Bulked Segregant RNA-Seq Provides Distinctive Expression Profile Against Powdery Mildew in the Wheat Genotype YD588

Wheat powdery mildew, caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a destructive disease leading to huge yield losses in production. Host resistance can greatly contribute to the control of the disease. To explore potential genes related to the powdery mildew (Pm) resistance, in this study, we used a resistant genotype YD588 to investigate the potential resistance components and profiled its expression in response to powdery mildew infection. Genetic analysis showed that a single dominant gene, tentatively designated PmYD588, conferred resistance to powdery mildew in YD588. Using bulked segregant RNA-Seq (BSR-Seq) and single nucleotide polymorphism (SNP) association analysis, two high-confidence candidate regions were detected in the chromosome arm 2B, spanning 453,752,054-506,356,791 and 584,117,809-664,221,850 bp, respectively. To confirm the candidate region, molecular markers were developed using the BSR-Seq data and mapped PmYD588 to an interval of 4.2 cM by using the markers YTU588-004 and YTU588-008. The physical position was subsequently locked into the interval of 647.1–656.0 Mb, which was different from those of Pm6, Pm33, Pm51, Pm52, Pm63, Pm64, PmQ, PmKN0816, MlZec1, and MlAB10 on the same chromosome arm in its position, suggesting that it is most likely a new Pm gene. To explore the potential regulatory genes of the R gene, 2,973 differentially expressed genes (DEGs) between the parents and bulks were analyzed using gene ontology (GO), clusters of orthologous group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Based on the data, we selected 23 potential regulated genes in the enriched pathway of plant-pathogen interaction and detected their temporal expression patterns using an additional set of wheat samples and time-course analysis postinoculation with Bgt. As a result, six disease-related genes showed distinctive expression profiles after Bgt invasion and can serve as key candidates for the dissection of resistance mechanisms and improvement of durable resistance to wheat powdery mildew.

Genome-wide and comparative phylogenetic analysis of senescence-associated NAC transcription factors in sunflower (Helianthus annuus)

Background Leaf senescence delay impacts positively in grain yield by maintaining the photosynthetic area during the reproductive stage and during grain filling. Therefore a comprehensive understanding of the gene families associated with leaf senescence is essential. NAC transcription factors (TF) form a large plant-specific gene family involved in regulating development, senescence, and responses to biotic and abiotic stresses. The main goal of this work was to identify sunflower NAC TF (HaNAC) and their association with senescence, studying their orthologous to understand possible functional relationships between genes of different species. Results To clarify the orthologous relationships, we used an in-depth comparative study of four divergent taxa, in dicots and monocots, with completely sequenced genomes (Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa). These orthologous groups provide a curated resource for large scale protein sequence annotation of NAC TF. From the 151 HaNAC genes detected in the latest version of the sunflower genome, 50 genes were associated with senescence traits. These genes showed significant differential expression in two contrasting lines according to an RNAseq assay. An assessment of overexpressing the Arabidopsis line for HaNAC001 (a gene of the same orthologous group of Arabidopsis thaliana ORE1) revealed that this line displayed a significantly higher number of senescent leaves and a pronounced change in development rate. Conclusions This finding suggests HaNAC001 as an interesting candidate to explore the molecular regulation of senescence in sunflower.

De novo Transcriptome Assembly and Comprehensive Annotation of Two Tree Tomato Cultivars (Solanum betaceum Cav.) with Different Fruit Color

The tree tomato (Solanum betaceum Cav.) is an underutilized fruit crop native to the Andean region and phylogenetically related to the tomato and potato. Tree tomato fruits have a high amount of nutrients and bioactive compounds. However, so far there are no studies at the genome or transcriptome level for this species. We performed a de novo assembly and transcriptome annotation for purple-fruited (A21) and an orange-fruited (A23) accessions. A total of 174,252 (A21) and 194,417 (A23) transcripts were assembled with an average length of 851 and 849 bp. A total of 34,636 (A21) and 36,224 (A23) transcripts showed a significant similarity to known proteins. Among the annotated unigenes, 22,096 (A21) and 23,095 (A23) were assigned to the Gene Ontology (GO) term and 14,035 (A21) and 14,540 (A23) were found to have Clusters of Orthologous Group (COG) term classifications. Furthermore, 22,096 (A21) and 23,095 (A23) transcripts were assigned to 155 and 161 (A23) KEGG pathways. The carotenoid biosynthetic process GO terms were significantly enriched in the purple-fruited accession A21. Finally, 68,647 intraspecific single-nucleotide variations (SNVs) and almost 2 million interspecific SNVs were identified. The results of this study provide a wealth of genomic data for the genetic improvement of the tree tomato.

