Uncovering interspecific genetic diversity and detecting gene exchange in Cochliobolus sativus

Abstract

Throughout fungal evolutionary history, interspecific gene exchange has significantly influenced rapid adaptation and the emergence of new pathogenic species. We enhanced the need to explore interspecific genetic diversity and gene flow in Cochliobolus sativus through both sexual and asexual stages. Morphological and
genotypic identification revealed isolates in both morphs exhibited 99 % identity in NCBI BLAST. Multi-locus
sequence analysis (ITS and GAPDH) confirmed all tested isolates as Bipolaris sorokiniana (teleomorph Cochliobolus sativus) with >98 % similarity to reference sequences, and phylogenetic analysis placed parental isolates
(BS-65 and BS-134) in a strongly supported clade with Bipolaris sorokiniana, clearly separated from other genera
such as Alternaria and Colletotrichum. DNA fingerprinting exhibited polymorphic fragments in 88.3 % of asexual
and 77.73 % of sexual isolates. Genetic distances of maternal and offspring isolates varied from 5 % to 71 % and
isolate CS-01 CS2 were at ancestral nodes. Identical parent-genetic similarity (94 %) was detected for CS 24 and
CS22, CS1 and CS11, CS3 and CS11. Phylogenetic analyses, using Tamura-Nei ML models in MEGA X with 1000
bootstraps reproducibility, indicated two distinct genetic clusters, confirming gene flow and introgression between F1 and F2 generations. F1-derived F2 isolates showed intermediate genotypes, supporting sexual
recombination and allele segregation. Genetic diversity parameters estimated by DnaSP6 software indicated a
high number of haplotypes (Hd = 1.0), a high number of nucleotide polymorphisms (Pi = 0.68152) and average
nucleotide differences (K = 413) for the F1s, and moderately high values for the F2s, indicating recombinationbased diversity. Chi-square and Hudson’s tests did not reveal significant differences between F1 and F2, while
Nei’s Gst and DeltaSt indicated relatively moderate to very high levels of gene flow (2.15 ≤ Nm ≤ 231.94) and
Fst and Nst were negative and indicated extensive sharing of alleles. Haplotype network analysis employing
PopART (version 1.7) identified six haplotypes, and none of the F2 isolates had the same haplotype as the
parents, visually showing meiotic recombination. These results are the first molecular evidence of gene flow via
sexual reproduction and demonstrate that hybridization reshuffled alleles, which led to new genotypes and a
contribution to genetic diversity. These results indicate that Cochliobolus sativus could possess evolutionary capacity by recombination in sexual and asexual phases that may be able to affect pathogenicity and prepare the
emergence of new genotypes.