分子标记参考资料.doc

分子标记技术及其应用I. Term Explanation (10%, 2 points for each)1. Probe：是一段具有特异碱基序列的单链DNA或RNA分子，经过放射性或非放射性标记、杂交，用于检测互补碱基序列（Single-stranded DNA or RNA molecules of specific base sequence, labeled either radioactively or non- labeled, that are used to detect the complementary base sequence by hybridization.）2. Marker-assisted selection (MAS)：通过对分子标记检测目标基因与某个分子标记的连锁，获知目标基因的基因型。借助分子标记对目标性状的基因型进行选择，这称为MAS。3. Gene pyramiding：通过分子标记，选择与目标基因连锁的基因型，将分散在不同品种中的有用基因聚合到同一个基因组中。4. Molecular marker：是指能反映生物个体或种群问基因组中某种差异特征的DNA片段。这种DNA片段的特性可通过将基因组DNA经限制性内切酶酶切、PCR扩增、分子杂交等技术在电泳凝胶上检测出来。DNA分子标记是遗传变异在DNA水平上的直接反映。5. Recombination inbred Lines (RIL)：由两个亲本杂交后，从F2代开始采用SSD等方法经连续多代自交或姊妹交而育成的近交系。6. Quantitative trait loci (QTL)：控制数量性状的基因在基因组中的位置称为数量性状基因座（QTL）。7. Primer：一段DNA或RNA序列。通过复性结合到模板单链DNA，在DNA聚合酶作用下，能使核苷酸数目得到增加。（A short DNA or RNA fragment annealed to a singled-stranded DNA and to which further nucleotides can be added by DNA polymerase. ）8. Genetic marker：指受基因控制的能够稳定遗传的，且能代表个体或群体的遗传特征，并可被用作遗传分析的物质。II. Fill in the blanks with the knowledge you have learned. (10%, one point for each)1. DAF (DNA Amplified Fingerprints ) is a method of general DNA amplification using a single primer of between 58 nucleotides in length.2. In transfer filter，the HCl-treatment is performed to 脱嘌呤（depurinate） large DNA- fragments and enhance their transfer rate to the membrane.3. RAPD (random amplified polymorphic DNA) is a method of general DNA amplification using a single primer about 10 nucleotides in length. 4. PCR reaction main involves 变性（denaturation）, 退火（annealing）and 延伸（extention） three steps.5. In plant DNA extraction, we need usually to add b- mercaptoethanol. Function of b- mercaptoethanol is抗氧化（preventing the browning of DNA）.6. In 1983, PCR was invented by Kary B. Mullis .7. AP-PCR (arbitrary primer PCR) is a method of DNA amplification using a single primer of between 20 or 30 nucleotides in length. 8. The restriction fragment length polymorphisms can be separated from one another by 凝胶电泳（gel electrophoresis）.9. SRAP marker include two sets of PCR primers, forward primer use CCGG sequence, reverse primer use AATT sequence.10. SCAR use 24 bp primers designed from the ends of cloned RAPD or AFLP markers.11. Theoretical basis of construction of linkage map is 染色体交换（chromosome crossover） and 重组（recombination） .12. STS is short (200 to 500 base pairs) DNA sequence that has a single occurrence in the genome and whose location and base sequence are known.13. ISSR primers based on 微卫星（microsatellites） are utilized to amplify inter-SSR DNA sequences.14. Expressed sequence tag (EST) is a small part of the active part of a gene, made from cDNA .15. In AFLP experiment, adaptor is about 1418 nucleotides in length.16. The AFLP technique detects polymorphisms is a combination of the RFLP and PCR technologies.17. In plant DNA extraction, we often need to add PVP for 除去多酚（糖）（keeping out of DNA Polyphenols from the samples） .III. Finish each of the following sentences by circling the letter of the correct response. (15%, one point for each)1. Primer used of RAPD marker is about c in length.a. 35bp b. 24bp c. 10bp d. 58bp2. Genomic tandem repeat include satellite DNA, minisatellite and microsatellite. Of these microsatellite is about b bp in