Polymorphism In Banding Patterns Research Articles

Genetic diversity of common bean (Phaseolus vulgaris L.) ecotypes from Pakistan using Simple Sequence Repeats

Common bean (Phaseolus vulgaris L.) is a legume crop grown all over the world and is a very important food of mountain population of Pakistan for protein intake. The Western Himalayan Mountains are rich in biodiversity including unexplored landraces of the common bean crop. Unfortunately, very little attention has been given to this valuable crop in Pakistan, and it is being exported, majorly from Ethiopia, to meet the country’s requirements. The exploitation, utilization, preservation and multiplication of existing germplasm within the area are very important for sustainable production of the crop and enhancing the nutrition value for the local community in mountain regions. A research study was conducted for evaluation of biological diversity of common bean landraces from Azad Kashmir and Northern areas of Pakistan using morpho-physiological and molecular markers. Thirty-five common bean ecotypes along with one check variety were collected from different altitudes of Azad Kashmir and Northern Pakistan and screened for biological diversity. Morphological characterization revealed high genetic diversity in parameters including stem anthocyanin, growth type, days to flowering, pods/plant and 100 seeds weight. Genomic characterization using SSR markers, for allelic diversity evaluation among germplasm, also provided diverse profile with 83.3% polymorphism in banding pattern. The bulk of gene pool diversity evaluated within bean landraces may help to initiate breeding program for common bean improvement.

Genetic diversity in several genotypes of Algerian lentil using biochemical markers

Abstract. Hammadi H, Hammouda-Bousbia D, Chaib G, Tir A. 2021. Genetic diversity in several genotypes of Algerian lentil using biochemical markers. Biodiversitas 22: 3494-2500. Studying seed storage proteins in legumes (Fabaceae) is very important besides its economic value; seed storage proteins play an important role in plant taxonomy because of the stability of their contents. The grain protein content and protein profiles of 12 lentil genotypes (Lens culinaris Medik.) were characterized based on protein profiling through SDS-PAGE. The obtained gel has shown forty bands ranged from 13 to 21 with molecular weight 10 to 120 KDa. Of which six monomorphic bands, thirty-four polymorphic bands and five unique bands in Flip90-31, Idlep3, idlep2, Idlep4 and Radjas genotypes. The protein banding pattern polymorphism (84%) revealed an inter-genotypic variability. The Idlep3 genotype is characterized by specific bands (or a specific marker), followed by the Flip90-31 genotype. So these two genotypes can be considered as valuable gene resources for further breeding programs.

Efficient In Vitro Regeneration System for Tecoma stans L., Using Shoot Tip and Assessment of Genetic Fidelity Among Regenerants

The present investigation was carried out to develop an efficient regeneration protocol for Tecoma stans L., a valuable antidiabetic plant, using shoot tip explants. The effect of different concentrations (1.0–10.0 µM) of benzyl adenine (BA), kinetin (Kin) or isopentyl adenine (2-iP) either alone or in combination with different concentrations (0.1–2.0 µM) of α-naphthalene acetic acid (NAA) was evaluated for in vitro shoot bud induction and proliferation. Of the tested concentrations, MS medium containing 7.5 µM BA + 0.5 µM NAA proved to be optimal for the maximum (91%) regeneration with mean shoot number (9.8 ± 0.48) and length (4.36 ± 0.33 cm) after 8 weeks of incubation. The best in vitro rooting in regenerated microshoots was achieved on MS medium supplemented with 1.0 µM IBA, which produced a mean root number (6.8 ± 0.58) and length (4.24 ± 0.29 cm) after 4 weeks of incubation. The regenerated plantlets with well-developed roots and shoots were successfully acclimatized in thermocol cups filled with soilrite, for 8 weeks, in growth chamber. Thereafter transfered to field conditions where they grew well in garden soil with 80% survival rate. During the acclimatization period (0–56 days), a subsequent increase in the content of photosynthetic pigments (chlorophyll a, b and carotenoid) was observed up to 56th day. On assessing genetic fidelity among regenerated plants through RAPD analysis, no polymorphism in banding pattern was observed with that of donor plant, thus indicating clonal stability among micropropagated plants.

