Nuclear microsatellite markers for the date palm (Phoenix dactylifera L.): characterization and utility across the genus Phoenix and in other palm genera

A (GA)_n microsatellite‐enriched library was constructed and 16 nuclear simple sequence repeat (SSR) loci were characterized in Phoenix dactylifera. Across‐taxa amplification and genotyping tests showed the utility of most SSR markers in 11 other Phoenix species and the transferability of some of them in Elaeis guineensis, 11 species of Pritchardia, Pritchardiopsis jeanneneyi and six species of Astrocaryum. The first to be published for P. dactylifera, these new SSR resources are available for cultivar identification, pedigree analysis, germplasm diversity as well as genetic mapping studies.     

A rapid and efficient method for the extraction of total DNA from mature leaves of the data palm (Phoenix dactylifera L.)

A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may be applicable to other species of palms.     

Insights into the historical biogeography of the date palm (Phoenix dactylifera L.) using geometric morphometry of modern and ancient seeds

Aim The main purpose of this work is to understand the origin, history, historical biogeography and mechanisms of date palm (Phoenix dactylifera L.) domestication. Location Seeds of uncultivated Phoenix individuals from isolated Oman populations, cultivated date palm varieties of various geographical origins and other related Phoenix species were analysed. Additionally, well‐preserved seeds from Egyptian archaeological sites (14th century bc to 8th century ad ) were compared with the morphometric reference model based on the analysis of modern material. Methods Elliptic Fourier transforms (EFT), a morphometric method applied to shape outline analysis, were used to characterize seed shape and to quantify morphological diversity in P. dactylifera and related species. Results Analysis of seed outlines by EFT (1) showed that P. dactylifera can be differentiated from other Phoenix species and (2) enabled the quantification of patterns of shape differentiation in the genus Phoenix at different taxonomic, geographical and chronological levels. Date palm agrobiodiversity, partitioned in distinct morphotypes, appeared to be complex in terms of geographical structure. Allocation of archaeological seeds to different modern Phoenix forms and date palm morphotypes allowed us to reveal ancient forms consumed and/or exploited in Egypt and finally to determine spatial and temporal changes in agrobiodiversity. Main conclusions Based on the morphological diversity quantified in P. dactylifera and related species, we characterized ancestral seed shape features present in uncultivated populations. The geographical distribution pattern of seed shapes points to human dispersal routes that spread cultivation from one or more initial ‘domestication centres’. Finally, this work provides a powerful tool to identify ancient forms as demonstrated by the analysis of well‐preserved Egyptian archaeological seeds, dating from the 14th century bc to the 8th century ad . Results open new and fascinating perspectives on the investigation of the origins and chrono‐geographical fluctuation of date palm agrobiodiversity.     

Influence of physical conditions of nutrient medium and sucrose on somatic embryogenesis of date palm

Shoot tips and leafy bud fragments removed from offshoots of adult date palms (Phoenix dactylifera L.) were cultured on a nutrient medium containing the Murashige and Skoog inorganic salts, 453 M 2,4-dichlorophenoxyacetic acid, 14.8 M N⁶-(2-isopentenyl)adenine and 3 g l^-1 activated charcoal to develop nodular callus after 8 months of culture. Callus was cultured in agar-solidified and stationary or shaken liquid media containing half-strength MS inorganic salts, 3 g l^-1 activated charcoal and different sucrose concentrations to study the influence of these factors on somatic embryogenesis. The best conditions for embryo development were culturing in liquid medium shaken at 100 rpm for a period of 2 weeks without sucrose, followed by culture on 3% sucrose.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP N⁶-(2-isopentenyl) adenine - MS Murashige & Skoog (1962) - rpm revolutions per minute     

Isolation and selection of virulent isolates of Beauveria bassiana for biological control of Ommatissus lybicus in Kerman province

Date palm (Phoenix dactylifera) is the most important economic crop in Kerman province. The palm date leafhopper (Ommatissus lybicus) is one of the most important and key pests that causes high level damage to date crop. Date is an organic fruit in this area, and biological control has been introduced as the best long-term strategy in this region. To investigate appropriate biological control agents in Kerman province during 2005–2007, 178 isolates were collected from naturally infected O. lybicus for the first time recorded in the world, and Beauveria bassiana was introduced as a suitable biological control agent for control of this pest.     

