Salim Khan

SCAR markers: a potential tool for authentication of herbal drugs.

Randomly Amplified Polymorphic DNA (RAPD) is easy to develop and simple molecular marker, but lack of reproducibility makes it less reliable for authentication of herbal drugs. Besides RAPD, other popular PCR and non-PCR based markers like AFLP, ISSR, SSR and RFLP are also used for authentication. However, these also have disadvantages like use of radioactive isotopes, high cost and absolute requirement of sequence information. Therefore, it is a better option to improve the reproducibility of RAPD by converting RAPD amplicons into Sequence Characterized Amplified Region (SCAR) markers. SCAR markers are easy, reliable and reproducible thus, have an advantage over RAPD markers for authentication of medicinal herbs used in the preparation of traditional medicines. These markers however, have been developed for only a few medicinal herbs. This review is an attempt to evaluate critically the role of SCAR markers in authentication of medicinal herbs used in traditional formulations.

Biotechnological approaches for conservation and improvement of rare and endangered plants of Saudi Arabia

Genetic variation is believed to be a prerequisite for the short-and long-term survival of the plant species in their natural habitat. It depends on many environmental factors which determine the number of alleles on various loci in the genome. Therefore, it is important to understand the genetic composition and structure of the rare and endangered plant species from their natural habitat to develop successful management strategies for their conservation. However, rare and endangered plant species have low genetic diversity due to which their survival rate is decreasing in the wilds. The evaluation of genetic diversity of such species is very important for their conservation and gene manipulation. However, plant species can be conserved by in situ and in vitro methods and each has advantages and disadvantages. DNA banking can be considered as a means of complimentary method for the conservation of plant species by preserving their genomic DNA at low temperatures. Such approach of preservation of biological information provides opportunity for researchers to search novel genes and its products. Therefore, in this review we are describing some potential biotechnological approaches for the conservation and further manipulation of these rare and endangered plant species to enhance their yield and quality traits.

Fusarium solani, P1, a new endophytic podophyllotoxin-producing fungus from roots of Podophyllum hexandrum

Podophyllotoxin, a well-known naturally occurring aryl tetralin lignan produced by few plant species is used as precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophos phosphate. The availability of this lignan is limited due to the scarce occurrence of its natural sources. Further, synthetic approaches for its production are still commercially unacceptable. This paper reports the synthesis of podophyllotoxin by an endophytic fungus Fusarium solani isolated from the roots of Podophyllum hexandrum. The presence of podophyllotoxin in fungal biomass was confirmed and quantified by HPLC and mass spectrometry. The fungus is able to produce 29.0 µg/g podophyllotoxin on dry weight basis.

Assessment of genetic diversity in the endangered populations of Breonadia salicina (Rubiaceae) growing in The Kingdom of Saudi Arabia using inter-simple sequence repeat markers

BackgroundBreonadia salicina (Rubiaceae) is a critically endangered plant at the local scale native to southwestern Saudi Arabia. To understand the levels and partitioning of genetic variation across populations and geographical regions of this species, we assessed its genetic diversity using inter-simple sequence repeat (ISSR) markers.ResultsFourteen ISSR primers selected from 43 primers gave rise to 211 amplified loci, of which 68 were polymorphic. The percentage of polymorphic loci (PPL) at the population level ranged from 17.1 to 23.7%, with an average of 21.3%. Nei’s gene diversity (h) and Shannon’s information index (I) were 0.086 and 0.125, respectively. At the species level, PPL was 32.2%, while h and I were 0.116 and 0.172, respectively. A hierarchical analysis of molecular variance revealed a high level of genetic differentiation among populations (17% of total variance, P = 0.001), consistent with the gene differentiation coefficient (GST = 0.256). Nevertheless, the evaluated genetic diversity was very low within populations; while relatively high among populations, levels were insufficient for long-term survival. Saudi Arabian accessions were also compared to accessions of a population from Yemen, where the species is more widespread. The Yemeni population also showed low genetic diversity but clustered separately.ConclusionsBreonadia salicina in Saudi Arabia is characterized by low within-population genetic diversity and high among-population genetic differentiation. Based on our findings, this locally endangered species is on the verge of local extinction. The species’ survival depends on successful implementation of suggested strategies for its long-term conservation.

Assessing Molecular Signature for Some Potential Date (Phoenix dactylifera L.) Cultivars from Saudi Arabia, Based on Chloroplast DNA Sequences rpoB and psbA-trnH

Phoenix dactylifera L. (date palm), being economically very important, is widely cultivated in the Middle East and North Africa, having about 400 different cultivars. Assessment of date cultivars under trading and farming is a widely accepted problem owing to lack of a unique molecular signature for specific date cultivars. In the present study, eight different cultivars of dates viz., Khodry, Khalas, Ruthana, Sukkari, Sefri, Segae, Ajwa and Hilali were sequenced for rpoB and psbA-trnH genes and analyzed using bioinformatics tools to establish a cultivar-specific molecular signature. The combined aligned data matrix was of 1147 characters, of which invariable and variable sites were found to be 958 and 173, respectively. The analysis clearly reveals three major groups of these cultivars: (i) Khodary, Sefri, Ajwa, Ruthana and Hilali (58% BS); (ii) Sukkari and Khalas (64% BS); and (iii) Segae. The economically most important cultivar Ajwa showed similarity with Khodary and Sefri (67% BS).The sequences of the date cultivars generated in the present study showed bootstrap values between 38% and 70% so these sequences could be carefully used as molecular signature for potential date cultivars under trading and selection of genuine cultivars at the seedling stage for farming.

