Experimental identification method for variety variety of soybean seed—ISSR
Some standard content:
ICS65.020.01
National Standard of the People's Republic of China
GB/T19563--2004
Experimental identification method for variety of soybean seed
Simple sequence repeat interval method
Experimental identification method for variety of soybean seed--IssR2004-06-22 Issued
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China Standardization Administration of China
2004-12-01 Implementation
The Appendix A of this standard is the normative appendix. Preface
This standard was proposed by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. GB/T19563-2004
The drafting units of this standard are: National Agricultural Standardization Blue Testing and Research Center (Longjiang), Heilongjiang Provincial Quality and Technical Supervision Bureau. The main drafters of this standard are: Wang Qinggui, Xu Jing, Jiang Xing, Li Guangyu, Shi Shaoye, Hao Zhaojun. GB/f19563-2004
Previously, the identification of crop seeds in my country mainly used planting identification, rapid determination method (phenol staining method, soybean seed coat guaiac staining method, sorghum seed potassium hydroxide-bleaching powder determination method, wheat seed fluorescence determination method, wheat seed potassium hydroxide determination method), acrylamide gel electrophoresis method to determine the purity of barley and wheat seeds, etc. These methods have a long inspection cycle. Although the electrophoresis method takes a short time, it does not have many advantages of molecular weight determination.
With the development of molecular biology, especially since the 1990s, molecular biology related technologies have been widely used. Its detection object is DNA, the genetic basis of organisms. DNA is used as the detection object, which has many advantages that other detection methods do not have: first, the number of DNA markers for detection is unlimited, which is incomparable to enzyme technology; second, DNA analysis technology is not like other technologies that vary with tissues or developmental stages. DNA provided by any part of the plant body and at any time can be used for analysis, and its detection results are the same. First, DNA analysis is not affected by the environment, and its variation only comes from the variation of the base DNA sequence in the year. This stability makes it easy to reveal the genetic variation between varieties, thereby excluding phenotypic variation caused by environmental variation. Based on the above advantages, INA analysis technology is an advanced method for variety identification in agriculture, forestry, animal husbandry, fishery, etc. 1 Scope
Experimental method for soybean seed variety identification,
Simple repeat sequence interval method
This standard specifies the experimental method for soybean seed variety identification. GB/T19563--2004
This standard applies to the experimental process of soybean seed variety identification using the simple sequence repeat inter-region (SSR) method. 2. Terms, definitions and abbreviations
2.1 Terms and definitions
The following terms and definitions apply to this standard. 2.1.1
Polymerase chain reactionpolymerase chain reaction is an enzyme-catalyzed reaction in which a DNA fragment with a specific base sequence of at least 100 copies can be amplified into millions of molecules in a few hours in vitro, i.e., DNA fusion reaction.
Simple sequence repeat inter-region is a molecular marker and detection method developed on the basis of PCR technology. A series of specific primers are designed based on a simple repeat sequence (microsatellite locus), and the base sequence of the microsatellite locus interval is amplified by P-type ruler reaction to detect the polymorphism of the length of the amplified fragment.
Microsatellite DNA is a type of tandem repeat sequence with a length of several nucleotides to several hundred nucleotides, which is composed of several nucleotides (usually 1 to 5) as repeating units.
Nucleotide
is the basic unit of nucleic acid, which is composed of three parts: pentose, phosphate and cyclic nitrogenous base. 2.1.5
Primer
A short single strand that binds to the template DNA chain, provides a 3-OH end as the starting point of DNA synthesis, and extends the complementary strand of the template DNA.
Primer amplified polymorphism A pair of primers amplifies DNA fragments of different numbers or lengths between two or more different materials.
2.2 Abbreviations
The following abbreviations apply to this standard.
