Random Amplified Polymorphic DNA Primers Research Articles

Screening of among seventeen sunflowers (Helianthus annuus L.) genotypes for oil content and the first flowering day using random amplified polymorphic DNA markers

Background Sunflower refers to the tribe Helianthus, subtribe Helianthinae, and family Asteraceae, which collectively contains 20 genera and 400 species. An important oilseed crop that yields edible oil is Helianthus annuus L. Objective The primary goal of the current study was to assess the genetic diversity of 17 genotypes of sunflower (Helianthus annuus L) To measure the oil content during the initial flowering period and to reach the highest percentage of oil can be obtained from the first flowering day. Materials and methods Five RAPD (random amplified polymorphic DNA) primers were used to detect the genetic diversity of the 17 sunflower hybrid genotypes obtained from Spain. Phylogenetic relationships of 17 sunflower genotypes were determined using three replications and 6 m lines on August 15, 2019, at the National Research Centre farm in Nubaria as part of a donation from the German corporation (strobe), Spain. To analyze the genetic diversity and phylogenetic linkages in sunflower germplasm, DNA fingerprinting and the Random Amplified Polymorphic DNA (RAPD) molecular marker approach were also used. Results and conclusion The oil content of 17 sunflower genotypes (Helianthus annuus L.) was assessed, with values ranging from 46 to 50%, with the highest values falling into five genotypes. However, the two genotypes were found to have the lowest oil percentage (46%). The early age and oil percentage differed among the varieties. In the Tornado and Elves genotypes, the longest and shortest days were 59 and 47, respectively. The means and standard errors for all statistical data are reported. Statistical significance was evaluated using the LSD. P values were considered statistically significant at P less than or equal to 0.05. According to the findings, RAPD primers generated 49 bands with a size range of 0.1–3 kb and an 87.75% polymorphism percentage. For RAPD, 43 polymorphic bands with distinct bands were observed. Morphological features and RAPD analysis separated the UPGMA Dendrogram into three groups. Jaccard’s coefficient was used to analyze the genetic similarity matrix, and a morphological study revealed that Tornado and Elvas, both from Spain, shared the most genetic similarity (0.970). RAPD analysis and morphological features are useful in identifying genetic variants. Conclusion, according to our findings, Helianthus annuus L. has a significant variation ratio. Indicating substantial diversity across the 17 sunflower genotypes, the genetic similarity index calculated using pooled data from RAPD markers showed an extensive range from 0.645 to 0.986. This study may be a reference for future research on Helianthus annuus L. and may support breeding initiatives and species concepts.

Genetic Diversity of West African Honey Bee (Apis ‎mellifera adansonii Latreille, 1804) from Rural and Urban ‎Areas of ‎Kwara State, North-Central Nigeria

Over one third of the world’s crops– including fruits, vegetables, nuts, spices, and oilseed–‎require insect pollination, and human reliance on ‎pollination services by honey bees (Apis ‎mellifera) to promote these crops continues to rise due to increasing demands from growing ‎human ‎populations. Identifying the effects of urbanization on genetic diversity on this ‎pollinating insect is important in the field of bioscience. This study aimed to investigate genetic diversity of A. mellifera in Kwara State, Nigeria, using the random amplified polymorphic DNA (RAPD) marker. ‎Thirty honey bees ‎were simultaneously collected from both rural and urban regions in ‎Kwara state, Nigeria. Samples were morphologically identified using ‎standard methods, ‎genomic DNA isolated and amplified using five RAPD primers. Data collected were ‎analysed using PyElph, ‎ARLEQUIN, and GeneAlEx version 6.501 software. The results ‎showed that the DNA fragment sizes produced per primer varied from 200 to ‎‎3000 bp. Percentages of polymorphic loci amplified by each primer varied from 17.33 to 33.33%. ‎Analysis of unbiased Nei genetic ‎distance values showed that Agbede (rural) and Adewole ‎‎(urban) showed the highest value of unbiased genetic distance (0.073), while ‎Amoyo ‎‎(rural) to Idofian (urban) exhibited the lowest value (0.027). Dendrogram analysis revealed ‎genetically close relationships among the sampled ‎A. mellifera‎ populations. The low level of genetic ‎polymorphisms observed among the honey bee populations in the two ‎regions ‎indicated that there is genetic relatedness among them. This study concluded that RAPD ‎marker is a useful method for ‎understanding population genetic structure of the African honey ‎bees. These results can be used as baseline information for future genetic ‎diversity ‎assessment of honey bees in Nigeria with larger samples. It is therefore recommended that ‎there is a need to safeguard the genetic ‎diversity of A. mellifera‎ to prevent extinction or ‎gradual loss of diversity‎‎‎.

