How to perform RT-qPCR accurately in plant species?: a case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

Background: Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. Results: An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. Conclusions: Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway

Anchoring linkage groups of the Rosa genetic map to physical chromosomes with tyramide-FISH and EST-SNP markers

In order to anchor Rosa linkage groups to physical chromosomes, a combination of the Tyramide-FISH technology and the modern molecular marker system based on High Resolution Melting (HRM) is an efficient approach. Although, Tyramide-FISH is a very promising technique for the visualization of short DNA probes, it is very challenging for plant species with small chromosomes such as Rosa. In this study, we successfully applied the Tyramide-FISH technique for Rosa and compared different detection systems. An indirect detection system exploiting biotinylated tyramides was shown to be the most suitable technique for reliable signal detection. Three gene fragments with a size of 1100 pb-1700 bp (Phenylalanine Ammonia Lyase, Pyrroline-5-Carboxylate Synthase and Orcinol O-Methyl Transferase) have been physically mapped on chromosomes 7, 4 and 1, respectively, of Rosa wichurana. The signal frequency was between 25% and 40%. HRM markers of these 3 gene fragments were used to include the gene fragments on the existing genetic linkage map of Rosa wichurana. As a result, three linkage groups could be anchored to their physical chromosomes. The information was used to check for synteny between the Rosa chromosomes and Fragaria

Multipoint-likelihood maximization mapping on 4 segregating populations to achieve an integrated framework map for QTL analysis in pot azalea (Rhododendron simsii hybrids)

<p>Abstract</p> <p>Background</p> <p>Azalea (<it>Rhododendron simsii </it>hybrids) is the most important flowering pot plant produced in Belgium, being exported world-wide. In the breeding program, flower color is the main feature for selection, only in later stages cultivation related plant quality traits are evaluated. As a result, plants with attractive flowering are kept too long in the breeding cycle. The inheritance of flower color has been well studied; information on the heritability of cultivation related quality traits is lacking. For this purpose, QTL mapping in diverse genetic backgrounds appeared to be a must and therefore 4 mapping populations were made and analyzed.</p> <p>Results</p> <p>An integrated framework map on four individual linkage maps in <it>Rhododendron simsii </it>hybrids was constructed. For genotyping, mainly dominant scored AFLP (on average 364 per population) and MYB-based markers (15) were combined with co-dominant SSR (23) and EST markers (12). Linkage groups were estimated in JoinMap. A consensus grouping for the 4 mapping populations was made and applied in each individual mapping population. Finally, 16 stable linkage groups were set for the 4 populations; the azalea chromosome number being 13. A combination of regression mapping (JoinMap) and multipoint-likelihood maximization (Carthagène) enabled the construction of 4 maps and their alignment. A large portion of loci (43%) was common to at least two populations and could therefore serve as bridging markers. The different steps taken for map optimization and integration into a reference framework map for QTL mapping are discussed.</p> <p>Conclusions</p> <p>This is the first map of azalea up to our knowledge. AFLP and SSR markers are used as a reference backbone and functional markers (EST and MYB) were added as candidate genes for QTL analysis. The alignment of the 4 maps on the basis of framework markers will facilitate in turn the alignment of QTL regions detected in each of the populations. The approach we took is thoroughly different than the recently published integrated maps and well-suited for mapping in a non-model crop.</p

Identification of quantitative trait loci controlling root and shoot traits associated with drought tolerance in a lentil (Lens culinaris Medik.) recombinant inbred line population

Drought is one of the major abiotic stresses limiting lentil productivity in rainfed production systems. Specific rooting patterns can be associated with drought avoidance mechanisms that can be used in lentil breeding programs. In all, 252 co-dominant and dominant markers were used for Quantitative Trait Loci (QTL) analysis on 132 lentil recombinant inbred lines based on greenhouse experiments for root and shoot traits during two seasons under progressive drought-stressed conditions. Eighteen QTLs controlling a total of 14 root and shoot traits were identified. A QTL-hotspot genomic region related to a number of root and shoot characteristics associated with drought tolerance such as dry root biomass, root surface area, lateral root number, dry shoot biomass and shoot length was identified. Interestingly, a QTL (QRSratioIX-2.30) related to root-shoot ratio, an important trait for drought avoidance, explaining the highest phenotypic variance of 27.6 and 28.9% for the two consecutive seasons, respectively, was detected. This QTL was closed to the co-dominant SNP marker TP6337 and also flanked by the two SNP TP518 and TP1280. An important QTL (QLRNIII-98.64) related to lateral root number was found close to TP3371 and flanked by TP5093 and TP6072 SNP markers. Also, a QTL (QSRLIV-61.63) associated with specific root length was identified close to TP1873 and flanked by F7XEM6b SRAP marker and TP1035 SNP marker. These two QTLs were detected in both seasons. Our results could be used for marker-assisted selection in lentil breeding programs targeting root and shoot characteristics conferring drought avoidance as an efficient alternative to slow and labor-intensive conventional breeding methods

Optimizing cutoff scores for the Barthel Index and the modified Rankin Scale for defining outcome in acute stroke trials