Monitoring of cultivar identity in tissue culture-derived date palms using RAPD and AFLP analysis

In the present study, two polymerase chain reaction (PCR)-based methods namely, randomly amplified polymophic DNA (RAPD) and amplification fragment length polymorphism (AFLP) were employed to assess genetic variations, which may appeared, in tissue culture-derived date palm (Phoenix dactylifera) offshoots. Analysis of RAPD banding patterns generated by PCR amplification using 37 random primers gave no evidences for somaclonal variations and the percentage of polymorphic bands in a total of 259 scored bands was zero. Meanwhile, analysis of AFLP banding patterns generated using 13 primer combinations pointed to minor genetic variations in the AFLP banding patterns. The percentage of genetic variations (polymorphism) in tissue culture-derived date palm offshoots belonging to cultivars Sakkoty, Gandila and Bertamoda was 2.6, 0.79 and 1 %, respectively, as revealed by AFLP analysis. The low percentage of genetic variations confirms the genetic stability of tissue culture-derived dry date palm cultivars.     

Identification of hybrids betweenQuercus petraea andQ. robur (Fagaceae): Results obtained with RAPD markers confirm allozyme studies based on theGot-2 locus

RAPDs were employed as genetic markers to detect interspecific hybridization between the closely related oak speciesQuercus robur andQ. petraea. Fourteen primers were used in order to check the genetic status (pure or hybrid) of individuals classified morphologically. Among the 147 PCR fragments obtained 11 appear to be species-specific. In the phenotypically intermediate individuals different combinations of these species-specific bands were obtained. The patterns in these putative hybrids were not additive, which may be either the result of repeated backcrossing and introgression between the two species or of heterozygosity within the parental species. The results of the RAPD study are consistent with morphological analyses and allozyme data obtained for theGot-2 locus. Thus the RAPD markers used in this study may provide a powerful genetic tool for the identification of hybrids and the discrimination between the two pure species.     

Biological studies of the predacious miteTyphlodromus pelargonicus a predator of the two spotted spider miteTetranychus urticae on cucumber plants [Acari: Phytoseiidae; Tetranychidae]

The predacious miteTyphlodromus pelargonicus El-Badry preyed on the different developmental stages of the two spotted spider miteTetranychus urticae Koch. The development was quickest and the number of prey consumed was highest when individuals were maintained on eggs compared with nymphs or adult female stages. The predator was able to develop and reproduce on the eriophyid miteEriophyes dioscoridis. Soliman & Abou-Awad, nymphs of the tobacco whiteflyBemisia tabaci (Genn.) and pollen grains ofPhoenix dactylifera andRicinus communis. The average number of eggs/♀/day was 1.4, 0.7, 1.2 and 0.9 on eriophyid, whitefly,Ph. dactylifera andR. communis respectively.      

Molecular phylogeny of date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia by DNA fingerprinting

Genetic diversity among 13 different cultivars of date palm (Phoenix dactylifera L.) of Saudi Arabia was studied using random amplified polymorphic DNA (RAPD) markers. The screening of 140 RAPD primers allowed selection of 37 primers which revealed polymorphism, and the results were reproducible. All 13 genotypes were distinguishable by their unique banding patterns produced by 37 selected primers. Cluster analysis by the unweighted paired group method of arithmetic mean (UPGMA) showed two main clusters. Cluster A consisted of five cultivars (Shehel, Om-Kobar, Ajwa, Om-Hammam and Bareem) with 0.59–0.89 Nei and Li's coefficient in the similarity matrix. Cluster B consisted of seven cultivars (Rabeeha, Shishi, Nabtet Saif, Sugai, Sukkary Asfar, Sukkary Hamra and Nabtet Sultan) with a 0.66–0.85 Nei and Li's similarity range. Om-Hammam and Bareem were the two most closely related cultivars among the 13 cultivars with the highest value in the similarity matrix for Nei and Li's coefficient (0.89). Ajwa was closely related with Om-Hammam and Bareem with the second highest value in the similarity matrix (0.86). Sukkary Hamra and Nabtet Sultan were also closely related, with the third highest value in the similarity matrix (0.85). The cultivar Barny did not belong to any of the cluster groups. It was 34% genetically similar to the rest of the 12 cultivars. The average similarity among the 13 cultivars was more than 50%. As expected, most of the cultivars have a narrow genetic base. The results of the analysis can be used for the selection of possible parents to generate a mapping population. The variation detected among the closely related genotypes indicates the efficiency of RAPD markers over the morphological and isozyme markers for the identification and construction of genetic linkage maps.Communicated by H.F. Linskens     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

The chloroplast DNA locus psbZ-trnfM as a potential barcode marker in Phoenix L. (Arecaceae)