ISSR Inter-Simple Sequence Repeat PCR Polymerase Chain Reaction DNA Deoxyribonucleic Acid GB/T19563-2004
RVA Ribonucleic Acid OD Optical Density
TBE Tris-Boric-Acid-EDTA 3 Experimental Methods
3. 1 Principle
DNA is the genetic base of organisms and carries all genetic materials. Identification at the DNA molecular level is the most effective method to distinguish differences between species, varieties and even individuals. Using PCR technology, the DVA fragment can be amplified tens or even millions of times in a short period of time, so as to achieve the purpose of detection. ISSR method is a detection method developed on the basis of PCR technology. It designs a series of specific primers based on simple repeat sequences (microsatellite loci), including microsatellite DNA fragments (usually 20 bases) with dinucleotides, trinucleotides and tetranucleotides as repeating units, and amplifies microsatellite loci and their spacers through PCR reaction to detect the polymorphism of the amplified fragments. 3.2 Environmental conditions
a Temperature. 15℃～25℃;
b) Humidity: The relative humidity (RH) should not be higher than 50%. 3.3 Receiver
a) PCR amplification instrument:
b) UV spectrophotometer;
High-speed desktop centrifuge: speed not less than 150001/min: d) Multi-purpose electrophoresis instrument and horizontal electrophoresis tank: UV transmission instrument:
Micropipette: specification is 0.1ul~2.5μl.2μL~20μ.20μL~200μL, 100mL1000μ; Microbalance: graduation value is (.0001g; g.
h) Magnetic heating stirrer;
) Gel imaging system:
Ultrapure water system:
k) Sterilizer;
Acidity meter: maximum allowable error is ±0.1pH; m) Microwave oven:
n) Constant temperature water bath: maximum allowable error is ±1℃ o) Constant condensation drying oven: maximum allowable error is +1℃. 3.4 Reagents and consumables
3.4.1 Reagents
TagDNA polymerase: storage conditions are -20℃±2℃; b)
10×Buffer: storage conditions are -20℃±2℃; c)
Four deoxyribonucleic acids (4×dNTP): storage conditions are -20℃±2℃ Mg+: storage conditions are -20℃±2℃; RNAse (DNAse): storage conditions are -20℃±2℃ e)
Agarose:
R) Tris (Tris): molecular formula is C.HuNO, h)
Disodium ethylenediaminetetraacetate (EDTANa·2HO): molecular formula is CHN,ONaz·2IIO Bromphenol blue (bromphenolbluesodiumsalt), molecular formula is CiH,Br.O,SNa) Xylene cyanol FF (xylen ecyanoleFF): molecular formula is CesH2rNzO,SN; 2
k) 8-hydroxyquinoline: molecular formula is C,H,No: 1) Sodium dodecyl sulfonate (SDS): molecular formula is CHsO,SNa Ethyl bromide (EB): molecular formula is CHaNBrm
Isoamyl alcohol: molecular formula is CuOH
Crystalline phenol: molecular formula is CHO, storage conditions are -20℃ to 2℃, 0
Trifluoromethane: molecular formula is CHCl, purity is analytical grade p
Boric acid: molecular formula is H,BO, purity is analytical grade; q)
Sucrase: molecular formula is Cr?H22O, purity is analytical grade, )
s) Anhydrous ethanol: molecular formula is C.HOH, purity is analytically pure:) Hydrochloric acid molecular formula is HCl, purity is analytically pure; Sodium chloride: molecular formula is NaCl purity is analytically pure:) Water: molecular formula is H, O. The water used in this experiment is deionized water. 3.4.2 Consumables
GB/T19563--2004
Centrifuge tube: specification is 1.5mL, 0.2mL. It needs to be autoclaved before use (according to the method specified in Chapter A1) a)
Grinding chain: specification is 7cm~10cm in diameter
Pipette warm head: specification is 20uL, 200μL and Wang 000μL. Chrysanthemum needs to be sterilized by pressure separation; e
d) Volumetric flask: specifications are 100mL and 1000mL; e) Beaker, specification is 100ml.
f) Erlenmeyer flask: specification is 250mlL
g) PE gloves;bZxz.net
h) Tin hoop paper t
i) Sealing film.
