Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

Genome and transcriptome analysis of the latent pathogen Lasiodiplodia theobromae, an emerging threat to the cacao industry.

Lasiodiplodia theobromae (Pat.) Griffon & Maubl., a member of the family Botryosphaeriaceae, is becoming a significant threat to crops and woody plants in many parts of the world, including the major cacao growing areas. While attempting to isolate Ceratobasidium theobromae, a causal agent of vascular streak dieback (VSD), from symptomatic cacao stems, 74% of isolated fungi were Lasiodiplodia spp. Sequence-based identification of 52 putative isolates of L. theobromae indicated that diverse species of Lasiodiplodia were associated with cacao in the studied areas, and the isolates showed variation in aggressiveness when assayed using cacao leaf discs. The present study reports a 43.75 Mb de novo assembled genome of an isolate of L. theobromae from cacao. Ab initio gene prediction generated 13 061 protein-coding genes, of which 2862 are unique to L. theobromae, when compared with other closely related Botryosphaeriaceae. Transcriptome analysis revealed that 11 860 predicted genes were transcriptionally active and 1255 were more highly expressed in planta compared with cultured mycelia. The predicted genes differentially expressed during infection were mainly those involved in carbohydrate, pectin, and lignin catabolism, cytochrome P450, necrosis-inducing proteins, and putative effectors. These findings significantly expand our knowledge of the genome of L. theobromae and the genes involved in virulence and pathogenicity.

Assessing hidden parentage and genetic integrity of the "United Fruit Clones" of cacao (Theobroma cacao) from Costa Rica using SNP markers.

The international cacao collection in CATIE, Costa Rica contains nearly 1200 accessions of cacao, mainly from the center of genetic diversity of this species. Among these accessions, the United Fruit clones (UF clones) were developed by the United Fruit Company in Costa Rica, and they represent one of the earliest groups of improved cacao germplasm in the world. Some of these UF clones have been used as key progenitors for breeding resistance/tolerance to Frosty Pod and Black Pod diseases in the Americas. Accurate information on the identity and background of these clones is important for their effective use in breeding. Using Single Nucleotide Polymorphism (SNP) markers, we genotyped 273 cacao germplasm accessions including 44 UF clones and 229 reference accessions. We verified the true-to-type identity of UF clones in the CATIE cacao collection and analyzed their population memberships using maximum-likelihood-based approaches. Three duplicate groups, representing approximately 30% of the UF clones, were identified. Both distance- and model-based clustering methods showed that the UF clones were mainly composed of Trinitario, ancient Nacional and hybrids between ancient Nacional and Amelonado. This result filled the information gap about the UF clones thus will improve their utilization for cacao breeding.

Association mapping of seed and disease resistance traits in Theobroma cacao L.

MAIN CONCLUSION: Microsatellite and single nucleotide polymorphism markers that could be used in marker assisted breeding of cacao were identified for number of filled seeds, black pod resistance and witches' broom disease resistance. An association mapping approach was employed to identify markers for seed number and resistance to black pod and witches' broom disease (WBD) in cacao (Theobroma cacao L.). Ninety-five microsatellites (SSRs) and 775 single nucleotide polymorphisms (SNPs) were assessed on 483 unique trees in the International Cocoa Genebank Trinidad (ICGT). Linkage disequilibrium (LD) and association mapping studies were conducted to identify markers to tag the phenotypic traits. Decay of LD occurred over an average 9.3 cM for chromosomes 1-9 and 2.5 cM for chromosome 10. Marker/trait associations were generally identified based on general linear models (GLMs) that incorporated principal components from molecular information on relatedness factor. Seven markers (mTcCIR 8, 66, 126, 212; TcSNP368, 697, 1370) on chromosomes 1 and 9 were identified for number of filled seeds (NSEED). A single marker was found for black pod resistance (mTcCIR280) on chromosome 3, whereas six markers on chromosomes 4, 5, 6, 8, and 10 were detected for WBD (mTcCIR91, 183; TcSNP375, 720, 1230 and 1374). It is expected that this association mapping study in cacao would contribute to the knowledge of the genetic determinism of cocoa traits and that the markers identified herein would prove useful in marker assisted breeding of cacao.

Genetic diversity and parentage in farmer selections of cacao from Southern Sulawesi, Indonesia revealed by microsatellite markers.

