菜豆基因组微卫星DNA的分离与鉴定_英文_

菜豆基因组微卫星DNA的分离与鉴定_英文_ () 植 物 学 报 2000 , 42 11:1179 - 1183 Acta B ota nica Si nica 菜豆基因组微卫星 D NA 的分离与鉴定 1 3122 郭建春胡新文柳原诚司江川宜伸 (1 . 中国热带农业科学院热带作物生物技术国家重点实验室 , 海口 571101 ; )2 . 日本国际农业科学研究中心 , 日本冲绳 907- 0002 ( ) 摘要 : 从耐高温 型 菜 豆 Phaseolus vulgaris L . 品 种“Haibushi”基 因 组 分 离 微 卫 星 DNA , 可 以 提 供 与 耐 热 性 QTL ( ) quantitative trait locus连锁的多态遗传标记 。为此 , 利用 lambda ZAP ?载体构建了一个包含 400,800 bp 插入片段 ( ) ( ) 的菜豆基因组文库 。利用地高辛末端标记寡聚核苷酸 GA和 CA作为探针筛选该文库 ,并对部分阳性克隆进 10106 4 () 行测序 分析 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 , 结果如下 : 获得了一个含 1. 125 ×10pfu plaque forming unit的基因组文库 , 其中约有 3. 72 ×10pfu 含 ( ) ( ) ( ) ( ) GA或 CA微卫星 DNA ,即 16. 7 kb 菜豆基因组 DNA 就含有一个 GA或 CA微卫星 DNA 。测序分析了 26 n n n n 个阳性克隆 ,每个克隆含有 1 个或 1 个以上的微卫星 DNA ,一共得到 35 个微卫星 DNA 。其中 17 个为单一重复子 , ( ) ( ) ( ) 18 个为复合重复子 。在 35 个微卫星 DNA 中 ,26 个为 GA重复子 ,占 81 % 。 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 明在菜豆基因组中 GA比 CAn n n ( ) ( ) 的丰度要高得多 。其中 13 个 GA的长度等于或大于 20 bp ,相当于每 33. 4 kb 基因组 DNA 含 1 个 。GA重复子 n n ( ) ( ) 的平均长度 n = 10. 4 ,明显高于 CA重复子的平均长度 n = 6. 5 ,表明 GA重复子更适合于用作遗传标记 。 关键n n 词 : 菜豆 ; 基因组文库 ; 微卫星 DNA ; 分子标记 ; DNA 序列分析 () 中图分类号 : Q943 文献标识码 : A 文章编号 : 057727496 20001121179205 Isolation and Characterization of Microsatellites in Sna p Bean 1 1 3 2 2GUO J ian- Chun, HU Xin- Wen, YANAGIHARA Seiji, YOSHINOBU Egawa (1 . National Key Biotechnology L aboratory f or Tropical Crops , Chinese Academy of Tropical Agricultural Sciences , Haikou 571101 , China ; )2 . J apan International Research Center f or Agricultural Sciences , Okinawa 907- 0002 , Japan Abstract : The objectives of this study were to isolate and characterize microsatellites from a heat tolerant va2 ( ) riety of snap bean Phaseolus vulgaris L . in order to generate polymorphic genetic markers linked to quantita2 tive trait loci for heat tolerance . A genomic library contained 400 - 800 bp inserts was constructed and ( ) ( ) screened for the presence of GA/ CTand CA/ GTrepeats. The proportion of positive clones yielded esti2 n n 4 mated of 3 . 72 ×10such dinucleotide repeats per genome , roughly comparable to the abundance reported inother eukaryotic genomes. Twenty- six positive clones were sequenced. In contrast to mammalian genomes , the ( ) ( ) ( ) GA/ CTmotif was much more abundant than the CA/ GTmotif in these clones. The GA/ CTrepeats n n n () also showed longer average repeat length mean n = 10 . 4 versus 6 . 5, suggesting that they are better candi2 dates for yielding polymorphic genetic markers in the snap bean genome . Key words : Phaseolus vulgaris ; genomic library ; microsatellite ; molecular marker ; DNA sequence analysis Microsatellites , also called “simple sequences re2 tural plant species such as soybean , sunflower and rape2 ( ) seed suggests that the relative abundance of motifs may peats”,“simple tandem repeats STRs”or“simple se2 differ dramatically from that observed in animal quences”consist of head- to- tail tandem arrays of short 1 - 3 ( ) genomes. In database searches of plants , the most DNA motifs usually 1 - 5 bases. They are a common common long dinucleotide repeat sequence was found to component of the eukaryotic genomes but are almost ab2 1 ,2 ,4 ( ) () ( be GA/ CTand AT/ TA. In contrast , CA/ sent in prokaryotes. In recent years , microsatellites have n n ) become the molecular marker of choice in target sequences GTsequences are the most common in mammals. Sur2 n veys of microsatellites in natural populations of higher for a wide range of application in genetic mapping and plants have not been reported. The only information on genome analysis. Research on microsatellites in agricul2 Received : 2000201224 Accepted : 2000207224 Foundation items : A Grant from Japan International Research Center for Agricultural Sciences ; fund partly from the Biooriented Technology Research Advancement Institution. 