Inter-simple Sequence Repeat Assay Research Articles

Isolation, characterization, and fingerprinting of some multidrug resistance clinical isolates from patients in Al-Qaseem Hospitals, Saudi Arabia

The aim of this study was to investigate the prevalence of antibiotic resistance genes in bacteria isolated from Saudi Arabian patients in the Al-Qassim region. We also examine the application of Random Amplified Polymorphic DNA- Polymerase chain reaction (RAPD-PCR) in genetic diversity of the strains in use. In this study, we have used 29 different clinical samples from Al-Qassim hospitals which consists of wound swabs, blood, sputum and urine. Clinical samples were streaked onto sterile Petri dishes containing nutrient agar media using sterile dry swabs. Additionally antibiotic sensitivity tests, micro scan walkway susceptibility tests and extraction of genomic DNA, amplification of RAPD and Inter simple sequence repeat assay were performed in this study. This study results confirmed from the clinical samples were streaked onto sterile Petri dishes containing nutrient agar media using sterile dry swabs. The PCR analysis in ISSR was confirmed that 30–70% of E. coli in Asia and Africa carried ESBLs in the GLASS 2020 study, which is on par with the 59% in VINARES 2016–2017. Comparing VINARES 2016–2017 to other nations, the prevalence of ESBL carriage among K. pneumoniae was 35%. However, RAPD results showed that all employed strains and primers could successfully fingerprint DNA. Between isolated bacterial strains, similarity dendrograms were produced. The technique generated bands with the same intensity as the typical PCR carried out using pure DNA, and it worked for all 29 bacterial strains examined. In conclusion, the results show that all of the isolates have at least one antibiotic-resistance gene, and that the PCR approach is a quick, easy, and reliable way to identify genes.

Genetic diversity and relationship analyses of mango (Mangifera indica L.) germplasm resources with ISSR, SRAP, CBDP and CEAP markers

Forty-eight mango accessions were compared for genetic diversity and relationships based on four molecular marker assays, namely, inter-simple sequence repeat (ISSR), sequence-related amplified polymorphism (SRAP), CAAT-box derived polymorphism (CBDP), and cis-element amplified polymorphism (CEAP). Comparative analyses of the marker assays based on Nei's genetic diversity (H), Shannon's information index (I), polymorphism information content (PIC), the marker index (MI), and resolving power (Rp) revealed that (i) the CEAP markers provided the most information according to the PIC, MI and Rp values; (ii) the CBDP and ISSR markers exhibited valuable information according to the H and I values. The correlation values (r) of the Mantel test ranged from 0.334 (ISSR and CEAP) to 0.819 (CEAP and the combination of the four marker assays). The unweighted pair group method with arithmetic mean (UPGMA) cluster results showed that a single-marker assay can accurately reflect the genetic relationships of some mango samples but cannot accurately reflect the genetic relationships of all mango samples and that when multiple markers are used to evaluate the genetic relationships of mango germplasm resources, the results are more accurate than those of a single-marker assay. The combined cluster analysis of the molecular marker types shows higher discrimination according to genetic relationships. Nine mango accessions were suggested to be likely parental or sister lines based on the combination of the ISSR, SRAP, CBDP, and CEAP marker assays.

Investigation of the genetic variation of anise (Pimpinella anisum L.) using RAPD and ISSR markers

