A physical map of the Escherichia coli K12 genome

A physical map of a genome is the structure of its DNA. Construction of such a map is a first step in the complete characterization of that DNA. The restriction endonuclease Not I cuts the genome of Escherichia coli K12 into 22 DNA fragments ranging from 20 kilobases (20,000 base pairs) to 1000 kilobases. These can be separated by pulsed field gel electrophoresis. The order of the fragments in the genome was determined from available E. coli genetic information and analysis of partial digest patterns. The resulting ordered set of fragments is a macrorestriction map. This map facilitates genetic and molecular studies on E. coli, and its construction serves as a model for further endeavors on larger genomes.     

Two new SINE insertion polymorphisms in pig Vertnin(VRTN) gene revealed by comparative genomic alignment

Despite one SINE retrotransposon insertion polymorphism(sRTIP) in the vertebrae development-associated(VRTN) gene was identified in pigs, the structural variations(SVs) in VRTN gene and its proximal flank regions were largely unknown. VRTN genic and flanking sequences from 14 breeds were assembled or downloaded from whole-genome shotgun contings(WGS) database, and aligned to identify the SVs with Clustalx, and retrotransposons in VRTN gene were annotated by RepeatMasker, the splicing patterns of VRTN gene were predicted by Genescan, and large SVs were evaluated by PCR. A total of 12 small SVs and three large SVs in intron of VRTN, derived from SINE insertion polymorphisms, were identified, and two of them(VRTN-sRTIP2 and VRTN-sRTIP3) were not reported before. These VRTN-sRTIPs may affect the splicing patterns of VRTN. They displayed polymorphisms in most detected eight breeds. VRTN-sRTIP2 and VRTN-sRTIP3 showed Hardy-Weinberg equilibrium distributions in most populations except the Chinese local Erhualian pigs, while VRTN-sRTIP1 showed genetic equilibrium in Erhualian pigs. Three VRTN-sRTIPs were identified, and displayed polymorphisms in pigs, and two of them were not reported before. These SVs provide a useful molecular markers for genetic analysis in pigs, and offer new information to facilitate the understanding the SVs of VRTN gene and their putative roles in the variation of vertebral number.     

The genome sequence of Drosophila melanogaster

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.     

QTL analysis for plant height and fine mapping of two environmentally stable QTLs with major effects in soybean

Plant height is an important agronomic trait, which is governed by multiple genes with major or minor effects. Of numerous QTLs for plant height reported in soybean, most are in large genomic regions, which results in a still unknown molecular mechanism for plant height. Increasing the density of molecular markers in genetic maps will significantly improve the efficiency and accuracy of QTL mapping. This study constructed a high-density genetic map using 4 011 recombination bin markers developed from whole genome re-sequencing of 241 recombinant inbred lines (RILs) and their bi-parents, Zhonghuang 13 (ZH) and Zhongpin 03-5373 (ZP). The total genetic distance of this bin map was 3 139.15 cM, with an average interval of 0.78 cM between adjacent bin markers. Comparative genomic analysis indicated that this genetic map showed a high collinearity with the soybean reference genome. Based on this bin map, nine QTLs for plant height were detected across six environments, including three novel loci (qPH-b_11, qPH-b_17 and qPH-b_18). Of them, two environmentally stable QTLs qPH-b_13 and qPH-b_19-1 played a major role in plant height, which explained 10.56–32.7% of the phenotypic variance. They were fine-mapped to 440.12 and 237.06 kb region, covering 54 and 28 annotated genes, respectively. Via the function of homologous genes in Arabidopsis and expression analysis, two genes of them were preferentially predicted as candidate genes for further study.     

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.     

