Unveiling unknown transcripts and exons: Insights into durian var. D24 (Durio zibethinus Murr.) fruit development and ripening

BackgroundDeep RNA sequencing (RNAseq) has opened a new horizon for understanding global gene expression. The functional annotation of non-model mammalian genomes including bovines is still poor compared to that of human and mouse. This particularly applies to tissues without direct significance for milk and meat production, like skin, in spite of its multifunctional relevance for the individual. Thus, applying an RNAseq approach, we performed a whole transcriptome analysis of pigmented and nonpigmented bovine skin to describe the comprehensive transcript catalogue of this tissue.ResultsA total of 39,577 unique primary skin transcripts were mapped to the bovine reference genome assembly. The majority of the transcripts were mapped to known transcriptional units (65%). In addition to the reannotation of known genes, a substantial number (10,884) of unknown transcripts (UTs) were discovered, which had not previously been annotated. The classification of UTs was based on the prediction of their coding potential and comparative sequence analysis, subsequently followed by meticulous manual curation. The classification analysis and experimental validation of selected UTs confirmed that RNAseq data can be used to amend the annotation of known genes by providing evidence for additional exons, untranslated regions or splice variants, by approving genes predicted in silico and by identifying novel bovine loci. A large group of UTs (4,848) was predicted to potentially represent long noncoding RNA (lncRNA). Predominantly, potential lncRNAs mapped in intergenic chromosome regions (4,365) and therefore, were classified as potential intergenic lncRNA. Our analysis revealed that only about 6% of all UTs displayed interspecies conservation and discovered a variety of unknown transcripts without interspecies homology but specific expression in bovine skin.ConclusionsThe results of our study demonstrate a complex transcript pattern for bovine skin and suggest a possible functional relevance of novel transcripts, including lncRNA, in the modulation of pigmentation processes. The results also indicate that the comprehensive identification and annotation of unknown transcripts from whole transcriptome analysis using RNAseq data remains a tremendous future challenge.

Populations of Noccaea caerulescens show tremendous differences in their capacity to hyperaccumulate and hypertolerate metals. To explore the differences that could contribute to these traits, we undertook SOLiD high-throughput sequencing of the root transcriptomes of three phenotypically well-characterized N. caerulescens accessions, i.e., Ganges, La Calamine, and Monte Prinzera. Genes with possible contribution to zinc, cadmium, and nickel hyperaccumulation and hypertolerance were predicted. The most significant differences between the accessions were related to metal ion (di-, trivalent inorganic cation) transmembrane transporter activity, iron and calcium ion binding, (inorganic) anion transmembrane transporter activity, and antioxidant activity. Analysis of correlation between the expression profile of each gene and the metal-related characteristics of the accessions disclosed both previously characterized (HMA4, HMA3) and new candidate genes (e.g., for nickel IRT1, ZIP10, and PDF2.3) as possible contributors to the hyperaccumulation/tolerance phenotype. A number of unknown Noccaea-specific transcripts also showed correlation with Zn(2+), Cd(2+), or Ni(2+) hyperaccumulation/tolerance. This study shows that N. caerulescens populations have evolved great diversity in the expression of metal-related genes, facilitating adaptation to various metalliferous soils. The information will be helpful in the development of improved plants for metal phytoremediation.

Durian (Durio zibethinus) is a tropical fruit that has a unique flavor and aroma. It occupies a significant phylogenetic position within the Malvaceae family. Extant core-eudicot plants are reported to share seven ancestral karyotypes that have undergone reshuffling, resulting in an abundant genomic diversity. However, the ancestral karyotypes of the Malvaceae family, as well as the evolution trajectory leading to the 28 chromosomes in durian, remain poorly understood. Here, we report the high-quality assembly of the durian genome with comprehensive comparative genomic analyses. By analyzing the collinear blocks between cacao and durian, we inferred 11 Malvaceae ancestral karyotypes. These blocks were present in a single-copy form in cacao and mainly in triplicates in durian, possibly resulting from a recent whole genome triplication (WGT) event that led to hexaploidization of the durian genome around 20 (17-24) million years ago. A large proportion of the duplicated genes in durian, such as those involved in the lignin biosynthesis module for phenylpropane biosynthesis, are derived directly from whole genome duplication, which makes it an important force in reshaping its genomic architecture. Transcriptome studies have revealed that genes involved in feruloyl-CoA formations were highly preferentially expressed in fruit peels, indicating that the thorns produced on durian fruit may comprise guaiacyl and syringyl lignins. Among all the analyzed transcription factors (TFs), members of the heat shock factor family (HSF) were the most significantly upregulated under heat stress. All subfamilies of genes encoding heat shock proteins (HSPs) in the durian genome appear to have undergone expansion. The potential interactions between HSF Dzi05.397 and HSPs were examined and experimentally verified. Our study provides a high-quality durian genome and reveals the reshuffling mechanism of ancestral Malvaceae chromosomes to produce the durian genome. We also provide insights into the mechanism underlying lignin biosynthesis and heat stress tolerance.

