De Novo Transcriptome Assembly and SNP Discovery for the Development of dCAPS Markers in Oat

Cultivated oat (Avena sativa L.) is an important cereal crop that has captured interest worldwide due to its nutritional properties and associated health benefits. Despite this interest, oat has lagged behind other cereal crops in genome studies and the development of DNA markers due to its large and complex genome. RNA-Seq technology has been widely used for transcriptome analysis, functional gene study, and DNA marker development. In this study, we performed the transcriptome sequencing of 10 oat varieties at the seedling stage using the Illumina platform for the development of DNA markers. In total, 31,187,392~41,304,176 trimmed reads (an average of 34,322,925) were generated from 10 oat varieties. All of the trimmed reads of these varieties were assembled and generated, yielding a total of 128,244 assembled unigenes with an average length of 1071.7 bp and N50 of 1752 bp. According to gene ontology (GO) analysis, 30.7% of unigenes were assigned to the “catalytic activity” of the parent term in the molecular function category. Of the 1273 dCAPS markers developed using 491 genotype-specific SNPs, 30 markers exhibiting polymorphism in 28 oat varieties were finally selected. The transcriptome data of oat varieties could be used for functional studies about the seedling stage of oat and information about sequence variations in DNA marker development. These 30 dCAPS markers will be utilized for oat genetic analysis, cultivar identification, and breeders’ rights protection.

Integrative Analysis of the Transcriptome and Metabolome Reveals the Mechanism of Saline–Alkali Stress Tolerance in Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge.) Hsiao

Saline–alkali stress is a major abiotic stress affecting the quality and yield of crops. Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus) is a well-known medicine food homology species with various pharmacological effects and health benefits that can grow well in saline–alkali soil. However, the molecular mechanisms underlying the adaptation of A. mongholicus plants to saline–alkali stress have not yet been clarified. Here, A. mongholicus plants were exposed to long-term saline–alkali stress (200 mmol·L -1 mixed saline–alkali solution), which limited the growth of A. mongholicus. The roots of A. mongholicus could resist long-term saline–alkali stress by increasing the activity of antioxidant enzymes and the content of osmolytes. Transcriptome analysis (via the Illumina platform) and metabolome analysis (via the Nexera UPLC Series QE Liquid Mass Coupling System) revealed that saline–alkali stress altered the activity of various metabolic pathways (e.g., amino acid metabolism, carbohydrate metabolism, lipid metabolism, and biosynthesis of other secondary metabolites). A total of 3,690 differentially expressed genes (DEGs) and 997 differentially accumulated metabolites (DAMs) were identified in A. mongholicus roots under saline–alkali stress, and flavonoid-related DEGs and DAMs were significantly up-regulated. Pearson correlation analysis revealed significant correlations between DEGs and DAMs related to flavonoid metabolism. MYB transcription factors might also contribute to the regulation of flavonoid biosynthesis. Overall, the results indicate that A. mongholicus plants adapt to saline–alkali stress by up-regulating the biosynthesis of flavonoids, which enhances the medicinal value of A. mongholicus.

RNA-combine: a toolkit for comprehensive analyses on transcriptome data from different sequencing platforms

Background Understanding the transcriptome has become an essential step towards the full interpretation of the biological function of a cell, a tissue or even an organ. Many tools are available for either processing, analysing transcriptome data, or visualizing analysis results. However, most existing tools are limited to data from a single sequencing platform and only several of them could handle more than one analysis module, which are far from enough to meet the requirements of users, especially those without advanced programming skills. Hence, we still lack an open-source toolkit that enables both bioinformatician and non-bioinformatician users to process and analyze the large transcriptome data from different sequencing platforms and visualize the results. Results We present a Linux-based toolkit, RNA-combine, to automatically perform the quality assessment, downstream analysis of the transcriptome data generated from different sequencing platforms, including bulk RNA-seq (Illumina platform), single cell RNA-seq (10x Genomics) and Iso-Seq (PacBio) and visualization of the results. Besides, this toolkit is implemented with at least 10 analysis modules more than other toolkits examined in this study. Source codes of RNA-combine are available on GitHub: https://github.com/dongxuemin666/RNA-combine. Conclusion Our results suggest that RNA-combine is a reliable tool for transcriptome data processing and result interpretation for both bioinformaticians and non-bioinformaticians.

