Whole-genome sequencing uncovers the structural and transcriptomic landscape of hexaploid wheat/Ambylopyrum muticum introgression lines

Wheat is a globally vital crop, but its limited genetic variation creates a challenge for breeders aiming to maintain or accelerate agricultural improvements over time. Introducing novel genes and alleles from wheat's wild relatives into the wheat breeding pool via introgression lines is an important component of overcoming this low variation but is constrained by poor genomic resolution and limited understanding of the genomic impact of introgression breeding programmes. By sequencing 17 hexaploid wheat/Ambylopyrum muticum introgression lines and the parent lines, we have precisely pinpointed the borders of introgressed segments, most of which occur within genes. We report a genome assembly and annotation of Am. muticum that has facilitated the identification of Am. muticum resistance genes commonly introgressed in lines resistant to stripe rust. Our analysis has identified an abundance of structural disruption and homoeologous pairing across the introgression lines, likely caused by the suppressed Ph1 locus. mRNAseq analysis of six of these introgression lines revealed that novel introgressed genes are rarely expressed and those that directly replace a wheat orthologue have a tendency towards downregulation, with no discernible compensation in the expression of homoeologous copies. This study explores the genomic impact of introgression breeding and provides a schematic that can be followed to characterize introgression lines and identify segments and candidate genes underlying the phenotype. This will facilitate more effective utilization of introgression pre-breeding material in wheat breeding programmes.     

Persistence of Mycobacterium avium subsp. paratuberculosis and Other Zoonotic Pathogens during Simulated Composting, Manure Packing, and Liquid Storage of Dairy Manure

Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis (Mycobacterium paratuberculosis). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated M. paratuberculosis, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55°C, manure packing at 25°C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with M. paratuberculosis at 10⁶ CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of M. paratuberculosis was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO₂, while no significant change in carbon content was observed in the liquid slurry. Escherichia coli, Salmonella, and Listeria were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55°C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days. M. paratuberculosis was detected by standard culture only on day 0 in all the treatments, but was undetectable in any treatment at 3 and 7 days. On days 14, 28, and 56, M. paratuberculosis was detected in the liquid storage treatment but remained undetectable in the compost and pack treatments. However, M. paratuberculosis DNA was detectable through day 56 in all treatments and up to day 175 in liquid storage treatments. Taken together, the results indicate that high-temperature composting is more effective than pack storage or liquid storage of manure in reducing these pathogens in dairy manure. Therefore, thermophilic composting is recommended for treatment of manures destined for pathogen-sensitive environments such as those for vegetable production, residential gardening, or application to rapidly draining fields.     

Impaired recycling of apolipoprotein E4 is associated with intracellular cholesterol accumulation

After internalization of triglyceride-rich lipoproteins (TRL) in hepatoma cells, TRL particles are immediately disintegrated in the early endosomal compartment. This involves the targeting of lipids and apoprotein B along the degradative pathway and the recycling of TRL-derived apoE through recycling endosomes. Re-secretion of apoE is accompanied by the concomitant association of apoE and cellular cholesterol with high-density lipoproteins (HDL). Since epidemiological data showed that apoE3 and apoE4 have differential effects on HDL metabolism, we investigated whether the intracellular processing of TRL-derived apoE4 differs from apoE3-TRL. In this study, we demonstrated by radioactive and immunofluorescence uptake experiments that cell-surface binding and internalization of TRL-derived apoE4 are increased compared with apoE3 in hepatoma cells. Pulse-chase experiments revealed that HDL-induced recycling, but not disintegration and degradation, of apoE4-enriched TRL is strongly reduced in these cells. Furthermore, impaired HDL-induced apoE4 recycling is associated with reduced cholesterol efflux. Studies performed in Tangier fibroblasts showed that apoE recycling does not depend on ATP-binding cassette transporter A1 activity. These studies provide initial evidence that impaired recycling of apoE4 could interfere with intracellular cholesterol transport and contribute to the pathophysiological lipoprotein profile observed in apoE4 homozygotes.     

Effect of osmotic desiccation on longevity and temperature tolerance of Phasmarhabditis hermaphrodita (Nematoda: Rhabditidae)

Limited storage stability severely restricts the biological control potential of slug-parasitic nematodes. In a series of experiments, we evaluated the effects of temperature and osmotic desiccation on the short- and long-term survival of the slug-parasitic nematode Phasmarhabditis hermaphrodita. Nematode survival in petri dishes at 1,500 infective juveniles/ml did not differ significantly at 5, 10, and 15 C but declined rapidly at 25 and 30 C. At 25 C about 50% of the nematodes survived for 4 wk, but at 30 C no nematode survived past 1 day. About 50% of the nematodes survived for 32 wk at 20 C. About 35-40% of the nematodes survived up to a year at 5, 10, and 15 C. Phasmarhabditis hermaphrodita showed poor survival under osmotic desiccation in glycerol with 15 and 20% glycerol significantly reducing survival at 5 and 15 C. Although the nematodes tolerated 10% glycerol, this level of desiccation also did not enhance long-term survival at either 5 or 15 C. There was a significant decrease in nematode survival in 10% glycerol at 25 C during the first 2-3 wk, but about 16% of the nematodes survived for 6 wk in 10% glycerol as compared with only 1% survival in water. The greatest benefit of osmotic desiccation in glycerol was observed in the enhanced survival of P. hermaphrodita at temperature extremes. Over 96% of the nematodes survived a 6-hr exposure to 35 C in 10% glycerol, whereas only 9% survived in water. Similarly, over 90% of the nematodes survived an exposure to -20 C for 4 hr in 10% glycerol, but less then 2% survived in water. We conclude that 5-15 C is an optimum temperature range for the storage of P. hermaphrodita. We also conclude that osmotic desiccation in 10% glycerol can substantially increase survival of P. hermaphrodita at temperature extremes (35 and -20 C) for short periods but has no effect on nematode longevity at the optimum temperature range of 5-15 C.     

