Phenology and fruit quality in sweet cherry (Prunus avium L.). Genetics, QTL analysis and marker assisted selection

La cereza (Prunus avium L.) es una fruta muy apreciada, con un alto valor nutricional y de interés económico, cuyo cultivo ha aumentado durante los últimos años. Para poder proporcionar nuevos cultivares adaptados a las demandas de los productores y consumidores, y que puedan hacer frente a desafíos como el calentamiento global, es necesario desarrollar herramientas que puedan ayudar a optimizar el proceso de mejora. Con este propósito, el objetivo de este trabajo es investigar la genética de algunos caracteres fenológicos y de calidad de fruto de interés en cerezo y avanzar en el conocimiento de los mecanismos biológicos que los regulan. Para alcanzar este objetivo se han utilizado siete familias intra-específicas de cerezo que derivan de polinizaciones cruzadas y autopolinizaciones de material vegetal local (‘Cristobalina’ y ‘Ambrunés’) que proporcionan variabilidad fenotípica adicional para éstos caracteres. Estas familias se genotiparon utilizando las plataformas genómicas ‘RosBREED cherry 6K y/o 15 SNP Illumina® Infinium’, lo que permitió desarrollar cinco mapas genéticos de alta densidad, que fueron utilizados para analizar la sintenia entre cerezo y melocotonero (P. persica), y para análisis de QTLs. Las siete familias fueron además fenotipadas para caracteres fenológicos (fecha de floración, período de desarrollo de fruto y fecha de maduración), y caracteres de calidad del fruto [firmeza, tamaño, color, contenido en sólidos solubles, acidez titulable y contenido en polifenoles (antocianinas y ácidos fenólicos)]. Estos datos se utilizaron para realizar análisis anuales y/o multianuales de QTLs utilizando diferentes estrategias de mapeo, el análisis de familias únicas se realizó utilizando MapQTL®, y el análisis de varias familias de manera combinada se realizó utilizando FlexQTL™. Estos análisis permitieron identificar QTLs principales y menores para todos los caracteres investigados, validar algunos QTLs previamente descritos para los mismos caracteres, identificar nuevos QTLs para caracteres estudiados por primera vez, e identificar nuevas variantes de QTLs de interés para la mejora. Se identificaron QTLs principales en los grupos de ligamientos (GL) 1 y 2 para fecha de floración, en el GL4 para el período de desarrollo de fruto y fecha de maduración, en los GL 1 y 2 para tamaño de fruto, en los GL 1 y 4 para firmeza, en el GL4 para el contenido en sólidos solubles, en GL6 para acidez titulable, en GL3 para color de fruto y contenido en antocianinas, y en GL1 para el contenido en ácidos fenólicos. Haplotipos de interés para la mejora en estos QTLs principales fueron identificados en algunos cultivares, como en los GL 1 y 2 de ‘Cristobalina’ para bajos requerimientos de frío y floración temprana, en el GL4 de ‘Cristobalina’ y ‘Burlat’ para período de desarrollo de fruto corto y fecha de maduración temprana, y en el GL1 de ‘Ambrunés’ para tamaño y firmeza. El período de desarrollo de fruto destacó como un carácter esencial para la fenología y la calidad del fruto, ya que la fecha de maduración, la firmeza y el contenido de sólidos solubles están correlacionados con el desarrollo de fruto y se asociaron a los mismos QTLs. La identificación de QTLs principales permitió proponer y confirmar genes candidatos para estos caracteres en estos QTL, y los genes candidatos para fecha de floración fueron investigados. Utilizando la secuencia del genoma de ‘Regina’, genes candidatos para requerimientos de frío y fecha de floración, PavDAM, ortólogos a los genes DAM de P. persica y P. mume, fueron identificados y caracterizados en el QTL principal de fecha de floración en GL1. La comparación de secuencias de los genes PavDAM de varios cultivares de cerezo con diferentes requerimientos térmicos y fechas de floración, permitió detectar polimorfismos que pueden estar