Indonesia is the third largest cocoa-producing country in the world. Knowledge of genetic diversity and parentage of farmer selections is important for effective selection and rational deployment of superior cacao clones in farmers' fields. We assessed genetic diversity and parentage of 53 farmer selections of cacao in Sulawesi, Indonesia, using 152 international clones as references. Cluster analysis, based on 15 microsatellite markers, showed that these Sulawesi farmer selections are mainly comprised of hybrids derived from Trinitario and two Upper Amazon Forastero groups. Bayesian assignment and likelihood-based parentage analysis further demonstrated that only a small number of germplasm groups, dominantly Trinitario and Parinari, contributed to these farmer selections, in spite of diverse parental clones having been used in the breeding program and seed gardens in Indonesia since the 1950s. The narrow parentage predicts a less durable host resistance to cacao diseases. Limited access of the farmers to diverse planting materials or the strong preference for large pods and large bean size by local farmers, may have affected the selection outcome. Diverse sources of resistance, harbored in different cacao germplasm groups, need to be effectively incorporated to broaden the on-farm diversity and ensure sustainable cacao production in Sulawesi.

Genome and secretome analysis of the hemibiotrophic fungal pathogen, Moniliophthora roreri, which causes frosty pod rot disease of cacao: mechanisms of the biotrophic and necrotrophic phases.

BACKGROUND: The basidiomycete Moniliophthora roreri is the causal agent of Frosty pod rot (FPR) disease of cacao (Theobroma cacao), the source of chocolate, and FPR is one of the most destructive diseases of this important perennial crop in the Americas. This hemibiotroph infects only cacao pods and has an extended biotrophic phase lasting up to sixty days, culminating in plant necrosis and sporulation of the fungus without the formation of a basidiocarp. RESULTS: We sequenced and assembled 52.3 Mb into 3,298 contigs that represent the M. roreri genome. Of the 17,920 predicted open reading frames (OFRs), 13,760 were validated by RNA-Seq. Using read count data from RNA sequencing of cacao pods at 30 and 60 days post infection, differential gene expression was estimated for the biotrophic and necrotrophic phases of this plant-pathogen interaction. The sequencing data were used to develop a genome based secretome for the infected pods. Of the 1,535 genes encoding putative secreted proteins, 1,355 were expressed in the biotrophic and necrotrophic phases. Analysis of the data revealed secretome gene expression that correlated with infection and intercellular growth in the biotrophic phase and invasive growth and plant cellular death in the necrotrophic phase. CONCLUSIONS: Genome sequencing and RNA-Seq was used to determine and validate the Moniliophthora roreri genome and secretome. High sequence identity between Moniliophthora roreri genes and Moniliophthora perniciosa genes supports the taxonomic relationship with Moniliophthora perniciosa and the relatedness of this fungus to other basidiomycetes. Analysis of RNA-Seq data from infected plant tissues revealed differentially expressed genes in the biotrophic and necrotrophic phases. The secreted protein genes that were upregulated in the biotrophic phase are primarily associated with breakdown of the intercellular matrix and modification of the fungal mycelia, possibly to mask the fungus from plant defenses. Based on the transcriptome data, the upregulated secreted proteins in the necrotrophic phase are hypothesized to be actively attacking the plant cell walls and plant cellular components resulting in necrosis. These genes are being used to develop a new understanding of how this disease interaction progresses and to identify potential targets to reduce the impact of this devastating disease.

The American Cherimoya Genome Reveals Insights into the Intra-Specific Divergence, the Evolution of Magnoliales, and a Putative Gene Cluster for Acetogenin Biosynthesis.

Annona cherimola (cherimoya) is a species renowned for its delectable fruit and medicinal properties. In this study, we developed a chromosome-level genome assembly for the cherimoya 'Booth' cultivar from the United States. The genome assembly has a size of 794 Mb with a N50 = 97.59 Mb. The seven longest scaffolds account for 87.6% of the total genome length, which corresponds to the seven pseudo-chromosomes. A total of 45,272 protein-coding genes (≥30 aa) were predicted with 92.9% gene content completeness. No recent whole genome duplications were identified by an intra-genome collinearity analysis. Phylogenetic analysis supports that eudicots and magnoliids are more closely related to each other than to monocots. Moreover, the Magnoliales was found to be more closely related to the Laurales than the Piperales. Genome comparison revealed that the 'Booth' cultivar has 200 Mb less repeats than the Spanish cultivar 'Fino de Jete', despite their highly similar (>99%) genome sequence identity and collinearity. These two cultivars were diverged during the early Pleistocene (1.93 Mya), which suggests a different origin and domestication of the cherimoya. Terpene/terpenoid metabolism functions were found to be enriched in Magnoliales, while TNL (Toll/Interleukin-1-NBS-LRR) disease resistance gene has been lost in Magnoliales during evolution. We have also identified a gene cluster that is potentially responsible for the biosynthesis of acetogenins, a class of natural products found exclusively in Annonaceae. The cherimoya genome provides an invaluable resource for supporting characterization, conservation, and utilization of Annona genetic resources.

