Ash tree genomics: an urgent need

该项目将对白蜡树（Fraxinus excelsior）的基因组进行测序。由于致命的真菌疾病Chalara fraxobacterium的快速传播，英国的白蜡树目前面临着最极端的威胁，可能带来毁灭性的经济后果。最近几天，人们已经清楚地看到，这种疾病在英国传播得太广，无法消除，也不太可能得到控制。因此，在这个国家，白蜡树的生存将取决于遗传性状赋予对Chalara fraxacetylum的抗性。在欧洲大陆，已经受到这种疾病的破坏，非常低比例的白蜡树对Chalara fraxalphus及其有性形式Hymenoscyphus pseudoalbidus表现出一定程度的抗性（Kjær等人，2012; McKinney等人，2011; McKinney等人，2012）。这种部分抗性的树木可能为将来在英国和欧洲大陆的白蜡树种群提供基础。目前还没有发现白蜡树的基因型对Chalara fraxalgae表现出完全抗性。然而，丹麦的白蜡树在其抗性方面表现出遗传变异，并且这种变异是加性的。这意味着培育具有高抗性的白蜡树是可能的。然而，所发现的耐药表型的确切遗传基础尚不清楚。这部分是由于缺乏关于白蜡树基因组的基本知识。目前还没有白蜡树的基因组序列，唯一一种有基因组序列的植物是猴花Mimulus guttatus，但这是一个不同的家族。目前迫切需要对白蜡树基因组进行基本鉴定。这样的资源是必不可少的未来的DNA，RNA和蛋白质为基础的研究灰病理学，生理和免疫反应，育种实验和重新引入战略。由于下一代DNA测序技术，对任何物种的整个基因组进行测序都相对简单。该项目的PI最近已经用这种方法对桦树的全基因组进行了测序，他的研究小组拥有英国独有的树木基因组学专业知识。白蜡树基因组的测序是发现抗Chalara fraxalae的遗传基础和制定育种计划的基础性步骤;它需要立即进行。该项目将于2013年1月21日开始。一棵用于基因组测序的白蜡树将与森林研究“遗传改良”项目组经理史蒂夫·李博士合作选出。将在PI实验室从芽和形成层组织中提取全基因组DNA，并将其送至Eurofins MWG Operon进行测序。测序将使用Illumina HiSeq 2000 v3平台进行，具有200 bp、300 bp、500 bp、800 bp、3 kb、20 kb和40 kb的插入片段大小的七个配对文库。白蜡树，原产于英国的白蜡树，基因组大小为954 Mbp（基尤C值数据库）;大约是人类基因组大小的三分之一。总共将生成143 Gb的DNA序列，从而提供150 X的基因组覆盖率。Illumina数据将由454 FLX++平台生成的更长读段补充，该平台将提供5x覆盖率，模式读段长度为950 bp。这些数据将由PI及其博士生在CLCbio，Aarhus，丹麦的服务器上根据各种参数设置进行汇总。将基于N50重叠群长度、低数量的Ns和保守的真核生物基因的高度完全覆盖来选择最佳组装。基因组将以电子方式注释。春天，将从正在组装基因组的白蜡树上收集叶子和花蕾。将从这些组织中提取RNA，并在QMUL进行测序。RNA-seq数据将用于注释基因组的基因区域。我们预计第一份基因组草图将于2013年8月完成，注释版将于2013年12月完成。这些数据将通过EBI数据库提供。

This project will sequence the genome of ash (Fraxinus excelsior).Ash trees in Britain currently face a most extreme threat due to the rapid spread of the deadly fungal disease Chalara fraxinea, with potentially devastating economic consequences. In recent days it has become clear that this disease is too widespread to be eliminated from Britain, and is unlikely to be contained. The survival of ash trees in this country will therefore depend upon genetic traits conferring resistance to Chalara fraxinea. In mainland Europe, already ravaged by this disease, a very low percentage of ash trees have shown a degree of resistance to Chalara fraxinea and its sexual form Hymenoscyphus pseudoalbidus (Kjær et al. 2012; McKinney et al. 2011; McKinney et al. 2012). Such partially resistant trees are likely to provide the basis for future populations of ash in Britain and mainland Europe.No genotype of ash has yet been found that shows complete resistance to Chalara fraxinea. However, ash trees in Denmark show genetic variation in their resistance, and this variation is additive. This means that the breeding of ash trees with a high level of resistance may be possible. However, the exact genetic basis of the resistant phenotypes found is unknown. This is partly due to the lack of fundamental knowledge about the genome of ash trees. Currently no genome sequence is available for ash, and the only plant in its order - the Lamiales - with a sequenced genome is the Monkey flower Mimulus guttatus, but this is in a different family. There is an urgent need for basic characterization of the ash genome. Such a resource is essential for future DNA, RNA and protein-based studies of ash pathology, physiological and immunological responses, breeding experiments and reintroduction strategies. Due to next generation DNA sequencing technologies, it is relatively straightforward to sequence the whole genome of any species. The PI of this project has recently sequenced the whole genome of birch in this way, and his research group has expertise in tree genomics which is unique to the UK. The sequencing of the ash genome is a foundational step towards discovering the genetic basis of resistance to Chalara fraxinea and developing a breeding programme; it needs to be conducted without delay.The project will begin on January 21, 2013. An ash tree for genome sequencing will be selected in collaboration with Dr Steve Lee, Programme Group Manager for 'Genetic Improvement' at Forest Research. Whole genomic DNA will be extracted from bud and cambial tissues in the PI's lab, and sent to Eurofins MWG Operon for sequencing. Sequencing will be carried out using the Illumina HiSeq 2000 v3 platform, with seven paired libraries of insert sizes of 200bp, 300bp, 500bp, 800bp, 3kb, 20kb and 40kb. Fraxinus excelsior, the ash tree native to Britain has a genome size of 954 Mbp (Kew C-value database); approximately one third of the size of the human genome. A total of 143 Gb of DNA sequence will be generated, giving 150X coverage of the genome. The Illumina data will be supplemented by longer reads generated by the 454 FLX++ platform which will provide 5x coverage with modal read length of 950bp. This data will be assembled by the PI and his PhD students on servers at CLCbio, Aarhus, Denmark under various parameter settings. The best assembly will be selected on the basis of N50 contig length, low number of Ns and high complete coverage of conserved eukaryote genes. The genome will be annotated electronically. In the spring, leaves and floral buds will be collected from the ash tree whose genome is being assembled. RNA will be extracted from these tissues and sequenced at QMUL. The RNA-seq data will be used to annotate the gene regions of the genome. We expect the first draft of the genome to be available by August 2013, and an annotated version by December 2013. This data will be made available through the EBI database.