Analysis of genetic diversity and population structure of Moroccan date palm (Phoenix dactylifera L.) using SSR and DAMD molecular markers.

BACKGROUND: Date palm, oasis pivot, plays a vital socio-economic part in the southern area of Morocco. However, with climate change and drought intensity and frequency increasing, the Moroccan palm grove is threatened with significant genetic degradation. Genetic characterization of this resource is key element for the development of effective conservation and management strategies in the current circumstances of climate change and various biotic and abiotic stresses. To evaluate the genetic diversity of date palm populations collected from different Moroccan oases, we used simple sequence repeats (SSR) and directed amplification of mini-satellite DNA (DAMD) markers. Our results showed that used markers could efficiently assess genetic diversity in Phoenix dactylifera L. RESULTS: A total of 249 and 471 bands were respectively scored for SSR and DAMD, of which 100% and 92.9% were polymorphic. The polymorphic information content (PIC = 0.95), generated by the SSR primer was nearly identical to that generated by the DAMD primer (PIC = 0.98). The resolving power (Rp) was higher in DAMD than SSR (29.46 and 19.51, respectively). Analysis of the molecular variance (AMOVA) based on the combined data sets for both markers revealed a higher variance within populations (75%) than among populations (25%). Principal coordinate analysis (PCoA) and the ascendant hierarchical classification showed that the population of Zagora and Goulmima regions were the closest populations. The STRUCTURE analysis clustering of the 283 tested samples into seven clusters based on their genetic composition. CONCLUSION: The results drawn from this study will orient genotypes selection strategies for a successful future breeding and conservation program, particularly under climate change context.

Assessment of genetic diversity among wild rose in Morocco using ISSR and DAMD markers.

BACKGROUND: Morocco is considered one of the main biodiversity hotspots in the Mediterranean region and contains various plant species including wild and domestic Rosa. This genus is the most important among cultivated ornamental plants in the world, with a high economic value in cosmetics, pharmaceutical industries, and floriculture. In the present study, genetic diversity among the collected accessions of wild Rosa species in Morocco was assessed using Inter-Simple Sequence Repeat (ISSR) and Directed Amplification of Minisatellites DNA (DAMD) markers. RESULTS: Results confirmed that both markers used have a good efficiency to assess genetic diversity in wild roses. Ten ISSR and eight DAMD primers amplified 276 and 203 loci, with an average of 27.4 and 25 polymorphic alleles per primer, respectively. The polymorphic information content (PIC) values were 0.34 with ISSR and 0.31 with DAMD. Analysis of molecular variance (AMOVA) showed that genetic variation in wild rose occurs mainly within populations (86%) rather than between populations (14%). The region of Azrou (Middle Atlas of Morocco) is the area that registered the highest genetic diversity in the present study with He = 0.21. The 39 rose accessions were divided into three main groups with among-group similarity of 30%. Principal component analysis and the hierarchical classification were consistent with genetic relationships derived by structure analysis. CONCLUSION: The findings revealed that the patterns of grouping are weakly correlated with geographical origin. ISSR and DAMD markers showed that the accessions have a good genetic diversity.

BuscoPhylo: a webserver for Busco-based phylogenomic analysis for non-specialists.

Here we present the BuscoPhylo tool that enables both students and established scientists to easily perform Busco-based phylogenomic analysis starting from a set of genomes sequences. BuscoPhylo is an efficient and user-friendly web server freely accessible at https://buscophylo.inra.org.ma/ . The source code, along with documentation, is freely available under an MIT license at https://github.com/alaesahbou/BuscoPhylo .

A Genome-Wide Association Study Identifying Single-Nucleotide Polymorphisms for Iron and Zinc Biofortification in a Worldwide Barley Collection.

Micronutrient deficiency affects half of the world's population, mostly in developing countries. Severe health issues such as anemia and inadequate growth in children below five years of age and pregnant women have been linked to mineral deficiencies (mostly zinc and iron). Improving the mineral content in staple crops, also known as mineral biofortification, remains the best approach to address mineral malnutrition. Barley is a staple crop in some parts of the world and is a healthy choice since it contains ß-glucan, a high dietary protein. Barley mineral biofortification, especially with zinc and iron, can be beneficial since barley easily adapts to marginalized areas and requires less input than other frequently consumed cereals. In this study, we analyzed zinc and iron content in 496 barley samples. The samples were genotyped with an Illumina 50 K SNP chip. Genome-wide association studies (GWAS) identified 62 SNPs and 68 SNPs (p < 0.001) associated with iron and zinc content in grains, respectively. After a Bonferroni correction (p < 0.005), there were 12 SNPs (single-nucleotide polymorphism) associated with Zn and 6 for iron. SNP annotations revealed proteins involved in membrane transport, Zn and Fe binding, linked to nutrient remobilization in grains. These results can be used to develop biofortified barley via marker-assisted selection (MAS), which could alleviate mineral malnutrition.

