Random Amplified Polymorphic DNA Markers Research Articles

Genetic relationships in wild olives (Olea europaea ssp. oleaster) by ISSR and RAPD markers

ABSTRACTWild olives or oleaster (Olea europaea ssp. oleaster) are found naturally in the Mediterranean basin. The changes in climate and environmental conditions have been gradually accelerating in the last decades. Since oleasters are more resistant to diseases, pests and unfavorable environmental conditions, they have become a very important source for resistance breeding studies that would be required in the future. Twelve inter simple sequence repeat (ISSR) and 12 random amplified polymorphic DNA (RAPD) markers were used; the effectiveness of these markers was studied and different parameters such as effective multiplex ratio, marker index, resolving power, and polymorphic information content, Shannon information index (I), effective allele number (ne), and Nei's genetic distance (h) were used in the determination of genetic varieties of 18 oleasters grown in Turkey. When both marker systems were analyzed in this study, it was observed that MI, RP and EMR parameters in ISSR gave higher values than RAPD while ne, I, and h were lower in ISSRs. Neighbour-joining dendrograms were developed by using Nei's genetic distance matrix, and principal coordinate analysis was performed.

Development of random amplified polymorphic DNA markers for authentication of Croton tiglium Linn.

Background: Croton tiglium Linn., commonly known as Jayapala, in Ayurvedic pharmacopoeia, is wellknown for its purgative action. In the herbal raw drug market, seeds of Baliospermum montanum Blume., Ricinus communis Linn. and Croton roxburghii Wall. etc. are sold in the name of Jayapla seeds due to their morphological similarities. Hence their identification through molecular characters is need of the hour. Aim: Present study aims with the molecular characterization of young leaves of C. tiglium Linn. Materials & Methods: Fresh young leaves of C. tiglium Linn. were collected from its natural habitat Udupi, Karnnataka, during January 2017. It was used for molecular characterization and DNA fingerprints, by standard and most convenient Random Amplified Polymorphic DNA (RAPD) markers at Food testing laboratory, Junagadh Agriculture University, Gujarat, India. Results: All the primers gave good band patterns. Primer 5, 6, 8, 11 and 13 showed more number of light and bright bands matching characters with plant. Conclusion: Observed RAPD marker can be used to differentiate genuine as well as adulterated samples. The results may be used for the further research purposes and also required DNA Barcoding studies for further authentication.

Production dynamics in relation to ontogenetic development and induction of genetic instability through in vitro approaches in Pelargonium graveolens: A potential essential oil crop of commercial significance

AbstractRose‐scented geranium (Pelargonium graveolens L’ Hért.) is a fragrant and bushy essential oil bearing crop of significant industrial and commercial importance. In an attempt to overcome the constraints imposed by sexual sterility for genetic improvement and restricted environmental adaptability of Pelargonium, the present study was aimed at exploring new niches for its cultivation in northern most sub‐tropical part of India (Jammu). The flowering stage was found to be the optimum stage for harvesting in relation to essential oil quantity (0.16–0.18%, Fresh Weight Basis; FWB) and quality (geraniol 18% and citronellol 37%) during different phenophases of plant growth. Dynamics of essential oil turnover correlated well with the glandular trichome ontogeny using scanning electron microscopy (SEM). The present study also entailed induction of somaclonal variations via in vitro organogenesis. Organogenetic callus induction was achieved on B5 (Gamborg's) basal medium supplemented with kinetin/2,4‐dichlorophenoxyacetic acid (Kn/2,4‐D) and kinetin/indole‐3‐butyric acid (Kn/IBA). Frequency of callus induction ranged from 54–85%. Field established in vitro regenerated plants/somaclones displayed appreciable variability in fresh leaf biomass (365–2020 g, FWB), essential oil content (0.06%–0.26%) and quality (geraniol and citronellol as major constituents). To evaluate the genetic instability of calliclones, RAPD (Random Amplification of Polymorphic DNA) markers were used to assess the genetic basis of variability. Further, the identity of Pelargonium was confirmed by DNA bar‐code analyses using three plant based barcoding loci (ITS, rbcL and psbA/trnH). They may be used as a standard reference system for varietal identification and protection of Intellectual Property Rights (IPR). Overall, the present investigation demonstrates the feasibility of Pelargonium cultivation in sub‐tropical niches as a short duration winter crop, with desirable essential oil content and composition for commercial purposes. It also establishes a viable method for induction of variability for genetic amelioration of sexually sterile crop.