严重急性呼吸道综合征(SARS)病毒基因组比较分析与进化研究

“严重急性呼吸道综合征”(SARS)由于危害严重，引起人们的高度重视，在各国科研人员努力下，目前已完成了十几个毒株的分离与全基因组测序工作。本研究对世界各地分离的SARS毒株的基因组全序列进行比较，发现不同毒株的同源性在99．8％以上，SARS病毒的遗传稳定性较高；将SARS病毒与其它冠状病毒基因组以及编码蛋白质序列进行比较分析，证实SARS病毒是一种新的冠状病毒，可能进化起源较早，并发现SARS病毒和牛冠状病毒同源程度最高。SARS病毒orflab、orfla、S、M、N编码区氨基酸序列和牛冠状病毒同源程度最高，鼠肝炎病毒次之。     

西瓜遗传图谱构建及果实相关性状QTL分析

【目的】利用CAPS及SSR标记构建西瓜遗传图谱,对西瓜果实相关性状进行QTL分析,为西瓜果实性状改良、主效基因精细定位及克隆奠定基础。【方法】授粉后40 d对母本PI186490、父本LSW-177以及两者杂交获得的F2群体的果实进行采摘,对每个果实的果形指数、中心和边缘可溶性固形物、中心和边缘果肉硬度、果皮硬度、种子长度、种子宽度、种子厚度以及种子百粒重进行调查,将所得数据用软件SPSS19进行统计分析。通过Illumina HiSeq 2000高通量测序平台对两亲本材料进行基因组重测序,每样品产出10 G数据量,覆盖西瓜基因组20×以上,所得数据以已经发布的基因组数据为参考基因组,用bwa软件进行基因组组装,组装后利用Samtools软件进行SNP发掘,利用perl语言自编脚本提取SNP位点前后1 000 bp的序列,将SNP及其侧翼序列输入软件SNP2CAPS以转化为CAPS标记。在每条染色体上平均选取20个CAPS酶切位点,利用Primer Premier 5软件在突变位点上下游100-500 bp左右设计CAPS引物,进行PCR扩增和酶切检验,酶切产物用1%琼脂糖凝胶电泳检测。SSR引物来源于前人发表文献,PCR扩增产物用聚丙烯酰胺凝胶电泳检测。对所有分子数据进行卡方检验,在其中选择符合1﹕2﹕1比例的标记用于构建遗传连锁图谱。利用Mapmaker/Exp version 3.0软件构建遗传连锁图谱,用Group命令对标记进行连锁分组,标记数目少于8的连锁群用Compare命令进行排序优化,标记数多于8的连锁群用Try命令排序。绘制遗传图谱使用Map Chart 2.1软件。QTL分析运用QTL Network 2.0软件,利用置换测验做1 000次重复,临界阈值为P=0.005,采用复合区间作图法,在每条染色体上以1.0 cM步行速度在全基因组范围内扫描,分析QTL加性效应和上位效应。【结果】本遗传连锁图谱共包含16个连锁群,涉及CAPS标记87个,SSR标记9个,覆盖基因组1 484.3 cM,平均图距15.46 cM。利用QTL Network 2.0分析,检测到6个西瓜果实相关性状的8个QTL位点和1对上位效应位点,其中包括果形指数QFSI 1、中心可溶性固形物QCBR、中心果肉硬度QCFF、边缘果肉硬度QEFF、种子长度QSL各1个,种子宽度QSWD 1、QSWD 2、QSWD 3 3个;上位效应位点包括果形指数FSI 2、FSI 3。表型贡献率大于等于10%的QTL有6个,可解释11.7%-18.8%的遗传变异。【结论】以CAPS标记为主要标记构建西瓜遗传图谱,并且定位了控制西瓜果实相关性状的8个加性QTL与1对上位性QTL,可用于进一步精细定位与克隆西瓜果实优良性状基因。     

The shaping of modern human immune systems by multiregional admixture with archaic humans

Whole genome comparisons identified introgression from archaic to modern humans. Our analysis of highly polymorphic human leukocyte antigen (HLA) class I, vital immune system components subject to strong balancing selection, shows how modern humans acquired the HLA-B*73 allele in west Asia through admixture with archaic humans called Denisovans, a likely sister group to the Neandertals. Virtual genotyping of Denisovan and Neandertal genomes identified archaic HLA haplotypes carrying functionally distinctive alleles that have introgressed into modern Eurasian and Oceanian populations. These alleles, of which several encode unique or strong ligands for natural killer cell receptors, now represent more than half the HLA alleles of modern Eurasians and also appear to have been later introduced into Africans. Thus, adaptive introgression of archaic alleles has significantly shaped modern human immune systems.     

