Inter-simple Sequence Repeat Analysis Research Articles

Molecular characterization of soybean (glycine max) accessions from the international collection of the plant gene resources of Canada: germplasm identification

ABSTRACT Soybean (Glycine max) is one of the most important crops in the world in terms of total production and usage. Evaluation of gene bank accessions such as the international collections of soybean (Glycine max) of the Plant Gene Resources of Canada has not been undertaken at the molecular level. The main objectives of the present study were to 1) determine the level of genetic variation and distance among G. max accessions from the international collections of the Plant Gene Resources of Canada and 2) develop and characterize accession-diagnostic inter simple sequence repeat (ISSR) and sequence-characterized amplified region (SCAR) markers that can be used in soybean molecular breeding programs. Genomic DNAs from 108 soybeans (G. max) accessions from 11 different countries were analyzed using ISSR primers. The levels of polymorphic loci detected were in general moderate varying from 29% to 52%. Overall, 82% of genetic distance values among accessions were above 0.40 based on ISSR analysis. This suggests that the genetic diversity among and within soybean accessions might not be as low as other studies have demonstrated. The soybean accessions from the Netherlands, China, France, and Sweden were the least genetically variable. The dendrograms constructed with ISSR data revealed that the South Korean accessions formed an out-group. The ISSR markers successfully distinguished soybean accessions within each group (country). In fact, several accession-diagnostic markers were identified in each of the 11 groups. They represent useful tools in soybean molecular breeding programs. One accession-diagnostic marker was cloned, sequenced, and converted to a SCAR marker.

Assessment of Genetic Diversity of Niger Plant (Guizotia abyssinica L.) in Moiben Sub County, Kenya, Using Inter Simple Sequence Repeat Markers

Niger plant (Guizotia abyssinica), exhibits phenotypic plasticity in different environments. There is need to assess its genetic diversity since guizotia species has a high number of species which may be confused amongst themselves. To achieve this, inter simple sequence repeat (ISSR) markers were used to estimate genetic diversity among 12 wild populations of Niger plant from Moiben sub-county. Total genomic DNA was extracted as per the cetyltrimethylammonium bromide (Ctab) method and subjected to ISSR analysis using 20 primers. None of the primers produced unique banding patterns. ISSR data were used to calculate a squared-euclidean distance matrix. All the twenty primers (100%) gave polymorphic bands thus they were all considered for further analysis. The allele frequency of all the primers was below 0.95 indicating that they were all polymorphic in character. Gene diversity was high ranging from 0.3550 to 0.7337 with a mean value of 0.6302. The ISSR based upgma clustering produced four clusters. Niger plant within Moiben sub-county was found to be genetically diverse though heterozygosity was not noticed. The study recommends further analysis of Niger plant so as to form a basis for further development of the plant.

Role of Biogenic Silver Nanoparticles on Hyperhydricity Reversion in Dianthus chinensis L. an In Vitro Model Culture

Hyperhydricity (HH) is one of the major problems in plant tissue culture. Increased frequency (75%) of HH in the cultures of. Dianthus chinensis L. cultivar telstar scarlet raised from nodal segments on agar (0.85%) gelled, benzyladenine (2.5 µM) supplemented Murashige and Skoog (MS) medium was noticed in the third subculture onwards. To reduce HH and to resume normal growth, the addition of silver nitrate is routinely recommended in the culture medium. However, the impact of biogenic silver nanoparticles (AgNPs) on HH reversion has not been well investigated. In this study, biogenic AgNPs prepared from leaf extract of D. chinensis were used to control HH in D. chinensis cultures. The characterization of bioynthesized AgNPs has been done by UV–Vis spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, energy-dispersive X-ray spectrum, transmission electron microscopy, and atomic force microscopy. Higher water content or addition of ethylene precursor, ethephon, in culture medium induced plantlet with unhealthy, pale green, glassy shoots (HH). Due to the biological activity of Ag+ ions and water regulating mechanism, AgNPs treatment on hyperhydric explant (4 weeks) resulted in high retroversion coupled with reduced relative water content. Supplementation of 100 µg L−1 AgNPs in MS medium significantly reduced the percentage of HH to 13.3%, in contrast to control (100%). Addition of AgNPs effectively reduced hydrogen peroxide (H2O2) content (50%) characterized by green, healthy shoots with proper stomata in contrast to hyperhydric shoots. The gene expression pattern of 1-aminocyclopropane-1-carboxylase synthase (ACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1) showed reduced expression after the retroversion of microshoots in 100 µg L−1 AgNPs medium compared to hyperhydric shoot. The relative gene expression profile of ACS1 and ACO1 at 100 µg L−1 AgNPs treatment was 9.8 and 1.8-fold over normal shoots (1), respectively. Inter simple sequence repeat (ISSR) analysis on AgNPs directed HH reverted shoots showed genetic stability thus proved safe to adopt this technique to produce true to type plants. Combined manipulation, coordination of water permeability, and anti-ethylene activity of AgNPs were responsible for effective HH reversion. These finding will put forward the future utilization of bioencapsulated AgNPs in plant tissue culture and agriculture practices.