Genetic Diversity Evaluation of Moringa oleifera, Lam from East Flores Regency Using Marker Random Amplified Polymorphic DNA (RAPD) and Its Relationship to Chemical Composition and In Vitro Gas Production

The research objective was to evaluate the genetic diversity of Moringa oleifera, Lam (MO) and its relationship to chemical composition and in vitro gas production (IVGP). Fresh MO leaves were kept frozen in ice gels pack until laboratory analysis. Four methods applied: RAPD marker for measuring DNA concentration and purification; Kjeldhal and HPLC for analysing proximate and amino acid (AA) composition; and IVGP. MO’s four distinct morphology found: green, red, reddish green and aromatic green. RAPD result analysis was 68.8-74.7 %, it means those MO had a close genetic similarity. The morphological differences are also related to leaves chemical composition variation. The highest protein and AAs content were found in aromatic green MO. Total IVGP at 96 hours reached 95.9, 99.3, 111, 115 mL per 500 mg DM in aromatic green, green, reddish green, red MO, respectively and statistically among those was highly significant difference (P<0.01). However, DM and OM digestibility did not differ significantly and estimated ME contents were similar suggesting MO leaves had sufficient fermentable nitrogen amount required to ensure rumen microbes normal activities. Conclusively, those MO has a close genetic relationship but the aromatic green MO more beneficial due its higher content of crude protein and AAs.

RAPD marker assisted evaluation of chloroplast DNA variation in twelve hosta taxa

Hostas are major ornamental plants for perennial shady nurseries. They have striking foliage with attractive flowers, and their leaves are sometimes used as vegetables in Japan and Korea. The objective of this study was to estimate the genetic identity and genetic distance of twelve hosta taxa and to build a UPGMA cluster for them using Random Amplified Polymorphic DNA (RAPD). Rhizomes of hostas were collected from Shikoku Island, Japan. Newly emerged unfurled hosta leaves were disrupted with Nuclei Lysis solution and then purified the genomic cpDNA. The cpDNA was dehydrated as a final point for extraction. We amplified the cpDNA of twelve taxa by means of 20 RAPD 10mer markers (Kit A: OPA-01 to OPA-20). The amplified DNA was run in 0.8% agarose gel using 1000 kb DNA ladder. Out of 20 RAPD markers, the genomic cpDNA was successfully amplified for fourteen. Fourteen markers generated a total of 102 fragments. We found maximum genetic distance (0.655) and minimum genetic identity (0.520) between H. sieboldiana - H. longissima taxa and between H. sieboldii - H. longissima. Estimated minimum genetic distance (0.071) and maximum genetic identity (0.931) were found between H. kikutii var. polyneuron - H. longipes var. caduca. The UPGMA dendrogram revealed the relationship among the 12 hostas. The results confirmed our hypothesis of large genetic variability among the taxa, which distinguishes the genotypes clearly. The RAPD 10mer markers were found useful in application to the evaluation of the genetic variability and diversity within taxa.

Molecular characterization of African marigold (Tagetes erecta) genotypes using RAPD markers

The present investigation was carried out to characterize 22 genotypes of marigold (Tagetes erecta L.) with RAPD (randomly amplified polymorphic DNA) markers. A total of 26 RAPD markers were utilized for assessment of genetic divergence in marigold which amplified a total of 241 amplicons, out of which 238 (98.80%) were found polymorphic. The average number of amplicon per primer was 9.27. The Jaccard's similarity coefficient ranged from0.18 to 0.80. The mean values of polymorphic information content (PIC), resolving power (RP) and marker index (MI) of RAPD primers were 0.38, 8.36 and 3.44 respectively. Cluster analysis was done by unweighted pair group method for arithmetic average (UPGMA) and Principal Component analysis (PCA) was also done for the grouping of genotypes. The broad range of Jaccard's similarity coefficient observed for the genotypic data generated suggest the wide genetic base for marigold genotypes which will be effectively explored for future breeding programmes.

Assessment of genetic diversity in Costus pictus accessions based on RAPD and ISSR markers.

