De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.).
De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.).
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使用 Illumina 测序对橡胶树 (Hevea brasiliensis Muell. Arg.) 中的树皮转录组进行从头组装和表征以及 EST-SSR 标记的开发
DOI：
10.1186/1471-2164-13-192
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发表时间：
2012-05-18
期刊：
影响因子：
4.4
通讯作者：
Men Z
中科院分区：
文献类型：
--
作者：
Li D;Deng Z;Qin B;Liu X;Men Z
在橡胶树中，树皮是重要的农业和生物器官之一。然而，橡胶树树皮形成和发育所涉及的分子机制在很大程度上仍然未知，这至少部分是由于缺乏树皮转录组和基因组信息。因此，有必要对橡胶树树皮进行高通量转录组测序，以产生大量转录本序列，用于功能鉴定和分子标记开发。
在这项研究中，使用Illumina双端测序技术产生了超过3000万条测序读段。通过从头组装，共获得了22756条平均长度为485bp的单基因。相似性搜索表明，分别有16520条和12558条单基因与来自美国国家生物技术信息中心（NCBI）非冗余蛋白数据库和瑞士蛋白数据库（Swissprot）的已知蛋白具有显著相似性。在这些已注释的单基因中，分别有6867条和5559条单基因被归类到基因本体论（GO）和直系同源簇（COG）。当将22756条单基因与京都基因与基因组百科全书通路（KEGG）数据库进行比对时，有12097条单基因被归类到5个主要类别，包括123条KEGG通路。在主要的KEGG类别中，代谢是最大的类别（9043条，占74.75%），这表明橡胶树树皮中存在活跃的代谢过程。此外，从22756条单基因中总共鉴定出39257个表达序列标签 - 简单重复序列（EST - SSRs），并且在橡胶树中进一步分析了EST - SSRs的特征。随机选择110个潜在的标记位点来验证组装质量并开发EST - SSR标记。在13份橡胶树种质中，本研究中110个标记的聚合酶链反应（PCR）成功率和多态性率分别为96.36%和55.45%。
通过从头组装和分析转录组测序数据，我们报道了橡胶树树皮的综合功能特征。这项研究产生了橡胶树转录组序列的很大一部分，这对于橡胶树的基因注释和发现、分子标记开发、基因组组装和注释以及微阵列开发是非常有用的资源。本研究中鉴定和开发的EST - SSR标记将促进橡胶树的标记辅助选择育种。此外，这项研究还表明，基于Illumina双端测序的转录组分析是对非模式物种（特别是那些具有大而复杂基因组的物种）进行转录组特征分析和分子标记开发的有力工具。
Background:In rubber tree, bark is one of important agricultural and biological organs. However, the molecular mechanism involved in the bark formation and development in rubber tree remains largely unknown, which is at least partially due to lack of bark transcriptomic and genomic information. Therefore, it is necessary to carried out high-throughput transcriptome sequencing of rubber tree bark to generate enormous transcript sequences for the functional characterization and molecular marker development.Results:In this study, more than 30 million sequencing reads were generated using Illumina paired-end sequencing technology. In total, 22,756 unigenes with an average length of 485 bp were obtained with de novo assembly. The similarity search indicated that 16,520 and 12,558 unigenes showed significant similarities to known proteins from NCBI non-redundant and Swissprot protein databases, respectively. Among these annotated unigenes, 6,867 and 5,559 unigenes were separately assigned to Gene Ontology (GO) and Clusters of Orthologous Group (COG). When 22,756 unigenes searched against the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database, 12,097 unigenes were assigned to 5 main categories including 123 KEGG pathways. Among the main KEGG categories, metabolism was the biggest category (9,043, 74.75%), suggesting the active metabolic processes in rubber tree bark. In addition, a total of 39,257 EST-SSRs were identified from 22,756 unigenes, and the characterizations of EST-SSRs were further analyzed in rubber tree. 110 potential marker sites were randomly selected to validate the assembly quality and develop EST-SSR markers. Among 13 Hevea germplasms, PCR success rate and polymorphism rate of 110 markers were separately 96.36% and 55.45% in this study.Conclusion:By assembling and analyzing de novo transcriptome sequencing data, we reported the comprehensive functional characterization of rubber tree bark. This research generated a substantial fraction of rubber tree transcriptome sequences, which were very useful resources for gene annotation and discovery, molecular markers development, genome assembly and annotation, and microarrays development in rubber tree. The EST-SSR markers identified and developed in this study will facilitate marker-assisted selection breeding in rubber tree. Moreover, this study also supported that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for transcriptome characterization and molecular marker development in non-model species, especially those with large and complex genomes.
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