Evolutionary Genomics of Drosophila

描述（申请人提供）：果蝇长期以来一直是分子进化研究的一个范例，不仅提供了重要的数据，而且促进了理论进步和分析方法的发展。它已经是进化基因组学研究的先驱生物之一。拟议研究的目标是超越基因表达微阵列的描述水平，开始对自然种群中表达变异的遗传和分子基础进行系统研究。我们将使用正式的遗传分析和实时定量逆转录酶PCR （rt-qPCR）相结合的方法来鉴定在纯合子系及其2号染色体和3号染色体替代系的互反杂交株中具有多态性的假定的顺式作用调控元件。对于这些基因的一个子集，我们将对编码和侧翼区域进行测序，以试图确定可能的调控差异，并将进行多态性和差异分析，以寻找氨基酸水平上选择的证据。这项研究特别关注在睾丸中表达的基因，因为作为一个群体，这些基因在物种内的表达比其他类别的基因更具多态性，在物种之间的表达更具差异性。出于速度、效率和经济的考虑，将对年轻男性进行初步的表达筛选，使用含有20,515个经过验证的PCR产品（Eurogentec）的微阵列，查询96%的开放阅读框。利用三龄流浪幼虫的睾丸，用rt-qPCR方法证实表达变异。假定的顺式调控变异将通过分离分析和分离的等位基因特异性PCR基因分型来确定。通过这些测试的候选基因将在更广泛的菌株中进行rt-qPCR分析，高表达和低表达等位基因将与其他物种的同源基因一起测序和分析，以寻找假定的顺式作用调节元件的多态性。分析编码序列的多态性和差异，以确定在表达水平上快速进化的睾丸表达基因是否也在氨基酸水平上快速进化，并进行正选择检验。单倍型分析也将进行，以确定是否有证据表明最近的选择性扫描。我们将立即启动遗传分析，使用rt-qPCR检测一小部分但不理想的睾丸表达基因，我们已经使用不完整和女性偏倚的cDNA微阵列在实验室菌株中确定为多态性。

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.