水稻基因组测序及基因功能的鉴定

水稻是重要的粮食作物。作为单子叶模式植物,水稻基因组的大规模测序具有巨大的理论价值和现实意义。目前已获得了籼稻“93—11”和粳稻“日本晴”高质量的基因组数据,这为在基因组水平上深入研究其生长、发育、抗病和高产等的遗传机理提供了便利,从而为进一步解决世界粮食危机提供了新的突破口和契机。随着水稻基因组计划的顺利结束,其研究重心也已由建立高分辨率的遗传、物理和转录图谱为主的结构基因组学转向基因功能的研究。结构基因组学研究获得的大量序列数据为揭示和开发功能基因开辟了广阔的前景。目前,利用图位克隆和电子克隆等方法已成功分离了多个水稻抗病、抗虫、抗逆境、抗倒伏、高产、优质等重要农艺性状相关的基因,对培育水稻新品种,促进农业的可持续发展意义重大。据估计,水稻至少拥有3．7万个非转座因子相关的蛋白编码基因。因此,完成全基因组序列测定后,重要基因功能的鉴定已成为当前基因组学研究的主要目标。反向遗传学、大规模基因功能表达谱分析和蛋白质组研究等策略已在研究水稻重要基因的功能方面发挥了重要作用。文章综述了水稻基因组测序及基因功能研究的现状,并就新基因发掘和基因功能注释的方法作了评述,期待为水稻遗传工程和育种实践提供参考。     

菌落PCR在大规模基因组测序中的应用

一种利用菌落直接PCR扩增DNA并用于测序的实验方法.通过对引物的设计和菌液浓度控制,使PCR反应后的内容物对测序干扰减到最小.与传统的测序过程比较,它省去了抽提模板DNA一步,节省了大量时间和实验成本.另外此方法可对BAC亚克隆库构建时由连接转化过程中导致的假阳性起筛选和鉴定作用.采用该法成功测定了籼稻（Oryza sativa indica）广陆矮4号的L3173号BAC DNA全长序列（约100 kb）,GenBank登录号：AL512542.     

微生物全基因组鸟枪法测序

全基因组测序主要有二种策略,一种是分级鸟枪法测序,另一种是全基因组鸟枪法测序。微生物是一种十分重要的遗传资源,运用全基因组鸟枪法可以方便、快捷地完成其基因组的测序任务。本文对微生物全基因组鸟枪法测序中文库构建、插入片段的长短比例、反应投入量、拼接以及补洞等问题作了较细致的描述,有些步骤作了举例说明。 Abstract:Two strategies introduced for whole genome sequencing,one is clone by clone method,the other is whole genome shotgun sequencing,for microbes which are very important to us,whole genome shotgun sequencing method is very convenient.In this article we discussed the library construction、long-to-short-ratio of insert,、total number of reads should be sequenced、assembly and gap filling technologies of the whole microbial genome shotgun sequencing method while some examples presented.     

Rice Proteome Database: A Step toward Functional Analysis of the Rice Genome

The technique of proteome analysis using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this study, the proteins of rice were cataloged, a rice proteome database was constructed, and a functional characterization of some of the identified proteins was undertaken. Proteins extracted from various tissues and subcellular compartments in rice were separated by 2D-PAGE and an image analyzer was used to construct a display of the proteins. The Rice Proteome Database contains 23 reference maps based on 2D-PAGE of proteins from various rice tissues and subcellular compartments. These reference maps comprise 13129 identified proteins, and the amino acid sequences of 5092 proteins are entered in the database. Major proteins involved in growth or stress responses were identified using the proteome approach. Some of these proteins, including a β-tubulin, calreticulin, and ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have unexpected functions. The information obtained from the Rice Proteome Database will aid in cloning the genes for and predicting the function of unknown proteins.     

