三倍体湘云鲫线粒体DNA序列变异性分析_英文_

三倍体湘云鲫线粒体DNA序列变异性 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 _英文_ 31 卷 第 6 期 Vol . 3 1 , No . 6 第 水 生 生 物 学 报 2 0 0 7 年 1 1 月 Nov. , 2 0 0 7 ACTA HYDROBIOLOGICA SINICA VARIABIL ITY OF MITOC HO ND RIAL D NA OF TRIPLOID HY BRIDS OF CRUCIAN CARP AND COMMO N CARP GUO Xin2Hong ,LIU Shao2J un ,Xiang Bing and LIU Yun ( Key L aboratory of Protein Chemistry and Fish Developmental Biology of National Education )Ministry , College of Life Sceinece , Hunan Normal University Changsha 410081 Abstract : The article presented the results of the first regular population study of triploid hybrids of crucian carp and com2 () mon carp by sequencing the tRNA2Thr gene ,tRNA2Pro gene ,and partial control region of mitochondrial DNA mtDNA. The ( ) 26 sequenced fragments ranged from 837 to 839 bp in length. The termination associated sequence TASmotifs , central ( ) ( ) conserved sequence blocks CSB2F ,CSB2E ,CSB2D,and conserved sequence block CSB1were identified in the mtDNA control region. Most heteroplasmic fish had 3 —5 TAS repeat units. The above sequences from 26 triploid hybrids were de2 tected 65 polymorphic sites , yielding 8 haplotypes. Sequence divergence among different haplotypes ranged from 0. 1 % to 613 %. This study provided some invaluable information for the reproducibility and genetic improvement of triploid hybrids. Key words : Triploid hybrids ; Mitochondrial DNA ; Control region ; Sequence variation () CLD number : S965 ; Q78 Document code :A Article ID :100023207 20070620855208 ( Red crucian carp Carassius auratus red var . , ?, to red crucian carp and common carp ,which can be stably inherited from generation to generation. The phenotype of ) ( Cyprinus carpio L . , 2n = 100and common carp ,2n Fto Fhybrids is a little different from that of F2F. It is ) = 100belong to different genera . The crossing between 3 15 12 the first example of creation of an allotetraploid population them is considered distal . The Fhybrids were derived 2 in a vertebrate by successive generations of hybridization. from crossing males with females of F, and Fhybrids 1 3 Our successful establishment of the allotetraploid popula2 were developed by crossing males with females of F. The 2 tion in vertebrate provided important diploid2gamete re2 cytological analysis revealed that the females and males of sources to create triploid hybrids through mating F2F315 Fhybrids were able to produce diploid eggs and diploid 2 ( ) ( hybrids with J apanese crucian carp s Carassius au2 sperms that fertilized each other to form tetraploid fish in ) ) ( ratu cuvieri ?. Triploid hybrids are sterile , grow F. Thus ,the hybrids of Fto Fof red crucian carp and 3 3 11 faster and have high survival rate , and thus greatly en2 () common carp were proved to be allotetraploids 4n = 2003 hance their annual production. It is necessary to reveal with two chromosome sets of red crucian carp and two the genetic diversity of triploid hybrids since they are so 1 ,2 chromosome sets of common carp . In the allotetraploid excellent fresh fish with high quality. population ,both females and males are fertile . Similar to The mtDNA of most animals ranges in size from 16 F2F, F2Fhybrids were also confirmed as allote2 311 1215 kbp to 20 kbp and encodes 13 protein subunits , 22 ( ) traploids data not published. The shape of the F2Fwas 12 tRNAs , and two rRNAs. The only substantial noncoding intermediate to red crucian carp and common carp . The segment ,the so2called control region or displacement loop tetraploids of Fto Fgenerations displayed similar mor2 3 15 () D2loop ,encompasses the sites of initiation of H2strand ( ) phological phenotype shape and coloralso intermediate replication and both H2and L2strand transcription. The Received date :2006207203 ; Accepted date :2007204206 ( ) ( ) Foundation item : National Natural Science Foundation of China No . 30170733 , No . 30330480 ; State Key Basic Research 973 Project of China () ()No . 2001CB109006; Training Project of Excellent Young Research of State Education Ministry of China No . 200248 () Brief introduction of the a uthor :Guo Xin2Hong 1976 —,female ,doctor ,associate professor ; major in molecular genetics ; E2mail :xinhong @hnu. cn Corresponding a uthor :Liu Shao2J un , E2mail :lsj @hunnu. edu. cn mammalian control region is organized into three major re2 trophoresis , and the final extracts were dissolved in TE gions ,or domains ,including the extended terminal associ2 ?for later use .buffer and kept in the 4 ( ) ated sequences ETAS,central ,and conserved sequence 113 PCR a mplif ication The polymerase chain reaction 4 ( ) ( ) μblock CSBdomains. The extended terminal associat2 PCRamplifications were carried out in a 50L final vol2 ( ) ed sequences ETAS,central conserved sequence blocks ume containing 100 —200 ng DNA , 1. 