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- Next-Generation Sequencing (NGS) identified Species-Specific SSR and SNP markers, allow the unequivocal identification of Strawberry Tree (Arbutus unedo L.) germplasm accessions and contribute to assess their genetic relationshipsPublication . Pereira, Ricardo; Anjos, Isabela Vera Dos; M. Reis, João; Dias, Carolina; M Leitão, JThe strawberry tree (Arbutus unedo L.), an evergreen bush to small tree of the Ericaceae family, is a main component of the natural flora of the Mediterranean basin that also grows profusely through the Iberian Peninsula, southwestern France, and Ireland. The small edible red fruits are usually used to produce preserves, jams, and liquors, as the Portuguese “aguardente de medronho”. The leaves and fruits have been used for a long time in traditional medicine, and their bioactive compounds are presently the subject of intense research. A strawberry tree germplasm collection was recently established by the company Corte Velada (Odiáxere, Portugal). A set of 50 germplasm accessions was selected for a breeding program. A next-generation sequencing project was performed, resulting in the establishment of the first strawberry tree genome assembly and further identification of 500 SSR and 500 SNP loci. Individual molecular fingerprints for the unequivocal identification of the selected 50 accessions were established based on 71 markers alleles amplified by 4 SSR and 9 SNP markers. The same species-specific markers alleles combined with 61 random amplified markers amplified by 5 RAPD and 5 ISSR primers were used to assess the genetic variability and genetic relationships among the selected accessions.
- Assessment of Wild Rocket (Diplotaxis tenuifolia (L.) DC.) Germplasm accessions by NGS identified SSR and SNP markersPublication . Reis, João M.; Pereira, Ricardo; Coelho, Paula S.; Leitão, JoséRocket is the common designation for two baby-leaf salad crops of the Brassicaceae family: Eruca sativa (L.) Cav., usually referred to as annual garden rocket, and Diplotaxis tenuifolia (L.) DC. commonly named to as perennial wild rocket. E. sativa is used for human consumption since antiquity. However, the growing consumer preference for D. tenuifolia is being accompanied by the fast increase in its production area and commercialization of new cultivars. Nevertheless, the worldwide number of wild rocket accessions maintained in germplasm collections is very reduced, the solution for which situation the project “REMIRucula” intends to contribute, establishing a germplasm collection at the INIAV, Oeiras, Portugal. Herein, we report on the establishment via next generation sequencing (NGS) of the first genome assembly of D. tenuifolia and the identification of specific single sequence repeat (SSR) and single nucleotide polymorphisms (SNP) loci for the establishment of specific DNA-markers for this species. A representative set of 87 D. tenuifolia and 3 E. sativa accessions were assessed by 5 SSR and 9 SNP-CAPS markers, allowing a drastic discrimination between both species and the establishment of unequivocal molecular fingerprints for the analyzed accessions. The non-discrimination within six pairs and one trio of D. tenuifolia accessions is discussed.
- Downy mildew resistance and genetic variability in a wild rocket germplasm collectionPublication . Coelho, Paula S.; Reis, João M.; Pereira, Ana L.; Vairinhos, Aliana; Lopes, Violeta; Leitão, JoséOne hundred accessions of a "core collection" of Diplotaxis tenuifolia (L.) DC. and Eruca spp. were screened at seedling stage for resistance to downy mildew. Accessions tested at the seedling stage were assigned to 0-6 interaction phenotypes. All cultivated rocket (Eruca spp.) accessions exhibited a resistant (R) response both in cotyledons and in young leaves. The wild rocket (D. tenuifolia) accessions exhibited higher susceptibility in cotyledons than in the 1st and 2nd leaves, with 16 and 47 accessions classified as resistant or partially resistant (PR) in the cotyledon and in leaves stages, respectively. Only three wild rocket accessions displayed an R phenotype in cotyledons and leaves. The most frequent response in cotyledons vs. leaves was the highly susceptible/susceptible (HS/S) combination (33 accessions), followed by the S/PR combination (18 accessions). A significant correlation (r = 0.917, P < .000) was observed between the disease index in cotyledons and leaves. The molecular markers analyses revealed a wide genetic distance between Diplotaxis and Eruca, which gather in two clearly separated species clusters. The molecular variability is accompanied by a wide diversity of interactions with the pathogen isolate. The closest similarities among D. tenuifolia accessions were found in accessions provided by the same breeding company. Future studies will be focused on two main objectives: (a) the assessment of the accessions behavior that have evidenced an R/R, S/PR, and HS/PR cotyledon and leaf response under greenhouse or field production and (b) the genome mapping of genetic features that provide downy mildew resistance.
