Inter-simple Sequence Repeat PCR Research Articles

Evaluating the genetic diversity of Erythropalum scandens based on using inter-simple sequence repeat markers

Erythropalum scandens Blume, an emerging medicinal plant with great potential for drug development, also possesses high edible value. In this study, we investigated the genetic diversity of the germplasm of E. scandens obtained from different geographical locations using inter simple sequence repeat (ISSR) markers. For this purpose, 18 ISSR primer pairs with a distinct background and adequate polymorphism were selected. We established an optimal ISSR–PCR reaction system (20 µL) with the following parameters: 1 µL DNA template (60 ng µL−1), 1.2 µL primers (10 µmol µL−1), 10 µL MasterMix, and 7.8 µL H2O. A total of 183 loci were amplified using the 18 primer pairs, of which 121 (66.12%) indicated polymorphism. Moreover, 34 germplasms of E. scandens exhibited genetic similarity coefficients ranging from 0.7104 to 0.9563, genetic distances ranging from 0.0447 to 0.3420, Nei’s genetic diversity index of 0.1946, and Shannon’s information index of 0.2982, suggesting high intraspecific genetic diversity. UPGMA cluster and PCoA analyses distinguished the germplasm of E. scandens obtained from Guangxi from those collected from Guangdong, Hainan, Fujian, and Guizhou. However, the Mantel correlation analysis revealed that the genetic variation among the 34 germplasms of E. scandens was not significantly related to geographical distance. The analysis of the genetic background of wild and cultivated germplasms of E. scandens can help guide variety selection and breeding. Furthermore, the present study revealed the genetic background and affinities among 34 germplasms of E. scandens. Overall, our findings lay the foundation for the conservation and utilization of germplasm resources, identification and classification of varieties, and variety selection and improvement of E. scandens at the molecular level.

Antagonistic activity and molecular characterization of biological control agent Trichoderma harzianum from Saudi Arabia

Trichodermaharzianum is one of the most commonly used fungal species used in the biological control of plant pathogenic fungi. Its ability to control biological pathogens is strengthened by the production of analytic enzymes. In the identification of these fungal species, it has been noted that morphological characteristics alone are insufficient. Hence, the aim of this study was to identify 12 Trichoderma isolates based on their molecular markers and to evaluate the antagonistic activity of these Trichoderma isolates against several plant pathogens. The 12 Trichoderma/Hypocrea isolates were harvested from the rhizosphere of healthy tomato plants in Abha region, Saudi Arabia. These isolates were identified by both morphological and molecular techniques, such as the sequencing of the 5.8S-ITS region. Furthermore, based on the taxonomy and phylogeny of these isolates, the genetic similarities based on the inter-simple sequence repeat-PCR markers separated the 12 Trichoderma isolates into 2 major clusters. The 12 isolates were then used in confrontational assays with Rhizoctonia solani, Pythium ultimum, and Alternaria solani. Most of the Trichoderma isolates showed a strong antagonistic activity, which could also be observed with the use of SEM. This study highlights the possibility of using T. harzianum in IPM programs as an effective biological agent against several pathogenic fungi.

An analysis of genetic diversity in Marphysa sanguinea from different geographic populations using ISSR polymorphisms

The genetic diversity of five wild populations of marine polychaete Marphysa sanguinea found in China was investigated using Inter-Simple Sequence Repeat-PCR (ISSR-PCR) polymorphisms. The results of the ISSR-PCR showed that 108 (90.8%) of the 119 ISSR loci tested were polymorphic. The Shannon's information index value was 0.4981, Nei's gene diversity was 0.3418, and the coefficient of gene differentiation (Gst) was 0.3671, which indicated that the among-population component accounted for 36.7% of the total variation, while the within-population component accounted for 63.3%. A UPGMA tree showed that the five populations clustered into two branches. Populations from Dalian, Xingcheng, and Rushan clustered together, while the two Guangxi populations, A and B, clustered into a unique group. The results indicated that the genetic diversity among the five populations of M. sanguinea is high, which will provide useful information for the protection of biodiversity among marine polychaetes.