The number of sequenced bacteriophage genomes is growing at an exponential rate. The majority of sequenced bacteriophage genomes are annotated by one or more of several freely available gene identification programs (Glimmer, GeneMark, RAST, Prodigal, etc.). No program has been shown to consistently outperform the others; thus, the choice of which program to use is not obvious. We present the Phage Commander application for rapid identification of bacteriophage genes using multiple gene identification programs. Phage Commander runs a bacteriophage genome sequence through nine gene identification programs (and an additional program for identification of tRNAs) and integrates the results within a single output table. Phage Commander also generates formatted output files for direct export to National Center for Biotechnology Information GenBank or genome visualization programs such as DNA Master. Users can select the threshold for which genes to export (genes identified by at least one program, genes identified by at least two programs, etc.). Phage Commander was benchmarked using eight high-quality bacteriophage genomes whose genes are backed by experimental data. Our results show that the most accurate annotations are obtained by exporting genes identified by at least two or three programs. Many groups opt to manually curate the annotations obtained from gene identification programs, and Phage Commander was designed to facilitate manual curation of genome annotations. Our benchmarking results show that manual curation does indeed produce more accurate annotations than any individual gene identification program. The authors thus recommend manually curating the output of Phage Commander to generate maximally accurate annotations. Phage Commander is currently being used in the corresponding author's bacteriophage genome annotation class and has reduced the labor cost and improved the quality of genome annotations.

Cocoa (Theobroma cacao L.) is a cash crop of huge economic significance in the world and the key raw material for chocolate manufacturing. Polyphenol oxidases (PPOs) are copper containing oxidoreductases that catalyze the hydroxylation and oxidation of phenolic compounds in the presence of molecular oxygen. The PPO presence and activity during fermentation and drying of cocoa beans is responsible for the development of flavor precursors. In this study, bioinformatics analysisof PPO from cocoa was done using standard bioinformatics tools such as Blastp, Hmmer, ClustalO, OMABrowser, EMBOSS and Swissmodel. The result showed that cocoa PPO homologs include Citrus sinensis, Populus euphratica, Gossypium raimondii, Litchi chinensis, Dimocarpuslongan, Canarium album, while the orthologsobtainedinclude Sorghum bicolor, Zea may, Manihot esculenta and Vitis vinifera among others.PPO of Solanum lycopersicum (tomato) was found to be a distant homolog of cocoa PPO, and that this evolutionary relationship implicated Witches’ Broom Disease (WBD). The computed physicochemical properties were in alignment with the experimental results. Keywords: Polyphenol Oxidase, Cocoa, Theobroma cacao L., Computational Analysis, Experimental Analysis.

Within developing countries, there is an appeal to use waste biomass for energy generation in the form of bio-briquettes. This study investigated the potential use of bio-briquettes that are produced from the waste biomass of the following tropical fruits: durian (Durio zibethinus), coconut (Cocos nucifera), coffee (Coffea arabica), cacao (Theobroma cacao), banana (Musa acuminata) and rambutan (Nephelium lappaceum). All fruit waste biomass samples exhibited an extremely high level of initial moisture content (78.22% in average). Fruit samples with the highest proportion of fruit waste biomass (of total unprocessed fruit mass) were represented by cacao (83.82%), durian (62.56%) and coconut (56.83%). Highest energy potentials (calorific value) of fruit waste biomass were observed in case of coconut (18.22 MJ∙kg−1), banana (17.79 MJ∙kg−1) and durian (17.60 MJ∙kg−1) fruit samples, whereas fruit waste biomass with the lowest level of ash content originated from the rambutan (3.67%), coconut (4.52%), and durian (5.05%) fruit samples. When investigating the energy demands to produce bio-briquettes from such feedstock materials, the best results (lowest amount of required deformation energy in combination with highest level of bio-briquette bulk density) were achieved by the rambutan, durian and banana fruit waste biomass samples. Finally, all investigated bio-briquette samples presented satisfactory levels of bulk density (>1050 kg∙m−3). In conclusion, our results indicated the practicability and viability of such bio-briquette fuel production, as well as supporting the fact that bio-briquettes from tropical fruit waste biomass can offer a potentially attractive energy source with many benefits, especially in rural areas.