Anise (Pimpinella anisum L.) is an annual spice and medicinal plant belonging to the family Apiaceae. Only a limited number of anise breeding studies are available. Therefore, genetic variation among anise landraces has not been extensively examined with molecular markers. In this study, the molecular diversity of 15 Turkish anise landraces collected from different regions of Turkey and four foreign landraces obtained from Cyprus, Syria and France were analyzed using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. The discriminatory power of these markers was assessed by calculating various marker parameters, namely percent polymorphism, polymorphism information content (PIC), resolving power (RP), and marker index (MI). The genomic DNA of 19 anise landraces was amplified with nine RAPD primers that generated 71 polymorphic bands and five ISSRs primers that produced 45 polymorphic bands. The percentage of polymorphic loci was 59.8% in RAPD profiling and 51.5% in ISSR profiling. The average PIC values obtained using RAPD and ISSR markers were 0.40 and 0.38, respectively. The mean RP (2.90 and 2.67) and MI values (1.23 and 1.05) of the RAPD assay were found to be somewhat higher than those of the ISSR assay, but ISSR was more favorable than the RAPD techniques in terms of presenting the phylogenetic relationships in anise landraces. A UPGMA cluster grouped the 19 anise landraces into two major clusters based on both markers. The RAPD and ISSR marker systems were found to be useful for determining genetic diversity in P. anisum L. and identifying variation among landraces.

Evaluation of genotoxicity of the aqueous extracts from wild Artemisia herba-alba and Jasmina montana against Zucchini yellow mosaic virus (ZYMV) in Cucurbita pepo plants

Squash or cucurbit (Cucurbita pepo) is one of the famous and important vegetable plants in most world countries. The squash is infected by the critical Zucchini yellow mosaic virus (ZYMV) in Egypt. Though pesticides can protect the plants of pest infections, there are no effective compounds that can be applied as virucides. In this work, the effects of aqueous extracts from Jasmina montana and Artemisia herba-alba on ZYMV infection in the squash plants were examined. In addition, SDS-PAGE protein patterns and enzyme activities were evaluated and induction of resistance by plant elicitors against ZYMV was studied The results observed that using of the aqueous extracts from A. herba-alba or J. montana extracts prior to ZYMV inoculation recorded 100% inhibition of virus infection. Also, complete inhibition was obtained by treatment of A. herba-alba extract with the virus inoculum led to destroy the virus particles due to presence of the effective compounds (terpens, phenolics and the essential oils) in the plant extract. SDS-PAGE protein profiles and enzyme activities were studied in treated and untreated plants. Genomic DNA variation was studied using random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) loci. The alterations in RAPD and ISSR assays of representative squash plants can be applied to comprehend of induced systemic resistance. These inducers stimulated resistance in the squash plants. Therefore, elicitors should be taken into consideration in the breeding programs for ZYMV control.

Use of promoter-anchored rapd analysis for divergence assessment in soybean as against conventionally used ISSR assay

The present study was aimed to compare the genetic diversity among 24 soybean genotypes as revealed by Promoter-anchored amplified polymorphism based on RAPD (PAAP-RAPD) primer-combinations analysis and Inter Simple Sequence Repeat (ISSR) analysis. All the 16 PAAP-RAPD primer-combinations, derived from 4 Promoter-based primers (TA1/G1/CA1/GC1) and 4 RAPD primers (OPE 1/5/7/9) showed amplification, of which 8 were polymorphic. They produced 63 loci, of which 52 were polymorphic. All 20 ISSR primers used were polymorphic generating 115 alleles of which 95 were polymorphic. The similarity coefficient based on ISSR and PAAP-RAPD analysis ranged from 0.67 to 0.93 and 0.41 to 1.00, respectively. The PAAP-RAPD consensus tree revealed 17 clusters; while ISSR revealed all genotypes as separate clusters. In 2D scatter plot, these were broadly classified into 5 major groups. Group-Ia had lowest divergence (MAUS71, KDS 344, KDS387, NRC94, KDS722, Bragg and KDS743) while Group-Ib had low divergence (Birsa Soya1, KDS749, KDS726, KDS739 and JS335). Moderate divergence was observed in Group II (JS9305 and RKS 18) and Group III (Himso 1684, Dsb22 and MACS450). Group IV (KDS741, VLS65, Rpp3 PI462312 variant, EC241778 and PI462312) had highest divergence. Only two genotypes (KDS753 and DS228) were placed separately in both the analyses. The study revealed that PAAP-RAPD has an advantage that it may specifically target regulatory regions of various candidate genes during molecular tagging for a specific trait.