- Downy mildew evaluation in wild rocket genotypes (Diplotaxis tenuifolia (L.) DC) under field and controlled conditionsPublication . Coelho, P. S.; Pereira, A. L.; Reis, João; Carranca, C.; Lopes, V. R.; Leitão, JoséWild rocket downy mildew (DM) is a foliar disease caused by the oomycete Hyaloperonospora spp. that limits the production of brassicas, especially in temperate climates, infecting plants at all growth stages. During 2021 autumn season, a field trial (polytunnel) with a set of 40 wild rocket accessions from different origins (harvest missions, gene banks, commercial cultivars) was installed in a commercial enterprise located in Odemira, southern Portugal. The plants were naturally infected by Hyaloperonospora spp. and were visually evaluated 71 days after sowing. Field observations of adult plants evidenced differences among accessions concerning the DM resistance and agronomic traits (e.g., flowering date, vigour, plant habit, leaf serration). Sixteen wild rocket accessions were identified with an interesting resistance response to DM in adult phase. In a previous study, the wild rocket accessions were tested to Hyaloperonospora isolate D5 at seedling stage under controlled conditions. A significant coefficient of correlation was observed (r=0.628, P=0.000, n=36) by comparing the plants in both growth phases. In general, the accessions were more resistant at adult than at seedling stage. No accessions were resistant at seedling and susceptible at adult stage. However, two accessions with a highly susceptible response at seedling stage presented a resistant and a partially resistant response at adult phase under field conditions. Our results suggest that DM resistance observed at the seedling stage is a good indication of wild rocket resistance behaviour at field adult plant. The most promising accessions identified as resistant at seedling stage may be further exploited in breeding programmes.
- Metabolic responses and resilience to environmental challenges in the sedentary batrachoid halobatrachus didactylusPublication . Molina, Juan M.; Kunzmann, Andreas; Reis, João; Guerreiro, Pedro MiguelThe Lusitanian toadfish, Halobatrachus didactylus is a marine teleost found in coastal lagoons and river estuaries, often exposed to important changes in salinity, temperature and reduced oxygen. Sedentary species, with strong site fidelity and low migratory ability along the temperature gradient such as this may be especially impacted by climate change. We aimed at establishing the tolerance limits to acute temperature and oxygen changes, and evaluate respiratory and metabolic responses in chronic control, warm and hypoxic (35% O2 ) conditions. Critical temperature maximum (CTmax) was determined in 12 individuals exposed to a temperature ramp of 3 ◦C per hour starting at 18 ◦C, and was found to be 34.8 ± 0.66 ◦C. Critical oxygen level (PO2crit) was determined in 8 fish at 18 ◦C while performing intermittent respirometry and oxygen depletion was created by nitrogen injection in the tank. PO2crit was calculated as the inflexion point between oxyregulation and oxyconformation, which was found to be around 1.2 mgO2/L, but fish survived down to 3% O2 , recovering from 0.2 mgO2/L but showing increased hematocrit (Hct), red blood cell (RBC) counts and blood pH. We also quantified routine aerobic scope and daily activity patterns, finding this fish to be extremely sedentary. H. didactylus showed one of the lowest daytime basal metabolic rates (MR) found in the literature but activity increased significantly at night (over two-fold when closed inside the metabolic chambers). The effect of temperature on metabolic rate (MR) was evaluated using a temperature ramp ranging from 8 to 32 ◦C (1 ◦C/h). Acute temperature changes resulted in a steady increase in MR up to circa 29 ◦C, beyond which MR become increasingly variable, especially among smaller individuals. Indeed, small fish appear to show high- and low-MR groups, and were more susceptible to heat and hypoxia than larger individuals. In chronic acclimation, the MR was increased by 3- and 4-fold (hypoxia vs. normoxia) in fish at 28 ◦C in relation to those at 12 ◦C. Standard MR were not statistically different between normoxia and hypoxia at 12 ◦C, but maximum MR in hypoxia was only about 2/3 of that in normoxia. Fish in high temperature lost weight (mean −3.1%) and had higher metabolism, while in low temperature, weight increased (mean +9.3%) and metabolism was low, and HIS was significantly lower in high temperature groups. Fish in hypoxic conditions showed consistently high Hct but not RBC or hemoglobin (Hb). Overall this study indicates that H. didactylus is highly tolerant to hypoxia and temperature variations. It remains to be seen if other populations along the Atlantic coast show similar metrics. The measured CTmax is close to the actual maximum temperature possible to experience in Ria Formosa ponds during summer, and it would not be unexpected to find this species establishing stable populations in other regions if climate change forces it out of its actual distribution.