Timoshevskiy Vladimir A, Sharma Atashi, Sharakhov Igor V, Sharakhova Maria V
Department of Entomology, Virginia Tech, USA.
J Vis Exp. 2012 Sep 17(67):e4215. doi: 10.3791/4215.
Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions. Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti and Cx. quinquefasciatus, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti, the accumulation of multiple chromosomal rearrangements in cell line chromosomes makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.
荧光原位杂交（FISH）是许多实验室常规使用的一种技术，用于确定DNA和RNA探针的染色体位置。该方法的一个重要应用是开发高质量的物理图谱，这有助于改进各种生物的基因组组装。多线染色体和有丝分裂染色体的天然带型为基因组超级重叠群的精确排序和定向提供了指导。在按蚊、伊蚊和库蚊这三个蚊属中，仅为按蚊开发了一种成熟的基于染色体的绘图技术，因为按蚊成员拥有可读的多线染色体。经过基因组绘图工作，88%的冈比亚按蚊基因组已被定位到精确的染色体位置。另外两个蚊属，伊蚊和库蚊，由于其基因组中转座元件的大量过度存在，多线染色体发育不良。只有31%和9%的基因组超级重叠群分别被无序或无定向地分配到埃及伊蚊和致倦库蚊的染色体上。此前，这两个物种的有丝分裂染色体制备仅限于脑神经节和细胞系。然而，从蚊子脑神经节制备的染色体玻片通常含有少量中期板。此外，虽然已经为埃及伊蚊细胞系的有丝分裂染色体开发了一种FISH技术，但细胞系染色体中多个染色体重排的积累使其无法用于基因组绘图。在这里，我们描述了一种简单、可靠的技术，可从四龄幼虫的成虫盘（ID）中获得高质量的有丝分裂染色体制备物，该技术可用于所有三个蚊属。针对使用基因组DNA的BAC克隆作为探针在埃及伊蚊和致倦库蚊的有丝分裂染色体上进行杂交，以及利用核糖体DNA（rDNA）的基因间隔区（IGS）作为探针在冈比亚按蚊染色体上进行杂交，对标准FISH方案进行了优化。除了物理绘图外，所开发的技术还可应用于蚊子和其他昆虫群体的群体细胞遗传学以及染色体分类学/系统学。