Field Performance and Genetic Stability of Micropropagated Gooseberry Plants (Ribes grossularia L.)

Gooseberry (Ribes grossularia L.) is a small fruit crop producing valuable fruits, which is constantly gaining importance. In vitro propagation of this species can significantly support the production of virus-free planting material and accelerate the introduction of new cultivars to the market. The aim of presented study was to assess field performance and genetic stability of micropropagated plants (MPs) of four gooseberry cultivars, “Captivator”, “Hinnonmaki Rot”, “Invicta”, and “Resika”. The growth vigor and yield of MPs and plants propagated by standard methods from softwood cuttings (ST) were evaluated in a field experiment. Microscopic observations of the number and length of the stomata of MP and ST plants were carried out. Two DNA-based techniques, amplified fragment length polymorphism (AFLP) and inter simple sequence repeat (ISSR), were used to assess genetic stability of MP plants. For analysis of genetic stability of ST plants, the ISSR technique was applied. For three cultivars, Captivator, Hinnonmaki Rot, and Invicta, the plants’ growth vigor and fruit yield were greater in MP plants than in ST plants. In the case of Resika, most of these parameters were higher in ST plants. Microscopic observations of the stomata indicated a lack of differences in the length between MP and ST plants, while the stomata frequency on leaves of MP plants was higher than that of ST plants. The genetic variability of MP plants, assessed by AFLP, ranged from 0.35% for Hinnonmaki Rot to 2.12% for Resika. The results of ISSR analysis of MP plants showed variability from 0% in the case of Hinnonmaki Rot and Resika to 4% and 8.69% for Captivator and Invicta, respectively. No polymorphism was detected among ST plants of all analyzed gooseberry cultivars.

Genetic assessment of the effects of self-fertilization in a Lilium L. hybrids using molecular cytogenetic methods (FISH and ISSR).

Self-fertilization (also termed selfing) is a mode of reproduction that occurs in hermaphrodites and has evolved several times in various plant and animal species. A transition from outbreeding to selfing in hermaphroditic flowers is typically associated with changes in flower morphology and functionality. This study aimed to identify genetic effects of selfing in the F2 progeny of F1 hybrid developed by crossing Lilium lancifolium with the Asiatic Lilium hybrid ‘Dreamland.’ Fluorescence in situ hybridization (FISH) and inter-simple sequence repeats (ISSR) techniques were used to detect genetic variations in plants produced by selfing. The FISH results showed that F1 hybrid were similar to the female parent (L. lancifolium) regarding the 45S loci, but F2 individuals showed variation in the number and location of the respective loci. In F2 progeny, F2-2, F2-3, F2-4, F2-5, and F2-8 hybrids expressed two strong and one weak 5S signal on chromosome 3, whereas F2-7 and F2-9 individuals expressed one strong and two weak signals. Only two strong 5S signals were detected in an F2-1 plant. The ISSR results showed a maximum similarity value of 0.6269 between the female parent and the F2-2 hybrid. Regarding similarity to the male parent, a maximum value of 0.6119 was found in the F2-1 and F2-2 hybrids. The highest genetic distance from L. lancifolium and the Asiatic Lilium hybrid ‘Dreamland’ was observed in the F2-4 progeny (0.6352 and 0.7547, respectively). Phylogenetic relationships showed that the F2 progeny were closer to the male parent than to the female parent. Self-fertilization showed effects on variation among the F2 progeny, and effects on the genome were confirmed using FISH and ISSR analyses.