Antagonistic potential of Moroccan entomopathogenic nematodes against root-knot nematodes, Meloidogyne javanica on tomato under greenhouse conditions.

The root-knot nematode, Meloidogyne javanica is a devastating pest affecting tomato production worldwide. Entomopathogenic nematodes (EPNs) are considered very promising biocontrol agents that could be used to effectively manage plant-parasitic nematode. The antagonistic activity of five EPN strains isolated from different fields in Morocco was evaluated against juvenile (J2s) antagonism in soil, the number of egg masses, and the galling index of M. javanica and J2s reproduction in the root. In greenhouse experiments, Steinernema feltiae strains (EL45 and SF-MOR9), Steinernema sp. (EL30), and those of Heterorhabditis bacteriophora (HB-MOR7 and EL27) were applied to the soil alongside RKN J2s. There was a significant reduction in M. javanica densities in the soil and roots by EPNs treatments when compared to the positive control. The EPNs decreased both egg masses formation and galling index by 80% compared to the positive control. The application of EPNs at a rate of 50 and 75 infective juveniles (IJs) cm-2 gave significant control of all studied nematological parameters compared to the positive control, which confirmed the importance of the doses applied. The applied dose was significantly correlated with M. javanica parameters according to polynomial regression models. The results also showed that S. feltiae strain (EL45) significantly increased plant height and root length, while H. bacteriophora strain (HB-MOR7) only enhanced root fresh weight. Therefore, both indigenous EPN strains; EL45 and SF-MOR9 have eco-friendly biological potential against M. javanica in vegetable crops.

Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of moroccan durum wheat

Drought is one of the major constraints in durum wheat production in the Mediterranean Basin. In order to overcome this problem, the genetic transformation of durum wheat is one of the choices for improvement. However, the recalcitrance to Agrobacterium-mediated transformation in durum wheat (Triticum turgidum L.) is one of the factors limiting a successful genetic transformation. The aim of this study was to investigate the effect of explant type and acetosyringone concentration for the efficient Agrobacterium-mediated genetic transformation of three Moroccan durum wheat varieties (Amria, Chaoui, and Marouane). The mature embryos (intact, halved and pieces) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pTF101.1 containing drought tolerance gene HVA1 from barley, and a selectable marker phosphinothricin (PPT) resistance (bar) gene. The explants were inoculated with A. tumefaciens (cell density OD650 at 0.7) at four different concentrations of acetosyringone (0, 100, 200, and 400 µM). The results showed that embryogenic calli from mature embryos showed higher regeneration and transformation than mature embryo halves and pieces. The integration of the transgene was confirmed by PCR amplification using primers specific to the bar gene, 2x35S promoter, and HVA1 gene. The transformation efficiency ranging from 0.33% to 2.33% was obtained in Amira variety using embryogenic calli and acetosyringone concentrations of 200 and 400 µM. The integration, as well as inheritance of the transgene, was confirmed by PCR amplification in T0 and T1 generations. This is the first report describing a genetic transformation of Moroccan durum wheat varieties via Agrobacterium tumefaciens.

In Vitro Root Induction from Argan (Argania spinosa (L.) Skeels) Adventitious Shoots: Influence of Ammonium Nitrate, Auxins, Silver Nitrate and Putrescine, and Evaluation of Plantlet Acclimatization.

Argania spinosa (L.) Skeels is an endangered plant species endemic to Morocco. In recent years, attempts to develop in vitro regeneration systems for this species were made. However, rooting and acclimatization of in vitro plants have been a bottleneck for successful propagation. In the present study, the effects of different concentrations of auxins, putrescine, silver nitrate (AgNO3) and ammonium nitrate on the in vitro rooting of adventitious shoots of two argan genotypes "Mejji" and "R'zwa", were evaluated. The highest rooting percentages (86.6% in "Mejji" and 84.4% in "R'zwa") were observed on Murashige and Skoog (MS) medium modified by reducing the ammonium nitrate concentration and supplemented with 1.5 mg L-1 indole-3-butyric acid (IBA), 0.5 mg L-1 1-naphthalene acetic acid (NAA), 2 mg L-1 AgNO3 and 160 mg L-1 putrescine. This medium resulted in the development of a good root system after only 10 days of culture. Plantlet acclimatization was carried out using different substrate mixtures, and high survival rates (100%) were observed when the substrate contained either peat alone or a sand-peat mixture (1:1, w/w). The high percentages of rooting and acclimatization reported in the present study are of high importance for rapid and large-scale propagation of this endangered species.