Assessment of Genetic Diversity among the Elite Rose (Rosa spp.) Accessions Using Rapd Markers

Aims: Owing to its export value in flower trade elsewhere in the world, Rose is the key commercial flower crop and the area under rose cultivation is ever increasing and the end-users always prefer new color variations. Hence, evolving new cultivars with novel color characteristics is the need of the hour, for which understanding genetic variation in the available cultivars is very much needed. 
 Study Design: This investigation was conducted to analyze the genetic diversity of 11 elite and commonly cultivated rose accessions in South India by using randomly amplified polymorphic DNA (RAPD) markers.
 Place and Duration of Study: Department of Floriculture and Landscape Architecture, Horticultural College and Research Institute, TNAU, Coimbatore.
 Methodology: A total of 10 RAPD primers were employed, which was sufficient to distinguish the investigated rose cultivars.
 Results: Among the 44 PCR products produced by these markers, 39 (88.64%) were found to be polymorphic bands. The number of amplified products per RAPD primer varied from 3 to 8 with a mean of 4.4 bands per primer. The Un weighted paired group of arithmetic means (UPGMA) dendrogram distinguished the rose accessions into two major clusters suggesting that the accessions were different from each other. The genetic similarity coefficients were determined with this RAPD data, and they were ranged from 0.59 to 0.89.
 Conclusion: Molecular profiling data of this study have contributed to characterize and catalogue the rose germplasm data, which will be useful to identify the diverse rose lines for further breeding program that have the potential to improve the color variations.

Genetic Diversity and Phylogenetic Relationships among Jatropha curcas L. Genotypes from Eastern India

Genetic diversity and relationships among 31 genotypes collected from wide geographical range in eastern India was studied employing Random Amplified Polymorphic DNA (RAPD) markers. 19 markers (identified out of 25) produced 139 band in total. Off these 139 bands, 131 bands were polymorphic exhibiting a high polymorphism of 94.24%. Similarity indices estimated on the basis of RAPD primers ranged widely from 0.44 to 0.83 which suggests that these accessions represent genetically diverse population possibly due to predominance of cross pollination and seed source variability. Genotyping data obtained for RAPD primer across collected accessions were used to generate the UPGMA - based phylogenetic tree which shows two major clusters. The cluster I consisted of 16 genotypes from two states Jharkhand and Odisha (Chotanagpur and Eastern plateau region) while 15 accessions from West Bengal and Bihar (Indo Gangetic plains region) were grouped together in the Cluster II. This clear alignment of accessions into two different clusters as per geographical regions was probably due to different growing conditions in two clusters.

Estimating the Genetic Diversity of Oranges Citrus spp. in South Sulawesi, Indonesia, Using RAPD Markers