length. a. 1060bp b. 16bp c. 100300bp d. 1000100000bp3. In linkage map construction, for gained similar precision, order of population required from large to small is d .a. BC1DH F2RI b. F2BC1DH RI c. BC1DH RIF2 d. F2RIBC1DH4. RFLP is based on two techniques that are widely used in modern molecular biology, it derive from b . a. Probe b. Probe and restriction endonuclease combinations c. Primer d. restriction endonuclease5. Salt concentration of AP-PCR reaction is a than classical PCR reaction.a. High b. Low c. Same d. uncertain6. CTAB can produce compound with nucleic acid，CTAB-DNA can dissolve and exist steadily in b .a. Middle salt concentration b. High salt concentrationc. Low salt concentration d. All of the above7. PCR is used to c . a. insert foreign DNA into a vectorb. create DNA without intronsc. amplify a single gene or smaller piece of DNA rd. all of the above8. During RAPD, AP-PCR, RFLP and DAF molecular markers, having highest polymorphic marker is d .a. RAPD b. AP-PCR c. SCAR d. DAF9. ISSR marker represent generally a .a. Dominant b. Codominant c. Dominant or codominant d. All of the above10. In DNA extraction, add chloroform-isoamyl alcohol (v/v, 24:1), c of the following is the function of the isoamyl alcohol. a. eliminate sugar b. Degrade cell membrane c. eliminate bubble d. precipitate DNA11. About character of DNA, following correct description is b . a. non-polar compound b. White in color c. Dissolve in ethanol d. All of the above12. In PCR reaction, primer extension temperature is usually performed at c . a. 37 b. 54 c. 72 d. 9413. SSR marker technology is one kind of effective techniques, High polymorphism of SSR marker derive from b . a. Type of tandem repeats b. Number of tandem repeats c. Structure of tandem repeats d. All of the above14. STS is a type of a molecular marker.a. Specific amplification b. Arbitrary amplificationc. Random amplification d. All of the above15. Primer of RGA derived from d .a. Unknown sequence b. Arbitrary sequencec. Random sequence d. Conserved sequence of resistant gene16. c of the following is NOT needed for PCR?a. Primers b. DNA of interest. c. DNA ligase d. DNA polymerase17. The steps involved in the RFLP technology should be performed in the following order d .1 = x-ray film2 = electrophoresis3 = digestion DNA with restriction enzyme4 = hybridization5 = Southern transfera. 3, 2, 4, 5, 1 b. 3, 4, 2, 5, 1 c. 2, 4, 3, 5, 1 d. 3, 2, 5, 4, 1IV. 匹配题（每小题1分，共15分）掌握下面缩写的英文全称及中文1. AFLP Amplified fragment length polymorphism 扩增片段长度多态性2. SSR Simple sequence repeat 简单序列重复 3. STS Sequence- tagged sites 序列锚定位点4. DArT Diversity array technology 多样性微阵列技术5. EST Expressed sequence tags 表达序列标签6. RFLP Restriction fragment length polymorphism 限制性片段长度多态性 7. SCAR Sequence characterized amplified regions 序列特征化扩增区域8. RAPD Random amplified polymorphic DNA 随机扩增多态性DNA9. CAPS Cleaved amplified polymorphic sequence切割扩增多态性序列10. PCR Polymerase chain reaction聚合酶链反应11. RIL Recombinant inbred lines 重组近交系12. DAF DNA Amplified Fingerprints DNA扩增指纹13. RGA Resistance Gene Analogs 抗病基因类似物 or 抗病基因同源序列14. SRAP Sequence-related amplified polymorphism 相关序列扩增多态性 15. TRAP Target Region Amplified Polymorphism 靶位区域扩增多态性16. SNP Single nucleotide polymorphism 单核苷酸多态性 17. DH Doubled haoloids 双单倍体18. AP-PCR arbitrary primer PCR 任意引物PCR19. ISSR Inter-simple sequence repeat 简单序列重复间区20. NIL Near-isogenic lines 近等基因系21. SSCP Single strand conformation polymorphism 单链构象多态性VI. Answer the following questions (10%, five point for each)1. Brief explain principles of DNA extraction.