asociados a las diferencias fenotipicas, y una deleción en el promotor de los PavDAM (DPD) en ‘Cristobalina’ que está asociado a floración temprana. También se desarrolló un marcador de ADN para esta mutación, que puede ser usado para la selección asistida por marcadores de floración temprana en ‘Cristobalina’. Sweet cherry (Prunus avium L.) is a very appreciated fruit, with a high nutritional value and economic interest, which cultivation has increased during last years. In order to provide new cultivars adapted to producer and consumer demands, and that confront challenges like global warming, is necessary to develop tools that can help optimizing the breeding process. For this purpose, the objective of this work is to investigate the genetics of some relevant phenology and fruit quality traits of sweet cherry and to advance in the understanding of the biological mechanisms that regulate them. To achieve this goal, seven sweet cherry intraspecific populations that derive from cross- and self-pollinations of local plant material (‘Cristobalina’ and ‘Ambrunés’), and that provide additional phenotypic variation for these traits, were used. These populations were genotyped with the whole genome RosBREED cherry 6K and/or 15K Illumina® Infinium SNP arrays, which allowed developing five high-density genetic maps that were then used for analyzing synteny between sweet cherry and peach (P. persica) and for QTL analyses. The seven populations were also phenotyped for phenology traits (bloom time, fruit development period and maturity date), and for fruit quality traits [firmness, size, color, solid soluble content, titratable acidity and polyphenols (anthocyanins and phenolic acids) content]. These data were used to perform single and/or multi-year QTL analysis, using different mapping strategies, which included single bi-parental analysis with MapQTL®, or combined multi-parental populations using FlexQTL™. These analyses allowed identifying major and minor QTLs for all the traits investigated, validating some QTLs previously reported for the same traits, reporting new QTLs for newly investigated traits, and identifying new QTLs variants of breeding interest. Major QTLs were identified on linkage groups (LGs) 1 and 2 for bloom time, on LG4 for fruit development period and maturity date, on LGs 1 and 2 for fruit size, on LGs 1 and 4 for fruit firmness, on LG4 for solid soluble content, on LG6 for titratable acidity, on LG3 for fruit color and anthocyanins content, and on LG1 for phenolic acids content. Relevant QTL haplotypes for breeding purposes were identified in these major QTLs in some cultivars, like for low chilling and early blooming in ‘Cristobalina’ LGs 1 and 2, for short development period and early maturity date in ‘Cristobalina’ and ‘Burlat’ LG4, and in ‘Ambrunés’ LG1 for size and firmness. Fruit development period reveal itself as an essential trait for phenology and fruit quality, as maturity date, firmness and soluble solids contents were correlated with fruit development and were associated to the same QTLs. The identification of major QTLs allowed proposing and confirming candidate genes for these traits at these QTLs, and candidate genes for bloom time were investigated. Using the ‘Regina’ sweet cherry genome sequence, candidate genes for chilling requirements and bloom time, PavDAM genes, orthologous to P. persica and P. mume DAM genes, were identified and characterized in the major bloom time QTL on LG1. Sequence comparison of PavDAM genes of various sweet cherry cultivars with different chilling requirements and bloom times allowed detecting sequence polymorphisms that may be associated to their phenotypic differences, and a deletion in the ‘Cristobalina’ PavDAM promoter (DPD) that is associated to early blooming. A DNA marker for this mutation, that can be used for marker-assisted selection of early blooming from ‘Cristobalina’, was also developed.<br /

