Inter-simple Sequence Repeat Amplification Research Articles

Genetic diversity and population structure of Distylium chinense revealed by ISSR and SRAP analysis in the Three Gorges Reservoir Region of the Yangtze River, China

Distylium chinense is a dominant native evergreen shrub or small tree in the riparian forests in the Three Gorges Reservoir zone in China. The genetic diversity and population structure of D. chinense within and among eight populations was analyzed by ISSR (Inter Simple Sequence Repeat) and SRAP (Sequence-Related Amplified Polymorphism) markers. A high level of genetic diversity was observed at the species level (H = 0.3465, I = 0.5039 in ISSR; H = 0.1924, I = 0.4123 in SRAP) and at the population level (H = 0.3047, I = 0.4471 in ISSR; H = 0.1543, I = 0.3415 in SRAP). The genetic differentiation among population (Gst = 0.1205, Nm = 1.8247 in ISSR and Gst = 0.1976, Nm = 1.015 in SRAP) indicated low levels of genetic variation among populations and high levels of genetic polymorphism within populations. AMOVA (Analyses of molecular variance) also suggested that the main source of variation was within populations (93.9% in ISSR and SRAP in 81.9%). UPGMA (Unweighted Pair Group Method with Arithmetic means) cluster analysis and PCA (Principal Component Analysis) based on ISSR and SRAP amplification showed that populations from the same or adjacent regions were classified together. However, Mantel's test demonstrated that there was no significant correlation between the genetic distance and the geographical distance of each population (P > 0.05). Thus, the declining status of this species is likely due to the destruction of natural habitats, anthropogenic activities and overharvesting, rather than a loss of genetic diversity.

Study of phylogenetic relationship of Turkish species ofMatthiola (Brassicaceae) based on ISSR amplification

Matthiola W.T.Aiton is a taxonomically complex genus in which there are many problems, mostly with Matthiola longipetala and M. Odoratissima. Matthiola species native to Turkey were collected from various locations in Anatolia, and their DNA was isolated. Revision studies performed on the basis of molecular data obtained from studies conducted in recent years have made the phylogenetic relationships and systematic positions of the taxa more apparent and reliable. Consequently, the remaining taxonomic problems among the species have been resolved through the use of DNA-based molecular analysis methods, which, unlike phenotype studies, are not affected by environmental factors. The inter-simple sequence repeat (ISSR) fingerprinting method was used in the study because its properties were considered to be more reliable and consistent than those of the randomly amplified polymorphic DNA method. DNA fragments were amplified through the use of ISSR primers. The phylogenetic relationships among the taxa were represented on a dendrogram constructed through means of NTSYSpc 2.02 software. The infrageneric and intergeneric phylogenetic relationships between Matthiola and other related genera were also characterized. It was determined that the taxa Matthiola odoratissima and M. Ovatifolia are separate but closely related. Moreover, it was observed that the Matthiola longipetala complex forms a separate group within the genera. Clearly, the genera Matthiola, Sterigmostemum, Strigosella, Malcolmia, and Chorispora are phylogenetically differentiated on the dendrogram.

English

Soil fungi, Fusarium solani and Fusarium oxysporum f. sp. passiflorae are among the pathogens of greatest economic importance to the passion fruit culture. Little is known about the genetic variability of these pathogens from different regions. The genetic variability of F. solani and F. oxysporum f. sp. passiflorae isolates from Pantanal, Amazon and Cerrado of Mato Grosso State, Brazil, was studied with the help of molecular markers inter-simple sequence repeat amplification (ISSR) and inter-retrotransposon amplified polymorphism (IRAP). The molecular identification of the isolates was performed by partial sequencing of the internal transcribed spacer (ITS) of the rDNA region. High genetic variability among isolates of F. solani and F. oxysporum f. sp. passiflorae was found. The dendrogram generated by UPGMA divided the fungi isolates in two large groups, however, with grouping tendency according to the biome of collection only for F. oxysporum f. sp. passiflorae. There was no significant correlation between geographic distance and genetic similarity for F. oxysporum f. sp. passiflorae and F. solani. Key words: Collar rot, Fusarium wilt, passionflower, Passiflora edulis, inter-simple sequence repeat amplification (ISSR), inter-retrotransposon amplified polymorphism (IRAP).