5 mmol/ L MgCl, 2 () CSB2F ,CSB2E ,CSB2Dand conserved sequence blocks μ012 mmol/ L of each dNTP ,1mol of each primer ,1 ×of ( ) CSB21 ,CSB22 ,CSB23of the mitochondrial DNA control amplification buffer and 1 unit of Taq2polymerase region in cyprinids with the different ploidy level were ( ) Promega . The following primers were used to amplify 5 identified. Because of the short conserved elements and fragments of the entire mtDNA tRNA2Thrgene , tRNA2Pro the propensity for rapid change ,the control region is one gene ,and partial control region sequences :forward primer L of the most interesting parts of the vertebrate mitochondri2 ( ) ( ) + 5′2GGACAAATTGCATCCGTCCT23′; backward al genome . Substantial length variation has been found in () ( ) primer H 25′2GTTTCGGGGTTTGACAAGGATA 23′. the control region of many mammalian and fish () The amplification conditions 30 cycleswere the follow2 6 —8 mtDNA. In addition to length variation , substantial ing :94 ?for 45s ,55 ?for 45s ,72 ?for 1min ,followed bynucleotide sequence variability in the control region has final 5min extension at 72 ?. PCR amplification was car29 ,10 been recorded both within and between species. Fur2 ( ried out on a programmable thermal controller GeneAmpR thermore ,the degree of genetic variability characteristic of ) μPCR System 2700. To detect PCR products ,5 L of the the control region has been widely exploited in studies of reaction mixture was applied to a gel of 1. 5 % agarose and 11 population structureand can be useful in identifying electrophoresed in the standard 1 × TAE buffer. The gel 12 μwas stained with 0. 1g/ mL ethidium bromide for 20min. meaningful population subdivisions. It is an important 114 mtD NA sequencing The final PCR products were prerequisite to effective conservation , management and run on a 1 . 5 % agarose gel at 120 V for 1 . 5h ,and puri2 monitoring. Such analyses of mitochondrial sequence data () fied by QIAquick Gel Extraction Kit Qiagen. Direct se2 have already proved invaluable in examining population quencing of the PCR product was performed with the genetic diversity and constructing phylogeny in a number above same PCR primers using an automated DNA se2 8 ,13 ,14 . In this study , we compared se2 of fish species( ) quencer AB I PRISM 377 , Pekin2Elmer following the quence information from the mitochondrial tRNA2Thr protocol for cycle sequencing in Shanghai Sangon , Inc . gene , tRNA2Pro gene , and part control region of triploid 115 Sequence analyses Sequence analyses were per2 hybrids to assess the degree of population genetic diversity. formed using the GCG package of computer programs At present ,the population structure of triploid hybrids has () Version 7 . 0 ; Genetics Computer Group Inc . ,Madison. attracted our considerable interests ,not only because of its Database searches were carried out using the J ellyfish pro2 importance for the management of freshwater fisheries , but gram. The initial points of the CSB2F and CSB21 were also because of fundamental interest in application for its used as the demarcations of the extended terminal associ2 popularization in national and international market . ated sequences ,central and conserved sequence block do2 mains , respectively. Localization of conserved sequences 1 Materials and methods was done using B ESTFIT. The mtDNA nucleotide se2 111 Fish sa mples The 28 triploid hybrids were cap2 quences in the triploid hybrid population were aligned us2 tured from Chinese National Tetraploid Fish Protection ing CLUSTAL W package . Station located in Hunan Normal University. 