3.5 Experimental Procedure
3. 5. 1 DNA Extraction
3.5.1.1 Take half a soybean seed, place it in a 1.5mL centrifuge tube, add 1000μL homogenization buffer (prepared according to the method specified in Chapter A2) and soak for 4 hours, pour it into a mortar and grind it thoroughly, pour the ground product into a 1.5ml centrifuge tube, take a small amount of homogenate and rinse the mortar with the flushing liquid, pour it into the centrifuge tube, centrifuge (speed is 40001/min) for 10 minutes, transfer the supernatant to a new 1.5mL centrifuge tube, and discard the precipitate. 3.5.3.2 Add an equal volume of phenol and phenol (prepared according to the method specified in Chapter A3) to the supernatant, slowly invert the centrifuge tube for 20 minutes, and avoid violent shaking.
3.5.1.3 Centrifuge (speed is 8000x/min) for 10 minutes, and transfer the supernatant to a new centrifuge tube. 3.5.1.4 Add phenol-chloroform-isoamyl alcohol (volume ratio of 24:231) equal to the supernatant, centrifuge (speed of 8000r/min) for 10min, and transfer the supernatant to a new centrifuge tube. 3.5.1.5 Add trifluoromethane-isoamyl alcohol (volume ratio of 23:1) equal to the supernatant, slowly pour for 10min, centrifuge (speed of 8000r/min) for 10min, and transfer the supernatant to a new centrifuge tube. 3.5.1.6 Add ice-cold ethanol twice the volume of the supernatant, spin the centrifuge for 50-100 times, and white flocculent DNA will appear. 3.5.1.7 Place the centrifuge tube in a 20℃ refrigerator for 30min, take it out and centrifuge (speed of 8000z/min) for 10min to form a white precipitate. 3.5.1.8 Pour out the liquid in the centrifuge tube, add 1.ml 75% ethanol, centrifuge (speed 15000/min) for 5min, pour out the ethanol, invert on clean absorbent paper, and absorb the body. 3.5.1.9 Place the centrifuge tube in a constant temperature drying oven (40℃~50℃) for 20min or in a ventilated place for 40min to evaporate the ethanol. 3.5.1.10 Add 100μ TE buffer (prepared according to the method specified in Chapter A.4) and 2L RNase (prepared according to the method specified in Chapter A.5) and place in a water bath (55℃±2℃) for 12h to fully dissolve the DNA precipitate, and store in a 4℃ refrigerator. GB/T19563—2004
3.5.2 DNA concentration detection
3.5.2.1 Ultraviolet absorption detection
The urine ring and pyrimidine ring in the DNA molecule can absorb ultraviolet light. The ultraviolet absorption peak of DNA is at a wavelength of 260nm, and the ultraviolet absorption peak of protein is at a wavelength of 280nm. Take 5uL of the DNA solution extracted in 3.5.1, add 995μ of water, put it in a colorimetric cup, detect it with an external spectrophotometer, record the OD values at 260 nm and 280 nm, calculate the DNA concentration and the ratio of OD and ODga. The OF26/OD2 of DNA is best around 1.8, and above 1.5 can also be used for ISSR-PCR analysis. If the ratio is too small, repeat steps 3.5.1.2~3.5.1.10 to continue extraction. The DNA concentration is calculated according to formula (I):
D=50X1000XDXs
Wherein,
D—DNA concentration, in nanograms per microliter50kb) and no degradation, a dense bright band appears near the sample well. If the DNA is partially degraded, it will be continuously distributed. If it is severely degraded, no large DNA fragments can be seen, and only RNA can be seen far away from the sample well. 3.5.3 ISSR amplification
