Project description:The goals of this study is understand the effect of the MiEFF18 nematode effector on alternative splicing. Methods: PolyA RNA from WT, MiEFF18 overexpressor and smd1b mutant seedlings were sequenced using the illumina NextSeq500 sequencer, in triplicate. Total RNA was extracted from the roots of the three Arabidopsis lines (Col0, OE-MiEFF18#13.6 and smd1b) with TriZol (Invitrogen), according to the Invitrogen protocol. The RNA was treated with DNAse treatment (Ambion), and its quality and integrity were assessed with a Bioanalyzer (Agilent). Libraries were constructed with the Tru-Seq Stranded mRNA Sample Prep kit (Illumina®). Paired-end sequencing with 75-bp reads was performed on a NextSeq500 perform. A minimum of 30 million paired-end reads per sample was generated. RNA-seq preprocessing included the trimming of library adapters and quality controls with Trimmomatic. Paired-end reads with a Phred Quality Score Qscore > 20 and a read length > 30 bases were retained, and ribosomal RNA sequences were removed with SortMeRNA. Processed reads were aligned using Tophat2 with the following arguments: --max-multihits 1 -i 20 --min-segment-intron 20 --min-coverage-intron 20 --library-type fr-firststrand --microexon-search -I 1000 --max-segment-intron 1000 --max-coverage-intron 1000 --b2-very-sensitive. Conclusions: MiEFF18 overexpression modifies the expression of genes important for the ontogenesis of giant cells, notably those involved in microtubule cytoskeleton reorganization and cell cycle regulation, strongly suggesting that MiEFF18-mediated SmD1 inactivation in plants leads to a loss of susceptibility to root-knot nematodes.
Project description:Cornus officinalis Sieb. et Zucc., a perennial woody plant which is recognized with high medicinal, economic and ecological values, has been used as traditional Chinese medicine (TCM) for thousands of years in China. Modern pharmacological research has revealed that cornel iridoid glycosides (CIGs, e.g. loganin and morroniside) in dried pericarp of C. officinalis have significant medicinal activities for strengthening immune functions. However, little is known on the molecular processes responsible for the medical properties of this species, owing to the absence of genomic resources such as available sequences of key enzyme genes in biosynthetic pathways. In this study, the RNA sequencing data of C. officinalis were first generated and used for transcriptome analysis. A total of 54,827 unigenes with an average length of 817 bp, an N50 of 1,379 bp, and an average GC content of 44.91% were yielded by de novo assembly, of which 31,780 unigenes were successfully annotated. As potential molecular markers, 121, 118, 96, 89, and 82 transcription factors belonged to bHLH, MYB, PHD, WRKY, and AP2-ERF were further analyzed, respectively. The results of qRT-PCR confirmed that geraniol 10-hydroxylase (G10H) and loganin synthase (SLS) were differentially expressed in fruits and leaves during different growing stages. Furthermore, we found that loganin accumulation was positively related to G10H expression but was negatively correlated with SLS expression. Collectively, the genomic information and gene expression results presented in this study will be helpful for future studies on gene discovery and molecular process of loganin synthesis in C. officinalis.
Project description:Purpose: The aim of this study is to determine the absolute and relative expresson levels of mRNA transcripts across two purbred (Angus and Brahman) cattle and their receipicalcross. Methods: Total RNA was extracted and purified from five tissues using the RiboZero Gold kit. Sequencing libraries were prepared with a KAPA Stranded RNA-Seq Library Preparation Kit following the Illumina paired-end library preparation protocol. Completed libraries were sequenced on HiSeq 2000 as 100 bp paired-end reads at the Australian Cancer Research Foundation (ACRF) Cancer Genomics Facility, Adelaide, Australia and USDA-ARS-US Meat Animal Research Center, Clay Center, USA. Approximately 60 million 100 bp single-end reads were obtained for each sample. Reads were aligned to the cattle reference genome UOA_brahman_1/UOA_angus_1 and mapped to known genomic features at the gene level using the Hisat2. Single reads were then summarized into gene-level counts using FeatureCounts.