ABR-PG: The Use of Pentaploid Surrogates for Assembly and Anchoring of Octoploid Strawberry Genomes

栽培草莓（Fragaria x ananassa）及其直系祖先F. chiloensis和F. virginiana是从进化、基因组学和园艺学角度研究的一个重要和有趣的作物系统。 栽培草莓起源于18世纪中期其两个直系祖先之间的杂交，是最年轻的作物物种之一。 它有四个基因组，每个基因组有两个拷贝，总共有八个基因组。 这些基因是从多达四个祖先传下来的，因此使栽培草莓成为基因组最复杂的作物之一。 到目前为止，还不可能得到栽培草莓的基因组序列，主要是因为有八个基因组的巨大复杂性。 该项目通过实施一项新战略来应对这一挑战，该战略将待测序的基因组数量从8个减少到4个。这种方法是通过首先将一个经过充分研究的具有两个基因组的品种（称为二倍体）与一个具有八个基因组的品种杂交，获得一个具有五个基因组的杂种-称为五倍体。 在对五倍体测序之后，二倍体的基因组被生物信息学地减去，仅留下四个基因组待组装。 结果将是第一个组装的基因组序列的直接祖先之一的栽培草莓，这将是一个巨大的价值资源草莓研究人员和育种有兴趣进一步改善这种经济上重要的和健康的水果作物。没有参考基因组尚未建立一个二倍体草莓。 这些努力所面临的挑战包括多倍体固有的挑战以及与杂合性相关的挑战。四倍体草莓品种包括栽培草莓（Fragaria x ananassa）及其直接祖先草莓（F. chiloensis和F.弗吉尼亚属植物遭受近亲繁殖衰退，并且在桃和苹果基因组计划中用于基因组组装的加倍单倍体克隆在草莓中还不是可用的选择。将实施一种大胆的方法来解决这一问题，即使用五倍体杂种作为“修饰的单倍体”受试者进行全基因组测序和连锁图谱绘制。该项目的目标是构建一个高分辨率的，基于五倍体的连锁图谱，并使用它来锚基于五倍体的基因组组装，从其中已知的二倍体基因组拷贝已被数字化扣除。将使用来自Axiom IStraw 90 SNP阵列的标记完成连锁作图。具体的项目目标是：1）基于新的五倍体作图群体构建高分辨率的四倍体连锁图; 2）基于来自单个五倍体植物的新产生的DNA序列和光学作图数据构建四倍体基因组组装体; 3）将基因组组装体锚在连锁图上;以及，4）比较新组装的二倍体基因组与现有二倍体基因组作为与二倍体种质中SNP发现的扩展程序相关的参考的效用。该项目将为随后的研究奠定必要的知识基础，旨在确定四倍体草莓属物种的亚基因组组成。只有在这一知识框架内，才有可能确定具有经济和基本科学意义的基因的同源物和亚基因组特异性。采用五倍体后代群体进行连锁图谱构建将提供对等位基因分离模式的详细了解，否则等位基因分离模式可能会被四倍体中基因型调用的挑战所掩盖，从而提供区分二体和多体遗传模式的可能性，并最终确定后者是否确实发生在草莓的四倍体水平。将通过国家生物技术信息中心（http：//www.ncbi.nlm.nih.gov/）和蔷薇科基因组数据库（http：//www.rosaceae.org/）公共数据库迅速传播待开发的基因组资源，包括第一个二倍体参考基因组。这些研究成果将推动草莓的基础和应用研究，并将加强实施草莓标记辅助育种的努力。

The cultivated strawberry (Fragaria x ananassa) and its immediate ancestors, F. chiloensis and F. virginiana, are an important and intriguing crop plant system from evolutionary, genomic and horticultural perspectives. Having originated from a cross between its two immediate ancestors in the mid-1700s, the cultivated strawberry is one of the youngest crop species. It has four genomes, each present in two copies, making a total of eight genomes. These were passed down from as many as four ancestors, thus making the cultivated strawberry among the most genomically complex crop plants. As yet, it has not been possible to get a genome sequence for the cultivated strawberry, primarily because of the great complexity of having eight genomes. This challenge is addressed in this project by implementation of a new strategy that reduces the number of genomes to be sequenced from eight to four. The approach is to get a hybrid with five genomes - called a pentaploid - by first crossing a well-studied variety with two genomes (termed a diploid) to one with eight. After sequencing the pentaploid, the genome of the diploid is subtracted out bioinformatically, leaving only four genomes to be assembled. The result will be the first assembled genome sequence for one of the immediate ancestors of the cultivated strawberry, which will be a resource of immense value to strawberry researchers and breeders interested in further improving this economically important and healthful fruit crop.No reference genome has yet been established for an octoploid strawberry. Challenges facing such efforts include those inherent to polyploidy, as well as those associated with heterozygosity. The octoploid strawberry species, including the cultivated Fragaria x ananassa and its immediate ancestors F. chiloensis and F. virginiana, are subject to inbreeding depression, and doubled haploid clones as used for genome assembly in the peach and apple genome projects have not been an available option in strawberry. A bold approach will be implemented to solve this problem by using pentaploid hybrids as "modified haploid" subjects for whole genome sequencing and linkage mapping. The project goals are to construct a high resolution, pentaploid-based linkage map and use it to anchor a pentaploid-based genome assembly, from which the known diploid genome copy has been digitally subtracted out. Linkage mapping will be accomplished using markers from the Axiom IStraw90 SNP array. The specific project objectives are to: 1) construct a high resolution, octoploid linkage map based upon a novel, pentaploid mapping population; 2) construct an octoploid genome assembly based upon newly generated DNA sequence and optical mapping data derived from a single pentaploid plant; 3) anchor the genome assembly to the linkage map; and, 4) compare the utility of the newly assembled octoploid genome to that of the existing diploid genome as references in relation to an expanded program of SNP discovery in octoploid germplasm. The project will establish a needed knowledge foundation for subsequent research aimed at defining the subgenome composition(s) of the octoploid Fragaria species. Only within this knowledge framework will it be possible to define the homoeologues and subgenomic specificities of genes of economic and basic scientific interest. The employment of a pentaploid progeny population for linkage map construction will provide detailed insight into patterns of allele segregation that might otherwise be obscured by the challenges of genotype calling in an octoploid, thereby offering the potential to differentiate disomic and polysomic inheritance patterns and to finally establish whether the latter do in fact occur at the octoploid level in strawberry. The genomic resources to be developed, including the first octoploid reference genome, will be rapidly disseminated via the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and Genome Database for Rosaceae (http://www.rosaceae.org/) public databases. The research products will advance both basic and applied research in strawberry, and will reinforce efforts to implement marker-assisted breeding in strawberry.