Analysis of genetic diversity of the critically endangered Onobrychis conferta subsp. conferta using cross-genera transferability of SSR markers developed from related legume species

The use of transferable cross-species/genera SSR markers is an alternative strategy to ensure availability of markers in genomic resources-limited crops, such as critically endangered species. Eighteen microsatellite markers derived from the genomes of Medicago truncatula Gaertn., Phaseolus vulgaris L. and Onobrychis viciifolia Scop. were tested for transferability and used to study the genetic diversity of the three remaining populations of the critically endangered species Onobrychis conferta subsp. conferta, collected from their natural habitats. All pairs of primers tested were found to be polymorphic and reproducible. A total of 257 alleles were obtained from 134 loci, resulting in an average of 1.93 alleles per locus. The average number of alleles per accession was 51.4, yielding an average of 14.27 alleles per SSR marker and accession. The lowest number of alleles was recorded in O. conferta from Aïn Dyssa, with 37 alleles, while the highest was observed in O. viciifolia, with 63 alleles. Each SSR amplified 3-16 alleles. The MTIC343 primer yielded the highest number of loci (16 loci). The mean Polymorphism Information Content (PIC), Marker Index (MI), and Resolving Power (Rp) were 0.36, 2.22, and 4.58, respectively, indicating a high level of polymorphism in the studied SSR markers. UPGMA cluster analysis grouped genotypes into two main clusters in corroboration with the morphological distinction of sections. Our study demonstrated that O. conferta subsp. conferta genomes could be successfully examined using other legume SSR markers, providing a valuable tool to detect polymorphism for future genetic studies, breeding programs, and conservation strategies, addressing the lack of available SSR markers in this genus. These microsatellite loci may help to further survey the adaptive evolution and genetic variation of Onobrychis conferta conservation

Responses to Salinity in Four Plantago Species from Tunisia

[EN] The genus Plantago is particularly interesting for studying the mechanisms of salt tolerance in plants, as it includes both halophytes and glycophytes, as well as species adapted to xeric environments. In this study, the salt stress responses of two halophytes, P. crassifolia and P. coronopus, were compared with those of two glycophytes, P. ovata and P. afra. Plants obtained by seed germination of the four species, collected in different regions of Tunisia, were subjected to increasing salinity treatments for one month under greenhouse conditions. Morphological traits and biochemical parameters, such as ion accumulation and the leaf contents of photosynthetic pigments, osmolytes, oxidative stress markers and antioxidant metabolites, were measured after the treatments. Salt-induced growth inhibition was more pronounced in P. afra, and only plants subjected to the lowest applied NaCl concentration (200 mM) survived until the end of the treatments. The biochemical responses were different in the two groups of plants; the halophytes accumulated higher Na+ and proline concentrations, whereas MDA levels in their leaves decreased, indicating a lower level of oxidative stress. Overall, the results showed that P. coronopus and P. crassifolia are the most tolerant to salt stress, and P. afra is the most susceptible of the four species. Plantago ovata is also quite resistant, apparently by using specific mechanisms of tolerance that are more efficient than in the halophytes, such as a less pronounced inhibition of photosynthesis, the accumulation of higher levels of Cl¿ ions in the leaves, or the activation of K+ uptake and transport to the aerial part under high salinity conditions.This research was supported by internal funds of Universitat Politecnica de Valencia to O.V. and M.B.Ltaeif, HB.; Sakhraoui, A.; González-Orenga, S.; Landa Faz, A.; Boscaiu, M.; Vicente, O.; Rouz, S. (2021). Responses to Salinity in Four Plantago Species from Tunisia. Plants. 10(7):1-20. https://doi.org/10.3390/plants10071392S12010

Habitat characterization and decline of Critically Endangered Onobrychis conferta subsp. conferta

