; 257-264) adopt 10 microsatellite locus to analyze 11 rice varieties and their parent; find that 9 microsatellite locus have polymorphic performance 11 cross-fertilize seed and make up finger printing, wherein 4 microsatellite locus (RM206, RM216; RM258 and RM263) can distinguish all cross-fertilize seed, can be used for identifying the protection of purity and kind.(Theoretical and Applied Genetics such as Smith, 1997,95,163-173) utilize 131 SSR primers and 80 RFLP probes 58 corn inbred lines and 4 cross-fertilize seed to be identified the result shows that SSR can more effectively identify corn germplasm than RFLP.Tan Jun etc. (southwestern agriculture journal, 2003,16,1-6) southwest has been made up the SSR finger printing and shown utilizing the SSR technology to carry out corn inbred line identifies it is feasible, effective producing 73 parts of corn inbred lines that using.(corn science such as Wang Fengge, 2003,11,3-6), (corn science, 2003,11 such as Zhao Jiuran, 3-5,8) utilize the SSR molecule marker that Chinese corn variety dna fingerprint storehouse is set up and carried out a series of systematic studyes, they optimize the SSR technology, finally set up the perfect SSR standards system that is applicable to the corn variety evaluation of a cover.In addition, crop such as cotton, wheat has also been set up the SSR finger printing of part kind.But at present, relatively lack, also do not set up a kind of construction process of perfect pimento dna fingerprinting in the research aspect the dna fingerprinting of the SSR of pimento.
Summary of the invention
One object of the present invention is to disclose a kind of construction process of sweet pepper SSR finger-print.
Another object of the present invention is to disclose a kind of sweet pepper SSR finger-print.
A further object of the present invention is to disclose the application of a kind of sweet pepper SSR finger-print in the plant gene type is identified, so that do not have the breeding material of accurate pedigree record to carry out genetic typing to those, remedies the disappearance of pedigree record; Hereditary feature to self-mating system is identified, avoids invading with the kind power of protecting these self-mating systems.
Pimento of the present invention is (Capsicum annuum L.)
The construction process of pimento dna fingerprinting of the present invention is as follows:
1, DNA extraction to pimento kind clip young leaflet tablet, is extracted total DNA by the SDS method;
2, the pcr amplification of SSR sequence carries out the polymorphism screening to specific primer, and uses the PCR thermal cycler respectively the genomic dna of pimento kind to be increased, in 35 cycles of PCR thermal cycler cocycle;
3, the PCR product separates and identifies that amplified production is electrophoresis in 8% (w/v) polyacrylamide gel, argentation dyeing back scanning instrument record;
4, data analysis is noted down according to each primer amplification situation of each material on the photo, and bands of a spectrum have band to count 1 by 0/1 system log (SYSLOG), do not have band and count 0, and the data disappearance counts 9, sets up database;
5, the structure of dna fingerprinting, according to primer amplification situation on the photo, the SSR polymorphism band that picking is representative is drawn the dna fingerprinting of pimento kind.
The method of DNA extraction is: get seed 6-9 grain from single experimental cultivar, in the matrix of being mixed thoroughly in proportion by vermiculite, black earth, perlite in the plantation, place the dark cultivation of constant incubator 7 days.With people such as Dellaporta (Plant Molecular Biology Reporter, 1983,1,19-21) the SDS method of Ti Chuing is extracted the total DNA of pimento from the blade of cultivating gained, and purifying (conventional molecular biology working method is with reference to molecular cloning document Sambrook J, Frets E F, Mannsdes Tet al.In:Molecular Cloning.2nd ed.Cold Spring Harbor Laboratory Press, 1989).Products therefrom is preserved with TE damping fluid (10mM Tris base, 0.1mM EDTA) earlier, detects quality and the concentration of DNA again with Beckman DU640B type ultraviolet-visible pectrophotometer, and is at last that the DNA diluted sample is standby to 10ng/ μ l.
The system of SSR sequence pcr amplification reaction is: contain 1 * Buffer, 2.5mmol/L MgCl in per 20 μ l volumes
2, 0.4mmol/L dNTP, each 0.25 μ mol/1 of SSR primer (preceding primer and back primer), 1U TaqDNA polysaccharase, the 20ng dna profiling is added a cover a mineral oil on the reaction solution, increase on carrying out on PTC-200 (MJ Research Inc.) the PCR instrument.Response procedures: 94 ℃ of pre-sex change 5min, 1 circulation; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, carried out 35 circulations altogether, extended 7 minutes at 72 ℃ at last.
