Polymorphic Random Amplified Polymorphic DNA Research Articles

Genetic diversity of Trichoderma atroviride strains collected in Poland and identification of loci useful in detection of within-species diversity.

Molecular markers that enable monitoring of fungi in their natural environment or assist in the identification of specific strains would facilitate Trichoderma utilization, particularly as an agricultural biocontrol agent (BCA). In this study, sequence analysis of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the ribosomal RNA (rRNA) gene cluster, a fragment of the translation elongation factor 1-alpha (tef1) gene, and random amplified polymorphic DNA (RAPD) markers were applied to determine the genetic diversity of Trichoderma atroviride strains collected in Poland, and also in order to identify loci and PCR-based molecular markers useful in genetic variation assessment of that fungus. Although tef1 and RAPD analysis showed limited genetic diversity among T. atroviride strains collected in Poland, it was possible to distinguish major groups that clustered most of the analyzed strains. Polymorphic RAPD amplicons were cloned and sequenced, yielding sequences representing 13 T. atroviride loci. Based on these sequences, a set of PCR-based markers specific to T. atroviride was developed and examined. Three cleaved amplified polymorphic sequence (CAPS) markers could assist in distinguishing T. atroviride strains. The genomic regions identified may be useful for further exploration and development of more precise markers suitable for T. atroviride identification and monitoring, especially in environmental samples.

STUDY OF DNA POLYMORPHISM OF THE CASTOR NEW LINES BASED ON RAPD MARKERS

The systematic evaluation of the molecular diversity encompassed in castor inbreds or parental lines offers an efficient means of exploiting the heterosis in castor as well as for management of biodiversity. Seventeen castor genotypes were assessed for genetic variability using Random Amplified Polymorphic DNA (RAPD) markers. Thirteen polymorphic RAPD primers amplified 102 DNA fragments, with an average of 7.85 fragments per primer. Number of amplified fragments ranged from 3 (OPE-07) to 13 (SIGMA-D-01), with the size of amplicons ranging from 100 to 1200 bp. The polymorphic information content (PIC) value ranged from 0.450 (OPE-07) to 0.892 (SIGMA-D-01) with an average of 0.771 and diversity index (DI) value ranged from 0.551 (OPE-07) to 0.894 (SIGMA-D-01) with an average of 0.787. The dendrogram based on hierarchical cluster analysis using UPGMA algorithm was prepared and analyzed genotypes were grouped into two main clusters and only two genotypes (RM-5 and RM-23) could not be distinguished.

Genotoxicity of Thermopsis turcica on Allium cepa L. roots revealed by alkaline comet and random amplified polymorphic DNA assays.

This study was undertaken to evaluate genotoxic potential of Thermopsis turcica aqueous extracts on the roots of onion bulb (Allium cepa L.) by comet assay and random amplified polymorphic DNA technique. The Allium root growth inhibition test indicated that the EC50 and 2×EC50 values were 8 and 16mg/ml concentrations of T. turcica aqueous extracts, respectively. The negative control (distilled water), positive control (methyl methane sulfonate, 10mg/l) and 8 and 16mg/ml concentrations of T. turcica extracts were introduced to the roots of onion bulbs for 24 and 96h. The root growth, DNA damage in root cells and randomly amplified polymorphic DNA (RAPD) profiles of root tissue were used as endpoints of the genotoxicity. The comet assay clearly indicated that dose-dependent single strand DNA breaks in the root nuclei of onions were determined for the treatment concentrations of T. turcica extracts. In comparison to RAPD profile of negative control group, RAPD polymorphisms became evident as disappearance and/or appearance of RAPD bands in treated roots. The diagnostic and phenetic numerical analyses of RAPD profiles obviously indicated dose-dependent genotoxicity induced by Thermopsis extracts. In conclusion, the results clearly indicated that water extract of T. turcica has genotoxic potential on the roots of onion bulbs as shown by comet assay and RAPD technique.

