Evaluation of diagnostic molecular markers for DUS phenotypic assessment in the cereal crop, barley (Hordeum vulgare ssp. vulgare L.)

The deployment of genetic markers is of interest in crop assessment and breeding programmes, due to the potential savings in cost and time afforded. As part of the internationally recognised framework for the awarding of Plant Breeders’ Rights (PBR), new barley variety submissions are evaluated using a suite of morphological traits to ensure they are distinct, uniform and stable (DUS) in comparison to all previous submissions. Increasing knowledge of the genetic control of many of these traits provides the opportunity to assess the potential of deploying diagnostic/perfect genetic markers in place of phenotypic assessment. Here, we identify a suite of 25 genetic markers assaying for 14 DUS traits, and implement them using a single genotyping platform (KASPar). Using a panel of 169 UK barley varieties, we show that phenotypic state at three of these traits can be perfectly predicted by genotype. Predictive values for an additional nine traits ranged from 81 to 99 %. Finally, by comparison of varietal discrimination based on phenotype and genotype resulted in correlation of 0.72, indicating that deployment of molecular markers for varietal discrimination could be feasible in the near future. Due to the flexibility of the genotyping platform used, the genetic markers described here can be used in any number or combination, in-house or by outsourcing, allowing flexible deployment by users. These markers are likely to find application where tracking of specific alleles is required in breeding programmes, or for potential use within national assessment programmes for the awarding of PBRs.

PCR-based markers diagnostic for spring and winter seasonal growth habit in barley

Toward the ultimate goal of replacing field-based evaluation of seasonal growth habit, we describe the design and validation of a multiplex polymerase chain reaction assay diagnostic for allelic status at the barley (Hordeum vulgare ssp. vulgare L.) vernalization locus, VRN-H1 By assaying for the presence of all known insertion–deletion polymorphisms thought to be responsible for the difference between spring and winter alleles, this assay directly tests for the presence of functional polymorphism at VRN-H1 Four of the nine previously recognized VRN-H1 haplotypes (including both winter alleles) give unique profiles using this assay. The remaining five spring haplotypes share a single profile, indicative of function-altering deletions spanning, or adjacent to, the putative “vernalization critical” region of intron 1. When used in conjunction with a previously published PCR-based assay diagnostic for alleles at VRN-H2, it was possible to predict growth habit in all the 100 contemporary UK spring and winter lines analyzed in this study. This assay is likely to find application in instances when seasonal growth habit needs to be determined without the time and cost of phenotypic assessment and during marker-assisted selection using conventional and multicross population analysis.

Integration of retrotransposons-based markers in a linkage map of barley

A deeper understanding of random markers is important if they are to be employed for a range of objectives. The sequence specific amplified polymorphism (S-SAP) technique is a powerful genetic analysis tool which exploits the high copy number of retrotransposon long terminal repeats (LTRs) in the plant genome. The distribution and inheritance of S-SAP bands in the barley genome was studied using the Steptoe × Morex (S × M) double haploid (DH) population. Six S-SAP primer combinations generated 98 polymorphic bands, and map positions were assigned to all but one band. Eight putative co-dominant loci were detected, representing 16 of the mapped markers. Thus at least 81 of the mapped S-SAP loci were dominant. The markers were distributed along all of the seven chromosomes and a tendency to cluster was observed. The distribution of S-SAP markers over the barley genome concurred with the knowledge of the high copy number of retrotransposons in plants. This experiment has demonstrated the potential for the S-SAP technique to be applied in a range of analyses such as genetic fingerprinting, marker assisted breeding, biodiversity assessment and phylogenetic analyses.

Non-concordance between genetic profiles of olive oil and fruit: a cautionary note to the use of DNA markers for provenance testing.