Data analysis press Smith etc. (Theoretical and Applied Genetics, 1997,95, the formula that 163-173) provides calculate every pair of SSR primer the polymorphism information amount (polymorphisminformation content, PIC).
By above-mentioned steps, sweet pepper SSR finger-print of the present invention can obtain through electrophoretic separation by being used alone or in combination primer shown in the table 1 to behind pcr amplification again.
Table 1: the primer of sweet pepper SSR finger-print correspondence
The application of sweet pepper SSR finger-print of the present invention in the plant gene type is identified, be by above-mentioned SSR primer to the total DNA of kind to be measured be template carry out pcr amplification and through polyacrylamide gel electrophoresis and dyeing after, compare with the electrophorogram of the SSR sequence pcr amplification of known pimento kind again, thereby determine the genetic typing of testing sample, and the evaluation that realizes the hereditary feature of self-mating system.
Concrete, the corresponding steps in the construction process of described total DNA extraction method, pcr amplification method and electrophoresis and painted method and pimento dna fingerprinting is identical.
Beneficial effect of the present invention is:
Pimento dna fingerprinting technology can be made clear and definite evaluation to the pimento genotype, does not particularly have the pimento breeding material of accurate pedigree record to carry out genetic typing to those, remedies the disappearance of pedigree record; The breeding expert can adopt the hereditary feature of this technical evaluation pimento self-mating system, protects the kind power of these self-mating systems to avoid invading.Its key band has been found out in the foundation of the dna fingerprinting of pimento, more effectively protects the intellecture property of pimento kind.
Description of drawings
Fig. 1 SSR primer CM28 is to the amplified production of pimento self-mating system;
Fig. 2 SSR primer CM96 is to the amplified production of pimento self-mating system.
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with accompanying drawing.Should be noted that, the embodiment of the invention is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.
Pimento kind and self-mating system are respectively from Vegetable ﹠ Flower Inst., Chinese Academy of Agriculture Science storehouse in germ plasm resource mid-term and gardening institute of Shanghai Academy of Agricultural Sciences.
Used pimento kind (material) is as follows:
If the used reagent of the present invention is unexplained reference, all available from Sigma-aldrich (Sigma-Aldrich) company.
Embodiment 1: the extraction of the total DNA of pimento
(1) test method:
1, collects fresh seedling (80 ℃ of preservations), in-20 ℃ of precooling mortars, use the liquid nitrogen grinding powdered.
2, with step 1 gained powder transfer in the 1.5ml centrifuge tube of-20 ℃ of precoolings, put into-20 ℃ of refrigerators and preserve stand-by (spending the night).
3, be equipped with to step 2 and add 600ul SDS extracting solution in the centrifuge tube of powder and shake up, 65 ℃, temperature is bathed 30min.Between or the vibration 3-4 time.
4, add afterwards 1/4 volume (about 100 μ l) KAC, shake up ice bath 30min.
5, the chloroform that adds 1/2 volume (about 300-400 μ l) afterwards: primary isoamyl alcohol (24: 1, volume ratio), fully shake up on the vibrator (120rpm, 30min).
6, desk centrifuge is centrifugal: 6000-8000rpm, 15min, get supernatant.
7, add equal-volume (about 400ul) chloroform afterwards: primary isoamyl alcohol (24: 1, volume ratio), the 80-90rpm 30min that vibrates.
8, the centrifugal 15min of 6000-8000rpm at room temperature again gets supernatant.
9, add the dehydrated alcohol of-20 ℃ of precoolings of 2 times of volumes (about 700 μ l) then, shake up.
10, place-20 ℃ of refrigerators, make suspended substance free setting 20min.
11, take out after, under the room temperature 12, the centrifugal 6min of 000rpm.
12, abandon supernatant, precipitation equal-volume (about 400 μ l) 70% (v/v) washing with alcohol 10min.
13, abandon 70% ethanol, after the DNA precipitation is air-dry, be dissolved in 200ul TE (1/10), 4 ℃ of preservations are standby.
(2) test-results:
The mensuration of DNA sample concentration
Measure with Perkin Elmer MBA 2000DNA concentration determination instrument, and the DNA concentration of each sample of balance is 10-20ng/ μ l.
The DNA sample quality detects
Adopt 1.5% (w/v) agarose electrophoresis to detect, the DNA sample is a master tape.
