Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

Hierarchical shotgun sequencing remains the method of choice for assembling high‐quality reference sequences of complex plant genomes. The efficient exploitation of current high‐throughput technologies and powerful computational facilities for large‐insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole‐genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high‐quality assemblies of a large number of clones to assemble map‐based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.     

Feed intake and performance of growing lambs raised on concentrate-based diets under cafeteria feeding systems

Two trials were undertaken to study the effects of cafeteria feeding systems on the feed intake, animal performance and carcass characteristics of growing lambs. Trial 1 was designed to compare conventional and cafeteria feeding systems in terms of the growth of individually reared lambs. For this assay, 26 weaned Merino lambs (15.5 ± 0.20 kg live weight) were assigned to three dietary treatment groups: (1) a control group fed barley straw and commercial concentrate under a conventional feeding system, (2) group W100S, fed soya-bean meal, whole barley grain and a mineral-vitamin supplement under a cafeteria feeding system, and (3) group W100S-T, fed as in the W100S treatment but allowing the lambs an initial training period so they could learn to identify a number of feeds. The feeding system had no significant effect ( P>0.05) on either average daily live-weight gain, carcass weight, or carcass conformation. The food conversion ratio was lower ( P < 0.05) for the cafeteria-reared animals (2.9 ± 0.16 v. 2.5 ± 0.08 g dry-matter intake per g average daily gain) than those of the control group. This might be related to the higher crude protein intake seen in the cafeteria groups (150 ± 5.6 v. 208 ± 12.5 g per animal per day; P < 0.001).In trial 2, cafeteria and conventional feeding system were compared in terms of the growth of feedlot lambs. Two hundred weaned Merino lambs (13.1 ± 0.10 kg) were divided into two experimental groups: (1) a control group, offered commercial concentrate and barley straw, and (2) a cafeteria group fed the same diet as W100ST in trial 1. The average daily gain (282 ± 5.8 and 309 ± 6.5; P < 0.01) was greater in the cafeteria than in the control group. Whereas neither carcass conformation nor fatness were affected by the feeding system, the dressing percentage was slightly higher ( P>0.001) in the conventional than in the cafeteria system lambs.The use of cafeteria systems for fattening lambs can improve the feed conversion efficiency and body growth rate over those achieved with conventional feeding systems, although the crude protein intake in these systems seems to be in excess of requirements.     

Partial purification and properties of nuclease 1 from barley malt

A sugar-unspecific nuclease has been purified 260-fold from barley malt diastase. The enzyme, a glycoprotein of 37 000 MW, is highly active on single-stranded polynucleotides at pH 5–6. The nuclease is inhibited by several adenine nucleotides, and it binds weakly to NADP-agarose and ATP-agarose.     

HvLsi1 is a silicon influx transporter in barley

Most plants accumulate silicon in their bodies, and this is thought to be important for resistance against biotic and abiotic stresses; however, the molecular mechanisms for Si uptake and accumulation are poorly understood. Here, we describe an Si influx transporter, HvLsi1, in barley. This protein is homologous to rice influx transporter OsLsi1 with 81% identity, and belongs to a Nod26-like major intrinsic protein sub-family of aquaporins. Heterologous expression in both Xenopus laevis oocytes and a rice mutant defective in Si uptake showed that HvLsi1 has transport activity for silicic acid. Expression of HvLsi1 was detected specifically in the basal root, and the expression level was not affected by Si supply. There was a weak correlation between Si uptake and the expression level of HvLsi1 in eight cultivars tested. In the seminal roots, HvLsi1 is localized on the plasma membrane on the distal side of epidermal and cortical cells. HvLsi1 is also located in lateral roots on the plasma membrane of hypodermal cells. These cell-type specificity of localization and expression patterns of HvLsi1 are different from those of OsLsi1. These observations indicate that HvLsi1 is a silicon influx transporter that is involved in radial transport of Si through the epidermal and cortical layers of the basal roots of barley.     

Sequential depolarization of root cortical and stelar cells induced by an acute salt shock - implications for Na(+) and K(+) transport into xylem vessels

Early events in NaCl-induced root ion and water transport were investigated in maize (Zea mays L) roots using a range of microelectrode and imaging techniques. Addition of 100 mm NaCl to the bath resulted in an exponential drop in root xylem pressure, rapid depolarization of trans-root potential and a transient drop in xylem K(+) activity (A(K+) ) within ～1 min after stress onset. At this time, no detectable amounts of Na(+) were released into the xylem vessels. The observed drop in A(K+) was unexpected, given the fact that application of the physiologically relevant concentrations of Na(+) to isolated stele has caused rapid plasma membrane depolarization and a subsequent K(+) efflux from the stelar tissues. This controversy was explained by the difference in kinetics of NaCl-induced depolarization between cortical and stelar cells. As root cortical cells are first to be depolarized and lose K(+) to the environment, this is associated with some K(+) shift from the stelar symplast to the cortex, resulting in K(+) being transiently removed from the xylem. Once Na(+) is loaded into the xylem (between 1 and 5 min of root exposure to NaCl), stelar cells become more depolarized, and a gradual recovery in A(K+) occurs.     

Palladium exposure of barley: uptake and effects

Motor vehicles are now equipped with exhaust gas catalytic converters containing rare metals, such as palladium (Pd), platinum and rhodium, as catalytic active materials, leading to significantly increased emission of these metals. Compared with platinum and rhodium, low concentrations of Pd have been shown to have more serious effects on cells and organisms. In the present study, uptake of Pd by barley and behaviour of Pd nanoparticles in nutrient solutions used to grow plants were observed in order to develop a model of Pd exposure of plant systems. Pd determination was performed using a selective separation and pre-concentration procedure, which was further developed for this study, and coupled to graphite furnace atomic absorption spectrometry. The results show that uptake of Pd depends on Pd particle diameter. Compared to other toxic metals, like mercury, Pd causes stress effects in leaves at lower concentrations in nutrient solutions. Furthermore, Pd particles are dissolved at different rates, depending on size, in the nutrient solution during plant growth.