Genome-Wide Analysis of NAC Transcription Factors in Sunflower (Helianthus Annuus), Their Comparative Phylogenetic Analysis and Association With Leaf Senescence

Background Leaf senescence delay impacts positively in grain yield by maintaining the photosynthetic area during the reproductive stage and during grain filling. Therefore a comprehensive understanding of the gene families associated with leaf senescence is essential. NAC transcription factors (TF) form a large plant-specific gene family involved in regulating development, senescence, and responses to biotic and abiotic stresses. The main goal of this work was to identify sunflower NAC TF (HaNAC) and their association with senescence, studying their orthologous to understand possible functional relationships between genes of different species. ResultsTo clarify the orthologous relationships, we used an in-depth comparative study of four divergent taxa, in dicots and monocots, with completely sequenced genomes (Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa). These orthologous groups provide a curated resource for large scale protein sequence annotation of NAC TF. From the 151 HaNAC genes detected in the latest version of the sunflower genome, 50 genes were associated with senescence traits. These genes showed significant differential expression in two contrasting lines according to an RNAseq assay. An assessment of overexpressing the Arabidopsis line for HaNAC001 (a gene of the same orthologous group of Arabidopsis thaliana ORE1) revealed that this line displayed a significantly higher number of senescent leaves and a pronounced change in development rate.ConclutionsThis finding suggests HaNAC001 as an interesting candidate to explore the molecular regulation of senescence in sunflower.

De Novo Transcriptomic Characterization Enables Novel Microsatellite Identification and Marker Development in Betta splendens

The wild populations of the commercially valuable ornamental fish species, Betta splendens, and its germplasm resources have long been threatened by habitat degradation and contamination with artificially bred fish. Because of the lack of effective marker resources, population genetics research projects are severely hampered. To generate genetic data for developing polymorphic simple sequence repeat (SSR) markers and identifying functional genes, transcriptomic analysis was performed. Illumina paired-end sequencing yielded 105,505,486 clean reads, which were then de novo assembled into 69,836 unigenes. Of these, 35,751 were annotated in the non-redundant, EuKaryotic Orthologous Group, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases. A total of 12,751 SSR loci were identified from the transcripts and 7970 primer pairs were designed. One hundred primer pairs were randomly selected for PCR validation and 53 successfully generated target amplification products. Further validation demonstrated that 36% (n = 19) of the 53 amplified loci were polymorphic. These data could not only enrich the genetic information for the identification of functional genes but also effectively facilitate the development of SSR markers. Such knowledge would accelerate further studies on the genetic variation and evolution, comparative genomics, linkage mapping and molecular breeding in B. splendens.

Phylostratigraphic analysis of gene networks of human diseases

Phylostratigraphic analysis is an approach to the study of gene evolution that makes it possible to determine the time of the origin of genes by analyzing their orthologous groups. The age of a gene belonging to an orthologous group is def ined as the age of the most recent ancestor of all species represented in that group. Such an analysis can reveal important stages in the evolution of both the organism as a whole and groups of functionally related genes, in particular gene networks. In addition to investigating the time of origin of a gene, the level of its genetic variability and what type of selection the gene is subject to in relation to the most closely related organisms is studied. Using the Orthoscape application, gene networks from the KEGG Pathway, Human Diseases database describing various human diseases were analyzed. It was shown that the majority of genes described in gene networks are under stabilizing selection and a high reliable correlation was found between the time of gene origin and the level of genetic variability: the younger the gene, the higher the level of its variability is. It was also shown that among the gene networks analyzed, the highest proportion of evolutionarily young genes was found in the networks associated with diseases of the immune system (65 %), and the highest proportion of evolutionarily ancient genes was found in the networks responsible for the formation of human dependence on substances that cause addiction to chemical compounds (88 %); gene networks responsible for the development of infectious diseases caused by parasites are signif icantly enriched for evolutionarily young genes, and gene networks responsible for the development of specif ic types of cancer are signif icantly enriched for evolutionarily ancient genes.