Embodiment 2:PCR amplification SSR molecule marker
(1) test method:
Agents useful for same
(1) SSR primer: Takara company is synthetic, and concentration is 2pmol/ μ l
(2) TEMED: Tetramethyl Ethylene Diamine
(3) DNA Ladder (50bp): Nanjing Sangon company
(4) indicator:
0.25% (w/v) bromjophenol blue (4 ℃ of preservations of Bromophenol Blue)
The blue or green FF (Xglue Cyanoll FF) of 0.25% (w/v) dimethylbenzene
The aqueous sucrose solution of 40% (w/v)
The Acri/bis-Acri gel storage liquid of (5) 40% (w/v)
Acrylamide (Acri) 38 grams
N,N methylene bis acrylamide (bis-Acri) 2 grams
Add ddH
24 ℃ of preservations of O to 100ml
The AP of (6) 10% (w/v) (ammonium persulphate)
The Sulfothiorine of (7) 10% (w/v) (mother liquor)
1.2PCR reaction
PCR reaction system (10 μ l)
DNA(10ng/μl) 1μl
Primer(2pmol/μl) 0.7μl
10×Buffer(freeMgCl
2) 1μl
dNTP(2.5mM) 0.2μl
MgCl
2(25mM) 0.6μl
Taq(5u/μl) 0.1μl
ddH
2O 6.4μl
The PCR response procedures:
94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension 1min carry out 35 circulations altogether; 72 ℃ are extended 7min.Use PTC-200 thermal cycler (MJ Research Inc.).
The detection of PCR product
Polyacrylamide gel electrophoresis
The native gel working fluid of 100ml 8%:
H
2O 70ml
10×TBE 10ml
40% (w/v) Acri/bis-Acri storage liquid 20ml
TEMED 100μl
10％(w/v)AP 1000μl
Cumulative volume 100ml
DNA Ladder with 50bp is the molecular weight size of comparing amplified production, and electrophoretic buffer is 0.5 * TBE, 200V constant voltage electrophoresis 90 minutes.
Silver dyes step
(1) contain 10% (v/v) alcohol, 0.5% (v/v) glacial acetic acid stationary liquid is fixed 6 minutes/time 2 times.
(2) 0.2% (w/v) AgNO
3Solution infiltration 10～12 minutes.
(3) ddH
2O cleans 2 times.
(4) 0.002% (w/v) hypo solution is put 30 seconds generations.
(5) contain NaOH 1.5% (w/v), the solution colour developing of formaldehyde 0.4% (v/v).
(6) 0.75% (w/v) Na
2CO
3The reaction of aqueous solution color development stopping.
(2) test-results:
Utilize 112 pairs of SSR primers that 10 pimento kinds and self-mating system are increased, it is clear finally to choose banding pattern, and 12 pairs of primer statisticses (seeing Table 1) of polymorphism are arranged.These 12 pairs of SSR primers are distributed on 6 linkage groups of pimento.These 12 pairs of SSR primers coamplification in 10 parts of materials goes out 35 polymorphic bandses, and the bands of a spectrum size is 50～1500bp.Minimum amplifies 1, and maximum amplifies 7, and average every pair of primer amplification goes out 2.9 polymorphism bands of a spectrum, and wherein the polymorphism bands of a spectrum that amplify of primer CM86 are maximum, reach 7.
Embodiment 3: data statistics and analysis
(1) test method:
According to the banding pattern of amplification, select and in most of material, all only amplify the primary spectrum band, and have polymorphism, good reproducibility to distinguish all primers for the examination material, be used to make up the finger printing of 10 the pimento kinds and the self-mating system of this experiment.
(2) test-results:
There are CM2, CM26, CM28, CM46, CM53, CM62, CM63, CM76, CM77, CM86, these 12 pairs of combination of primers of CM96 will separate for the examination material in the SSR primer.Primer CM63 has characteristic spectrum belt to material 4; Primer CM28 has the characteristic spectrum belt (see figure 1) to material 5; Primer CM76 has characteristic spectrum belt to material 4,6; Primer CM86 has characteristic spectrum belt to material 4 and 5; Primer CM96 has the characteristic spectrum belt (see figure 2) to material 6; Primer CM46 and CM62 have characteristic spectrum belt to material 10 separately; Material 1 is distinguished in primer CM2 and primer CM77 combination; Material 8 is distinguished in primer CM86 and CM96 combination; Material 2 is distinguished in primer CM2, CM63 and CM77 combination; Primer CM46, material 3 is distinguished in the combination of CM76 and CM77; Material 7 is distinguished in primer CM46, CM86 and CM96 combination; And material 9 can be made up by primer CM52, CM86 and CM96 and distinguishes.
The result shows that 12 pairs of combination of primers can be used among the evaluation of plant gene type, so that do not have the breeding material of accurate pedigree record to carry out genetic typing to those.Can be applied to identify the hereditary feature of pimento self-mating system, protect the kind power of these self-mating systems to avoid invading.