Multifaceted Analysis of Barley Landraces Collected during Gene Bank Expeditions in Poland at the End of the 20th Century

The extensive genetic diversity of crop landraces deposited in gene banks is an important part of future breeding programs. However, in order to exploit this potential, it is necessary both to preserve germplasm and to collect and share a wide range of data on its characteristics and evaluation. The main objective of the presented paper was to evaluate the diversity of spring barley landraces originating from Poland. Historical data from standard field evaluation of agronomic traits, genetic data obtained from Inter Simple Sequence Repeat (ISSR) analysis and grain morphometric data were used. The analysis showed that groups of hulled and hulls barley accessions were substantially separate. Genetic and grain morphological variation did not show any relationship with eco-geographical conditions in the place of origin. There was also no clear division into forms with two- and six-row ears. The combination of the results of various types of analyzes describing this collection makes us believe that its chances of being used in research and breeding will increase.

High Efficiency In vitro Regeneration and Genetic Stability of Corallocarpus epigaeus - An Endangered Medicinal Plant

Indirect regeneration of plantlets from multiple shoot induction of Corallocarpus epigaeus was obtained from leaf and nodal explants on MS with different concentrations of BAP in combination with IAA/IBA or IBA alone. Among all the combinations, BAP and IBA exhibited maximum regeneration. High frequency of multiple shoots (89%) was obtained on BAP (2.0 mg/l) and IBA (1.5 mg/l) in nodal explants. Maximum mean shoot length of 6.8 ± 0.33 cm was obtained in nodal explants cultured on BAP (1.0 mg/l) + IBA (0.5 mg/l), followed by leaf explants with 6.7 ± 0.47 cm on BAP (3.0 mg/l) + IAA (2.5 mg/l). The highest frequency of rooting (88.3%) was obtained on NAA (1.0 mg/l) and IBA (2.0 mg/l) with 21.83 ± 0.57 mean number of roots. The well-rooted healthy plantlets were acclimatized with a survival rate of 80%. Inter simple sequence repeat (ISSR) analysis revealed the genetic similarity of in vitro raised plants with the mother plant.
 Plant Tissue Cult. & Biotech. 30(2): 219-229, 2020 (December)

Ex vitro Rooting, Acclimatization and Genetic Stability of Lonicera caerulea var. kamtschatica

Abstract Ex vitro rooting and acclimatization of two cultivars ‘Wojtek’ and ‘Zojka’ of blue honeysuckle (Lonicera caerulea var. kamtschatica Sevast.) were studied. To the ex vitro conditions were transferred rooted and unrooted shoots. The post-effect of auxin type and concentration as well as microcutting and soil substrate types were tested. The genetic stability of the plantlets in relation to the mother plants by using amplified fragment length polymorphism (AFLP) and inter simple sequence repeat (ISSR) markers has been also determined. It has been found that in vitro rooted cuttings of both cultivars showed a higher survival rate (max. 88%) and better growth and development when they were rooted on a medium containing a low auxin level (1.0 mg·dm−3). The results of the second experiment showed successful ex vitro rooting of blue honeysuckle shoots without auxin treatment. Higher ex vitro rooting and survival rate in the greenhouse have been observed for ‘Wojtek’ (max. 96%) than ‘Zojka’ (max. 88%). Better growth and development of shoots and roots were observed on peat alone or a mixture of peat and perlite as compared to a mixture of peat and sand. The micropropagated plantlets appeared similar to mother plants. Molecular analysis confirmed a high level of genetic stability of blue honeysuckle after 2 years of in vitro propagation. However, among the cultivars studied, ‘Wojtek’ showed slightly higher genetic stability than ‘Zojka’ (99.5% and 97.7%, respectively). For ‘Zojka’ plants, the degree of variation was comparable for AFLP and ISSR markers. For ‘Wojtek’, no polymorphism was detected using the ISSR analysis in contrast to the AFLP analysis.

Genetic Stability of the Endangered Species Salix lapponum L. Regenerated In Vitro during the Reintroduction Process.