Identification of Neofabraea species causing bull's eye rot of apple in Poland and their direct detection in apple fruit using multiplex PCR

Based on partial sequence analysis of the β‐tubulin gene, 19 isolates of fungi causing bull's eye rot on apple in Poland were classified into species: Neofabraea alba, N. perennans and N. kienholzii. To the authors’ knowledge, the detection of N. kienholzii is the second in Europe and the first in Poland. Species affiliation of these fungi was confirmed by a new species‐specific multiplex PCR assay developed on the basis of previously published methods. The new protocol allowed for the specific identification of bull's eye rot‐causing species, both from pure cultures and directly from the skin of diseased or apparently healthy apples. In 550 samples of diseased fruits collected from nine cold storage rooms located in three regions of Poland, in 2011 and 2012, N. alba was detected as the predominant species causing bull's eye rot, occurring on average in 94% of the tested samples. Neofabraea perennans was found in a minority of apple samples, N. kienholzii was found only in two apple samples, while N. malicorticis was not detected in any sample tested. In tests on 120 apparently healthy fruits, only N. perennans was detected in a single sample. The results of genetic diversity analyses of bull's eye rot‐causing fungi based on the β‐tubulin gene sequence and an ISSR (inter‐simple sequence repeat) PCR assay with two primers were consistent, showing the expected segregation of tested isolates with respect to their species boundaries. However, the genetic distance between N. perennans and N. malicorticis was very low, as reported previously.

Genetic characterisation of microsporidia infecting Indian tasar silkworm, Antheraea mylitta, using morphology and molecular tools.

The utility of inter simple sequence repeat-PCR (ISSR-PCR) assay in the genetic characterisation and elucidation of the phylogenetic relationship of different microsporidian isolates infecting tropical tasar silkworm, Antheraea mylitta Drury, is demonstrated. A total of 22 different microsporidians collected from the diseased tasar silkworms from Jharkhand state of India were analysed using morphological characters and ISSR-PCR. Observations spores under phase contrast microscope revealed oval to elongate in shape with length ranging from 3.8 μm to 5.1 μm and width from 2.6 μm to 3.3 μm. All the microsporidian isolates except MIJ-1gC showed gonadal infection and transovarial transmission in infected tasar silkworms. Fourteen out of 20 ISSR primers tested generated reproducible profiles and yielded a total of 281 fragments, of which 273 were polymorphic (97%). The degree of banding pattern was used to evaluate genetic distances and for phylogenetic analysis. The results demonstrated that ISSR analysis may be a useful and efficient tool for taxonomical grouping and phylogenetic classification of different microsporidians in general.

English

Inter-simple sequence repeat (ISSR)-PCR technique was used to assess genetic variation and phylogenetic relationships between Lymnaea natalensis collected from Giza, Ismailia, Damietta, and Beheira governorates in Egypt and compared with lab-bred snail in addition to characterization of watercourses from these sites. Five ISSR primers generated 47 amplified bands, of which 63.63% showed high polymorphism. All tested primers detected the common band (approximately 455 bp) in all L. natalensis studied. Three bands (318, 782 and 2013 bp for primers HB8, HB12 and HB13, respectively) are characteristic for L. natalensis collected from Ismailia, El Behira and Damietta governorates, respectively. These markers were used to estimate genetic similarity among the varieties using Jaccard’s similarity coefficient. The similarity matrix was used to construct a dendrogram. The most abundant snails Physa acuta (56.0%) and the lowest abundant snails Planorbis planorbis, and Melanoides tuberculata (0.5) were found associated with L. natalensis in many governorates. Eichhornia crassipes is the only aquatic plant which grows and is found associated with L. natalensis in all canals of governorates. Also, dragon fly is the only macroinvertebrate collected from all canals, then shrimps and true bugs. Heavy metals were detected in water samples of all tested governorates with different significant differences. In conclusion, the present study used for the first time the ISSR PCR technique for studying genetic variations of L. natalensis snails in Egypt. L. natalensis snails can survive when associated with other snails, plants, and insects and can tolerate the heavy metals in water.   Key words: Lymnaea natalensis, inter-simple sequence repeat (ISSR)-PCR, dendrogram, heavy metals, macroinvertebrates. &nbsp