Embodiment 4: the repeatability checking
(1) test method:
Get 12 pairs of SSR primers of this polymorphism, carry out the variation of differential responses system and response procedures and carry out amplification of PCR product and spectral band analysis.
(2) test-results:
This experiment has also been done checking to the repeatability of these 12 pairs of SSR primers, show no matter be conditioned reaction system or response procedures, can only cause the variation of non-specific amplified production, but the banding pattern of specificity product and the relative position on offset plate can not change, illustrate that the SSR molecule marker has stability and repeated preferably, the finger printing that utilizes SSR molecule marker constructed dna is reliable.
Sequence table
＜110〉Academy of Agricultural Sciences, Shanghai City
＜120〉creation method of sweet pepper SSR finger-print and application thereof
<130>0811544
<160>24
<170>PatentIn?version?3.3
<210>SEQ?ID?No?1
<211>21
<212>DNA
＜213〉primer before the CM2 of sweet pepper SSR finger-print correspondence
<400>1
cagcagtaac?agaggcaggt?c 21
<210>SEQ?ID?No?2
<211>26
<212>DNA
＜213〉primer behind the CM2 of sweet pepper SSR finger-print correspondence
<400>1
cacaagtgag?tttattcata?tcacca 26
<210>SEQ?ID?No?3
<211>25
<212>DNA
＜213〉primer before the CM26 of sweet pepper SSR finger-print correspondence
<400>1
tttctgcagt?gttaccaata?tttca 25
<210>SEQ?ID?No?4
<211>19
<212>DNA
＜213〉primer behind the CM26 of sweet pepper SSR finger-print correspondence
<400>1
cccatgggtc?ctacctcag 19
<210>SEQ?ID?No?5
<211>20
<212>DNA
＜213〉primer before the CM28 of sweet pepper SSR finger-print correspondence
<400>1
tcatggaaaa?ttaacgcata 20
<210>SEQ?ID?No?6
<211>23
<212>DNA
＜213〉primer behind the CM28 of sweet pepper SSR finger-print correspondence
<400>1
gggggttgga?gaagaagaaa?gtt 23
<210>SEQ?ID?No?7
<211>20
<212>DNA
＜213〉primer before the CM46 of sweet pepper SSR finger-print correspondence
<400>1
ggtggaaact?tgcttggaga 20
<210>SEQ?ID?No?8
<211>20
<212>DNA
＜213〉primer behind the CM46 of sweet pepper SSR finger-print correspondence
<400>1
cccagaacca?tccacctact 20
<210>SEQ?ID?No?9
<211>20
<212>DNA
＜213〉primer before the CM52 of sweet pepper SSR finger-print correspondence
<400>1
acggtcgatt?cctgtattgc 20
<210>SEQ?ID?No?10
<211>22
<212>DNA
＜213〉primer behind the CM52 of sweet pepper SSR finger-print correspondence
<400>1
gcatgctaaa?cccaattttc?tc 22
<210>SEQ?ID?No?11
<211>19
<212>DNA
＜213〉primer before the CM53 of sweet pepper SSR finger-print correspondence
<400>1
ttggtgtggt?tagggagag 19
<210>SEQ?ID?No?12
<211>20
<212>DNA
＜213〉primer behind the CM53 of sweet pepper SSR finger-print correspondence
<400>1
ggcgttcgaa?cttgtgaaat 20
<210>SEQ?ID?No?13
<211>20
<212>DNA
＜213〉primer before the CM62 of sweet pepper SSR finger-print correspondence
<400>1
ccttcttctt?tgccaccttc 20
<210>SEQ?ID?No?14
<211>20
<212>DNA
＜213〉primer behind the CM62 of sweet pepper SSR finger-print correspondence
<400>1
tagcagcagc?tgatggagaa 20
<210>SEQ?ID?No?15
<211>20
<212>DNA
＜213〉primer before the CM63 of sweet pepper SSR finger-print correspondence
<400>1
tgcattggtg?ggctaacata 20
<210>SEQ?ID?No?16
<211>20
<212>DNA
＜213〉primer behind the CM63 of sweet pepper SSR finger-print correspondence
<400>1
gctcttgaca?caaccccaat 20
<210>SEQ?ID?No?17
<211>19
<212>DNA
＜213〉primer before the CM76 of sweet pepper SSR finger-print correspondence
<400>1
cgcatgaagc?aatgtacca 19
<210>SEQ?ID?No?18
<211>20
<212>DNA
＜213〉primer behind the CM76 of sweet pepper SSR finger-print correspondence
<400>1
acctgcagtt?tgttgttgga 20
<210>SEQ?ID?No?19
<211>20
<212>DNA