Heterochromatin: new possibilities for the inheritance of structure

Significant portions of the eukaryotic genome are heterochromatic, made up largely of repetitious sequences and possessing a distinctive chromatin structure associated with gene silencing. New insights into the form of packaging, the associated histone modifications, and the associated nonhistone chromosomal proteins of heterochromatin have suggested a mechanism for providing an epigenetic mark that allows this distinctive chromatin structure to be maintained following replication and to spread within a given domain.     

Endotoxin Activity of Moraxella osloensis against the Grey Garden Slug, Deroceras reticulatum

Moraxella osloensis is a gram-negative bacterium associated with Phasmarhabditis hermaphrodita, a slug-parasitic nematode that has prospects for biological control of mollusk pests, especially the grey garden slug, Deroceras reticulatum. This bacterium-feeding nematode acts as a vector that transports M. osloensis into the shell cavity of the slug, and the bacterium is the killing agent in the nematode-bacterium complex. We discovered that M. osloensis produces an endotoxin(s), which is tolerant to heat and protease treatments and kills the slug after injection into the shell cavity. Washed or broken cells treated with penicillin and streptomycin from 3-day M. osloensis cultures were more pathogenic than similar cells from 2-day M. osloensis cultures. However, heat and protease treatments and 2 days of storage at 22°C increased the endotoxin activity of the young broken cells but not the endotoxin activity of the young washed cells treated with the antibiotics. This suggests that there may be a proteinaceous substance(s) that is structurally associated with the endotoxin(s) and masks its toxicity in the young bacterial cells. Moreover, 2 days of storage of the young washed bacterial cells at 22°C enhanced their endotoxin activity if they were not treated with the antibiotics. Furthermore, purified lipopolysaccharide (LPS) from the 3-day M. osloensis cultures was toxic to slugs, with an estimated 50% lethal dose of 48 μg per slug, thus demonstrating that the LPS of M. osloensis is an endotoxin that is active against D. reticulatum. This appears to be the first report of a biological toxin that is active against mollusks.     

High-resolution mapping of mouse Chromosome 8 identifies an evolutionary chromosomal breakpoint

The central region of mouse Chromosome (Chr) 8, containing the myodystrophy (myd) locus, is syntenic with human Chr 4q28-qter. The human neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) maps to Chr 4q35, and myd has been proposed as a mouse homolog of FSHD. We have employed a comparative mapping approach to investigate this relationship further by extending the mouse genetic map of this region. We have ordered 12 genes in a single cross, 8 of which have human homologs on 4q28-qter. The results confirm a general relationship between the most distal genes on human 4q and the most proximal genes in the mouse 8 syntenic region. Despite chromosomal rearrangements of syntenic groups in this region, conservation of gene order is maintained between the group of genes in the human telomeric region of 4q35 and MMU8. Furthermore, this conserved telomeric HSA4q35 syntenic group maps proximal to the myd mutation and is flanked by genes with homologs on HSA8p22. At the proximal boundary of the MMU8 linkage group we have identified a single 300-kb YAC containing the genes Frgl and Pcml, which have human homologs on 4q35 and 8p22, respectively. Thus, this YAC spans an evolutionary chromosomal breakpoint. As well as providing clues about chromosomal evolution, this map of the FSHD syntenic mouse region should prove invaluable in the isolation of candidate genes for this disease. Received: 20 January 1998 / Accepted: 10 April 1998     

Regulation of heterochromatin by histone methylation and small RNAs

Heterochromatin mediates various nuclear processes including centromere function, gene silencing and nuclear organization. Although it was discovered nearly 75 years ago, the pathways involved in heterochromatin establishment, assembly and epigenetic maintenance have been elusive. Recent reports have demonstrated that distinct and novel chromatin-associated factors, including DNA, RNA and histone modifications, are involved in each of these events. These new findings define a novel conserved mechanism of heterochromatin formation that is likely to have an impact on all eukaryotic silencing pathways.     

Biochemical interactions between proteins and mat1 cis-acting sequences required for imprinting in fission yeast

DNA recombination required for mating type (mat1) switching in Schizosaccharomyces pombe is initiated by mat1 imprinting. The imprinting event is regulated by mat1 cis-acting elements and by several trans-acting factors, including swi1 (for switch), swi3, swi7, and sap1. swi1 and swi3 were previously shown to function in dictating unidirectional mat1 DNA replication by controlling replication fork movement around the mat1 region and, second, by pausing fork progression around the imprint site. With biochemical studies, we investigated whether the trans-acting factors function indirectly or directly by binding to the mat1 cis-acting sequences. First, we report the identification and DNA sequence of the swi3 gene. swi3 is not essential for viability, and, like the other factors, it exerts a stimulatory effect on imprinting. Second, we showed that only Swi1p and Swi3p interact to form a multiprotein complex and that complex formation did not require their binding to a DNA region defined by the smt-0 mutation. Third, we found that the Swi1p-Swi3p complex physically binds to a region around the imprint site where pausing of replication occurs. Fourth, the protein complex also interacted with the mat1-proximal polar terminator of replication (RTS1). These results suggest that the stimulatory effect of swi1 and swi3 on switching and imprinting occurs through interaction of the Swi1p-Swi3p complex with the mat1 regions.