Alternative Approaches to Genome Mapping and Sequencing

The main methods used for large-scale mapping of the human and other genomes are reviewed. These methods comprise two procedures of random mapping/sequencing and an approach using linking and jumping libraries. Importantly, no method used up to now has proved efficient in comparative genome analysis. A new method is presented basing on slalom libraries. These libraries provide 10–100 times higher efficiency and may be used for mapping and sequencing whole genomes by small research groups.     

稻属植物的基因组进化

水稻所在的稻属(Oryza)共有24个左右的物种。由于野生稻含有大量的优良农艺性状基因, 在水稻遗传学研究中日益受到重视。随着国际稻属基因组计划的开展, 越来越多的稻属基因组序列被测定, 稻属成为进行比较、功能和进化基因组学研究的模式系统。近期开展的一系列研究对稻属不同基因组区段以及全基因组序列的比较分析, 揭示了稻属在基因组大小、基因移动、多倍体进化、常染色质到异染色质的转化以及着丝粒区域的进化等方面的分子机制。转座子的活性以及转座子因非均等重组或非法重组而造成的删除, 对稻属基因组的扩增和收缩具有重要作用。DNA双链断裂修复介导的基因移动, 特别是非同源末端连接, 是稻属基因组非共线性基因形成的主要来源。稻属基因组从常染色质到异染色质的转换过程, 伴随着转座子的大量扩增、基因片段的区段性和串联重复以及从基因组其他位置不断捕获异染色质基因。对稻属不同物种间基因拷贝数、特异基因和重要农艺性状基因的进化等研究, 可揭示稻属不同物种间表型和适应性差异的分子基础, 将加速水稻的育种和改良。     

鸡基因组研究新进展

鸡基因组测序草图的完成标志着禽类功能基因组时代的到来。鸡不仅是全世界广泛饲养且有重要经济价值的禽类,而且是极具生命科学研究价值的模式动物。因此,鸡基因组测序草图的完成将对遗传育种和生物学研究有重要的影响。本文综述了近年来鸡基因组研究的最新进展,主要内容包括鸡基因组的有关数据、物理图谱、遗传连锁图谱、比较基因组学、序列表达标签、生物信息学等方面所取得的成绩,同时对鸡基因组研究结果的应用前景进行了展望。     

Draft De Novo Genome Sequence of Agapornis roseicollis for Application in Avian Breeding

In aviculture, lovebirds are considered one of the most popular birds to keep. This African parakeet is known for its range of plumage colors and ease to tame. Plumage variation is the most important price-determining trait of these birds, and also the main selection criterion for breeders. Currently, no genetic screening tests for traits of economic importance or to confirm pedigree data are available for any of the nine lovebird species. As a starting point to develop these tests, the de novo genome of Agapornis roseicollis (rosy-faced lovebird) was sequenced, assembled, and annotated. Sequencing was done on the Illumina HiSeq 2000 platform and the assembly was performed using SOAPdenovo v2.04. The genome was found to be 1.1?Gb in size and 16,044 genes were identified and annotated. This compared well with other previously sequenced avian genomes, such as the chicken, zebra finch, and budgerigar. To assess genome completeness, the number of benchmarking universal single-copy orthologs were identified in the genome. This was compared to other previously assembled avian genomes and the results indicated that the genome will be useful in the development of genetic screening tests to aid lovebird breeders in selecting breeding pairs.     

Rice functional genomics research in China

Rice functional genomics is a scientific approach that seeks to identify and define the function of rice genes, and uncover when and how genes work together to produce phenotypic traits. Rapid progress in rice genome sequencing has facilitated research in rice functional genomics in China. The Ministry of Science and Technology of China has funded two major rice functional genomics research programmes for building up the infrastructures of the functional genomics study such as developing rice functional genomics tools and resources. The programmes were also aimed at cloning and functional analyses of a number of genes controlling important agronomic traits from rice. National and international collaborations on rice functional genomics study are accelerating rice gene discovery and application.     