112 Total D NA extraction The total DNA was isolat2 2 Results ed from blood samples collected from the triploid hybrid caudal veins by using DNA Extraction Kit from Shanghai 211 Direct sequencing Sangon , Inc . The extraction method was performed accord2 Almost for all samples , PCR products generated clear ing to the manufactureπs instructions. The concentration sequencing results ,though a few samples showed“messy” and quality of DNA were assessed by the agarose gel elec2 sequences at the beginning. This messy start was likely due 857 6 期 郭新红等 :三倍体湘云鲫线粒体 DNA 序列变异性分析 ) to incomplete removal of primer dimmer during the purifi2 —838bp ,143 —839bpafter removing of areas of ambi2 143 ( ) guity as well as missing data Fig. 1. The control region of cation of PCR products or the presence of nonspecific PCR products , resulting in mixed and confusing base callings. the above 26 fish included the extended terminal associated ( ) All sequences were therefore double checked on elec2 sequence ETAS domain , central conserved sequence block domain ,partial conserved sequence block domain. In trophoregrams. Among 28 triploid hybrids ,the PCR products the triploid hybrids ,the central conserved sequence block of 26 fish were successfully sequenced except an unexpect2 domain contained three central conserved sequence blocks ed “accident ” for two individuals. Obviously , BLAST () CSB2F , CSB2E , CSB2Didentified in the 5′2end of the searches showed that the sequenced fragments of 26 fish in2 control regions. The CSB21 conserved sequence block was () cluded mtDNA tRNA2Thr gene 1 —72bp,tRNA2Pro gene ( ) (identified in the 3′2end of the control regions Fig. 1. ) ( 71 —142bp , and partial control region 143 —837bp , 859 6 期 郭新红等 :三倍体湘云鲫线粒体 DNA 序列变异性分析 Fig11 The nucleotide sequence comparison of the mtDNA tRNA2Thr genes ,tRNA - Pro genes and partial control regions in the 8 triploid cruian carp haplo2 types. The alphabets ahead every row are abbreviations of the triploid cruian carp haplotypes , dashes are gaps required for alignment ,and“ 3 ”indicates the ( ) ( ) same base site . The borders of the tRNAs are indicated by arrows. The conserved sequence block CSBis underlined. The TAS motifs TACATare framed , () and the palindromic motifs ATGTAare indicated by shadows 212 Sequence diversity and heteroplasmy , was located near 5πend of the control 15 region . In contrast to some other fish species,these re2 For the 26 samples examined , the length of the mtDNA tRNA2Thr gene ,tRNA2Pro gene ,and partial con2 peat units contained the terminal associated sequence ( ) TASmotifs. TAS sequences were also present in the 8 ( ) trol region sequences ranged from 837 to 839 bp Fig. 1. ( ) triploid hybrid haplotypes Fig. 1. There were three TAS On average ,base composition was A 31 . 9 % ,T 33 . 1 % ,C motifs in the TC24 haplotype ,four TAS motifs in the TC2 19 . 5 % and G 15 . 5 %. Among the 26 triploid hybrids ,65 polymorphic sites were detected ,and yielded 8 haplotypes 1 , TC22 , TC23 , TC25 , TC26 and AT27 haplotypes ,and five that were named TC21 , TC22 , TC23 , TC24 , TC25 , TC26 , TAS motifs in the TC28 haplotype . Moreover , TC26 and ( ) TC27 , and TC28 , respectively Fig. 1 . Sequence diver2 TC27 haplotypes had 1 bp deletion at position 193 , and gence calculated by nucleotide diversity among different TC24 and TC25 haplotypes had 1 bp deletion at position ( ) haplotypes ranged from 0 . 1 % to 6 . 3 % Tab. 1. When 417 in the conserved sequence block domain , however , all the DNA sequences were aligned together , variations TC26 , TC27 and TC28 haplotypes had 1 bp insert at posi2 ( ) can clearly be seen among different haplotypes Fig. 1. ( tion 156 in the terminal associated sequence domain Fig. As expected ,individual variations were mainly observed in ) 1. amplified control region fragments. We observed 64 poly2 3 Discussion morphic sites of sequence in mtDNA control region and 1 polymorphic sites of sequence in the mtDNA tRNA2Thr The control region is unique because of a faster rate gene , tRNA2Pro gene . Thus , more than 98 . 5 % polymor2 of evolution as compared with the rRNA and protein2cod2 phic sites occurred in mtDNA control region. Especially , ing genes of mitochondrial genome . This region in the about 60 % polymorphic sites were concentrated at posi2 triploid hybrid follows the general structure previously de2 4 tion 143 —395 of the 5’end sequence of mtDNA control scribed. Concerning the selection of an appropriate sec2 region ,which was an active hypervariable region. There2 tion of the control region for maximum variability , the fore the terminal associated sequence domain showed a ETAS and central CSB domains provide the greatest vari2 greater number of variable sites ,while the less variability ability for population analyses. The ETAS has been shown 16 —20 was found in conserved blocks between pair position 143 to be effective for population analysis. If small in2 and 839 . All these polymorphisms included 48 transitions , dels are a concern , the CSB region should be avoided. 