Mohammad Shobrak

A Brief Review of Molecular Techniques to Assess Plant Diversity

Massive loss of valuable plant species in the past centuries and its adverse impact on environmental and socioeconomic values has triggered the conservation of plant resources. Appropriate identification and characterization of plant materials is essential for the successful conservation of plant resources and to ensure their sustainable use. Molecular tools developed in the past few years provide easy, less laborious means for assigning known and unknown plant taxa. These techniques answer many new evolutionary and taxonomic questions, which were not previously possible with only phenotypic methods. Molecular techniques such as DNA barcoding, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), microsatellites and single nucleotide polymorphisms (SNP) have recently been used for plant diversity studies. Each technique has its own advantages and limitations. These techniques differ in their resolving power to detect genetic differences, type of data they generate and their applicability to particular taxonomic levels. This review presents a basic description of different molecular techniques that can be utilized for DNA fingerprinting and molecular diversity analysis of plant species.

DNA marker technology for wildlife conservation

Use of molecular markers for identification of protected species offers a greater promise in the field of conservation biology. The information on genetic diversity of wildlife is necessary to ascertain the genetically deteriorated populations so that better management plans can be established for their conservation. Accurate classification of these threatened species allows understanding of the species biology and identification of distinct populations that should be managed with utmost care. Molecular markers are versatile tools for identification of populations with genetic crisis by comparing genetic diversities that in turn helps to resolve taxonomic uncertainties and to establish management units within species. The genetic marker analysis also provides sensitive and useful tools for prevention of illegal hunting and poaching and for more effective implementation of the laws for protection of the endangered species. This review summarizes various tools of DNA markers technology for application in molecular diversity analysis with special emphasis on wildlife conservation.

Application of RAPD for molecular characterization of plant species of medicinal value from an arid environment

The use of highly discriminatory methods for the identification and characterization of genotypes is essential for plant protection and appropriate use. We utilized the RAPD method for the genetic fingerprinting of 11 plant species of desert origin (seven with known medicinal value). Andrachne telephioides, Zilla spinosa, Caylusea hexagyna, Achillea fragrantissima, Lycium shawii, Moricandia sinaica, Rumex vesicarius, Bassia eriophora, Zygophyllum propinquum subsp migahidii, Withania somnifera, and Sonchus oleraceus were collected from various areas of Saudi Arabia. The five primers used were able to amplify the DNA from all the plant species. The amplified products of the RAPD profiles ranged from 307 to 1772 bp. A total of 164 bands were observed for 11 plant species, using five primers. The number of well-defined and major bands for a single plant species for a single primer ranged from 1 to 10. The highest pair-wise similarities (0.32) were observed between A. fragrantissima and L. shawii, when five primers were combined. The lowest similarities (0) were observed between A. telephioides and Z. spinosa; Z. spinosa and B. eriophora; B. eriophora and Z. propinquum. In conclusion, the RAPD method successfully discriminates among all the plant species, therefore providing an easy and rapid tool for identification, conservation and sustainable use of these plants.

Interpretation of electrophoretograms of seven microsatellite loci to determine the genetic diversity of the Arabian Oryx.

Microsatellite markers are commonly used for examining population structure, especially inbreeding, outbreeding and gene flow. An array of microsatellite loci, preferably with multiallelic presentation, is preferable for ensuring accurate results. However, artifact peaks or stutters in the electrophoretograms significantly hamper the reliable interpretation of genotypes. We interpreted electrophoretograms of seven microsatellite loci to determine the genetic diversity of the Arabian Oryx. All the alleles of different loci exhibited good peak resolutions and hence were clearly identified. Moreover, none of the stutter peaks impaired the recognition or differentiation between homozygote and heterozygote. Our findings suggest that correct identification of alleles in the presence of co-amplified nonspecific fragments is important for reliable interpretation of microsatellite data.

A Simple Method for DNA Extraction from Mature Date Palm Leaves: Impact of Sand Grinding and Composition of Lysis Buffer

Molecular marker techniques have been widely used for cultivar identification of inbred date palms (Phoenix dactylifera L.; Arecaceae) and biodiversity conservation. Isolation of highly pure DNA is the prerequisite for PCR amplification and subsequent use such as DNA fingerprinting and sequencing of genes that have recently been developed for barcoding. To avoid problems related to the preservation and use of liquid nitrogen, we examined sterile sand for grinding the date palm leaves. Individual and combined effects of sodium chloride (NaCl), polyvinylpyrrolidone (PVP) and lithium chloride (LiCl) with the cetyltrimethylammonium bromide (CTAB) method for a DNA yield of sufficient purity and PCR amplification were evaluated in this study. Presence of LiCl and PVP alone or together in the lysis buffer did not significantly improve the DNA yield and purity compared with the addition of NaCl. Our study suggested that grinding of date palm leaf with sterile sand and inclusion of NaCl (1.4 M) in the lysis buffer without the costly use of liquid nitrogen, PVP and LiCl, provides a DNA yield of sufficient purity, suitable for PCR amplification.

Cytochrome c oxidase subunit I barcoding of the green bee-eater (Merops orientalis).