Molecular authentication of the medicinal herb Ruta graveolens (Rutaceae) and an adulterant using nuclear and chloroplast DNA markers.

Dried parts of different plant species often look alike, especially in powdered form, making them very difficult to identify. Ruta graveolens, sold as a dried medicinal herb, can be adulterated with Euphorbia dracunculoides. The genomic DNA was isolated from the leaf powder (100 mg each) using the modified CTAB method. Internal transcribed spacer sequences of nuclear ribosomal DNA (nrDNA-ITS), and chloroplast spacer sequences (rpoB and rpoC1) are regarded as potential genes for plant DNA barcoding. We amplified and sequenced these spacer sequences and confirmed the sequences with a BLAST search. Sequence alignment was performed using ClustalX to look for differences in the sequences. A DNA marker was developed based on rpoB and rpoC1 of the nrDNA-ITS for the identification of the adulterant E. dracunculoides in samples of R. graveolens that are sold in local herbal markets. Sequence-characterized amplified region markers of 289 and 264 bp for R. graveolens and 424 bp for E. dracunculoides were developed from dissimilar sequences of this nrDNA-ITS to speed up the authentication process. This marker successfully distinguished these species in extracted samples with as little as 5 ng DNA/μL extract.

Development of genetic markers for Ochradenus arabicus (Resedaceae), an endemic medicinal plant of Saudi Arabia

Some species of the genus Ochradenus are difficult to identify based on morphological markers. Similar limitations are found for biochemical markers. We developed genetic markers based on DNA sequences for Ochradenus arabicus, which is an endemic plant to Saudi Arabia, locally utilized as a medicinal shrub. The internal transcribed spacer sequence of nuclear ribosomal DNA and chloroplast (rpoB and rpoC1) markers were more informative than other chloroplast DNA markers. Based on these markers, we were able to discriminate this species from another species of the same genus (O. baccatus) that is widely distributed in Saudi Arabia, despite a high degree of morphological similarity. These genetic markers facilitate its identification, even when acquired in a dried state from local markets.

Inheritance of grain filling duration in spring wheat (Triticum aestivum L. em thell)

To understand the genetic control of grain filling duration (GFD), i.e., the number of days from anthesis to physiological maturity, we studied the F1, F2, BC1 and BC2 generations of six spring wheat crosses from nine varieties/genotypes. Generation mean analysis for gene effects indicated that one or more types of epistasis were significant in all crosses. In each pairing, the F1 and F2 means were either intermediate or closer to the mean of the parent having the longer GFD. Our narrow-sense heritability estimate was reasonably high, at 47.67 (based on diallel analysis). This demonstrated that progress could be made from the selection in these crosses for either long or short GFD. The two early varieties that had identical maturity durations differed in their GFD values, indicating that maturity dates are not good criteria when choosing parents for modifying GFD. To utilize favorable additive × additive effects during this selection, we suggest that a single seed descent (SSD) or bulk popula-tion approach be adopted. In comparison, dominance effects would prove quite useful in hybrid wheat breeding programs.

Molecular Identification of Sex in Phoenix dactylifera Using Inter Simple Sequence Repeat Markers

Early sex identification of Date Palm (Phoenix dactylifera L.) at seedling stage is an economically desirable objective, which will significantly increase the profits of seed based cultivation. The utilization of molecular markers at this stage for early and rapid identification of sex is important due to the lack of morphological markers. In this study, a total of two hundred Inter Simple Sequence Repeat (ISSR) primers were screened among male and female Date palm plants to identify putative sex-specific marker, out of which only two primers (IS_A02 and IS_A71) were found to be associated with sex. The primer IS_A02 produced a unique band of size 390 bp and was found clearly in all female plants, while it was absent in all male plants. Contrary to this, the primer IS_A71 produced a unique band of size 380 bp and was clearly found in all male plants, whereas it was absent in all the female plants. Subsequently, these specific fragments were excised, purified, and sequenced for the development of sequence specific markers further in future for the implementation on dioecious Date Palm for sex determination. These markers are efficient, highly reliable, and reproducible for sex identification at the early stage of seedling.

DNA fingerprinting for the authentication of Ruta graveolens

Ruta graveolens is a small aromatic shrub and has been used medicinally and magically, since ancient times. In this study, random amplified polymorphic DNA (RAPD) was employed to develop reproducible markers for authentication of this species from its adulterant Euphorbia dracunculoides . The random decamer oligonucleotide primers (42) were screened for identification of genuine and adulterant samples using the DNA isolated from the dried leaf, seed and stem of both samples. Out of 42 primers, 10 gave faint band, 12 gave species-specific reproducible unique band and the remaining did not amplify the DNA. RAPD could thus, serve as a complementary tool for quality control.