Direct and indirect in vitro regeneration of Miscanthus × giganteus cultivar Freedom: effects of explant type and medium on regeneration efficiency

Miscanthus × giganteus (giant miscanthus; Mxg) is a seed-sterile, perennial bioenergy crop with the potential to produce liquid fuel from lignocellulosic biomass. A new cultivar, Freedom, is being commercially grown in the USA on increasing acreage. To determine this genotype’s regeneration responses in tissue culture, three explant sources were screened on media proven successful for other genotypes. Four callus induction media contained 13.6–22.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with 0.44–4.4 μM 6-benzyladenine (BA). Callus induction percentages for all explants ranged from 93 to 97%. Media yielding the greatest percentages of explants producing regenerable calli for shoot apices (from in vitro and greenhouse plant sources) were media containing either 13.6 μM 2,4-D plus 0.44 μM BA or 22.6 μM 2,4-D plus 0.44 μM BA. After culture on a regeneration medium containing 22 μM BA plus 1.3 μM naphthaleneacetic acid (NAA), 3.59–3.74 regenerants were obtained per explant. Immature inflorescence explants (from field-maintained plants) gave up to 77% regenerable calli and 6.99 regenerants per explant. Direct regenerants (shoots) arose from immature inflorescence explants on a medium containing 9.0 μM 2,4-D. Intact plants could be generated within 16–18 wk after culture initiation. Extensive visual assessments, and molecular assessments via inter-simple sequence repeat (ISSR) PCR analysis using 21 different primers, did not reveal distinguishable somaclonal variation among regenerants or when compared to rhizome-propagated transplants under field conditions. We believe that this is the first extensive in vitro and ex vitro analysis on a commercially grown Mxg genotype.

Plant genotyping using fluorescently tagged inter-simple sequence repeats (ISSRs): basic principles and methodology.

Inter-simple sequence repeat PCR (ISSR-PCR) is a fast, inexpensive genotyping technique based on length variation in the regions between microsatellites. The method requires no species-specific prior knowledge of microsatellite location or composition. Very small amounts of DNA are required, making this method ideal for organisms of conservation concern, or where the quantity of DNA is extremely limited due to organism size. ISSR-PCR can be highly reproducible but requires careful attention to detail. Optimization of DNA extraction, fragment amplification, and normalization of fragment peak heights during fluorescent detection are critical steps to minimizing the downstream time spent verifying and scoring the data.

Genetic population structure in Nacella magellanica: Evidence of rapid range expansion throughout the entire species distribution on the Atlantic coast

Nacella magellanica, the most abundant limpet in the Magellanic biogeographic Province of Argentina and Chile, is characterized by a noticeable variability in shell morphology and color patterns. Ecological as well as evolutionary features make this limpet an interesting species for evolutionary and population genetic studies. Here, arbitrary nuclear molecular markers, inter-simple sequence repeat-PCR, are used to analyze the population genetic structure in 14 localities of N. magellanica along 2900km on the Atlantic coast of the Magellanic Province. We compare the present results, based on nuclear markers, with previous findings of a phylogeographic study that used the mitochondrial COI gene. All the results presented here suggest little genetic structure within N. magellanica, with moderate to high genetic connectivity among populations. The short time elapsed since the expansion of this species plus a possible long larval lifespan and the oceanographic and environmental conditions of the Magellanic Province might explain the lack of genetic structuring and the low levels of genetic differentiation in the species throughout its distribution range along the Argentine coast.

