Moolhuijzen Paula M, Lew-Tabor Ala E, Morgan Jess A T, Valle Manuel Rodriguez, Peterson Daniel G, Dowd Scot E, Guerrero Felix D, Bellgard Matthew I, Appels Rudi
Centre for Comparative Genomics, Murdoch University, South St,, Perth, Western Australia, 6150, Australia.
BMC Res Notes. 2011 Jul 22;4:254. doi: 10.1186/1756-0500-4-254.
Rhipicephalus (Boophilus) microplus (Rmi) a major cattle ectoparasite and tick borne disease vector, impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crustaceans. Model arthropod genomes such as Drosophila and Anopheles are too taxonomically distant for a reference in tick genomic sequence analysis. This study focuses on the de-novo assembly of two R. microplus BAC sequences from the understudied R microplus genome. Based on available R. microplus sequenced resources and comparative analysis, tick genomic structure and functional predictions identify complex gene structures and genomic targets expressed during tick-cattle interaction.
In our BAC analyses we have assembled, using the correct positioning of BAC end sequences and transcript sequences, two challenging genomic regions. Cot DNA fractions compared to the BAC sequences confirmed a highly repetitive BAC sequence BM-012-E08 and a low repetitive BAC sequence BM-005-G14 which was gene rich and contained short interspersed elements (SINEs). Based directly on the BAC and Cot data comparisons, the genome wide frequency of the SINE Ruka element was estimated. Using a conservative approach to the assembly of the highly repetitive BM-012-E08, the sequence was de-convoluted into three repeat units, each unit containing an 18S, 5.8S and 28S ribosomal RNA (rRNA) encoding gene sequence (rDNA), related internal transcribed spacer and complex intergenic region.In the low repetitive BM-005-G14, a novel gene complex was found between to 2 genes on the same strand. Nested in the second intron of a large 9 Kb papilin gene was a helicase gene. This helicase overlapped in two exonic regions with the papilin. Both these genes were shown expressed in different tick life stage important in ectoparasite interaction with the host. Tick specific sequence differences were also determined for the papilin gene and the protein binding sites of the 18S subunit in a comparison to Bos taurus.
In the absence of a sequenced reference genome we have assembled two complex BAC sequences, characterised novel gene structure that was confirmed by gene expression and sequencing analyses. This is the first report to provide evidence for 2 eukaryotic genes with exon regions that overlap on the same strand, the first to describe Rhipicephalinae papilin, and the first to report the complete ribosomal DNA repeated unit sequence structure for ticks. The Cot data estimation of genome wide sequence frequency means this research will underpin future efforts for genome sequencing and assembly of the R. microplus genome.
微小牛蜱是牛的主要体表寄生虫和蜱传疾病媒介，对动物福利和产业生产力有影响。在节肢动物研究中，缺乏包括蜱、螨、蜘蛛、蝎子和甲壳类动物在内的完整螯肢动物基因组。果蝇和按蚊等模式节肢动物基因组在分类学上与蜱的基因组序列分析参考距离太远。本研究聚焦于从未充分研究的微小牛蜱基因组中对两个微小牛蜱BAC序列进行从头组装。基于现有的微小牛蜱测序资源和比较分析，蜱基因组结构和功能预测确定了蜱与牛相互作用期间表达的复杂基因结构和基因组靶点。
在我们的BAC分析中，通过BAC末端序列和转录序列的正确定位，组装了两个具有挑战性的基因组区域。与BAC序列相比，Cot DNA组分证实了一个高度重复的BAC序列BM - 012 - E08和一个低重复的BAC序列BM - 005 - G14，后者富含基因且包含短散在元件（SINEs）。直接基于BAC和Cot数据比较，估计了SINE Ruka元件在全基因组中的频率。对高度重复的BM - 012 - E08采用保守的组装方法，将该序列解卷积为三个重复单元，每个单元包含一个18S、5.8S和28S核糖体RNA（rRNA）编码基因序列（rDNA）、相关的内部转录间隔区和复杂的基因间区域。在低重复的BM - 005 - G14中，在同一条链上的两个基因之间发现了一个新的基因复合体。在一个9 Kb大的papilin基因的第二个内含子中嵌套着一个解旋酶基因。这个解旋酶在两个外显子区域与papilin重叠。这两个基因在蜱与宿主体表寄生虫相互作用重要的不同蜱生活阶段均有表达。与牛相比，还确定了papilin基因和18S亚基的蛋白质结合位点的蜱特异性序列差异。
在没有测序参考基因组的情况下，我们组装了两个复杂的BAC序列，鉴定了通过基因表达和测序分析证实的新基因结构。这是首次为两个在同一条链上外显子区域重叠的真核基因提供证据的报告，首次描述微小牛蜱亚科papilin，也是首次报告蜱的完整核糖体DNA重复单元序列结构。全基因组序列频率的Cot数据估计意味着这项研究将为微小牛蜱基因组测序和组装的未来工作奠定基础。