3 Author for correspondence . Abbreviations : pfu , plaque forming unit ; QTL , quantitative trait locus. 5 woody species comes from Condit and Hubbell, in hybridization. The library was screened by plaque hy2 which DNA libraries of five tropical tree species were bridization. Plaque lifts were performed according to the + ( ) ( ) screened for GA/ CTand CA/ GTmotifs. They n n procedure supplied with the Hybond- N membrane found those repeats to be relatively abundant in higher () Amersham. 3 5 plants and estimated that there were 10- 10such sites ( ) ( ) Oligonucleotide probes GAand CAwere hy2 10 10 5 bridized to the membranes to select plaques containing in the genomes of the species examined. This is con2 similar sequences. The hybridization solution for oligonu2 cordant with the results obtained from grapevine , Quercus ( ) macrocarpa , bread wheat , Eucalyptus grandis and E. cleotide probes was 5 ×SSC , 1 . 0 % W/ Vblocking 6 - 9 ( ) reagent for nucleic acid hybridization , 0 . 1 % N- lauroyl2 urophylla . The objectives of this study were to 1 isolate and characterize microsatellites from snap bean ; sarcosine , and 0 . 02 % sodium dodecyl sulfate . The probe () 2determine the sequences and lengths of both types of concentration was 2 pmol/ mL hybridization solution. Hy2 ( ) ( ) () microsatellites GAand CA; 3estimate the rela2 bridization temperature for the individual oligonucleotides n n ( ) was 5 ?below the Tm values of 56 ?for CAand 50 tive abundance of microsatellites in the snap bean 10 ( ) ?for GA. The wash and detection step s were con2genome . 10 () ducted following the protocol Boehringer-Mannheim. 1 Materials and Methods 1 . 4 I n vivo excision of pBluescript phagemid from the la mbda ZAP ? vector1 . 1 Plant materials ( A heat tolerant variety of snap bean Phaseolus vul2 For efficient excision , the following components were μcombined in a sterile tube : 200 L of XL1-Blue MRF’ ) garis L . cv. Kentudky Wondernamed Haibushi was cul2 μ(cells at an ODof 1 . 0 , 250L of phage stock contain2 600 tivated in a greenhouse at J IRCAS Okinawa Subtropical 5 μ) ing > 1 ×10phage particles, 1 L of the ExAssist Station and used for DNA extraction. 6 ( μ) helper phage > 1 ×10pfu/L. The tube was incubat2 ed 1 . 2 Genomic library construction at 37 ? for 15 min. After 3 mL of LB broth was Genomic DNA was extracted from fresh leaves with added , the mixture was incubated at 37 ?for 3 h , then heated at 65 - 70 ? for 20 min and centrifuged at CTAB method and sonicated with Ultrasonicator VP- 5T 1 000 g for 15 min. The supernatant was collected into a ( ) TAITEC Co . Ltd. . The sonication conditions were as sterile tube and stored at 4 ?. This stock contained the follows : probe size in 2 mm , power scale select switch set excised pBluescript phagemid packaged as filamentous at 25 W , sonication time for 6 min. The DNA sample particles. contained in a 1 . 5 mL microcentrifuge tube was immersed μμ To plate the excised phagemids , 100L and 10 Lin an ice- salt-water- alcohol bath throughout the sonication μof phage supernatant was mixed respectively with 200 L procedure . of freshly grown SOLR cells in microcentrifuge tubes and (DNA fragments of the desired size range 400 - 800 μincubated at 37 ? for 15 min. One hundred L of cell ) bp were size- selected twice by low melting agarose mixture from each microcentrifuge tube was spread on LB- () 1 . 5 %gel electrophoresis. The DNA fragment ends ( ) ampicillin 50 mg/ mL agar plate and incubated were filled in by Mung Bean Nuclease and Klenow , then overnight at 37 ?. The colonies were selected for plas2 ligated to EcoR ?