Simple SummarySalix lapponum, a downy willow, is a boreal relict species, threatened with extinction in Poland. In order to save populations in their refugia on the southern limit of the specie’s range, some activities were undertaken to rebuild their resources. The in vitro propagation was chosen to produce new plants, as it allows obtaining many individuals in a relatively short time with no harm to natural populations. The collected shoot pieces were multiplicated on a special growing media, containing all the necessary macro- and micronutrients, with addition of plant growth regulators to make them form shoots and roots. The obtained plants were then planted into soil and acclimated to natural habitat conditions. On the basis of the conducted genetic analysis and flow cytometry, it was stated that the new plants were genetically unchanged in comparison to the mother plants. The research results confirmed that the tissue culture may be applied in the propagation of the endangered willow species and the obtained plants may be used to build new populations or to strengthen the present ones. Salix lapponum L. is a boreal relict species, threatened with extinction in Poland. An 80% decrease in the number of its stands was confirmed in the last half-century, so that to prevent the loss of downy willow, attempts were made to reintroduce this species in natural habitats. Micropropagation was chosen as a first stage of its active conservation. S. lapponum shoots were collected and disinfected with NaOCl, AgNO3, or HgCl2 or with a two-step disinfection with NaOCl and then placed on MS medium with BA 1 mg·dm−3 and IBA 0.1 mg·dm−3. Regenerated shoots were cultivated with addition of BA, KIN, or 2iP, alone or in combination with auxins, to find the highest multiplication rate. Inter-simple sequence repeat (ISSR) analysis and flow cytometric analyses were conducted on in vitro regenerated plants to check their genetic stability. Disinfection was quite difficult and the use of HgCl2 was the most efficient. The highest multiplication rate was obtained in presence of KIN at 0.5 mg·dm−3 + IAA at 0.5 mg·dm−3. The analysis confirmed the genome size stability, which is in agreement with the results obtained by ISSR, revealing no somaclonal variation in plantlets and therefore allowing the use of the obtained plants for reintroduction.

English

Girardinia diversifolia (Link) Friis is a perennial herb commonly known as Himalayan giant nettle which belongs to the family Urticaceae. The plant has cultural, medicinal and economic importance. The plant contains high concentration of polysaccharides, polyphenols and secondary metabolites which obstructs the process of isolation of the Deoxyribonucleic Acid (DNA) and inhibits downstream Polymerase Chain Reaction (PCR) amplifications. This study protocol developed a DNA extraction protocol from leaf-tissue based on the Cetyltrimethylammonium bromide, and optimized the PCR protocol for Inter Simple Sequence Repeat (ISSR) analysis. Genomic DNA extraction process was conducted using modified Doyle and Doyle method to obtain good quality DNA. The method yielded 445 ng/µL of DNA, where the purity ranged from 1.8-2.0 indicating minimum contamination of metabolites. The optimum condition for ISSR analysis was established using 4 mM MgCl2 , 0.6 mM dNTPs, 2.0 U Taq polymerase, 50 ng template DNA, and 0.7 µM primer. PCR program was optimized in the sequence of denaturation at 94°C for 3 min, subsequently followed by 45 cycles at 94°C for 30 s, annealing temperature at 45°C for 30 s, extension at 72°C for 2 min, and final extension at 72°C for 10 min. The modified technique was found to be ideal for isolation of genomic DNA and optimization of PCR process for ISSR analysis of G. diversifolia. The results of the research are beneficial for future molecular characterization and genetic diversity analysis of allied taxa. Key words: Girardinia diversifolia, Himalayan nettle, DNA isolation.

Regeneration of Viburnum dentatum L. from Alginate-Encapsulated Shoot Explants after Short-Term Cold Storage and Assessment of Genetic Stability Using ISSR Analysis