Costus pictus, belonging to the family Costaceae, is one of the valuable medicinal plants with its anti-diabetic property. Despite ever-increasing demand from the pharmaceutical industry, this species is being less exploited at molecular level. Hence, an effort has been made in the present study to characterize the 15 accessions of C. pictus collected from different geographical regions of India through random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. A total of 25 RAPD and 20 ISSR primers were used in the present study. The RAPD analysis generated 343 loci, of which 124 were polymorphic with an average of 4.96 loci per primer. While, ISSR primers produced 177 loci, of which 77 were polymorphic with an average of 3.85 loci per primer. The similarity coefficients ranged from 0.86-0.99, 0.84-0.95 and 0.86-0.96 for RAPD, ISSR and combined RAPD-ISSR, respectively. The UPGMA dendrogram generated using these data showed low level of divergence among the accessions from South and West regions. Further, accession-specific bands were also revealed by RAPD and ISSR markers which might be contributed to specific trait. This investigation was an understanding of genetic variation within the C. pictus accessions. The present finding indicates that both the marker tools RAPD and ISSR combined or individually can be used in determining the genetic relationship between the accessions. It may be concluded that data of hereditary differences appeared among the C. pictus accessions could be utilized for their conservation and reproducing programs.

Polimorfisme Penanda RAPD untuk Analisis Keragaman Genetik Pinusmerkusii di Hutan PendidikanUnhas

The aims of Genetic study of pinus identified stand in Unhas Experimental Forest is to analyses of genetic characteristics of stand, based on RAPD (Random Amplified Polymorphic DNA) marker. The study was conducted in Biotechnology and Tree Breeding Laboratory, Forestry Faculty, Hasanuddin University. The method are DNA isolation, Primers selection and RAPD analyses. DNA analyses of Pine with ten RAPD marker showed number of alel varietied and polymorphic. Coefficient of similarity in population have number of 0.15-0.73.Highest genetic distance is 0.9630 and lowest genetic distance is 0.2698. Number of genetic diversity of Pine in Experimental Forest Hasanuddin University is 0,489 and categorized highly.

Genetic Relationship and Diversity in Some Ornamental Palms Based on Proteins and Randomly Amplified Polymorphic DNA Markers

For the first time, genetic diversity among 14 ornamental palm accessions originating from different countries and grown in different regions in Egypt were examined. Identification of genetic variation and phylogenetic relationships in ornamental palms would be useful for its genetic identification, improvement, and conservation. Genetic polymorphism was analyzed using the randomly amplified polymorphic DNA (RAPD) as well as protein markers. The electrophoretic pattern of protein analysis produced 21 bands distributed in all accessions with molecular sizes ranging from 11.8 to 99.3 KDa. Some accessions possessed some bands, which were absent in other accessions and could be used for their identification. Furthermore, 10 RAPD selected primers were employed to determine genetic variation among the 14 palm genotypes as well as to test the effectiveness of RAPD primers as a genetic marker. RAPD analysis revealed a high level of polymorphism (100%) among the studied accessions. A total number of 310 amplified bands were generated across the studied genotypes with an average of 30 bands per primer. Cluster analysis using sequence alignment was done to generate a dendrogram verifying the relationship among the 14 studied ornamental palms, with an average similarity matrix range of 0.00 to 0.08 and 0.39 to 0.93 for RAPD and protein markers, respectively. It is concluded that, both SDS-protein and RAPD markers are equally important for genetic analysis and are suitable for the characterization of ornamental palm collection.

Genetic divergence and allelic-specificity in relation to expression of voltinism in silkworm using ISSR and RAPD fingerprinting