To investigate the contribution of paternal alleles to the DNA content of olive oil, genetic analyses of olive DNA samples from fruits, leaves, and oil derived from the same tree (cv. Leccino) were carried out. DNA extracted from maternal tissues--leaves and flesh--from different fruits showed identical genetic profiles using a set of DNA markers. Additional simple sequence repeat (SSR) alleles, not found in the maternal samples, were amplified in the embryos (stone), and they were also detected in DNA extracted from the paste obtained by crushing whole fruits and from the oil pressed from this material. These results demonstrate that the DNA profile obtained from olive oil is likely to represent a composite profile of the maternal alleles juxtaposed with alleles contributed by various pollen donors. Therefore, care needs to be taken in the interpretation of DNA profiles obtained from DNA extracted from oil for resolving provenance and authenticity issues.

Markers for nutrition studies: review of criteria for the evaluation of markers

The following criteria were identified as essential elements in the evaluation of markers: (1) the marker has a causal biological link with the endpoint, (2) there is a significant association between marker and endpoint in the target population, (3) marker changes consistently with the endpoint, e.g., in response to an intervention, and (4) change in the marker explains a substantial proportion of the change in the endpoint in response to the intervention.

Gene expression changes in phosphorus deficient potato (Solanum tuberosum L.) leaves and the potential for diagnostic gene expression markers

Background: There are compelling economic and environmental reasons to reduce our reliance on inorganic phosphate (Pi) fertilisers. Better management of Pi fertiliser applications is one option to improve the efficiency of Pi fertiliser use, whilst maintaining crop yields. Application rates of Pi fertilisers are traditionally determined from analyses of soil or plant tissues. Alternatively, diagnostic genes with altered expression under Pi limiting conditions that suggest a physiological requirement for Pi fertilisation, could be used to manage Pifertiliser applications, and might be more precise than indirect measurements of soil or tissue samples. Results: We grew potato (Solanum tuberosum L.) plants hydroponically, under glasshouse conditions, to control their nutrient status accurately. Samples of total leaf RNA taken periodically after Pi was removed from the nutrient solution were labelled and hybridised to potato oligonucleotide arrays. A total of 1,659 genes were significantly differentially expressed following Pi withdrawal. These included genes that encode proteins involved in lipid, protein, and carbohydrate metabolism, characteristic of Pi deficient leaves and included potential novel roles for genes encoding patatin like proteins in potatoes. The array data were analysed using a support vector machine algorithm to identify groups of genes that could predict the Pi status of the crop. These groups of diagnostic genes were tested using field grown potatoes that had either been fertilised or unfertilised. A group of 200 genes could correctly predict the Pi status of field grown potatoes. Conclusions: This paper provides a proof-of-concept demonstration for using microarrays and class prediction tools to predict the Pi status of a field grown potato crop. There is potential to develop this technology for other biotic and abiotic stresses in field grown crops. Ultimately, a better understanding of crop stresses may improve our management of the crop, improving the sustainability of agriculture.

Evaluation of genetic markers as instruments for Mendelian randomization studies on vitamin D

Combining SNPs into allele scores provides a more powerful instrument for MR analysis than a single SNP in isolation. Population stratification and the potential for pleiotropic effects need to be considered in MR studies on vitamin D.

Effects of increased wholegrain consumption on immune and inflammatory markers in healthy low habitual wholegrain consumers

Purpose Wholegrain (WG) consumption is associated with reduced risk of cardiovascular disease, but clinical data on inflammation and immune function is either conflicting or limited. The objective of this study was to assess the impact of increasing WG consumption to at least 80 g/d on markers of inflammation and glucose metabolism and on phenotypic and functional aspects of the immune system, in healthy, middle-aged adults with low habitual WG intake. Methods Subjects consumed a diet high in WG (> 80 g/d) or low in WG (< 16 g/d, refined grain diet) in a crossover study, with 6-week intervention periods, separated by a 4-week washout. Adherence to the dietary regimes was achieved by dietary advice and provision of a range of food products, with compliance verified through analysis of plasma alkylresorcinols (ARs). Results On the WG intervention, WG consumption reached 168 g/d (P < 0.001), accompanied by an increase in plasma ARs (P < 0.001) and fibre intake (P < 0.001), without affecting other aspects of dietary intake. On the WG arm there were trends for lower ex vivo activation of CD4+ T cells and circulating concentrations of IL-10, C-reactive protein, C-peptide, insulin and plasminogen activator inhibitor-1. The percentage of CD4+ central memory T cells and circulating levels of adipsin tended to increase during the WG intervention. Conclusions Despite the dramatic increase in WG consumption, there were no effects on phenotypic or functional immune parameters, markers of inflammation or metabolic markers.