Rare endemic plant species are very susceptible to anthropogenic threats and natural changes, thereby facing a high risk of extinction. The Mediterranean Basin supports a rich flora with high percentages of endemic species. An iconic case of Mediterranean narrow endemic plant taxon is represented by Onobrychis conferta subsp. conferta (Desf.) Desv. (Fabaceae). Management of this species requires better information on its habitat requirements and population status. Field surveys and historical records were used to characterize the distribution of O. conferta in Tunisia. Habitats were characterized using topography, soils, climate, and land cover variables. Expeditions on ten previously reported sites revealed the presence of O. conferta in only one of the previous recorded localities and, for the first time, in two new localities. We recorded a sharp decreasing trend for the total number of plants per population in all locations over 6-year period. Our findings showed that the current status of O. conferta in Tunisia is Critically Endangered (CR). Onobrychis conferta faces an extremely high risk of extinction in the wild. Therefore, prompt comprehensive conservation actions are needed to ensure its survival. We propose a conservation and recovery recommendations including in situ and ex situ conservation actions. In situ actions are aimed at minimizing the effects of the identified threats and to create new populations of O. conferta in its characterized habitat. Ex situ actions include an environmental education campaign and seed preservation.Laboratory of Agricultural Production Systems and Sustainable Development (LR03AGR02

Medicago sativa and M. tunetana reveal contrasting physiological and metabolic responses to drought

Alfalfa production is frequently constrained by drought, indicating the importance of assessing species biodiversity in endemic close relatives to enhance forage production under future global change conditions. In the present study, plants of two ecotypes of M. tunetana, native to Tunisia, and four commercial cultivars of M. sativa were subjected to two water regimes (control vs drought [15% field capacity]). Physiological, isotopic and metabolic analyses were used to characterize leaf and nodule profiles of the plants. Biomass, gas exchange and the maximum carboxylation rate (Vc) indicated significant decreases in photosynthetic capacity under drought in M. sativa cultivars. However, M. tunetana ecotypes maintained photosynthetic performance and aboveground biomass under drought conditions. Furthermore, nitrogen isotope composition (δN) in nodules and leaves was significantly decreased, which reveals a reduction in the N fixing activity of nodules under drought conditions that was not translated into lower leaf N content but was probably due to lower N demand. Analyses of starch, soluble sugar, and amino acid content in leaves and nodules have clearly proven the ability of Medicago spp. cultivars to increase the accumulation of osmo-protectors under drought. This study demonstrated the genetic variability of the strategy adopted among the studied cultivars in response to drought. In this sense, M. tunetana, and in part the M. sativa cultivar adapted to Mediterranean conditions, seem capable of maintaining adequate biomass, photosynthesis and biological N fixation in comparison to the other M. sativa cultivars

Potential use of wild Onobrychis species for climate change mitigation and adaptation

Climate change is threatening environmental and nutritional security. In this scenario, forage crops and their wild relatives may contribute to maintain ecosystem services and minimize the effects of global warming. We reviewed the literature regarding the ecological, agronomic, and nutraceutical values of wild Onobrychis Miller species (sainfoins, Family Fabaceae). We also discussed their application prospects and the existing problems hampering their widespread domestication. Wild species of the genus Onobrychis have some interesting agronomic and ecological features such as perenniality, deep root system, and high-stress tolerance, which make them suitable for future cultivation under climate change. Wild Onobrychis species are multipurpose species, which can be utilized for fodder and honey production under harsh environmental conditions. In addition, wild Onobrychis species water, ethanol, and methanol extracts exhibit antioxidant, antidiabetic, anti-inflammatory, antitumor, antimicrobial, anti-stressor, and many other pharmacological effects. Wild Onobrychis species have not been the subject of detailed studies. Compared with the cultivated forage legumes, wild Onobrychis species are still underexploited despite their potential to alleviate forage and food insecurity, either by domestication or by providing novel alleles for breeding purposes of the cultivated species Onobrychis viciifolia. Our review aims to increase scientific awareness toward exploring the potential uses of wild Onobrychis species to tackle current climate change.University of Carthage LR03AGR0

Germination and early seedling growth in four Plantago species in response to Zn, Cu and Fe

Heavy metal contamination is an increasingly pressing global ecological concern adversely affecting plant growth. Phytoremediation is an eco-friendly and low-cost approach to help solve this problem by using plants to remove metals. This study aimed to evaluate the phytoremediation potential of four Plantago species, exposing them to different concentrations (0, 150, 300, 600 and 900 ppm) of zinc, copper, and iron during germination and early seedling growth. These are generally the phases of the plant life cycle most sensitive to stress. The germination percentage (GP), mean germination time (MGT), radicle, hypocotyl and cotyledons length, biomass, water content and tolerance index (TI) were recorded under controlled conditions. The results indicated that metal-induced stress significantly reduced GP, increased MGT, and inhibited seedling growth with increased metal concentration. The relative toxicity of the tested metals could be ranked as Fe > Cu > Zn. Regarding the Plantago species, P. tunetana and P. lanceolata could be considered highly tolerant, P. albicans moderately tolerant, and P. afra low-tolerant to metal toxicity during germination. Therefore, P. tunetana and P. lanceolata present an excellent potential for phytoremediation of metal-contaminated zones.University of Carthage LR03AGR0

Plantago tunetana Murbeck, Contrib.