Coronarin-D Production from Tissue Cultured Plantlets of Hedychium coronarium

Hedychium coronarium known as white ginger lily, an endangered medicinal plant species belonging to family Zingiberaceae. Coronarin D, a labdane diterpene isolated from rhizomes of H.coronarium inhibits NF-kB activation induced by different inflammatory stimuli and carcinogenesis. In the international market the price of coronarin D is USD 260/5 mg. Callus cultures were initiated, plantlets were regenerated and experimental study of in vitro cultures of H. coronarium was done to estimate the highest presence of coronarin D in lines using different growth regulators as compared to that of the mother plant and its genetic study. Explants were inoculated on Murashige and Skoog media and cultures were maintained. The enhanced coronarin D content from matured rhizomes of established cultures were quantified using High Performance Thin Layer Chromatography with the standard. Inter simple sequence repeats study was performed to characterize the plants genetically. The maximum presence of coronarin D content was found to be 3.51 % whereas in control (mother plant) was 2.35 %. The molecular characterization showed no polymorphism in banding pattern as compared to the control. The present protocol reports somaclone with high coronarin D content which could be further analyzed to be used in large scale for commercial exploitation.

Assessment of genetic diversity in 48 landraces of Momordica dioica Roxb. ex Willd. from Odisha, India using RAPD and ISSR markers

Spine gourd (Momordica dioica) is an underutilized cucurbitaceous vegetable crop. Forty eight landraces of spine gourd from Odisha were analyzed for genetic diversity using RAPD and ISSR markers which showed an average variation of 86.20 % polymorphism in banding patterns of the total score of 144 RAPD and 46 ISSR amplicons. Cluster analysis of combined RAPD and ISSR markers revealed that landraces of spine gourd studied from Odisha belongs to three distinct groups i.e. cluster I, II and III. All the landraces showed narrow genetic diversity except cluster-III (OR-2 of Larasara, Sambalpur and OR-9 of Pratappur, Mayurbunj) and cluster II with 7 landraces (OR-4 of Chandanpur, OR-6 of Kakatpur, OR-13 of Chilka, OR-24 of Raghunathpur, OR-32 of Kanpur, OR-33 of Dharmagarh and OR-46 of Tangi) which could be used as a potential source of genetically diverse germplasm of spine gourd for improved crop breeding. The genetic diversity among the landraces might contribute to intraspecific crosses among landraces of this spine gourd collection having high potential for genetic improvement of this vegetable crop.

Molecular Characterization of Commercial Varieties of Phyllanthus emblica Using RAPD and Nuclear rDNA SNPs

The Indian gooseberry, Phyllanthus emblica is an important minor fruit crop having commercial significance. Due to great tolerance to salinity and sodicity it is well adapted to arid conditions of Rajasthan. Genetic diversity studies of eight varieties Anand-2, Banarasi, Chakaiya, Francis, Krishna, Kanchan, NA7 and NA10 were carried out. The morpho-physiological characterization proved insufficient to distinguish studied varieties of P. emblica. Ten RAPD primers exhibited an average of 56.18 % polymorphism in banding pattern and 83.1 % PIC. They can be used as powerful markers to reveal genetic diversity in P. emblica. Present study validates the utility of ITS rDNA region being third generation molecular marker as a reliable indicator of phylogenetic interrelationships, especially for ITS regions as DNA barcode at higher levels can serve as an additional approach for identification and genetic cataloguing of P. emblica germplasm for crop improvement.

Assessment of Genetic Diversity Among Indian Jujube Varieties Based on Nuclear Ribosomal DNA and RAPD Polymorphism

Indian jujube germplasm characterization, evaluation and improvement are fundamentally based on morpho-physiological traits. The lack of break through has been due to under utilization of genetic variability for superior quality and high yield potential. We report the extent of genetic diversity and relationships among 22 putative Ziziphus mauritiana varieties using nuclear ribosomal DNA and RAPD polymorphism. A hybrid protocol of DNA isolation was developed to obtain high molecular weight DNA. Ten RAPD primers generated 101 markers and detected intra-specific variation amounting to 85.15 % polymorphism in banding patterns delineating them into four main clusters and variety Tikadi as a distinct out group. Besides, internal transcribed spacer (ITS) length variations, single nucleotide polymorphism and insertions/deletions (INDELS) in nuclear rDNA region delineated test varieties and revealed lineages with GenBank reference sequences of Z. mauritiana. The novel gene sequences exhibiting nucleotide variations have been submitted to NCBI database and assigned accession numbers from JQ627029 to JQ627050. Higher values of Nei’s genetic diversity (h), Shannon information index (i) and genetic distance analysis validate higher genetic diversity among Indian jujube varieties. The genetically distinct varieties of different phylogenetic clades of RAPD and ITS phylogram can be effectively utilized as parents for crop improvement.