The silkworm B. mori is a multicellular organism revealing genetic resources which makes an ideal model for lepidoptera for the present investigation. With the objective of targeting distinctive markers for utilization in future breeding programmes, Bivoltine and Polyvoltine silkworm strains were used by inter-simple sequence repeats (ISSR) and random amplified polymorphic-DNA (RAPD) fingerprinting to detect their genetic versatility and volatility. Six ISSR primers generated 99 markers, of which 76.76% were found to be polymorphic with an average number of observed alleles (Na) (1.86 ± 0.40), an effective number of alleles (Ne) (1.43 ± 0.30) as well as six RAPD primers that produced a total of 95 bands, developing 61.05% polymorphism with Na (1.93 ± 0.51) and Ne (1.18 ± 0.30). The dendrogram produced by UPGMA analysis, based on Dice’s coefficient, clustered four races into two major groups which accurately segregated them according to their inheritance of voltinism. In this research, the ISSR markers were more accurate than the RAPD markers and ISSR also displayed better polymorphism. The outcome showed that the bivoltine strains exhibited higher allelic expressions with ISSR primers when compared to the polyvoltine strains. Despite exhibiting their unique race by certain DNA markers, most of the primers represented voltinism-specificity. Hence molecular marker amplification is a beneficial approach to reveal genetic divergence among closely related strains, and molecular characterization of phylogenetic relationships in addressing evolutionary evidences of individuals.

Genetic Diversity Assessment of a Critically Endangered Medicinal Plant Populations from Shorea tumbuggaia Roxb.

A successful assessment of genetic diversity is consider vital for formulating conservation of the rare, endangered and threatened (RET) species. Our aim was to assess the genetic diversity in Shorea tumbuggaia Roxb. population (Dipterocarpaceae family)—a critically endangered medicinal plant of conservative interest using Random Amplified Polymorphic DNA (RAPD) technique. According to International Union for Conservation of Nature (IUCN) and Conservation Assessment and Management Planning (CAMP) S. tumbuggaia is considered as an endemic and globally endangered tree species. RAPD markers were used to estimate the genetic variability and similarity among the S. tumbuggaia. Eighteen (18) primers of OPU (1-20) series generated a total of 137 polymorphic bands out of 735 total bands (19.86% polymorphism), with an average of 40.83 amplified bands per primers and 598 bands showed monomorphic banding pattern with an average of 33.22 per primers. Genetic similarity coefficients calculated from RAPD data ranged from 0.82 to 0.95, with the highest value of 0.95 in ST10 and ST11 and the lowest value of 0.82 between accessions ST2 and ST9, ST3 and ST4, ST4 and ST10 and ST5 and ST8.

A comparative analysis of genetic diversity in Portuguese grape germplasm from ampelographic collections fit for quality wine production

Grapevine cultivars diversity is vast and full of synonyms and homonyms. Up to few decades ago characterization of grapevine was based on morphological characters. In the last decades, molecular markers were developed and have been used as tools to study genetic diversity in a range of different plant species. Fifty-six Portuguese accessions representative of ‘Vinhos Verdes’ and ‘Douro’ Controlled Designations of Origin (DOC) were analysed through DNA fingerprints generated by Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). The study aimed to compare the effectiveness of RAPD and ISSR molecular techniques in the detection of synonyms, homonyms and misnames. RAPD and ISSR analysis enabled the detection of 36 different band patterns, reducing in about 36% the initial material. Several accessions grown under different names, between and within collections, were confirmed as the same genotype, namely Gouveio/Verdelho, Sousão Douro/Vinhão and Arinto Oeste/Pedernã. Similarly, some homonyms/misnames were also identified, namely within Azal Tinto and Rabigato accessions. RAPD and ISSR markers revealed to be adequate molecular techniques for grapevine varieties fingerprinting with advantages over other molecular procedures, contributing for a good management of grapevine collections.

Phylogenetic analysis among some pome fruit trees of Rosaceae family using RAPD markers

ABSTRACTTo evaluate the phylogenetic relationships among subtribe Pyrinae of Rosaceae, 50 different genotypes and cultivars of pome fruit trees were collected from various locations in Iran, and analysed using random amplified polymorphic DNA (RAPD) markers. Altogether, 85 polymorphic fragments were produced by 11 random 10-mer primers. The number of produced bands varied from 4 to 12 for each primer, 7.72 on average. The lowest Jaccard's genetic similarity coefficient was scored between apple cv. ‘Akan’ and a wild Pyrus syriaca pear (0.04), and the highest similarity was observed between two genotypes of P. syriaca (0.97). Cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) properly separated the accessions and divided them into three major groups, including Malus, Pyrus, Mespilus and Crataegus. Members of Mespilus and Crataegus were further separated and formed two subgroups. Analysis of the genetic structure, using STRUCTURE and phylogenetic relationship using TreeView software confirmed the results of the UPGMA dendrogram and properly distinguished different genera. Among different genera studied in our experiment, Malus was the most distinct one and showed high genetic distance from other genera. Generally, the results from the present study provide valuable information about the genetic relationship among important pome fruit trees in Iran and indicate that, although Mespilus is genetically close to the Crataegus genus, it has relatively high similarity with Pyrus and Malus. Moreover, outcomes of this research confirmed that RAPD molecular markers are an appropriate means for evaluation of the genetic relationship among various genera of pome fruit trees.

