Development of genome-wide insertion/deletion markers in rice based on graphic pipeline platform

DNA markers play important roles in plant breeding and genetics.The Insertion/Deletion(InDel) marker is one kind of co-dominant DNA markers widely used due to its low cost and high precision.However,the canonical way of searching for InDel markers is time-consuming and laborintensive.We developed an end-to-end computational solution(InDel Markers Development Platform,IMDP) to identify genome-wide InDel markers under a graphic pipeline environment.IMDP constitutes assembled genome sequences alignment pipeline(AGA-pipe) and next-generation resequencing data mapping pipeline(NGS-pipe).With AGA-pipe we are able to identify 12,944 markers between the genome of rice cultivars Nipponbare and 93-11.Using NGS-pipe,we reported 34,794 InDels from re-sequencing data of rice cultivars Wu-Yun-Geng7 and Guang-Lu-Ai4.Combining AGApipe and NGS-pipe,we developed 205,659 InDels in eight japonica and nine indica cultivars and 2,681 InDels showed a subgroup-specific pattern.Polymerase chain reaction(PCR)analysis of subgroup-specific markers indicated that the precision reached 90%(86 of 95).Finally,to make them available to the public,we have integrated the InDels/markers information into a website(Rice InDel Marker Database,RIMD,http://202.120.45.71/).The application of IMDP in rice will facilitate efficiency for development of genome-wide InDel markers,in addition it can be used in other species with reference genome sequences and NGS data.     

胡杨无性系幼苗响应盐胁迫的miRNA表达差异研究

植物miRNA在调控基因表达、细胞周期、生物体发育、抗逆等方面起重要作用。为研究胡杨(Populus euphratica Oliv.)的耐盐机制,以1年生胡杨无性系幼苗为材料,构建具有空间代表性的盐胁迫胡杨cDNA文库,利用二代测序技术测定NaCl胁迫下和正常培养条件下胡杨叶和根miRNA表达情况。结果表明,不同的miRNA之间表达量存在明显差异,表达丰度最高的miRNA有miR156、miR157、miR165、miR166和miR167等,合计占总表达量的90%以上。胡杨根部存在特异表达的miRNA,在整个耐盐调控机制中发挥着生理调节、分子调控和信号传导等极为重要的作用。盐处理样品中发现大量响应盐胁迫的miRNA,对这些转录因子进行靶基因预测和注释后,发现很多盐胁迫响应的miRNA与NAC和SPL等重要转录因子家族相关,与前人的结论一致,另外还发现许多miRNA的调控对象是ATP酶和激素响应因子。     

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.     

Using phylogenomics to resolve mega-families: An example from Compositae

Next-generation sequencing and phylogenomics hold great promise for elucidating complex relationships among large plant families. Here, we performed targeted capture of low copy sequences followed by next-generation sequencing on the Illumina platform in the large and diverse angiosperm family Compositae (Asteraceae). The family is monophyletic, based on morphology and molecular data, yet many areas of the phylogeny have unresolved polytomies and interpreting phylogenetic patterns has been historically difficult. In order to outline a method and provide a framework and for future phylogenetic studies in the Compositae, we sequenced 23 taxa from across the family in which the relationships were well established as well as a member of the sister family Calyceraceae. We generated nuclear data from 795 loci and assembled chloroplast genomes from off-target capture reads enabling the comparison of nuclear and chloroplast genomes for phylogenetic analyses. We also analyzed multi-copy nuclear genes in our data set using a clustering method during orthology detection, and we applied a network approach to these clusters—analyzing all related locus copies. Using these data, we produced hypotheses of phylogenetic relationships employing both a conservative (restricted to only loci with one copy per targeted locus) and a multigene approach (including all copies per targeted locus). The methods and bioinformatics workflow presented here provide a solid foundation for future work aimed at understanding gene family evolution in the Compositae as well as providing a model for phylogenomic analyses in other plant mega-families.     

基于高通量测序的全基因组关联研究策略

全基因组关联研究（Genome-wide association study, GWAS）是人类复杂疾病研究的重要组成部分之一,在群体水平检测全基因组范围的遗传变异与可观测性状间的遗传关联。传统的GWAS是以芯片（Array）技术获得高密度的遗传变异,尽管硕果累累,但也存在不少问题。如：所谓的“缺失的遗传力”,即利用关联分析检测达到全基因组水平显著的遗传变异位点只能解释小部分遗传力;在某些性状上不同研究的结果一致性较弱;显著关联的遗传变异位点的功能较难解释等。高通量测序技术,也称第二代测序（Next-generation sequencing, NGS）技术,可以快速、准确地产出高通量的变异位点数据,为解决以上问题提供了可行的方案。基于NGS技术的GWAS方法（NGS-GWAS）,可在一定程度上弥补传统GWAS的不足。文章对NGS-GWAS策略和方法进行了系统性调研,提出了目前较为可行的NGS-GWAS的实施策略和方法,并对NGS-GWAS如何应用于个体化医疗（Personalized medicine, PM）进行了展望。     