Transcriptome analyses of female black tiger shrimps (Penaeus monodon) for reproductive trait using RNA-sequencing approach

The black tiger shrimp (Penaeus monodon) is the widely cultured aquaculture species in Vietnam and worldwide. Production of P. monodon postlarvae still relies on the wild broodstock due to their higher fecundity and larval quality in comparison with the domesticated broodstock. Therefore, the current study applied an RNA-sequencing approach by the Illumina platform to generate the transcriptomic resources for the wild and domesticated females at the previtellogenic stage of ovaries (stage 0). Total clean reads were 20,977,708 for the wild female and 31,185,197 for the domesticated female. De novoassembly was employed to generate 35,870 contigs with an average length of 1,018 bp and N50 length of 1,488 bp. The ratios of contigs possessing significant similarity through annotation across seven databases ranged from 19.74 to 47.77%. Top hit species from BLASTx searches included Hyalella azteca, Cryptotermes secundus, Zootermopsis nevadensis, followed by Penaeus sp.. We identified a total of 5,788 differentially expressed transcripts between the ovaries of wild and domesticated shrimps. The differentially expressed transcripts were further enriched according to the classification terms of Gene Ontology (GO). Results of GO enrichment analysis in the wild female indicated that many genes such as hemolymph clottable, peritrophin, ecdysteroid were up-regulated while the following genes, including serine protease, alpha-L-fucosidase-like, actin, catenin alpha were down-regulated. The current study provides more transcriptomic resources for the reproductive trait in P. monodon. These resources are potentially applied for the improvement of reproduction in the domesticated shrimp.

Transcriptome Analysis of Salt Stress in Hibiscus hamabo Sieb. et Zucc Based on Pacbio Full-Length Transcriptome Sequencing

Hibiscus hamabo Sieb. et Zucc is an important semi-mangrove plant with great morphological features and strong salt resistance. In this study, by combining single molecule real time and next-generation sequencing technologies, we explored the transcriptomic changes in the roots of salt stressed H. hamabo. A total of 94,562 unigenes were obtained by clustering the same isoforms using the PacBio RSII platform, and 2269 differentially expressed genes were obtained under salt stress using the Illumina platform. There were 519 differentially expressed genes co-expressed at each treatment time point under salt stress, and these genes were found to be enriched in ion signal transduction and plant hormone signal transduction. We used Arabidopsis thaliana (L.) Heynh. transformation to confirm the function of the HhWRKY79 gene and discovered that overexpression enhanced salt tolerance. The full-length transcripts generated in this study provide a full characterization of the transcriptome of H. hamabo and may be useful in mining new salt stress-related genes specific to this species, while facilitating the understanding of the salt tolerance mechanisms.

Smilax auriculata: A new host for Orchid fleck dichorhavirusvirus identified in Florida, USA

In June 2020, Orchid fleck virus (OFV) was detected in a species of Liriope in Leon and Alachua County, Florida (Fife et al; 2021). In October of the same year, four adjacent dune/ear-leaf greenbrier vines, Smilax auriculata (Smilaceae: Liliales), showed yellowing and mottling symptoms (Figure 1). Infected and healthy S. auriculata leaves samples were collected in Alachua County by the Florida Department of Agriculture and Consumer Services, Gainesville, Florida. OFV primers successfully detected in four Smilax samples by conventional RT-PCR assay. Amplicon sequences (Acc. No. MZ645935 and MZ645938) shared 99% nucleotide identity with OFV infecting orchids (LC222629) and citrus (MK522804). The OFV subgroup I (OFV-Orc1) and subgroup II (OFV-Orc2) specific primers (Kondo et al 2017) were utilized to confirm the presence of OFV type strains infecting Smilax. Sanger sequencing of subgroup I specific amplicons (MZ645934) shared 99% nucleotide identity with OFV-Orc1 (LC222629) whereas subgroup II specific amplicon sequence (MZ645930) shared 98-99 % nucleotide identity with OFV-Orc2 (AB244417). Further confirmation was done by USDA-APHIS-PPQ-Plant Pathogen Confirmatory Diagnostics Laboratory utilizing optimized conventional RT-PCR protocols (Roy et al. 2020) and deep sequencing on a on a NextSeq550 Illumina platform. Assembled reads identified seven non-overlapping viral contigs. Five RNA1 and two RNA2 contigs covered more than 97% of the bipartite OFV genome with average coverage depth of 5297.61 and 5186.04, respectively. Contigs of RNA1 and RNA2 shared 98-99% nt identity to OFV-Orc2-RNA1 (AB244417) and OFV-Orc-RNA2 (AB244418 and LC222630). No other pathogen sequences were identified. This is the first time the genus Smilax has been identified as a natural host of OFV. Very recent findings of OFV-Orc in Florida in Liriope, Aspidistra, and Ophiopogon among the Asparagaceae family members (Fife et al; 2021) and now in the Smilacaceae suggest a broader host range of the virus than previously known; further research should be conducted to better characterize the potential risk of introduction into citrus in Florida.