3.5.3.1 Reaction components
25μL reaction system contains 1×Buffer, 1.5mnol/LMg+200umol/LdNTP, 1UTagDNA polymerase, 1μumol/L primer. 100ng template DNA, add water to 25μl (the preparation of each reaction component is carried out according to the method specified in Chapter A.8). Add the reaction solution to 0.2mL centrifuge tubes in the above proportions, put it in the PCR instrument, and perform PCR cycles. 3.5.3.2 Cycling process
The cycling process is shown in Figure 1:
94℃ pre-denaturation for 1 min
94℃ denaturation 30%
48℃ annealing 45 s
T extension S
72℃ extension 7 min
4 cycles
45 cycles
Figure 1: PCR reverse cycling process
3.5.3.3 Electrophoresis detection
Put the prepared 2% agarose gel (prepared according to the method specified in Chapter A.6) into the electrophoresis well, and make the sample well at the negative end of the power supply. Take 10L of PCR amplification product and 2ul of gel loading buffer (prepared according to the method specified in Chapter A.7) and pipette up and down on the sealing film to mix, and add it to the sample well. Turn on the power supply, control the voltage at 3V/cm, electrophoresis for 4 hours, cut off the power supply, take out the gel, and scan on the gel imaging. 3.5.3.4 Identification
Compare the gel imaging results of the variety to be tested with the standard spectrum band of the original variety to identify the authenticity of the variety. A1 Operation method of high-pressure sterilization
Appendix A
(Normative Appendix)
Sterilization method and preparation of buffer and main reagents GB/T19563—2004
Put the centrifuge tube to be sterilized in a beaker, seal it with tin foil paper, and place the pipette tip in the tip tray: put the prepared reagent into the reagent bottle, cover it, and put it in the sterilizer. First, raise the pressure to 1Pa, release the air, and then raise the pressure to 1Pa again, start timing, and turn off the power supply after 20 minutes. When the pressure reaches zero, take out the centrifuge tube, pipette tip and reagent bottle for standby use. A. 2 Preparation of homogenate buffer
Take 200 mL of 1 mol/L TrisCl (pH 8.0), 50 mL of 0.5 mol/L DIA (pH 8.1), and 0.5 g SDS, and add water to 100 mL. Before use, ensure that the SDS is fully dissolved. If crystals appear, place it in a 50°C water bath and dissolve it for 10 min. A. 2. 11 mol/ Tris ·Cl (pH 8.0), weigh 12.11 g Tris, dissolve it in 80 mL of water, and adjust the pH value of the solution to about 8.0 with concentrated HCl. Add water to make up to 100 mL, and sterilize it by high pressure. A.2.2 0.5 mol/L EDTA (pH8.0). Weigh 18.61 g EDTA Na * 2H2O, dissolve in 80 mL water, stir vigorously on a magnetic heating stirrer, adjust the pH value of the solution to 8.0 with NaOH (about 2 g), add water to make up to 100 mL, and autoclave. A.3 Preparation of saturated phenol
The preparation of saturated phenol should be carried out in a fume hood or under well-ventilated conditions. A.3.1 Place the phenol at room temperature in a 60℃ water bath to melt, then pour it into a condenser and heat it, and collect the liquid at about 180℃. The redistilled phenol is packaged and stored in a 20°C refrigerator for later use. A.3.2 Melt the redistilled phenol in a 60°C water bath, add 8-hydroxylindole to a natural concentration of 0.1%, add 0.5% isopropyl alcohol (Tris·Cl) to the drop solution (pH 8.0), mix well with a magnetic heating stirrer and let it stand for a while, remove the upper aqueous phase, and repeat this process until the pH value of the phenol phase is greater than 7.8, which is a saturated phenol solution. Put it into a brown bottle and store it in a 4°C refrigerator. A.4 Preparation of TE buffer