Oranges hold significant economic importance, being cultivated extensively worldwide and having a large global market. Indonesia, ranked eighth globally as a producer of oranges, is one of the countries with high genetic diversity of oranges. This diversity is distributed across various regions of Indonesia, including South Sulawesi. Despite the advancements in DNA-based molecular marker techniques for assessing genetic diversity, information on orange diversity in South Sulawesi is currently unavailable and under-researched. In this study, random amplified polymorphic DNA (RAPD) markers were utilized to analyze the genetic diversity of oranges in five production centers in South Sulawesi. Leaf samples of 13 orange varieties were collected from the five production centers: Pangkep, Sidrap, Bantaeng, North Luwu, and Selayar in South Sulawesi, Indonesia. Genomic DNA extraction from the orange leaves followed the protocol of the DNA Mini Kit Geneaid. DNA amplification was carried out using the RAPD method with 14 primers: OPE-04, OPH-04, OPH-15, OPN-14, OPN-16, OPR-08, OPR-20, OPW-06, OPW-09, OPX-07, OPX-11, OPX-17, UBC-18, and UBC-51. The RAPD primers yielded 109 amplified fragments ranging in size from 200 to 2000 base pairs (bp), and all RAPD primers showed 100% polymorphism. The genetic diversity value (He) of oranges in South Sulawesi was moderate (0.236). Cluster analysis based on a similarity coefficient of 77% divided the 175 orange genotypes into five groups. The most closely related genotypes were SB6 and SB7, exhibiting 100% similarity, followed by genotypes JS8 and JS9 and JS13 and JS17, with genetic similarities exceeding 99% for each pair. Genotypes P9 and SI5 displayed the highest genetic distance, with a similarity coefficient of 57%. The dendrogram diagram can serve as a basis for selecting desired plant traits in the improvement of plant characteristics through both conventional breeding and genetic engineering activities.

Revealing genetic diversity of wild Phalaenopsis orchids in Thailand through Random Amplified Polymorphic DNA markers

Abstract. Kate-ngam S, Jantasuriyarat C, Lakote P, Promchot T, Chueakaew C. 2023. Revealing genetic diversity of wild Phalaenopsis orchids in Thailand through Random Amplified Polymorphic DNA markers. Biodiversitas 24: 5409-5417. Wild Phalaenopsis orchids are widely distributed in Thailand, especially along the banks of the Mekong River. The tremendous decrease in the population of these orchids is a matter of concern for maintaining and preventing genetic loss in the natural ecosystem. This study aimed to determine the genetic diversity of 50 wild Phalaenopsis accessions collected from the northeastern of Thailand using random amplified polymorphic DNA (RAPD) markers. The 204 polymorphic bands generated from 27 RAPD primers were employed for fingerprinting these orchid accessions. Dice’s similarity coefficients ranged from 0.43 to 0.98, with an average of 0.65. In general, a dendrogram constructed based on the unweighted pair group method with arithmetic average (UPGMA) grouped these wild Phalaenopsis accessions into two clusters, namely P. pulcherrima and P. ubonensis clusters. The UPGMA clustering pattern and principal component analysis (PCA) corresponded well with their morphological classification and ploidy level. Our results suggest that this wild Phalaenopsis orchid exhibits a moderate level of relatedness. This might be a consequence of habitat destruction and human over-exploitation which limit gene flow and cause genetic drift among populations. The present finding supports the urgent need for a systematic conservation effort of this orchid species in Thailand.

Evaluation of Biological Control of Sorghum Strains Using Bacillus Thuringiensis and Pseudomonas Aeruginosa Under Drought Stress

