Whole-genome sequencing of a year-round fruiting jackfruit (Artocarpus heterophyllus Lam.) reveals high levels of single nucleotide variation.

Jackfruit (Artocarpus heterophyllus Lam.) is the national fruit of Bangladesh and produces fruit in the summer season only. However, jackfruit is not commercially grown in Bangladesh because of an extremely high variation in fruit quality, short seasonal fruiting (June-August) and susceptibility to abiotic stresses. Conversely, a year-round high yielding (ca. 4-fold higher than the seasonal variety) jackfruit variety, BARI Kanthal-3 developed by the Bangladesh Agricultural Research Institute (BARI) derived from a wild accession found in Ramgarh of Chattogram Hiltracts of Bangladesh, provides fruits from September to June. This study aimed to generate a draft whole-genome sequence (WGS) of BARI Kanthal-3 to obtain molecular insights including genes associated with year-round fruiting trait of this important unique variety. The estimated genome size of BARI Kanthal-3 was 1.04-gigabase-pair (Gbp) with a heterozygosity rate of 1.62%. De novo assembly yielded a scaffolded 817.7 Mb genome while a reference-guided approach, yielded 843 Mb of genome sequence. The estimated GC content was 34.10%. Variant analysis revealed that BARI Kanthal-3 included 5.7 M (35%) and 10.4 M (65%) simple and heterozygous single nucleotide polymorphisms (SNPs), and about 90% of all these polymorphisms are in inter-genic regions. Through BUSCO assessment, 97.2% of the core genes were represented in the assembly with 1.3% and 1.5% either fragmented or missing, respectively. By comparing identified orthologous gene groups in BARI Kanthal-3 with five closely and one distantly related species of 10,092 common orthogroups were found across the genomes of the six species. The phylogenetic analysis of the shared orthogroups showed that A. heterophyllus was the closest species to BARI Kanthal-3 and orthogroups related to flowering time were found to be more highly prevalent in BARI Kanthal-3 compared to the other Arctocarpus spp. The findings of this study will help better understanding the evolution, domestication, phylogenetic relationships, year-round fruiting of this highly nutritious fruit crop as well as providing a resource for molecular breeding.

Genome structural evolution in Brassica crops.

The cultivated Brassica species include numerous vegetable and oil crops of global importance. Three genomes (designated A, B and C) share mesohexapolyploid ancestry and occur both singly and in each pairwise combination to define the Brassica species. With organizational errors (such as misplaced genome segments) corrected, we showed that the fundamental structure of each of the genomes is the same, irrespective of the species in which it occurs. This enabled us to clarify genome evolutionary pathways, including updating the Ancestral Crucifer Karyotype (ACK) block organization and providing support for the Brassica mesohexaploidy having occurred via a two-step process. We then constructed genus-wide pan-genomes, drawing from genes present in any species in which the respective genome occurs, which enabled us to provide a global gene nomenclature system for the cultivated Brassica species and develop a methodology to cost-effectively elucidate the genomic impacts of alien introgressions. Our advances not only underpin knowledge-based approaches to the more efficient breeding of Brassica crops but also provide an exemplar for the study of other polyploids.

The tepary bean genome provides insight into evolution and domestication under heat stress.

Tepary bean (Phaseolus acutifolis A. Gray), native to the Sonoran Desert, is highly adapted to heat and drought. It is a sister species of common bean (Phaseolus vulgaris L.), the most important legume protein source for direct human consumption, and whose production is threatened by climate change. Here, we report on the tepary genome including exploration of possible mechanisms for resilience to moderate heat stress and a reduced disease resistance gene repertoire, consistent with adaptation to arid and hot environments. Extensive collinearity and shared gene content among these Phaseolus species will facilitate engineering climate adaptation in common bean, a key food security crop, and accelerate tepary bean improvement.

Long noncoding miRNA gene represses wheat ß-diketone waxes.

The cuticle of terrestrial plants functions as a protective barrier against many biotic and abiotic stresses. In wheat and other Triticeae, ß-diketone waxes are major components of the epicuticular layer leading to the bluish-white glaucous trait in reproductive-age plants. Glaucousness in durum wheat is controlled by a metabolic gene cluster at the WAX1 (W1) locus and a dominant suppressor INHIBITOR of WAX1 (Iw1) on chromosome 2B. The wheat D subgenome from progenitor Aegilops tauschii contains W2 and Iw2 paralogs on chromosome 2D. Here we identify the Iw1 gene from durum wheat and demonstrate the unique regulatory mechanism by which Iw1 acts to suppress a carboxylesterase-like protein gene, W1-COE, within the W1 multigene locus. Iw1 is a long noncoding RNA (lncRNA) containing an inverted repeat (IR) with >80% identity to W1-COE The Iw1 transcript forms a miRNA precursor-like long hairpin producing a 21-nt predominant miRNA, miRW1, and smaller numbers of related sRNAs associated with the nonglaucous phenotype. When Iw1 was introduced into glaucous bread wheat, miRW1 accumulated, W1-COE and its paralog W2-COE were down-regulated, and the phenotype was nonglaucous and ß-diketone-depleted. The IR region of Iw1 has >94% identity to an IR region on chromosome 2 in Ae. tauschii that also produces miRW1 and lies within the marker-based location of Iw2 We propose the Iw loci arose from an inverted duplication of W1-COE and/or W2-COE in ancestral wheat to form evolutionarily young miRNA genes that act to repress the glaucous trait.

