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Table of contents

There is mounting evidence that different marker systems vary in their mechanisms of detecting polymorphism and genome coverage. Therefore, they could complement each other to generate accurate sex-specific markers in date palm. Also described is how to characterize the identified markers by Sanger sequencing and to explore their functions through alignment of their sequences with the Genbank databases. Breeding of date palm is complicated because of its long life cycle and heterozygous nature.

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Sexual propagation of date palm does not produce true-to-type plants. Sex of date palms cannot be identified until the first flowering stage.

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Molecular markers such as random amplified polymorphic DNA RAPD , sequence-characterized amplified regions SCAR , and simple sequence repeats SSR have successfully been used to identify the sex-linked loci in the plant genome and to isolate the corresponding genes. Female date palm plants are of economic importance as they bear the fruit. Therefore, sex identification at an early stage is highly desirable. DNA-based markers are useful for early sex detection.

In this chapter, we describe male-specific sequence-characterized amplified region SCAR markers to identify sex in date palm at the seedling stage. Genomic DNA is isolated separately from both male and female date palm genotypes. Based on this amplification pattern, the sex of date palm seedlings can be predicted. Ambiguity in the total number of date palm cultivars across the world is pointing toward the necessity for an enumerative study using standard morphological and molecular markers.

Among molecular markers, DNA markers are more suitable and ubiquitous to most applications. They are highly polymorphic in nature, frequently occurring in genomes, easy to access, and highly reproducible.

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This chapter explains a stepwise protocol for extracting total genomic DNA from date palm leaves. A user-friendly protocol for RAPD analysis and a table showing the primers used in different molecular techniques that produce polymorphisms in date palm are also provided. Molecular markers are credible for the discrimination of genotypes and estimation of the extent of genetic diversity and relatedness in a set of genotypes. Inter-simple sequence repeat ISSR markers rapidly reveal high polymorphic fingerprints and have been used frequently to determine the genetic diversity among date palm cultivars.

This chapter describes the application of ISSR markers for genotyping of date palm cultivars. The application involves extraction of genomic DNA from the target cultivars with reliable quality and quantity. Subsequently the extracted DNA serves as a template for amplification of genomic regions flanked by inverted simple sequence repeats using a single primer.

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The similarity of each pair of samples is measured by calculating the number of mono- and polymorphic bands revealed by gel electrophoresis. Matrices constructed for similarity and genetic distance are used to build a phylogenetic tree and cluster analysis, to determine the molecular relatedness of cultivars.

The protocol describes 3 out of 9 tested primers consistently amplified 31 loci in 6 date palm cultivars, with 28 polymorphic loci. Investigation of genetic variation and phylogenetic relationships among date palm Phoenix dactylifera L. Various molecular markers such as restriction fragment length polymorphisms RFLPs , simple sequence repeat SSR , representational difference analysis RDA , and amplified fragment length polymorphism AFLP have been developed to molecularly characterize date palm cultivars.

Analysis of data generated from these two techniques and the use of these data to reveal phylogenetic relationships among date palm cultivars are also discussed. Date palm cultivars are classified based on the fruit moisture content, as dry, semidry, and soft dates. There are a number of biochemical and molecular techniques available for characterization of the date palm variation. These techniques coupled with appropriate statistical tools proved useful for determining phylogenetic relationships among date palm cultivars and provide information resources for date palm gene banks.

Box , Egypt. Molecular marker technologies which rely on DNA analysis provide powerful tools to assess biodiversity at different levels, i. A range of different molecular marker techniques have been developed and extensively applied for detecting variability in date palm at the DNA level. Recently, the employment of gene-targeting molecular marker approaches to study biodiversity and genetic variations in many plant species has increased the attention of researchers interested in date palm to carry out phylogenetic studies using these novel marker systems.

Molecular markers are good indicators of genetic distances among accessions, because DNA-based markers are neutral in the face of selection. Box , Baghdad, Iraq. The tree has been and still is at the center of the comprehensive agricultural development. The number of known date palm cultivars, distributed worldwide, is approximately The success of genetic diversity conservation or any breeding program depends on an understanding of the amount and distribution of the genetic variation already in existence in the genetic pool.

Development of suitable DNA molecular markers for this tree may allow researchers to estimate genetic diversity, which will ultimately lead to the genetic conservation of date palm. Simple sequence repeats SSRs are DNA strands, consisting of tandemly repeated mono-, di-, tri-, tetra-, or penta-nucleotide units that are arranged throughout the genomes of most eukaryotic species. Microsatellite markers, developed from genomic libraries, belong to either the transcribed region or the non-transcribed region of the genome, and there is rarely available information on their functions.

Microsatellite sequences are especially suited to distinguish closely related genotypes due to a high degree of variability making them ideally suitable in population studies and the identification of closely related cultivars. This chapter focuses on the methods employed to characterize date palm genotypes using SSR markers. Genetic fingerprinting using molecular markers is an important tool for the analysis of genetic diversity and cultivar identification. Here, we present an improved DNA extraction protocol using leaf tissue, based on the standard cetyltrimethyl ammonium bromide CTAB protocol, which yields large amounts of high-quality amplifiable DNA.