Multimerization variants as potential drivers of neofunctionalization

Whole-genome duplications are common during evolution, creating genetic redundancy that can enable cellular innovations. Novel protein-protein interactions provide a route to diversified gene functions, but, at present, there is limited proteome-scale knowledge on the extent to which variability in protein complex formation drives neofunctionalization. Here, we used protein correlation profiling to test for variability in apparent mass among thousands of orthologous proteins isolated from diverse species and cell types. Variants in protein complex size were unexpectedly common, in some cases appearing after relatively recent whole-genome duplications or an allopolyploidy event. In other instances, variants such as those in the carbonic anhydrase orthologous group reflected the neofunctionalization of ancient paralogs that have been preserved in extant species. Our results demonstrate that homo- and heteromer formation have the potential to drive neofunctionalization in diverse classes of enzymes, signaling, and structural proteins.

De Novo Transcriptome Assembly of Two Microsorum Fern Species Identifies Enzymes Required for Two Upstream Pathways of Phytoecdysteroids

Microsorum species produce a high amount of phytoecdysteroids (PEs), which are widely used in traditional medicine in the Pacific islands. The PEs in two different Microsorum species, M. punctatum (MP) and M. scolopendria (MS), were examined using high-performance liquid chromatography (HPLC). In particular, MS produces a high amount of 20-hydroxyecdysone, which is the main active compound in PEs. To identify genes for PE biosynthesis, we generated reference transcriptomes from sterile frond tissues using the NovaSeq 6000 system. De novo transcriptome assembly after deleting contaminants resulted in 57,252 and 54,618 clean transcripts for MP and MS, respectively. The clean Microsorum transcripts for each species were annotated according to gene ontology terms, UniProt pathways, and the clusters of the orthologous group protein database using the MEGAN6 and Sma3s programs. In total, 1852 and 1980 transcription factors were identified for MP and MS, respectively. We obtained transcripts encoding for 38 and 32 enzymes for MP and MS, respectively, potentially involved in mevalonate and sterol biosynthetic pathways, which produce precursors for PE biosynthesis. Phylogenetic analyses revealed many redundant and unique enzymes between the two species. Overall, this study provides two Microsorum reference transcriptomes that might be useful for further studies regarding PE biosynthesis in Microsorum species.

Universal and differential transcriptional regulatory pathways involved in the preparation of summer and winter diapauses in Pieris melete

Much progress has been made in understanding the environmental and hormonal systems regulating winter diapause. However, transcriptional regulation of summer diapause is still largely unknown, making it difficult to understand an all-around regulation profile of seasonal adaptation. To bridge this gap, comparison RNA-seq to profile the transcriptome and to examine differential gene expression profiles between non-diapause, summer diapause, and winter diapause groups were performed. A total number of 113 million reads were generated and assembled into 79,117 unigenes, with 37,492 unigenes categorized into 58 functional gene ontology groups, 25 clusters of orthologous group categories, and 256 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG analysis mapped 2108 differentially expressed genes to 48 and 67 pathways for summer and winter diapauses, respectively. Enrichment statistics showed that 11 identical pathways similarly overlapped in the top 20 enriched functional groups both related to summer and winter diapauses. We also identified 35 key candidate genes for universal and differential functions related to summer and winter diapause preparation. Furthermore, we identified some genes involved in the signaling and metabolic pathways that may be the key drivers to integrate environmental signals into the summer and winter diapause preparation. The current study provided valuable insights into global molecular mechanisms underpinning diapause preparation.