Background and Purpose - There is little agreement on how to assess outcome in acute stroke trials. Cutoff scores for the Barthel Index (BI) and modified Rankin Scale (mRS) are frequently arbitrarily chosen to dichotomize favorable and unfavorable outcome. We investigated sensitivity and specificity of BI cutoff scores in relation to the mRS to obtain the optimal corresponding BI and mRS scores. Methods - BI and mRS scores were collected from 1034 ischemic stroke patients. Sensitivity and specificity were calculated for BI cutoff scores from 45 to 100 in mRS score 1, 2, and 3 and were plotted in receiver operator characteristic (ROC) curves. Results - The cutoff scores for the BI with the highest sum of sensitivity and specificity were 95 (sensitivity 85.6%; specificity 91.7%), 90 (sensitivity 90.7%; specificity 88.1%), and 75 ( sensitivity 95.7%; specificity, 88.5%) for, respectively, mRS 1, 2, and 3. The area under the ROC curve was 0.933 in mRS 1, 0.960 in mRS 2, and 0.979 in mRS 3. Conclusions - The optimal cutoff scores for the BI were 95 for mRS 1, 90 for mRS 2, and 75 for mRS 3. For future acute stroke trials that assess stroke outcome with the BI and mRS, we recommend the use of these BI cutoff score(s) with the corresponding mRS cutoff score(s), to ensure the use of consistent and uniform end points

Molecular variance and population structure of lentil (Lens culinaris Medik.) landraces from Mediterranean countries as revealed by simple sequence repeat DNA markers : implications for conservation and use

The Mediterranean region has a rich history of domestication and cultivation of lentil (Lens culinaris Medik.). Landraces have been grown and repeatedly selected by local farmers under different agro-environments. Characterization of molecular variation and genetic differentiation helps to ensure enhanced valorization, conservation and use of these genetic resources. Nineteen Simple Sequence Repeat DNA markers were used for molecular variance analysis (AMOVA) and population structure assessment underlying 74 lentil landraces from four Mediterranean countries: Morocco, Italy, Greece and Turkey. Based on AMOVA, presence of population structure and genetic differentiation at different levels were evidenced. Genetic diversity among Turkish landraces was higher than that of other countries. These landraces were more homogeneous as shown by low genetic differentiation among individuals within each landrace. Whereas Moroccan landraces followed by Italian and Greek provenances showed higher diversity and differentiation among individuals within landraces. The wide genetic variability of these landraces could help to better adaptation to biotic and abiotic stresses. Moreover, they could provide useful alleles related to adaptive traits for breeding purposes. Based on structure analysis, we obtained indications of possible presence of two major gene pools: a northern gene pool composed of Turkish, Italian and Greek landraces, and a southern gene pool composed of Moroccan landraces. Our results could be of interest when designing future diversity studies, collection missions, conservation and core collection construction strategies on Mediterranean lentil landraces

Admission hyperglycemia and outcome after intravenous thrombolysis:is there a difference among the stroke-subtypes?

Background: The prognostic influence of hyperglycemia in acute stroke has been well established. While in cortical stroke there is a strong association between hyperglycemia and poor outcome, this relation is less clear in lacunar stroke. It has been suggested that this discrepancy is present among patients treated with intravenous tissue plasminogen activator (tPA), but confirmation is needed. Methods: In two prospectively collected cohorts of patient treated with intravenous tPA for acute ischemic stroke, we investigated the effect of hyperglycemia (serum glucose level > 8 mmol/L) on functional outcome in lacunar and non-lacunar stroke. Poor functional outcome was defined as modified Rankin Scale score >= 3 at 3 months. Results: A total of 1012 patients was included of which 162 patients (16 %) had lacunar stroke. The prevalence of hyperglycemia did not differ between stroke subtypes (22 % vs 21 %, p = 0.85). In multivariate analysis hyperglycemia was associated with poor functional outcome in non-lacunar stroke (OR 2.1, 95 % CI 1.39-3.28, p = 0.001). In patients with lacunar stroke, we did not find an association (OR 1.8, 95 % CI 0.62-4.08, p = 0.43). Conclusion: This study confirms a difference in prognostic influence of hyperglycemia between non-lacunar and lacunar ischemic stroke

Feasibility of AmbulanCe-Based Telemedicine (FACT) Study:Safety, Feasibility and Reliability of Third Generation Ambulance Telemedicine

Telemedicine is currently mainly applied as an in-hospital service, but this technology also holds potential to improve emergency care in the prehospital arena. We report on the safety, feasibility and reliability of in-ambulance teleconsultation using a telemedicine system of the third generation.A routine ambulance was equipped with a system for real-time bidirectional audio-video communication, automated transmission of vital parameters, glycemia and electronic patient identification. All patients ( ≥ 18 years) transported during emergency missions by a Prehospital Intervention Team of the Universitair Ziekenhuis Brussel were eligible for inclusion. To guarantee mobility and to facilitate 24/7 availability, the teleconsultants used lightweight laptop computers to access a dedicated telemedicine platform, which also provided functionalities for neurological assessment, electronic reporting and prehospital notification of the in-hospital team. Key registrations included any safety issue, mobile connectivity, communication of patient information, audiovisual quality, user-friendliness and accuracy of the prehospital diagnosis.Prehospital teleconsultation was obtained in 41 out of 43 cases (95.3%). The success rates for communication of blood pressure, heart rate, blood oxygen saturation, glycemia, and electronic patient identification were 78.7%, 84.8%, 80.6%, 64.0%, and 84.2%. A preliminary prehospital diagnosis was formulated in 90.2%, with satisfactory agreement with final in-hospital diagnoses. Communication of a prehospital report to the in-hospital team was successful in 94.7% and prenotification of the in-hospital team via SMS in 90.2%. Failures resulted mainly from limited mobile connectivity and to a lesser extent from software, hardware or human error. The user acceptance was high.Ambulance-based telemedicine of the third generation is safe, feasible and reliable but further research and development, especially with regard to high speed broadband access, is needed before this approach can be implemented in daily practice