Genomics,genetics and breeding of common bean in Africa: A review of tropical legume project

Common bean (Phaseolus vulgaris L.) is an important legume crop worldwide. The International Centre for Tropical Agriculture (CIAT) and its national partners in Africa aim to overcome production constraints of common bean and address the food, nutrition needs and market demands through development of multitrait bean varieties. Breeding is guided by principles of market‐driven approaches to develop client‐demanded varieties. Germplasm accessions from especially two sister species, P. coccineus and P. acutifolius, have been utilized as sources of resistance to major production constraints and interspecific lines deployed. Elucidation of plant mechanisms governing pest and disease resistance, abiotic stress tolerance and grain nutritional quality guides the selection methods used by the breeders. Molecular markers are used to select for resistance to key diseases and insect pests. Efforts have been made to utilize modern genomic tools to increase scale, efficiency, accuracy and speed of breeding. Through gender‐responsive participatory variety selection, market‐demanded varieties have been released in several African countries. These new bean varieties are a key component of sustainable food systems in the tropics.     

Maturity and storage influence on the apple (Malus domestica) allergen Mal d 3, a nonspecific lipid transfer protein

Consumption of apples can provoke severe allergic reactions, in susceptible individuals, due to the presence of the allergen Mal d 3, a nonspecific lipid transfer protein, found largely in the fruit skin. Levels of Mal d 3 were determined in peel as a function of apple cultivar, position of the fruit growing on the tree, apple maturity, and postharvest storage by ELISA. As the apples mature, Mal d 3 levels increased, although the rate was dependent on cultivar and tree position. During storage, levels of Mal d 3 decreased in all cultivars (cvs. Cox, Jonagored, and Gala), the rate of overall decrease being greatest under controlled atmosphere conditions. There was no correlation between Mal d 3 levels and total apple peel protein, indicating specific alterations in Mal d 3 expression. Thus pre- and postharvest treatments (i.e., storage) can modify the allergen load in apple peel, the highest levels being found in overly mature and freshly harvested fruits.     

DORSAL DENS ANGULATION AND A CHIARI TYPE MALFORMATION IN A CAVALIER KING CHARLES SPANIEL

A nineteen month old Cavalier King Charles had clinical signs consistent with a cervical myelopathy. Radiologically there was abnormal angulation of the dens with cervical spinal cord compression and a Chiari type malformation of the atlanto-occipital region. The condition and method of investigation is discussed.     

Digested Ara h 1 loses sensitizing capacity when separated into fractions

The major peanut allergen Ara h 1 is an easily digestible protein under physiological conditions. The present study revealed that pepsin digestion products of Ara h 1 retained the sensitizing potential in a Brown Norway rat model, while this sensitizing capacity was lost by separating the digest into fractions by gel permeation chromatography. Protein chemical analysis showed that the peptide composition as well as the aggregation profiles of the fractions of Ara h 1 digest differed from that of the whole pool. These results indicate that the sensitizing capacity of digested Ara h 1 is a consequence of the peptides being in an aggregated state resembling the intact molecule or that most peptides of the digests need to be present in the same solution, having a synergistic or adjuvant effect and thereby augmenting the immune response against other peptides.     

Transglutaminase polymerization of peanut proteins

Transglutaminase promotes protein cross-linking reactions through an acyl transferase mechanism involving protein-bound glutaminyl residues and primary amines including the epsilon-amino group of lysine residues in soy, myosin, gluten, oat globulin, casein, and whey. Herein, we present a first report of exogenous transglutaminase catalysis of several peanut protein fractions, including purified Ara h 1. In most cases, SDS-PAGE banding patterns revealed the formation of high molecular weight polymers while catalysis of Ara h 1 resulted in distinct dimer formation. Cross-linking effects were accomplished in the presence and absence of the reducing reagent, dithiothreitol. Ortho-phthaldialdehyde assays, used to quantify the degree of polymerization, indicated approximately 21% and approximately 30% coupling over a similar time interval, using either cold hexane extracted peanut protein fractions or lightly roasted flour dispersions, respectively. Rheological measurements established that transglutaminase-modified peanut extracts exhibited lowered viscosity readings compared to nontreated dispersions. Peanut protein polymers and glycoprotein conjugates, created by covalent linkage between protein substrates and monosaccharide amino sugars, exhibited similar IgE binding activity, compared to control solutions. These results suggested that potential allergic responses were not enhanced after enzymatic modification. Ultimately, these approaches may provide novel peanut-based food ingredients with unique functional characteristics for expanded applications within the world marketplace.     