/ Bst X ? adapters of Invitrogen. The ( ) mid preparations mini prep . and the inserts were adapter ends were kinased by adding Tpolynucleotide ki2 4 checked by PCR or digestion with EcoR ?. μ( ) nase Ta KaRa , 10 U/L . Excess adapters were re2 1 . 5 PCR screening of positive clones moved by Seakem agarose gel electrophoresis and the tar2 In order to minimize the amount of unnecessary se2 ( get DNA was recovered by Geneclean ? kit Strategene , quencing , a PCR screen was used to check each putative ) LaJolla , CA , USA. positive SSR- bearing clone for the presence and position A genomic library was constructed by using the of a microsatellite . This enabled the determination of the lambda ZAP ? Predigested EcoR ?/ CIAP- Vector Kit approximate size of the clone , as well as the position of ( ) Strategene , LaJolla , CA , USA, and was packaged in a ( ) the SSR simple sequence repeatwithin the clone . PCR high efficiency system such as Gigapack ? Gold packag2 screens were performed using three primer pairs , i . e . ( ) ing extract Strategene , LaJolla , CA , USA. Reaction No . 1 : M13 Forward + M13 Reverse ; Reaction 1 . 3 Screening of genomic library No . 2 : M13 Forward + SSR primer A ; Reaction No . 3 : ( ) ( ) Two oligonucleotide probes GAand CAwere 10 10 SSR primer B + M13 Reverse . SRR primers A and B digoxigenin- labeled by using the Genius 6 Oligonucleotide ( ) Tailing Kit Boehringer Mannheim and used for ()1181 11 期 郭建春等 : 菜豆基因组微卫星 DNA 的分离与鉴定 英 Fig. 1. PCR amplification of 26 positive clones by using M13 Forward and M13 Reverse . 100 bp ladder was used as DNA molecular weight marker. were the microsatellite probe repeat sequence and its com2 or 1 every 16 . 7 kb if randomly distributed. Twenty- six positive plaques were picked out and plementary sequence , respectively. This procedure checked by PCR. The expected amplification effectively determined whether the clone contained a mi2 products ( ) were obtained from all of these plaques Fig. 1. After crosatellite , the position of the microsatellite within the excision , the plasmid samples were digested by EcoR ? clone and , in some cases , the type of microsatellite . and the DNA bands showed exactly the insert size , re2 1 . 6 D NA sequence analysis () spectively 650 bp , see Fig. 2, which was about 200 bp Plasmid DNA of positive clones was prepared by mi2 shorter than their individual PCR product amplified by us2 ni alkaline lysis preparation procedure and purified by ing M13 Forward and M13 Reverse . () Quantum Prep Plasmid Miniprep Kit Bio- Radand se2 quenced by the dideoxynucleotide chain termination method using T7 DNA polymerase sequencing kit the ( )Pharmacia ’s instruc2 following the manufacturer10 tions. 2 Results 2 . 1 Genomic library construction 6 A library with 1 . 125 ×10plaque forming units was obtained , and checked by PCR using M13 Forward and ( ) M13 Reverse Fig. 1. Sixteen plaques were picked up randomly from agar plates and used as templates. Among them , 12 plaques displayed the expected amplification Fig. 2. The insert sizes of 16 positive clones separated by excision of pBluescript phagemid from lambda ZAP ? vector and digested products. This meant that the library contained about 8 . with EcoR ?. 5 100 bp ladder was used as DNA molecular weight marker. 44 ×10recombinant plaques , i . e . about 88 . 5 % ge2 nomic DNA of snap bean using 650 bp as the average size 8 2 . 3 Sequence characterization of sna p bean SSRs of the inserted DNA and 6 . 2 ×10bp as the approximate Sequences of the genomic insert for 26 positive genome size . plaques were obtained , and each contained 1 or more mi2 2 . 