Take 1 mL of 1 mol/L Tris Cl (pH7.6), 0.2 mL of 0.5 mol/L EDTA (pH8.0), and add water to 100 mL. A. 4.11 mol/L Tris ·Cl (pH7.6): weigh 12.11 g Tris, dissolve in 80 mL water, adjust the pH value of the solution to 7.6 with concentrated HCl, add water to 100 mL, and sterilize by autoclave. A.4.20.5 mol/L EDTA (pH8.0). A.2.2. A.5 Preparation of RNase (10 mg/mL)
Weigh 1 mg of RNase, dissolve in 100 μL TE buffer, place in a water bath (70°C) for 10 min, and then slowly cool to room temperature. A, 6 Preparation of agarose gel
To prepare agarose gel with a concentration of 0.7% (or 2%), weigh 0.7 g (or 2 g) of agarose and place it in a 200 mL conical flask, dissolve it in 100 mL 0.5×TBE, and melt it in a microwave until the solution is transparent. Cool it to 50℃~60℃, add ethidium bromide (10 mg/mL) and adjust it to a final concentration of 0.5 mg/ml. After mixing, pour the gel onto a sealed and combed gel plate (about 1.0 mm from the bottom plate). The gel thickness should be 3 mm~5 mm. After the gel is completely solidified, place it in the electrophoresis tank for use. A, 6.1 Ethyl bromide (EB) stock solution (10 mg/mL): weigh 1 g of ethidium bromide and dissolve it in 100 ml. water, stir with a magnetic heating stirrer for several hours to ensure that it is completely dissolved, transfer to a brown bottle, and store in a refrigerator at 4°C. GB/T 195632004
A, 6.2 5X TBE, weigh 54 Tris, dissolve in 800 mL. water, add 20 mL 0.5 m01/L EDTA (pH8.0) and 27.5 g dicalcium, dilute to 1000 ml, sterilize by high pressure, and store at room temperature. A, 6.3 0.5×TBE: weigh 100 mL 5×TBE, add water to dilute to 1 000 mL, and set aside. A.7 Gel loading buffer
0.25% phenol blue, 0.25% xylene cyanol FF, 40% (mass concentration) sucrose aqueous solution. A, Preparation of each reaction component in the reaction system A, 8. 11U TaqDNA polymerase
25 The volume of TagDNA polymerase required to be added to the μL reaction system is calculated according to formula (A1): V
Where,
-The volume of TaqDNA sanghe alcohol, in microliters (μI); D-The concentration of TaqDNA sanghe alcohol, in units per microliter (U/μL). A. 8. 21. 5 mmol/L Mg +
The volume of Mg+ required to be added to the 25μL reaction system is calculated according to formula (A.2): V=1.5×25
Where,
V-The volume of Mg2+, in microliters (μL); D-The concentration of Mg+, in units per liter (mmol/L). A, 8. 310 μmol/L Primer
First centrifuge the primer, then dilute with water. The volume of water added is calculated according to formula (A.3), and stored in a 20°C refrigerator. Dilute three times with water before each use, and the final concentration is 10 Amol/LV
Wherein, the calculation of M in formula (A.3) is carried out according to formula (A, 4): .*( A.3)
M nA X 313. 22 + nG X 329, 22 + nC X 289, 19 + nT X 304. 19 - 61, 97 -**-+( A, 4 ) In the formula,
is the volume of water added, in microliters (tL);
is the molecular weight of the primer, in grams per milliliter (g/ml): - number of bases 1
A is adenine
is guanine;
C is cytosine;
is thymine.