Plantago tunetana Murbeck, Contrib. ä la Connaiss. de la Flore du Nord-Ouest de l’Afr. et plus spec. de la Tunisie III: 2, T. 10 Fig. 5 –8 (1899) Type (lectotype, designated here):— TUNISIA. Souk-el-Djema, in campis argillosis, 5 June 1896, Murbeck s.n. [LD1221559! (Fig. 1), isolectotype P00466350! (image of the isolectotype available at https://science.mnhn.fr/taxon/species/plantago/tunetana?lang=en_ US)]. Notes on typification:— Murbeck (1899: 2–3) did not cite any original specimen in the protologue of Plantago tunetana. A provenance was provided: “ Tunisie centrale: Collines calcaires, champs argileux, etc. entre Maktar et Souk-el-Djema, c. 1000 m.”. In addition, an illustration (“Tab. Nostra X figs. 5–8) was provided and it is part of the original material for the name. We traced two specimens at LD (barcode LD1221559) and P (barcode P00466350) which are also original material. Svante Samuel Murbeck (1859–1946) lived and worked in or around Lund during most of his life, including the time when P. tunetana was published. For this reason, we here designate the specimen in LD as the lectotype (Fig. 1). LD1221559 bears two plants that are well-preserved and morphologically complete, which match the Murbeck’s description and correspond to the current application of the name. Description:—Perennial, roots thick woody. Leaves ovate or lanceolate–oblong, obtuse or short blade (4.0– 7.6 cm × 0.6–2.0 cm) with sinews 7 raising transmitted by base petiole broad. These leaves are obtuse at the apex, or suddenly contracted into a very short deflecting blade often slightly wavy at the edges, mostly entire or remotely denticulate especially at the veins, which are covered with very fine, soft hairs, the younger silky white, the more mature green. Scapes (3.6–12.0 cm) arched or subrogate, densely pubescent. Spikes cylindrical elongated or oblong, shorter than the scapes (1.4–3.6 cm long). Bracts lanceolate (3.51–3.83 mm × 2.13–2.33 mm) and villous on the outside, pale green behind, and white or brownish towards the margins. Sepals equal, lanceolate or ovate-lanceolate (5.23–6.09 mm × 1.93–2.28 mm), pointed, villous behind and at the apex, towards the margins with a whitish membrane, all free with an unkeeled back. Corolla tube glabrous, 5.0– 5.5 mm long. Corolla lobes very broad or reflexed, triangular or ovate-lanceolate (3.25–3.76 mm × 2.07–2.54 mm), gradually tapering in acuity, dark brown above, below with long, densely hairy silky hairs. Seeds smooth (3.25–3.54 mm × 1.32–1.42 mm), deeply grooved inner surface with sub-cymbiform. Etymology:—The specific epithet refers to “Tunetia”, a Latin name for Tunisia. Taxonomic relationships:— Plantago tunetana belongs to P. subg. Albicans Rahn (1996: 185) sect. Albicantes Barnéoud (1844: 18) ser. Ciliatae Rahn (1996: 188). According to Murbeck (1899), this species shares remarkable similar affinities with P. albicans and P. cylindrica (Forsskal 1775: 31). These two species differ from P. tunetana by the presence of three to five raising veins on the abaxial face of blade leaves, less rapidly acuminate, linear or linearlanceolate. In addition, P. albicans differs considerably by its glabrous broadly oval corolla and by their seeds at most 2.5 mm long (Figures 1−6) (Table 2) (Murbeck 1899, Shipunov 2020). Phenology:—Flowering in late spring (April–)May–July, fruiting in July–August(–October). Distribution and ecology:— Rahn (1996) stated that Plantago tunetana was found in restricted areas in the mountains in north-west Africa. In Tunisia, P. tunetana was found only within roadsides in Gouraïa (about 100 plants), Kef (35°51’30.39” N; 8°41’04.72” E; 720 m a.s.l.) (Fig. 7). In Gouraïa, P. tunetana was observed growing on a silty clay soil, 10% of pebbles, nitrogen (33.83–34.87 ppm), phosphorus (36.67−38.71 pmm), potassium (99.47−102.19 ppm) content, organic matter (0.31−0.47%), soil pH (8.15−8.39); electrical conductivity (0.97−1.02 g /L), total limestone (42.77−44.43%), and active limestone (19.86−20.88%), being either totally exposed to the sun or partially exposed due to the presence of Olea europaea Linnaeus (1753: 8) subsp. europaea. We found only one population composed of a few adult individuals growing as isolated rosettes. The site was cultivated with O. europaea subsp. europaea. We recoded the presence of Avena fatua Linnaeus (1753: 80), Silybum marianum Linnaeus (1753: 823) Gaertner (1791: 378), Bromus scoparius Linnaeus (1755: 6). The climate of the area is typically high semi-arid with fresh winter (Verner et al. 2018). Conservation status:—According to the IUCN Red List criteria (IUCN 2019), this species should be considered as Critically Endangered (CR B2a, b ii, iii, iv, v; C1a ii, b) in Tunisia. The habitat of Plantago tunetana is currently threatened by the expansion works of the road linking Dahmani to Djérissa (code P18), which passes through Gouraïa. Because this construction will require more space to widen the road, at least a part of the roadside will have to be removed, threatening the species with extinction. Therefore, the inclusion of P. tunetana in the “Taxon’s at risk of disappearance through serious rarefaction” of the Tunisian Flora (Le Floc’h et al. 2010) is crucial in our opinion. We recommend more collection efforts in the neighbouring area, in search for other possible populations of P. tunetana. This species should be the target of in-situ conservation efforts (microreserves, recovery of the habitats, use of targets to assess the success or failure of interventions in biodiversity conservation) and its seeds must be collected and cryopreserved ex-situ to increase their survival and natural regeneration. Notes on the associated species with Plantago tunetana in Tunisia :— Plantago tunetana occurs in degraded shrub community, dominated by some woody species. These communities consist of a tree layer dominated by Olea europaea subsp. europaea with a canopy cover of approximately 60%, whereas on herb layer several species which are common in opened, degraded, grazing Mediterranean areas take place, such as Avena fatua, Silybum marianum, and Bromus scoparius (Table 2). The full records of plants abundance/dominance are reported in Table 2. All the recorded species were native.Published as part of Ltaeif, Hela Belhaj, Sakhraoui, Anis, Hassemer, Gustavo, Castillo, Jesús M., Elimem, Mohamed & Rouz, Slim, 2023, Plantago tunetana (Plantaginaceae) in Tunisia: notes on its morphology, distribution, and ecology, pp. 195-205 in Phytotaxa 600 (3) on pages 196-200, DOI: 10.11646/phytotaxa.600.3.6, http://zenodo.org/record/808083

