Laboratory Information Management Software for genotyping workflows: Applications in high throughput crop genotyping

Background With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow. Results A laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics. Conclusion A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from http://www.icrisat.org/gt-bt/lims/lims.asp

Interaction of inflammatory cytokines and erythropoeitin in iron metabolism and erythropoiesis in anaemia of chronic disease

In chronic inflammatory conditions increased endogenous release of specific cytokines (TNFα, IL-1, IL-6, IFNγ and others) is presumed. It has been shown that those of monocyte lineage play a key role in cytokine expression and synthesis. This may be associated with changes in iron metabolism and impaired erythropoiesis and may lead to development of anaemia in patients with rheumatoid arthritis. Firstly, increased synthesis of acute phase proteins, like ferritin, during chronic inflammation is proposed as the way by which the toxic effect of iron and thereby the synthesis of free oxy-radicals causing the damage on the affected joints, may be reduced. This is associated with a shift of iron towards the mononuclear phagocyte system which may participate in the development of anaemia of chronic disease. Secondly, an inhibitory action of inflammatory cytokines (TNFα, IL-1), on proliferation and differentiation of erythroid progenitors as well as on synthesis of erythropoietin has been shown, thereby also contributing to anaemia. Finally, chronic inflammation causes multiple, complex disturbances in the delicate physiologic equilibrium of interaction between cytokines and cells (erythroid progenitors, cells of mononuclear phagocyte system and erythropoietin producing cells) leading to development of anaemia of chronic disease (Fig. 1)

Genetic diversity, hybrid performance and combining ability for yield in Musa germplasm

Genome size variation occurs within and across generations in Musa spp., which reduces the predictive accuracy of parental performance on progeny value for yield and other traits with complex inheritance. Parental selection through progeny testing of prospective parents is required to achieve further genetic gains. This was carried out in this study, using a factorial mating design involving five 4x females and five 2x males. Genetic differences among offspring families were essentially due to differences in additive effects of the parents. Thus, little recombinative heterosis can be expected upon 4x2x crossbreeding, and breeding strategies should target the development of 4x and 2x cultivars by accumulation of favorable alleles through recurrent selection within each ploidy pool. Offspring yield was positively correlated with parental GCAs but not with midparent values. Hybrid performance was also associated but not significantly correlated with genetic similarity indices based on both pedigree and molecular data. This study further suggests that current genetic models may not be adequate for populations with intergeneration genome size polymorphism

Ploidy determination in Musa germplasm using pollen and chloroplast characteristics

We attempted to determine ploidy level in the gametophyte and the sporophyte of Musa using pollen and chloroplast characteristics, respectively. In the gametophyte, interploidy differences accounted for 63.8 % of the genetic variance for pollen diameter and 87.5% for pollen stainability, the remainder being attributable to intraploidy differences among clones. While pollen count and stainability effectively separated triploid accessions from diploids or tetraploids, they did not discriminate between diploids and tetraploids. In the sporophyte, the relative contributions of interploidy and intraploidy differences to genetic variation in the number of chloroplasts in stomatal guard cells were 70.8% and 29.2%, respectively. Although pollen diameter and chloroplast number increased with ploidy, the use of the sporophytic parameter appears to provide a more satisfactory means of estimating ploidy status in Musa

Comparative analysis of phenotypic and genoytpic diversity among plantain landraces (Musa spp., AAB group)

Genetic diversity amongst 76 plantain landraces has been studied using RAPD analysis at two levels of intensity and compared with groupings based on phenotypic indices and morphotype. There was a good correlation (R2=0.78) between estimates of genetic diversity based on 76 RAPD bands and 164 RAPD bands. However, there was a poor correlation between RAPD-based estimates of genetic diversity and a phenotypic index based on agronomic characters. There was also a poor correlation between RAPD analyses and morphotype group (based on bunch type and stature). These results suggest that the traditional designations of plantain landraces based on morphotype do not provide a true reflection of overall genetic divergence. Similarly, classification systems using phenotypic indices based on agronomic characters may not provide accurate taxonomic differentiation. The level of genetic divergence within morphogroups based on bunch type suggests that True Horn plantains are derived from False Horn plantains which in turn are derived from French plantains. Genetic divergence was found to be generally quite low within the plantain landrace genepool, which is consistent with the proposed evolution of this germplasm through somatic mutation of a relatively small number of introductions. However, putative synonyms/duplicates have been shown to be genetically distinct. In contrast, a group of 12 landraces have been identified that are highly distinct from one another (showing 20–35% dissimilarity). Fertile members of this group may be useful for generating genetically diverse 2x and 4x breeding populations that can be used in breeding secondary triploid hybrid plantain varieties

Segregation at microsatellite loci in haploid and diploid gametes of Musa

The triploid genome of plantain (Musa spp., AA group) has been considered intractable to genetic improvement because of the production of putatively homogeneous 2n (= 3.r) gametes. Plantain breeding schemes have been based on the hybridization of 2n megaspores from the triploid plantain and haploid micr08pores from a wild diploid banana. The resultant full-sib populations of tetraploid hybrids exhibited extreme variation in phenotypic characteristics. This study was conducted to analyze the genetic constitution of tetraploid hybrids in order to characterize the nature of the gametes from which they were derived. Parental genotypes were screened with 31 primer pairs specific to different Musa microsatellite loci. Primers which detected polymorphisms between the parental genotypes were used to screen a population of 14 full-sib tetraploid hybrids. A subset of primers was also used to screen a population generated by self-pollination of the parental diploid banana. During analysis of the tetraploid hybrid population, 75% of the alleles donated by the maternal genotype (Obino I'Ewai) were observed to segregate. These data demonstrate the occurrence of recombination during the formation of 2n megaspores in triploid plantain. Analysis of the paternal diploid banana accession (Calcutta 4) and its selfed progeny suggests that this accession is highly heterozygous. These data demonstrate the importance of genetic characterization to Musa breeding, and also indicate that microsatellite markers are well suited for marker-assisted selection systems in Musa

La détection des patrons de réponses problématiques dans le contexte des tests informatisés

Prehospital cervical spine (c-spine) immobilisation is common, despite c-spine injury being relatively rare. Unnecessary immobilisation results in a significant burden on limited prehospital and emergency department (ED) resources. This study aimed to determine whether the incidence of unnecessary c-spine immobilisation by ambulance personnel could be safely reduced through the implementation of an evidence-based algorithm. Following a training programme, complete forms on 103 patients were identified during the audit period, of which 69 (67%) patients had their c-spines cleared at scene. Of these, 60 (87%) were discharged at scene, with no clinical adverse events reported, and 9 (13%) were taken to the local ED with non-distracting minor injuries, all being discharged home the same day. 34 (33%) patients could not have their c-spines safely cleared at scene according to the algorithm. Of these, 4 (12%) patients self-discharged at scene and 30 (88%) were conveyed to an ED as per the normal procedure. C-spine clearance at scene by ambulance personnel may have positive impacts on patient care, efficient use of resources and cost to healthcare organisations