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RAPD and ISSR markers reveal sufficient genetic diversity as well as give some unique markers in some genotypes with a maximum number of bands. Fungal contamination of in vitro cultures of date palm Phoenix dactylifera L. Different molecular approaches have been applied successfully to analyze both inter- and intraspecific variation among fungal species as well as determine their identity. This chapter describes step-by-step procedures of molecular identification of fungal contaminants by internal transcribed spacer ITS products of the most common fungal contaminants of date palm tissue culture.

To begin with, samples of genera Alternaria, Aspergillus, Cladosporium, Epicoccum, and Penicillium were collected to isolate each fungal genus and extraction of genomic DNA. The molecular identification herein is a rapid and reliable procedure to identify date palm fungal contaminants which is very important in their control and treatment. Date palm is a fruit-bearing tree commonly found in arid and semiarid regions.

It is a dioecious plant, producing fruit on female plants and a limited number of basal offshoots for propagation. To produce large numbers of uniform plantlets, tissue culture techniques are required. It is highly advisable to detect genetic variation that may occur through micropropagation techniques as it may lead to phenotypic alterations. Screening of markers requires repeated confirmation of the pattern obtained in individual samples.

Synthetic seed or encapsulated somatic embryos may be used for propagation, storage, and exchange of plant germplasm and have many diverse applications in date palm cultivation. They have advantages over conventional use of offshoot material for germplasm propagation, maintenance, exchange, and transportation. This chapter describes a protocol for date palm synthetic seed production by encapsulation of somatic embryos with sodium alginate. This protocol is promising for in vitro conservation and international exchange of date palm germplasm. In vitro conservation is carried out to maintain disease-free genetic materials, in a small area, protected against pests, insects, soil problems alkaline, acidic, excess salinity, lack of organic matter, too dry, or too wet , climatic changes, and high-multiplication potential.

A requirement of successful in vitro conservation is that the plants can be regenerated into complete plants rapidly when desired. The current work describes in vitro propagation and conservation techniques employing slow-growth conditions of date palm somatic embryo cultures. Successfully conserved somatic embryos multiply and germinate to regenerate plants with well-developed shoots and roots, which survive acclimatization and field transfer.

Date palm fruit production has great economic significance for many countries. There is a fundamental necessity to conserve valuable date palm germplasm, but there are various problems with in vivo and ex situ conservation. In vitro storage has several advantages over conventional germplasm conservation methods. The in vitro technique offers a developed method of slow-growth storage, which is considered as an alternate solution for short- and medium-term storage of date palm germplasm under controlled conditions.

Minimal growth conditions for germplasm conservation are generally achieved by reducing growth rate through modification of environmental growing conditions and culture, by using low temperatures, and the addition of growth retardants and osmotic agents. Leuven , Leuven, Belgium. Cryopreservation is the technology of choice not only for plant genetic resource preservation but also for virus eradication and for the efficient management of large-scale micropropagation.

In this chapter, we describe three cryopreservation protocols standard vitrification, droplet vitrification, and encapsulation vitrification for date palm highly proliferating meristems that are initiated from vitro-cultures using plant growth regulator-free MS medium.

The positive impact of sucrose preculture and cold hardening treatments on survival rates is significant. All regenerated plants from non-cryopreserved or cryopreserved explants don't show morphological variation by maintaining genetic integrity without adverse effect of cryogenic treatment. Cryopreservation of date palm vitro-cultures enables commercial tissue culture laboratories to move to large-scale propagation from cryopreserved cell lines producing true-to-type plants after clonal field-testing trials.

When comparing the cost of cryostorage and in-field conservation of date palm cultivars, tissue cryopreservation is the most cost-effective. Moreover, many of the risks linked to field conservation like erosion due to climatic, edaphic, and phytopathologic constraints are circumvented.

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Using this protocol, In vitro technology offers a potential solution for the conservation of date palm germplasm. Slow growth induced by low temperature allows storage from several months up to few years. Pollen storage and viability are very important for pollination, breeding, biodiversity, biotechnology, conservation, and other biological and non-biological studies of the date palm.

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Optimizing procedures and duration of storage are important for effective and long-term date palm pollen storage and viability. Curr Biol. Electronic address: nathan. In a new study, previously unknown populations of wild date palm have been identified in remote areas of Oman. Genomic analyses indicate date palm domestication occurred in the eastern portion of the Arabian Peninsula and reveal substantial subsequent gene flow with African palm populations.

Epub Jul Electronic address: muriel. Electronic address: daniel. For many crops, wild relatives constitute an extraordinary resource for cultivar improvement [1, 2] and also help to better understand the history of their domestication [3]. However, the wild ancestor species of several perennial crops have not yet been identified.

Perennial crops generally present a weak domestication syndrome allowing cultivated individuals to establish feral populations difficult to distinguish from truly wild populations, and there is frequently ongoing gene flow between wild relatives and the crop that might erode most genetic differences [4]. Here we report the discovery of populations of the wild ancestor species of the date palm Phoenix dactylifera L.