Authentication of Akebia quinata DECNE. from its common adulterant medicinal plant species based on the RAPD-derived SCAR markers and multiplex-PCR

Akebiae Caulis, ‘Mutōng’ in Chinese, refers to the stem of Akebia quinata in the Korean modern pharmacopoeia. The stems of Aristolochia manshuriensis and Clematis armandii have also been traditionally used for clinical purposes, and are often marketed as ‘Mutōng’ under the names ‘Guān-Mutōng and Chuān-Mutōng’, respectively. Furthermore, due to the morphological similarity of the herbal medicines, their correct identification by traditional subjective methods is difficult. Therefore, to develop a reliable method for discriminating these three herbal medicines, we applied the tools of molecular genetics, including random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) marker development using samples of Akebia quinata, Akebia trifoliata, Aristolochia manshuriensis, and Clematis armandii. By comparative analysis of RAPD polymorphisms, we identified several potential PCR products that can be used to distinguish the herbal materials of each species. Using these RAPD products, we developed unique sequence characterized amplified region (SCAR) markers for determination of the species of plant materials; the products were 590 bp for A. quinata, 300 bp for A. trifoliata, 373 bp for A. manshuriensis, and 432 bp for C. armandii. Furthermore, we optimized SCAR markers for simultaneous discrimination of the four species by multiplex-PCR. These markers allow efficient identification of closely related herbal medicines originating from Akebia, Aristolochia, and Clematis species, and have potential uses in preventing the distribution of adulterants, as they can be used to identify each species and distinguish them from inauthentic substitutes.

English

Knowledge of genetic distances between genotypes is important for efficient organization and conservation of plant genetic resources for crop improvement programs. In this study genetic distances between genotype pairs (complements of Jaccard's similarity coefficient) were estimated from Random Amplified Polymorphic DNA (RAPD) data collected from 48 Zimbabwean sorghum landraces. These varieties showed variation in their seed proanthocyanidin (PAs) levels with 16 and 29 of them having detectable and non-detectable PA levels respectively. RAPDs revealed considerable genetic variation between the varieties used and 2.7 polymorphisms per primer were obtained.  Ninety nine polymorphic RAPD bands were used to calculate genetic distances and the mean genetic distance between the genotypes was 0.494 (± 0.113) with a range of 0.051 to 0.761. A multidimensional scaling (MDS) plot of the distance matrix revealed two distinct clusters of cultivated and wild sorghums. No clustering of genotypes according to their seed proanthocyanidin levels was revealed by MDS analysis; also the mean genetic distances of genotypes in the low, medium and high PA categories were not different from each other and none of them was significantly different from the mean genetic distances between all the groups. The RAPD markers used in the present study could not distinguish between sorghums with different PA levels in their seeds; however, the protocol established could be useful in further analysis of this trait in near isogenic lines.   Key words:  Genetic distances, multidimensional scaling, proanthocyanidins, Random Amplified Polymorphic DNA (RAPD), Sorghum bicolor, Zimbabwe.

Genetic diversity of Vicia faba L. based on random amplified polymorphic DNA and simple sequence repeat markers

The genetic diversity of 13 Tunisian faba bean (Vicia faba L.) accessions, and the commercial variety “Aguadulce”, were studied using simple sequence repeats (SSRs) and random amplified polymorphic DNA (RAPD) markers. In comparison to RAPD markers, SSRs showed a higher level of polymorphism and greater information content, as assessed by the expected heterozygosity. One hundred and fourteen bands were amplified using six SSR primer combinations and 376 bands were observed using four RAPD primers. The percentages of polymorphic fragments were 100% and 60.63% for SSR and RAPD markers, respectively. The polymorphism information content, gene diversity and population differentiation test were 0.370, 0.490 and 0.746, respectively, for SSR markers and 0.319, 0.406 and 0.775 for RAPD markers. The correlation coefficients of similarity based on the Mantel test were statistically significant for the both marker systems used, but were higher for SSR data than for RAPD. Dendrogram topologies, conducted separately on polymorphic RAPD and SSR markers, showed minor differences in the grouping of accessions. The dendrogram based on combined RAPD and SSR data sets identified clustering of accessions according to their geographic diffusion. Collectively, these results will aid Tunisian faba bean germplasm management, conservation and breeding.