The present study demonstrates an efficient protocol for alginate encapsulation, interim cold storing of artificial seeds and conversion to genetically stable plants of Viburnum dentatum L. “Lucidum Aiton”. Explants of shoot tips and first-node segments, excised from in vitro-derived viburnum microshoots, were encapsulated in 2.5% sodium alginate mixed with liquid MS nutrient medium and hardened in 50 mM of calcium chloride producing solid, soft and uniform beads. These artificial seeds achieved 28.9% germination under light, forming 4.3 microshoots per bead. However, with 100 mM of calcium chloride for hardening, the beads were firm and of a uniform globular shape and suitable for handling and exhibited a germination response of 48.9%. Encapsulated shoot tip explants of viburnum, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in regeneration response (73.3, 62.2, 51.1%, respectively), while non-encapsulated explants, stored under same conditions, did not survive after the fourth week of cold storage. Microshoots from cold-stored encapsulated explants, which were rooted in solid MS nutrient medium with 0.5 μΜ of Indole-3-acetic acid (IAA) and transplanted to a substrate of peat-perlite (3:1, v/v), acclimatized successfully after application of 75 or 50% shading, which was gradually reduced, and were established with minimum losses in a greenhouse. For the genetic stability of the artificial seed-derived plantlets and compared with the mother plant, an assessment was conducted using Inter Simple Sequence Repeats (ISSRs) analysis. The ISSR profiles proved the genetic uniformity and clonal stability of the regenerated plantlets and their genetic resemblance to the mother plant. The present regeneration procedure could be used as an alternative method for the micropropagation of V. dentatum.

Assessment of Inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers to reveal genetic diversity among Tamarix ecotypes

Tamarix is an important genus of Tamarieaceae family, known for its medicinal importance. Its taxonomy is tricky, and a few molecular makers are available for species identification. The present study was designed to evaluate the genetic diversity among 21 Tamarix specimens collected from Thal desert, Punjab, Pakistan. Ten Inter-Simple Sequence Repeats (ISSR) and six Simple Sequence Repeats (SSR) markers were used. Results revealed that ISSR markers produced 131 bands, of which 116 were polymorphic (88.5%) with a mean Polymorphic Information Content (PIC) value of 0.34. The SSR markers yielded 30 amplified bands, of which 24 were polymorphic (80.28%) with mean PIC value of 0.35. Cluster and factorial analysis revealed a high level of genetic variability among tested Tamarix ecotypes. Dissimilarity index indicated a genetic variability among tested specimens ranging from 0.00 to 0.77. The ISSR analysis reflected that Tamarix specimens stemming from Kalurkot and Bhakkar region revealed the highest dissimilarity level with respect to the rest of the specimens.

Genetic Diversity Assessment of a Chinese Medicinal Endemic Species, Aspidopterys obcordata var. obcordata, by Combined Molecular Marker Methods (ISSR & SRAP).

Aspidopterys obcordata var. obcordata, a medicinal plant endemic to China, is a narrowly distributed species and wild resources are extremely limited. To evaluate the genetic variability and degree of genetic divergence of A. obcordata var. obcordata, and to make rational scientific decisions on its harvest and germplasm conservation, we collected 122 samples from across nearly all of its distribution area and studied genetic diversity using inter-simple sequence repeats (ISSRs), sequence-related amplified polymorphisms (SRAPs), and a method combining the two techniques. The results revealed the high genetic diversity of A. obcordata var. obcordata, mainly due to its intra-population diversity, and the top two populations with the highest levels of intra-population diversity were ML and DH, individuals of which can serve as excellent germplasm candidates during the processing of germplasm screening and conservation. In general, the combining method was prior to the ISSR analyses and SRAP analyses results, except for a slight difference in the genetic structure of individual populations. Therefore, we suggest that a combination analysis of the two marker methods is ideal for evaluating the genetic diversity and genetic relationships of A. obcordata var. obcordata.

Generation of novelties in the genus Ocimum as a result of natural hybridization: A morphological, genetical and chemical appraisal

The genus Ocimum is a boutique of a diverse set of specialized metabolites such as terpenoids and phenylpropanoids. Each Ocimum species and its cultivars represent a characteristic chemical profile. The present study explored the two interspecific Ocimum hybrids originating through a serendipitous natural cross between O. kilimandscharicum and O. basilicum. These two novel Ocimum hybrids exhibited intermediate morphological features of two parental species. Inter simple sequence repeats (ISSR) analysis and DNA barcoding with the plastid non-coding trnH-psbA intergenic spacer region reaffirmed unambiguous parental identification and differentiation of these natural hybrids from other available Ocimum species. Consequently, gas chromatography-mass spectrometry-based metabolite profiling of two hybrids identified them as specific chemotypes with the presence of a unique blend of specialized metabolites from the parental species, which are either rich in terpenes or phenylpropanoids. Additionally, expression analysis of key genes from terpenoid and phenylpropanoid pathways corroborated with differential metabolite accumulation. Thus, these two Ocimum hybrids represented the novel chemotypes, which could be useful in commercial cultivation to produce novel essential oil and bioactive constituents. Further, the resulting metabolite diversity could have potential industrial applications in the areas of healthcare, bioremediation, and crop protection.

