Genome size estimation of Phymatotrichopsis omnivora, the causal agent of cotton root rot

Phymatotrichopsis omnivora (Duggar) Hennebert is a destructive root pathogen of dicotyledonous plants and is endemic to Oklahoma, Texas, New Mexico and Arizona. The disease caused by this pathogen, best known as cotton root rot, has greatly hindered the production of cotton and alfalfa in this region. Currently, no practical disease management methods are available and no cotton or alfalfa varieties are known to be resistant or tolerant to this fungus. To gain insight into the genome structure and organization of this fungus, sequencing of the P. omnivora genome is currently underway. The draft genome sequence, obtained predominantly by '454' pyrosequencing, assembled into 75 Mb, considerably larger than initially expected. However, this large assembly could result from the multinucleate, heterokaryotic nature of this presumably asexual fungus. To support sequencing efforts, we are determining the genome size of P. omnivora using electrophoretic chromosome separations and size estimation based on a BAC library. At least seven chromosomal DNA bands, ranging in size from 3.8 to 610 Mb and tot ling 640 Mb, were observed by pulse-field gel electrophoresis. The chromosome number was also determined by hybridization of P. omnivora DNA with a telomere probe. Probes detecting single copy genes, such as beta tubulin and RNA polymerase II subunit 2, were hybridized against an arrayed P. omnivora BAC library that was estimated to represent 10x genome coverage based on a 40 Mb genome size. The number of hybridizing clones represented by these genes was less than expected, inferring a genome size much larger than 40 Mb. The genome sequence of P. omnivora should enhance our understanding of its intriguing biology and should help in the development of strategies to control cotton root rot.