July 7, 2019  |  

Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of Hypericum perforatum

Hypericum perforatum is a widely known medicinal herb used mostly as a remedy for depression because of its abundant secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is very necessary for the interpretation of qRT-PCR data. In this study, we investigated the expression of fourteen candidate genes, including nine housekeeping genes and five potential candidate genes. Additionally, the HpHYP1 gene, belonging to the PR-10 family associated with stress control, was used for validation of the candidate reference genes. Three programs were applied to evaluate the gene expression stability across four different plant tissues, three developmental stages and a set of abiotic stress and hormonal treatments. The candidate genes showed a wide range of Ct values in all samples, indicating that they are differentially expressed. Integrating all of the algorithms and evaluations, ACT2 and TUB-ß were the most stable combination overall and for different developmental stages samples. Moreover, ACT2 and EF1-a were considered to be the two most applicable reference genes for different tissues and for stress samples. Majority of the conventional housekeeping genes exhibited better than the potential reference genes. The obtained results will contribute to improving credibility of standardization and quantification of transcription levels in future expression research of H. perforatum.

July 7, 2019  |  

De novo genome assembly of the olive fruit fly (Bactrocera oleae) developed through a combination of linked-reads and long-read technologies

Long-read sequencing has greatly contributed to the generation of high quality assemblies, albeit at a high cost. It is also not always clear how to combine sequencing platforms. We sequenced the genome of the olive fruit fly (Bactrocera oleae), the most important pest in the olive fruits agribusiness industry, using Illumina short-reads, mate-pairs, 10x Genomics linked-reads, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT). The 10x linked-reads assembly gave the most contiguous assembly with an N50 of 2.16 Mb. Scaffolding the linked-reads assembly using long-reads from ONT gave a more contiguous assembly with scaffold N50 of 4.59 Mb. We also present the most extensive transcriptome datasets of the olive fly derived from different tissues and stages of development. Finally, we used the Chromosome Quotient method to identify Y-chromosome scaffolds and show that the long-reads based assembly generates very highly contiguous Y-chromosome assembly.

July 7, 2019  |  

Whole-Genome and Expression Analyses of Bamboo Aquaporin Genes Reveal Their Functions Involved in Maintaining Diurnal Water Balance in Bamboo Shoots.

Water supply is essential for maintaining normal physiological function during the rapid growth of bamboo. Aquaporins (AQPs) play crucial roles in water transport for plant growth and development. Although 26 PeAQPs in bamboo have been reported, the aquaporin-led mechanism of maintaining diurnal water balance in bamboo shoots remains unclear. In this study, a total of 63 PeAQPs were identified, based on the updated genome of moso bamboo (Phyllostachys edulis), including 22 PePIPs, 20 PeTIPs, 17 PeNIPs, and 4 PeSIPs. All of the PeAQPs were differently expressed in 26 different tissues of moso bamboo, based on RNA sequencing (RNA-seq) data. The root pressure in shoots showed circadian rhythm changes, with positive values at night and negative values in the daytime. The quantitative real-time PCR (qRT-PCR) result showed that 25 PeAQPs were detected in the base part of the shoots, and most of them demonstrated diurnal rhythm changes. The expression levels of some PeAQPs were significantly correlated with the root pressure. Of the 86 sugar transport genes, 33 had positive co-expression relationships with 27 PeAQPs. Two root pressure-correlated PeAQPs, PeTIP4;1 and PeTIP4;2, were confirmed to be highly expressed in the parenchyma and epidermal cells of bamboo culm, and in the epidermis, pith, and primary xylem of bamboo roots by in situ hybridization. The authors’ findings provide new insights and a possible aquaporin-led mechanism for bamboo fast growth.

July 7, 2019  |  

The Draft Genome of the MD-2 Pineapple

The main challenge in assembling plant genome is its ploidy level, repeats content, and polymorphism. The second-generation sequencing delivered the throughput and the accuracy that is crucial to whole-genome sequencing but insufficient and remained challenging for some plant species. It is known that genomes produced by next-gen- eration sequencing produced small contigs that would inflate the number of annotated genes (Varshney et al. 2011) and missed on the transposable elements that are abun- dant in plant genome due to their repetitive nature (Michael and Jackson 2013).

July 7, 2019  |  

The complete genome sequence of a marine sponge-associated bacteria, Bacillus safensis KCTC 12796BP, which produces the anti-allergic compounds

The full genome sequence of Bacillus safensis KCTC 12796BP which had been isolated from the marine sponge in the seawater of Jeju Island, was determined by Pac-Bio next- generation sequencing system. A circular chromosome in the length of 3,935,874 bp was obtained in addition to a circular form of plasmid having 36,690 bp. The G + C content of chromosome was 41.4%, and that of plasmid was 37.3%. The number of deduced CDSs in the chromosome was 3,980, whereas 36 CDS regions were determined in a plasmid. Among the deduced CDSs in chromosome, 81 tRNA genes and 24 rRNA genes in addition to one tmRNA were allocated. More than 30 CDSs for sporulation, 16 CDSs for spore coat, and 20 CDSs for germination were also assigned in the chromosome. Several genes for capsular polysaccharide biosynthesis and for flagella biosynthesis and chemotaxis in addition to genes for osmotic tolerance through glycine-choline betaine pathway were also identified. Above all, the biosynthetic gene cluster for anti- allergic compounds seongsanamides were found among two non-ribosomal peptide synthetase (NRPS) gene clusters for secondary metabolites.

January 23, 2017  |  

Tutorial: HGAP4 de novo assembly application

This tutorial provides an overview of the Hierarchical Genome Assembly Process (HGAP4) de novo assembly analysis application. HGAP4 generates accurate de novo assemblies using only PacBio data. HGAP4 is suitable…

June 29, 2015  |  

Human Genome Sequenced without Cloning Steps

A collaboration of scientists led by researchers from Icahn School of Medicine at Mt. Sinai has created a comprehensive analysis of a diploid human genome using two complementary single DNA molecule methods for sequencing and genome mapping, and without the need for any DNA amplification techniques.

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