Complex Genetics of D-M Incompatibilities

描述（由申请人提供）：杂交雄性不育因子是理解动物物种形成的关键，也是霍尔丹规则的分子基础，即异种交配性的生殖受损是生殖隔离的第一阶段。关于生殖隔离起源的Dobzhansky-Muller模型假设，分离的同种种群经历了独立的基因替换，当这些基因替换在杂交中结合在一起时，会导致杂交不育或不育。实际上，我们对杂交不亲和因素的遗传或分子基础一无所知。一系列性别比例减数分裂驱动系统及其抑制因子重塑了精子发生，这被认为是果蝇杂交雄性不育的一种机制。本论文主要对3号染色体右臂上至少5-6个杂交雄性不育因子进行精细遗传定位、定位克隆、测序、分子分析和进化研究。利用毛里求斯龙骨3号染色体上存在的许多不同位置的遗传标记p -元件转基因基因，每个基因都被定位到大约1-2厘米的区域。这些标记的基因组区域已渗入到标准的遗传背景和纯合的渗入杂交种的育性测试。在合适的遗传背景下，在第三条染色体上有19个因素与杂交雄性不育有关。其中一个因素，被称为tmy，不仅与杂交男性生育能力严重降低有关，而且与性别比例减数分裂驱动有关。利用一种新的遗传作图策略，在感兴趣的区域进行交换，可以很容易地对重组进行表型鉴定，tmy基因被定位到一个10 kb或更小的区域。同样的定位策略将用于细化其他杂交雄性不育因子的定位，使其具有足够的分辨率来支持位置克隆。每个杂交雄性不育因子将被克隆和测序，并确定其分子组织，如果可行，将用于种系转化救援。将对该系统进行细胞学上的详细分析。初步证据表明，二次分裂Y染色体的丢失和断裂率很高。其他杂交雄性不育因子的下游功能分析超出了本提案的范围，但当然是一个长期目标。将分离的5-6个杂交雄性不育因子中的每一个都将对其分子进化的速率和模式进行进化分析，使用系统发育分析、D. melanogaster、D. simulans和D. mauritiana之间的多态性和差异分析，以及种群内多态性和单倍型分析来寻找最近选择性扫描的证据。

DESCRIPTION (provided by applicant): Hybrid male sterility factors are the key to understanding speciation in animals and the molecular basis of Haldane's rule that impaired reproduction of the heterogametic sex is the first stage in reproductive isolation. The Dobzhansky-Muller model for the origin of reproductive isolation assumes that separated conspecific populations undergo independent gene substitutions which, when combined together in the hybrid, result in hybrid sterility or inviability. Virtually nothing is known about the genetic or molecular basis of hybrid incompatibility factors. A succession of sex-ratio meiotic drive systems and their suppressors that remodel spermatogenesis has been suggested as one mechanism associated with hybrid male sterility in Drosophila. In this amended proposal, we focus on the refined genetic mapping, positional cloning, sequencing, molecular analysis and evolutionary studies of at least 5-6 hybrid male sterility factors on the right arm of chromosome 3. These have each been mapped to approximately 1-2 cM regions using genetically marked P-element transgenes present at many different locations in chromosome 3 of D. mauritiana. These marked genomic regions have been introgressed into a standard genetic background of D. simulans and the homozygous introgression hybrids tested for fertility. Across the third chromosome, 19 factors are associated with hybrid male sterility in suitable genetic backgrounds. One of these factors, denoted tmy, is associated not only with severely reduced hybrid male fertility but also with sex ratio meiotic drive. Using a novel genetic mapping strategy that allows easy phenotypic identification of recombinants with an exchange in the region of interest, the tmy gene has been mapped to a region of 10 kb or smaller. This same mapping strategy will be used to refine the map positions of the other hybrid male sterility factors, which will have sufficient resolution to support positional cloning. Each hybrid male sterility factor will be cloned and sequenced and its molecular organization determined, and if feasible will be used for germline transformation rescue. The tmy system will be analyzed in detail cytologically. Preliminary evidence suggests a high rate of second-division Y chromosome loss and fragmentation. Downstream functional analysis of the other hybrid male sterility factors is beyond the scope of the present proposal but is of course a long-range goal. Each of the 5-6 hybrid male sterility factors that will be isolated will be subjected to an evolutionary analysis for its rate and pattern of molecular evolution, using phylogenetic analysis, polymorphism and divergence analysis between D. melanogaster, D. simulans and D. mauritiana, and intrapopulation analysis of polymorphisms and haplotypes to look for evidence of recent selective sweeps.