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r=0.78) was better than that of the RAPD marker system (r=0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.

Genetic Diversity and Identification of Variety-specific RAPD and ISSR Markers in Foeniculum vulgare

Inter simple sequence repeats (ISSRs) and random amplification of polymorphic DNA (RAPD) were used to determine genetic relationships and variety specific markers in fennel varieties grown in Rajasthan. In this study, the percentage polymorphism revealed by RAPD and ISSR was 50 and 39.1%, respectively. Dendrogram constructed from unweighted pair group method with arithmetic mean cluster analysis based on RAPD and ISSR data showed clear genetic relationships among these germplasms. Similar clustering was obtained when another software DARwin 5 was used to construct tree plot from factorial analysis based on RAPD and ISSR data. Besides, from the bands amplified, there were three independent ways to identify the fennel varieties, such as unique RAPD and ISSR markers, unique band patterns and a combination of the band patterns provided by different primers. Finally, the molecular fingerprints have been generated for five fennel varieties, namely FNL-41, 46, RF-125, 143 and 178. These could be distinguished by using specific 10 RAPD and four ISSR markers, which showed either presence or absence of bands generated by seven RAPD and four ISSR primers.

Genetic Diversity Analysis of Date Palm Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR).

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are PCR-based molecular techniques used for evaluation and characterization of date palm to find the best genotype and male/female identification at an early stage. Genetic fingerprinting using molecular markers is an important tool for the analysis of genetic diversity and cultivar identification. Here, we present an improved DNA extraction protocol using leaf tissue, based on the standard cetyltrimethyl ammonium bromide (CTAB) protocol, which yields large amounts of high-quality amplifiable DNA. RAPD and ISSR markers reveal sufficient genetic diversity as well as give some unique markers in some genotypes with a maximum number of bands.

Development and Validation of Scar Marker for Stem Rust Resistance Gene Sr26 in Wheat (Triticum aestivum L.)

The stem rust resistance gene Sr26 imparts resistance to all the virulent pathotypes of stem rust (Puccinia graminis f. sp. tritici) including a new race of pathogen named TTKSK (syn.Ug99). Wheat genotypes including non-carrier Kalyan Sona (-Sr26) and carrier Kite (+Sr26) of the Sr26 gene were analysed using polymerase chain reactions and a RAPD marker OPAE07620 was found to link with the desired trait. Linkage between the marker OPAE07620 and rust resistance phenotype was confirmed by analyzing F2 population obtained from a cross between a resistant and susceptible genotype. The Random Amplified Polymorphic DNA (RAPD) marker was converted into sequence characterized amplified region (SCAR) marker SCOPAE07620. The marker was validated in another segregating population and 45 wheat genotypes including carrier and non-carriers of Sr26 gene. The marker developed in this study can be used for pyramiding of the Sr26 gene with other major resistance genes that are effective against the TTKSK lineage (Ug99) which will help in attaining durable resistance against a disease that poses a serious threat to global wheat production.

Diallel analysis and genetic diversity of some yellow maize inbred lines (Zea mays L.) using RAPD and SSR markers

