Inter-simple Sequence Repeat Marker Analysis Research Articles

Study of Genetic Diversity Among Crataegus Species (Hawthorn) Using ISSR Markers in Northwestern of Iran

Background: Crataegus spp. (Hawthorn) are used for horticultural and medicinal purposes.
 Objectives: In the present study, the genetic relationships among the five Iranian Crataegus species were evaluated by Inter Simple Sequence Repeats (ISSR) molecular marker
 Methods: PCR reactions were performed using six primers (811, 827, 834, 845, 856, and 868) and genetic similarity was calculated based on Jacquard’s similarity coefficient.
 Results: Six primers generated 79 products in total, of which 71 were polymorphic (89.9%), with an average of 13.1 bands per primer. The percentage of polymorphic bands ranged from 77 to 100. Primer 856 produced the highest number of bands, while the lowest was generated by primer 845. The Jacquard’s similarity coefficient, derived from ISSR marker analysis, ranged from 0.164 to 0.337, indicating high genetic variation among Crataegus species in Iran.
 Conclusion: This study provides important data for identifying species relationships and helps develop plant breeding strategies to improve the medicinal properties of this genus in the future.

The utility of ISSRs for the identification of interspecific hybrids between pearl millet (Pennisetum glaucum [L.] R.Br.) × napier grass (Pennisetum purpureum Schumach)

AbstractInterspecific hybrids between pearl millet (Pennisetum glaucum) and napier grass (Pennisetum purpureum) give rise to perennial fodder crops characterized by high biomass, broad clumps and good palatability. These hybrids are triploid and developed by hand pollination of napier grass pollen on pearl millet panicles. The progeny shows a high percentage of pearl millet genotype due to self-pollination in the female parent. Identification of hybrids at a young stage based on morphological characters is difficult. DNA-based molecular markers have high discriminating power and were used to assess genetic differences between hybrids and their parents. Genetic diversity was studied in 18 pearl millet × napier grass hybrids along with their parents and two released national checks using inter simple sequence repeat (ISSR) markers. Eight ISSR primers gave rise to 125 bands, of which 120 bands were polymorphic. Polymorphic information content and ISSR primer index ranged from 0.40 to 0.49 and 8.88 to 11.14, respectively. The hybrids showed the presence of unique bands, besides those shared with male and female parents. Female (pearl millet) parents formed a separate group in the dendrogram constructed based on ISSR polymorphism. The male (napier grass) parents formed a separate group along with hybrids, indicating a higher similarity of hybrids with the male parents. Principal component analysis and STRUCTURE analyses showed a similar grouping. The close resemblance of hybrids to the male parents confirmed their interspecific origin. The study revealed that ISSR marker analysis could be a quick and reliable method to identify interspecific hybrids at an early stage of growth.

Genetic variations and dog breed identification using inter-simple sequence repeat markers coupled with high resolution melting analysis.

The identification of differing physical characteristics of dogs is an uncomplicated and straightforward way to categorize dog breeds. However, many dog owners and veterinarians still struggle to distinguish between pure breed and mixed variations in certain breeds of dogs. Presently, the absence of the tools and methods needed to confirm a pure breed dog is a significant problem since the only method available to validate pure or mongrel breeds is the official pedigree system. Inter-simple sequence repeat markers have been successfully used to assess genetic variations and differentiations. Notably, inter-simple sequence repeat markers coupled with high resolution melting analysis were effectively used for the breed identification of 43 breeds of dogs (total 463 dogs). The 10 primers chosen for analysis resulted in a range of 31–78.6% of breed discrimination when using one primer, while a combination of two primers was able to successfully discriminate between all of the 43 dog breeds (100%). Shannon’s index information (I = 2.586 ± 0.034) and expected heterozygosity (He = 0.908 ± 0.003) indicated a high level of genetic diversity among breeds. The fixation index (Fst) revealed a value of 10.4%, demonstrating that there was a high level of genetic subdivision between populations. This study showed that inter-simple sequence repeat marker analysis was effective in demonstrating high genetic diversity among varying breeds of dogs, while a combination of Inter-simple sequence repeat marker analysis and high resolution melting analysis could provide an optional technique for researchers to effectively identify breeds through genetic variations.

