Evolutionary Genomics of Drosophila

描述(申请人提供)：长期以来，果蝇一直是分子进化研究的范例，不仅提供了重要的数据，而且刺激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。这项拟议研究的目标是超越基因表达微阵列的描述性水平，开始对自然种群表达差异的遗传和分子基础进行系统研究。我们将结合正式的遗传分析和实时定量逆转录聚合酶链式反应(RT-qPCR)来鉴定在2号染色体和3号染色体替代系的纯合品系及其正反交品系中具有多态的可能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，试图确定可能的调控差异，并将进行多态和分歧分析，以寻找在氨基酸水平上进行选择的证据。这项研究特别关注睾丸中表达的基因，因为初步数据显示，这些基因作为一个群体，在物种内的表达更具多态，在物种之间的表达比其他类别的基因更具差异性。出于速度、效率和经济的原因，将使用微阵列对年轻男性进行初始表达筛选，微阵列具有20,515个经过验证的聚合酶链式反应产物(Eurogentec)，查询所有开放阅读框架的96%。利用三龄游荡幼虫的睾丸标本，用RT-qPCR方法证实基因表达的差异。可能的顺式作用调控变异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。通过这些测试的候选基因将在更广泛的菌株中通过RT-qPCR进行检测，高表达和低表达的等位基因将被测序，并与其他物种的同源基因一起进行分析，以寻找可能的顺式作用调节元件的多态。将对编码序列进行多态和差异分析，以确定在表达水平上进化迅速的睾丸表达基因是否也在氨基酸水平上快速进化，并进行正选择测试。还将进行单倍型分析，以确定是否有证据表明最近进行了选择性扫描。我们将立即开始使用RT-qPCR对一小部分但不理想的睾丸表达基因进行遗传分析，我们已经使用不完整的和偏向女性的cDNA微阵列在实验室菌株中鉴定出这些基因是多态的。

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.