Information about combining ability and genetic diversity among maize inbred lines is fundamental in designing breeding strategies for improving grain yield. Twenty one F1 hybrids were generated by crossing seven yellow maize inbred lines in a half diallel mating scheme in 2013 season. The 21 F1 hybrids plus the two check hybrids (SC166 and SC173) were evaluated in a randomized complete block design with three replications during 2014 season, to estimate combining ability effects and identify type of gene action governing the inheritance of grain yield and other important agronomic traits. Results showed that both general (GCA) and specific (SCA) combining ability mean squares were highly significant for all the studied characters. The GCA/SCA ratio was more than unity for all the studied traits, except days to 50% silking, plant height and grain yield, indicating the preponderance of the additive gene effects in the inheritance of these traits. The inbred line P1 appeared to be the best general combiner for earliness, grain yield and its components. The crosses P1×P2, P3×P7, P4×P7, and P5×P6 had the best SCA effects for grain yield and most of its component traits. The cross P1×P2 significantly out-yielded the two check hybrids (SC166 and SC173). The genetic diversity among the seven yellow parental inbred lines was assessed using two types of molecular markers; Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSR). Seven random primers were used in the RAPD reactions, resulting in the amplification of 82 bands with 95.12% polymorphism. While, eight pairs of SSR primers were used and resulted in 49 fragments with 100% polymorphism. Genetic similarity among all possible pairs of inbred lines varied from 0.12 to 0.69, with an average of 0.34, for RAPD markers, and from 0.01 to 0.56, with an average of 0.24, for SSR markers. The similarity matrices for RAPD and SSR data were not significantly correlated (r=0.34, p >0.05). Genetic distances based on RAPD and SSR markers were insignificantly correlated with F1 hybrids grain yield, the correlation values being (r =0.02 and 0.429 P>0.05), respectively. Therefore, the parental genetic distance could not be used to predict the grain yield of the F1 hybrids in this study.

Efficient in vitro plant regeneration from seedling-derived explants and genetic stability analysis of regenerated plants of Simarouba glauca DC. by RAPD and ISSR markers

Simarouba glauca DC. is a multipurpose tree species known for oil, timber, and medicinal properties. The application of biotechnological methods for genetic improvement of this species depends on the availability of an efficient plant regeneration system. In this study, the shoot regeneration potential of various seedling-derived explants was assessed after culturing on Murashige and Skoog (MS) and woody plant (WP) medium containing different growth regulators. The explants differed in their capacity for shoot bud formation and subsequent shoot elongation on the media tested. Shoot bud induction was achieved at a high frequency (44.8–76.2%) from different explants on MS medium with 2 mg L−1 6-benzylaminopurine (BAP) as compared to other media tested. Cotyledons exhibited the highest capacity for shoot bud induction (76.2%) and shoot elongation (9.1 elongated shoots per explant). The in vitro-regenerated shoots rooted at a frequency of 66.7% after pulse treatment in 10 mg mL−1 indole-3-butyric acid (IBA) solution for 5 min followed by culture on half-strength WP medium with 0.2 mg L−1 IBA. The regenerated plants were acclimatized and established in the glasshouse with a survival rate of 80%. Molecular characterization of regenerated plants using 14 random amplified polymorphic DNA (RAPD) and 15 intersimple sequence repeat (ISSR) primers revealed a high number of monomorphic bands, with only 1.6–2.6% of the bands being polymorphic. The regeneration system established in the study has the potential to be used for rapid multiplication, conservation, and genetic transformation of this species.

Evaluation of Genetic Diversity amongthe Pakistani Wheat (Triticumaestivum L.) Lines through Random Molecular Markers

ABSTRACT The presence of geneticdiversity is of great importance in improving wheat traits and developing strategies for optimal conservation of germplasm. Genetic diversity was assessed among common wheat cultivars using RAPD (Random Amplified Polymorphic DNA) markers at the Center of Agriculture, Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad. RAPD primers were used among 14 Pakistani wheat cultivars, to screen the progenies and for the identification of the genes of interest. The polymorphic information content (PIC), was measured as the percentage of polymorphic fragments for all primers. A total of 583 bands(84% polymorphic) in all 14 wheat cultivars was amplified and discriminated all the wheat genotypes. The number of fragments amplified per primer ranged from 35 to 69 with an average of 48.52 fragments per primer averagely was observed. Population structure analysis anddandrogram showed distinct clustering among different wheat genotypes. Millat-11, Punjab-11, PBW-222 generated the maximum level of polymorphism, standing alone in the cluster while others are scatteredin different group.As a result, genetically numerous progenies are known, increasing the quality of sorts collections by broadening the genetic base of wheat cultivars. This study additionally indicates that RAPD markers allow quicker response and provide high throughputprocedure of accessions from a variety assortment to assess genetic diversity among wheat genotypes.