基于简化基因组测序的越橘杂交后代鉴定

【目的】针对遗传群体测序数据开发一种杂交后代鉴定方法,以获得继承双亲基因的真杂种后代,为果树杂交育种、遗传分析及遗传图谱构建奠定基础。【方法】本研究以越橘正反交群体共计318个F₁子代和2个亲本为试材,利用SLAF技术进行简化基因组测序并比对越橘参考基因组获得群体SNP数据,通过稀有等位变异分析和基于PCA、K-means聚类的遗传关系分析鉴定供试群体中的非杂交后代,结果利用双亲纯合显性SNP标记进行验证。【结果】SLAF简化测序共获得65.89 Gb数据,GC含量39.72%,平均Q30为95.04%,亲本和子代平均测序深度为12.86×和5.41×。参照四倍体越橘基因组信息,正、反交组合分别获得73 543个和114 851个SNP,利用次等位基因频率（MAF）>0.05的SNP数据集分别对正、反交群体进行PCA和K-means聚类分析,鉴定出4个离群个体;利用MAF<0.05的SNP数据集对正、反交群体进行个体稀有等位变异和个体特有的稀有等位变异数统计,共鉴定出10个离群个体（包含了MAF>0.05的SNP数据集鉴定的4个离群个体）。通过双亲纯合显性SNP标记进行验证,正、反交群体双亲纯合SNP位点分别占群体总SNP的34.56%和38.95%,除H194-123个体外,其余非杂交后代在验证结果中同为离群个体,即准确通过验证。【结论】对于有参考基因组物种的杂交群体,利用基于测序的SNP次等位基因频率（MAF）数据集,采用遗传关系和个体特有的稀有等位变异分析方法,从不同角度反映群体子代间的遗传关系以鉴别离群个体,是一种鉴定群体假杂交后代的有效方法。     

Evidence for DNA loss as a determinant of genome size

Eukaryotic genome sizes range over five orders of magnitude. This variation cannot be explained by differences in organismic complexity (the C value paradox). To test the hypothesis that some variation in genome size can be attributed to differences in the patterns of insertion and deletion (indel) mutations among organisms, this study examines the indel spectrum in Laupala crickets, which have a genome size 11 times larger than that of Drosophila. Consistent with the hypothesis, DNA loss is more than 40 times slower in Laupala than in Drosophila.     