References
GB/F 19563—2004
[.1] 3. Sambrook, E, F. Fritsch, T. Manninetis. Molecular cloning. Beijing, Science Press, 1996 [2] Benjamin Levwin. Genes.New York: Oxford University Press, 2000[3J Blair. MW, O. Panaud, S. R, MeCouch, inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza satina L,).Thear Appl Genet 98,1999.780792E4 Davis. JI, DL Childers, D, N. Kuhn, Clonal variation in a Florida Bay Thalassia testudi-nun meadow: tolecular genetic assessment of populatian structure. Mar Ecol Prog Ser l86.1999.127~136.|| tt||[s] Fang. DQ, R, R. Krueger,M, L. Ronse, Phylogenetic relationships among selected citrusgermplasm accessions revealed by inter-simple sequence repeat (1SsR) markers. J Am SocHort Sci 123,1998. 612~~617
L6] Ge. .Mol.Ecol.8,J999.20612069L7] Godwin, I. D, .EA B, Aitken,L, W. Smith. Application of inter-sinple sequence repeat (IS-SR) markers to plant genetics, Electrophoresis 18,1997.1524 -~1528[8] Huang. J. ,S. M, Genetic diversity and relstionships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis af chloroplast DNA. Thear Appl Genet 100,2000. 1050--1060[9] Kojima, T., T. Nagaoka,K, Noda,et al. Genetie linkage map of ISSR and RAPD markers inEinkorn wheat in relatiun to that of RFLP rnarkers. Theor. Appl. Genet, 96.1998, 37-~45E1oJ Prevost, A., MJ Wilkinson, A new syslem of comparing PCR primners applied to ISSR fin\gerprinting of potato cultivars. Theor Appl Genet 98,1999. l07-112 [11J Ratnaparkhe, M, B., M. Tekeoglu, FJ Muehlbauer. Intersimple sequence repeat (ISSR)polymorphisms are useful for finding markers associated with disease resistance gene clusters.Theor.Appl.Genet. 97,1998.515~~519[12 Yang. W, AC de Oliveire, I. Godwin, et al. Comparison of DNA marker lechnologies in characterizing plant genome diversity: variability in Chinese sorghums. Crop Sci 36, 1996.1669~1676
[13'] Zavodna, M, ,J. Kraic,G.Paglia,et al. Differentiation between closely related lentil (Lens cu-linaris MFDIK.) cultivars using DNA merkers, Seed Sei Tech 28 2000.217~219[l4] Zhou. ZH ,M, Miwa, T. Hogetsu. Analysis of genetic structure of a Suillus grevillei popu-lation in a Larix kaemferi stand by polymorphism of inter-simple sequence repeat (ISSR).NewPhbytol144.1999.55~63
[15] Zietkiewicx. E. , A. Rafalski, D. Labude, Genome fingerprinting by simple sequence repeat(SSR)-anehored polymerase chain reaction amplification, Genomics 20,1994, 176-183Clonal variation in a Florida Bay Thalassia testudi-nun meadow: tolecular genetic assessment of populatian structure. Mar Ecol Prog Ser l86.1999.127~136.
[s] Fang. DQ, R, R. Krueger,M, L . Ronse, Phylogenetic relationships among selected citrusgermplasm accessions revealed by inter-simple sequence repeat (1SsR) markers. J Am SocHort Sci 123,1998. 612~~617
L6] Ge. X J. ,M. Sun, Reproductive biology and gcnetic diversity of a cryptoviviparous mangroveAegiceras corniculatum (Myrtinaceae) using allozyme and intersimple sequence repeat (1s-SR)analyais.Mol.Ecol.8,J999.20612069L7] Godwin, I. D, .EA B, Aitken,L, W. Smith. Application of inter-sinple sequence repeat (IS-SR) markers to plant genetics, Electrophoresis 18,1997.1524-~1528[8] Huang. J. , S. M, Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis af chloroplast DNA. Thear Appl Genet 100,2000. 1050--1060[ 9] Kojima, T., T. Nagaoka, K, Noda, et al. Genetie linkage map of ISSR and RAPD markers in Einkorn wheat in relatiun to that of RFLP rnarkers. Theor. Appl. Genet, 96.1998, 37-~45E1oJ Prevost, A., MJ Wilkinson, A new syslem of comparing PCR primners applied to ISSR fin\gerprinting of potato cultivars. Theor Appl Genet 98,1999. l07-112[11J Ratnaparkhe, M, B., M. Tekeoglu, FJ Muehlbauer. Intersimple sequence repeat (ISSR)polymorphisms are useful for finding markers associated with disease resistance gene clusters.Theor.Appl.Genet. 97,1998.515~~519[12 Yang. W, , AC de Oliveire, I . Godwin, et al. Comparison of DNA marker lechnologies incharacterizing plant genome diversity: variability in Chinese sorghums. Crop Sci 36, 1996.1669~1676
[13'] Zavodna, M, ,J. Kraic,G.Paglia,et al. Differentiation between closely related lentil (Lens cu-linaris MFDIK.) cultivars using DNA merkers,Seed Sei Tech 28 2000.217~219[l4] Zhou. ZH ,M, Miwa, T. Hogetsu. Analysis of genetic structure of a Suillus grevillei popu-lation in a Larix kaemferi stand by polymorphism of inter-simple sequence repeat (ISSR).NewPhbytol144.1999.55~ 63
[15] Zietkiewicx. E. , A. Rafalski, D. Labude, Genome fingerprinting by simple sequence repeat(SSR)-anehored polymerase chain reaction amplification, Genomics 20,1994, 176-183Clonal variation in a Florida Bay Thalassia testudi-nun meadow: tolecular genetic assessment of populatian structure. Mar Ecol Prog Ser l86.1999.127~136.