Draft Genome Sequence of Fusarium oxysporum f. sp. albedinis Strain Foa 133, the Causal Agent of Bayoud Disease on Date Palm.

Fusarium oxysporum f. sp. albedinis is the causal agent of vascular wilt of date palm. Here, we report the genome assembly of the Foa 133 strain, which consists of 3,325 contigs with a total length of 56,228,901 bp, a GC content of 47.42%, an N 50 value of 131,587 bp, and 3,684 predicted genes.

First draft genome assembly of the Argane tree ( Argania spinosa).

Background: The Argane tree ( Argania spinosa L. Skeels) is an endemic tree of southwestern Morocco that plays an important socioeconomic and ecologic role for a dense human population in an arid zone. Several studies confirmed the importance of this species as a food and feed source and as a resource for both pharmaceutical and cosmetic compounds. Unfortunately, the argane tree ecosystem is facing significant threats from environmental changes (global warming, over-population) and over-exploitation. Limited research has been conducted, however, on argane tree genetics and genomics, which hinders its conservation and genetic improvement. Methods: Here, we present a draft genome assembly of A. spinosa. A reliable reference genome of  A. spinosa was created using a hybrid  de novo assembly approach combining short and long sequencing reads. Results: In total, 144 Gb Illumina HiSeq reads and 7.2 Gb PacBio reads were produced and assembled. The final draft genome comprises 75 327 scaffolds totaling 671 Mb with an N50 of 49 916 kb. The draft assembly is close to the genome size estimated by k-mers distribution and covers 89% of complete and 4.3 % of partial Arabidopsis orthologous groups in BUSCO. Conclusion: The A. spinosa genome will be useful for assessing biodiversity leading to efficient conservation of this endangered endemic tree. Furthermore, the genome may enable genome-assisted cultivar breeding, and provide a better understanding of important metabolic pathways and their underlying genes for both cosmetic and pharmacological purposes.

Allelic variation at high-molecular weight and low-molecular weight glutenin subunit genes in Moroccan bread wheat and durum wheat cultivars.

Glutenin is a major protein fraction contributing to the functional properties of gluten and dough. The glutenin constitutes 30-40% of the protein in wheat flour and about half of that in gluten. It is essential to identify correct glutenin alleles and to improve wheat quality by selecting alleles that exert favorable effects. Moroccan wheat cultivars are unique in West Asia and North Africa region, since many of them possess resistance to Hessian fly, a pest, which is becoming important in other countries in the region. Hence, these cultivars are being used as donor for the resistance in the breeding program. Here, we determine the allelic variation in high-molecular weight glutenin subunits (HMW-GS) and low-molecular weight glutenin subunits (LMW-GS) in Moroccan cultivars of bread and durum wheat using the gene-specific PCR markers. In 20 cultivars of bread wheat, 9 different allele variants were detected at HMW-GS and 13 different allele variants were detected at LMW-GS, in which the alleles Glu-A1b (2*), Glu-B1i (17 + 18), Glu-B1c (7*/7 + 9), Glu-D1d (5 + 10), Glu-A3c, Glu-B3 h, and Glu-D3b were the most frequents. In 26 cultivars of durum wheat, less allelic variation was found: seven different allele variants at HMW-GS and six different allele variants at LMW-GS were identified, in which the major alleles were Glu-A1c (null), Glu-B1b (7 + 8), Glu-B1e (20), Glu-A3c, and Glu-B3d. The mean value of the genetic diversity for the glutenin loci was 0.502 in bread wheat and 0.449 in durum wheat. Most of the glutenin alleles carried by Moroccan bread wheat cultivars impart good bread-making quality. Most of the durum wheat glutenin alleles were related to low strength dough or poor quality and need to be improved. To improve quality of Moroccan durum wheat, essentially, Glu-A1c and Glu-B3d alleles of the genes should be replaced with the better alleles through breeding.

Correlation between the Chemical and Genetic Relationships among Thymus saturejoides Genotypes Cultured under in vitro and in vivo Environments.