Cacao pod transcriptome profiling of seven genotypes identifies features associated with post-penetration resistance to Phytophthora palmivora.

The oomycete Phytophthora palmivora infects the fruit of cacao trees (Theobroma cacao) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora, yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue-P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis-regulatory modules likely to be involved in cacao defense against P. palmivora. This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora-resistant cacao.

Three de novo assembled wild cacao genomes from the Upper Amazon.

Theobroma cacao, the chocolate tree, is indigenous to the Amazon basin, the greatest biodiversity hotspot on earth. Recent advancement in plant genomics highlights the importance of de novo sequencing of multiple reference genomes to capture the genome diversity present in different cacao populations. In this study, three high-quality chromosome-level genomes of wild cacao were constructed, de novo assembled with HiFi long reads sequencing, and scaffolded using a reference-free strategy. These genomes represent the three most important genetic clusters of cacao trees from the Upper Amazon region. The three wild cacao genomes were compared with two reference genomes of domesticated cacao. The five cacao genetic clusters were inferred to have diverged in the early and middle Pleistocene period, approximately 1.83-0.69 million years ago. The results shown here serve as an example of understanding how the Amazonian biodiversity was developed. The three wild cacao genomes provide valuable resources for studying genetic diversity and advancing genetic improvement of this species.

Global analyses of Ceratocystis cacaofunesta mitochondria: from genome to proteome.

BACKGROUND: The ascomycete fungus Ceratocystis cacaofunesta is the causal agent of wilt disease in cacao, which results in significant economic losses in the affected producing areas. Despite the economic importance of the Ceratocystis complex of species, no genomic data are available for any of its members. Given that mitochondria play important roles in fungal virulence and the susceptibility/resistance of fungi to fungicides, we performed the first functional analysis of this organelle in Ceratocystis using integrated "omics" approaches. RESULTS: The C. cacaofunesta mitochondrial genome (mtDNA) consists of a single, 103,147-bp circular molecule, making this the second largest mtDNA among the Sordariomycetes. Bioinformatics analysis revealed the presence of 15 conserved genes and 37 intronic open reading frames in C. cacaofunesta mtDNA. Here, we predicted the mitochondrial proteome (mtProt) of C. cacaofunesta, which is comprised of 1,124 polypeptides - 52 proteins that are mitochondrially encoded and 1,072 that are nuclearly encoded. Transcriptome analysis revealed 33 probable novel genes. Comparisons among the Gene Ontology results of the predicted mtProt of C. cacaofunesta, Neurospora crassa and Saccharomyces cerevisiae revealed no significant differences. Moreover, C. cacaofunesta mitochondria were isolated, and the mtProt was subjected to mass spectrometric analysis. The experimental proteome validated 27% of the predicted mtProt. Our results confirmed the existence of 110 hypothetical proteins and 7 novel proteins of which 83 and 1, respectively, had putative mitochondrial localization. CONCLUSIONS: The present study provides the first partial genomic analysis of a species of the Ceratocystis genus and the first predicted mitochondrial protein inventory of a phytopathogenic fungus. In addition to the known mitochondrial role in pathogenicity, our results demonstrated that the global function analysis of this organelle is similar in pathogenic and non-pathogenic fungi, suggesting that its relevance in the lifestyle of these organisms should be based on a small number of specific proteins and/or with respect to differential gene regulation. In this regard, particular interest should be directed towards mitochondrial proteins with unknown function and the novel protein that might be specific to this species. Further functional characterization of these proteins could enhance our understanding of the role of mitochondria in phytopathogenicity.