Background: Sorghum is an economically significant staple food crop for more than half a billion people in developing nations, especially in arid and semi-arid locations where drought stress is a significant limiting factor. Despite usually being regarded as tolerant, sorghum suffers severely from drought stress, which lowers its productivity and nutritional quality throughout its principal cultivation areas. Objective: Improvements in DNA fingerprinting by ISSRs, SSRs, and RAPD markers have also been employed in sorghum genetic modification (GMOs) to enhance the economic characteristics of this crop. Materials and methods: To provide a natural defence against pests, the most tolerant plants among the seven varieties of sorghum bicolour were selected and planted in the second season of 2020–2021 under treatment with two microorganisms, B. thuringiensis and P. aeruginosa. This study considered seven varieties of sorghum bicolour planted under 50% water deficiency in 2019–2020. Genetic variability analysis of sorghum genotypes was performed using seven Inter-Simple Sequence Repeat (ISSR) primers, six Simple Sequence Repeat (SSR) primers, and five Random Amplified Polymorphic DNA (RAPD) primers. Seven Sorghum bicolour accessions were collected from various regions of Egypt and their phylogenetic relationships were evaluated. Additionally, DNA fingerprinting and analyses of the genetic diversity and evolutionary linkages in the sorghum germplasm employed the (ISSR) molecular marker technique. Results and conclusion: The Fisher Least Significant difference test (LSD) at P < 0.05, based on RAPD, ISSR, and SSR markers demonstrated a significant connection. The findings demonstrated that 51 bands with a size range of 100–1500 bp and polymorphism percentage of 72.5% were created using five RAPD primers. Seven ISSR primers generated 45 bands With a 57.8(%) polymorphism percentage, ranging in size from 100 to 3000 bp. six SSR primers generated 28 bands with (67.86%) polymorphism percentage of 67.86 %, ranging in size from 100 to 1500 bp. Morphological characteristics and ISSR, SSR, and RAPD analyses were used to group the UPGMA Dendrogram into groups. Jaccard's coefficient was used to analyse the genetic similarity matrix. The maximum similarity was observed for ISSR between Hybrid Sh1 and Hybrid Sh306 (0.984%), SSR between Hybrid Sh306 and Sudan grass (0.964%), and RAPD between Giza 15 and Indian Millet (0.706%). The classification of sorghum germplasm, breeding initiatives, and conservation efforts rely heavily on the determination of the genetic diversity among sorghum species. Identification of genetic variants, morphological features, and genetic analysis of ISSR, SSR, and RAPD are useful techniques. These findings demonstrate a large ratio of variation in sorghum. This work could serve as a guide for future research on sorghum and aid in the understanding of species and breeding initiatives.

Variation of Fruit Color in Cakra Hijau, G1/M8 and HV-149 Chilli Pepper Cultivar: Physiology and Molecular Approach

The fruit color of chili pepper is an important characteristic in identification and classification and is often used as the basis for determining consumer preferences. Information on the relationship between chili fruit color and its molecular profile is very important in supporting selection activities in plant breeding. This study aims to identify genetic diversity associated with the fruit color of three genotypes of chili (Capsicum frutescens L.): Cakra Hijau, HV-149 and G1/M8, using Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR). Morphological confirmation was carried out according to Capsicum descriptors. Nineteen RAPD markers and six SSR markers were used for genetic variability assessment. Genetic variation was analyzed using the unweighted pair group method with the arithmetic mean and the Jaccard similarity index. The three chili genotypes had different fruit colors at each maturation stage. The immature Cakra Hijau fruit is dark green and turns dark red as it ripens. The immature fruits of the G1/M8 line are light green and turn red when ripe. Finally, immature HV-149 fruits are dark green and yellow when ripe. The SSR markers used in this study were unable to show polymorphism. On the other hand, the RAPD marker successfully detected genetic variation in the three chili genotypes and resulted in a total of 49 alleles. The average value of polymorphic information content of the RAPD primers used ranged from 0 to 0.296, with the highest index indicated by OPA-1. The dendrogram shows the separation of the three genotypes into two main clusters, with the first cluster consisting of the HV-149 variety and the second cluster consisting of Cakra Hijau and G1/M8 lines. This study revealed that there are genetic variations based on the morphological characteristics of fruit color at each ripening stage and RAPD band profile. The RAPD marker was more effective than the SSR marker for identifying the genetic diversity of fruit color in the three chilies studied.

Association between quantitative morphological traits and RAPD molecular markers in pomegranate (Punica granatum L.).

Pomegranate (Punica granatum L.) is very important in terms of horticulture and food around the world. The present research aimed to identify the random amplified polymorphic DNA (RAPD) markers associated with morphological traits in pomegranate genotypes. Significant differences were observed among the studied genotypes based on the recorded traits. The 18 RAPD primers produced a total of 154 polymorphic fragments among genotypes. Using multiple regression analysis between each of the morphological traits and 154 RAPD polymorphic bands, RAPD markers associated with each of the morphological traits were identified. In total, 11 markers showed significant correlations with fruit weight, 9 markers with 100-aril weight, 11 markers with anthocyanin, and 8 markers with total soluble solids. Some markers were associated with more than one morphological trait, showing that the association of a marker with more than one trait can be caused by the pleiotropic effects of quantitative trait loci related to each other in different traits. For instance, the BA6-1 marker showed positive correlations with fruit weight, fruit crown width, and leaf length. Also, OPG13-3 and BA6-10 markers showed positive correlations with total soluble solids and anthocyanin content. The informative markers identified related to morphological characteristics in pomegranate can be a suitable guide to identify the genotypes with valuable fruit traits. Also, these markers can be used in selecting suitable parents for population generation for mapping purposes.