Bazı Soya Fasulyesi [Glycine Max (L.) Merr.] Genotipleri Arasında Agronomik Özelliklerin ve ISSR Yönteminin Karşılaştırmalı Analizi

In this study, the genetic diversity was investigated among 12 soybeans genotypes using inter simple sequence repeats (ISSR) and agronomic traits. DNA was isolated from the leaves of the genotypes. For molecular characterization, a total of 26 primers of ISSRs and eight agronomic characteristics were evaluated. ISSR analysis revealed 88 polymorphic bands. The genetic diversity among the genotypes according to ISSR analysis and agronomic traits were estimated based on Nei homology and Euclidian distance, respectively, and dendrograms reflecting genetic similarity were constructed using UPGMA and NTSYSpc, respectively. Nei’s homology coefficient values used for ISSR analysis ranged from 78%-84%, and the average Euclidean distance used for agronomic data ranged from 1.96-9.77. Although soybean genotypes evaluated in this study were highly similar, dendrograms showed that these genotypes could be distinguished both morphologically and genetically.

Evaluation of the Genetic Fidelity of <i>In-Vitro</i> Raised Plants of <i>Nardostachys jatamansi</i> (D.Don) dc. (Valerianaceae) using ISSR Markers

Genetic fidelity analysis of in vitro established N. jatamansi plants has been carried out. Inter Simple Sequence Repeats (ISSR) marker was used to assess the genetic fidelity of tissue culture products with the mother plant. Among 17 ISSR primers screened, nine produced clear, reproducible and scorable bands. A total of 26 distinct bands in the size range of 200 to 2900 bp were produced. Most of the banding profiles from micropropagated plants were monomorphic and similar to those of the control plant, however few samples produced polymorphic bands also. The percentage of polymorphic bands in the ISSR analysis was 11.53%. The genetic similarity was calculated on the basis of ISSR data among the selected plant materials which ranged from 0.93 to 1.00 (mean = 0.965). The similarity coefficient by ISSR analysis revealed that differences between tissue culture raised plants and mother plant was not remarkable, thus ascertaining the true nature of the in vitro raised plants. This study is likely to suggest an alternate method for large scale production carried forward with an in situ participatory approach which will thereby enhance the germplasm conservation. In future, with this approach N. jatamansi is likely to become a potent ethnomedicine in the pharma industry.

Morphological, cytological and molecular variations induced by gamma rays in Chrysanthemum morifolium ‘Donglinruixue’

Abstract The current study investigated the effects of gamma radiation on the death rate, morphological traits and meiotic abnormalities in ground-grown chrysanthemum ‘Donglinruixue’, and inter-simple sequence repeat (ISSR) markers were used to identify the DNA polymorphism among mutants. The results showed that the death rate significantly increased with increase in radiation dose. Semi-lethal (LD50) dose was approximately 35 Gy. Compared with unirradiated control, plant growth was significantly inhibited. After irradiation, a series of morphological variations and cytological aberrations occurred in radiated plants. The peak in variation frequency appeared at 35 Gy. In total, ISSR analysis produced 72 scorable bands, of which 64 (88.89%) were polymorphic. The current study demonstrated that gamma irradiation generates a sufficient number of induced mutations and that ISSR analysis offered a useful molecular marker analysis for the identification of mutants.