Nuclear DNA, DNA finger printing and essential oil content variation in callus derived regenerants of Curcuma longa L.

Callus cultures were initiated from shoot base explants of turmeric (Curcuma longa L.) on Murashige and skoog (MS) media supplemented with 2 mg/l 2, 4-dichlorophenoxy acetic acid. Plantlets were regenerated from 90 days old calli on MS media supplemented with 3 mg/l benzyl adenine. Regenerated plants were subsequently transferred to the field. 24 regenerants were analyzed for possible occurrence of somaclonal variation through cytophotometric estimation of 4C nuclear DNA content, analysis of essential oil content and molecular characterization. Significant variation was observed in 4C nuclear DNA content with mean value 8.82 pg and 8.33 pg in 2 regenerants. Analysis of essential oil of 3 regenerants showed significant variation with mean value (0.93 %, 0.73 %, 0.8 %) respectively when compared with the mean oil content 0.48 % of source plant. Inter simple sequence repeat (ISSR) based DNA fingerprinting could also detect variation in regenerants with polymorphism in banding pattern as compared to control. These somaclonal variations detected in callus derived regenerants have enough significance for producing improved varieties of turmeric.

Morphology, flow cytometry and molecular assessment of ex-vitro grown micropropagated anthurium in comparison with seed germinated plants

Micropropagated and seed propagated plantlets of anthurium ( Anthurium andreanum Lind. cv. CanCan) were transferred to similar field condition and growth stage. A comparative study was conducted using morphological parameters, ploidy level assessment as well as deoxyribonucleic acid (DNA) fingerprinting using inter simple sequence repeat (ISSR) markers. The in vitro generated anthurium plants exhibited comparable vegetative growth and more sucker production when compared to plants propagated through seeds. No variation in ploidy level was established through flow cytometric study. Genetic assessment through ISSR showed no polymorphism in banding pattern. It was revealed that there was no significant variation between micropropagated and seed propagated plants at ploidy and molecular level assuring the trueness of the micropropagated anthurium clones and their commercial applicability. Key words: Anthurium andreanum Lind, Ex vitro performance, ISSR, morphological competence, ploidy level.

English

Micropropagated and seed propagated plantlets of anthurium (Anthurium andreanum Lind. cv. CanCan) were transferred to similar field condition and growth stage. A comparative study was conducted using morphological parameters, ploidy level assessment as well as deoxyribonucleic acid (DNA) fingerprinting using inter simple sequence repeat (ISSR) markers. The in vitro generated anthurium plants exhibited comparable vegetative growth and more sucker production when compared to plants propagated through seeds. No variation in ploidy level wasestablished through flow cytometric study. Genetic assessment through ISSR showed no polymorphism in banding pattern. It was revealed that there was no significant variation between micropropagated and seed propagated plants at ploidy and molecular level assuring the trueness of the micropropagated anthurium clones and their commercial applicability.   Key words:  Anthurium andreanum Lind, Ex vitro performance, ISSR, morphological competence, ploidy level.

Molecular characterization of Vigna radiata (L.) Wilczek genotypes based on nuclear ribosomal DNA and RAPD polymorphism