The potential of gamma irradiation on antioxidant capacity and genomic alterations in Calendula officinalis

There are lines of evidence that ionizing radiations such as gamma rays can cause different biological effects on plants. Marigold (Calendula officinalis L.) is a member of the family Asteraceae. It possesses profound amounts of active ingredients. The aim of this study was to evaluate the changes imposed upon different dose levels of gamma radiation on some features of Calendula officinalis such as antioxidant activity, total phenolic compounds and flavonoid contents, antibacterial activity and genomic alterations. Calendula officinalis seeds were exposed to different doses of Gamma radiation (0, 10, 15, 20 and 25 GY). Total phenolics, flavonoids, antioxidant activity (measured by DPPH assay) using methanolic extracts of plants and antibacterial activity measured by the disc diffusion assay showed significant differences to the control samples. The samples treated with 10 GY gamma rays showed the highest total phenol and flavonoid contents. Antioxidant activity significantly differed between Gamma rays dose levels and it was the highest at 25 GY. Four bacterial strains including E. coli, Bacillus subtilis and Pseudomonas aeroginosa were used for the antibacterial assay. Extracts from plants treated with 25 GY gamma rays showed the highest antibacterial activity against the 4 bacterial strains. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the genetic variation. The polymorphism information content (PIC) for RAPD primers ranged from 3% to 13% and ranged from 6 to 13% for ISSR primers. Results indicated that ISSR markers were more efficient than RAPD markers, as they detected 25.57% polymorphic DNA bands compared to 21.31% polymorphism for RAPD markers.

DNA barcodes, ISSR, RAPD and SCAR markers as potential quality control tools for molecular authentication of black and white mulberry

Morus species, especially black mulberry (Morus nigra L.), are well known for their nutritive and economic values. Black mulberry is often a subject for deliberate and undeliberate substitution with the morphologically similar white mulberry (Morus alba L.). In the current study, the genetic diversity of Morus nigra L. and Morus alba L. was assessed using different genetic profiling and fingerprinting techniques namely namely DNA barcoding, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). DNA barcoding was implemented using 2 plastid loci (matK, and rbcL-1) and 2 nuclear spacers (ITS and ITS2) aiming at accurate authentication of the studied species. DNA barcoding, targeting the selected loci succeeded to identify and authenticate the studies Morus species but failed to differentiate them, owing to their close genetic resemblance. Out of 52 screened RAPD primers, 27 were able to produce reproducible and clear profiles with 115 amplified fragments of which 37 were polymorphic accounting for 32.17% polymorphism. The number of amplified bands varied from 2 to 7 ranging in size from 100 to 700 bp. Moreover, 55 ISSR primers were evaluated, 108 fragments were produced, using 20 primers, of which 28 were polymorphic accounting for 25.92% polymorphism with size range of 200–800 bp. Sequence Characterized Amplified Region (SCAR) marker was developed based on reproducible banding pattern obtained by RAPD primer (OPG-03), for the purpose of accelerating the initial screening of Morus batches and enabling the preventative rejection of adulterated samples. The polymorphic band obtained was sequenced and specific primers were designed to develop a SCAR marker of approximately 558 bp exclusively for M. nigra, allowing detection of down to 1 ng, of M. nigra. The SCAR marker showed 100% success in differentiating authentic M. nigra and M. alba samples. In addition, the application of the developed SCAR marker was extended to distinguish M. nigra from its four common adulterants namely Ficus carica L., Fragaria vesca L., Ficus racemosa L. and Vitis vinifera L.. The proposed protocol was successfully implemented for the evaluation of 47 commercial M. nigra samples collected from different Egyptian locations.