BackgroundThe African clawed frogs Xenopus laevis and Xenopus tropicalis are prominent animal model organisms. Xenopus research contributes to the understanding of genetic, developmental and molecular mechanisms underlying human disease. The Xenopus Anatomy Ontology (XAO) reflects the anatomy and embryological development of Xenopus. The XAO provides consistent terminology that can be applied to anatomical feature descriptions along with a set of relationships that indicate how each anatomical entity is related to others in the embryo, tadpole, or adult frog. The XAO is integral to the functionality of Xenbase (http://www.xenbase.org), the Xenopus model organism database.ResultsWe significantly expanded the XAO in the last five years by adding 612 anatomical terms, 2934 relationships between them, 640 synonyms, and 547 ontology cross-references. Each term now has a definition, so database users and curators can be certain they are selecting the correct term when specifying an anatomical entity. With developmental timing information now asserted for every anatomical term, the ontology provides internal checks that ensure high-quality gene expression and phenotype data annotation. The XAO, now with 1313 defined anatomical and developmental stage terms, has been integrated with Xenbase expression and anatomy term searches and it enables links between various data types including images, clones, and publications. Improvements to the XAO structure and anatomical definitions have also enhanced cross-references to anatomy ontologies of other model organisms and humans, providing a bridge between Xenopus data and other vertebrates. The ontology is free and open to all users.ConclusionsThe expanded and improved XAO allows enhanced capture of Xenopus research data and aids mechanisms for performing complex retrieval and analysis of gene expression, phenotypes, and antibodies through text-matching and manual curation. Its comprehensive references to ontologies across taxa help integrate these data for human disease modeling.

Cotton leafroll dwarf virus (genus Polerovirus, family Solemoviridae) has been commonly reported affecting cotton plants (Gossypium spp., family Malvaceae) and several weed species (Ramos-Sobrinho et al., 2021; Sedhain et al., 2021). During a recent survey, cacao (Theobroma cacao L.) trees exhibiting virus-like symptoms such as leaf mosaic, vein clearing, and yellow spot were observed in the south part of the state of Bahia, northeastern Brazil, in 2022. Leaf samples were randomly collected from symptomatic cacao plants (n=30) growing in an affected area of approximately 30 ha. Total RNA obtained from pooled cacao samples were subjected to Illumina HiSeq 2500 sequencing as previously described (Keith et al., 2021), and partial sequences of cotton leafroll dwarf virus (CLRDV), and other virus-specific sequence contigs, were de novo assembled according to Ramos-Sobrinho et al. (2021). To further investigate the presence of CLRDV in cacao leaves, total RNA was individually extracted using a modified silica protocol (Rott and Jelkmann, 2001) and used as template for cDNA synthesis with random hexamers using the SuperScript™ IV First-Strand Synthesis System (Invitrogen, CA, USA) following the manufacturer´s protocol. Detection of CLRDV was carried out by reverse transcription-polymerase chain reaction (RT-PCR) with the primers PL4F and o3-R, which amplify the open reading frame 3 (ORF3) encoding the capsid protein (Corrêa et al., 2005). Expected size amplicons (~0.6 kb) were observed from 16 out of 30 symptomatic plants, indicating ~53% of the cacao trees were infected by CLRDV. Considering 14 symptomatic plants tested negative for CLRDV, the symptoms observed here could also be caused by other viral groups or abiotic stress. To confirm the detection of CLRDV, the first half (~3.5kb) of the viral genome was amplified from two representative cacao samples using the primers P20F and P22R (Avelar et al., 2020). The RT-PCR products were gel-purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, WI, USA) and Sanger sequenced. The RNA Illumina sequencing from pooled cacao samples (n=30) yielded 34,610,572 million trimmed reads. Two contigs of 868 and 839 nucleotides (nt) in length, and sharing high nt identity with CLRDV isolates, were assembled from 6,903 and 10,271 reads, at a coverage depth of 795 and 1,224x, respectively. Together, these contigs represent ~29% of the complete viral genome and included part of the 5´-untraslated region, ORF0 and the second half of ORF1-ORF2. Additional CLRDV-like contigs were observed across the viral genome, but they were not considered for further analyses due to the poor sequence quality. The Illumina- and Sanger-derived ORF0 and partial ORF1-ORF2 sequences shared >97% nt identity, suggesting they were congruent. Pairwise sequence comparisons for ORF0, encoding the gene silencing suppressor P0, indicated the cacao-associated isolates shared 99.7 and 99.2% nt and amino acid (aa) identity one with another, respectively. The ORF0 nt sequences showed 91.9-93.8 and 90.7-93.6% identity, while the aa sequences shared 85.8-88.5 and 86.2-90.0% similarity, with CLRDV isolates previously reported in South America and the USA, respectively. Finally, the ~3.5kb nt sequences of cacao-infecting CLRDV isolates shared 92.9-95.8% identity with CLRDV genomes deposited in NCBI-GenBank. The Bayesian phylogenetic tree reconstructed based on ORF0 nt sequences showed the new sequences were more closely related to CLRDV-atypical isolates (GenBank accession nos. KF359946, KF359947, KF906260, and KF906261). Together, these results suggest the new ORF0 sequences belong to CLRDV and were deposited in GenBank under accession nos. ON954058-ON954059. To our knowledge, this is the first report of CLRDV infecting cacao plants, expanding the range of malvaceous hosts of this polerovirus. CLRDV is largely known for causing yield losses in cotton crops, but additional studies are needed to determine if CLRDV infection is detrimental to cacao production.