Density-weighted Algorithms for Similarity Computation and Cluster Tree Construction in the RAPD Analysis of Natural Cordyceps sinensis

Objective: The goal of this study was to develop and validate density-weighted arithmetic methods for the analysis of the dynamic maturational changes in random amplified polymorphic DNA (RAPD) polymorphisms in Cordyceps sinensis containing multiple fungi. Methods: Ten random primers were used for PCR amplification to monitor changes in the RAPD molecular marker polymorphisms in caterpillar body, stroma and ascocarp portion samples of C. sinensis collected at different stages of maturation. We compared (1) the density-unweighted Nei-Li similarity equation and new similarity equations considering the densities of DNA bands and (2) the density-unweighted and weighted arithmetic methods for cluster analysis. Results: The algorithm using the Nei-Li similarity equation did not account for the differences in the density of the matched randomly amplified amplicons, whereas the new similarity equations were capable of integrating different amplicon densities into the similarity computation. With improvements in the similarity computation and cluster construction, the new methods revealed dynamic maturational changes in the RAPD polymorphisms of molecular markers in C. sinensis caterpillar bodies and stromata. The polymorphism analysis revealed similarities of 74%-88% between the RAPD polymorphisms of the ascocarp portion versus premature and mature C. sinensis stromata, but low similarities of<70% versus the caterpillar bodies and maturing stromata. Apparent dissimilarities (similarity <66%) were also found between Hirsutella sinensis and C. sinensis samples. Conclusions: These new algorithmic methods represent an advance in similarity computations when comparing polymorphisms of RAPD molecular markers in C. sinensis and provide accurate analytic tools to capture the molecular information related to the dynamic

KARAKTERISASI BEBERAPA STRAIN GURAMI Osphronemus gouramy Lac. MENGGUNAKAN MARKA RAPD

Giant gouramy, Osphronemus gouramy Lac. is a popular fish species in Indonesia, especially in Java and Sumatera as this freshwater fish species has a high economic value of stable price. Fish farmers in Bogor divide giant gouramy into six strains based on egg productivity, growth rate, and maximum weight of the adult. They are soang, jepang, blue saphire, paris, bastar, and porselin. These various strains lead to the need of study on the genetic relationship among them, which can be performed by the use of RAPD (Random Amplified Polymorphic DNA) marker. This study aims to determine primers producing consistent and polymorphic RAPD markers, determine specific RAPD markers, and know the genetic relationship among several giant gouramy strains. The strains used in this study are soang, jepang, and blue saphire. Survey method was applied employing purposive random sampling technique. Total genomic DNA was isolated using Genjettm genomic DNA purification kit (Fermentas), which was then used as template to amplify RAPD markers with primers OPA-07, OPA-09, OPA-11, OPA-20, OPAH-01, OPAH-08, OPAH-09, and OPAC-14. The variables examined were patterns and numbers of specific DNA fragments as the PCR amplification products. Selected primers were determined descriptively on the basis of specific DNA bands appearing on the agarose gel. Genetic diversity was predetermined by changing qualitative band pattern into quantitative binnary data. Genetic relationship was analyzed using cladistic method with PAUP software. The results showed that only five of the eight primers produce consistent and polymorphic RAPD markers, i.e. OPA-11, OPA-20, OPAH-1, OPAH-8, and OPAH-9. Specific RAPD markers which can be used to distinguish several gouramy strains are those amplified with OPA-20 of 786 bp, OPA-20 of 1,176 bp, OPAH-8 of 1,000 bp and OPAC-14 of 1,607 bp. Nervertheless, it was found that RAPD markers cannot be used to clearly determine genetic relationship among gouramy strains.

