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.

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.

Clonal reproduction of Moniliophthora roreri and the emergence of unique lineages with distinct genomes during range expansion.

The basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogen's range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens' expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

Bruchid beetle ovipositioning mediated defense responses in black gram pods.

BACKGROUND: Black gram [Vigna mungo (L)] seeds are a rich source of digestible protein and dietary fibre, both for human and animal consumption. However, the quality and quantity of the Vigna seeds are severely affected by bruchid beetles during storage. Therefore, analyses of the expression of the bruchid induced transcript dynamics in black gram pods would be helpful to understand the underlying defense mechanism against bruchid oviposition. RESULTS: We used the RNAseq approach to survey the changes in transcript profile in the developing seeds of a moderately resistant cultivar IC-8219 against bruchid oviposition using a susceptible cultivar T-9 as a control. A total of 96,084,600 and 99,532,488 clean reads were generated from eight (4 each) samples of IC-8219 and T-9 cultivar, respectively. Based on the BLASTX search against the NR database, 32,584 CDSs were generated of which 31,817 CDSs were significantly similar to Vigna radiata, a close relative of Vigna mungo. The IC-8219 cultivar had 630 significantly differentially expressed genes (DEGs) of which 304 and 326 genes up and down-regulated, respectively. However, in the T-9 cultivar, only 168 DEGs were identified of which 142 and 26 genes up and down-regulated, respectively. The expression analyses of 10 DEGs by qPCR confirmed the accuracy of the RNA-Seq data. Gene Ontology and KEGG pathway analyses helped us to better understand the role of these DEGs in oviposition mediated defense response of black gram. In both the cultivars, the most significant transcriptomic changes in response to the oviposition were related to the induction of defense response genes, transcription factors, secondary metabolites, enzyme inhibitors, and signal transduction pathways. It appears that the bruchid ovipositioning mediated defense response in black gram is induced by SA signaling pathways and defense genes such as defensin, genes for secondary metabolites, and enzyme inhibitors could be potential candidates for resistance to bruchids. CONCLUSION: We generated a transcript profile of immature black gram pods upon bruchid ovipositioning by de novo assembly and studied the underlying defense mechanism of a moderately resistant cultivar.

Changes in Gene Expression in Leaves of Cacao Genotypes Resistant and Susceptible to Phytophthora palmivora Infection.

Black pod rot, caused by Phytophthora palmivora, is a devastating disease of Theobroma cacao L. (cacao) leading to huge losses for farmers and limiting chocolate industry supplies. To understand resistance responses of cacao leaves to P. palmivora, Stage 2 leaves of genotypes Imperial College Selection 1 (ICS1), Colección Castro Naranjal 51 (CCN51), and Pound7 were inoculated with zoospores and monitored for symptoms up to 48 h. Pound7 consistently showed less necrosis than ICS1 and CCN51 48 h after inoculation. RNA-Seq was carried out on samples 24 h post inoculation. A total of 24,672 expressed cacao genes were identified, and 2,521 transcripts showed induction in at least one P. palmivora-treated genotype compared to controls. There were 115 genes induced in the P. palmivora-treated samples in all three genotypes. Many of the differentially expressed genes were components of KEGG pathways important in plant defense signal perception (the plant MAPK signaling pathway, plant hormone signal transduction, and plant pathogen interactions), and plant defense metabolite biosynthesis (phenylpropanoid biosynthesis, α-linolenic acid metabolism, ethylene biosynthesis, and terpenoid backbone biosynthesis). A search of putative cacao resistance genes within the cacao transcriptome identified 89 genes with prominent leucine-rich repeat (LRR) domains, 170 protein kinases encoding genes, 210 genes with prominent NB-ARC domains, 305 lectin-related genes, and 97 cysteine-rich RK genes. We further analyzed the cacao leaf transcriptome in detail focusing on gene families-encoding proteins important in signal transduction (MAP kinases and transcription factors) and direct plant defense (Germin-like, ubiquitin-associated, lectin-related, pathogenesis-related, glutathione-S-transferases, and proteases). There was a massive reprogramming of defense gene processes in susceptible cacao leaf tissue after infection, which was restricted in the resistant genotype Pound7. Most genes induced in Pound7 were induced in ICS1/CCN51. The level of induction was not always proportional to the infection level, raising the possibility that genes are responding to infection more strongly in Pound7. There were also defense-associated genes constitutively differentially expressed at higher levels in specific genotypes, possibly providing a prepositioned defense. Many of the defense genes occur in blocks where members are constitutively expressed at different levels, and some members are induced by Ppal infection. With further study, the identified candidate genes and gene blocks may be useful as markers for breeding disease-resistant cacao genotypes against P. palmivora.

Transcript profiling of chickpea pod wall revealed the expression of floral homeotic gene AGAMOUS-like X2 (CaAGLX2).

The differentially expressed genes in the chickpea pod wall have been identified for the first time using a forward suppression subtractive hybridization (SSH) library. In all, 226 clones of SSH library were sequenced and analyzed. A total of 179 high-quality expressed sequence tags (ESTs) were generated and based on the CAP3 assembly of these ESTs, 126 genes (97 singletons and 29 contigs) were computationally annotated. The mapping of 88.26% ESTs by gene ontology (GO) annotation distributed them into 751 GO terms of three categories, cellular location, molecular function, and biological process. The KEGG pathway analysis revealed 45 ESTs are involved in 49 different biological pathways. Also, 67 ESTs encodes four different classes of enzymes such as oxidoreductases (29), transferase (20), hydrolases (16) and isomerase (2). Six genes were selected and subjected to qPCR analysis, of these, two genes (FHG Floral homeotic AGAMOUS-like isoform X2, MADS1 MADS-box transcription factor) showed significant up-regulation in the pod wall compared to leaves. Surprisingly, one of the MADS1 box gene, FHG (CaAGLX2), responsible for flower development expressed in the pod wall. Therefore, understanding its specific role in the pod wall could be interesting. Thus, the transcript dynamics of the chickpea pod wall revealed differentially expressed genes in the pod wall, which may be participating in the metabolic build-up of both pod wall and seeds.

Bruchid egg induced transcript dynamics in developing seeds of black gram (Vigna mungo).

Black gram (Vigna mungo) seeds are a rich source of digestible proteins, however, during storage these seeds are severely damaged by bruchids (Callosobruchus spp.), reducing seed quality and yield losses. Most of the cultivated genotypes of black gram are susceptible to bruchids, however, few tolerant genotypes have also been identified but the mechanism of tolerance is poorly understood. We employed Suppression Subtractive Hybridization (SSH) to identify specifically, but rarely expressed bruchid egg induced genes in black gram. In this study, Suppression Subtractive Hybridization (SSH) library was constructed to study the genes involved in defense response in black gram against bruchid infestation. An EST library of 277 clones was obtained for further analyses. Based on CAP3 assembly, 134 unigenes were computationally annotated using Blast2GOPRO software. In all, 20 defense related genes were subject to quantitative PCR analysis (qPCR) out of which 12 genes showed up-regulation in developing seeds of the pods oviposited by bruchids. Few major defense genes like defensin, pathogenesis related protein (PR), lipoxygenase (LOX) showed high expression levels in the oviposited population when compared with the non-oviposited plants. This is the first report on defense related gene transcript dynamics during the bruchid-black gram interaction using SSH library. This library would be useful to clone defense related gene(s) such as defensin as represented in our library for crop improvement.