Identification of Highly Variable Supernumerary Chromosome Segments in an Asexual Pathogen

Abstract

Supernumerary chromosome segments are known to harbor different transposons from their essential counterparts. The aim of this study was to investigate the role of transposons in the origin and evolution of supernumerary segments in the asexual fungal pathogen Fusarium virguliforme. We compared the genomes of 11 isolates comprising six Fusarium species that cause soybean sudden death syndrome (SDS) or bean root rot (BRR), and identified significant levels of genetic variation in A+T-rich repeat blocks of the essential chromosomes and in A+T-neutral regions of the supernumerary segments. The A+T-rich repeat blocks in the essential chromosomes were highly variable between F. virguliforme and non-F. virguliforme isolates, but were scarcely variable between F. virguliforme isolates. The A+T-neutral regions in the supernumerary segments, however, were highly variable between F. virguliforme isolates, with a statistically significant number (21 standard deviations above the mean) of single nucleotide polymorphisms (SNPs). And supernumerary sequence types and rearrangement patterns of some F. virguliforme isolates were present in an isolate of F. cuneirostrum but not in the other F. virguliforme isolates. The most variable and highly expressed region in the supernumerary segments contained an active DNA transposon that was a most conserved match between F. virguliforme and the unrelated fungus Tolypocladium inflatum. This transposon was absent from two of the F. virguliforme isolates. Furthermore, transposons in the supernumerary segments of some F. virguliforme isolates were present in non-F. virguliforme isolates, but were absent from the other F. virguliforme isolates. Two supernumerary P450 enzymes were 43% and 57% identical to their essential counterparts. This study has raised the possibility that transposons generate genetic variation in supernumerary chromosome segments by frequent horizontal transfer within and between closely related species.

Fig 1. Maximum likelihood tree of 13 duplicated sequences (length 3,772 bp) in the Fv Mont-1 assembly.

Each sequence was named based on its A+T content followed by its contig name. For example, sequence P61.mc37.31 indicates an A+T content of 61% and mc37.31 as its source contig. The four most recently duplicated sequences were P69.mc53.3, P70.mc175.2, P69.mc29.3 and P70.mc147.3, with the highest A+T content range of 69% to 70%. Support values from 100 bootstrap replicates are provided at internodes.

Fig 2. Maximum-likelihood midpoint rooted tree of 11 SDS/BRR Fusarium isolates, inferred from genome-wide SNP data with 200 bootstrap samples.

Fig 3. Chromosomal rearrangement between Fv Mont-1 and Fv Clinton-1B.

Each horizontal line represents a contig with its name and orientation (+ denotes forward) given on the right. A unique significant match between contig regions in opposite orientations is indicated by a pair of cross lines; one in the same orientation by a pair of parallel lines. In each case, the percent identity of the match is shown next to the lines. The beginning and end of each contig region in the match are marked with vertical arrows along with their positions in bp. A red box in contig mc28.4 and a black box in contig cc26.1 represent different islands surrounded by the match; the black box is part of the match with contig mc184.2.

Fig 4. Eight sequence alignments with SNPs and small indels (4 to 166 bp).

Each alignment is composed of two or three sequence types (denoted by Types a, b and c): a reference contig, a contig in the Fv Clinton-1B assembly, and sometimes short reads from one of the ten isolates, which were mapped to one of the two contigs. The name of each contig along with its orientation (+ denotes forward and—denotes reverse), or the name of the isolate if present, is shown to the right of its sequence type. Every allele in the contig is marked with an arrow and a number in bp showing its position. Notation: mc184.2, Fv Mont-1 contig 184.2; cc26.1, Fv Clinton-1B contig 26.1.

Fig 5. Proteins encoded by two supernumerary segments.

The related proteins between the segments were P450 enzymes and reverse transcriptase-like (RT-like) enzymes. The larger segment contained a gene encoding a G1/S-specific cyclin protein.