Integrating the genome sequence and genetic linkage map of Physcomitrella patens: a platform for map based gene cloning

我们的研究：在利兹大学30年的BBSRC支持下，我们率先建立了苔藓小立碗藓作为国际上采用的植物细胞和发育生物学研究的“模式生物”。这种支持使我们能够（i）开发分离基本植物过程异常突变体的技术-这是确定生物过程遗传基础的第一步（ii）通过“基因靶向”优化遗传工程技术-这是任何其他植物都不可能实现的精确工具（iii）启动了一项基因发现计划，最终由一个国际财团完成了立碗藓基因组的测序。现在，我们将建立最后的工具，使快速和常规识别基因的基础异常突变体：整合立碗藓基因组序列与其遗传连锁图谱。这将使国际和英国植物科学界能够通过应用“比较功能基因组学”（BBSRC的研究重点）从新发布的立碗藓基因组中提取最大价值。比较研究的价值：我们对复杂的生物过程的理解已经通过使用少量具有良好特征的模式生物进行的遗传实验而发生了革命性的变化。在动物王国中，对线虫、果蝇、斑马鱼和老鼠的研究使我们能够分析动物在广泛的进化谱中的发育，包括不断增加的复杂性。这种广泛的比较分析使我们能够辨别出产生这些不同生物的进化过程，以及在进化过程中基本相似的基因被不同地利用的方式。在植物王国中，允许实现类似的广泛进化视角的可比较的模式生物是开花植物拟南芥和苔藓小立碗藓。选择模式生物是因为它们在实验中易于处理。所有这些生物体的全基因组序列都已确定。所有这些都可以通过引入外源DNA来操纵：在小鼠和苔藓的情况下，外源DNA可以被精确地引入到它们基因组中的特定位点。在所有这些生物体中，可以诱导随机突变，导致其正常发育过程中的根本缺陷。通过鉴定发生这种突变的基因，我们可以鉴定出正常调节这些过程的基因。只有当测序的基因组得到“遗传连锁图”的支持时，这种识别才是可能的：遗传连锁图是基于大群体中大量突变的遗传的基因组表示。位于基因组相邻部分的突变通常是一起遗传的，并且被称为“遗传连锁”。如果突变性状与相邻的已知“遗传标记”（通常是序列已知的DNA长度）共同遗传（遗传连锁），则可以分离出导致突变性状的基因。这种强有力的实验方法允许基因的功能鉴定，而无需事先知道它们的序列。这种方法在拟南芥中是常规的。这一提议将使它在立碗藓属中成为常规，并为所有植物科学家所接受。立碗藓不是开花植物。它是最早的陆地植物群的一员-比开花植物早3亿年。因此，它代表了一个祖先的参考，在植物基因结构和功能的分歧已经发生。它还提供了对“原始”植物性状的见解，例如增强的脱水耐受性（对土地的成功殖民至关重要）和植物多细胞性的起源，这些性状对未来的植物改良具有价值。

Our research: BBSRC support of Leeds University over a period of 30 years has led us to pioneer the establishment of the moss Physcomitrella patens as an internationally adopted 'model organism' for the study of plant cell and developmental biology. This support enabled us to (i) develop techniques for the isolation of mutants aberrant in basic plant processes - the first step to identify the genetic basis of biological processes (ii) optimise a technique for genetic engineering by 'gene targeting' - a precision tool not possible in any other plant (iii) initiate a programme of gene discovery that has culminated in the complete sequencing of the Physcomitrella genome by an international consortium. Now we shall establish the final tool to enable the rapid and routine identification of genes that underlie abnormal mutants: the integration of the Physcomitrella genome sequence with its genetic linkage map. This will enable the international and UK plant science community to extract maximum value from the newly released Physcomitrella genome through the application of 'comparative functional genomics' - a BBSRC research priority. The value of comparative studies: Our understanding of complex biological processes has been revolutionised by genetic experiments conducted using a small number of well characterised model organisms. In the Animal Kingdom, the study of the nematode worm, the fruit fly, the zebra fish and the mouse allows analysis of animal development across a wide evolutionary spectrum encompassing increasing levels of complexity. Such wide comparative analysis enables us to discern the evolutionary processes that have generated these disparate creatures, and the ways in which essentially similar genes have been differently utilised in the course of evolution. Within the Plant Kingdom, the comparable model organisms that allow a similarly wide evolutionary perspective to be achieved are the flowering plant, Arabidopsis thaliana, and the moss, Physcomitrella patens. Model organisms are chosen for their experimental tractability. All these organisms have had their whole genome sequences determined. All can be manipulated by the introduction of foreign DNA: in the case of the mouse and the moss, foreign DNA can be introduced to specific sites within their genomes with exquisite precision. In all these organisms, random mutations can be induced that cause radical defects in their normal developmental processes. By identifying the genes in which such mutations have occurred, we identify the genes that normally regulate these processes. This identification is possible only where the sequenced genome is supported by a 'genetic linkage map': a representation of the genome based on the inheritance of large numbers of mutations in a large population. Mutations that lie in adjacent sections of the genome are typically inherited together, and are said to be 'genetically linked'. A gene that is responsible for a mutant trait can be isolated if that trait is co-inherited (genetically linked) with neighbouring, known 'genetic markers' - typically lengths of DNA whose sequence is known. This potent experimental approach allows the functional identification of genes without prior knowledge of their sequence. Such an approach is routine in Arabidopsis. This proposal will make it routine in Physcomitrella and accessible to all plant scientists. Physcomitrella is not a flowering plant. It is a member of the earliest group of land plants - the bryophytes - an ancient and biodiverse group which emerged ca. 300 million years before the flowering plants. It therefore represents an ancestral reference by which divergence in plant gene structure and function has occurred. It also provides insights into 'primitive' plant traits such as enhanced dehydration tolerance (essential for a successful colonisation of the land) and the origins of plant multicellularity, that retain their value for future plant improvement.