Indonesia is the third largest cacao (Theobroma cacao L.) producer in the world and also well-known for its fine cacao varieties (Java fine-flavor cacao). Indonesian fine cacao breeding program will be accelerated by early detection of its specific trait through the use of molecular marker. One of the traits thatcould differentiate fine and bulk cacao, in this case Criollo and Forastero, respectively, is the pod color. Previous research reported that MYB transcription factor gene regulated cacao pod color and was able to differentiate Criollo from Forastero. The gene involved in the control of plant-specific processes including primary and secondary metabolism, cell fate and identity, developmental processes and responses to biotic and abiotic stresses. This research aimed to identify the diversity of Indonesian fine and bulk cacao based on MYB nucleotide sequence fragments. Identification of the MYB nucleotide sequence was conducted by DNA isolation from cacao leaves and specific primer design based on two cacao MYB transcription factor ene accessions. These primers were used to evaluate the diversity of three Indonesian fine cacao (DRC 16, PNT 16, and ICCRI 01) and two bulk cacao (PA 191 and ICCRI 03) clones. The cluster analysis showed that this specific primer is similar to other MYB gene accessions in Malvaceae family (Theobroma, Herrania, Gossypium). It is also able to differentiate bulk and fine cacao in accordance to their pedigree. The primer developed in this study could be used for further analysis of Indonesian fine cacao molecular marker.

Bacteriophages are the most numerous entities on Earth. The number of sequenced phage genomes is approximately 8000 and increasing rapidly. Sequencing of a genome is followed by annotation, where genes, start codons, and functions are putatively identified. The mainstays of phage genome annotation are auto-annotation programs such as Glimmer and GeneMark. Due to the relatively small size of phage genomes, many groups choose to manually curate auto-annotation results to increase accuracy. An additional benefit of manual curation of auto-annotated phage genomes is that the process is amenable to be performed by students, and has been shown to improve student recruitment to the sciences. However, despite its greater accuracy and pedagogical value, manual curation suffers from high labor cost, lack of standardization and a degree of subjectivity in decision making, and susceptibility to mistakes. Here, we present a method developed in our lab that is designed to produce accurate annotations while reducing subjectivity and providing a degree of standardization in decision-making. We show that our method produces genome annotations more accurate than auto-annotation programs while retaining the pedagogical benefits of manual genome curation.

A comprehensive identification and function analysis of the ATBS1 Interacting Factors (AIFs) gene family of Gossypium species in fiber development and under multiple stresses