Molecular genetic homogeneity assessment of micropropagated Dendrobium moschatum Sw. - A rare medicinal orchid, using RAPD and ISSR markers

Generation of true-to-type plant is one of the most important aspects in germplasm conservation through micropropagation. In this study, reliable in vitro protocols were established for the generation of genetically stable plants of Dendrobium moschatum using seed explants via protocorm formation. Thidiazuron (TDZ) and 6-benzyl aminopurine (BAP) induced effective shooting when incorporated individually or in combination with auxins in the medium. High shooting response was observed in Mitra medium either with 2.4 mgL−1BAP or 1.2 mgL−1TDZ and 1.2 mgL−11-naphthyl acetic acid (NAA). Indole-3-butyric acid (IBA) or NAA produced better root multiplication in the culture than indole -3- acetic acid (IAA). Medium with 1.2 mg L−1 TDZ + 1.2 mgL−1 IBA or 1.2 mg L−1 TDZ + 1.2 mg L−1 NAA generated a good rooting response. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers analysis showed 95.2% and 98% genetic monomorphism respectively among in vitro regenerants and the mother plant. Nei’s genetic distance ranged from 0.00 to 0.044 disclosing high genetic similarity among the regenerants. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) based dendrograms generated from RAPD and ISSR marker analysis also revealed high genetic proximity between mother and micropropagated plants. Principal Coordinate Analysis (PCoA) further confirmed the grouping of plants in accordance with UPGMA dendrograms. Correlation analysis between the matrices of markers revealed higher efficiency of ISSR over RAPD markers. This is the first report of genetic homogeneity testing of in vitro propagated Dendrobium moschatum using molecular markers.

Molecular markers based genetic fidelity assessment of micropropagated Dendrobium chrysotoxum Lindl

Plant tissue culture is one of the most important methods used for effective conservation of many rare and endangered orchids. Genetic variation may appear in regenerants due to numerous factors associated with in vitro culture conditions. Assessment of genetic stability of in vitro regenerants is highly essential if true to type plants are the desired end product. In the present investigation, genetic homogeneity testing of micropropagated Dendrobium chrysotoxum was performed using 12 random amplified polymorphic DNA (RAPD) and 11 inter-simple sequence repeat (ISSR) primers. Molecular marker analysis unveiled high genetic monomorphism (96.30%) and low genetic polymorphism (3.6%) among the in vitro clones and mother plant. Nei's genetic distance ranged from 0.00 to 0.037 disclosing high genetic similarity between the clones. Dendrograms generated from RAPD and ISSR marker analysis also revealed high genetic proximity with mother plant and micropropagated clones. Principal coordinate analysis (PCoA) further confirmed the grouping of plants in accordance with the cluster analysis determined by unweighted pair group method using arithmetic averages (UPGMA) analysis. The study reported the successful clonal fidelity assessment of micropropagated D. chrysotoxum using RAPD and ISSR markers.

Determination of the effects of two feed supplements on Drosophila melanogaster

Pleurotus spp. are considered extremely rich mushroom species from the nutritional pointof view, providing immune-enhancement effects when consumed. However, few mushroomshave been tested for their phenotypic and genotypic responses in animal modelsto ensure the proper dosage for their use. This study aimed to evaluate the effects of twomushroom species on the reproductive capacity of Drosophila melanogaster . Pleurotuscitrinopileatus Singer and Lentinus sajor-caju (Fr.) Fr. were provided separately, in specificconcentrations, as feed supplements to the fruit flies. The total numbers of developedlarvae, pupae, and adults were then measured for each treatment. Inter simple sequencerepeats marker analysis was carried out to infer genotypic changes in the mushroom-fedflies. Our results suggest that Pleurotus spp. can cause positive changes to the flies’ reproductivecapacity, since Pleurotus citrinopileatus in particular accelerated the life cycle andrevealed a higher genetic dissimilarity of the diet-supplemented flies. Keywords: fruitflies, reproductive fitness, edible mushrooms.