Mungbean germplasm characterization, evaluation and improvement are fundamentally based on morpho-agronomic traits. The lack of break-through in mungbean production has been due to non-availability of genetic variability for high yield potential. Forty-four genotypes of mungbean [Vigna radiata (L.)Wilczek] were subjected to random amplified polymorphic DNA (RAPD) analysis to assess the genetic diversity and relationships among the genotypes. Multilocus genotyping by twelve RAPD primers generated 166 markers and detected an average of intraspecific variation amounting to 82% polymorphism in banding patterns. Dendrogram obtained from cluster analysis delineated all the 44 genotypes into six clusters. Higher values of Nei's gene diversity (h) and Shannon information index (i) and genetic distance analysis validate existence of wide genetic diversity among mungbean genotypes tested. Besides internal transcribed spacer (ITS) length variations, single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELS) were detected at number of sites in nuclear rDNA region and the sequences of representatives of each sub-cluster and all distinct genotypes have been submitted to NCBI database and assigned Gen accession numbers HQ 148136-148147. Multiple sequence alignment revealed further lineages of distinct genotypes with main RAPD clusters. The measures of relative genetic distances among the genotypes of mungbean did not completely correlate the geographical places of their development. The homogeneous phenotypic markers proved insufficient in exhibiting genetic divergence among mungbean genotypes studied. RMG-62, RMG-976, and NDM-56 have been identified as potential source of parents for crop improvement. RAPD primers, OPA-9 and OPA-2 as polymorphic genetic markers and number of pods/plant and number of seeds/plant as dependable phenotypic markers have been identified for improving yield potentials. This genetic diversity will be of significance in developing intraspecific crosses in mungbean crop improvement programme.

Assessment of Genetic Diversity in Trigonella foenum-graecum Based on Nuclear Ribosomal DNA, Internal Transcribed Spacer and RAPD Analysis

Fenugreek (Trigonella foenum-graecum) is receiving global attention due to rare medicinal properties of significance to human health. Gene banks possess scanty germplasm and very little background information regarding its genetic variability that has hampered its improvement. We investigated the extent of variability among 17 Indian varieties of fenugreek using phenotypic and genetic markers. Multilocus genotyping by ten random amplified polymorphic DNA (RAPD) primers detected an average of intraspecific variations amounting to 64.7% polymorphism in banding patterns. Analysis of molecular variance indicated that a greater proportion of total genetic variation exists within population (91%) rather than among populations. Higher values of Nei’s gene diversity (h) and Shannon Information Index (i) and genetic distance analysis validate higher genetic diversity among Indian fenugreek varieties. SNPs at 14 sites of rDNA region revealed further lineages of distinct varieties with main RAPD clusters. The representative sequences of each subgroup and all distinct varieties have been submitted to NCBI database and assigned Gen Accession numbers HM 176640–176649. The measures of relative genetic distances among varieties of fenugreek did not completely correlate with the geographical distances of places of their development. The homogeneous phenotypic markers proved insufficient in exhibiting genetic divergence among fenugreek varieties studied. Eventually, the knowledge of their genetic relationships, DNA bar coding and phylogenies might contribute for the designing of intraspecific crosses between cultivars of this fenugreek collection with potential interest in seed spices breeding programme.

Systematic revision of the arboreal snail Satsuma albida species complex (Mollusca: Camaenidae) with descriptions of 14 new species from Taiwan

The taxonomy of the endemic arboreal snail Satsuma albida species complex from Taiwan was unclear due to the animals' highly similar morphology, and their nocturnal and strict arboreal behaviour, leading to difficulties in collecting living specimens. This article is the first comprehensive comparative study on the systematics and taxonomy of this species complex using external morphology, anatomy of the reproductive system and molecular phylogeny. Consequently, two subspecies of S. albida are raised to species status, namely S. insignis and S. mollicula. Fourteen new species are also described. Fourteen of the 17 species showed polymorphism in banding pattern amongst populations and other species retained the whitish unity as seen in S. albida. Distributions of almost all taxa are geographically limited, with the exception of S. polymorpha sp. nov. The phylogeny of these species was reconstructed using 20 morphological characters and molecular data from the partial sequences of mtDNA CO1 and 16S rRNA genes, and the complete ITS2 sequence. The molecular phylogeny revealed three subclades (west, east and polymorpha clade) and revealed that these snails are monophyletic, originating from a ground-dwelling ancestor. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 154, 437–493.