BackgroundJute is considered one of the most important crops for fiber production and multipurpose usages. Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a crucial enzyme involved in lignin biosynthesis in plants. The potential functions of CCoAOMT in lignin biosynthesis of jute have been reported in several studies. However, little is known about the evolution of the CCoAOMT gene family, and either their expression level at different developing stages in different jute cultivars, as well as under abiotic stresses including salt and drought stress.ResultsIn the present study, 66 CCoAOMT genes from 12 species including 12 and eight CCoAOMTs in Corchorus olitorius and C. capsularis were identified. Phylogenetic analysis revealed that CCoAOMTs could be divided into six groups, and gene expansion was observed in C. olitorius. Furthermore, gene expression analysis of developing jute fibers was conducted at different developmental stages (15, 30, 45, 60, and 90 days after sowing [DAS]) in six varieties (Jute-179 [J179], Lubinyuanguo [LB], and Qiongyueqing [QY] for C. capsularis; Funong No.5 [F5], Kuanyechangguo [KY], and Cvlv [CL] for C. olitorius). The results showed that CCoAOMT1 and CCoAOMT2 were the dominant genes in the CCoAOMT family. Of these two dominant CCoAOMTs, CCoAOMT2 showed a constitutive expression level during the entire growth stages, while CCoAOMT1 exhibited differential expression patterns. These two genes showed higher expression levels in C. olitorius than in C. capsularis. The correlation between lignin content and CCoAOMT gene expression levels indicated that this gene family influences the lignin content of jute. Using real-time quantitative reverse transcription PCR (qRT-PCR), a substantial up-regulation of CCoAOMTs was detected in stem tissues of jute 24 h after drought treatment, with an up to 17-fold increase in expression compared to that of untreated plants.ConclusionsThis study provides a basis for comprehensive genomic studies of the entire CCoAOMT gene family in C. capsularis and C. olitorius. Comparative genomics analysis among the CCoAOMT gene families of 12 species revealed the close evolutionary relationship among Corchorus, Theobroma cacao and Gossypium raimondii. This study also shows that CCoAOMTs are not only involved in lignin biosynthesis, but also are associated with the abiotic stress response in jute, and suggests the potential use of these lignin-related genes to genetically improve the fiber quality of jute.

Lipid droplet-associated proteins (LDAPs) play essential roles in tissue growth and development and in drought stress responses in plants. Cotton is an important fiber and cash crop; however, the LDAP family has not been characterized in cotton. In this study, a total of 14, six, seven, and seven genes were confirmed as LDAP family members in Gossypium hirsutum, Gossypium raimondii, Gossypium arboreum, and Gossypium stocksii, respectively. Additionally, expansion in the LDAP family occurred with the formation of Gossypium, which is mirrored in the number of LDAPs found in five Malvaceae species (Gossypioides kirkii, Bombax ceiba, Durio zibethinus, Theobroma cacao, and Corchorus capsularis), Arabidopsis thaliana, and Carica papaya. The phylogenetic tree showed that the LDAP genes in cotton can be divided into three groups (I, II, and III). The analysis of gene structure and conserved domains showed that LDAPs derived from group I (LDAP1/2/3) are highly conserved during evolution, while members from groups II and III had large variations in both domains and gene structures. The gene expression pattern analysis of LDAP genes showed that they are expressed not only in the reproductive organs (ovule) but also in vegetative organs (root, stem, and leaves). The expression level of two genes in group III, GhLDAP6_At/Dt, were significantly higher in fiber development than in other tissues, indicating that it may be an important regulator of cotton fiber development. In group III, GhLDAP2_At/Dt, especially GhLDAP2_Dt was strongly induced by various abiotic stresses. Decreasing the expression of GhLDAP2_Dt in cotton via virus-induced gene silencing increased the drought sensitivity, and the over-expression of GhLDAP2_Dt led to increased tolerance to mannitol-simulated osmotic stress at the germination stage. Thus, we conclude that GhLDAP2_Dt plays a positive role in drought tolerance.

Hibiscus syriacus (L.) (rose of Sharon) is one of the most widespread garden shrubs in the world. We report a draft of the H. syriacus genome comprised of a 1.75 Gb assembly that covers 92% of the genome with only 1.7% (33 Mb) gap sequences. Predicted gene modeling detected 87,603 genes, mostly supported by deep RNA sequencing data. To define gene family distribution among relatives of H. syriacus, orthologous gene sets containing 164,660 genes in 21,472 clusters were identified by OrthoMCL analysis of five plant species, including H. syriacus, Arabidopsis thaliana, Gossypium raimondii, Theobroma cacao and Amborella trichopoda. We inferred their evolutionary relationships based on divergence times among Malvaceae plant genes and found that gene families involved in flowering regulation and disease resistance were more highly divergent and expanded in H. syriacus than in its close relatives, G. raimondii (DD) and T. cacao. Clustered gene families and gene collinearity analysis revealed that two recent rounds of whole-genome duplication were followed by diploidization of the H. syriacus genome after speciation. Copy number variation and phylogenetic divergence indicates that WGDs and subsequent diploidization led to unequal duplication and deletion of flowering-related genes in H. syriacus and may affect its unique floral morphology.

From the raw seed to chocolate: Volatile profile of Blanco de Criollo in different phases of the processing chain