Genetic characterization of microsporidians infecting Indian non-mulberry silkworms (Antheraea assamensis and Samia cynthia ricini) by using PCR based ISSR and RAPD markers assay

This study established the genetic characterisation of 10 microsporidian isolates infecting non-mulberry silkworms (Antheraea assamensis and Samia cynthia ricini) collected from biogeographical forest locations in the State of Assam, India, using PCR-based markers assays: inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD). A Nosema type species (NIK-1s_mys) was used as control for comparison. The shape of mature microsporidian spores were observed oval to elongated, measuring 3.80 to <TEX>$4.90{\mu}m$</TEX> in length and 2.60 to <TEX>$3.05{\mu}m$</TEX> in width. Fourteen ISSR primers generated reproducible profiles and yielded 178 fragments, of which 175 were polymorphic (98%), while 16 RAPD primers generated reproducible profiles with 198 amplified fragments displaying 95% of polymorphism. Estimation of genetic distance coefficients based on dice coefficients method and clustering with un-weighted pair group method using arithmetic average (UPGMA) analysis was done to unravel the genetic diversity of microsporidians infecting Indian muga and eri silkworm. The similarity coefficients varied from 0.385 to 0.941 in ISSR and 0.083 to 0.938 in RAPD data. UPGMA analysis generated dendrograms with two microsporidian groups, which appear to be different from each other. Based on Euclidean distance matrix method, 2-dimensional distribution also revealed considerable variability among different identified microsporidians. Clustering of these microsporidian isolates was in accordance with their host and biogeographic origin. Both techniques represent a useful and efficient tool for taxonomical grouping as well as for phylogenetic classification of different microsporidians in general and genotyping of these pathogens in particular.

ISSR에 의한 잔디속 식물의 DNA 다형성과 유전적 관계 평가

The genetic variability of four species of the genus Zoysia collected from South Korea was analyzed using an inter-simple sequence repeat (ISSR) marker system. Polymerase chain reactions (PCR) with eight ISSR primers generated 86 amplicons, 76 (87.1%) of which were polymorphisms. The polymorphism information content (PIC) value of the ISSR marker system was 0.848. The percentage of polymorphic loci (Pp) ranged from 41.2% to 44.7%. Nei’s gene diversity (H) ranged from 0.149 to 0.186, with an average overall value of 0.170. The mean of Shannon’s information index (I) value was 0.250. Total genetic diversity values (H T ) varied between 0.356 (ISSR-1) and 0.418 (ISSR-16), for an average overall polymorphic loci of 0.345. Interlocus variation in within-species genetic diversity (H S ) was low (0.170). On a per-locus basis, the proportion of total genetic variation due to differences among species (G ST ) was 0.601. This indicated that about 60.1% of the total variation was among species. Thus, about 39.9 of genetic variation was within species. The estimate of gene flow, based on GST, was very low among species of the genus Zoysia (N m = 0.332). The phylogenic tree showed three distinct groups: Z. macrostachya and Z. tenuifolia clades and other species were formed the separated clusters. In conclusion, the ISSR assay was useful for detecting genetic variation in the genus Zoysia, and its discriminatory power was comparable to that of other genotyping tools.

Efficiency of ISSR and RAPD markers in genetic divergence analysis and conservation management of Justicia adhatoda L., a medicinal plant