Discrimination of the sibling species Apodemus flavicollis and A. sylvaticus (Rodentia, Muridae)

Karyotyping and several molecular methods have allowed successful identification of two morphologically similar wide-ranging Western Palearctic species, the yellow-necked field mouse Apodemus flavicollis (Melchior, 1934) and the long-tailed wood mouse A. sylvaticus (Linnaeus, 1758), but reliable species diagnosis on the basis of morphometric characters is particularly problematic. Although they are easily morphologically distinguishable in Central and Northern Europe, this is not the case in southern parts of their distribution areas. Despite that, we have successfully discriminated A. flavicollis and A. sylvaticus from Serbia (Southern Europe) using geometric and traditional morphometric methods on a data set for ventral crania of specimens previously genotyped by the Inter Simple Sequence Repeat-PCR (ISSR-PCR). Discrimination power of applied approaches was more or less similar. The majority of our results were consistent with those obtained for specimens collected across the Czech Republic (Central Europe). Morphological differences observed herein, as well as those already reported between A. flavicollis and A. sylvaticus from the central and northern parts of their distribution areas, could be the outcome of their biology, i.e. ecological discrepancies, different assumed evolutionary scenarios considering biogeography, phylogeny, history and ontogeny.

Molecular Characterization ofOnobrychis altissima(Fabaceae) Populations from Iran, with the Description ofO. chaldoranensis sp. nova

Seventy specimens from 14 populations of Onobrychis altissima (Fabaceae) were collected from their natural habitats in Iran. In order to assess the genetic variation, we carried out genomic fingerprinting using inter-simple sequence repeat PCR (ISSR). We identified a population which is distinguishable from all other populations genetically as well as morphologically by several unique characters, most notably by having the keels longer than standards (rarely in other members of sect. Onobrychis in Iran), wings ± large and pods with four to five very short teeth on the crest. We describe this taxon as Onobrychis chaldoranensis Toluei, Ranjbar & Wink sp. nova (sect. Onobrychis).

A local dormancy cline is related to the seed maturation environment, population genetic composition and climate.

Seed dormancy varies within species in response to climate, both in the long term (through ecotypes or clines) and in the short term (through the influence of the seed maturation environment). Disentangling both processes is crucial to understand plant adaptation to environmental changes. In this study, the local patterns of seed dormancy were investigated in a narrow endemic species, Centaurium somedanum, in order to determine the influence of the seed maturation environment, population genetic composition and climate. Laboratory germination experiments were performed to measure dormancy in (1) seeds collected from different wild populations along a local altitudinal gradient and (2) seeds of a subsequent generation produced in a common garden. The genetic composition of the original populations was characterized using intersimple sequence repeat (ISSR) PCR and principal co-ordinate analysis (PCoA), and its correlation with the dormancy patterns of both generations was analysed. The effect of the local climate on dormancy was also modelled. An altitudinal dormancy cline was found in the wild populations, which was maintained by the plants grown in the common garden. However, seeds from the common garden responded better to stratification, and their release from dormancy was more intense. The patterns of dormancy variation were correlated with genetic composition, whereas lower temperature and summer precipitation at the population sites predicted higher dormancy in the seeds of both generations. The dormancy cline in C. somedanum is related to a local climatic gradient and also corresponds to genetic differentiation among populations. This cline is further affected by the weather conditions during seed maturation, which influence the receptiveness to dormancy-breaking factors. These results show that dormancy is influenced by both long-and short-term climatic variation. Such processes at such a reduced spatial scale highlight the potential of plants to adapt to fast environmental changes.

Floral size variation causes differentiation of pollinators and genetic parameters in Alpinia nieuwenhuizii, a flexistylous ginger (Zingiberaceae)

Floral size dimorphism, pollination, and genetic variation of Alpinia nieuwenhuizii (Zingiberaceae), a flexitylous ginger, were studied. This study revealed that floral size differed among habitats (i.e., roadsides/riversides vs. forest floors). The effective pollinators of small-flowered populations of the species on a forest floor were different from those of large-flowered populations along roadsides/riversides. Using inter-simple sequence repeat (ISSR) PCR, considerable genetic differentiation was detected between small- and large-flowered populations. These results indicate that reproductive isolation in A. nieuwenhuizii owing to the differentiation of pollen vectors between two floral size morphs may lead to genetic differentiation between the two morphs.

