Radiation hybrid mapping: a somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes

Radiation hybrid (RH) mapping, a somatic cell genetic technique, was developed as a general approach for constructing long-range maps of mammalian chromosomes. This statistical method depends on x-ray breakage of chromosomes to determine the distances between DNA markers, as well as their order on the chromosome. In addition, the method allows the relative likelihoods of alternative marker orders to be determined. The RH procedure was used to map 14 DNA probes from a region of human chromosome 21 spanning 20 megabase pairs. The map was confirmed by pulsed-field gel electrophoretic analysis. The results demonstrate the effectiveness of RH mapping for constructing high-resolution, contiguous maps of mammalian chromosomes.     

A physical map of the 1-gigabase bread wheat chromosome 3B

As the staple food for 35% of the world's population, wheat is one of the most important crop species. To date, sequence-based tools to accelerate wheat improvement are lacking. As part of the international effort to sequence the 17-billion-base-pair hexaploid bread wheat genome (2n = 6x = 42 chromosomes), we constructed a bacterial artificial chromosome (BAC)-based integrated physical map of the largest chromosome, 3B, that alone is 995 megabases. A chromosome-specific BAC library was used to assemble 82% of the chromosome into 1036 contigs that were anchored with 1443 molecular markers, providing a major resource for genetic and genomic studies. This physical map establishes a template for the remaining wheat chromosomes and demonstrates the feasibility of constructing physical maps in large, complex, polyploid genomes with a chromosome-based approach.     

A genetic linkage map with 178 SSR and 1901 SNP markers constructed using a RIL population in wheat (Triticum aestivum L.)

The construction of high density genetic linkage map provides a powerful tool to detect and map quantitative trait loci(QTLs) controlling agronomically important traits. In this study, simple sequence repeat(SSR) markers and Illumina 9K i Select single nucleotide polymorphism(SNP) genechip were employed to construct one genetic linkage map of common wheat(Triticum aestivum L.) using 191 recombinant inbred lines(RILs) derived from cross Yu 8679×Jing 411. This map included 1 901 SNP loci and 178 SSR loci, covering 1 659.9 c M and 1 000 marker bins, with an average interval distance of 1.66 c M. A, B and D genomes covered 719.1, 703.5 and 237.3 c M, with an average interval distance of 1.66, 1.45 and 2.9 c M, respectively. Notably, the genetic linkage map covered 20 chromosomes, with the exception of chromosome 5D. Bioinformatics analysis revealed that 1 754(92.27%) of 1 901 mapped SNP loci could be aligned to 1 215 distinct wheat unigenes, among which 1 184(97.4%) were located on o ne single chromosome, and the rest 31(2.6%) were located on 2 to 3 chromosomes. By performing in silico comparison, 214 chromosome deletion bin-mapped expressed sequence tags(ESTs), 1 043 Brachypodium genes and 1 033 rice genes were further added onto the genetic linkage map. This map not only integrated genetic and physical maps, SSR and SNP loci, respectively, but also provided the information of Brachypodium and rice genes corresponding to 1 754 SNP loci. Therefore, it will be a useful tool for comparative genomics analysis, fine mapping of QTL/gene controlling agronomically important traits and marker-assisted selection breeding in wheat.     

Polymorphism analysis of microsatellites and construction of linkage map in part regions of four chromosomes in chicken

Based on chicken' consensus map issued in 2000,17 microsatellites near 4 candidate genes such as IGF2,OBR,GDF8 and APOA1 in 4 chromosomes(chromosome 5,7,8 and 24)were chosen for polymorphism analysis and construction of linkage map.Combining the technique of PCR and the fluorescent semi-automated detection,genome scanning was performed for 440 chickens,which was derived from China Agricultural University chicken resource families within three generations.The individuals of this resource families were genotyped.The results showed that the number of alleles ranged from 4 to 14;heterozygosity(H) of markers was between 0.3116 and 0.9148.Polymorphic information content(PIC)varied from 0.2672 to 0.8679.Microsatellites along with above-mentioned 4 candidate genes doing as general markers were used to construct linkage map.The spans of 4 linkage maps constructed in the part region of chromosome 5,7,8 and 24 were 263.5,79.9,206.2 and 104.2 cM,respectively.The order of markers was consistent with that of counterpart of reported consensus map.However,The spans of linkage map were larger than that of consensus map.The constructed linkage maps laid the foundation for mapping quantitative trait loci(QTL)responsible for economically important traits in chicken.     