Genetic variation within and among population is the basis for survival of the population both in short and long term. Thus, studying the plant genetic diversity is essential for any conservation program. Indigenous medicinal plants like Justicia adhatoda L. which are facing high rate of depletion from the wild population need immediate attention. DNA-based dominant molecular marker techniques, random amplification of polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) were used to unravel the genetic variability and relationships across thirty-two wild accessions of J. adhatoda L., a valuable medicinal shrub widespread throughout the tropical regions of Southeast Asia. Amplification of genomic DNA using 38 primers (18 RAPD and 20 ISSR) yielded 434 products, of which 404 products were polymorphic revealing 93.11 % polymorphism. The average polymorphic information content value obtained with RAPD and ISSR markers was 0.25 and 0.24, respectively. Marker index (RAPD = 3.94; ISSR = 3.53) and resolving power (RAPD = 4.24; ISSR = 3.94) indicate that the RAPD markers were relatively more efficient than the ISSR assay revealing the genetic diversity of J. adhatoda. The Shannon diversity index obtained with RAPD and ISSR markers was 0.40 and 0.38, respectively. The similarity coefficient ranged from 0.26 to 0.89, 0.33 to 0.93 and 0.31 to 0.90 with RAPD, ISSR and combined UPGMA dendrogram, respectively. PCA derived on the basis of pooled data of both the markers illustrated that the first three principal coordinate components accounted 79.27 % of the genetic similarity variance. The mantel test between two Jaccard’s similarity matrices gave r = 0.901, showing the fit correlation between ISSR- and RAPD-based similarities. Based on the results, ex-situ methods may be the most suitable and efficient measure for long-term conservation.

Molecular variability of plantain ecotypes from the genus Musa (Musaceae)

Twelve plantain ecotypes were characterized for molecular variability using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. Out of the 60 RAPD and 40 ISSR primers tested, 16 RAPD and 14 ISSR primers were found polymorphic, and were used for molecular profiling of the plantain ecotypes. The average number of bands per primer, average number of polymorphic bands per primer, mean polymorphic information content (PIC) value, resolving power (Rp), and marker indices (MI) for RAPD primers were 8.63, 3.25, 0.87, 15.49, and 2.86, whereas the respective values for the ISSR assay were 7.93, 4.14, 0.85, 12.49, and 3.55. The genetic similarity coefficient was calculated using the Jaccard coefficient. The unweighted pair group method with arithmetic averages (UPGMA)-based clustering pattern remained more or less similar for RAPD, ISSR, and combined RAPD and ISSR data. Clustering was strongly supported by high bootstrap values. The genotypes Njockkon and Changalikodan (similarity coefficient > 0.94) and Manjeri Nendran (a) and Manjeri Nendran (b) (similarity coefficient > 0.89) were more closely related than the other ecotypes. The principal component analysis (PCA) showed results similar to those of the dendrogram. The results revealed that ISSR would be a better tool than RAPD for evaluation of genetic diversity in plantain ecotypes. Further, huge variability exists among the plantain ecotypes, which can be utilized for germplasm collection, in situ conservation, and development of high yielding nutritionally superior cultivars in Musa L. breeding programs.

Genotoxicity of 1-1-Butyl-3-Methylimidazolium Ionic Liquids on Pakchoi (&lt;i&gt;Brassica campestris&lt;/i&gt; L. ssp. Chinensis) Using ISSR Markers

Ionic liquids (ILs) are one class of important industrial chemicals that may replace conventional organic solvents. Due to the solubility, the toxicity study of ILs focused on aquatic mediums. However, there is seldom research for the the toxicity on terrestrial organism. In this paper, we studied the toxicity of three imidazolium ionic liquids 1-butyl-3-methyl imidazolium chloride ([Bmim][CL]), 1-butyl-3-methyl imidazolium tetrafluoroborate ([Bmim][BF4]), and 1-butyl-3-methyl imidazolium trifluoroacetate ([Bmim][CF3COOH]) on seed germination and seedling growth of pakchoi. Inter-simple sequence repeat (ISSR) marker technique was applied to detect DNA damage induced by imidazolium ILs. The results showed that all of three ILs could inhibit seed germination and seedling growth of pakchoi significantly at higher concentration (≥16 mM/L). At same concentration (1 mM/L and 4 mM/L), the genotoxicity of three ILs was following the trends: [Bmim][BF4]&gt; [Bmim][CF3COOH]&gt; [Bmim][CL]. It was concluded that ISSR assay offered a useful way to detect toxicity of ILs contamination on plant at low concentration even without transformation of physiological feature, such as germination and growth parameters and so on.