Molecular phylogeny and ecogeography of Onobrychis viciifolia Scop. (Fabaceae) based on nrDNA ITS sequences and genomic ISSR fingerprinting

AbstractIn this study 40 specimens from 8 representative Iranian populations of Onobrychis viciifolia SCOP. were collected from their natural habitats. The specimens were biometrically assessed using 43 quantitative and 15 qualitative morphological characters. At each sample site we recorded ecogeographical variables. In order to evaluate the difference between the variation due to phenotypic responses and genetic adaptation, we generated nucleotide sequence data from the internal transcribed spacer of the nuclear rDNA genes (ITS) and carried out genomic fingerprints using inter‐simple sequence repeat PCR (ISSR). Cluster analysis of morphological characters divided the eight populations into three major groups. Leaf length, leaflet length and width, calyx length, plant height, and stem length were introduced as diagnostic characters for three different morphological groups. Furthermore, canonical correspondence analysis (CCA) of ecogeographical data showed correlations between morphological variations and ecogeographical factors. Clay%, saturation percentage (SP), total neutralizing value (TNV%), texture, minimum temperature, and geographic separation were the main environmental variables associated with morphological groups of O. viciifolia. ISSR analysis revealed three main groups which are in correspondence with morphological groups, indicating that the three morphological groups have been shaped by genetic and phenotypic responses to environmental conditions. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

고도에 따른 지역별 복숭아혹진딧물 집단 변이 마커 개발

This study focused on the green peach aphid, Myzus persicae, as an indicator pest in Chinese cabbage cultivation to develop a genetic marker. We hypothesized that M. persicae gene flow is related to climate change. Genetic variation was analyzed using five local populations collected at different altitudes (157 m, 296 m, 560 m, 756 m and 932 m above sea level) in Hoengseong, Pyeongchang, and Gangneung areas, plus a laboratory strain used as an outgroup. There were no differences in ecological characteristics among strains. Esterase isozyme pattern and inter-simple sequence repeat (ISSR) PCR results showed significantly different bands between laboratory and wild, local populations. However, there was no difference among local populations. Partial fragments of ribosomal RNA (rRNA) and mitochondrial cytochrome oxidase I (mtCO I) were amplified and their nucleotide sequence was analyzed. Single nucleotide polymorphisms (SNPs) were detected in internal transcribed spacer-2 (ITS-2) and mtCO I regions among the five local populations. These SNPs can be use to discriminate different populations of M. persicae to monitor gene flow.

Global population structure of Aspergillus terreus inferred by ISSR typing reveals geographical subclustering

BackgroundAspergillus terreus causes invasive aspergillosis (IA) in immunocompromised individuals and can be the leading cause of IA in certain medical centers. We examined a large isolate collection (n = 117) for the presence of cryptic A. terreus species and employed a genome scanning method, Inter-Simple Sequence Repeat (ISSR) PCR to determine A. terreus population structure.ResultsComparative sequence analyses of the calmodulin locus revealed the presence of the recently recognized species A. alabamensis (n = 4) in this collection. Maximum parsimony, Neighbor joining, and Bayesian clustering of the ISSR data from the 113 sequence-confirmed A. terreus isolates demonstrated that one clade was composed exclusively of isolates from Europe and another clade was enriched for isolates from the US.ConclusionsThis study provides evidence of a population structure linked to geographical origin in A. terreus.

