An ultrastructural karyotype for the fungus Eocronartium muscicola using epifluorescence preselection of pachytene nuclei

Abstract

Mithramycin-stained nuclei in midpachynema of prophase I were preselected with epifluorescence microscopy as judged by their degree of chomatin condensation. The pachytene nuclei were processed for transmission electron microscopy (TEM) to generate ultrastructural karyotypes for Eocronartium muscicola (Fr.) Fitz. using three-dimensional reconstructions from serial sections of synaptonemal complexes. This is the first report of the use of epifluorescence to preselect fungal pachytene nuclei prior to serial sectioning for TEM and the first ultrastructural karyotype for a heterobasidiomycete. Preselection helped ensure that nuclei were in comparable states of chromatin condensation and influenced the accuracy of cross-correlating bivalent length measurements. Seventeen synapsed homologues were resolved in each of six fully reconstructed pachytene nuclei, originating from two different moss hosts, and four yielded comparable total genomic length measurements. Six of the 17 bivalents were cross-correlated among the four nuclei based on calculated length and centromeric index: the shortest and longest most extreme metacentric and submetacentric bivalents, and the bivalent associated with the nucleolus and the spindle pole body (SPB). The SPB-associated bivalent may relate to the heterochromatin found to subtend the SPB in nonpachytene stages. Key words: chromosomes, Eocronartium, epifluorescence, heterobasidiomycete, karyotype, pachynema, synaptonemal complex, spindle pole body, ultrastructure, Uredinales.