DNA barcoding using mitochondrial cytochrome c oxidase subunit I (COI) is regarded as a standard method for species identification. Recent reports have also shown extended applications of COI gene analysis in phylogeny and molecular diversity studies. The bee-eaters are a group of near passerine birds in the family Meropidae. There are 26 species worldwide; five of them are found in Saudi Arabia. Until now, GenBank included a COI barcode for only one species of bee-eater, the European bee-eater (Merops apiaster). We sequenced the 694-bp segment of the COI gene of the green bee-eater M. orientalis and compared the sequences with those of M. apiaster. Pairwise sequence comparison showed 66 variable sites across all the eight sequences from both species, with an interspecific genetic distance of 0.0362. Two and one within-species variable sites were found, with genetic distances of 0.0005 and 0.0003 for M. apiaster and M. orientalis, respectively. This is the first study reporting barcodes for M. orientalis.

DNA Barcodes of Arabian Partridge and Philby's Rock Partridge: Implications for Phylogeny and Species Identification

Recently, DNA barcoding based on mitochondrial cytochrome c oxidase subunit I (COI) has gained wide attention because of simplicity and robustness of these barcodes for species identification including birds. The current GenBank records show the COI barcodes of only one species, chukar partridge (Alectoris chukar), of the Alectoris genus. In this study, we sequenced the 694 bp segment of COI gene of the two species including, Arabian partridge (Alectoris melanocephala) and Philbys rock partridge (Alectoris philbyi) of the same genus. We also compared these sequences with earlier published barcodes of chukar partridge. The pair-wise sequence comparison showed a total of 53 variable sites across all the 9 sequences from 3 species. Within-species variable sites were found to be 4 (Alectoris chukar), 0 (Alectorisphilbyi) and 3 (Alectoris melanocephala). The genetic distances among the 9 individuals varied from 0.000 to 0.056. Phylogenetic analysis using COI barcodes clearly discriminated the 3 species, while Alectoris chukar was found to be more closely related to Alectoris philbyi. Similar differentiation was also observed using 1155 bp mitochondrial control region (CR) sequences suggesting the efficiency of COI gene for phylogenetic reconstruction and interspecific identification. This is the first study reporting the barcodes of Arabian partridge and Philbys rock partridge.

Skin lipids from Saudi Arabian birds

Skin lipids play an important role in the regulation of cutaneous water loss (CWL). Earlier studies have shown that Saudi desert birds exhibit a tendency of reduced CWL than birds from temperate environment due to adaptive changes in composition of their skin lipids. In this study, we used thin-layer chromatography (TLC) for separation and detection of non-polar and polar lipids from the skin of six bird species including sooty gull, brown booby, house sparrow, Arabian waxbill, sand partridge, and laughing dove. The lipids were separated and detected on Silica gel G coated TLC plates and quantified by using densitometric image analysis. Rf values of the non-polar lipids were as follows: cholesterol (0.29), free fatty acids (0.58), triacylglycerol (0.69), fatty acids methyl esters (0.84) and cholesterol ester (0.97). Rf values for the polar lipids were: cerebroside (0.42), ceramide (0.55) and cholesterol (0.73). The results showed the abundance of fatty acids methyl esters (47.75-60.46%) followed by triacylglycerol (12.69-24.14%). The remaining lipid compositions were as follows: cholesterol (4.09-13.18%), ceramide (2.18-13.27%), and cerebroside (2.53-12.81%). In conclusion, our findings showed that TLC is a simple and sensitive method for the separation and quantification of skin lipids. We also reported a new protocol for lipid extraction using the zirconia beads for efficient disruption of skin tissues. This study will help us better understand the role of skin lipids in adaptive physiology towards adverse climatic conditions.

DNA Barcodes of Asian Houbara Bustard (Chlamydotis undulata macqueenii)

Populations of Houbara Bustards have dramatically declined in recent years. Captive breeding and reintroduction programs have had limited success in reviving population numbers and thus new technological solutions involving molecular methods are essential for the long term survival of this species. In this study, we sequenced the 694 bp segment of COI gene of the four specimens of Asian Houbara Bustard (Chlamydotis undulata macqueenii). We also compared these sequences with earlier published barcodes of 11 individuals comprising different families of the orders Gruiformes, Ciconiiformes, Podicipediformes and Crocodylia (out group). The pair-wise sequence comparison showed a total of 254 variable sites across all the 15 sequences from different taxa. Three of the four specimens of Houbara Bustard had an identical sequence of COI gene and one individual showed a single nucleotide difference (G > A transition at position 83). Within the bustard family (Otididae), comparison among the three species (Asian Houbara Bustard, Great Bustard (Otis tarda) and the Little Bustard (Tetrax tetrax)), representing three different genera, showed 116 variable sites. For another family (Rallidae), the intra-family variable sites among the individuals of four different genera were found to be 146. The COI genetic distances among the 15 individuals varied from 0.000 to 0.431. Phylogenetic analysis using 619 bp nucleotide segment of COI clearly discriminated all the species representing different genera, families and orders. All the four specimens of Houbara Bustard formed a single clade and are clearly separated from other two individuals of the same family (Otis tarda and Tetrax tetrax). The nucleotide sequence of partial segment of COI gene effectively discriminated the closely related species. This is the first study reporting the barcodes of Houbara Bustard and would be helpful in future molecular studies, particularly for the conservation of this threatened bird in Saudi Arabia.