Correlación y análisis genético de maduración y calidad de fruto en cerezo

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Control genético del color y el contenido en compuestos fenólicos en cerezo

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Evaluación del contenido de azúcares y ácidos orgánicos en familias de cerezo mediante técnicas cromatográficas

La cereza es una fruta de alto valor nutricional y gran interés económico. Su calidad está determinada en gran medida por sus propiedades organolépticas. Así, el dulzor y la acidez son dos de los principales atributos relacionados con la aceptación de esta fruta por parte de los consumidores. A pesar de la importancia de estos caracteres, los mecanismos genéticos que los regulan son aún desconocidos en esta especie. Este estudio tiene como objetivo avanzar en el conocimiento de estos caracteres mediante el estudio del contenido de azúcares y ácidos en cinco familias de cerezo. Este trabajo se enmarca en un proyecto de investigación nacional que pretende avanzar en el conocimiento del control genético de estos caracteres mediante el análisis de este material vegetal. En este trabajo se analizó la acidez titulable y el contenido en sólidos soluble de 259 individuos de cerezo, y además se realizó de la extracción, identificación y cuantificación del perfil de azúcares simples y ácidos orgánicos mediante cromatografía de capa líquida de alto rendimiento (UPLC) para cada individuo de la muestra. Los resultados mostraron valores de acidez titulable entre 0,58-1,39% y un contenido en sólidos solubles entre 15-27 ºBrix. Cinco ácidos orgánicos fueron identificados (ácido málico, quínico, oxálico, cítrico y shikímico), siendo el ácido málico el que presentó un contenido más alto. Además se observó una elevada correlación positiva de este ácido y la acidez titulable, confirmando que el ácido málico es el principal compuesto relacionado con la acidez de las cerezas. De igual forma, cuatro azúcares simples fueron detectados: glucosa, fructosa, sacarosa y sorbitol. Los dos primeros azúcares, glucosa y fructosa, fueron las más abundantes. Además, estos azucares presentaron las correlaciones más altas con el contenido en sólidos solubles, destacando su importancia en el dulzor del fruto. En la mayoría de los ácidos orgánicos y azúcares simples detectados en los individuos de las cinco familias se observó una distribución ajustad a la normalidad, así como diferentes rangos de concentraciones entre familias. Estos resultados revelan una herencia cuantitativa de estos compuestos en cerezo, confirmando las observaciones en otras especies del mismo género. No se observó correlación entre azúcares y ácidos, confirmando la regulación independiente de ambos tipos de compuestos. <br /

Identificación y cuantificación de azúcares simples y ácidos orgánicos en familias de cerezo para análisis genéticos

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Identification of Key Genes Related to Dormancy Control in Prunus Species by Meta-Analysis of RNAseq Data

Bud dormancy is a genotype-dependent mechanism observed in Prunus species in which bud growth is inhibited, and the accumulation of a specific amount of chilling (endodormancy) and heat (ecodormancy) is necessary to resume growth and reach flowering. We analyzed publicly available transcriptome data from fifteen cultivars of four Prunus species (almond, apricot, peach, and sweet cherry) sampled at endo- and ecodormancy points to identify conserved genes and pathways associated with dormancy control in the genus. A total of 13,018 genes were differentially expressed during dormancy transitions, of which 139 and 223 were of interest because their expression profiles correlated with endo- and ecodormancy, respectively, in at least one cultivar of each species. The endodormancy-related genes comprised transcripts mainly overexpressed during chilling accumulation and were associated with abiotic stresses, cell wall modifications, and hormone regulation. The ecodormancy-related genes, upregulated after chilling fulfillment, were primarily involved in the genetic control of carbohydrate regulation, hormone biosynthesis, and pollen development. Additionally, the integrated co-expression network of differentially expressed genes in the four species showed clusters of co-expressed genes correlated to dormancy stages and genes of breeding interest overlapping with quantitative trait loci for bloom time and chilling and heat requirements.Publishe

Análisis de QTLs para azúcares simples en cerezo

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Healthcare workers hospitalized due to COVID-19 have no higher risk of death than general population. Data from the Spanish SEMI-COVID-19 Registry

Aim To determine whether healthcare workers (HCW) hospitalized in Spain due to COVID-19 have a worse prognosis than non-healthcare workers (NHCW). Methods Observational cohort study based on the SEMI-COVID-19 Registry, a nationwide registry that collects sociodemographic, clinical, laboratory, and treatment data on patients hospitalised with COVID-19 in Spain. Patients aged 20-65 years were selected. A multivariate logistic regression model was performed to identify factors associated with mortality. Results As of 22 May 2020, 4393 patients were included, of whom 419 (9.5%) were HCW. Median (interquartile range) age of HCW was 52 (15) years and 62.4% were women. Prevalence of comorbidities and severe radiological findings upon admission were less frequent in HCW. There were no difference in need of respiratory support and admission to intensive care unit, but occurrence of sepsis and in-hospital mortality was lower in HCW (1.7% vs. 3.9%; p = 0.024 and 0.7% vs. 4.8%; p<0.001 respectively). Age, male sex and comorbidity, were independently associated with higher in-hospital mortality and healthcare working with lower mortality (OR 0.211, 95%CI 0.067-0.667, p = 0.008). 30-days survival was higher in HCW (0.968 vs. 0.851 p<0.001). Conclusions Hospitalized COVID-19 HCW had fewer comorbidities and a better prognosis than NHCW. Our results suggest that professional exposure to COVID-19 in HCW does not carry more clinical severity nor mortality

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030