Analysis of Genotyping-by-Sequencing (GBS) Data.

The development of genotyping-by-sequencing (GBS) to rapidly detect nucleotide variation at the whole genome level, in many individuals simultaneously, has provided a transformative genetic profiling technique. GBS can be carried out in species with or without reference genome sequences yields huge amounts of potentially informative data. One limitation with the approach is the paucity of tools to transform the raw data into a format that can be easily interrogated at the genetic level. In this chapter we describe bioinformatics tools developed to address this shortfall together with experimental design considerations to fully leverage the power of GBS for genetic analysis.

Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea.

BACKGROUND: Brassica oleracea is a valuable vegetable species that has contributed to human health and nutrition for hundreds of years and comprises multiple distinct cultivar groups with diverse morphological and phytochemical attributes. In addition to this phenotypic wealth, B. oleracea offers unique insights into polyploid evolution, as it results from multiple ancestral polyploidy events and a final Brassiceae-specific triplication event. Further, B. oleracea represents one of the diploid genomes that formed the economically important allopolyploid oilseed, Brassica napus. A deeper understanding of B. oleracea genome architecture provides a foundation for crop improvement strategies throughout the Brassica genus. RESULTS: We generate an assembly representing 75% of the predicted B. oleracea genome using a hybrid Illumina/Roche 454 approach. Two dense genetic maps are generated to anchor almost 92% of the assembled scaffolds to nine pseudo-chromosomes. Over 50,000 genes are annotated and 40% of the genome predicted to be repetitive, thus contributing to the increased genome size of B. oleracea compared to its close relative B. rapa. A snapshot of both the leaf transcriptome and methylome allows comparisons to be made across the triplicated sub-genomes, which resulted from the most recent Brassiceae-specific polyploidy event. CONCLUSIONS: Differential expression of the triplicated syntelogs and cytosine methylation levels across the sub-genomes suggest residual marks of the genome dominance that led to the current genome architecture. Although cytosine methylation does not correlate with individual gene dominance, the independent methylation patterns of triplicated copies suggest epigenetic mechanisms play a role in the functional diversification of duplicate genes.

The emerging biofuel crop Camelina sativa retains a highly undifferentiated hexaploid genome structure.

Camelina sativa is an oilseed with desirable agronomic and oil-quality attributes for a viable industrial oil platform crop. Here we generate the first chromosome-scale high-quality reference genome sequence for C. sativa and annotated 89,418 protein-coding genes, representing a whole-genome triplication event relative to the crucifer model Arabidopsis thaliana. C. sativa represents the first crop species to be sequenced from lineage I of the Brassicaceae. The well-preserved hexaploid genome structure of C. sativa surprisingly mirrors those of economically important amphidiploid Brassica crop species from lineage II as well as wheat and cotton. The three genomes of C. sativa show no evidence of fractionation bias and limited expression-level bias, both characteristics commonly associated with polyploid evolution. The highly undifferentiated polyploid genome of C. sativa presents significant consequences for breeding and genetic manipulation of this industrial oil crop.

MicroRNAs and their putative targets in Brassica napus seed maturation.

BACKGROUND: MicroRNAs (miRNAs) are 20-21 nucleotide RNA molecules that suppress the transcription of target genes and may also inhibit translation. Despite the thousands of miRNAs identified and validated in numerous plant species, only small numbers have been identified from the oilseed crop plant Brassica napus (canola) - especially in seeds. RESULTS: Using next-generation sequencing technologies, we performed a comprehensive analysis of miRNAs during seed maturation at 9 time points from 10 days after flowering (DAF) to 50 DAF using whole seeds and included separate analyses of radicle, hypocotyl, cotyledon, embryo, endosperm and seed coat tissues at 4 selected time points. We identified more than 500 conserved miRNA or variant unique sequences with >300 sequence reads and also found 10 novel miRNAs. Only 27 of the conserved miRNA sequences had been previously identified in B. napus (miRBase Release 18). More than 180 MIRNA loci were identified/annotated using the B. rapa genome as a surrogate for the B.napus A genome. Numerous miRNAs were expressed in a stage- or tissue-specific manner suggesting that they have specific functions related to the fine tuning of transcript abundance during seed development. miRNA targets in B. napus were predicted and their expression patterns profiled using microarray analyses. Global correlation analysis of the expression patterns of miRNAs and their targets revealed complex miRNA-target gene regulatory networks during seed development. The miR156 family was the most abundant and the majority of the family members were primarily expressed in the embryo. CONCLUSIONS: Large numbers of miRNAs with diverse expression patterns, multiple-targeting and co-targeting of many miRNAs, and complex relationships between expression of miRNAs and targets were identified in this study. Several key miRNA-target expression patterns were identified and new roles of miRNAs in regulating seed development are suggested. miR156, miR159, miR172, miR167, miR158 and miR166 are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development. miR156 may regulate the developmental transition to germination.