English

Amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat (ISSR) markers were used to study the DNA polymorphism in elite mungbean genotypes. A total of nine AFLP primer combination and 22 ISSR primers were used. Amplification of genomic DNA of the 30 genotypes, using AFLP analysis, yielded 300 fragments that could be scored, of which 192 were polymorphic, with an average of 21.3 polymorphic fragments per primer. Number of amplified fragments with AFLP primers ranged from 29 (E-AAC: M-CAG) to 10 (E-ACG: M-CAT). Percentage polymorphism ranged from 46.3% (E-AAC: M-CCA) to a maximum of 100% (E-AAC: M-CAC), with an average of 64%. The 22 ISSR primers used in the study produced 108 bands across 30 genotypes, of which 68 were polymorphic. The number of amplified bands varied from two UBC820) to ten URP 6F). The average numbers of bands per primer and polymorphic bands per primer were 4.9 and 3.1, respectively. Percentage polymorphism ranged from 25% (UBC844) to 85% (UBC846, UBC864, UBC895), with an average percentage polymorphism of 58.3% across all the genotypes. AFLP markers were more efficient than the ISSR assay, as they detected 64% polymorphic DNA markers in Vigna radiata as compared to 58.3% for ISSR markers. The Mantel test between the two Jaccard&#39;s similarity matrices gave r = 0.19, showing low correlation between AFLP- and ISSR-based similarities. Clustering of genotypes within groups was not similar when AFLP and ISSR derived dendrograms were compared. Key words: AFLP, ISSR, Vigna radiata (mung bean), marker index, unweighted pair-group method with arithmetic averages (UPGMA).

Use of Intersimple Sequence Repeat Assay for Detection of DNA Polymorphism Induced by Gamma Rays in Curcuma alismatifolia

In plant breeding programs with vegetatively propagated plants, induced mutations with low physiological effects and strong genetic effects are important approaches for broadening the genetic variation. In the present study, intersimple sequence repeat (ISSR) assays were used to identify DNA polymorphism among the mutant varieties of Curcuma alismatifolia (Chiang Mai Red, Sweet Pink, Kimono Pink) and one hybrid (Doi Tung 554) exposed to 0-, 10-, and 20-Gy optimum doses of acute gamma irradiation. To identify DNA polymorphism among the mutants (10 and 20 Gy) and non-treated (0 Gy) samples through ISSR assay, a total of 25 random primers were screened and 14 gave reproducible polymorphic bands. The number of presumed alleles revealed by the ISSR analysis ranged from three to 19 alleles with mean values of 6.5, 7.8, and 9.2 alleles per locus for doses of 0 (control), 10, and 20 Gy, respectively. The average effective number of alleles, Nei’s gene diversity, Shannon’s information index, and polymorphic information content (PIC) were 1.41 to 1.48, 0.24 to 0.33, 0.35 to 0.45, and 0.21 to 0.25 for the three doses, respectively. Percentages of polymorphic loci for non-irradiated (0 Gy), 10 Gy, and 20 Gy individual plants were 76.4%, 83.7%, and 85.8%, respectively, with an average of 81.9%. Jaccard’s genetic similarity coefficient varied from 0.19 to 1.0 indicating the level of genetic variation among the mutants studied. In terms of variety, the range of genetic variation (0.27 to 0.4) among individuals of the Doi Tung population was more than that observed in the other three varieties. In a dendrogram constructed based on genetic similarity coefficients, the 44 C. alismatifolia individual plants fell into seven major clusters at a coefficient level of 0.35. The study revealed that DNA polymorphism detected by ISSR analysis offered a useful molecular marker for the identification of mutants in gamma radiation-treated plants. Additionally, this research demonstrated that the DNA of the hybrid (Doi Tung 554) showed a greater response in induced mutation compared with the other varieties.

ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793

With the current trends in high density plantations of fruit trees, numerous clonal rootstocks of apple have been developed through various breeding programs. Among them, Merton 793 is the most popular in India because of the desirable traits of vigorous growth and resistance to woolly apple aphid and collar rot. The planting material of this rootstock cannot be multiplied at a desirable rate by means of conventional vegetative propagation methods, so micropropagation techniques are being explored to augment scarce planting material. Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, it is advisable to check the clonal fidelity of in vitro raised plants, especially of perennials prior to their field transplantation. The genetic stability of in vitro raised plants of apple rootstock Merton 793, multiplied through enhanced axillary bud proliferation up to 22 subculture passages, was tested by intersimple sequence repeat (ISSR) assay. Of 24 ISSR primers screened, 15 primers produced clear reproducible bands, resulting in a total of 134 distinct bands with an average of 8.9 bands per primer. Apple rootstock MM 111 and scion Jonathan, taken as outliers with tissue culture-raised progenies of Merton 793, ruled out the possibility that the invariant banding pattern occurred because of inefficiency of ISSR primers in detecting variations. The homogenous amplification profile observed for all the micropropagated plants compared to the donor plant confirmed the clonal fidelity of the tissue culture-raised Merton 793 plants. This suggests that axillary bud multiplication is the safest mode for multiplication of true-to-type plants. This is the first study that evaluates the applicability of ISSR markers in establishing clonal fidelity of tissue culture-raised apple plants.

Comparison of RAPD and ISSR markers for genetic diversity analysis among different endangered Mangifera indica genotypes of Indian Gir forest region

The genetic variability and relationships among 20 Mangifera indica genotypes representing 15 endangered and 5 cultivars, obtained from Indian Gir forest region, were analyzed using 10 random amplified polymorphic DNA (RAPD) and 21 inter simple sequence repeat (ISSR) markers. RAPD markers were more efficient than the ISSR assay with regards to polymorphism detection. Also, the average numbers of polymorphic loci per primer, average polymorphic information content (PIC) and primer index (PI) values were more for RAPD than for ISSR. But, total number of genotype specific marker loci, Nei’s genetic diversity (h), Shannon’s information index (I), total heterozygosity (Ht), average heterozygosity (Hs) and mean coefficient of gene differentiation (Gst) were more for ISSR as compared to RAPD markers. The regression test between the two Nei’s genetic diversity indexes showed low regression between RAPD and ISSR based similarities but maximum for RAPD and RAPD + ISSR based similarities. The pattern of clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared. Thus, both the markers were equally important for genetic diversity analysis in M. indica.

Genetic Variability of Thermal Nymphaea (Nymphaeaceae) Populations Based on ISSR Markers: Implications on Relationships, Hybridization, and Conservation

The globally widespread genus Nymphaea exhibits a wide range of morphological and taxonomical diversity. The intrusion of a cultivated variety by progressive propagation and its affect on aquatic habitat is demonstrated in this case study. We have studied the genetic diversity, population, and stand structure of the neophyte Nymphaea × ‘Panama Pacific’ as well as other species found in Lake Hévíz and dikes nearby using inter-simple sequence repeat (ISSR) markers. The ISSR assay revealed a low genetic variability for the small populations of Nymphaea caerulea, Nymphaea lotus var. thermalis, and a medium level for Nymphaea alba, Nymphaea rubra var. longiflora, and Nymphaea × ‘Panama Pacific’. The evolutionary genetic status of individuals found in the overlapping cultivation area of Nymphaea × ‘Panama Pacific’ and N. caerulea was affirmed to be of hybrid origin by reticulate network analysis and with morphological parameters. The Bayesian analysis of hybrid classes and the segregation of the ISSR markers also confirmed the hybrid origin of the individuals in question and revealed that they are falling into F2 or latter genotype frequency classes, indicating the viability and fertility of the hybrids. The set of analyzed species by phylogenetic network analysis of ISSR data has been divided into three major groups according to their evolutionary patterns (subg. Barachyceras, Lotos, and Nymphaea). Our results are in accordance with these three major subgenera within Nymphaea.