Genome-wide characterization and evolutionary analysis of the AP2/ERF gene family in lettuce (Lactuca sativa).

The APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) gene family plays vital roles in plants, serving as a key regulator in responses to abiotic stresses. Despite its significance, a comprehensive understanding of this family in lettuce remains incomplete. In this study, we performed a genome-wide search for the AP2/ERF family in lettuce and identified a total of 224 members. The duplication patterns provided evidence that both tandem and segmental duplications contributed to the expansion of this family. Ka/Ks ratio analysis demonstrated that, following duplication events, the genes have been subjected to purifying selection pressure, leading to selective constraints on their protein sequence. This selective pressure provides a dosage benefit against stresses in plants. Additionally, a transcriptome analysis indicated that some duplicated genes gained novel functions, emphasizing the contribution of both dosage effect and functional divergence to the family functionalities. Furthermore, an orthologous relationship study showed that 60% of genes descended from a common ancestor of Rosid and Asterid lineages, 28% from the Asterid ancestor, and 12% evolved in the lettuce lineage, suggesting lineage-specific roles in adaptive evolution. These results provide valuable insights into the evolutionary mechanisms of the AP2/ERF gene family in lettuce, with implications for enhancing abiotic stress tolerance, ultimately contributing to the genetic improvement of lettuce crop production.

Genotyping of Jujube (Ziziphus spp.) Germplasm in New Mexico and Southwestern Texas.

Since the early 19th century, a substantial amount of jujube (Ziziphus spp.) germplasm has been introduced from China and Europe into the United States. However, due to a lack of passport data, cultivar mislabeling is common and the genetic background of the introduced germplasm remains unknown. In the present study, a low-density SNP array was employed to genotype 204 jujube trees sampled from multiple locations in New Mexico, Texas, Missouri, and Kentucky. Multilocus matching of SNP profiles revealed a significant rate of genetic redundancy among these jujube samples. A total of 14 synonymous groups were detected, comprising 48 accessions. Bayesian clustering analysis and neighbor-joining tree partitioned the US jujube germplasm into two major clusters. The first cluster included cultivated genotypes (Ziziphus jujuba Mill.), whereas the other major cluster comprised the wild/sour jujube (Ziziphus spinosa Hu.). The results also revealed a unique jujube population at Fabens/Tornillo, Texas, and a semi-naturalized population at Tucumcari, NM. These findings will provide valuable guidance to jujube growers and researchers on the effective utilization of jujube germplasm in the horticultural industry.

A potential role for an extracellular methanol oxidase secreted by Moniliophthora perniciosa in Witches' broom disease in cacao.

The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, the causal agent of Witches' broom disease (WBD) in cacao, is able to grow on methanol as the sole carbon source. In plants, one of the main sources of methanol is the pectin present in the structure of cell walls. Pectin is composed of highly methylesterified chains of galacturonic acid. The hydrolysis between the methyl radicals and galacturonic acid in esterified pectin, mediated by a pectin methylesterase (PME), releases methanol, which may be decomposed by a methanol oxidase (MOX). The analysis of the M. pernciosa genome revealed putative mox and pme genes. Real-time quantitative RT-PCR performed with RNA from mycelia grown in the presence of methanol or pectin as the sole carbon source and with RNA from infected cacao seedlings in different stages of the progression of WBD indicate that the two genes are coregulated, suggesting that the fungus may be metabolizing the methanol released from pectin. Moreover, immunolocalization of homogalacturonan, the main pectic domain that constitutes the primary cell wall matrix, shows a reduction in the level of pectin methyl esterification in infected cacao seedlings. Although MOX has been classically classified as a peroxisomal enzyme, M. perniciosa presents an extracellular methanol oxidase. Its activity was detected in the fungus culture supernatants, and mass spectrometry analysis indicated the presence of this enzyme in the fungus secretome. Because M. pernciosa possesses all genes classically related to methanol metabolism, we propose a peroxisome-independent model for the utilization of methanol by this fungus, which begins with the extracellular oxidation of methanol derived from the demethylation of pectin and finishes in the cytosol.

The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development.