English

Citrus is an economically important fruit crop with a long history of cultivation worldwide. A great number of varieties of citrus are extensively grown in the Middle East including Iraq for domestic consumption and exports. However, the genetic diversity of this genus in Iraq has not been reported. Therefore, the objective of this study was to evaluate genetic relationships of Iragi citrus genotypes to provide useful information for germplasm conservation and planning of breeding strategies. Twenty decamer primers were used to generate RAPD markers to evaluate genetic relationship among 16 genotypes (14 species and hybrids) of cultivated Citrus in Iraq. Based on RAPD polymorphisms, the citrus genotypes were classified into two main groups; the first consisted of citron (Citrus medica) and its hybrids (lime and lemon). The second group contained the remaining genotypes including three sub-groups; the first being formed of sour orange (Citrus aurantium), sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi), the second the mandarins (Citrus reticulata) and the third the pummelo (Citrus grandis). The RAPD-based classification was consistent with previous studies based on other types of molecular markers. &nbsp; Key words: Citrus, genetic diversity, Iraq, oranges, random amplified polymorphic DNA (RAPD) markers.&nbsp

Genetic diversity in Monilinia laxa populations in stone fruit species in Hungary

The objectives of this study were firstly, to determine the genetic diversity of Monilinia laxa isolates from Hungary, using the PCR-based inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) technique; secondly, to prepare genetic diversity groups based on the dendrograms; and finally, to select some relevant isolates to study their fungicide sensitivity. 55 and 77 random amplified polymorphic ISSR and RAPD markers, of which 23 and 18 were polymorphic and 32 and 59 monomorphic, respectively, were used to assess the genetic diversity and to study the structure of M. laxa populations in Hungary. 27 isolates out of 57 ones were confirmed as M. laxa from several orchards (subpopulations) in three geographical regions, in various inoculum sources and in various hosts, were used. 10 fungicides and 12 isolates selected from genetic diversity groups based on the ISSR dendrograms were used to determine the fungicide sensitivity of the selected isolates. The analysis of population structure revealed that genetic diversity within locations, inoculum sources and host (H(S)) accounted for 99 % of the total genetic diversity (H(T)), while genetic diversity among locations, inoculum sources and host represented only 1 %. The relative magnitude of gene differentiation between subpopulations (G(ST)) and the estimate of the number of migrants per generation (Nm) averaged 0.005-0.009 and 53.9-99.2, respectively, for both ISSR and RAPD data set. The results obtained in dendrograms were in accordance with the gene diversity analysis. Grouping of isolates in the dendrograms was irrespective of whether they came from the same or different geographical locations. There was no relationship between clustering among isolates from inoculum sources and hosts. In the fungicide sensitivity tests, five isolates out of 12 were partly insensitive to boscalid+piraclostrobin, cyprodinil, fenhexamid or prochloraz. Obtained results in genetic diversity of M. laxa populations are discussed together with implications for the management of brown rot.

Identification and authentication of Rosa species through development of species-specific SCAR marker(s).

Roses (Rosa indica) belong to one of the most crucial groups of plants in the floriculture industry. Rosa species have special fragrances of interest to the perfume and pharmaceutical industries. The genetic diversity of plants based on morphological characteristics is difficult to measure under natural conditions due to the influence of environmental factors, which is why a reliable fingerprinting method was developed to overcome this problem. The development of molecular markers will enable the identification of Rosa species. In the present study, randomly amplified polymorphic DNA (RAPD) analysis was done on four Rosa species, Rosa gruss-an-teplitz (Surkha), Rosa bourboniana, Rosa centifolia, and Rosa damascena. A polymorphic RAPD fragment of 391 bp was detected in R. bourboniana, which was cloned, purified, sequenced, and used to design a pair of species-specific sequence-characterized amplified region (SCAR) primers (forward and reverse). These SCAR primers were used to amplify the specific regions of the rose genome. These PCR amplifications with specific primers are less sensitive to reaction conditions, and due to their high reproducibility, these species-specific SCAR primers can be used for marker-assisted selection and identification of Rosa species.