Genetic diversity and chemical characterization of selected Polish and Russian cultivars and clones of blue honeysuckle (Lonicera caerulea)

In traditional medicine, Lonicera caerulea has a notable place, and it has been used for thousands of years in East Asian countries for treating cancer, inflammation, hepatic complications, influenza, and wounds. However, the molecular and chemical characterization of this plant is not well defined. In this study, inter-simple sequence repeat (ISSR) markers were used to determine the genetic variability of 7 blue honeysuckle cultivars and 3 clones. To characterize these 10 accessions, total flavonoids, phenolic acids contents, and antioxidant activities were estimated. Twelve primers generated 101 polymorphic ISSR-polymerase chain reaction bands. Based on the ISSR amplification profiles, a cluster dendrogram was obtained, which showed that the similarity coefficients among the 10 genotypes of L. Caerulea ranged from 0.53 to 0.69. The study also identified promising genotypes in terms of flavonoid and phenolic acid contents and high antioxidant activities, as follows: cultivar Atut and clone P. A systematic cluster analysis conducted on the basis of chemical data showed that the 10 tested genotypes can be classified into 2 groups. The ISSR analysis together with data for antioxidant activities as well as flavonoid and phenolic acid contents in honeysuckle could be used to evaluate the genetic diversity and to make more efficient choices of parents in current blue honeysuckle breeding programs.

Assessment of Genetic Diversity in Sugarcane Germplasm Using ISSR and SSR Markers

Molecular diversity within and between Saccharum species clones and elite commercial hybrid varieties was studied using intersimple sequence repeat (ISSR) and simple sequence repeat (SSR) markers. The present study was performed to characterize 81 sugarcane genotypes. A total of 13 ISSR primers used and produced 65 amplified fragments, of which 63 (96.5 %) were polymorphic. The Polymorphic Information Content (PIC) value ranged from 0.11 (UBC824) to 0.45 (UBC825) primers with an average value of 0.28. The primer UBC 817 and UBC 825 exhibited highest resolving power (Rp) value 3.8 among thirteen primers. Genetic similarity (GS) by Jaccard’s similarity coefficient ranged from 0.23 to 0.95 with a mean of 0.59. Of the 79 alleles amplified by 28 SSRs primers showed 76 alleles, which were found to be polymorphic (96.2 %). The PIC value ranged from 0.06 (VSICRAD4) to 0.55 (VSICRAD26) primers with an average value of 0.17. The primer VSICRAD23 exhibited highest resolving power (Rp) value 4.3 among 28 primers. The GS by Jaccard’s similarity coefficient ranged from 0.11 to 0.91 with a mean of 0.51. Dendrograms constructed using the UPGMA cluster analysis revealed low level of correlation between genetic similarities based on pedigree and DNA profiles. The ISSR and SSR amplification proved to be valuable method for assessing genetic diversity among sugarcane complex and their related wild varieties and for identification of the cultivars.

Genetic Diversity among Indian Oak Tasar Silkworm, Antheraea proylei J. Revealed by ISSR Markers

The Indian Oak Tasar silkworm, Antheraea proylei J. is a beneficial insect with great economic importance in India for its silk production. In this study, six populations of Antheraea proylei and A. frithi Moore (as an out group) were subjected to inter simple sequence repeat (ISSR) marker analysis in order to assess its genetic diversity. Fifteen ISSR primers produced 91 markers among different breeds of A. proylei and A. frithi of which 89 are polymorphic, generating 97.8% polymorphism. The dendrogram constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and cluster analysis made using Nei's genetic distance resulted in the formation of one major group containing four sub-groups separating the breeds. This result suggests that ISSR amplification is potentially useful for molecular characterization of oak tasar silkworm genotypes.

Sequence-related amplified polymorphism (SRAP) molecular marker system and its applications in crop improvement

Plant biotechnology has great potential for improving target traits in crops. This can be achieved by the production of transgenic crops and marker-assisted breeding. Marker-assisted breeding has gained momentum in recent times since it does not need biosafety regulations. Several kind of molecular markers are available for use in crop breeding, such as restriction fragment length polymorphism, microsatellites, sequence characterized amplified region, sequence-tagged site, inter-simple sequence repeat amplification, amplified fragment length polymorphism and single nucleotide polymorphism. Sequence-related amplified polymorphism is a novel molecular marker system which is based on open reading frames (ORFs) developed from genome sequence data of Arabidopsis. It provides a unique combination of forward and reverse primers which can be selected arbitrarily giving a large number of primer combinations. Since this is an ORF-based marker system, it targets functional genes and has potential for their application in crop breeding. This marker system was first used and demonstrated by Li and Quiros in Brassica oleracea in 2001, and since then there have been several reports in different plant species ranging from field crops to tree species for assessing genetic diversity, mapping and tagging of genes, hybrid identification and sex determination. This review provides an overview of SRAP markers and their applications in crop improvement.

Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves

Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds—namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 μg/μL and the purity, evaluated by the ratio A260/A280, was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR.

The application of ISSR-PCR to determine the genetic relationship and genetic diversity between narrow leaved bluegrass (Poa angustifolia) and rough bluegrass (Poa trivialis) accessions

: The present study aimed to determine the genetic relationship between Poa angustifolia and Poa trivialis as well as intraspecies genetic diversity using inter simple sequence repeat (ISSR) markers. ISSR amplification was evaluated for its applicability to narrow leaved bluegrass and rough bluegrass accessions identification. A total of 401 bands, 363 of which were polymorphic (90.52%), were generated using selected 20 primers. Genetic similarity between accessions belonging to Poa species was estimated using Nei's genetic similarity and UPGMA (unweighted pair group method with arithmetic) cluster analysis was used to construct a dendrogram. UBC836 and M6 primers were observed to be the most suitable for distinguishing between species. Because interspecies differences are especially important in plant breeding, M5 and UBC812 primers were ideal in distinction of P. trivialis and P. angustifolia accessions, respectively. Primer UBC856 was determined to be ideal in discrimination both intraspecies and interspecies. The ISSR fingerprinting technique used was confirmed to be a reproducible and sensitive tool for the identification of Poa accessions and for determination of the correct starting material for plant breeding.

Phylogenetic relationships between Malcolmia, Strigosella, Zuvanda, and some closely related genera (Brassicaceae) from Turkey revealed by inter-simple sequence repeat amplification

The genus Malcolmia W.T.Aiton is taxonomically problematic, and some of its species have recently been transferred to the genera Strigosella Boiss. and Zuvanda Dvorák. Three species of this genus are native to Turkey. The revision studies based on molecular data, which were collected in the past few years, display the phylogenetic relations and the systematic positions of the taxa more reliably and apparently. Thus taxonomic problems of species are resolved through DNA-based molecular analyses, which are not affected by environmental factors compared to phenotypic studies. In the present study, the amplifications of the DNA fragments were carried out using ISSR primers, and the phylogenetic relationship among the taxa was revealed through a dendrogram produced as the outcome of the NTSYSpc 2.1 software. The infrageneric and intergeneric phylogenetic relationships among Malcolmia and other related genera are determined. A very close relationship was determined between Malcolmia chia and M. flexuosa. Strigosella and Zuvanda species were determined to be phylogenetically different from these 2 species. The phylogenetic relationships among the Malcolmia, Strigosella, Zuvanda, Leptaleum DC., Neotorularia Hedge & J.Léonard, and Sisymbrium L. taxa were investigated. The phylogenetic separation of Malcolmia, Strigosella, Zuvanda Leptaleum, Neotorularia, and Sisymbrium genera and their specimens constituted separate clades on the dendrogram.

Genetic Variation in the Qinghai-Tibetan Plateau Endemic and Endangered Conifer Cupressus gigantea, Detected Using RAPD and ISSR Markers

Abstract Assessing the level and distribution of genetic diversity of rare tree species is essential for their management and the development of effective conservation strategies. Cupressus gigantea is a long-lived endemic cypress of the west Qinghai-Tibetan Plateau and the tallest tree in its genus. The current populations of this species are fragmented and highly disturbed. We used RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat amplification) markers to assess the genetic variation and population structure of this endangered cypress. The 15 RAPD primers used in this study amplified 108 reproducible bands, 49 (45.4%) of which were polymorphic, while the 12 ISSR primers amplified 94 bands, 65 (69.2%) of which were polymorphic. Analysis of Molecular Variance (AMOVA) indicated that 49.7% and 38.3% of the variation was attributable to differences between populations for the RAPD and ISSR markers, respectively; relatively high compared to values reported for other conifer species. These estimates were also similar to Gst values obtained from Nei’s gene diversity analyses (RAPD = 0.41 and ISSR = 0.36), and suggest that there is a high degree of population differentiation in this narrowly-distributed conifer. The genetic structure of this species has probably been shaped by its long life cycle and climatic changes during the Quaternary. The high degree of population differentiation in this species highlights the need for additional conservation measures, including measures to protect of all of the remaining populations. The substantial similarities between the results of the RAPD and ISSR analyses of samples from the same individuals indicate that they can be interpreted with high levels of confidence.