Evaluation of altered miRNA expression pattern to predict COVID-19 severity.

Outbreak of COVID-19 pandemic in December 2019 affected millions of people globally. After substantial research, there is no specific and reliable biomarker available till date. Present study was designed to identify specific biomarkers to predict COVID-19 severity and tool for formulating treatment. A small cohort of subjects (n=43) were enrolled and categorized in four study groups; Dead (n=16), Severe (n=10) and Moderate (n=7) patients and healthy controls (n=10). Small RNA sequencing was done on Illumina platform after isolation of microRNA from peripheral blood. Differential expression (DE) of miRNA (patients groups compared to control) revealed 118 down-regulated and 103 up-regulated known miRNAs with fold change (FC) expression ≥2 folds and p≤0.05. DE miRNAs were then subjected to functional enrichment and network analysis. Bioinformatic analysis resulted in 31 miRNAs (24 Down-regulated; 7 up-regulated) significantly associated with COVID-19 having AUC>0.8 obtained from ROC curve. Seventeen out of 31 DE miRNAs have been linked to COVID-19 in previous studies. Three miRNAs, hsa-miR-147b-5p and hsa-miR-107 (down-regulated) and hsa-miR-1299 (up-regulated) showed significant unique DE in Dead patients. Another set of 4 miRNAs, hsa-miR-224-5p (down-regulated) and hsa-miR-4659b-3p, hsa-miR-495-3p and hsa-miR-335-3p were differentially up-regulated uniquely in Severe patients. Members of three miRNA families, hsa-miR-20, hsa-miR-32 and hsa-miR-548 were significantly down-regulated in all patients group in comparison to healthy controls. Thus a distinct miRNA expression profile was observed in Dead, Severe and Moderate COVID-19 patients. Present study suggests a panel of miRNAs which identified in COVID-19 patients and could be utilized as potential diagnostic biomarkers for predicting COVID-19 severity.

First report of Fusarium oxysporum f. sp. cubense Tropical Race 4, causing Fusarium wilt in Cavendish bananas in Peru

Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), the causal agent of Fusarium wilt of banana (FWB), is currently the major threat to the banana industry worldwide (Dita et al. 2018). Restricted to South Asia for more than 20 years, Foc TR4 has spread in the last years to the Middle East, Mozambique, and Colombia (García-Bastidas et al. 2019; https://pestdisplace.org/embed/news/map/disease/11). The incursion of Foc TR4 in Colombia increased awareness and prevention efforts across Latin American and the Caribbean (LAC). However, new Foc TR4 outbreaks in LAC countries were considered a matter of time. In April 2021, banana (Musa spp., Cavendish, AAA) plants (30% of incidence) showing typical symptoms of FWB, such as leave yellowing, wilting and vascular discoloration were observed in one farm (about 1 ha) located in Querecotillo, Peru (4°43’54.84”S 80°33’45.00”W). Mycological analyses of samples (pseudostem strands) collected from 10 symptomatic plants were performed as described by Dita et al. (2010). These analyses revealed a continuous presence of fungal colonies identified as Fusarium oxysporum species complex. Molecular diagnostics targeting two different genome regions (Dita et al 2010; Li et al. 2013) identified nine of these isolates as Foc TR4. These results were further confirmed by qPCR analyses using the commercial Clear Detections TR4 kit. The genome of four single-spore isolates (PerS1, PerS2, PerS3 and PerS4) was sequenced using the Illumina platform (MiSeq Kit, 2x151 bp Paired-End). The strain PerS4 was also sequenced using Oxford Nanopore (FLOW-MIN111; R10.3 chemistry) as described by Lopez-Alvarez et al., (2020). The generated draft assembly yielded 533 contigs for a size of 47 Mbp (BioProject: PRJNA755905), which is comparable with sizes of previously reported Foc TR4 strains (Asai et al. 2019; García-Bastidas et al. 2019; Maymon et al. 2020; Warmington et al. 2019; Zheng et al. 2018). The sequence assembly showed high contiguity (94.9%) and high similarity (95.48%) with the high-quality genome sequence of the Foc TR4 isolate ‘UK0001’ (Warmington et al. 2019). Further analyses to identify the presence/absence of full sequences for the putative effector genes (Secreted In Xylem - SIX) and their allelic copies, also revealed that the SIX genes profile of the strains isolated from Querecotillo matched with previously reported Foc TR4 isolates (Czislowski et al. 2017). Pathogenicity tests with three isolates and water controls were performed as described by Dita et al. (2010), using five Cavendish plantlets per treatment. Four weeks after the inoculation typical external and internal symptoms of FWB were observed only in the inoculated plants. Fungal isolates recovered from inoculated plants tested positive for Foc TR4 when analyzed with PCR diagnostics as mentioned above. No fungal isolates were recovered from water-control plants which did not show any symptoms. Altogether, our results confirm the first incursion of Foc TR4 in Peru. Currently, Foc TR4 has the phytosanitary status of a present pest with restricted distribution in Peru and it is under official control of the National Plant Protection Organization – SENASA. Reinforced prevention and quarantine measures, disease monitoring and capacity building to detect, contain and manage eventual new outbreaks of Foc TR4 are strongly encouraged across LAC banana producing countries, especially for those bordering Peru with larger banana plantations, such as Ecuador and Brazil.