Morphologic and genetic analysis for geographic populations of greenbug Schizaphis graminum (Hemiptera: Aphididae) in Egypt

Greenbug Schizaphis graminum is one of widespread aphid species in most wheat growing regions of Egypt that causes severe damages to crops. Studying morphological and genetic variations within insect pest populations are essential for identifying species and population distinctions and subsequently developing integrated pest management. Sixteen geographic populations of S. graminum were sampled from wheat fields in Egypt. Specimens were characterized by morphological screening for 19 quantitative morphological characters, examination of genomic DNA polymorphism with 11 inter simple sequence repeats (ISSR) primers, and measuring melting temperature (Tm) for cDNA of the mitochondrial cytochrome oxidase subunit I (COI) gene. The greatest morphological variance was observed among aphid populations from Domiata, El-Menia, and Beni-Suif. Geographic location of sampled populations had significant effect on melting temperature of the COI gene. The highest value was observed in populations from Assuit and the lowest from El-Gharbia. Populations from El-Monofia and Kafer El-Shikh were most similar based a combination of COI melting temperatures and quantitative morphological features. Tested ISSR primers showed a polymorphism rate of 49.43% among geotypes. The UBC814 primer recognized a population-specific band of 950 bp that characterized the Domiata population. Moreover, UBC811 and UBC868 primers generated bands with molecular sizes 440 and 340 bp characterizing only populations of Lower and Upper Egypt, respectively. ISSR analysis revealed that geographic populations from El-Behera, Domiata, and Kafer El-Shikh are genetically homologous with identities of 90.4–90.7%. In contrast, populations of Luxor and El-Behera are genetically analogous. Finally, a molecular branching key was constructed to discriminate among tested populations.

Discrimination of common Iris species from Egypt based on their genetic and metabolic profiling.

Irises have been medicinally used in Ancient Egyptians, Anatolian, Chinese, British and Irish folk medicine. They are also well-known ornamental plants that have economic value in the perfume industry. The main obvious diagnostic difference between the different species is based on the morphology of the flowers. The flowering cycle is very short as well as the persistence of the fully opened flowers extends for a few days only. Moreover, the climatic conditions significantly causes fluctuation in their blooming time from year to year. This makes the morphological discrimination very difficult. The discrimination of different iris species is of a great importance, as each species is reported to possess different folk medicinal activities. Finding genetic and metabolic markers for differentiation between Iris confusa Sealy (Subgen. Limniris Sect. Lophiris), I. pseudacorus L. (Subgen. Limniris Sect. Limniris) and I. germanica L. (Subgen. Iris Sect. Iris) on levels other than traditional taxonomic features. Inter-simple sequence repeat (ISSR) and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The highest similarity was found between I. pseudacorus L. and I. germanica L. and the least similarity was between I. confusa Sealy and I. pseudacorus L. The metabolic profiling of the leaves confirmed genetic profiling discriminating I. confusa from the other two species. The primary metabolites of the underground parts showed clear discrimination between the three species. This study represents the sole complete map for distinguishing the three Iris species on genetic and metabolic bases.

Applicability of inter-simple sequence repeat (ISSR), start codon targeted (SCoT) markers and ITS2 gene sequencing for genetic diversity assessment in Moringa oleifera Lam

Moringa oliefera Lam. is the most widely cultivated species among the members of Moringa genus in tropics for its nutritional and medicinal values. Almost all plant parts have been used in human's nutrition and therapy. The molecular characterizations are required for effective improvement for better utilization. In the present study, genetic variation in ten individual Moringa oleifera plants were investigated using ten primers of each inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) markers. In current study, SCoT is used for the first time to assess the genetic diversity in moringa. The results of ISSR and SCoT analysis for the ten local M. oleifera revealed approximately 91 and 108 different banding patterns, 73 and 87 of them consider as monomorphic bands and other 18 and 21 fragments consider as polymorphic bands with a percentage about 19.7 and 19.4%, respectively. Low levels of polymorphism were observed between ISSR and SCoT which means that they are useful tools for genetic similarity detection in M. oleifera species. Sequence identity of M. oleifera plants in relation to NCBI data base were 96, 100, 99 and 99 with ITS2, matK, psbA and rbcL genes, respectively, with lower E value (≤1e). Moreover, the phylogenetic Neighbor-joining (NJ) tree was constructed using rbcL, matK, psbA and ITS gene sequences. M. oleifera clustered into two clades with 99% similarity. The results suggest that the neighbor-joining phylogeny tree could effectively identify M. oleifera from its adulterants, including its closely related species. M. oleifera showed a sister relationship to M. oleifera MG548808.1 and M. oleifera KT737756.1. The results confirmed the usefulness of molecular techniques such ISSR, SCoT and ITS barcoding markers to assess the genetic diversity among the selected M. oleifera types for genetic conservation and plant improvement.