Microsatellite Marker Identification Using Genome Screening and Restriction-Ligation

We have identified a fast and easy method for finding microsatellite markers that utilizes genome screening with inter-simple sequence repeat (ISSR) primers to detect microsatellite regions and to obtain sequence information flanking one side of the microsatellites and a restriction-ligation technique with a specific adaptor to allow sequence walking to obtain sequence information flanking the other side of the microsatellites. Two main alternatives of the method (with or without cloning) are presented. We successfully utilized the method when identifying microsatellite markers for 21 bryophyte species, three algal species, and for the raccoon dog. The proportion of polymorphic markers equaled 95%. We observed that microsatellites are commonly found within the sequenced ISSR amplification products (54% in the present study), in which case specific primers can be identified for the microsatellite without a further restriction-ligation step. It is evident that the DNA regions amplified by ISSR markers commonly represent microsatellite hotspots. We propose that the identified method and the knowledge of the common presence of additional microsatellite repeats within ISSR amplification products are especially attractive to researchers who conduct small-scale microsatellite identification, such as researchers in population genetics and conservation biology.

Analyzing Genetic Relationships in <I>Bombyx mori</I> Using Intersimple Sequence Repeat Amplification

Intersimple sequence repeat (ISSR) amplification was used to analyze genetic relationships among silkworm, Bombyx mori L., strains. Nineteen primers containing simple sequence repeat (SSR) motifs were tested for amplification on a panel of 42 strains, representative of the diversity of silkworm germplasm; 12 of the primers amplified distinct, reproducible bands. The primers amplified a total of 108 bands, of which 85 (78.7%) were polymorphic. The ISSR results suggested that within the dinucleotide class, the poly(CA) motif was more common than the poly(CT) motif. The ISSR amplification pattern was used to group the silkworm strains into seven subclusters based on their origin in an unweighted pair-group method with arithmetic average cluster analysis by using Nei's genetic distance. Seven major ecotypic silkworm groups were analyzed. Principal component analysis of the ISSR data supported the unweighted pair-group method with arithmetic average clustering. Therefore, ISSR amplification is a valuable method for determining genetic variability among silkworm varieties. This efficient genetic fingerprinting technique should be useful for characterizing the large numbers of silkworm strains held in national and international germplasm centers.

Analyzing Genetic Relationships in Bombyx mori Using Intersimple Sequence Repeat Amplification

Intersimple sequence repeat (ISSR) amplification was used to analyze genetic relationships among silkworm, Bombyx mori L., strains. Nineteen primers containing simple sequence repeat (SSR) motifs were tested for amplification on a panel of 42 strains, representative of the diversity of silkworm germplasm; 12 of the primers amplified distinct, reproducible bands. The primers amplified a total of 108 bands, of which 85 (78.7%) were polymorphic. The ISSR results suggested that within the dinucleotide class, the poly(CA) motif was more common than the poly(CT) motif. The ISSR amplification pattern was used to group the silkworm strains into seven subclusters based on their origin in an unweighted pair-group method with arithmetic average cluster analysis by using Nei’s genetic distance. Seven major ecotypic silkworm groups were analyzed. Principal component analysis of the ISSR data supported the unweighted pair-group method with arithmetic average clustering. Therefore, ISSR amplification is a valuable method for determining genetic variability among silkworm varieties. This efficient genetic fingerprinting technique should be useful for characterizing the large numbers of silkworm strains held in national and international germplasm centers.

A genetic linkage map for tef [Eragrostis tef (Zucc.) Trotter

Tef [Eragrostis tef (Zucc.) Trotter] is the major cereal crop in Ethiopia. Tef is an allotetraploid with a base chromosome number of 10 (2n = 4x = 40) and a genome size of 730 Mbp. Ninety-four F(9) recombinant inbred lines (RIL) derived from the interspecific cross, Eragrostis tef cv. Kaye Murri x Eragrostis pilosa (accession 30-5), were mapped using restriction fragment length polymorphisms (RFLP), simple sequence repeats derived from expressed sequence tags (EST-SSR), single nucleotide polymorphism/insertion and deletion (SNP/INDEL), intron fragment length polymorphism (IFLP) and inter-simple sequence repeat amplification (ISSR). A total of 156 loci from 121 markers was grouped into 21 linkage groups at LOD 4, and the map covered 2,081.5 cM with a mean density of 12.3 cM per locus. Three putative homoeologous groups were identified based on multi-locus markers. Sixteen percent of the loci deviated from normal segregation with a predominance of E. tef alleles, and a majority of the distorted loci were clustered on three linkage groups. This map will be useful for further genetic studies in tef including mapping of loci controlling quantitative traits (QTL), and comparative analysis with other cereal crops.

