Identification of Genetic Diversity, Pyrrocidine-Producing Strains and Transmission Modes of Endophytic Sarocladium zeae Fungi from Zea Crops.

Genotyping by sequencing (GBS) was used to reveal the inherent genetic variation within the haploid fungi Sarocladium zeae isolated from diverse Zea germplasm, including modern Zea mays and its wild progenitors-the teosintes. In accordance with broad host relationship parameters, GBS analysis revealed significant host lineages of S. zeae genetic diversity, indicating that S. zeae genetic variation may associate with different evolutionary histories of host species or varieties. Based on a recently identified PKS-NRPS gene responsible for pyrrocidine biosynthesis in S. zeae fungi, a novel PCR assay was developed to discriminate pyrrocidine-producing S. zeae strains. This molecular method for screening bioactive strains of S. zeae is complementary to other approaches, such as chemical analyses. An eGFP-labelled S. zeae strain was also developed to investigate the endophytic transmission of S. zeae in Z. mays seedlings, which has further improved our understanding of the transmission modes of S. zeae endophytes in maize tissues.

Outlier analyses and genome-wide association study identify glgC and ERD6-like 4 as candidate genes for foliar water-soluble carbohydrate accumulation in Trifolium repens.

Increasing water-soluble carbohydrate (WSC) content in white clover is important for improving nutritional quality and reducing environmental impacts from pastoral agriculture. Elucidation of genes responsible for foliar WSC variation would enhance genetic improvement by enabling molecular breeding approaches. The aim of the present study was to identify single nucleotide polymorphisms (SNPs) associated with variation in foliar WSC in white clover. A set of 935 white clover individuals, randomly sampled from five breeding pools selectively bred for divergent (low or high) WSC content, were assessed with 14,743 genotyping-by-sequencing SNPs, using three outlier detection methods: PCAdapt, BayeScan and KGD-FST. These analyses identified 33 SNPs as discriminating between high and low WSC populations and putatively under selection. One SNP was located in the intron of ERD6-like 4, a gene coding for a sugar transporter located on the vacuole membrane. A genome-wide association study using a subset of 605 white clover individuals and 5,757 SNPs, identified a further 12 SNPs, one of which was associated with a starch biosynthesis gene, glucose-1-phosphate adenylyltransferase, glgC. Our results provide insight into genomic regions underlying WSC accumulation in white clover, identify candidate genomic regions for further functional validation studies, and reveal valuable information for marker-assisted or genomic selection in white clover.

Breaking Free: The Genomics of Allopolyploidy-Facilitated Niche Expansion in White Clover.

The merging of distinct genomes, allopolyploidization, is a widespread phenomenon in plants. It generates adaptive potential through increased genetic diversity, but examples demonstrating its exploitation remain scarce. White clover (Trifolium repens) is a ubiquitous temperate allotetraploid forage crop derived from two European diploid progenitors confined to extreme coastal or alpine habitats. We sequenced and assembled the genomes and transcriptomes of this species complex to gain insight into the genesis of white clover and the consequences of allopolyploidization. Based on these data, we estimate that white clover originated ∼15,000 to 28,000 years ago during the last glaciation when alpine and coastal progenitors were likely colocated in glacial refugia. We found evidence of progenitor diversity carryover through multiple hybridization events and show that the progenitor subgenomes have retained integrity and gene expression activity as they traveled within white clover from their original confined habitats to a global presence. At the transcriptional level, we observed remarkably stable subgenome expression ratios across tissues. Among the few genes that show tissue-specific switching between homeologous gene copies, we found flavonoid biosynthesis genes strongly overrepresented, suggesting an adaptive role of some allopolyploidy-associated transcriptional changes. Our results highlight white clover as an example of allopolyploidy-facilitated niche expansion, where two progenitor genomes, adapted and confined to disparate and highly specialized habitats, expanded to a ubiquitous global presence after glaciation-associated allopolyploidization.

Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing.

BACKGROUND: The recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or "orphan" crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6-$9/sample) for consumables. RESULTS: We modified a non-organic solvent protocol to extract microgram quantities (1-13 µg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384-576 samples/day), and is inexpensive at US$0.62/sample for consumables. CONCLUSIONS: This versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

Randomized control of extracorporeal shock wave therapy versus placebo for chronic decubitus ulceration.

OBJECTIVE: To assess whether extracorporeal shock wave therapy increases the rate of healing in chronic decubitus ulceration. DESIGN: Double-blind randomized cross-over study. SETTING: A large, long-stay hospital specializing in the management of people with complex neurological disabilities. SUBJECTS: The total population of available patients with chronic neurological conditions and chronic decubitus ulceration who met the inclusion criteria. INTERVENTIONS: Ulcers were randomized into receiving either the extracorporeal shock wave therapy or the placebo for a four-week period, followed by a two-week 'washout' period followed by a four-week period of the cross-over treatment/ placebo. MAIN MEASURES: Measurement of the area of the ulceration. For each observation the average of three measurements were taken. RESULTS: Nine ulcers (in eight patients) were included in the study: five on the buttocks/sacrum/trochanter and four on the feet/ankles. All those with static chronic ulcers showed improved healing starting 6-8 weeks after the start of extracorporeal shock wave therapy, whether treated first with the placebo or the therapy. CONCLUSIONS: Extracorporeal shock wave therapy has a potential part to play in the treatment of chronic skin ulceration.

Do patients expect too much from extracorporeal shock wave lithotripsy?

BACKGROUND: The era of patient-focused care has brought a new dimension to medical treatment. Now, as well as looking for therapies that work effectively, they must also be acceptable to patients. Previous studies in this department have shown that stone clearance rates with lithotripsy compare favourably with national standards, but very little work has been done regarding patient's own perception of the experience. METHODS: A postal survey was conducted of all patients attending the lithotripsy unit over a period of 1 year, questioning their opinion of all aspects of the treatment. The results were analysed by conversion to a numerical score. RESULTS: On the whole, patients expressed satisfaction, but they were more dissatisfied with the stone clearance than any other aspect of their treatment. In the light of a previous study, it was felt that the patients' expectations were unrealistic. As a result, we changed our practice such that patients attending the unit have a better idea of the likelihood of success. CONCLUSIONS: This was a simple and cost-effective exercise, that has generated some valuable data which can be used to improve the entire patient experience.

Sticks and stones: use of acupuncture in extracorporeal shockwave lithotripsy.

BACKGROUND AND PURPOSE: Extracorporeal shockwave lithotripsy (SWL) is an effective noninvasive, outpatient method of stone clearance. In our unit, it is performed using a combination of oral analgesia and intravenous sedation, which allows us to treat to therapeutic levels in the vast majority of our patients. However, we have encountered patients who do not tolerate various elements of the analgesia protocol and thus cannot be treated to full effect. The options for these people are currently limited to either SWL under formal sedation or epidural or general anesthetic or the use of another technique of stone clearance, such as percutaneous nephrolithotomy, which may not be as appropriate, and again necessitates an anesthetic, an inpatient stay, or both. PATIENTS AND METHODS: We describe three patients who had previously failed SWL who received acupuncture in place of standard analgesia prior to the next treatment. RESULTS: All three patients were able to tolerate the procedure better and were treated at a higher level with more shocks than in the previous session. No side effects were noted. CONCLUSIONS: We propose that acupuncture may be considered in patients unable to take standard sedoanalgesia. It is a cost-effective, safe method of inducing sedation with analgesia and had no demonstrable side effects in our series. It provides an attractive alternative to the use of general or regional anesthetics in these patients.