14 transversions and 3 indels. So the main variation de2 However ,if indels are of interest for the analysis ,the CSB tected in the population was due to nucleotide substitu2 domain is an ideal region for examination. The mtDNA tions ,rather than insertion/ deletion mutational events. The control region can provide an appropriate region for exami2 distribution showed a large bias towards transitional nation for a number of different types of studies. However , changes rather than transversional changes. the use of a single domain may provide all of the resolu2 Tab11 The sequence variation rate of the mtDNA t RNA2Thr genes ,t RNA2 tion necessary for a specific analysis , thus reducing the Pro genes and partial control regions in the 8 triploid hybrid haplotypes cost of a research project by reducing the required amount Haplotypes TC21 TC22 CTC23 TC24 TC25 TC26 TC27 of DNA sequencing. TC21 In the present study , summarizing the distribution of TC22 12 0repeat units in the 8 haplotypes of the 26 triploid hybrids , TC23 016 14 0there had three TAS motifs in the TC24 ,four TAS motifs TC24 218 216 10 3in the TC21 , TC22 , TC23 , TC25 , TC26 and TC27 , respec2 TC25 211 118 212 12 2tively ,and five TAS motifs in the TC28 . This indicated a TC26 417 417 413 613 12 5selective mechanism within the distribution of tandem ar2 TC27 418 416 412 612 511 11 0rays because without such a driving force a more or less TC28 217 214 213 415 316 218 217 even magnitude of distribution of tandem arrays would be expected or theoretically the number of repeat units should 213 Repeat sequences and length variations be unlimited. The TAS motif and its palindromic motif The repeat region , responsible for length variations 861 6 期 郭新红等 :三倍体湘云鲫线粒体 DNA 序列变异性分析 ( )can form the stable hairpin2loop structure , triploid hybrids possessed many advantages such as sterili2 ATGTA ty ,faster growth ,high survival rate ,facile fishing and deli2 2 which may be the main body of conserved extended termi 1 ( ) nation2associated sequences ETASs,suggesting it was a cious taste. The present study showed the triploid hy2 conserved secondary structure that may contribute to TAS brid population had the abundant genetic diversity by function. Generally ,in some TAS tandem arrays ,only one analysis of mtDNA tRNA2Thr genes ,tRNA2Pro genes ,and partial control regions from the molecular level ,providing TAS was the functional motif , and other TASes were the results of the intra2and intermolecular recombination or il2 the supporting evidence that this population may have a greater ability to adapt to changing environments. In con2 legitimate elongation without performing function. Among the 26 triploid hybrids ,65 polymorphic sites clusion ,the triploid hybrid is a species of excellent fresh ( were detected and 8 haplotypes TC21 , TC22 , TC23 , TC2 fish with high quality. It is worth popularizing in the na2 ) ( )4 , TC25 , TC26 , TC27 and TC28 , respectivelyFig. 1 tional and international markets. We thus are amplifying the breeding area of triploid hybrids to increase production were yielded. Sequence divergence among different haplo2 of the fresh fish that not only supply the actual require2 ( ) types ranged from 0 . 1 % to 6 . 3 % Tab11. The results ment in the national market , but also provide the wide indicated that the triploid hybrid population had the rela2 foreground to make profit in foreign markets. tively abundant genetic diversity. Triploid hybrids were ) ( produced through mating allotetraploid hybrids with References : ( ) J apanese crucian carp s ?. More generally ,the mtDNA 1 Liu S J ,Liu Y ,Zhou G J , et al . The formation of tetraploid stocks of is inherited maternally. According to the mtDNA maternal red crucian carp ×common carp hybrids as an effect of interspecific heritance characteristic ,in fact ,the analysis of the mtDNA hybridization J . Aquaculture ,2001 ,192 :171 —186 polymorphism of triploid hybrids is equivalent to study on Sun Y D ,Liu S J , Zhang C , et al . 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Chapman & Hall ,NY ,1996 三倍体湘云鲫线粒体 D NA 序列变异性分析 郭新红 刘少军 向 兵 刘 筠 ( )湖南师范大学生命科学学院蛋白质化学与鱼类发育生物学教育部重点实验室 ,长沙 410081 摘要 :对 26 尾三倍体湘云鲫的线粒体 tRNA2Thr 基因 、tRNA2Pro 基因和部分控制区的核苷酸序列进行了测定 ,获得 26 条长度为 837 —839 bp 的同源基因序列 ,共发现 65 个多态性核苷酸变异位点 ,多态位点比例为 0. 077 ,定义了 8 种 ( )单元型 。在湘云鲫 8 种单元型中确认了 DNA 复制终止相关的序列 TAS、中央保守区序列 CSB2F、CSB2E 和 CSB2D 和保守序列 CSB1 ,8 种单元型含有 3 —5 个 TAS 序列 。在 65 个变异位点中 ,大部分序列变异为转换 ,8 种单元型之 间的序列差异在 0. 1 % —6. 3 %之间 。该研究为三倍体湘云鲫的繁殖和遗传改良提供了一些有价值的信息 。 关键词 :三倍体湘云鲫 ;线粒体 DNA ;控制区 ;序列变异