Comparative genomics using microarrays studied in Metarhizium anisopliae 2575 and 324

The entomopathogen Metarhizium anisopliae contains strains with wide host ranges and specialist strains adapted to particular hosts. Patterns of gene duplication, divergence and deletion in three generalist and three specialist strains were investigated by heterologous hybridization of genomic DNA to genes from the generalist strain ARSEF 2575. Many sequences from 2575 that are highly conserved in fungi showed rapid evolution and loss in specialist Metarhizium genomes. Some poorly hybridizing genes in specialists were functionally coordinated, including several involved in toxin biosyntheses and sugar metabolism in root exudates, indicative of reductive evolution. This suggests that specialists are loosing genes required to live in alternative hosts or as saprophytes. Several components of mobile genetic elements were also highly divergent or lost in specialists. Exceptionally, the genome of the specialist strain ARSEF 443 contained extra insertion elements that might play a role in generating evolutionary novelty. Three microarray slides were used in comparison (cDNAs are replicated in triplicate on each slide). 324 strain’s DNA was co-hybridized with strain ARSEF 2575 DNA in dye swapping replicate experiments and the relative hybridization efficiency (fluorescence ratio) of their DNA for strain ARSEF 2575 genes was compared. This array harbors PCR amplified fragments from the unique cDNA clones from M. anisopliae var. anisopliae ARSEF 2575 and a few genes from M. anisopliae var. acridum ARSEF 324 absent from the libraries of ARSEF 2575. In total, 1730 amplified clones were printed in triplicates on the slides. Additional background control was provided by 30 randomly distributed spots of 3×SSC buffer. Printing, hybridization, and scanning of slides was as described before (Freimoser et al., 2005).

昆虫病原真菌金龟子绿僵菌（Metarhizium anisopliae）包含寄主范围宽泛的广寄主菌株，以及特化适应特定寄主的专化型菌株。本研究以广寄主菌株ARSEF 2575的基因为参照，通过基因组DNA异源杂交的方法，对3株广寄主菌株与3株专化型菌株的基因重复、趋异演化及基因缺失模式展开了分析。在真菌中高度保守的ARSEF 2575的诸多序列，在专化型绿僵菌基因组中呈现快速演化与丢失的特征。专化型菌株中杂交信号较弱的部分基因存在功能协同现象，其中包含多个参与毒素生物合成以及根系分泌物中糖代谢的基因，这提示专化型菌株发生了简化演化。该结果表明，专化型菌株正在丢失其在替代寄主中生存或作为腐生生物所必需的基因。专化型菌株中，可移动遗传元件的多个组分同样呈现高度趋异或丢失的特征。值得注意的是，专化型菌株ARSEF 443的基因组中携带额外的插入元件，这类元件可能在产生演化新性状的过程中发挥作用。本研究共使用3张基因芯片玻片开展比较分析，每张玻片上的互补DNA（cDNA）克隆均设置三次重复点样。将ARSEF 324菌株的DNA与ARSEF 2575菌株的DNA进行互换染料标记的重复杂交实验，并比较二者DNA针对ARSEF 2575菌株基因的相对杂交效率（荧光比值）。该基因芯片包含两类序列：一是源自金龟子绿僵菌黏虫变种（M. anisopliae var. anisopliae）ARSEF 2575的独特cDNA克隆的PCR扩增片段；二是ARSEF 2575文库中缺失的部分蝗绿僵菌变种（M. anisopliae var. acridum）ARSEF 324的基因。总计1730个扩增克隆在每张玻片上以三次重复的方式点样。实验还设置了额外的背景对照：30个随机分布的3×SSC缓冲液点样。玻片的点样、杂交与扫描流程参照此前发表的研究（Freimoser等，2005）进行。