[s] Fang. DQ, R, R. Krueger,M, L . Ronse, Phylogenetic relationships among selected citrusgermplasm accessions revealed by inter-simple sequence repeat (1SsR) markers. J Am SocHort Sci 123,1998. 612~~617
L6] Ge. X J. ,M. Sun, Reproductive biology and gcnetic diversity of a cryptoviviparous mangroveAegiceras corniculatum (Myrtinaceae) using allozyme and intersimple sequence repeat (1s-SR)analyais.Mol.Ecol.8,J999.20612069L7] Godwin, I. D, .EA B, Aitken,L, W. Smith. Application of inter-sinple sequence repeat (IS-SR) markers to plant genetics, Electrophoresis 18,1997.1524-~1528[8] Huang. J. , S. M, Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis af chloroplast DNA. Thear Appl Genet 100,2000. 1050--1060[ 9] Kojima, T., T. Nagaoka, K, Noda, et al. Genetie linkage map of ISSR and RAPD markers in Einkorn wheat in relatiun to that of RFLP rnarkers. Theor. Appl. Genet, 96.1998, 37-~45E1oJ Prevost, A., MJ Wilkinson, A new syslem of comparing PCR primners applied to ISSR fin\gerprinting of potato cultivars. Theor Appl Genet 98,1999. l07-112[11J Ratnaparkhe, M, B., M. Tekeoglu, FJ Muehlbauer. Intersimple sequence repeat (ISSR)polymorphisms are useful for finding markers associated with disease resistance gene clusters.Theor.Appl.Genet. 97,1998.515~~519[12 Yang. W, , AC de Oliveire, I . Godwin, et al. Comparison of DNA marker lechnologies incharacterizing plant genome diversity: variability in Chinese sorghums. Crop Sci 36, 1996.1669~1676
[13'] Zavodna, M, ,J. Kraic,G.Paglia,et al. Differentiation between closely related lentil (Lens cu-linaris MFDIK.) cultivars using DNA merkers,Seed Sei Tech 28 2000.217~219[l4] Zhou. ZH ,M, Miwa, T. Hogetsu. Analysis of genetic structure of a Suillus grevillei popu-lation in a Larix kaemferi stand by polymorphism of inter-simple sequence repeat (ISSR).NewPhbytol144.1999.55~ 63
[15] Zietkiewicx. E. , A. Rafalski, D. Labude, Genome fingerprinting by simple sequence repeat(SSR)-anehored polymerase chain reaction amplification, Genomics 20,1994, 176-183Application of inter-sinple sequence repeat (IS-SR) markers to plant genetics, Electrophoresis 18,1997.1524-~1528[8] Huang. J. ,S. M, Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas ( Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis af chloroplast DNA. Thear Appl Genet 100,2000. 1050--1060[9] Kojima, T. , T. Nagaoka,K, Noda,et al. Genetie linkage map of ISSR and RAPD markers inEinkorn wheat in relatiun to that of RFLP rnarkers. Theor. Appl. Genet, 96.1998, 37-~45E1oJ Prevost, A. , MJ Wilkinson, A new syslem of comparing PCR primners applied to ISSR fin\gerprinting of potato cultivars. Theor Appl Genet 98,1999. l07-112[11J Ratnaparkhe, M, B., M. Tekeoglu, FJ Muehlbauer. Intersimple sequence repeat (ISSR)polymorphisms are useful for finding markers associated with disease resistance gene clusters.Theor.Appl.Genet. 97,1998.515~~519[12 Yang. W, AC de Oliveire, I. Godwin,et al. Comparison of DNA marker lechnologies incharacterizing plant Genome diversity: variability in Chinese sorghums. Crop Sci 36, 1996.1669~1676
