CONSTRUCTION OF A GENETIC LINKAGE MAP OF ZEBRAFISH

此拨款申请是为响应基因组RFA而提交的 为斑马鱼提供资源，申请资金以 在斑马鱼身上构建一个0.3厘米的地图。我们应对此RFA的目标 是为了生成斑马鱼Danio的密集微卫星连锁图谱 Rerio，这将对克隆基因的简易性产生戏剧性的影响 通过位置，并以一种现成的方式对整个 使用这种有机体的科学家社区。 已定义了600多个对发育具有重要意义的基因 斑马鱼经化学诱变筛选。我们学院是一个专业 这类筛选和中断基因的克隆的站点，特别是 那些与器官发育相关的基因。这些突变说明了 重要的人类疾病，包括心力衰竭和心律失常， 癌症、造血功能缺陷、视网膜变性、多囊 肾脏疾病和神经退化。分离这些基因将会 需要一份密集和强大的遗传图谱，由可靠的、易于操作的 使用可以在许多品种的鱼之间杂交的标记。 基于简单序列长度多态性的微卫星标记 (SSLP)在整个基因组中重复现在是标准的开始 在大多数基因中定位克隆和锚定其他图谱的点 有机体。它们在基因组中含量丰富，分布均匀； 它们在近交系之间是高度多态的；与RAPD不同的是， 它们共同支配的高发生率使它们在 二倍体和非自交系；它们是单基因座扩增 产品，使其成为固定辐射的必要工具 基于杂交(RH)的表达序列标签(EST)映射到遗传图谱， 这是本RFA的另一个组成部分目标。 在我们测绘项目的最初几年里，我们开发了一种低- 密度完整的地图，有足够的标记(600-700)覆盖全部25个 染色体的平均间隔为5厘米。许多 世界各地的调查人员已经使用了我们目前的低密度 微卫星图谱启动重要基因的定位克隆， 但我们同意，作为一个领域，我们必须增加 我们地图上的微卫星标记，并使它们迅速可用。我们的 实验室，以取得的经验为基础 密度贴图，已经开发了一种简化的方法来做到这一点。

This grant application is submitted in response to the RFA for Genomic Resources for the Zebrafish, requesting applications for funds to construct a 0.3 cM map in zebrafish. Our goal in responding to this RFA is to generate a dense microsatellite linkage map of the zebrafish Danio rerio, one that will have a dramatic effect on the ease of cloning genes by position, and to do so in a manner of ready utility for the entire community of scientists using this organism. More than 600 genes of developmental importance have been defined in zebrafish by chemical mutagenesis screens. Our institute is one major site for such screens and the cloning of the disrupted genes, especially those which are relevant to organ development. The mutations speak to important human diseases, including heart failure and arrhythmias, cancer, hematopoietic deficiency, retinal degeneration, polycystic kidney disease, and neural degeneration. Isolation of these genes will require a dense and robust genetic map, composed of reliable, easy-to- use markers that can be used in crosses between many strains of fish. Microsatellite markers based on simple sequence length polymorphisms (SSLPs) repeats throughout the genome are now the standard starting point for positional cloning and anchoring of other maps in most genetic organisms. They are abundant and well distributed through the genome; they are highly polymorphic et between outbred strains; unlike RAPDs, their high incidence of codominance makes them especially useful in diploids and non-inbred strains; and they are single locus amplification products, making them an essential tool for the anchoring of a radiation hybrid (RH)-based expressed sequence tag (EST) map to the genetic map, which is the other component goal of this RFA. During the initial years of our mapping project, we developed a low- density complete map with sufficient markers (600-700) to cover all 25 chromosomes at an average interval of 5 centiMorgans (cM). Many investigators around the world have used our current low-density microsatellite map to initiate positional cloning of important genes, but agree that we, as a field, must increase the number of microsatellite markers on our map and make them rapidly available. Our laboratory, building on the experience gained while making the low density map, has developed a streamlined method to do so.