Flanking SNP markers for vicine–convicine concentration in faba bean (Vicia faba L).

The pyrimidine glycosides, vicine and convicine, limit the use of faba bean (Vicia faba L.) as food and feed. A single recessive gene, vc-, is responsible for a lowered vicine–convicine concentration. The biosynthetic pathway of these closely related compounds is not known, and the nearest available markers are several cM away from vc-. Improved markers would assist breeding and help to identify candidate genes. A segregating population of 210 F5 recombinant inbred lines was developed from the cross of Mélodie/2 (low vicine–convicine) × ILB 938/2 (normal vicine–convicine), and vicine–convicine concentrations were determined twice on each line. The population was genotyped with a set of 188 SNPs. A strong, single QTL for vicine–convicine concentration was identified on chromosome I, flanked by markers 1.0 cM away on one side and 2.6 cM on the other. The interval defined by these markers in the model species Medicago truncatula includes about 340 genes, but no candidate genes were identified. Further fine mapping should lead to the identification of tightly linked markers as well as narrowing down the search for candidate regulatory or biosynthetic genes which could underlie the vc- locus.

Xylo-oligosaccharides alone or in synbiotic combination with Bifidobacterium animalis subsp. lactis induce bifidogenesis and modulate markers of immune function in healthy adults: a double-blind, placebo-controlled, randomised, factorial cross-over study.

Prebiotics, probiotics and synbiotics are dietary ingredients with the potential to influence health and mucosal and systemic immune function by altering the composition of the gut microbiota. In the present study, a candidate prebiotic (xylo-oligosaccharide, XOS, 8 g/d), probiotic (Bifidobacterium animalis subsp. lactis Bi-07, 109 colony-forming units (CFU)/d) or synbiotic (8 g XOS+109 CFU Bi-07/d) was given to healthy adults (25–65 years) for 21 d. The aim was to identify the effect of the supplements on bowel habits, self-reported mood, composition of the gut microbiota, blood lipid concentrations and immune function. XOS supplementation increased mean bowel movements per d (P= 0·009), but did not alter the symptoms of bloating, abdominal pain or flatulence or the incidence of any reported adverse events compared with maltodextrin supplementation. XOS supplementation significantly increased participant-reported vitality (P= 0·003) and happiness (P= 0·034). Lowest reported use of analgesics was observed during the XOS+Bi-07 supplementation period (P= 0·004). XOS supplementation significantly increased faecal bifidobacterial counts (P= 0·008) and fasting plasma HDL concentrations (P= 0·005). Bi-07 supplementation significantly increased faecal B. lactis content (P= 0·007), lowered lipopolysaccharide-stimulated IL-4 secretion in whole-blood cultures (P= 0·035) and salivary IgA content (P= 0·040) and increased IL-6 secretion (P= 0·009). XOS supplementation resulted in lower expression of CD16/56 on natural killer T cells (P= 0·027) and lower IL-10 secretion (P= 0·049), while XOS and Bi-07 supplementation reduced the expression of CD19 on B cells (XOS × Bi-07, P= 0·009). The present study demonstrates that XOS induce bifidogenesis, improve aspects of the plasma lipid profile and modulate the markers of immune function in healthy adults. The provision of XOS+Bi-07 as a synbiotic may confer further benefits due to the discrete effects of Bi-07 on the gut microbiota and markers of immune function.