基于ChIP-Seq进行肿瘤睾丸抗原XAGE-1b基因表达蛋白的DNA结合位点鉴定

XAGE-1b（X antigen family member 1B）属于XAGE亚家族,是一种肿瘤 睾丸抗原（cancer/testis antigen,CTA）,表达于正常人睾丸组织和多种类型的肿瘤细胞中.本实验室前期研究发现,该基因在涎腺腺样囊性癌高转移细胞系中呈高表达.为了进一步研究XAGE-1b下游调控基因,本实验采用ChIP Seq技术筛查XAGE 1b蛋白质可能存在的DNA结合片段. 结果发现,XAGE-1b下游调控基因富集于细胞分裂（cell division,P-Value＝7.95e-04）、细胞周期调控（cell cycle,P-Value＝5.532e-03）、及癌症相关基因（GESA/MSigDB module_11,P-Value＝2.010e-06）中．同时发现,XAGE-1b下游调控多个基因的表达产物（NCBI/interactions 22827,P-Value＝4.678e-06）能与原癌基因c-Myc的启动子抑制蛋白PUF60发生蛋白质相互作用,并通过qPCR进行了验证．这些研究对阐明XAGE-1b在肿瘤细胞的增殖和转移中的作用有重要意义.     

flg22诱导的拟南芥转录组分析及芥子油苷代谢途径的变化

flg22是细菌鞭毛蛋白N端的一段保守性极高的区域,能够诱导植物天然的免疫反应,为全面了解植物在受到细菌性病原菌侵害后的系统响应,利用Illumina Hiseq2000对flg22处理和未处理的拟南芥幼苗进行转录组测序。对两组数据进行差异表达分析,共获得1 200个差异表达基因,包括290个下调基因和910个上调基因。对差异表达基因进行GO富集分析和KEGG pathway富集分析,结果显示,flg22处理后,拟南芥在能量代谢、氨基酸代谢及次生代谢产物的生物合成等方面产生了巨大变化。芥子油苷是一类在植物防御病原菌的天然免疫反应中起重要作用的次生代谢产物,因此对芥子油苷代谢途径的变化进行了深入分析。根据测序结果,Flg22处理后吲哚族芥子油苷合成途径的基因表达水平显著提高,而脂肪族芥子油苷代谢途径几乎没有变化,进一步对吲哚族芥子油苷合成途径的关键酶基因进行Real Time RT-PCR的分析,验证了测序结果的正确性,证明了吲哚族芥子油苷在植物抗病防御反应中的重要作用。这为深入理解病原菌诱导的植物防御性反应及吲哚族芥子油苷的抗病机制提供了大量参考数据。     

癌症基因组测序方案制定的研究进展

癌症的基因组测序对于癌症的预防、诊断、预后、治疗以及基础生物学研究都有巨大的潜在的应用价值,正是由于其方向广泛,所以基因组测序的方案制定对于实现特定的研究目标,就显得尤为重要。同时,了解了基因组测序方案制定的规则,也有助于评估如今快速增长的发表文献的正确性和重要性。主要论述高通量测序技术在癌症基因组测序中的实际应用,并讨论癌症基因组测序在方案设计和方法学上如何调整,才能更好地实现特定的研究目的。     

miRDOA:集数据存储与在线分析于一体的综合型microRNA数据库

miRNA(microRNA)是一类小的非编码RNA,普遍存在于动物、植物等多个物种中,研究表明在原生生物以及病毒中也有miRNA。miRNA在生物的成长、发育、细胞凋亡、神经紊乱、癌症等多个方面都起到至关重要的作用,miRNA还可作为Biomarker应用于癌症等疾病的诊断和治疗。miRDOA (miRNA Database and Online Analysis)数据库存储了二百多个物种的miRNA相关信息,提供了基本的浏览和搜索功能。用户可以通过物种来浏览miRNA相关数据,也可以通过miRNA、靶基因或者序列进行检索。除此之外,miRDOA还提供了在线分析模块,主要对高通量测序数据进行初步分析,在此基础上,添加了靶基因预测、表达谱数据差异分析,以及在线BLAST功能。miRDOA是一个完全免费的开源的数据库网站,并实时更新。