Transcriptome profiling and methyl homeostasis of an Arabidopsis mutant deficient in S-adenosylhomocysteine hydrolase1 (SAHH1).

Transcriptome profiling was conducted to detect genes whose expression is significantly changed in an Arabidopsis mutant deficient in S-adenosylhomocysteine hydrolase1 (SAHH1) during early seedling development when mutant phenotypes could be clearly observed. A total of 2,040 differentially expressed genes were identified, representing approximately 6.7% of the 30,385 DNA oligonucleotide targets on the microarray. Among these differential expressed genes, many were mapped to pathways essential to plant growth and development including those of primary, secondary and hormone metabolisms. A significant proportion of up-regulated genes encoded transposable elements which were mapped to the centromeric and pericentromeric regions of the Arabidopsis chromosomes that were analyzed. A number of down-regulated genes were found to be involved in root hair formation, which might have contributed to the root hair defective phenotype of the mutant. Analysis of genes encoding transposable elements and those associating with root hair development indicated that these genes were highly co-expressed during seedling development. Despite SAHH1 deficiency, the expression of genes encoding methyltransferase remained largely unchanged in the sahh1 mutant. Bisulfite sequencing analysis of the transposable elements and the FWA gene revealed that their sequences in the mutant were deficient of 5-methylcytosines. Analysis of mutant genomic DNA using restriction endonucleases that were unable to cut methylated DNA suggested a genome-wide hypomethylation had occurred in the mutant. These results indicated that SAHH1 plays a critical role in methyl homeostasis, and its deficiency is a major contributing factor to the change of global gene expression, metabolic pathways and activation of transposable elements in the sahh1 mutant.

The genome of the mesopolyploid crop species Brassica rapa.

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.

Using a state-space model and location analysis to infer time-delayed regulatory networks.

Computational gene regulation models provide a means for scientists to draw biological inferences from time-course gene expression data. Based on the state-space approach, we developed a new modeling tool for inferring gene regulatory networks, called time-delayed Gene Regulatory Networks (tdGRNs). tdGRN takes time-delayed regulatory relationships into consideration when developing the model. In addition, a priori biological knowledge from genome-wide location analysis is incorporated into the structure of the gene regulatory network. tdGRN is evaluated on both an artificial dataset and a published gene expression data set. It not only determines regulatory relationships that are known to exist but also uncovers potential new ones. The results indicate that the proposed tool is effective in inferring gene regulatory relationships with time delay. tdGRN is complementary to existing methods for inferring gene regulatory networks. The novel part of the proposed tool is that it is able to infer time-delayed regulatory relationships.

Cancer-associated regulation of alternative splicing.

Alternative splicing of pre-mRNA increases the diversity of protein functions. Here we show that about half of all active alternative splicing events in ovarian and breast tissues are changed in tumors, and many seem to be regulated by a single factor; sequence analysis revealed binding sites for the RNA binding protein FOX2 downstream of one-third of the exons skipped in cancer. High-resolution analysis of FOX2 binding sites defined the precise positions relative to alternative exons at which the protein may function as either a silencer or an enhancer. Most of the identified targets were shifted in the same direction by FOX2 depletion in cell lines as they were in breast and ovarian cancer tissues. Notably, we found expression of FOX2 itself is downregulated in ovarian cancer and its splicing is altered in breast cancer samples. These results suggest that the decreased expression of FOX2 in cancer tissues modulates splicing and controls proliferation.

Identification of alternative splicing markers for breast cancer.

Breast cancer is the most common cause of cancer death among women under age 50 years, so it is imperative to identify molecular markers to improve diagnosis and prognosis of this disease. Here, we present a new approach for the identification of breast cancer markers that does not measure gene expression but instead uses the ratio of alternatively spliced mRNAs as its indicator. Using a high-throughput reverse transcription-PCR-based system for splicing annotation, we monitored the alternative splicing profiles of 600 cancer-associated genes in a panel of 21 normal and 26 cancerous breast tissues. We validated 41 alternative splicing events that significantly differed in breast tumors relative to normal breast tissues. Most cancer-specific changes in splicing that disrupt known protein domains support an increase in cell proliferation or survival consistent with a functional role for alternative splicing in cancer. In a blind screen, a classifier based on the 12 best cancer-associated splicing events correctly identified cancer tissues with 96% accuracy. Moreover, a subset of these alternative splicing events could order tissues according to histopathologic grade, and 5 markers were validated in a further blind set of 19 grade 1 and 19 grade 3 tumor samples. These results provide a simple alternative for the classification of normal and cancerous breast tumor tissues and underscore the putative role of alternative splicing in the biology of cancer.