Draft Genome Sequencing and Comparative Analysis of Aspergillus sojae NBRC4239

We conducted genome sequencing of the filamentous fungus Aspergillus sojae NBRC4239 isolated from the koji used to prepare Japanese soy sauce. We used the 454 pyrosequencing technology and investigated the genome with respect to enzymes and secondary metabolites in comparison with other Aspergilli sequenced. Assembly of 454 reads generated a non-redundant sequence of 39.5-Mb possessing 13 033 putative genes and 65 scaffolds composed of 557 contigs. Of the 2847 open reading frames with Pfam domain scores of >150 found in A. sojae NBRC4239, 81.7% had a high degree of similarity with the genes of A. oryzae. Comparative analysis identified serine carboxypeptidase and aspartic protease genes unique to A. sojae NBRC4239. While A. oryzae possessed three copies of α-amyalse gene, A. sojae NBRC4239 possessed only a single copy. Comparison of 56 gene clusters for secondary metabolites between A. sojae NBRC4239 and A. oryzae revealed that 24 clusters were conserved, whereas 32 clusters differed between them that included a deletion of 18 508 bp containing mfs1, mao1, dmaT, and pks-nrps for the cyclopiazonic acid (CPA) biosynthesis, explaining the no productivity of CPA in A. sojae. The A. sojae NBRC4239 genome data will be useful to characterize functional features of the koji moulds used in Japanese industries.     

Comprehensive Draft Genome Analyses of Three Rockfishes (Scorpaeniformes,Sebastiscus) via Genome Survey Sequencing

Sebastiscus species, marine rockfishes, are of essential economic value. However, the genomic data of this genus is lacking and incomplete. Here, whole genome sequencing of all species of Sebastiscus was conducted to provide fundamental genomic information. The genome sizes were estimated to be 802.49 Mb (S. albofasciatus), 786.79 Mb (S. tertius), and 776.00 Mb (S. marmoratus) by using k-mer analyses. The draft genome sequences were initially assembled, and genome-wide microsatellite motifs were identified. The heterozygosity, repeat ratios, and numbers of microsatellite motifs all suggested possibly that S. tertius is more closely related to S. albofasciatus than S. marmoratus at the genetic level. Moreover, the complete mitochondrial genome sequences were assembled from the whole genome data and the phylogenetic analyses genetically supported the validation of Sebastiscus species. This study provides an important genome resource for further studies of Sebastiscus species.     

Separation and Identification of Rice Prolamins by Two-Dimensional Gel Electrophoresis and Amino Acid Sequencing

There are difficulties in detecting and separating rice prolamin polypeptides by 2D-PAGE analysis because prolamin polypeptides are insoluble, and the amino acid sequences show high homology among them. In this study, we improved the prolamin extraction method and the 2D-PAGE procedure, and succeeded in separating prolamin polypeptide species by 2D-PAGE and in identifying major prolamin polypeptide sequences.     

Sequencing Strategies for Fusion Gene Detection

Fusion genes formed by chromosomal rearrangements are common drivers of cancer. Recent innovations in the field of next-generation sequencing (NGS) have seen a dynamic shift from traditional fusion detection approaches, such as visual characterization by fluorescence, to more precise multiplexed methods. There are many different NGS-based approaches to fusion gene detection and deciding on the most appropriate method can be difficult. Beyond the experimental approach, consideration needs to be given to factors such as the ease of implementation, processing time, associated costs, and the level of expertise required for data analysis. Here, the different NGS-based methods for fusion gene detection, the basic principles underlying the techniques, and the benefits and limitations of each approach are reviewed. This article concludes with a discussion of how NGS will impact fusion gene detection in a clinical context and from where the next innovations are evolving.     