High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.     

芥菜型油菜A9染色体黄籽基因区域BAC重叠群的构建

A9染色体是芸薹属植物A基因组最长的染色体(序列约37 Mb),具有控制种子大小、种皮颜色、硫苷合成、油脂合成等重要性状的基因。利用芥菜型油菜A9染色体黄籽性状共分离标记A9-88和A9-32对已经构建的ZBjH BAC文库进行PCR步移筛选,共筛选出BAC 752个,测序395个,得到BAC末端序列674条,利用BAC末端序列设计引物533对,此外利用白菜已经公布的序列设计SSR引物38对、STS引物15对,构建了芥菜型油菜黄籽基因区域估算长约2.2 Mb的BAC重叠群。     

A genetic map and recombination parameters of the human malaria parasite Plasmodium falciparum

Genetic investigations of malaria require a genome-wide, high-resolution linkage map of Plasmodium falciparum. A genetic cross was used to construct such a map from 901 markers that fall into 14 inferred linkage groups corresponding to the 14 nuclear chromosomes. Meiotic crossover activity in the genome proved high (17 kilobases per centimorgan) and notably uniform over chromosome length. Gene conversion events and spontaneous microsatellite length changes were evident in the inheritance data. The markers, map, and recombination parameters are facilitating genome sequence assembly, localization of determinants for such traits as virulence and drug resistance, and genetic studies of parasite field populations.     

Identification of molecular markers linked to rice bacterial blight resistance genes from Oryza meyeriana

Y73 is a progeny of asymmetric somatic hybridization between Oryza sativa cv. Dalixiang and the wild rice species Oryza meyeriana. Inoculation with a range of strains of Xanthomonas oryzae pv. oryzae showed that Y73 had inherited a high level of resistance to rice bacterial blight (BB) from its wild parent. An F₂ population of 7125 individuals was constructed from the cross between Y73 and a BB-susceptible cultivar IR24. After testing 615 SSR and STS markers covering the 12 rice chromosomes, 186 markers were selected that showed polymorphism between Y73 and IR24. Molecular markers linked to the BB resistance genes in Y73 were scanned using the F₂ population and the polymorphic markers. The SSR marker RM128 on chromosome 1, the STS marker R03D159 on chromosome 3 and the STS marker R05D104 on chromosome 5 were found to be linked to the rice BB resistance genes in Y73.     

The genetic linkage map of the human X chromosome

A database useful for mapping the human X chromosome has been established. The data consist of the genotypic characterizations obtained at more than 20 DNA marker loci from a set of 38 selected families. Multilocus linkage analysis has provided an initial genetic map completely spanning the distance from the distal short arm to the distal long arm of the chromosome, for a total genetic length of at least 185 recombination units. Analysis of the recombinational behavior of fully marked chromosomes suggests that the number of recombination events on the X chromosome may be nonrandom. Linkage studies of six families that carry the mutation which causes Duchenne muscular dystrophy were combined with linkage data from a large number of normal families. This permitted mapping of the locus for Duchenne muscular dystrophy with greater precision and statistical confidence than studies in which disease families alone provided the genotypic database. This observation suggests that the normal linkage map of this chromosome should be especially valuable in the mapping of rare X-linked diseases.     