ISSRs 마크에 의한 고려 인삼의 분자적 인증과 유전적 다형현상

ISSR마크를 사용하여 고려 인삼의 품종 및 계통간 분자적 인증과 유전적 다형현상을 조사하였다. 56개의 ISSR 프라이머 중 5개가 일곱 품종 및 계통간 명확하고 재현성이 높은 DNA분절을 나타내는 최적 프라이머로 선택되었다. 전체 43밴드는 250 bp - 1,700 bp의 분자량을 가지며 프라이머당 8.6개의 밴드를 나타내었다. 고려 인삼에서 다형현상 정도는 20.9%였다. 특히 천풍 품종이 가장 높은 다형현상을 나타낸 반면 다른 품종은 거의 다형현상을 나타내지 않았다. 결론적으로 DNA수준에서 ISSR마크로 천풍이 다른 고려 인삼의 품종 및 계통인 연풍, 황숙종, 자경종과 구분에 이용될 수 있음이 판명되었다. Molecular authentication and genetic polymorphism of Korean ginseng cultivars and accessions were investigated using ISSR (inter-simple sequence repeat amplification) markers. Five primers among 56 produced clear and reproducible DNA fragments among seven cultivars and accessions. A total of 43 bands ranging from 250 bp to 1,700 bp from five primers were scored. Average number of bands per primer was 8.6 and only nine bands were polymorphic across the six Panax ginseng from Korea. Especially Chunpoong cultivar exhibited the highest level of polymorphism, whereas other accessions did not showed almost any polymorphism. Consequently, these ISSR markers will be available to differentiate Chunpoong cultivar from other major Korean ginseng cultivars and accessions, such as Yunpoong, Hwangsukjong and Jakyungjong, at the DNA level.

Use of ISSR fingerprints to detect microsatellites and genetic diversity in several related Brassica taxa and Arabidopsis thaliana.

Use of ISSR fingerprints to detect microsatellites and genetic diversity in several related Brassica taxa and Arabidopsis thaliana.

Highly informative nature of inter simple sequence repeat (ISSR) sequences amplified using tri- and tetra-nucleotide primers from DNA of cauliflower (Brassica oleracea var. botrytis L.).

Inter simple sequence repeat (ISSR) sequences as molecular markers can lead to the detection of polymorphism and also be a new approach to the study of SSR distribution and frequency. In this study, ISSR amplification with nonanchored primer was performed in closely related cauliflower lines. Fourty-four different amplified fragments were sequenced. Sequences of PCR products are delimited by the expected motifs and number of repeats, which validates the ISSR nonanchored primer amplification technique. DNA and amino acids homology search between internal sequences and databases (i) show that the majority of the internal regions of ISSR had homologies with known sequences, mainly with genes coding for proteins implicated in DNA interaction or gene expression, which reflected the significance of amplified ISSR sequences and (ii) display long and numerous homologies with the Arabidopsis thaliana genome. ISSR amplifications revealed a high conservation of these sequences between Arabidopsis thaliana and Brassica oleracea var. botrytis. Thirty-four of the 44 ISSRs had one or several perfect or imperfect internal microsatellites. Such distribution indicates the presence in genomes of highly concentrated regions of SSR, or "SSR hot spots." Among the four nonanchored primers used in this study, trinucleotide repeats, and especially (CAA)5, were the most powerful primers for ISSR amplifications regarding the number of amplified bands, level of polymorphism, and their nature.

Intersimple sequence repeat (ISSR) polymorphisms as a genetic marker system in cotton

AbstractWe studied the applicability of intersimple sequence repeat (ISSR) polymorphism in cotton. We found that: (i) the resolving power of agarose gels is poor relative to that provided by sequencing gels; (ii) fluorescent labelling of ISSR amplification primers produced numerous scorable bands; (iii) primer mixing (double priming) generated more bands than the sum of fragments resulting from two single primers, although an unexplained disappearance of several larger fragments also reproducibly occurred; (iv) ISSR fingerprinting patterns are highly heritable; and (v) double priming ISSR is an easy and informative genetic marker system in cotton for revealing both inter‐ and intraspecific variations.

POTATO CULTIVARS IDENTIFICATION USING MOLECULAR MARKERS

Cultivar identification becomes determinant for potato breeders. The increasing number of potato cultivars and the importance of their choice make necessary to strengthen user's guarantees concerning purity and identity. Today, identification mainly based on phenotypic characters implies culture inspection at different stages and is often doubtful. RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeat) molecular markers are considered to produce genetic fingerprints for a reliable and rapid cultivar identification. 37 cultivars are chosen to represent the total variability of the French catalogue. The use of 3 RAPD primers allows their molecular identification. However, ISSR amplification appear to be more polymorphic and reliable than RAPD.