[13'] Zavodna, M, ,J. Kraic,G.Paglia,et al. Differentiation between closely related lentil (Lens cu-linaris MFDIK.) cultivars using DNA merkers,Seed Sei Tech 28 2000.217~219[l4] Zhou. ZH ,M, Miwa, T. Hogetsu. Analysis of genetic structure of a Suillus grevillei popu- lation in a Larix kaemferi stand by polymorphism of inter-simple sequence repeat (ISSR). NewPhbytol144.1999.55~63
[15] Zietkiewicx. E., A. Rafalski, D. Labude, Genome fingerprinting by simple sequence repeat (SSR)-anehored polymerase chain reaction amplification, Genomics 20,1994, 176-183Application of inter-sinple sequence repeat (IS-SR) markers to plant genetics, Electrophoresis 18,1997.1524-~1528[8] Huang. J. ,S. M, Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas ( Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis af chloroplast DNA. Thear Appl Genet 100,2000. 1050--1060[9] Kojima, T. , T. Nagaoka,K, Noda,et al. Genetie linkage map of ISSR and RAPD markers inEinkorn wheat in relatiun to that of RFLP rnarkers. Theor. Appl. Genet, 96.1998, 37-~45E1oJ Prevost, A. , MJ Wilkinson, A new syslem of comparing PCR primners applied to ISSR fin\gerprinting of potato cultivars. Theor Appl Genet 98,1999. l07-112[11J Ratnaparkhe, M, B., M. Tekeoglu, FJ Muehlbauer. Intersimple sequence repeat (ISSR)polymorphisms are useful for finding markers associated with disease resistance gene clusters.Theor.Appl.Genet. 97,1998.515~~519[12 Yang. W, AC de Oliveire, I. Godwin,et al. Comparison of DNA marker lechnologies incharacterizing plant Genome diversity: variability in Chinese sorghums. Crop Sci 36, 1996.1669~1676
[13'] Zavodna, M, ,J. Kraic,G.Paglia,et al. Differentiation between closely related lentil (Lens cu-linaris MFDIK.) cultivars using DNA merkers,Seed Sei Tech 28 2000.217~219[l4] Zhou. ZH ,M, Miwa, T. Hogetsu. Analysis of genetic structure of a Suillus grevillei popu- lation in a Larix kaemferi stand by polymorphism of inter-simple sequence repeat (ISSR). NewPhbytol144.1999.55~63
[15] Zietkiewicx. E., A. Rafalski, D. Labude, Genome fingerprinting by simple sequence repeat (SSR)-anehored polymerase chain reaction amplification, Genomics 20,1994, 176-183Analysis of genetic structure of a Suillus grevillei popu-lation in a Larix kaemferi stand by polymorphism of inter-simple sequence repeat (ISSR). NewPhbytol144.1999.55~63
[15] Zietkiewicx. E. , A. Rafalski, D. Labude, Genome fingerprinting by simple sequence repeat(SSR)-anehored polymerase chain reaction amplification, Genomics 20,1994, 176-183Analysis of genetic structure of a Suillus grevillei popu-lation in a Larix kaemferi stand by polymorphism of inter-simple sequence repeat (ISSR). NewPhbytol144.1999.55~63
[15] Zietkiewicx. E. , A. Rafalski, D. Labude, Genome fingerprinting by simple sequence repeat(SSR)-anehored polymerase chain reaction amplification, Genomics 20,1994, 176-183
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