Giemsa C-banded karyotypes of three subspecies of Taeniatherum caput-medusae and of two intergeneric hybrids with Psathyrostachys spp. (Poaceae)

Three subspecies of Taeniatherum caput-medusae (2n=2x=14) had the same karyotype with five pairs of metacentrics, one pair of submetacentrics, and one pair of metacentric SAT-chromosomes with small satellites. The rather similar C-banded karyotypes were characterized by banding patterns with a limited number of mostly small or very small bands randomly distributed. Plants of the same population had identical banding patterns. Banding pattern polymorphism was prevalent, but apparently no larger among than within taxa. The similarity of the C-banded karyotypes supports a close relationship of taxa. Differences in chromosome size and C-banding patterns identified parental genomes in two Taeniatherum×Psathyrostachys hybrids. Psathyrostachys SAT-chromosomes exerted nucleolar dominance. A hierarchy of nucleolar dominance: Hordeum vulgare > P. fragilis > T. caput-medusae is indicated.

Giemsa C-banded karyotypes of Hordeum secalinum, H. capense and their interspecific hybrids with H. vulgare

The European H. secalinum (2n=4x=28) and the South African H. capense (2n=4x=28) had similar karyotypes with ten pairs of metacentrics, three of submetacentrics, and one of SAT-chromosomes. The C-banded karyotypes of H. secalinum from northern Europe were characterized by banding patterns with few bands, those of H. secalinum from Spain and H. capense by banding patterns with more bands. The bands were mostly small or very small and had no preferential disposition. Intraplant banding pattern polymorphism was observed in North European H. secalinum, in agreement with an outbreeding reproductive system. No banding pattern polymorphism was present within plants of H. secalinum from Spain and H. capense, suggesting self-pollination. In both species banding pattern polymorphism was prevalent among plants. Together with other evidence the fairly similar basic C-banded karyotypes of the two species indicate a rather close relationship.

Comparison of the Giemsa C-banded karyotypes of Dasy-pyrum villosum (2x) and D. breviaristatum (4x) from Greece

The karyotypes of one plant of Dasypyrum villosum (2n = 2x= 14), of two plants of D. breviaristanam (2n = 4x = 28) and an intraspecific hybrid, and of a D. breviaristatum x D. villosum hybrid were studied by Giemsa C-banding of somatic metaphases and Giemsa and AgNO3-staining of interphases. The karyo-type of D. villosum had 10 metacentrics, 2 submetacentries, and 2 metacentric SAT-chromosomes with small satellites. The C-banding patlerns were characterized by conspicuous distal and/or terminal, in some cases double bands in most chromosome arms, and inconspicuous centromeric and juxtacentromeric bands, D. breviaristatum had two cytotypes. one with 20 metacentrics, 2 submetacentrics. and 6 metacentric SAT-chromosomes, 4 similar ones with Large and 2 with small satellites. In the other cytotype the latter pair was replaced by a pair without visible satellites. The banding patterns had most conspicuous bands at centromeric or proximal positions in about half the chromosomes. Banding pattern polymorphism was observed in both species in agreement with their outbreeding nature. Reliable identification of homology was only possible in D. villosum. In D. breviaristatum it was prevented by the overall similarily of banding patterns and chromosome morphology, but genomic relationships of chromosomes was established in the intraspecific hybrid. The C-banded karyolype of D. breviaristatum did not support an autoploid origin based on D. villosum.

Giemsa C-Banded Karyotypes of two Cytotypes (2x, 4x) of Psathyrostachys Lanuginosa (Poaceae; Triticeae)

The karyotypes of the diploid (2n = 2x = 14) and tetraploid (2n = 4x = 28) cytotypes of the outbreeding P. lanuginosa collected from one population in China were investigated by Giemsa C- and N-banding and AgNO3 staining. The complement of the diploid consisted of 14 large chromosomes: 8 metacentrics and 6 SAT-chromosomes, one metacentric and 2 submetacentric pairs, with small to minute, polymorphic, completely heterochromatic satellites. Except that only eight SAT-chromosomes were observed, the complement of the tetraploid matched a doubling of that of the diploid and indicated an autoploid origin. The existence of 12 chromosomes with nucleolus organizers was established through AgNO3 staining demonstrating nucleoli. C-banding patterns comprised from zero to three (2x), or four (4x) small bands per chromosome giving a low content of constitutive heterochromatin (< 3 percnt;). The level of banding pattern polymorphism was low. N-banding produced no bands. Variation in the size of nucleoli demonstrated a differential nucleolus forming capacity of nucleolus organizers among SAT-chromosomes. The basic karyotype was similar to those of P. fragilis, P. juncea and P. stoloniformis, indicating a close relationship and the presence of genome N. Meiotic analysis suggested the presence of a chromosomal rearrangement in the diploid. The frequency of interstitial chiasmata was high. Differences in pollen diameters differentiated the two cytotypes.