基于SLAF-seq技术的甘薯SNP位点开发

【目的】单核苷酸多态性（SNP）是基因组中最普遍的遗传变异,是构建遗传图谱、完成分子标记辅助育种的一种非常重要的遗传标记。新一代高通量测序平台为SNP位点的检测提供了强有力的技术支持。多倍体作物通常表现为基因组序列大且重复比例高,一直以来多倍体作物的SNP位点挖掘面临巨大的挑战。【方法】共收集300份甘薯种质资源,利用SLAF-seq测序技术进行测序。首先以马铃薯基因组为参照,通过生物信息学分析进行实验方案的系统设计,筛选特异长度的DNA片断,构建SLAF-seq文库。后通过高通量测序的方式获得海量序列,进而通过软件分析比对,获得多态性SLAF标签,最后在多态性SLAF标签上开发大量特异性SNP位点。【结果】对照拟南芥的测序数据与其参考基因组进行比对的结果表明,双端比对效率在本试验中为87.71%,酶切效率为93.22%,说明本试验SLAF建库正常。通过测序共产生498.14 Mb的读长数据,测序后各样品所获得的读长数目在441 595-2 731 920范围内,其中,冀薯4号获得的数据量最大,共计获得2 731 920个读长,对照拟南芥数据量最小,共计获得441 595个读长。测序质量值Q30的范围在88.37%-90.67%,样品3043 Q30测序值仅为87.31%,样品鄂紫1号Q30测序值为91.31%,所有样品Q30值均在80%以上。测序获得GC含量的范围在37.23%-38.09%,样品苏薯9号和浙薯2号存在极端值,样品苏薯9号的GC含量在39.80%,样品浙薯2号GC含量在37.10%,所有样品GC含量均值为37.64%,GC含量普遍不高,说明达到测序要求。本研究共计获得597 094 个SLAF标签,样品的平均测序深度为11.77×,其中多态性SLAF标签260 000个,占SLAF标签总数的43.54%。根据所获得的260 000个多态性SLAF标签来统计SNP位点信息,共计获得795 794个群体SNP位点。【结论】共获得498.14 Mb的读长数据,597 094个高质量的SLAF标签,260 000个多态性SLAF标签,从260 000个多态性SLAF标签上共计开发获得795 794个高质量SNP位点。表明SLAF-seq技术可以很好地应用于甘薯SNP位点的开发,其效率远远高于SSR、AFLP、RAPD等分子标记技术。从全基因组范围获得的SNP位点可以进一步的用来完成后续群体进化分析和特异性SNP标记的开发。     

Integrating the physical and genetic map of bread wheat facilitates the detection of chromosomal rearrangements

The bread wheat genome harbors a high content of repetitive DNA, which is amenable to detection and characterization using fluorescence in situ hybridization(FISH) karyotyping. An integrated genetic map was derived from a recombinant inbred population bred from a cross between a synthetic hexaploid wheat and a commercial Chinese bread wheat cultivar, based on 28 variable FISH sites and 150 000 single nucleotide polymorphism(SNP) loci. The majority(20/28) of the variable FISH sites were physically located within a chromosomal region consistent with the genetic location inferred from that of their co-segregating SNP loci. The eight exceptions reflected the presence of either a translocation(1 R/1 B, 1 A/7 A) or a presumptive intra-chromosomal inversion(4 A). For eight out of the nine FISH sites detected on the Chinese Spring(CS) karyotype, there was a good match with the reference genome sequence, indicating that the most recent assembly has dealt well with the problem of placing tandem repeats. The integrated genetic map produced for wheat is informative as to the location of blocks of tandemly repeated DNA and can aid in improving the quality of the genome sequence assembly in regions surrounding these blocks.     

Genome-wide insertional mutagenesis of Arabidopsis thaliana

Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.     

A fine-scale map of recombination rates and hotspots across the human genome

Genetic maps, which document the way in which recombination rates vary over a genome, are an essential tool for many genetic analyses. We present a high-resolution genetic map of the human genome, based on statistical analyses of genetic variation data, and identify more than 25,000 recombination hotspots, together with motifs and sequence contexts that play a role in hotspot activity. Differences between the behavior of recombination rates over large (megabase) and small (kilobase) scales lead us to suggest a two-stage model for recombination in which hotspots are stochastic features, within a framework in which large-scale rates are constrained.     

Permuted tRNA genes expressed via a circular RNA intermediate in Cyanidioschyzon merolae

A computational analysis of the nuclear genome of a red alga, Cyanidioschyzon merolae, identified 11 transfer RNA (tRNA) genes in which the 3' half of the tRNA lies upstream of the 5' half in the genome. We verified that these genes are expressed and produce mature tRNAs that are aminoacylated. Analysis of tRNA-processing intermediates for these genes indicates an unusual processing pathway in which the termini of the tRNA precursor are ligated, resulting in formation of a characteristic circular RNA intermediate that is then processed at the acceptor stem to generate the correct termini.     