Characterization of para-Nitrophenol-degrading bacterial communities in river water by using functional markers and stable isotope probing

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [13C6]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.

Investigating subtypes of reward processing deficits as trait markers for depression

Background: Anhedonia, the loss of pleasure in usually enjoyable activities, is a central feature of major depressive disorder (MDD). The aim of the present study was to examine whether young people at a familial risk of depression display signs of anticipatory, motivational or consummatory anhedonia, which would indicate that these deficits may be trait markers for MDD. Methods: The study was completed by 22 participants with a family history of depression (FH+) and 21 controls (HC). Anticipatory anhedonia was assessed by asking participants to rate their anticipated liking of pleasant and unpleasant foods which they imagined tasting when cued with images of the foods. Motivational anhedonia was measured by requiring participants to perform key presses to obtain pleasant chocolate taste rewards or to avoid unpleasant apple tastes. Additionally, physical consummatory anhedonia was examined by instructing participants to rate the pleasantness of the acquired tastes. Moreover, social consummatory anhedonia was investigated by asking participants to make preference-based choices between neutral facial expressions, genuine smiles, and polite smiles. Results: It was found that the FH+ group’s anticipated liking of unpleasant foods was significantly lower than that of the control group. By contrast, no group differences in the pleasantness ratings of the actually experienced tastes or in the amount of performed key presses were observed. However, controls preferred genuine smiles over neutral expressions more often than they preferred polite smiles over neutral expressions, while this pattern was not seen in the FH+ group. Conclusion: These findings suggest that FH+ individuals demonstrate an altered anticipatory response to negative stimuli and show signs of social consummatory anhedonia, which may be trait markers for depression.

Characterisation of microsatellite markers for fig-pollinating wasps in the Pleistodontes imperialis species complex

We characterised a set of nine polymorphic microsatellite loci for Pleistodontes imperialis sp. 1, the pollinator wasp of Port Jackson fig (Ficus rubiginosa) in south-eastern Australia. Characterisation was performed on 30 female individuals collected from a population in Sydney, Australia. The average number of alleles per locus was 7.33, and eight loci were not in Hardy–Weinberg equilibrium. This was expected as fig wasps are known to be highly inbred. A test of genetic differentiation between two natural populations of P. imperialis sp. 1 (Sydney and Newcastle, Australia – some 120 km apart) yielded a very low FST value of 0.012, suggesting considerable gene flow. Bayesian clustering analysis using TESS 2.3.1, which does not assume Hardy–Weinberg equilibrium, however, indicated potential spatial substructuring between the Sydney and Newcastle populations, as well as within the Sydney population. The described loci were also characterised for two other species in the P. imperialis complex: P. imperialis sp. 2 (Townsville, Australia) and P. imperialis sp. 4 (Brisbane, Australia). Seven and six of the nine loci were polymorphic for P. imperialis sp. 2 and P.imperialis sp. 4, respectively.

Microsatellite markers for hoop-petticoat daffodils (Narcissus sect. Bulbocodii; Amaryllidaceae)

• Premise of the study: Microsatellite markers were developed using hoop-petticoat daffodils ( Narcissus sect. Bulbocodii ; Amaryllidaceae) to aid in the taxonomic revision of the section, and further to evaluate their broad applicability for daffodil cultivar identification. • Methods and Results: Three hundred fifty-one primer pairs were developed using a commercial service. Nineteen polymorphic and repeatable markers were developed by screening 67 of these primer pairs. Of these, 11 chosen markers were used to screen 317 samples; the number of alleles per locus ranged from four to 21, and the observed heterozygosity ranged from 0.101 to 0.297. There were null genotypes in some samples for six of the markers. All the microsatellites were transferable to other Narcissus sections. • Conclusions: The results indicate that these new markers have sufficient potential variation to be used for taxonomic revision of the genus and to distinguish many commercial daffodil cultivars.