2 L ibrary screening crosatellite sequences. Table 1 shows the lengths and A total of approximately 7 000 recombinant plaques types of the microsatellites. Approximately 34 . 6 % of the resulted from the cloning of short genomic inserts into clones contained more than one type of microsatellite re2 lambda ZAP ?vectors were used for screening. Of these , peats , and usually the different repeat types were very () 170 2 . 4 %hybridized to the simple- sequence probes. similar to one another in the clones. DNA sequences of The number of microsatellites in the snap bean genome 5 clone SBHM 16 and SBHM 24 containing a simple repeat was roughly estimated following Condit and Hubbell, and compound repeats respectively are shown in Fig. 3 . using 650 bp as the average size of the inserted DNA 12 8 Using the classification of Weber, we determined that () Fig. 2and 6 . 2 ×10bp as the approximate genome (17 of the inserts contained single“perfect”repeats with size . This calculation showed that the snap bean genome 4 ) no interruptions of the core motif and 9 were ( ) ( ) regions ,CA/ GThas about 3 . 72 ×10and GA/ CTn n (“compound”repeats with different motifs located in close ) proximity, yielding a total of 35 dinucleotide repeats. ( ) (Five of these were TA/ ATrepeats for which we had n ) not screenedthat were part of the compound repeats. Of ( ) () the remaining 30 , 26 were GA/ CT81 %. The aver2 n ( ) age length of GA/ CTwas n = 10 . 4 , substantially n () higher than the average number of either the AT/ TAn () ( ) ( repeats mean n = 5 . 6or CA/ GTrepeats mean n n ) = 6 . 5. The sequence analyses showed that 13 of the 26 positive plaques contained a microsatellite longer than 20 ( ) Fig. 3. Microsatellites in the clones SBHM 16 and 24 Table 1, bp in length , respectively. It was estimated that on aver2 showing simple and compound repeats respectively. age a repeat longer than 20 bp in length occurred every 33 . 4 kb in nuclear of snap bean of genome . 3 Discussion The entire sequences for 5 genomic inserts containing It has been estimated that on average a repeat longer microsatellites were obtained. The A + T contents for than 20 bp in length occurs every 33 . 4 kb in plant nucle2 these inserts were relatively high , approximately 65 %. In 4 a number of cases , the composition of the flanking se2 ar genomes compared with every 6 kb in mammals. The relatively low frequency of microsatellites in plant genomes quences made it difficult for designing PCR primers. presents technical problems for the large- scale isolation of Ta ble 1 Length and type of microsatellites from sequenced frag2 SSRs. Standard methods for the isolation of SSRs involve : ments of snap bean DNA the creation of a small insert genomic library ; library ) ( Number of repeat units nfor each motif screening by hybridization ; DNA sequencing of positive ( ) ( ) () GA/ CTCA/ GTAT/ TA nnnclones ; primer design and locus- specific PCR analysis ; Simple repeats and quantification of polymorphisms. SBHM 1 10 For development of microsatellites , the small insert SBHM 2 8 genomic libraries were always constructed by digesting ge2 SBHM 3 11 nomic DNA with restriction enzymes or sonication. In the SBHM 4 10 enzymatic digestion the restriction sites are fixed , while SBHM 5 8 SBHM 6 13 sonication can degrade DNA randomly. So the library SBHM 7 11 constructed with sonication possibly contained the entire SBHM 8 7 genome even though DNA fragments were size-fractionat2 SBHM 9 17 ed. SBHM 10 12 Our estimate for the abundance of microsatellite re2 SBHM 11 10 peats in snap bean is similar to Condit and Hubbell’s es2SBHM 12 7 4 5 timate of 10- 10simple sequence repeats in five species SBHM 13 7 5 SBHM 14 14 of tropical treesand comparable to