马铃薯品种和组织对基因组测序深度分布模式的影响

【目的】探讨品种和组织对马铃薯基因组测序深度分布的影响.【方法】对‘布尔班克’和‘云薯107’2个品种的薯块芽端和茎端进行了深测序,建立了在参照基因组上的测序深度分布图.【结果】发现这些分布在品种间有明显差别(例如在三号染色体中部和尾部),在同一品种的DNA样本间很类似,但芽端和茎端在二号染色体有明显差别.这些结果说明测序深度的分布主要受品种基因组本身所影响,也受薯块上部位影响,而且在很大程度上可以重复.【结论】这些结果能帮助设计试验和合适利用测序深度.     

基于CAPS标记的西瓜果实与种子相关性状QTL分析

【目的】基于西瓜全基因组重测序数据,挖掘SNP位点并将其转变为CAPS标记,构建遗传连锁图谱,对西瓜果实与种子相关性状进行QTL分析,为西瓜果实与种子相关性状主效基因精细定位及克隆奠定理论基础。【方法】以普通栽培西瓜品系(Citrullus lanatus ssp.vulgaris)‘W1-1’和黏籽西瓜品系(Citrullus lanatus ssp.mucosospermus)‘PI186490’为亲本杂交获得F_1代,以‘W1-1’为轮回亲本,构建BC_1P_1群体。采摘成熟果实,对每个西瓜果实中心及边缘可溶性固形物、中心及边缘果肉硬度、种子百粒重、种皮底色进行调查及数据分析。对亲本材料进行覆盖度约为20×的全基因组重测序,用BWA、SAMTOOLS及VCFTOOLS等软件比对并检测亲本材料在全基因组范围内的SNP位点。运用SNP2CAPS软件选择7种在西瓜基因组上具有丰富酶切位点的限制性内切酶,对包含SNP位点的序列进行酶切位点分析,转化CAPS标记。选取全基因组范围内均匀分布的450个CAPS分子标记,筛选多态性CAPS标记,对BC_1P_1群体内225株分别进行基因分型,使用QTL Ici Mapping及Windows QTL Cartographer V2.5等软件进行遗传图谱的构建和西瓜果实与种子相关性状的QTL分析。【结果】在两亲本间共获得SNP位点751 532个,根据7种限制性内切酶位点信息共开发了450个CAPS分子标记,筛选出其中具有多态性的200个CAPS标记,在225株BC1P1群体构建一张包含11个连锁群(分别对应11条染色体)的遗传连锁图谱,覆盖长度1 376.95 c M,标记间的平均遗传距离为6.88 c M。QTL分析定位到与果实与种子相关性状QTL位点15个,其中包括中心可溶性固形物含量相关位点3个(CTSS2.1、CTSS2.2、CTSS8.1);边缘可溶性固形物含量相关位点1个(ETSS2.1);中心果实硬度相关位点3个(CFF6.1、CFF6.2、CFF8.1);边缘果实硬度相关位点2个(EFF6.1、EFF6.2);种皮底色相关位点4个(SCC8.1、SCC8.2、SCC8.3、SCC8.4);种子百粒重相关位点2个(SHW6.1、SHW9.1)。15个QTL位点的贡献率范围为5.25%—74.59%,贡献率大于15%的位点有5个(CTSS8.1、SCC8.1、SCC8.2、SCC8.3、SCC8.4)。【结论】共开发出SNP位点751 532个,QTL分析检测到西瓜果实与种子相关性状QTL位点15个,其中主效QTL位点5个,分别为果实中心可溶性固形物及种皮底色相关位点(CTSS8.1、SCC8.1、SCC8.2、SCC8.3、SCC8.4),为进一步精细定位及克隆西瓜果实与种子优良性状基因奠定了基础。     

Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.