Race-specific genetics of resistance to black rot disease [Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson] and the development of three random amplified polymorphic DNA markers in cauliflower

SummaryIn order to understand the genetics of resistance to black rot disease caused by Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson in cauliflower (Brassica oleracea var. botrytis L.) and to identify random amplified polymorphic DNA (RAPD) markers that segregate with the resistance genes, susceptible (‘Pusa Himjyoti’, female parent) and resistant (‘BR-161’, pollen parent) plants were crossed. Six generations of plants (30 P1, 30 P2,30 F1, 120 F2, 90 B1, and 90 B2) were evaluated for the presence or absence of black rot disease in a randomised block design with three replications. The pattern of segregation of resistance was tested by the χ2 test at the 5% level of significance. All F1 progeny plants were resistant, and the segregation of resistant and susceptible plants in the F2 and two backcross generations (B1 and B2) showed that a single dominant gene caused resistance to the black rot pathogen in ‘BR-161’. Three polymorphic RAPD markers (OPO-04833, OPAW-202538, and OPG-25625) were found by bulk segregant analysis, which produced unique amplicons 833 bp, 2,538 bp, and 625 bp in length, respectively. These markers were associated in coupling phase to the resistance allele. Best fit ratios of 3:1 (resistant:susceptible) in the F2 plants with the three RAPD markers, suggested that the markers were linked to the single gene controlling black rot resistance. These markers will be useful to identify more closely-linked markers and to develop black rot-resistant hybrid cauliflower varieties.

Genetic diversity among some canola cultivars as revealed by RAPD, SSR and AFLP analyses.

To assess the genetic diversity among four canola cultivars (namely, Serw-3, Serw-4, Misser L-16 and Semu 249), random amplified polymorphic DNA (RAPD), simple sequence repeat polymorphism (SSR) and amplified fragment length polymorphism (AFLP) analyses were performed. The data indicated that all of the three molecular markers gave different levels of polymorphism. A total of 118, 31 and 338 markers that show 61, 67.7 and 81 % polymorphism percentages were resulted from the RAPD, SSR and AFLP analyses, respectively. Based on the data obtained the three markers can be used to differentiate between the four canola cultivars. The genotype-specific markers were determined, 18 out of the 72 polymorphic RAPD markers generated were found to be genotype-specific (25 %). The highest number of RAPD specific markers was scored for Semu 249 (15 markers), while Serw-4 scored two markers. On the other hand, Serw-3 scored one marker. The cultivar Semu 249 scored the highest number of unique AFLP markers, giving 57 unique markers, followed by Misser L-16 which was characterized by 40 unique AFLP markers, then Serw-3 giving 31 unique markers. While Serw-4 was characterized by the lowest number producing 14 unique positive markers. The dendrogram built on the basis of combined data from RAPD, SSR and AFLP analysis represents the genetic distances among the four canola cultivars. Understanding the genetic variability among the current canola cultivars opens up a possibility for developing a molecular genetic map that will lead to the application of marker-assisted selection tools in genetic improvement of canola.

Stem fasciation in cacti and succulent species — tissue anatomy, protein pattern and RAPD polymorphisms

Fasciated and normal stem segments of Opuntia microdasys, Opuntia cylindrica, Huernia primulina and Euphorbia lactea were collected from the same plant and compared for their anatomy, water relations and genetic variations. Anatomical differences in terms of thickness of cuticle, vascular bundle, xylem and phloem were analyzed in both normal and fasciated stems. The mucilage cells were higher in the fasciated form of Opuntia microdasys than that in the normal form. Water status in terms of total water content (TWC), water deficit and relative water content (RWC) was influenced by fasciation. Genetic variations were tested in normal and fasciated stems using randomly amplified polymorphic DNA (RAPD) fingerprints and SDS-PAGE of soluble protein extracts. SDS-PAGE protein and RAPD analysis confirmed that normal and fasciated tissues were genetically different. Polymerase chain reaction (PCR) yielded different polymorphic banding patterns that were unique to each primer and distinguishable over all samples. The PCR results of normal and fasciated samples were significantly different in cases of primers P1, P2 and P3. These results indicate that occurrence of fasciation in Opuntia microdasys, Opuntia cylindrica, Huernia primulina and Euphorbia lactea is an epigenetic mutation of tissues.