The tropical pathogen Moniliophthora perniciosa causes witches' broom disease in cacao. As a hemibiotrophic fungus, it initially colonizes the living host tissues (biotrophic phase), and later grows over the dead plant (necrotrophic phase). Little is known about the mechanisms that promote these distinct fungal phases or mediate the transition between them. An alternative oxidase gene (Mp-aox) was identified in the M. perniciosa genome and its expression was analyzed througout the fungal life cycle. In addition, the effects of inhibitors of the cytochrome-dependent respiratory chain (CRC) and alternative oxidase (AOX) were evaluated on the in vitro development of M. perniciosa. Larger numbers of Mp-aox transcripts were observed in the biotrophic hyphae, which accordingly showed elevated sensitivity to AOX inhibitors. More importantly, the inhibition of CRC prevented the transition from the biotrophic to the necrotrophic phase, and the combined use of a CRC and AOX inhibitor completely halted fungal growth. On the basis of these results, a novel mechanism is presented in which AOX plays a role in the biotrophic development of M. perniciosa and regulates the transition to its necrotrophic stage. Strikingly, this model correlates well with the infection strategy of animal pathogens, particularly Trypanosoma brucei, which uses AOX as a strategy for pathogenicity.

The complete genome sequences of Erythroxylum coca and Erythroxylum novogranatense.

The flowering plant genus Erythroxylum contains approximately 300 species, including the economically and socially consequential crops called coca. We present the genome sequences of Erythroxylum coca and E. novogranatense, two cultigens produced for medicinal and quotidian use in the Andes and Amazon regions of South America, as well as the international cocaine industry. Sequencing was performed on an Illumina X-Ten platform, and reads were assembled by a de novo method followed by finishing via comparison with several species from the same genus. The BioProject, raw and assembled data can be accessed in GenBank for E. coca (PRJNA676123; JAJMLV000000000) and E. novogranatense (PRJNA675212; JAJKBF000000000).

United States tea: A synopsis of ongoing tea research and solutions to United States tea production issues.

Tea is a steeped beverage made from the leaves of Camellia sinensis. Globally, this healthy, caffeine-containing drink is one of the most widely consumed beverages. At least 50 countries produce tea and most of the production information and tea research is derived from international sources. Here, we discuss information related to tea production, genetics, and chemistry as well as production issues that affect or are likely to affect emerging tea production and research in the United States. With this review, we relay current knowledge on tea production, threats to tea production, and solutions to production problems to inform this emerging market in the United States.

The chromosome-level genome of dragon fruit reveals whole-genome duplication and chromosomal co-localization of betacyanin biosynthetic genes.

Dragon fruits are tropical fruits economically important for agricultural industries. As members of the family of Cactaceae, they have evolved to adapt to the arid environment. Here we report the draft genome of Hylocereus undatus, commercially known as the white-fleshed dragon fruit. The chromosomal level genome assembly contains 11 longest scaffolds corresponding to the 11 chromosomes of H. undatus. Genome annotation of H. undatus found ~29,000 protein-coding genes, similar to Carnegiea gigantea (saguaro). Whole-genome duplication (WGD) analysis revealed a WGD event in the last common ancestor of Cactaceae followed by extensive genome rearrangements. The divergence time between H. undatus and C. gigantea was estimated to be 9.18 MYA. Functional enrichment analysis of orthologous gene clusters (OGCs) in six Cactaceae plants found significantly enriched OGCs in drought resistance. Fruit flavor-related functions were overrepresented in OGCs that are significantly expanded in H. undatus. The H. undatus draft genome also enabled the discovery of carbohydrate and plant cell wall-related functional enrichment in dragon fruits treated with trypsin for a longer storage time. Lastly, genes of the betacyanin (a red-violet pigment and antioxidant with a very high concentration in dragon fruits) biosynthetic pathway were found to be co-localized on a 12 Mb region of one chromosome. The consequence may be a higher efficiency of betacyanin biosynthesis, which will need experimental validation in the future. The H. undatus draft genome will be a great resource to study various cactus plants.

Mitochondrial Genomics of Six Cacao Pathogens From the Basidiomycete Family Marasmiaceae.