Multiple and specific mRNA processing targets for the major human hnRNP proteins.

Alternative splicing is a key mechanism regulating gene expression, and it is often used to produce antagonistic activities particularly in apoptotic genes. Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins form a family of RNA-binding proteins that coat nascent pre-mRNAs. Many but not all major hnRNP proteins have been shown to participate in splicing control. The range and specificity of hnRNP protein action remain poorly documented, even for those affecting splice site selection. We used RNA interference and a reverse transcription-PCR screening platform to examine the implications of 14 of the major hnRNP proteins in the splicing of 56 alternative splicing events in apoptotic genes. Out of this total of 784 alternative splicing reactions tested in three human cell lines, 31 responded similarly to a knockdown in at least two different cell lines. On the other hand, the impact of other hnRNP knockdowns was cell line specific. The broadest effects were obtained with hnRNP K and C, two proteins whose role in alternative splicing had not previously been firmly established. Different hnRNP proteins affected distinct sets of targets with little overlap even between closely related hnRNP proteins. Overall, our study highlights the potential contribution of all of these major hnRNP proteins in alternative splicing control and shows that the targets for individual hnRNP proteins can vary in different cellular contexts.

Development of a Brassica seed cDNA microarray.

Brassica species represent several important crops including canola (Brassica napus). Understanding of genetic elements that contribute to seed-associated functions will impact future improvements in the canola crop. Brassica species share a very close taxonomic and molecular relationship with Arabidopsis thaliana. However, there are several subtle but distinct seed-associated agronomic characteristics that differ among the oil seed crop species. To address these, we have generated 67,535 ESTs predominately from Brassica seeds, analyzed these sequences, and identified 10,642 unigenes for the preparation of a targeted seed cDNA array. A set of 10,642 PCR primer pairs was designed and corresponding amplicons were produced for spotting, along with relevant controls. Critical quality control tests produced satisfactory results for use of this microarray in biological experiments. The microarray was also tested with specific RNA targets from embryos, germinating seeds, and leaf tissues. The hybridizations, signal intensities, and overall quality of these slides were consistent and reproducible. Additionally, there are 429 ESTs represented on the array that show no homology with any A. thaliana annotated gene or any gene in the Brassica genome databases or other plant databases; however, all of these probes hybridized to B. napus transcripts, indicating that the array also will be useful in defining expression patterns for genes so far unique to Brassica species.

Multiple alternative splicing markers for ovarian cancer.

Intense efforts are currently being directed toward profiling gene expression in the hope of developing better cancer markers and identifying potential drug targets. Here, we present a sensitive new approach for the identification of cancer signatures based on direct high-throughput reverse transcription-PCR validation of alternative splicing events. This layered and integrated system for splicing annotation (LISA) fills a gap between high-throughput microarray studies and high-sensitivity individual gene investigations, and was created to monitor the splicing of 600 cancer-associated genes in 25 normal and 21 serous ovarian cancer tissues. Out of >4,700 alternative splicing events screened, the LISA identified 48 events that were significantly associated with serous ovarian tumor tissues. In a further screen directed at 39 ovarian tissues containing cancer pathologies of various origins, our ovarian cancer splicing signature successfully distinguished all normal tissues from cancer. High-volume identification of cancer-associated splice forms by the LISA paves the way for the use of alternative splicing profiling to diagnose subtypes of cancer.

Deletion of many yeast introns reveals a minority of genes that require splicing for function.

Splicing regulates gene expression and contributes to proteomic diversity in higher eukaryotes. However, in yeast only 283 of the 6000 genes contain introns and their impact on cell function is not clear. To assess the contribution of introns to cell function, we initiated large-scale intron deletions in yeast with the ultimate goal of creating an intron-free model eukaryote. We show that about one-third of yeast introns are not essential for growth. Only three intron deletions caused severe growth defects, but normal growth was restored in all cases by expressing the intronless mRNA from a heterologous promoter. Twenty percent of the intron deletions caused minor phenotypes under different growth conditions. Strikingly, the combined deletion of all introns from the 15 cytoskeleton-related genes did not affect growth or strain fitness. Together, our results show that although the presence of introns may optimize gene expression and provide benefit under stress, a majority of introns could be removed with minor consequences on growth under laboratory conditions, supporting the view that many introns could be phased out of Saccharomyces cerevisiae without blocking cell growth.