Structural and functional analysis of rice genome

Rice is an excellent system for plant genomics as it represents a modest size genome of 430 Mb. It feeds more than half the population of the world. Draft sequences of the rice genome, derived by whole-genome shotgun approach at relatively low coverage (4-6 X), were published and the International Rice Genome Sequencing Project (IRGSP) declared high quality (> 10 X), genetically anchored, phase 2 level sequence in 2002. In addition, phase 3 level finished sequence of chromosomes 1, 4 and 10 (out of 12 chromosomes of rice) has already been reported by scientists from IRGSP consortium. Various estimates of genes in rice place the number at >50,000. Already, over 28,000 full-length cDNAs have been sequenced, most of which map to genetically anchored genome sequence. Such information is very useful in revealing novel features of macroand micro-level synteny of rice genome with other cereals. Microarray analysis is unraveling the identity of rice genes expressing in temporal and spatial manner and should help target candidate genes useful for improving traits of agronomic importance. Simultaneously, functional analysis of rice genome has been initiated by marker-based characterization of useful genes and employing functional knock-outs created by mutation or gene tagging. Integration of this enormous information is expected to catalyze tremendous activity on basic and applied aspects of rice genomics.     

基因组时代的昆虫学研究

昆虫种类繁多,它与生态系统中的生物多样性,以及人类的日常生活和生产密切相关。自2000年黑腹果蝇Drosophila melanogaster全基因组测序完成以来,至今已先后开展了88种昆虫全基因组测序工作,这标志着昆虫学研究进入了基因组时代。本文综述了近年来昆虫基因组测序进展,以及基于基因组的昆虫学研究方法及应用等两方面的研究成果。同时,着重介绍了昆虫全基因组测序进程,昆虫基因组在个体生物学、多物种间及种群,及系统生物学研究中的应用等方面的内容。最后,还探讨了基因组时代昆虫学研究所面临的挑战。     

Selective Whole Genome Amplification for Resequencing Target Microbial Species from Complex Natural Samples

Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The selective whole genome amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA, which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratory-prepared mixtures of cultured microbes as well as a natural host–microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in >10⁵-fold amplification of the target genomes with <6.7-fold amplification of the background. SWGA-treated genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput resequencing reads mapping to the W. pipientis genome. By contrast, 2–9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs.     

Sequencing and annotated analysis of an Estonian human genome

In present study we describe the sequencing and annotated analysis of the individual genome of Estonian. Using SOLID technology we generated 2,449,441,916 of 50-bp reads. The Bioscope version 1.3 was used for mapping and pairing of reads to the NCBI human genome reference (build 36, hg18). Bioscope enables also the annotation of the results of variant (tertiary) analysis. The average mapping of reads was 75.5% with total coverage of 107.72 Gb. resulting in mean fold coverage of 34.6. We found 3,482,975 SNPs out of which 352,492 were novel. 21,222 SNPs were in coding region: 10,649 were synonymous SNPs, 10,360 were nonsynonymous missense SNPs, 155 were nonsynonymous nonsense SNPs and 58 were nonsynonymous frameshifts. We identified 219 CNVs with total base pair coverage of 37,326,300 bp and 87,451 large insertion/deletion polymorphisms covering 10,152,256 bp of the genome. In addition, we found 285,864 small size insertion/deletion polymorphisms out of which 133,969 were novel. Finally, we identified 53 inversions, 19 overlapped genes and 2 overlapped exons. Interestingly, we found the region in chromosome 6 to be enriched with the coding SNPs and CNVs. This study confirms previous findings, that our genomes are more complex and variable as thought before. Therefore, sequencing of the personal genomes followed by annotation would improve the analysis of heritability of phenotypes and our understandings on the functions of genome.     

水稻突变体与功能基因组学

突变体是功能基因组学研究的重要材料。本文综述了水稻突变体的创制方法、变异的机制、水稻突变体,基因分类和数量、克隆的水稻重要基因的研究进展,包括1698个水稻突变体,基因的分类和43个克隆的水稻突变体基因,并提出了利用水稻突变体的展望。