＜213〉primer before the CM77 of sweet pepper SSR finger-print correspondence
<400>1
cggattcggt?tgagtcgata 20
<210>SEQ?ID?No?20
<211>20
<212>DNA
＜213〉primer behind the CM77 of sweet pepper SSR finger-print correspondence
<400>1
gtgctttggt?tcggtctttc 20
<210>SEQ?ID?No?21
<211>21
<212>DNA
＜213〉primer before the CM86 of sweet pepper SSR finger-print correspondence
<400>1
ccaggatggt?gttaagggtt?t 21
<210>SEQ?ID?No?22
<211>20
<212>DNA
＜213〉primer behind the CM86 of sweet pepper SSR finger-print correspondence
<400>1
gtcgcatcaa?tgagcatagg 20
<210>SEQ?ID?No?23
<211>21
<212>DNA
＜213〉primer before the CM96 of sweet pepper SSR finger-print correspondence
<400>1
aacgaaaaac?aaacccaatc?a 21
<210>SEQ?ID?No?24
<211>22
<212>DNA
＜213〉primer behind the CM96 of sweet pepper SSR finger-print correspondence
<400>1
ttgaaattgc?tgaaactctg?aa 22
Claims (5)
1. a sweet pepper SSR finger-print is characterized in that obtaining as follows: the extraction of the total DNA of pimento, the pcr amplification of SSR sequence and the evaluation of pcr amplification product.
2. sweet pepper SSR finger-print according to claim 1, the pcr amplification primer that it is characterized in that described SSR sequence is to being one of the following or the right combination of several primer, described primer is all represented in proper order with 5 ' end to 3 ' end.
Primer is to 1: preceding primer CAGCAGTAACAGAGGCAGGTC
Back primer CACAAGTGAGTTTATTCATATCACCA
Primer is to 2: preceding primer TTTCTGCAGTGTTACCAATATTTCA
Back primer CCCATGGGTCCTACCTCAG
Primer is to 3: preceding primer TCATGGAAAATTAACGCATA
Back primer GGGGGTTGGAGAAGAAGAAAGTT
Primer is to 4: preceding primer GGTGGAAACTTGCTTGGAGA
Back primer CCCAGAACCATCCACCTACT
Primer is to 5: preceding primer ACGGTCGATTCCTGTATTGC
Back primer GCATGCTAAACCCAATTTTCTC
Primer is to 6: preceding primer TTGGTGTGGTTAGGGAGAG
Back primer GGCGTTCGAACTTGTGAAAT
Primer is to 7: preceding primer CCTTCTTCTTTGCCACCTTC
Back primer TAGCAGCAGCTGATGGAGAA
Primer is to 8: preceding primer TGCATTGGTGGGCTAACATA
Back primer GCTCTTGACACAACCCCAAT
Primer is to 9: preceding primer CGCATGAAGCAATGTACCA
Back primer ACCTGCAGTTTGTTGTTGGA
Primer is to 10: preceding primer CGGATTCGGTTGAGTCGATA
Back primer GTGCTTTGGTTCGGTCTTTC
Primer is to 11: preceding primer CCAGGATGGTGTTAAGGGTTT
Back primer GTCGCATCAATGAGCATAGG
Primer is to 12: preceding primer AACGAAAAACAAACCCAATCA
Back primer TTGAAATTGCTGAAACTCTGAA
3. sweet pepper SSR finger-print according to claim 1, the application in the pimento genotype identification.
4. sweet pepper SSR finger-print according to claim 1, the application in pimento breeding material genetic typing.
5. sweet pepper SSR finger-print according to claim 1, the application in the hereditary feature of identifying the pimento self-mating system.
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| CN107190089A (en) * | 2017-07-19 | 2017-09-22 | 福建省农业科学院生物技术研究所 | The method that using molecular labeling 3 kinds of strawberry cultivars are carried out with quick discriminating |
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| CN108300798A (en) * | 2018-04-10 | 2018-07-20 | 新疆农业科学院园艺作物研究所 | A kind of primer pair of walnut microsatellite DNA mark fingerprint map construction method and its application |
| CN108486275A (en) * | 2018-04-16 | 2018-09-04 | 中国科学院昆明植物研究所 | A kind of SSR finger-prints differentiate the method and its application of begonia kind |
| CN108486275B (en) * | 2018-04-16 | 2021-08-31 | 中国科学院昆明植物研究所 | Method for identifying begonia varieties by SSR (simple sequence repeat) fingerprint spectrum and application of method |
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