In this study, the in vitro and in vivo essential oil (EO) composition and genetic variability in six micropropagated genotypes of Thymus saturejoides Coss., a Mediterranean medicinal and aromatic plant, were analyzed by GC/MS and randomly amplified polymorphic DNA (RAPD). Yield and composition of the EO varied between genotypes. Cluster analysis based on RAPD data and EO grouped the six genotypes in three groups in both culture conditions, thus showing considerable intraspecific genetic and chemical variations. Applying the Mantel test, the result showed a significant correlation between the two proximity matrices RAPD and EO obtained from in vitro genotypes, whereas this correlation was not observed when using the EO obtained from the in vivo genotypes.

Identification of Differentially Expressed Genes bycDNA-AFLP Technique in Response to Drought Stressin Triticum durum.

Drought is the single largest abiotic stress factor leading to reduced crop yields. The identification of differentially expressed genes and the understanding of their functions in environmentally stressful conditions are essential to improve drought tolerance. Transcriptomics is a powerful approach for the global analysis of molecular mechanisms under abiotic stress. To identify genes that are important for drought tolerance, we analyzed mRNA populations from untreated and drought-stressed leaves of Triticum durum by cDNA- -amplified fragment length polymorphism (cDNA-AFLP) technique. Overall, 76 transcript- -derived fragments corresponding to differentially induced transcripts were successfully sequenced. Most of the transcripts identified here, using basic local alignment search tool (BLAST) database, were genes belonging to different functional categories related to metabolism, energy, cellular biosynthesis, cell defense, signal transduction, transcription regulation, protein degradation and transport. The expression patterns of these genes were confirmed by quantitative reverse transcriptase real-time polymerase chain reaction (qRT- -PCR) based on ten selected genes representing different patterns. These results could facilitate the understanding of cellular mechanisms involving groups of genes that act in coordination in response to stimuli of water deficit. The identification of novel stress-responsive genes will provide useful data that could help develop breeding strategies aimed at improving durum wheat tolerance to field stress.

Sucrose utilization during somatic embryo development in black spruce: involvement of apoplastic invertase in the tissue and of extracellular invertase in the medium.

The involvement of apoplastic invertase (Ap Inv) and sucrose synthase (SuSy) in the somatic embryo development of black spruce (Picea mariana) was investigated under different maturation conditions. Replacing 6% sucrose with 3% or 1% sucrose in the maturation medium drastically decreased Ap Inv activity and amount in embryogenic tissues. This was accompanied by a decrease in the hexose pool that resulted in a lower starch deposition and protein amount in embryogenic tissues together with a lower embryo production. Conversely, SuSy activity was stable during maturation regardless of the sucrose concentration used in the medium. The presence of an extracellular enzyme responsible for sucrose hydrolysis in the maturation medium was also verified. An immunodetection experiment with anti-acid invertase antibodies revealed the presence of an active 53 kDa polypeptide in the medium, which had a similar molecular mass to that of the Ap Inv polypeptide found in embryogenic tissues. Utilization of sucrose from the medium by the tissues was also studied using labelled 14C-sucrose. Distribution of the radioactivity between tissular sucrose, glucose, and fructose showed that sucrose was diffused into the cell wall of embryogenic tissues and partly hydrolyzed by Ap Inv. These results show that the utilization of sucrose from the medium, the Ap Inv activity in embryogenic tissues, and the release of an active invertase into the medium operate together for the utilization of the carbohydrates during somatic embryo development in black spruce.

The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos.

The present study was conducted to understand the role of sucrose in the medium on the maturation of black spruce and white spruce somatic embryos. A maturation medium containing 6% sucrose, which hydrolyzed into glucose and fructose, gave significantly more embryos than a medium containing 3.16% of each glucose and fructose. Preventing the complete sucrose hydrolysis by a daily transfer of the tissues onto fresh medium significantly decreased the yield of somatic embryos compared to when sucrose was allowed to complete its hydrolysis. This reduction was not due to the manipulation of the tissues during the transfer, since a daily in situ transfer did not affect embryo production. To verify if the better embryo production observed on a medium containing 6% sucrose was due to the increasing osmotic pressure of the medium, this increasing osmotic pressure was simulated with a sequence of media containing different concentrations of glucose and fructose. Unexpectedly and for both species, this simulation did not improve somatic embryo production, which stayed similar to the one obtained on constant osmotic pressure. To understand these results, embryos produced on the different treatments were analyzed in terms of sucrose, glucose, fructose and starch levels and protein contents. The embryo carbohydrate content was independent from the carbohydrate used in the maturation medium. However, embryos matured on 6% sucrose allowed to hydrolyze during the maturation period contained significantly more soluble and insoluble proteins than embryos matured on any other treatment. Furthermore, embryos with a higher protein content also exhibited a higher epicotyl appearance frequency. The role of sucrose as a regulatory factor during the maturation of spruce somatic embryos is discussed.