绿豆高密度分子遗传图谱的构建

【目的】在前期研究的基础上,进一步利用绿豆基因组SSR、EST-SSR、STS和普通菜豆基因组SSR等标记构建绿豆遗传连锁图谱,为绿豆重要性状相关基因的定位、克隆及分子标记辅助选育新品种等研究搭建技术平台。【方法】利用澳大利亚引进的Berken（高感豆象绿豆栽培种）× ACC41（高抗豆象绿豆野生种）及其重组自交系（recombinant inbreed line,RIL）群体,对6 686对引物进行PCR扩增及多态性筛选,包括6 100对绿豆基因组SSR、149对EST-SSR、13对STS和424对普通菜豆基因组SSR引物,将亲本间多态性引物,进一步分析重组自交系群体。结合前期研究的分子标记数据,利用Mapmarker/Exp 3.0软件构建遗传图谱,并设置LOD≥3.0,最大图距50.00 cM。用Joinmap 4.0软件进行图谱整合。【结果】用2个亲本共筛选了6 686对SSR引物,共有3 691对引物有稳定的扩增产物,得到有多态的引物有588对。其中,通过磁珠富集法开发的绿豆SSR引物6 100对,有效扩增3 459对,有效扩增率56.7%,得到多态性引物559对;通过转录组测序开发的绿豆MGCP引物149对,有效扩增126对,有效扩增率84.6%,得到多态性引物21对;通过磁珠富集法开发的菜豆SSR引物424对,有效扩增97对,有效扩增率22.9%,得到多态性引物6对;绿豆STS引物13对,有效扩增9对,有效扩增率69.2%,得到多态性引物2对。表明不同来源和种类的SSR引物在RIL群体亲本中的有效扩增率有明显差别,绿豆EST-SSR引物（84.6%）最高,绿豆STS引物（69.2%）和SSR引物（55.7%）次之,菜豆SSR引物（22.9%）最低。获得一张含有585个标记（499个SSR标记、74个RFLP标记、9个STS标记和3个RAPD标记）的绿豆遗传图谱,图谱总长732.9 cM,包括11个连锁群,每个标记间的平均距离为1.25 cM,平均长度为66.63 cM。每个连锁群长度为45.2-112.8 cM,每条染色体上面的标记数为35-92个,平均53.18个。标记位点数最多的连锁群LG1含92个标记,长度为112.8 cM;标记位点数最少的连锁群LG11仅含有35个标记,长度为48.7 cM。对图谱的585个标记位点进行χ2测验,在P＜0.05和P＜0.01条件下,分别有79个和151个标记表现为偏分离,占总标记位点数的39.3%。【结论】构建了一张目前国内外发表的标记数最多、密度最高的绿豆遗传连锁图谱。     

Assignment of the gene for myelin proteolipid protein to the X chromosome: implications for X-linked myelin disorders

Several inherited disorders in humans and in rodents result in myelin dysgenesis and a deficiency of the molecular constituents of myelin. A complementary DNA to one of the two major myelin proteins, myelin proteolipid protein (also known as lipophilin), has been used with Southern blot analysis of somatic cell hybrid DNA to map the human proteolipid protein gene to the middle of the long arm of the human X chromosome (bands Xq13-Xq22) and to assign the murine proteolipid protein gene to the mouse X chromosome. Comparison of the gene maps of the human and mouse X chromosomes suggests that myelin proteolipid protein may be involved in X-linked mutations at the mouse jimpy locus and has implications for Pelizaeus-Merzbacher disease, a human inherited X-linked myelin disorder.     

萝卜D染色体在7号连锁群的定位研究

在已知萝卜D染色体附加系对线虫具有抗性和萝卜分子图谱的7号连锁群定位有抗线虫基因Hs1Raph的基础上,利用(dp)RAPD分子标记技术,以甘蓝型油菜异源萝卜附加系为材料,筛选到90个萝卜D染色体的特异标记,选择4个在萝卜构图亲本间表现多态性的D染色体标记进行F2群体(包括245个单株)分离检测,并与构图用的505个AFLP和RAPD标记一起用Join-map3.0进行连锁定位分析。结果萝卜D染色体标记全部定位于7号连锁群,并且7号连锁群上只有D染色体的特异标记,说明萝卜7号连锁群就是D染色体在分子水平的反映。证实了定位有抗线虫基因Hs1Raph的7号连锁群对应于具线虫抗性效应的萝卜D染色体。实验结果对研究萝卜D染色体控制的农艺性状或抗逆性以及萝卜抗线虫育种和将萝卜抗线虫基因转入其它芸薹属作物的研究都将会起到重要的指导作用。     