Efficiency of RAPD and ISSR markers in assessing genetic diversity and relationships in black gram (Vigna mungo L. Hepper) vari

The DNA fingerprinting methodologies, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), were used to estimate genetic diversity and relationships among 20 black gram (Vigna mungo L. Hepper) varieties. Thirty selected RAPD primers amplified 255 bands, 168 of which were polymorphic (66.5%). On average, these primers produced 8.5 bands, 5.6 of which were polymorphic. Polymorphic band number varied from 2 (A-05) to 10 (OPA-02), with sizes ranging from 100 to 2550 bp. Twenty-four selected ISSR primers produced 238 amplified products, 184 of which were polymorphic (77.8%). On average, these primers generated 9.8 bands, with 7.7 polymorphic bands ranging in number from 4 (ISSR-13) to 11 (ISSR-03), and size from 100-2650 bp. Genetic relationships were estimated using similarity coefficient (Jaccard’s) values between different accession pairs; these varied from 30.7 to 85.0 for RAPD, and from 37.2 to 88.4 with ISSR. UPGMA analysis indicated that the varieties ranged in similarity from 0.50 to 1.00 (mean of 0.75) for RAPD, and from 0.47 to 1.00 (mean of 0.76) with ISSR. Cluster analysis of RAPD and ISSR results identified three clusters with significant bootstrap values, which revealed greater homology between the varieties. Principal coordinates analysis also supported this conclusion. Among the black gram varieties, WBU-108 and RBU-38 were highly divergent, whereas LBG-648 and LBG-623 were genetically similar. The markers generated by RAPD and ISSR assays can provide practical information for the management of genetic resources and these results will also provide useful information for the molecular classification and breeding of new black gram varieties.Key words: Black gram, cluster analysis, genetic diversity, ISSR, molecular markers, RAPD

A Comparative Analysis of ISSR and RAPD Markers for Study of Genetic Diversity in Shisham (Dalbergia sissoo)

Shisham (Dalbergia sissoo) is one of the most preferred timber tree species of South Asia. Two DNA-based molecular marker techniques, intersimple sequence repeat (ISSR) and random amplified polymorphism DNA (RAPD), were compared to study the genetic diversity in this species. A total of 30 polymorphic primers (15 ISSR and 15 random) were used. Amplification of genomic DNA of 22 genotypes, using ISSR analysis, yielded 117 fragments, of which 64 were polymorphic. Number of amplified fragments with ISSR primers ranged from five to ten and varied in size from 180 to 1,900 bp. Percentage polymorphism ranged from 0 to 87.5. The 15 RAPD primers produced 144 bands across 22 genotypes, of which 84 were polymorphic. The number of amplified bands varied from five to 13, with size range from 180 to 2,400 bp. Percentage polymorphism ranged from 0 to 100, with an average of 58.3 across. RAPD markers were relatively more efficient than the ISSR assay. The mental test between two Jaccard’s similarity matrices gave r ≥ 0.90, showing very good fit correlation in between ISSR- and RAPD-based similarities. Clustering of isolates remained more or less the same in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.734 to 0.939, 0.563 to 0.946, and 0.648 to 0.920 with ISSR, RAPD, and combined dendrogram, respectively.

Inter-simple sequence repeat (ISSR) variation in populations of the cutgrass Leersia hexandra

Genetic variability of a perennial clonal aquatic weed Leersia hexandra was estimated by the ISSR assay. A total of 225 L. hexandra individuals collected from 10 populations in China were analyzed. The 12 used ISSR primers generated 175 bands, with an average of 14.6 per primer. The average values of percentage of polymorphic bands (PPB), effective number of alleles ( A e), and gene diversity ( H e) in these populations were 49.7%, 1.258, and 0.154, respectively, indicating a considerable genetic diversity in this species. Significant genetic differentiation was found among populations with the major genetic variability within populations based on Nei's genetic diversity analysis ( G st = 0.417) and AMOVA (40.1%). Genetic variation is independent of geographical distance. This pattern of genetic variation may be associated with reproductive mode of its mixed breeding systems although mainly through vegetative propagation.