Comparative mitogenomic analysis of Sporisorium reilianum f. sp. zeae suggests recombination events during its evolutionary history

Abstract

Introduction: Modern understanding of the concept of genetic diversity must include the study of both nuclear and organellar DNA, which differ greatly in terms of their structure, organization, gene content and distribution. This study comprises an analysis of the genetic diversity of the smut fungus Sporisorium reilianum f. sp. zeae from a mitochondrial perspective.

Methods: Whole-genome sequencing data was generated from biological samples of S. reilianum collected from different geographical regions. Multiple sequence alignment and gene synteny analysis were performed to further characterize genetic diversity in the context of mitogenomic polymorphisms.

Results: Mitochondria of strains collected in China contained unique sequences. The largest unique sequence stretch encompassed a portion of cox1, a mitochondrial gene encoding one of the subunits that make up complex IV of the mitochondrial electron transport chain. This unique sequence had high percent identity to the mitogenome of the related species Sporisorium scitamineum and Ustilago bromivora.

Discussion: The results of this study hint at potential horizontal gene transfer or mitochondrial genome recombination events during the evolutionary history of basidiomycetes. Additionally, the distinct polymorphic region detected in the Chinese mitogenome provides the ideal foundation to develop a diagnostic method to discern between mitotypes and enhance knowledge on the genetic diversity of this organism.

Keywords: horizontal gene transfer; mitochondria; mitogenome evolution; population mitogenomics; smut fungi.

FIGURE 1

Comparison of concatenated alignments of 14 mitochondrial protein-coding genes of different fungi (A) or strains (B) were made based on (A) annotated reference mitogenomes (Um = Ustilago maydis (NCBI Accession No. NC_008368.1), Ss = S. scitamineum (NCBI Accession No. CP010939), Sr = S. reilianum (NCBI Accession No. FQ311469.1)) and (B) the raw WGS data obtained in this study. Mitogenome sizes (bp) are indicated next to each alignment. The presence of unique polymorphisms in a single gene is indicated by shade variation. Inverted triangles are used to indicate polymorphisms within the coding regions of cob, nad6, and cox1 (B).

FIGURE 2

Sequence alignment of samples tested against the annotated reference genome of SRZ2 (NCBI Accession No. FQ311469.1) confirmed the point mutation previously detected for nad6 in the raw WGS data. Only 40 bp of DNA was included for sequence alignment and position 642 of the reference genome (indicated in orange) highlights the single base-pair deletion. For representative electropherograms of these comparisons, see Supplementary Figure S2.

FIGURE 3

MUSCLE analysis of sequenced nad6 against the annotated version reveals a truncation at the C-terminus of the protein. The predicted polypeptide sequences for nad6 in Ustilago maydis (Um) and Ustilago bromivora (Ub) were included for comparison. Amino acids with similar physico-chemical properties are represented by the same color. Bars represent amino acid conservation across all sequences analyzed, with darker and taller bars corresponding to higher conservation indexes. Amino acids in bold make up the consensus sequence and represent 100% conservation in that position.

FIGURE 4

Map of diagnostic PCR for cox1. Sequence is based on SRZ2 reference genome. (A) Additional PCR experiments were carried out around the cox1 polymorphic region. Primer pairs binding outside of the indicated region were designed to inspect mtDNA integrity. These primer combinations should work in any SRZ strain, regardless of the mitotype. Predicted amplicon sizes are indicated. (B) Complementary primer combinations are color coded and should yield the following amplicons: oHM114/115 = 2,507 bp in German or 1,158 bp in Chinese, oHM119/120 = 4,143 bp in German or 2,795 bp in Chinese, oHM112/128 = 1,347 bp in German only. For gel electrophoresis results, refer to Supplementary Figure S3. (C) Table of diagnostic primer combinations for haploid sporidial German and Chinese strains. NA, not applicable, i.e., no band was observed, as expected.

FIGURE 5

The primer walking sequencing approach previously described revealed unique sequences in cox1, with a 1,611 bp fragment in German strains matching SRZ2 mtDNA (black, red, and yellow) and a 3,356 bp fragment in Chinese strains matching S. scitamineum and Ustilago bromivora (red). The updated gene sizes are indicated in bold. Exons (“e”) are indicated as yellow arrows; the position of primers used in Primer Walking sequence analysis of the polymorphic region in the Chinese isolates is illustrated below the map (Seq Primers oHM133-139).

FIGURE 6

Characterization of mitotypes from teliospore segregants after maize infections. Teliospores were germinated on YPS agar and colonies selected 48–72 h after growth at 28 C; DNA was isolated from re-streaked individual colonies. Four independent colonies from the teliospores germinated for each cross were used for DNA isolation. PCR experiments using primers that work for both German and Chinese strains (114 + 115, 127 + 131) were carried out to determine genetic traceability of the cox1 polymorphism previously detected. For this figure, agarose gel electrophoresis of PCR products using primers oHM114 and oHM115 are shown, including both control haploid strains (Lanes 2 and 3, SRZ2 and SRZCXI2, respectively), as well as for one segregant colony from each teliospore source: Lane 4, SRZ1 x SRZ2 #1; lane 5, SRZ1 x SRZCXI2 #1; lane 6, SRZ2 x SRZCXI2 #1; lane 7, SRZ2 x SRZCXII2 #1; lane 8, SRZ1 x SRZCXI3 #1; lane 9, SRZCXII2 x SRZCXI3 #1. Lane 1, Quick DNA 1 kb Size standard, New England Biolabs, lane 10, no DNA negative control. Mating types of strains and those in crosses are indicated above each lane; strain names for Chinese isolates are abbreviated CXI2, CXII2, and CXI3. Results for PCR reactions using this primer combination, as well as oHM127/oHM131 on additional independent segregant colonies are shown in Supplementary Figure S8.