小麦品种偃展1号与品系早穗30重组自交系群体遗传连锁图谱构建及重要农艺性状的QTL分析

【目的】构建重组自交系(recombinant inbred line,RIL)群体及其遗传连锁图谱,对小麦重要农艺性状进行数量性状位点(quantitative trait locus,QTL)分析,为发现小麦新基因与分子标记辅助育种奠定基础。【方法】配制普通小麦品种(系)早穗30和偃展1号的杂交组合,通过一粒传的方法培育重组自交系群体;利用SSR(simple sequence repeat)标记、DarT(diversity arrays technology)标记、ISBP(insertion site-basedpolymorphism)标记以及抽穗期和株高的功能标记绘制其遗传连锁图谱并通过复合区间作图法(Compositeinterval mapping,CIM)对多个环境下的抽穗期、株高、千粒重、穗粒数、每穗小穗数、穗长等农艺性状进行QTL定位分析。【结果】培育出由219个F7家系组成的重组自交系群体;构建了含481个分子标记的遗传连锁图谱;检测出分布在12条染色体上的26个与重要农艺性状相关的QTL,其中9个QTL能够在至少2个环境下重复;研究还发现了3个QTL聚集的"QTL簇",其中4D染色体上的矮秆基因Rht2所在区段控制株高与千粒重,5D染色体上的Vrn-D1-WMS212区间控制抽穗期、穗粒数与每穗小穗数,7B染色体上wPt4230-wPt4814区段控制抽穗期、穗粒数、株高与穗长。【结论】构建的小麦遗传作图群体可成功地用于重要农艺性状分析;矮秆基因Rht2与春化基因Vrn-D12个发育相关基因均与多个重要农艺性状有关;在7B上可能存在与发育相关的重要新基因。     

An Integrated Map of Soybean Physical Map and Genetic Map

Soybean is a major crop in the world, and it is a main source of plant proteins and oil. A lot of soybean genetic maps and physical maps have been constructed, but there are no integrated map between soybean physical map and genetic map. In this study, soybean genome sequence data, released by JGI （US Department of Energy＇s Joint Genome Institute）, had been downloaded. With the software Blast 2.2.16, a total of 161 super sequences were mapped on the soybean public genetic map to construct an integrated map. The length of these super sequences accounted for 73.08% of all the genome sequence. This integrated map could be used for gene cloning, gene mining, and comparative genome of legume.     

Characterization of the supernumerary chromosome in cat eye syndrome

Most individuals with cat eye syndrome (CES) have a supernumerary bisatellited chromosome which, on the basis of cytogenetic evidence, has been reported to originate from either chromosome 13 or 22. To resolve this question, a single-copy DNA probe, D22S9, was isolated and localized to 22q11 by in situ hybridization to metaphase chromosomes. The number of copies of this sequence was determined in CES patients by means of Southern blots and densitometry analysis of autoradiographs. In patients with the supernumerary chromosome, four copies were found, whereas in one patient with a duplication of part of chromosome 22, there were three copies. Therefore, the syndrome results from the presence of either three or four copies of DNA sequences from 22q11; there is no evidence that sequences from other chromosomes are involved. This work demonstrates how DNA sequence dosage analysis can be used to study genetic disorders that are not readily amenable to standard cytogenetic analysis.     