Reduced grazing and the decline of Pulsatilla vulgaris Mill. (Ranunculaceae) in England, UK

In England Pulsatilla vulgaris is a threatened herb that declined from 130 to 33 sites between 1750 and the 1960s due to ploughing-up of calcareous grassland. We examined the subsequent fate of these populations using documentary evidence and field survey. Demographic trends were related to changes in grassland composition, structure and management and responses to increased above-ground competition (caused by reduced grazing) were simulated in a 10-year shading experiment. Since 1968 P. vulgaris has been lost from 16 sites and gradually declined on four others. However, the total population size increased by 258% due to the reintroduction of winter grazing on three sites. This produced a significantly shorter, more herb-rich sward, with a lower cover of Bromopsis erecta (c. 10%), than on sites where populations remained stable or declined. Experimental shading had a significant negative effect on plant survival and flowering performance. These results confirmed that reduced grazing is now one of the major threats to species dependent on short swards, especially on isolated sites where livestock farming is no longer economically viable. That many of these declines took place on nature reserves highlights the difficulties of managing isolated grasslands, and the urgent need to re-instate grazing on reserves supporting populations of threatened species in otherwise intensively managed lowland landscapes.     

Effects of soaking on the water-soluble carbohydrate and crude protein content of hay

The aim of this study was to determine the amounts of water-soluble carbohydrate (WSC), WSC constituents and protein leached from a range of U.K. hays soaked according to common practice. Initial hay WSC content ranged from 123 to 230 g/kg dry matter (DM). Soaking the hays for up to 16 hours in water at a mean temperature of 8°C resulted in a mean loss of 27 per cent (range 6 to 54 per cent) of hay WSC. The mean percentage losses of WSC constituents were 24 per cent (range 14 to 31) for fructan, 41 per cent (range 21 to 70) for fructose, 45 per cent (range 28 to 100) for sucrose and 56 per cent (range 29 to 100) for glucose. The mean crude protein content of the initial hays was 58.7 g/kg DM (range 30 to 86 g/kg DM) and this value was not affected significantly by soaking. Despite a mean WSC loss of 27 per cent, the WSC contents of seven of the hays remained above the suggested upper limit for laminitic animals of 100 g/kg DM.     

Myokymia and neuromyotonia in 37 Jack Russell terriers

The clinical and clinicopathological characteristics, treatment and outcome of vermicular muscle contractions (myokymia) and generalized muscle stiffness (neuromyotonia) in 37 Jack Russell terriers were evaluated retrospectively. Thirty dogs were affected by both disorders, whereas seven were presented with myokymia and never developed neuromyotonia. Clinical signs started at the mean age of 8 months. Except for signs of myokymia and neuromyotonia, clinical and neurological examination was normal in all dogs. Thirty dogs demonstrated typical signs of hereditary ataxia.Changes in serum chemistry included increased creatine kinase, aspartate aminotransferase and alanine aminotransferase concentrations. Electromyographic abnormalities, especially in muscles showing macroscopically visible myokymia, consisted of semirhythmic bursts of doublet, triplet, or multiplet discharges of a single motor unit. The amplitudes varied between 80 μV and 1 mV and occurred with an interburst frequency between 10 and 40 Hz and an intraburst frequency between 150 and 280 Hz.Most dogs were treated with a sodium channel blocker with variable results. Seven dogs died (most likely because of hyperthermia) or were euthanased during a neuromyotonic attack; 15 dogs were euthanased due to worsening of clinical signs, or lack of or no long-lasting effect of medication, and three were euthanased for unknown or unrelated reasons. Nine dogs were lost to follow-up and three were still alive 5–10.5 years after the start of clinical signs. In conclusion, young Jack Russell terriers with myokymia and neuromyotonia should undergo a complete blood and electrophysiological examination. Long-term prognosis is not favourable.     

Universal endogenous gene controls for bisulphite conversion in analysis of plant DNA methylation

Background

Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once.

Results

We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca²⁺ chelator and detergent. Cell extracts prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method was also applicable to genomic DNA extraction for PCR analysis. Our method was applied to many other plant species, and worked well for some of them.

Conclusions

Our method is rapid and economical, and allows many samples to be prepared simultaneously for protein analysis. Our method is useful not only for Arabidopsis research but also research on certain other species.