GENOTYPING AND ASSESSMENT OF GENETIC RELATIONSHIPS AMONG CULTIVARS, GENETIC LINES AND SPECIES OF LONICERA L. USING RAPD

A Random Amplified Polymorphic DNA (RAPD) analysis of 73 accessions, including species, cultivars and genetic lines of the genus Lonicera was carried out to genotype accessions and to assess relationships within the honeysuckle germplasm collection of the Botanical Garden of Vilnius University. The twelve selected primers identified 149 polymorphic RAPD loci. A high level of DNA polymorphism was found in the whole collection and within each group of accessions. Unique RAPD band profiles for all studied genotypes were obtained. An UPGMA dendrogram grouped all accessions of taxa producing edible fruits and closely related to L. caerulea into one cluster, separate from other species of Lonicera. The genetic relationships indicated by RAPD band profiles of the studied species are, at least in part, consistent with previously published taxonomic studies of Lonicera. RAPD bands identified in this work can be used as markers in the management of germplasm collections of Lonicera.

Genetic variability inCitrussp., related genera and hybrids from germplasm collection by random amplified polymorphic DNA (RAPD)

The tools of molecular biology have enabled the improvement of citrus with significant reduction in identification and selection of new materials, and are usually preferred for genetic diversity assessment, population genetic structure and mapping. Relationships among 24 accessions of Citrus species, related genera and hybrids were investigated using random amplified polymorphic DNA (RAPD). A dendrogram based on the unweighted pair-group method for the arithmetic mean method was constructed using a similarity matrix derived from 96 polymorphic RAPD fragments. High polymorphism was detected among the accessions and the number of fragments/primer/accession ranged from 2 to 8. Accession-specific DNA fragments were observed for Murraya and Feroniella oblata. Within the subtribe Citrinae, the genera Poncirus, Microcitrus, Severinia and Atalantia were more distant from Citrus, and Fortunella was the most closely related. The Citrus species clustered in one group, including possible hybrid species, and the data did not support a separation into the subgenera Citrus and Papeda. Although RAPD technique sometimes has been criticized, it still can be used with successfully and this study confirmed these markers are useful and efficient for analysis of variability in citrus species. The information generated will be important for breeding programs using citrus germplasm.

Genetic characterization of Fusarium graminearum and F. culmorum isolates from Turkey by using random-amplified polymorphic DNA

Five Fusarium graminearum and 12 F. culmorum isolates, primarily pathogenic species of Fusarium head blight, were obtained from naturally infected wheat from various agro-ecological regions of Turkey. Genotyping of the isolates was carried out using random-amplified polymorphic DNA (RAPD). Sixty-five 10-mer oligonucleotide primers were used to amplify the RAPD markers. Among them, 50 primers produced strong and reproducible DNA amplicons. The remaining primers generated either insufficient or no amplification patterns. In total, 1200 fragments were scored, 311 of which were determined to be polymorphic and unique to the isolates. The produced RAPD markers ranged from 0.2 to 5 kb. The mean genetic similarity values of the F. graminearum and F. culmorum isolates were 61.5 and 65%, respectively. The similarity coefficient was 43 to 76.1% among F. graminearum isolates and 49 to 81.1% among F. culmorum isolates. Genetically, the most similar F. graminearum isolates were F6 and F7 (76.1%), which originated from the same agro-ecological region (Sakarya). The most similar F. culmorum isolates were F20 and F21 (81.1%), which were from different geographic regions (Bilecik and UÅŸak, respectively). Moreover, interspecific variation between the two species was determined to be 86.3 to 93.3%. Cluster analysis generated two branched groups, each containing isolates of one species, except F13 of F. culmorum. The sequencing of stable and reproducible monomorphic and polymorphic RAPD markers indicated that the Fusarium genome shared high similarity (105-625 bit scores) with the genomes of other organisms as well as with the F. graminearum reference genome.