Evaluation of genetic relationship between 15 bamboo species of North-East India based on ISSR marker analysis.

The classification of bamboos based on floral morphology and reproductive characters is very hard due to erratic flowering behavior and unusually long reproductive cycle. The application of reliable and effective DNA molecular markers is highly essential to address this problem. In the present investigation, inter-simple sequence repeats (ISSR) markers were employed to study phylogenetic relationship of 15 different bamboos of North-East India. The 10 ISSR primers generated 116 polymorphic loci or scorable bands with average of 11.6 bands per primer. The genetic similarity coefficient ranged from 0.41-0.76 showing high genetic polymorphism among different bamboo species. The phylogenetic tree constructed based on genetic similarity matrix revealed genetic proximity of endemic Bambusa mizorameana to other five Dendrocalamus species by clustering into a single group, while Dendrocalamus manipureanus segregated from the cluster indicating its genetic divergent character. Except for Schizotachyum fuchsianum, the three Schizotachyum species viz., S. dullooa, S. pergracile and S. munroi exhibited close genetic affinity by grouping into a single minor clade. Principal coordinates analysis (PCoA) showed distribution of different bamboos species in the plot in accordance to unweighted pair-group method with arithmetic average (UPGMA) cluster analysis. Genetic relationship of 15 different bamboos as revealed from the dendrogram and PCoA analysis reasonably conformed to traditional system of classification though slight disagreement existed. This is the first report on the successful use of ISSR markers in the phylogenetic and genetic variation studies of 15 important bamboos of the region including the endemic bamboo species of B. mizorameana, B. manipureana, D. sikkimensis and D. manipureanus.

Genetic Diversity and Variation in Captive Asian Elephants (Elephas maximus) in Thailand

Numbers of wild Asian elephants ( Elephas maximus) have been decreasing gradually throughout Asia due primarily to human activities, such as poaching, and habitat encroachment and destruction that lead to human–elephant conflict. Sustainability problems exist in captive populations as well, where morbidity and mortality rates are high and reproduction is low. Determining the genetic diversity of these populations is essential for conservation and sustainable utilization efforts. Intersimple sequence repeat markers were used to assess the genetic variation and differentiation in 97 captive Asian elephants from seven elephant camps in Chiang Mai, Thailand. The nine primers chosen for the analysis revealed 88 bands in male and 115 bands in female elephants, of which 37 (42.05%) and 83 (63.64%) were polymorphic, respectively. Shannon’s index information ( I = 2.415 ± 0.054) and expected heterozygosity (He = 0.892 ± 0.008) indicated high species-level genetic diversity. The fixation index ( Fst) was −0.130 ± 0.016, demonstrating there was no genetic subdivision between populations. A cluster analysis was performed using Unweight Pair-Group Method with Arithmetic Mean and dendrograms, which illustrated genetic relationships among captive Asian elephants that included 2 main clusters across the seven camps and 27 clusters for the 97 individual elephants. This high variability may be due to the different origins of these individuals, including originating from other Asian countries. Thus, this study showed that intersimple sequence repeat marker analysis was effective in demonstrating high genetic diversity among captive Asian elephants in Chiang Mai province and found cluster differences that could be used to guide breeding management to decrease the risk of inbreeding among Asian elephant groups.