The Giemsa C-banded Karyotype of Crithopsis Delileana (Poaceae; Triticeae)

Three populations of Crithopsis delileunu had similar karyotypes. They included 14 large chromosomes; five pairs of metacentrics, one pair of submetacentrics, and one pair of metacentric SAT-chromosomes with rather small satellites. The karyotypes of two populations were investigated in detail through application of Giemsa C-banding, Giemsa N-banding and AgN0,-staining to meta- and interphases. The C-banding patterns included a few small to very small, mainly centromeric or telomeric bands. The chromosomes of the two populations were differentiated by banding pattern polymorphism. Banding patterns identified homologous and discriminated among non-homologous chromosomes within the same population. The scarcity of diagnostic bands prevented identification of homologous chromosomes between populations beyond the level obtained from morphology, i.e., of submetdcentrics and SAT chromosomes. N-banding produced no or few weak bands. Silver nitrate staining produced two NORs and two nucleoli corresponding to the number of SAT-chromosomes. The data give some support to a hypothesis of a closer relationship between the genera Critlzopsis and Tueniutherum, than between either of these and the genera Psuthyrostuchys and Hordeum.

The Karyotype of Elymus Mendodnus (Poaceae; Triticeae) from Argentina studied by Banding Techniques

The karyotype of Elymus mendodnus (2n = 8x = 56) was investigated by Giemsa C-banding and N-banding, and AgNO3 staining. The chromosomes were large (7–13 μm). The chromosome complement consisted of 22 metacentric and 6 submetacentric pairs. Two metacentric and one submetacentric pairs had satellites on the short arms. One of the metacentric pairs had large satellites. The other satellites were small. The number of SAT-chromosomes was verified through AgNO3 staining confirming the presence of up to 6 nucleoli at interphase. The C-banding pattern comprised small to large C-bands at telomeric positions in all short arms except one and in most of the long arms. A few, mostly small bands were located at distal, intercalary positions. The small satellites were entirely heterochromatic. The amount of constitutive heterochromatin (14 %) was among the highest recorded in the Triticeae. Except in 12 chromosome pairs, incl. the 3 satellited ones, the overall similarity of the C-banding patterns of most chromosomes prevented a safe identification of homologues. Additionally, the level of identified banding pattern polymorphism was low, but supports an outbreeding nature of the species. N-banding produced no bands. A similar distribution of large C-bands and lack of N-banding have previously been observed in Psathyrostachys huashanica. This agrees with the presence of the Ns genome and the absence of the H genome in E. mendodnus (DUBCOVSKY et al. 1997), and suggests the absence of the pivotal St genome of Elymus. However, lacking further evidence we propose to retain the species in Elymus.

DNA banding pattern polymorphism in vancomycin-resistant Enterococcus faecium and criteria for defining strains.

The degree of DNA banding pattern polymorphism exhibited by vancomycin-resistant Enterococcus faecium (VREM) strains isolated on a renal unit over an 11-month period was investigated. Thirty VREM strains from different patients were analyzed by pulsed-field gel electrophoresis (PFGE; with extended run and optimal pulse times), ribotyping, plasmid profile analysis, biotyping, pyrolysis mass spectrometry, and antibiogram analysis. PFGE resolved 17 banding patterns which formed four distinct clusters at the 82% similarity level. Intercluster band differences ranged from 14 to 31 bands. The strains in one cluster, which contained seven patterns that differed from each other by one to seven bands and from the common pattern by five bands, were confirmed to be a single strain by four of the five other typing methods. The strains in a second cluster with eight patterns, which differed from each other by 1 to 12 bands, contained two subclusters. This subdivision was supported by ribotyping and biotyping. However, it was unclear whether these subclusters represented distinct strains. In one strain, marked polymorphism (patterns that differed from each other by up to four bands) was observed in the ribotype pattern. This study demonstrates the high degree of DNA banding pattern polymorphism found for some strains of VREM and illustrates the complexity involved in defining such strains.