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Merino sheep are a breed of choice across the world, popularly kept for their wool and mutton value. They are often reared as a pure breed or used in crossbreeding and are a common component in synthetic breed development. This study evaluated genetic diversity, population structure, and breed divergence in 279 animals of Merino and Merino-based sheep breeds in South Africa using the Illumina Ovine SNP 50K BeadChip. The sheep breeds analysed included the three Merino-derived breeds of Dohne Merino (n = 50); Meatmaster (n = 47); and Afrino (n = 52) and five presumed ancestral populations of Merinos (Merino (n = 46); South African Merino (n = 10); and South African Mutton Merino (n = 8)); and the non-Merino founding breeds of Damara (n = 20); Ronderib Afrikaner (n = 17); and Nguni (n = 29). Highest genetic diversity values were observed in the Dohne Merino (DM), with H o = 0.39 ± 0.01, followed by the Meatmaster and South African Merino (SAM), with H o = 0.37 ± 0.03. The level of inbreeding ranged from 0.0 ± 0.02 (DM) to 0.27 ± 0.05 (Nguni). Analysis of molecular variance (AMOVA) showed high within-population variance (>80%) across all population categories. The first principal component (PC1) separated the Merino, South African Mutton Merino (SAMM), DM, and Afrino (AFR) from the Meatmaster, Damara, Nguni, and Ronderib Afrikaner (RDA). PC2 aligned each Merino-derived breed with its presumed ancestors and separated the SAMM from the Merino and SAM. The iHS analysis yielded selection sweeps across the AFR (12 sweeps), Meatmaster (four sweeps), and DM (29 sweeps). Hair/wool trait genes such as FGF12; metabolic genes of ICA1, NXPH1, and GPR171; and immune response genes of IL22, IL26, IFNAR1, and IL10RB were reported. Other genes include HMGA, which was observed as selection signatures in other populations; WNT5A, important in the development of the skeleton and mammary glands; ANTXR2, associated with adaptation to variation in climatic conditions; and BMP2, which has been reported as strongly selected in both fat-tailed and thin-tailed sheep. The DM vs. SAMM shared all six sweep regions on chromosomes 1, 10, and 11 with AFR vs. SAMM. Genes such as FGF12 on OAR 1:191.3-194.7 Mb and MAP2K4 on OAR 11:28.6-31.3 Mb were observed. The selection sweep on chromosome 10 region 28.6-30.3 Mb harbouring the RXFP2 for polledness was shared between the DM vs. Merino, the Meatmaster vs. Merino, and the Meatmaster vs. Nguni. The DM vs. Merino and the Meatmaster vs. Merino also shared an Rsb-based selection sweep on chromosome 1 region 268.5-269.9 Mb associated with the Calpain gene, CAPN7. The study demonstrated some genetic similarities between the Merino and Merino-derived breeds emanating from common founding populations and some divergence driven by breed-specific selection goals. Overall, information regarding the evolution of these composite breeds from their founding population will guide future breed improvement programs and management and conservation efforts.

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Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.

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BACKGROUND: Proteins in the Glycoside Hydrolase family 32 (GH32) are carbohydrate-active enzymes known as invertases that hydrolyse the glycosidic bonds of complex saccharides. Fungi rely on these enzymes to gain access to and utilize plant-derived sucrose. In fungi, GH32 invertase genes are found in higher copy numbers in the genomes of pathogens when compared to closely related saprophytes, suggesting an association between invertases and ecological strategy. The aim of this study was to investigate the distribution and evolution of GH32 invertases in the Ceratocystidaceae using a comparative genomics approach. This fungal family provides an interesting model to study the evolution of these genes, because it includes economically important pathogenic species such as Ceratocystis fimbriata, C. manginecans and C. albifundus, as well as saprophytic species such as Huntiella moniliformis, H. omanensis and H. savannae. RESULTS: The publicly available Ceratocystidaceae genome sequences, as well as the H. savannae genome sequenced here, allowed for the identification of novel GH32-like sequences. The de novo assembly of the H. savannae draft genome consisted of 28.54 megabases that coded for 7 687 putative genes of which one represented a GH32 family member. The number of GH32 gene family members appeared to be related to the ecological adaptations of these fungi. The pathogenic Ceratocystis species all contained two GH32 family genes (a putative cell wall and a putative vacuolar invertase), while the saprophytic Huntiella species had only one of these genes (a putative cell wall invertase). Further analysis showed that the evolution of the GH32 gene family in the Ceratocystidaceae involved transposable element-based retro-transposition and translocation. As an example, the activity of a Fot5-like element likely facilitated the assembly of the genomic regions harbouring the GH32 family genes in Ceratocystis. CONCLUSIONS: This study provides insight into the evolutionary history of the GH32 gene family in Ceratocystidaceae. Our findings suggest that transposable elements shaped the evolution of the GH32 gene family, which in turn determines the sucrolytic activities and related ecological strategies of the Ceratocystidaceae species that harbour them. The study also provides insights into the role of carbohydrate-active enzymes in plant-fungal interactions and adds to our understanding of the evolution of these enzymes and their role in the life style of these fungi.