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Biospotlight

Microsatellite Discovery by Deep Sequencing of Enriched Genomic Libraries

Robust molecular markers such as microsatellites are important tools used to understand the dynamics of natural populations, but their identification and development are typically time consuming and labor intensive. The recent emergence of so-called next-generation sequencing raised the question as to whether this new technology might be applied to microsatellite development. Following this view, we considered whether deep sequencing using the 454 Life Sciences/Roche GS-FLX genome sequencing system could lead to a rapid protocol to develop microsatellite primers as markers for genetic studies. For this purpose, genomic DNA was sourced from three unrelated organisms: a fungus (the pine pathogen Fusarium circinatum), an insect (the pine-damaging wasp Sirex noctilio), and the wasp's associated nematode parasite (Deladenus siricidicola). Two methods, FIASCO (fast isolation by AFLP of sequences containing repeats) and ISSR-PCR (inter-simple sequence repeat PCR), were used to generate microsatellite-enriched DNA for the 454 libraries. From the resulting 1.2-1.7 megabases of DNA sequence data, we were able to identify 873 microsatellites that have sufficient flanking sequence available for primer design and potential amplification. This approach to microsatellite discovery was substantially more rapid, effective, and economical than other methods, and this study has shown that pyrosequencing provides an outstanding new technology that can be applied to this purpose.

Production and characterization of arboreous and fertile Solanum melongena + Solanum marginatum somatic hybrid plants

In crop plants the shift from being annuals to perennials may allow future agricultural systems requiring less energy inputs. The practicability of this was tested for Solanum melongena. Leaf protoplasts of S. melongena (2n = 2x = 24) and one of the related arborescent species Solanum marginatum (2n = 2x = 24) were electrofused and fertile somatic hybrids with arborescent habit regenerated. The magnetic cell sorter (MACS) technique was used for the selection of heterokaryons. The hybrid nature of 18 regenerated plants was assessed on the banding patterns generated by inter-simple sequence repeat PCR. When taken to maturity in the greenhouse, hybrids grew more vigorously compared to the parental species. Their morphological traits were intermediate between those of S. melongena and S. marginatum. Hybrids flowered and produced an average of 85% stainable viable pollen and fertile fruits. The somatic hybrids were maintained in the greenhouse for more than 3 years and continued to produce flowers developing into two types of fruits with plentiful seeds. Fruits were either striated green containing non-germinable seeds or yellow with fully germinable seeds. Their S(1) progenies showed common features with S(0) hybrids, including fertility and arborescent habit. Cytologically, somatic hybrids exhibited the expected chromosome number of 2n = 4x = 48, while chromosome pairing during microsporogenesis was associated with a low frequency of intergenomic pairing. It is concluded that an arborescent perennial species has been obtained by somatic hybridization. The usefulness of this species per se or in eggplant breeding will depend not only on the transmission of the arborescent habit to cultivated eggplant varieties, but also on the variability that should be created from backcrossing the S. melongena + S. marginatum hybrids to S. melongena.

Genetic characterization ofVibrio choleraestrains by inter simple sequence repeat-PCR

The utility of inter simple sequence repeat-PCR (ISSR-PCR) assay in the characterization and elucidation of the phylogenetic relationship between the pathogenic and nonpathogenic isolates of Vibrio cholerae is demonstrated. A total of 45 V. cholerae strains including 15 O1 El Tor, nine O139 and 21 non-O1/non-O139 strains were analyzed using eight ISSR primers. These primers, which are essentially simple sequence repeats (SSR) with additional nonrepeat bases at the 5' or 3' end, amplify genomic regions interspersed between closely spaced SSRs. Neighbor-joining analysis showed that the strains belonging to the same serogroup clustered together with the exception of one O1 and two O139 strains. The absence of pathogenicity islands in these strains, as confirmed by PCR, suggested their non-O1/non-O139 origin. Thus the ISSR-PCR-based phylogeny was consistent with the classification of V. cholerae based on serological methods. A finer resolution of the clustering of the toxinogenic O1 El Tor and toxinogenic O139 subtypes was obtained by ISSR-PCR analysis as compared with the Enterobacterial Repetitive Intergenic Consensus sequences-based PCR analysis for the same set of strains. Thus, it is proposed that ISSR-PCR is an efficient tool in phylogenetic classification of prokaryotic genomes in general and diagnostic genotyping of microbial pathogens in particular.