The genus Phoenix (Arecaceae) comprises 14 species distributed from Cape Verde Islands to SE Asia. It includes the economically important species Phoenix dactylifera. The paucity of differential morphological and anatomical useful characters, and interspecific hybridization, make identification of Phoenix species difficult. In this context, the development of reliable DNA markers for species and hybrid identification would be of great utility. Previous studies identified a 12 bp polymorphic chloroplast minisatellite in the trnG (GCC)-trnfM (CAU) spacer, and showed its potential for species identification in Phoenix. In this work, in order to develop an efficient DNA barcode marker for Phoenix, a longer cpDNA region (700 bp) comprising the mentioned minisatellite, and located between the psbZ and trnfM (CAU) genes, was sequenced. One hundred and thirty-six individuals, representing all Phoenix species except P. andamanensis,were analysed. The minisatellite showed 2-7 repetitions of the 12 bp motif, with 1-3 out of seven haplotypes per species. Phoenix reclinata and P. canariensis had species-specific haplotypes. Additional polymorphisms were found in the flanking regions of the minisatellite, including substitutions, indels and homopolymers. All this information allowed us to identify unambiguously eight out of the 13 species, and overall 80% of the individuals sampled. Phoenix rupicola and P. theophrasti had the same haplotype, and so had P. atlantica, P. dactylifera, and P. sylvestris (the “date palm complex” sensu Pintaud et al. 2013). For these species, additional molecular markers will be required for their unambiguous identification. The psbZ-trnfM (CAU) region therefore could be considered as a good basis for the establishment of a DNA barcoding system in Phoenix, and is potentially useful for the identification of the female parent in Phoenix hybrids.     

Yellow Death: A Disease of Date Palm in Iran Caused by Fusarium solani

In December 2003, a severe general yellowing and death of the fronds of date palm (Phoenix dactylifera) occurred in a grove in the vicinity of Kazeron district, west of Fars province, Iran. Fusarium solani was isolated from the crown, and xylem rays sampled from the trunk 1.5 m above soil level. In pathogenicity tests using artificially infested soil and 1‐year‐old date palm seedlings, an isolate from the trunk (FST) induced general chlorosis and death of seedlings 25–28 days after inoculation. Similar results were obtained when seedlings were planted in naturally infested soil. In both procedures, distal portions of the roots and crown were affected. The fungus was re‐isolated from the crown and leaf bases of the inoculated seedlings. This is the first report of a serious disease of date palm, which we call yellow death, incited by F. solani in Iran.     

The effect of biofungicide Mycostop on Ceratocystis radicicola, the causal agent of black scorch on date palm

In vitro studies were conducted to assess the efficacy of the biofungicide Mycostop on the fungus Ceratocystis radicicola (= Ceratostomella radicicola), which causes black scorch on date palm (Phoenix dactylifera) and the effect salinity on the growth of Streptomyces griseoviridis, the activecomponent of Mycostop. C. radicicola wasisolated from roots of diseased date palm inlandscape settings, nurseries, andplantations. In vitro, the effectiveconcentration (EC₅₀) of Mycostop thatreduced the mycelial growth by 50% was 0.35 g/l of PDA. Mycostop at the rate of 0.35 g/lor greater reduced spore germination,plasmolyzed germlings and reduced sporulationof C. radicicola. In essence it reducedthe inoculum potential of C. radicicola. In sterile soil treated with Mycostop, rootpieces of date palm inoculated with C.radicicola were less necrotic than those inuntreated soil. Streptomycesgriseoviridis in Mycostop did not grow on PDAamended with 5.0% NaCl (–4.28 MPa), but thenumber of colony formed units on PDA amendedwith 1.0 to 4.0% NaCl was not significantlydifferent from a factor that was considered ifMycostop is to be used to manage black scorchin Kuwait. The observations showed the effectsof Mycostop on C. radicicola inlaboratory studies but its efficacy on blackscorch disease of date palms in nurseries andplantations is yet to be determined.     

Genetic isolation of Cape Verde Island Phoenix atlantica (Arecaceae) revealed by microsatellite markers

Increasing human pressure on the environment in the isolated Macaronesian island group of Cape Verde is threatening many endemic species with extinction. The status of Phoenix atlantica, the Cape Verde Island date palm, is one of the unresolved taxonomic issues not only of the archipelago’s flora but also in the genus Phoenix. We applied 15 nuclear microsatellite markers and one chloroplast minisatellite marker to individuals of Phoenix from the Cape Verde Islands, P. dactylifera, P. canariensis and P. sylvestris, in order to assess the taxonomic position of P. atlantica within the genus. Our analysis showed that P. atlantica is clearly distinct from its close relatives and that its closest relative is likely to be its nearest geographical neighbour, P. dactylifera. Comparable levels of genetic diversity were found in insular P. atlantica and continental P. dactylifera despite the large difference in geographic range size. Our findings highlight the importance of conserving the relatively fragmented and isolated populations of P. atlantica as one of only␣two endemic trees on the islands and emphasise the need for further studies into its evolution and relationship with P. dactylifera.     