答：(1)DNA在细胞中是以与蛋白质结合的状态存在（DNP）； (2) DNP在NaCl溶液中的溶解度，是随着NaCl的浓度的变化而改变的。当NaCl的物质的量浓度为0.14 molL时，DNP的溶解度最低。利用这一原理，可以使溶解在NaCl溶液中的DNP析出。(3) DNA不溶于酒精溶液，但是细胞中的某些物质则可以溶于酒精溶液。利用这一原理，可以进一步提取出含杂质较少的DNA。2. Brief explain principles of DNA extraction using CTAB method.答：(1)DNA在细胞中是以与蛋白质结合的状态存在（DNP）； (2) DNP在NaCl溶液中的溶解度，是随着NaCl的浓度的变化而改变的。当NaCl的物质的量浓度为0.14 molL时，DNP的溶解度最低。利用这一原理，可以使溶解在NaCl溶液中的DNP析出。(3) DNA不溶于酒精溶液，但是细胞中的某些物质则可以溶于酒精溶液。利用这一原理，可以进一步提取出含杂质较少的DNA。3. Whats foreground selection and background selection? Then explain function of them respectively. 答：foreground selection：对目标基因的选择称为前景选择，这是标记辅助选择的主要方面。前景选择的可靠性主要取决于标记与目标基因间连锁的紧密程度。（前景选择的作用：保证从每一回交世代选出的作为下一轮回交亲本的个体都包含目标基因；避免或减轻连锁累赘避免或减轻连锁累赘。Background selection：对基因组中除了目标基因之外的其它部分（即遗传背景）的选择，称为背景选择（background selection）。与前景选择不同的是，背景选择的对象几乎包括了整个基因组，因此，这里牵涉到一个全基因组选择的问题。背景选择的作用：加快遗传背景恢复成轮回亲本基因组的速度，缩短育种年限。4.VII. Calculation(10%, five point for each)1. If oligonucleotide primers are generally supplied as “4 OD260 and 20-mer”, now we need to make a 50uM stock solution for PCR. Please Calculate that how many microlitre (ul) ddH2O added. (Note: Average molecular weight of oligonucleotide 324.5)答：分子量（MW）=20324.5=6490质量数（mass number）=433=132g摩尔数（Molarity）=132/6490=0.020mol=20nmol所需水的量（l）=20nmol/50M=0.4ml=400l2. Following tabulation is preparing the mastermix for PCR, please calculate and complete the blanks.Tab. Mastermix of different DNA (Reaction volume:20l)ReagentsStock. Conc.React. Conc.ml /sampleSterile ddH2O mlRE buffer1012.0mlMgCl225mM1.5mM dNTP-mix4mM200mM Primer5M250nM Taq polymerase5U/ml1U Template DNA (10100ng)1ml用公式：(Stock concentration) ( volume needed ) = (Reaction concentration) (volume of sample) 进行计算。如：MgCl2 25 x =1.520 则 x (volume needed)=(1.520)/25=1.2mlVIII. Analytic question (20%, ten point for each)P1 P2 B1 B2P1 P2 B1 B2P1 P2 B1 B2P1 P2 B1 B2ABCD1. Bulked segregant analysis( BSA, 集团分离分析法) is a effective method for gene mapping, the following figure show results of four markers on resistant gene. According to results conclude preliminary linked markes with resistant gene. Then brief explain the basic principles of BSA. ( Note: P1 represent genotype of resistant parent; P2 represent genotype of sensitive parent; B1 represent resistant gene pool; B2 represent sensitive gene pool; A, B, C, D represent markers）解题要点：先看亲本间有无多态性，在亲本间存在多态性的情况下，看与亲本P1性状构建的基因池带型是否与亲本P1相同、与亲本P2性状构建的基因池带型是否与亲本P2相同，如完全一致，即可判断为紧密连锁。如一致，但与另一亲本构建的基因池存在弱带，说明有交换，连锁但不紧密。答：由图可初步判断标记B、D两个标记与抗病基因连锁（2分），其中标记B与抗病基因紧密连锁，标记D连锁不紧密（2分）。BSA基本原理：在分离群体中，依据目标性状表型的相对差异（如抗病与感病），将个体分成两组，然后分别将两组中的个体的DNA混合，形成相对的DNA池（2分）。可以推测两DNA池间差异仅在目标区域上不同，而整个遗传背景是相同的（2分）。因此，在这两个DNA池间表现出多态性的DNA标记，就有可能与目标基因连锁。（2分）2. Graphic genotype（图示基因型） is a intuitionistic method in marker-assisted selection (MAS,标记辅助选择). If A is a common cultivar(contain 2 resistant gene), but B is a elite cultivar(no resistant gene). Now we need transfer resistant gene from A to B by backross, following figure is graphic genotype of 3 plants, acording to results sele