RAPD와 ITS 영역에 의한 민자주방망이 버섯의 유전적 변이

A genetic variation of Lepista nuda and two genus Lepista species (L. irina and L. sordida) were analyzed by random amplified polymorphic DNA (RAPD) and internal transcribed spacer (ITS) sequence analysis. In the resulting RAPD analysis, 22 out of 40 random primers amplified polymorphic RAPD fragment patterns, the amplified bands were 355, and DNA fragment sizes were 200-400bp. Intraspecific genetic dissimilarity of the 10 L. nuda strains were calculated to range from 0% to 21.60%, L. sordida from 16.93% to 24.82%, L. irina were 20.62% to 25.54%, and intraspecific genetic dissimilarity of L. sordida and L. irina was 23.49%. The 673 base pairs were sequenced during the analysis of the ITS I and II region; six L. nuda strains intraspecific genetic dissimilarities ranged from 1.58% to 11.47%, L. nuda and L. sordida from 3.83% to 12.88%, L. nuda and L. irina from 7.11% to 15.61%, and intraspecific genetic variation between L. sordida and L. irina was 4.79%. The findings showed that RAPD and ITS sequencing could be used for developing molecular genetic markers and screening of unidentified genus Lepista species.

Molecular Characterization of Selected Local and Exotic Cattle Using RAPD Marker.

In order to develop specific genetic markers and determine the genetic diversity of Bangladeshi native cattle (Pabna, Red Chittagong) and exotic breeds (Sahiwal), randomly amplified polymorphic DNA (RAPD) analysis was performed using 12 primers. Genomic DNA was extracted from 20 cattle (local and exotic) blood samples and extracted DNA was observed by gel electrophoresis. Among the random primers three were matched and found to be polymorphic. Genetic relations between cattle’s were determined by RAPD polymorphisms from a total of 66.67%. Statistical analysis of the data, estimating the genetic distances between cattle and sketching the cluster trees were estimated by using MEGA 5.05 software. Comparatively highest genetic distance (0.834) was found between RCC-82 and SL-623. The lowest genetic distance (0.031) was observed between M-1222 and M-5730. The genetic diversity of Red Chittagong and Sahiwal cattle was relatively higher for a prescribed breed. Adequate diversity in performance and adaptability can be exploited from the study results for actual improvement accruing to conservation and development of indigenous cattle resources.

Efficiency of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers for genotype fingerprinting and genetic diversity studies in canola (Brassica napus)

Genetic diversity evaluations among 10 canola ( Brassica napus ) genotypes were determined using RAPD and ISSR markers. The RAPD and ISSR primers with the highest degree of polymorphism were selected. A total of 67 bands of polymorphic RAPD bands were detected out of 77 bands, with an average of 13.4 polymorphic fragments per primer. The number of amplified fragments with RAPD primers ranged from 8 to 21, with the size of amplicons ranging from 162 to 3154 bp. The polymorphism ranged from 68.4 to 100.0, with an average of 87%. The five ISSR primers produced 94 bands across 10 genotypes, of which 76 were polymorphic, with an average of 15.2 polymorphic fragments per primer. The number of amplified bands varied from 4 to 32, with size of amplicons ranging from 127 to 3011 bp. The percentage of polymorphism using ISSR primers ranged from 25 to 100.0 with an average of 78.8%. Dice similarity coefficient was calculated for all pairwise comparisons and was used to construct a UPGMA dendrogram. Clustering of genotypes within the groups was not similar when RAPD and ISSR derived dendrograms were compared, whereas, the pattern of clustering of the genotypes remained akin in ISSR and the combined data of RAPD and ISSR. The similarity coefficient ranged from 0.44 to 0.78, 0.41 to 0.72 and 0.47 to 0.73 with RAPD, ISSR and combined dendrogram, respectively. These results provided valuable information for fingerprinting that can be used to create wider genetic base for future canola breeding program.