Validation of Genomic Selection Approach for Predicting Sheath Blight Resistance in Rice (Oryza sativa L.,)

Rice sheath blight (ShB) is one of the most serious fungal diseases caused by Rhizoctonia solani, instigating significant yield losses in many rice-growing regions of the world. Intensive studies indicated that resistance for sheath blight is controlled possibly by polygenes. Because of complex inheritance, it’s very difficult to exploit and tap all the genomic regions conferring resistance using classical approaches of QTL mapping, it’s very important to have a different strategy to harness such resistance mechanism. One promising approach that can potentially provide accurate predictions of the resistance phenotypes is genomic selection (GS). The research was undertaken with an objective to validate genomic selection approach for predicting sheath blight resistance involving 1545 Recombinant inbred lines (RILs) derived from eleven crosses between resistant and susceptible parents (Jasmine 85XTN1, Jasmine 85XSwarnaSub1, Jasmine 85XII32B, Jasmine 85XIR54, TetepXTN1, TetepXSwarna Sub1, TetepXII32B, TetepXIR54, MTU 9992XTN1, MTU 9992XII32B and MTU 9992XIRBB4). Where, Jasmine 85, Tetep & MTU 9992 were resistant parents and TN1, Swarna Sub1, II32B, IR54 & IRBB4 were susceptible parents. During rainy season (2020) the F7 RILs were screened for their reaction to sheath blight in two hot spot locations. The genotyping was done with Illumina platform having 6564 SNP markers. Bayesian B approach was used to train the statistical model for calculation of marker effects and GEBVs. The prediction accuracy of training set (data fit analysis) obtained was 0.70 and random cross validation with different approaches, the prediction accuracy ranged from 0.67 to 0.74. The results are lucrative, all in all, high prediction accuracies observed in this study suggest genomic selection as a very promising breeding strategy for predicting sheath blight resistance in Rice.

Comprehensive Transcriptome and Metabolic Profiling of Petal Color Development in Lycoris sprengeri

Lycoris sprengeri (L. sprengeri) is an important ornamental bulbous plant, and its numerous varieties in different color forms are widely planted. Multiple color types of petals in L. sprengeri provide us with possibilities to delineate the complicated metabolic networks underlying the biochemical traits behind color formation in this plant species, especially petal color. In this study, we sequenced and annotated a reference transcriptome of pink and white petals of L. sprengeri and analyzed the metabolic role of anthocyanin biosynthesis in regulating color pigment metabolism. Briefly, white and pink petal samples were sequenced with an Illumina platform, to obtain the reads that could be assembled into 100,778 unique sequences. Sequences expressed differentially between white vs. pink petals were further annotated with the terms of Gene Ontology (GO), Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and eggNOG. Gene expression analyses revealed the repression of anthocyanin and steroid biosynthesis enzymes and R2R3 MYB transcription factor (TF) genes in white petals compared to pink petals. Furthermore, the targeted metabolic profiling of anthocyanins revealed that color-related delphinidin (Del) and cyanidin (Cy) pigments are lower in white petals, which correlate well with the reduced gene expression levels of anthocyanin biosynthesis genes. Taken together, it is hypothesized that anthocyanin biosynthesis, steroid biosynthesis, and R2R3 MYB TFs may play vital regulatory roles in petal color development in L. sprengeri. This work provides a valuable genomic resource for flower breeding and metabolic engineering in horticulture and markers for studying the flower trait evolution of L. sprengeri.