RESUMO
Vascular wilt, caused by the pathogen Fusarium oxysporum f. sp. physali (Foph), is a major disease of cape gooseberry (Physalis peruviana L.) in Andean countries. Despite the economic losses caused by this disease, there are few studies related to molecular mechanisms in the P. peruviana-Foph pathosystem as a useful tool for crop improvement. This study evaluates eight candidate genes associated with this pathosystem, using real-time quantitative PCR (RT-qPCR). The genes were identified and selected from 1,653 differentially expressed genes (DEGs) derived from RNA-Seq analysis and from a previous genome-wide association study (GWAS) of this plant-pathogen interaction. Based on the RT-qPCR analysis, the tubuline (TUB) reference gene was selected for its highly stable expression in cape gooseberry. The RT-qPCR validation of the candidate genes revealed the biological variation in their expression according to their known biological function. Three genes related to the first line of resistance/defense responses were highly expressed earlier during infection in a susceptible genotype, while three others were overexpressed later, mostly in the tolerant genotype. These genes are mainly involved in signaling pathways after pathogen recognition, mediated by hormones such as ethylene and salicylic acid. This study provided the first insight to uncover the molecular mechanism from the P. peruviana-Foph pathosystem. The genes validated here have important implications in the disease progress and allow a better understanding of the defense response in cape gooseberry at the molecular level. Derived molecular markers from these genes could facilitate the identification of tolerant/susceptible genotypes for use in breeding schemes.
RESUMO
Abstract Cape gooseberry (Physalis peruviana, L.) is a herbaceous plant belonging to the Solanaceae family that produces an edible berry appreciated for its nutraceutical and pharmaceutical properties. Its production is often limited by diseases and reproducible fruit quality. Recent studies have reported genes associated with fruit quality and resistance response to the root-infecting fungus Fusarium oxysporum f. sp. physali (Foph,) which causes vascular wilt. In order to standardize a method to validate the biological function of candidate genes in the non-model species P. peruviana, we tested the robust approach in reverse genetics, virus-induced gene silencing (VIGS). In this study, we validated and optimized VIGS using an insert of the phytoene desaturase (PDS) gene in a silencing viral vector generated from tobacco rattle virus (TRV). Leaves infiltrated with Agrobacterium (GV3101 strain) showed photo-bleached segments, which were distinctive for PDS suppression at 7 days post-infection (dpi). More than half of the treated plants showed photo-bleaching, indicating an efficiency rate of 50 % of the VIGS protocol. The results of this study showed that VIGS can be used for future functional gene characterization implicated in the immune response, disease resistance and fruit quality in capegooseberry.
Resumo A physalis (Physalis peruviana, L.) é uma planta herbácea pertencente à família Solanaceae, que produz uma baga comestível apreciada por suas propriedades nutracêuticas e farmacêuticas. Sua produção com frequência se vê limitada devido a enfermidades e baixa reprodutibilidade na qualidade do fruto. Estudos recentes reportaram genes associados com a qualidade do fruto e com a resposta de resistência ao fungo radicular Fusarium oxysporum f. sp. physali (Foph.), que causa esmorecimento vascular. Com a finalidade de padronizar um método para validar a função biológica de genes candidatos na espécie não-modelo P. p ruviana, avaliamos uma aproximação robusta em genética invertida, o sil nciamento de genes induzidos por vírus (VIGS). Neste estudo, validamos e otimizamos o VIGS usando um inserto da fitoeno desaturase (PDS) em um vetor viral de silenciamento produzido a partir do vírus do chocalho do tabaco (TRV). As folhas infiltradas com Agrobacterium (cepa GV3101) mostraram segmentos fotobranqueados, que foram distintivos para a supressão de PDS a 7 dias pós-infecção (dpi). Mais da metade das plantas tratadas mostraram fotobranqueamento, o que indica uma taxa de eficiência de 50 % do procotolo VIGS. Os resultados de este estudo mostraram que o VIGS pode ser usado em caracterizações futuras de genes funcionais implicados na resposta imune, na resistência a enfermidades e na qualidade do fruto de physalis.
Resumen La uchuva (Physalis peruviana, L.) es una planta herbácea perteneciente a la familia de las solanáceas, que produce una baya comestible apreciada por sus propiedades nutracéuticas y farmacéuticas. Su producción con frecuencia se ve limitada debido a enfermedades y a falta de reproducibilidad en la calidad del fruto. Estudios recientes han reportado genes asociados con la calidad del fruto y con la respuesta de resistencia al hongo radicular Fusarium oxysporum f. sp. physali (Foph,), que causa marchitamiento vascular. Con el fin de estandarizar un método para validar la función biológica de genes candidatos en la especie no-modelo P. peruviana, evaluamos la aproximación robusta en genética inversa, el silenciamiento génico inducido por virus (VIGS). En este estudio, validamos y optimizamos el VIGS usando un inserto de la fitoeno desaturasa (PDS) en un vector viral de silenciamiento producido a partir del virus del cascabeleo del tabaco (TRV). Las hojas infiltradas con Agrobacterium (cepa GV3101) mostraron segmentos fotoblanqueados, que fueron distintivos para la supresión de PDS a 7 días pos-infeccion (dpi). Más de la mitad de las plantas tratadas mostraron fotoblanqueo, lo cual indica una tasa de eficiencia del 50 % del protocolo VIGS. Los resultados de este estudio mostraron que el VIGS se puede usar en futuras caracterizaciones de genes funcionales implicados en la respuesta inmune, la resistencia a enfermedad y la calidad del fruto en la uchuva.
RESUMO
Association mapping has been proposed as an efficient approach to assist plant breeding programs to investigate the genetic basis of agronomic traits. In this study, we evaluated 18 traits related to yield, (FWP, NF, FWI, and FWII), fruit size-shape (FP, FA, MW, WMH, MH, HMW, DI, FSI, FSII, OVO, OBO), and fruit quality (FIR, CF, and SST), in a diverse collection of 100 accessions of Physalis peruviana including wild, landraces, and anther culture derived lines. We identified seven accessions with suitable traits: fruit weight per plant (FWP) > 7,000 g/plant and cracked fruits (CF) < 4%, to be used as parents in cape gooseberry breeding program. In addition, the accessions were also characterized using Genotyping By Sequencing (GBS). We discovered 27,982 and 36,142 informative SNP markers based on the alignment against the two cape gooseberry references transcriptomes. Besides, 30,344 SNPs were identified based on alignment to the tomato reference genome. Genetic structure analysis showed that the population could be divided into two or three sub-groups, corresponding to landraces-anther culture and wild accessions for K = 2 and wild, landraces, and anther culture plants for K = 3. Association analysis was carried out using a Mixed Linear Model (MLM) and 34 SNP markers were significantly associated. These results reveal the basis of the genetic control of important agronomic traits and may facilitate marker-based breeding in P. peruviana.