dinucleotide repeat 11 SBHM 15 6 frequencies reported in other higher eukaryotesand SBHM 16 4 20 some plants. The number of microsatellites found in SBHM 17 7 snap bean contradicts the results of Lahercrantz et al , Compound repeats who reported that microsatellites are 5 times less abundant SBHM 18 6 5 1 in the genomes of plants than in mammals . SBHM 19 8 5 The frequency of each class of SSR is highly variable SBHM 20 8 5 12 SBHM 21 11 5 among plant species. Even though it was based on a SBHM 22 18 8 limited sample of 26 SSR- containing clones , our results SBHM 23 13 5 indicate that both GA and CA repeats appear to be well- SBHM 24 9 , 8 ( dispersed throughout the genome of snap bean. The GA/ SBHM 25 5 6 ) CTmotifs were seen to be more abundant than the n SBHM 26 6 5 () CA/ GTmotif , and also to have longer average repeat n Mean number of repeats 10. 4 6. 5 5 . 6 () length mean n = 10 . 4 versus 6 . 5, suggesting that they Total number of microsatellites 26 4 5 ( ) are better candidates for yielding polymorphic genetic All microsatellites are perfect no substitutions. 8markers in snap bean genomes. Byrne et al also ()1183 11 期 郭建春等 : 菜豆基因组微卫星 DNA 的分离与鉴定 英 Condit R , Hubbell S P. Abundance and DNA sequence of 5 ] reported that GA repeats were more abundant in high two- base repeat regions in tropical tree genomes. Genome , plants. Our results demonstrate that in snap bean , as in 1991 , 34 :61 - 71. the majority of other plant species surveyed to date , GA Thomas M R , Matsumoto S , Cain P , Scott N S. Repetitive 6 ] repeats are more abundant than CA repeats , as distinct DNA of grapevine :classes present and sequences suitable for from the reports showing that CA repeats are more fre2 cultivar identification. Theor Appl Genet , 1993 , 86 : 173 - 13 quent in mammalian genomes. 180. The high efficiency of recovery of SSR- containing Dow B D , Ashley M V , Howe H F. Characterization of 7 ] ( ) clones from genomic library constructed by sonication and highly variable GA/ CTmicrosatellites in the bur oak , n Quercus macrocarpa. Theor Appl Genet , 1995 , 91 : 137 - the occurrence of two different classes of repeats suggest 141. that a large number of dinucleotide repeats is available in Bryan G J , Collins A J , Stephenson P. Isolation and char2 8 ] the snap bean genome for the development of molecular acterisation of microsatellites from hexaploid bread wheat . markers. Theor Appl Genet , 1997 , 94 :557 - 563. Brondani R P V , Brondani C , Tarchini R. Development , 9 ] References : characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla. Theor Appl Genet , 1 ] Lagercrantz U , Ellegren H , Andersson L . The abundance of 1998 , 97 :816 - 827. various polymorphic microsatellite motifs differs between Sanger F , Nicklen S , Coulson A R. DNA sequencing with 10 ] plants and vertebrates. N ucl Acids Res , 1993 , 21 : 1111 - chain- terminating inhibitors. Proc N atl Acad Sci USA , 1115. 1977 , 74 :5463 - 5467. Akkaya M S , Bhagwat A A , Cregan P B. Length polymor2 2 ] Powell W , Machray G C. Polymorphism revealed by simple 11 ] phisms of simple sequence repeat DNA in soybean. Genet2 sequence repeats. Trends Plant Sci , 1996 , 1 :209 - 245. ics , 1992 , 132 :1131 - 1139. ( ) ( ) Weber J L . Informativeness of human dC- dAdG- dT12 ] n n Brunel D. A microsatellite marker in Helianthus annuus L . 3 ] polymorphisms. Genomics , 1990 , 7 :524 - 530. Plant Mol Biol , 1994 , 24 :397 - 400. Weissenbach J , Gyapay G , Dib C , Vignal A. A second- 13 ] Wang Z , Weber J L , Zhong G , Tanksley S D. Survey of 4 ] generation linkage map of the human genome . N ature , plant short tandem repeats. Theor Appl Genet , 1994 , 88 : 1 1992 , 359 :794 - 801. - 6. ()责任编辑 : 谢 巍