What are palm groves of <Emphasis Type="Italic">Phoenix</Emphasis>? Conservation of <Emphasis Type="Italic">Phoenix</Emphasis> palm groves in the European Union

There are three species of Phoenix (Arecaceae) in the territory of the European Union, P. canariensis, P. dactylifera and P. theophrasti, found in wild-native populations, feral, planted and intermediate states, accounting each for thousands of individuals. The EU Habitats Directive has addressed the conservation of P. theophrasti and P. canariensis under the habitat type 9370, ‘Palm groves of Phoenix,’ but neglected to include the wild-growing populations of P. dactylifera palms in southern Spain. In this paper, we survey the habitats and status of both representative native and naturalized populations of Phoenix, in total 103, through fieldwork, image analysis and review of literature. We underline the significance of feral populations and palms originating from ancient abandoned plantations, existing in protected areas as a reservoir of genetic variation. We conclude that, in order to improve their conservation status by adequate protection and conservation management, the concept of Phoenix palm groves in the Habitats Directive should be redefined to include the western group of P. dactylifera and the various habitats of P. canariensis and P. theophrasti that do not appear in the current definition.     

Random amplified polymorphic DNA variation in the eggplant,Solanum melongena L. (Solanaceae)

RAPD analysis was carried out on 52 accessions of Solanum melongena (eggplant) and related weedy forms known as insanum. Twenty-two primers amplified 130 fragments. Solanum melongena exhibited 117 of the fragments, all of which were also present in insanum. Insanum displayed an additional 13 fragments not found in S. melongena. Overall, the insanum accessions were more diverse than those of S. melongena. The calculated similarity between them was 0.947. The RAPD results were closely concordant with the results of an electrophoretic isozyme survey performed on the same accessions. The concordance of the results shows that even though S. melongena and insanum are highly diverse morphologically, it is no longer appropriate to distinguish them taxonomically.     

Historical biogeography, phylogenetic relationships and intraspecific diversity of agamid lizards in the Central Asian deserts of Kazakhstan and Uzbekistan

The Central Asian agamid lizards are ecologically and morphologically diverse, occurring across a broad range of desert environments in this biogeographically important region. It is probable that past climatic shifts have significantly influenced the diversification patterns and distributions of the agamid lizards of this region. To assess this within a phylogenetic framework we sequenced a 1200 bp region of mitochondrial DNA and a 1200 bp nuclear gene (RAG-1), incorporating both inter- and intraspecific sampling across Central Asian agamids. Our topology and divergence time estimates support an Eocene origin of the Agaminae subfamily on the Indian subcontinent, coinciding with the collision of India into Eurasia. The onset of aridification in Central Asia during the Late Oligocene, resulting from the retreat of the Paratethys Sea and the intensified uplift of the Tibetan–Himalayan complex, probably played an important role in the diversification of Phrynocephalus, one of the three genera studied. Intensification of aridity and geologic events in the Plio-Pleistocene and Quaternary glacial cycling probably had a significant influence on intraspecific diversification patterns within Phrynocephalus.     

A genetic and structural analysis of the -glycosylation capabilities

The recent draft sequencing of the rice (Oryza sativa) genome has enabled a genetic analysis of the glycosylation capabilities of an agroeconomically important group of plants, the monocotyledons. In this study, we have not only identified genes putatively encoding enzymes involved in N-glycosylation, but have examined by MALDI-TOF MS the structures of the N-glycans of rice and other monocotyledons (maize, wheat and dates; Zea mays, Triticum aestivum and Phoenix dactylifera); these data show that within the plant kingdom the types of N-glycans found are very similar between monocotyledons, dicotyledons and gymnosperms. Subsequently, we constructed expression vectors for the key enzymes forming plant-typical structures in rice, N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101), core 1,3-fucosyltransferase (FucTA; EC 2.4.1.214) and 1,2-xylosyltransferase (EC 2.4.2.38) and successfully expressed them in Pichia pastoris. Rice GlcNAc-TI, FucTA and xylosyltransferase are therefore the first monocotyledon glycosyltransferases involved in N-glycan biosynthesis to be characterised in a recombinant form.     

Phenolic compounds in date palm cultivars sensitive and resistant toFusarium oxysporum

In date palm (Phoenix dactylifera L.) leaves, the main compounds of the phenolic pool were quercetin and isorhamnetin heterosides, (+)-catechin and (-)-epicatechin. Although previously observed only in date palm fruits, 5-caffeoylshikimic acid (dactylifric acid) and its positional isomers (3-caffeoylshikimic acid and 4-caffeoylshikimic acid) were detected also in the leaves and roots. Quantitative, but not qualitative, differences between cultivars resistant and susceptible toFusarium oxysporum f. sp.albedinis during growth period were observed Acknowledgements: The authors are very grateful to Dr. Janati, Director of SCAS (INRA, Marrakech) for supplying the plant material used in this study. The study was funded by the Programme de Cooperation Franco-Marocaine (Al 473/90) between Cadi Ayyad University (Marrakech, Maroc) and Montpellier II University (France)     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric ( _{2 2}) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3 and 5 parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.