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Sexual reproduction in fungi is controlled by genes present at the mating type (MAT) locus, which typically harbors transcription factors that influence the expression of many sex-related genes. The MAT locus exists as two alternative idiomorphs in ascomycetous fungi and sexual reproduction is initiated when genes from both idiomorphs are expressed. Thus, the gene content of this locus determines whether a fungus is heterothallic (self-sterile) or homothallic (self-fertile). Recently, a unique sub-class of homothallism has been described in fungi, where individuals possessing a single MAT idiomorph can reproduce sexually in the absence of a partner. Using various mycological, molecular and bioinformatic techniques, we investigated the sexual strategies and characterized the MAT loci in two tree wound-infecting fungi, Huntiella moniliformis and Huntiella omanensis. H. omanensis was shown to exhibit a typically heterothallic sexual reproductive cycle, with isolates possessing either the MAT1-1 or MAT1-2 idiomorph. This was in contrast to the homothallism via unisexual reproduction that was shown in H. moniliformis, where only the MAT1-2-1 gene was present in sexually reproducing cultures. While the evolutionary benefit and mechanisms underpinning a unisexual mating strategy remain unknown, it could have evolved to minimize the costs, while retaining the benefits, of normal sexual reproduction.

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Intersterility (IS) is thought to prevent mating compatibility between homokaryons that belong to different species. Although IS in Heterobasidion is regulated by the genes located at the IS loci, it is not yet known how the IS genes influence sexual compatibility and heterokaryon formation. To increase our understanding of the molecular events underlying IS, we studied mRNA abundance changes during IS compatible and incompatible interactions over time. The clustering of the transcripts into expression profiles, followed by the application of Gene Ontology (GO) enrichment pathway analysis of each of the clusters, allowed inference of biological processes participating in IS. These analyses identified events involved in mating and sexual development (i.e., linked with IS compatibility), which included processes associated with cell-cell adhesion and recognition, cell cycle control and signal transduction. We also identified events potentially involved in overriding mating between individuals belonging to different species (i.e., linked with IS incompatibility), which included reactive oxygen species (ROS) production, responses to stress (especially to oxidative stress), signal transduction and metabolic biosynthesis. Our findings thus enabled detection and characterization of gene expression changes associated with IS in Heterobasidion, as well as identification of important processes and pathways associated with this phenomenon. Overall, the results of this study increase current knowledge regarding the molecular mechanisms underpinning IS in Heterobasidion and allowed for the establishment of a vital baseline for further studies.

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In filamentous fungi a system known as somatic incompatibility (SI) governs self/non-self recognition. SI is controlled by a regulatory signaling network involving proteins encoded at the het (heterokaryon incompatible) loci. Despite the wide occurrence of SI, the molecular identity and structure of only a small number of het genes and their products have been characterized in the model fungi Neurospora crassa and Podospora anserina. Our aim was to identify and study the distribution and evolution of putative het gene homologs in the Basidiomycota. For this purpose we used the information available for the model fungi to identify homologs of het genes in other fungi, especially the Basidiomycota. Putative het-c, het-c2 and un-24 homologs, as well as sequences containing the NACHT, HET or WD40 domains present in the het-e, het-r, het-6 and het-d genes were identified in certain members of the Ascomycota and Basidiomycota. The widespread phylogenetic distribution of certain het genes may reflect the fact that the encoded proteins are involved in fundamental cellular processes other than SI. Although homologs of het-S were previously known only from the Sordariomycetes (Ascomycota), we also identified a putative homolog of this gene in Gymnopus luxurians (Basidiomycota, class Agaricomycetes). Furthermore, with the exception of un-24, all of the putative het genes identified occurred mostly in a multi-copy fashion, some with lineage and species-specific expansions. Overall our results indicated that gene duplication followed by gene loss and/or gene family expansion, as well as multiple events of domain fusion and shuffling played an important role in the evolution of het gene homologs of Basidiomycota and other filamentous fungi.

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In filamentous fungi, vegetative compatibility among individuals of the same species is determined by the genes encoded at the heterokaryon incompatibility (het) loci. The hyphae of genetically similar individuals that share the same allelic specificities at their het loci are able to fuse and intermingle, while different allelic specificities at the het loci result in cell death of the interacting hyphae. In this study, suppression subtractive hybridization (SSH) followed by pyrosequencing and quantitative reverse transcription PCR were used to identify genes that are selectively expressed when vegetatively incompatible individuals of Amylostereum areolatum interact. The SSH library contained genes associated with various cellular processes, including cell-cell adhesion, stress and defence responses, as well as cell death. Some of the transcripts encoded proteins that were previously implicated in the stress and defence responses associated with vegetative incompatibility. Other transcripts encoded proteins known to be associated with programmed cell death, but have not previously been linked with vegetative incompatibility. Results of this study have considerably increased our knowledge of the processes underlying vegetative incompatibility in Basidiomycetes in general and A. areolatum in particular.

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Amylostereum areolatum is a filamentous fungus that grows through tip extension, branching and hyphal fusion. In the homokaryotic phase, the hyphae of different individuals are capable of fusing followed by heterokaryon formation, only if they have dissimilar allelic specificities at their mating-type (mat) loci. In turn, hyphal fusion between heterokaryons persists only when they share the same alleles at all of their heterokaryon incompatibility (het) loci. In this study we present the first genetic linkage map for A. areolatum, onto which the mat and het loci, as well as quantitative trait loci (QTLs) for mycelial growth rate are mapped. The recognition loci (mat-A and het-A) are positioned near QTLs associated with mycelial growth, suggesting that the genetic determinants influencing recognition and growth rate in A. areolatum are closely associated. This was confirmed when isolates associated with specific mat and het loci displayed significantly different mycelial growth rates. Although the link between growth and sexual recognition has previously been observed in other fungi, this is the first time that an association between growth and self-recognition has been shown.

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