Rooibos (Aspalathus linearis) Genome Size Estimation Using Flow Cytometry and K-Mer Analyses

Plant genomes provide information on biosynthetic pathways involved in the production of industrially relevant compounds. Genome size estimates are essential for the initiation of genome projects. The genome size of rooibos (Aspalathus linearis species complex) was estimated using DAPI flow cytometry and k-mer analyses. For flow cytometry, a suitable nuclei isolation buffer, plant tissue and a transport medium for rooibos ecotype samples collected from distant locations were identified. When using radicles from commercial rooibos seedlings, Woody Plant Buffer and Vicia faba as an internal standard, the flow cytometry-estimated genome size of rooibos was 1.24 ± 0.01 Gbp. The estimates for eight wild rooibos growth types did not deviate significantly from this value. K-mer analysis was performed using Illumina paired-end sequencing data from one commercial rooibos genotype. For biocomputational estimation of the genome size, four k-mer analysis methods were investigated: A standard formula and three popular programs (BBNorm, GenomeScope, and FindGSE). GenomeScope estimates were strongly affected by parameter settings, specifically CovMax. When using the complete k-mer frequency histogram (up to 9 × 105), the programs did not deviate significantly, estimating an average rooibos genome size of 1.03 ± 0.04 Gbp. Differences between the flow cytometry and biocomputational estimates are discussed.

POST-VACCINATED ALTERATIONS IN THE MARKERS OF LIPID AND PROTEIN OXIDATION IN THE GILLS OF RAINBOW TROUT (ONCORHYNCHUS MYKISS WALBAUM) IMMUNIZED AGAINST THE ENTERIC REDMOUTH DISEAS

The aim of the study was the evaluation of the content of oxidative stress biomarkers (2-thiobarbituric-acid-reacting substances as a biomarker of lipid peroxidation, aldehydic and ketonic derivatives of oxidatively modified proteins) in the gills of rainbow trout (Oncorhynchus mykissWalbaum) vaccinated by a vaccine against Yersiniaruckeri. Rainbow trout (Oncorhynchus mykiss Walbaum) with a mean body mass of (107.9±3.1) g were used in the experiments. The study was carried out in a Department of Salmonid Research, Inland Fisheries Institute in Rutki (Poland). Experiments were performed at a water temperature of 14.5±0.5°C and the pH was 7.5. The dissolved oxygen level was about 12 ppm with additional oxygen supply with a water flow of 25 L per min, a photoperiod of 12 hours per day. The fish were fed with a commercial pelleted diet at an optimal level, using 12-hour belt feeders for fish. All enzymatic assays were carried out at the Department of Zoology and Animal Physiology, Institute of Biology and Earth Sciences, Pomeranian University in Słupsk (Poland).The fish were kept for 60 days after vaccination at a water temperature of 14.5±0.5°C and pH 7.5. In our study, 15 rainbow trout from unhandled control and 15 vaccinated trout were used. Two months after immunization, samples from rainbow trout were collected. The fish were captured and killed 61 days post-vaccination (n = 15 in each group). Gills were removed in situ. The organs were rinsed clear of blood with cold isolation buffer and homogenized using a glass homogenizer H500 with a motor-driven pestle immersed in an ice water bath to yield a homogenate in proportion 1:9 (weight/volume). The isolation buffer contained 100 mMTris-HCl; a pH of 7.2 was adjusted with HCl. Homogenates were centrifuged at 3,000g for 15 min at 4°C. After centrifugation, the supernatant was collected and frozen at −20°C until analyzed. Protein contents were determined using the method of Bradford (1976) with bovine serum albumin as a standard. Absorbance was recorded at 595 nm. All enzymatic assays were carried out at 22±0.5°C using a Specol 11 spectrophotometer (Carl Zeiss Jena, Germany) in duplicate. The enzymatic reactions were started by the addition of the tissue supernatant. Our results demonstrated that immunization by the anti-Yersinia vaccine does not alter the gills of rainbow trout. Oxidative stress parameters examined in gills homogenate, i.e., lipid peroxidation as measured by the amount of TBARS, as well as aldehydic (increased by 18.9%) and ketonic derivatives of OMP (decreased by 6.5 %) were non-significantly changed (p>0.05) in gills of vaccinated fish. Thus, immunization by anti-Yersinia vaccine does not alter oxidative stress markers compared to unhandled control in the second month after immunization. Our results confirm that the vaccine against Y. ruckeri has no adverse effect on the condition and metabolism in the gills of the fish. Alterations in the content of oxidative stress biomarkers recorded in our studies are proof that the vaccine against Y. ruckeri has no negative effects.

Nuclei Release Methods Comparison for Fresh Leaves of Rice (Oryza sativa) for Efficient High Throughput Flow Cytometry Ploidy Studies

Flow cytometry trituration methods and the efficiency of isolation buffer solutions are compared in this study for extraction of nuclei from fresh leaves of rice. The razor blade sample trituration procedure has been widely used to release nuclei from tissues in many plant species, and combined with different isolation buffers for low throughput analysis. In contrast, the bead beating trituration method has rarely been used for DNA ploidy determination, despite it being proposed as a less tedious alternative procedure to prepare nuclear suspensions. In this study, bead beating was assessed and compared with the traditional chopping procedure. Each trituration method was combined with one of three nuclear isolation buffers (i.e. Hanson’s, Otto’s and LB01 buffer). Bead beating was applied for the first time using all three of the buffers, resulting in a rapid and effective procedure for ploidy determination in fresh rice leaves. In addition, bead beating saved, while reducing the exposure of the user to harmful substances. The best results were obtained when Hanson’s nuclear isolation buffer was combined with the bead beating trituration method.