Mutagenesis in olive (Olea europaea L.) calli caused by sodium azide and detection of mutants using ISSR and RAPD markers

SummaryIn vitro callus formation was established in the olive (Olea europaea L.) cultivar ‘Kronaki’ and in vitro mutagenesis of calli was carried out using five different concentrations (1.0 – 5.0 mM) of sodium azide (NaN3). Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to detect genetic polymorphism among the olive mutants produced. Six RAPD and seven ISSR primers were used to amplify DNA segments from the genomes of untreated and NaN3-treated calli. The optimum medium for callus formation was 0.5× Murashige and Skoog (MS) medium, supplemented with 2 mg l–1 αnaphthaleneacetic acid and 0.5 mg l–1 2-isopentenyl-adenine. A total 60 RAPD amplicons and 60 ISSR fragments were detected. The sizes of the PCR amplicons varied from 200 – 3,000 bp in length. Fifteen of the 60 RAPD (40%) and ten of the 60 ISSR (16.6%) fragments were polymorphic. The results showed that 4.0 mM and 5.0 mM NaN3 were the most effective doses for mutagenesis. Based on RAPD, ISSR, and combined RAPD and ISSR marker analysis, it was shown that all NaN3 treatments caused detectable levels of mutation. The use of NaN3 for in vitro mutagenesis in olive calli resulted in genetic dissimilarity values of 0.14 – 0.21, and represents a useful way to induce mutants in olive which could be exploited in the future for trait improvement and in olive breeding programmes.

Genetic diversity of Salix koreensis based on inter-simple sequence repeat (ISSR) in South Korea

Background Salix koreensisis native to Korea, Japan and China, is a deciduous broadleaved tall tree growing throughout Korea. This species is dioecius and planted along the streets and for landscaping. Along the rivers the species grows well reaching up to 20 meters in height. Trees bloom in April and fruits ripe in May. This study was carried out to investigate the genetic diversity of S. koreensis within and among 10 populations throughout South Korea using ISSR marker analysis.

Genetic diversity analysis of Monascus strains using SRAP and ISSR markers

In this study, sequence-related amplification polymorphism (SRAP) and inter-simple sequence repeat (ISSR) were analyzed for accessing the genetic diversity of 37 Monascus isolates and 14 control strains. According to the dendrogram produced by SRAP data, all the tested strains were grouped into four clusters at a 78% similarity level. Comparatively, 51 tested strains were divided into four major groups at a similarity level of 74% based on the dendrogram generated via ISSR marker analysis. Based on the two sets of dendrograms, Monascus aurantiacus, M. purpureus, M. serorubescens, M. anka, and M. ruber were clustered in the same clade; M. albidus, M. fuliginosus, and M. barkeri were clustered with M. pilosus in a second clade; and M. lunisporas and M. argentinensis occurred together in a third cluster distinct from the other Monascus species. The cluster result produced by SRAP data shared great similarity with that by ISSR data with minor differences in the subgroups, which is basically in agreement with morphological observations. In general, SRAP and ISSR are more simple, rapid, and efficient, which may provide alternative molecular approaches to studying genetic diversity, classification, and identification of Monascus strains.

Genetic diversity analysis of <i>Monascus</i> strains using SRAP and ISSR markers

In this study, sequence-related amplification polymorphism (SRAP) and inter-simple sequence repeat (ISSR) were analyzed for accessing the genetic diversity of 37 Monascus isolates and 14 control strains. According to the dendrogram produced by SRAP data, all the tested strains were grouped into four clusters at a 78% similarity level. Comparatively, 51 tested strains were divided into four major groups at a similarity level of 74% based on the dendrogram generated via ISSR marker analysis. Based on the two sets of dendrograms, Monascus aurantiacus, M. purpureus, M. serorubescens, M. anka, and M. ruber were clustered in the same clade; M. albidus, M. fuliginosus, and M. barkeri were clustered with M. pilosus in a second clade; and M. lunisporas and M. argentinensis occurred together in a third cluster distinct from the other Monascus species. The cluster result produced by SRAP data shared great similarity with that by ISSR data with minor differences in the subgroups, which is basically in agreement with morphological observations. In general, SRAP and ISSR are more simple, rapid, and efficient, which may provide alternative molecular approaches to studying genetic diversity, classification, and identification of Monascus strains.