小麦2D染色体上多酚氧化酶（PPO）基因STS标记的开发与应用

 【目的】开发出小麦籽粒PPO活性的分子标记,并对新标记的应用进行研究,为面制食品外观品质的改良提供参考。【方法】根据一条由小麦2D染色体上PPO基因编码的mRNA序列（GenBank：AY515506）设计引物,对7个高PPO活性和7个低PPO活性小麦品种进行PCR扩增,筛选出有差异的引物,对130份小麦品种资源进行检测,验证不同带型与PPO活性的相关性,并利用一套中国春缺体、四体及双端体对STS标记进行染色体定位。在此基础上,结合一个位于小麦2A染色体上的PPO基因分子标记（PPO18）,对新开发的标记在小麦低PPO活性分子标记辅助选择中的作用进行评价。【结果】在AY515506的不同位置共设计了8对引物,其中一对引物（STS01）在高、低PPO活性材料中表现出多态性。该引物在7个低PPO活性的材料中能扩增出560 bp的目标片段,在7个高PPO活性的材料中没有扩增出目标片段,利用中国春缺体、四体及双端体最终将该标记定位在2D染色体长臂上。利用该标记检测130份小麦品种,结果表明有75个品种可以扩增出560 bp的目标片段,其PPO活性均值为221.08 A475/（min•g•103）;55个品种没有扩增出目标片段,PPO活性均值为309.98 A475/（min•g•103）,方差分析表明两者的差异达极显著水平（P＜0.01）。利用STS01和PPO18共同检测后发现,130份品种中,有37个品种的双标记扩增均表现出高活性带型（H1H2）,这些品种PPO活性的均值为337.82 A475/（min•g•103）,显著高于其它几种扩增带型品种的PPO活性均值（P＜0.01）。32个双标记扩增均表现为低活性带型（L1L2）的小麦品种PPO活性值普遍较低,可作为改良面制食品外观品质的候选亲本。【结论】STS01是一个位于小麦2D染色体长臂上PPO基因分子标记,可以在小麦PPO活性分子标记辅助选择中加以应用。     

Natural selection shapes genome-wide patterns of copy-number polymorphism in Drosophila melanogaster

The role that natural selection plays in governing the locations and early evolution of copy-number mutations remains largely unexplored. We used high-density full-genome tiling arrays to create a fine-scale genomic map of copy-number polymorphisms (CNPs) in Drosophila melanogaster. We inferred a total of 2658 independent CNPs, 56% of which overlap genes. These include CNPs that are likely to be under positive selection, most notably high-frequency duplications encompassing toxin-response genes. The locations and frequencies of CNPs are strongly shaped by purifying selection, with deletions under stronger purifying selection than duplications. Among duplications, those overlapping exons or introns, as well as those falling on the X chromosome, seem to be subject to stronger purifying selection.     

Development of EST-PCR markers specific to the long arm of chromosome 6V of Dasypyrum villosum

Expressed sequence tags-derived polymerase chain reaction (EST-PCR) molecular markers specific for alien chromosomes can be used to not only monitor the introgressed alien chromatin in wheat background, but also provide the evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers specific for chromosome 6VL of Dasypyrum villosum, 297 primer pairs were designed based on the expressed sequence tags (EST) sequences, which were previously mapped in different bins of the long arms of wheat homoeologous 6AL, 6BL, and 6DL. By using the Triticum aestivum, D. villosum, T. durum-D. villosum amphiploid, and T. aestivum-D. villosum alien chromosome lines involving chromosome 6V, it was found that 32 (10.77%) primers could amplify specific bands for chromosome 6V, and 31 could be allocated to chromosome arm 6VL. These 6VL specific markers provided efficient tools for the characterization of structural variation involving the chromosome 6VL in common wheat background as well as for the selection of useful genes located on 6VL in breeding programs.     

染色体研究的新方法与新进展

从染色体显微分离与显微克隆、染色体连锁图谱、染色体原位杂交、染色体涂染、染色体的基因定位以及染色体的片段转移等几个方面,分别综述了染色体研究的新方法和新进展,并展示了分子细胞遗传学的发展前景。