Evolutionary Genomics of Drosophila

描述（由申请人提供）：果蝇长期以来一直是分子进化研究的典范，不仅提供了重要的数据，而且促进了理论进步和分析方法。它已经是进化基因组学研究的先驱生物之一。拟议的研究的目标是超越基因表达微阵列的描述性水平，开始在自然人群中表达变异的遗传和分子基础的系统研究。我们将使用正式的遗传分析和实时定量逆转录酶PCR（rt-qPCR）相结合，以确定假定的顺式作用的调控元件，是多态性的纯合线和它们的相互杂交的染色体2和3置换线。对于这些基因的一个子集，我们将测序编码区和侧翼区，试图确定推定的监管差异，并将进行多态性和分歧的分析，寻找在氨基酸水平上的选择的证据。这项研究特别关注睾丸中表达的基因，因为作为一个群体，这些基因在初步数据中显示出在物种内的表达更具多态性，并且与其他类别的基因相比，在物种之间存在差异。出于速度、效率和经济的原因，将使用具有20，515个验证的PCR产物（Eurogentec）的微阵列对年轻男性进行初始表达筛选，查询所有开放阅读框的96%。将使用从三龄游走幼虫解剖的睾丸，用rt-qPCR确认表达变化。将通过分离分析和分离体的等位基因特异性PCR基因分型来鉴定推定的顺式作用调控变异。通过这些检测的候选基因将在更广泛的菌株中通过rt-qPCR进行分析，并将对高表达和低表达等位基因进行测序，并与其他物种的直系同源物一起进行沿着分析，以寻找推定的顺式作用调控元件中的多态性。将分析编码序列的多态性和趋异性，以确定在表达水平上快速进化的测试表达基因是否也在氨基酸水平上快速进化，并进行阳性选择试验。还将进行单倍型分析，以确定是否有最近选择性扫描的证据。我们将立即开始使用一个小的，但不理想的一组测试表达的基因，我们已经确定为多态性的实验室菌株使用不完整的和女性偏见的cDNA微阵列的rt-qPCR分析的遗传分析。

DESCRIPTION (provided by applicant): Drosophila has long been a paradigm for studies in molecular evolution, providing not only important data but serving as a stimulus for theoretical advances and analytical methods. Already it is among the pioneer organisms for studies in evolutionary genomics. The goal of the proposed research is to move beyond the descriptive level of gene-expression microarrays to begin a systematic study of the genetic and molecular basis of expression variation in natural populations. We will use a combination of formal genetic analysis and real-time quantitative reverse transcriptase PCR (rt-qPCR) to identify putative cis-acting regulatory elements that are polymorphic among homozygous lines and their reciprocal hybrids of chromosome 2 and 3 substitution lines. For a subset of these genes, we will sequence coding and flanking regions to try to identify putative regulatory differences, and will carry out analyses of polymorphism and divergence to look for evidence of selection at the amino acid level. The research focuses specifically on genes expressed in testes because as a group these genes have been shown in the preliminary data to be more polymorphic in expression within species and divergent between species than other classes of genes. For reasons of speed, efficiency and economy, initial expression screening will be carried out with young males using microarrays with 20,515 verified PCR products (Eurogentec) querying 96% of all open reading frames. Expression variation will be confirmed with rt-qPCR using testes dissected from third instar wandering larvae. Putative cis-acting regulatory variation will be identified by segregation analysis and by allele-specific PCR genotyping of segregants. Candidate genes that pass these tests will be assayed by rt-qPCR in a wider set of strains, and high and low expression alleles will be sequenced and analyzed along with orthologs from other species to look for polymorphisms in putative cis-acting regulatory elements. The coding sequences will be analyzed for polymorphism and divergence to ascertain whether testes-expressed genes that evolve rapidly at the expression level also evolve rapidly at the amino acid level, and to carry out tests for positive selection. Haplotype analysis will also be carried out to determine whether there is evidence for recent selective sweeps. We will initiate the genetic analysis immediately using rt-qPCR assays of a small but not ideal set of testes-expressed genes that we have identified as polymorphic in laboratory strains using incomplete and female-biased cDNA microarrays.