Thread blight disease has recently been described as an emerging disease on cacao (Theobroma cacao) in Ghana. In Ghana, thread blight disease is caused by multiple species of the Marasmiaceae family: Marasmius tenuissimus, M. crinis-equi, M. palmivorus, and Marasmiellus scandens. Interestingly, two additional members of the Marasmiaceae; Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches' broom disease), are major pathogens of cacao in the Western hemisphere. It is important to accurately characterize the genetic relationships among these economically important species in support of their disease management. We used data from Illumina NGS-based genome sequencing efforts to study the mitochondrial genomes (mitogenomes) of the four cacao thread blight associated pathogens from Ghana and compared them with published mitogenomes of Mon. roreri and Mon. perniciosa. There is a remarkable interspecies variation in mitogenome size within the six cacao-associated Marasmiaceae species, ranging from 43,121 to 109,103 bp. The differences in genome lengths are primarily due to the number and lengths of introns, differences in intergenic space, and differences in the size and numbers of unidentified ORFs (uORF). Among seven M. tenuissimus mitogenomes sequenced, there is variation in size and sequence pointing to divergent evolution patterns within the species. The intronic regions show a high degree of sequence variation compared to the conserved sequences of the 14 core genes. The intronic ORFs identified, regardless of species, encode GIY-YIG or LAGLIDADG domain-containing homing endonuclease genes. Phylogenetic relationships using the 14 core proteins largely mimic the phylogenetic relationships observed in gene order patterns, grouping M. tenuissimus with M. crinis-equi, and M. palmivorus with Mon. roreri and Mon. perniciosa, leaving Mar. scandens as an outlier. The results from this study provide evidence of independent expansion/contraction events and sequence diversification in each species and establish a foundation for further exploration of the evolutionary trajectory of the fungi in Marasmiaceae family.

Genes acquired by horizontal transfer are potentially involved in the evolution of phytopathogenicity in Moniliophthora perniciosa and Moniliophthora roreri, two of the major pathogens of cacao.

Moniliophthora perniciosa and Moniliophthora roreri are phytopathogenic basidiomycete species that infect cacao causing two important diseases in this crop: "Witches' Broom" and "Frosty Pod Rot", respectively. The ability of species from this genus (Moniliophthora) to cause disease is exceptional in the family Marasmiaceae. Species in closely related genera including, Marasmius, Crinipellis, and Chaetocalathus, are mainly saprotrophs and are not known to cause disease. In this study, the possibility that this phytopathogenic lifestyle has been acquired by horizontal gene transfer (HGT) was investigated. A stringent genome comparison pipeline was used to identify potential genes that have been obtained by Moniliophthora through HGT. This search led to the identification of three genes: a metallo-dependent hydrolase (MDH), a mannitol phosphate dehydrogenase (MPDH), and a family of necrosis-inducing proteins (NEPs). Phylogenetic analysis of these genes suggests that Moniliophthora acquired NEPs from oomycetes, MDH from actinobacteria and MPDH from firmicutes. Based on the known gene functions and on previous studies of M. perniciosa infection and development, a correlation between gene acquisition and the evolution of the phytopathogenic genus Moniliophthora can be postulated.

Effectiveness of Single Nucleotide Polymorphism Markers in Genotyping Germplasm Collections of Coffea canephora Using KASP Assay.

Accurate genotype identification is imperative for effective use of Coffea canephora L. germplasm to breed new varieties with tolerance or resistance to biotic and abiotic stresses (including moisture stress and pest and disease stresses such as coffee berry borer and rust) and for high yield and improved cup quality. The present study validated 192 published single nucleotide polymorphism (SNP) markers and selected a panel of 120 loci to examine parentage and labeling errors, genetic diversity, and population structure in 400 C. canephora accessions assembled from different coffee-producing countries and planted in a field gene bank in Ghana. Of the 400 genotypes analyzed, both synonymous (trees with same SNP profiles but different names, 12.8%) and homonymous (trees with same name but different SNP profiles, 5.8%) mislabeling were identified. Parentage analysis showed that 33.3% of the progenies derived from controlled crossing and 0% of the progenies derived from an open pollinated biclonal seed garden had parentage (both parents) corresponding to breeder records. The results suggest mislabeling of the mother trees used in seed gardens and pollen contamination from unwanted paternal parents. After removing the duplicated accessions, Bayesian clustering analysis partitioned the 270 unique genotypes into two main populations. Analysis of molecular variance (AMOVA) showed that the between-population variation accounts for 41% of the total molecular variation and the genetic divergence was highly significant (Fst = 0.256; P < 0.001). Taken together, our results demonstrate the effectiveness of using the selected SNP panel in gene bank management, varietal identification, seed garden management, nursery verification, and coffee bean authentication for C. canephora breeding programs.