Reassessment of Practical Subspecies Identifications of the USDA Daucus carota L. Germplasm Collection: Morphological Data

The genus Daucus includes about 20 recognized species. The most widespread and economically important species, Daucus carota L., occurs on almost every continent. The cultivated carrot, subsp. sativus (Hoffm.) Schübl. and G. Martens, has been selected from wild populations that are extremely diverse, especially in the western Mediterranean. The predominant outcrossing and the lack of sexual isolating mechanisms among recognized infraspecific taxa complicate the taxonomy and identification of the wild populations, resulting in widely different interpretations of the number of infraspecific taxa. We measured 36 morphological characters from multiple individuals within each of 155 accessions of D. carota and from the morphologically similar species D. capillifolius (both species 2n = 18) alongside other species for comparison (D. aureus Desf., 2n = 22; D. broteri Ten., 2n = 20;D. involucratus Sm., 2n = 20; and D. littoralis Sm., 2n = 20) in an experimental field plot. Within D. carota, multivariate analyses were able to identify only two subspecies, but even these showed great overlap of individual characters. Because of the ease of crossability of wild D. carota to the domestic landraces and cultivars and because of the taxonomic challenges, the purpose of our study is to explore morphological support for subspecies within D. carota, including the phenetically similar D. capillifolius, which is part of the same clade as D. carota, with the long-term goal of resolving taxonomic disagreements and developing a practical system to classify variation within this economically important species.This article is from Crop Science 54 (2014): 706–718, doi:10.2135/cropsci2013.04.0231.</p