Evaluation of the Genetic Stability of Amaranthus viridis L. Species in Selected Regions of Western Ghats using Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA is cheap and fast molecular technique to identify the genetic resemblances or variations in DNA in various plants. The proposed research work wasintendedto compare the genetic diversification of Amaranthus viridis species in selected regions of Western Ghats in Tamilnadu and Kerala by RAPD analysis.The biometric morphological data among the five A. viridis accessions were compared. Their genomic DNA was isolated and finger prints were obtained using three RAPD markers. The percentage polymorphism, polymorphism information content, effective multiplex ration and resolving power were calculated. UPGMA dendrogram was constructed and their genetic relatedness was compared using Jaccard coefficient.Upon finger printing, 66 bands were counted for the three RAPD primers used,among which 46 bands were polymorphic band numbers from 8 to 21. Percentage of polymorphic bands ranged between 44.44% and 80.77%. EMRvalues for 66 polymorphic loci ranged between 3.56 and 17.01. The UPGMA dendrogram comparing the genomic profiles of the 5 A. viridis accessions using Jaccard coefficient revealed their genetic distances where the maximum Jaccard’s coefficient value observed was 0.96 between AVKATN and AVNEK. Among the 5 selections the peak similarity index (0.9565) was witnessed between AVKATN and AVNEK. The cuurent study revealed that AVKATN and AVNEK had the highest their genetic distances, whereas AVKATN and AVNEK were genetically similar among the 5 A. viridis accessions. Several polymorphic bands which ranged up to 80.77% have proved their genetic variations.

Relationship between molecular markers and important fruit‐related traits in almond (Prunus dulcis [Mill.] D.A. Webb syn. P. amygdalus Batsch) as revealed using multiple regression analysis (MRA)

AbstractAlmond (Prunus dulcis [Mill.] D.A. Webb syn. P. amygdalus Batsch) is one of the most important nut crops, and its kernel is the edible part that has a high nutritional value and is used in the confectionery and cosmetics industries. The present research aimed to identify random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) molecular markers associated with important fruit traits in late‐blooming almond genotypes through multiple regression analysis (MRA). The studied genotypes showed significant differences from each other in terms of the measured fruit‐related traits. The ISSR primers used produced a total of 125 bands in the studied germplasm, of which 112 showed polymorphic bands. The RAPD primers produced a total of 190 DNA fragments, of which 172 fragments showed polymorphism among genotypes. Some polymorphic fragments of ISSR and RAPD showed significant correlations with the fruit traits measured. Some of these informative markers were associated with more than one trait, which could be caused by the pleiotropic effects of quantitative trait loci related to each other in different traits. For instance, some of the markers showed significant correlations with both nut weight and kernel weight, which indicates a positive correlation between these two traits. Informative markers identified in this study can be used to select suitable parents for population generation for mapping. It is also useful for selecting superior genotypes, especially when information about their genetic basis, such as a linkage map, is not available.

Evaluation in Genetic Variation of Krachai by Sequence Related Amplified Polymorphism (SRAP) and Random Amplified Polymorphic DNA (RAPD) Techniques

Krachais are local name of Thai herb which are identified within 2 genera, namely Bosenbergia (white or red krachai) and Kaempferia (black krachai). The rhizome of Bosenbergia rotunda (L.) Mansf. contains major aromatic compounds against COVID19 but its many morphological characteristics such as leaves, stems and rhizome are like other species in same genus or family. In this study, a total of 13 accessions containing 5 samples of B. rotunda, 2 samples of Boesenbergia spp. and 3 samples of Kaempferia parviflora including 3 samples of other species in same family as outgroups were analyzed and clustered using sequence-related amplified polymorphism (SRAP) and random amplified polymorphism DNA (RAPD) markers. The results showed that 8 SRAP primers (M1E1, M1E2, M1E7, M2E10, M3E9, M5E1, M5E2 and M6E10) could generate 79 polymorphism bands with an average of 92.15% whereas a total of 5 RAPD primers (OPY04, OPY02, OPY04, JAT11 and JAT12) could give 64 polymorphisms with 100% as a percentage of the polymorphism band. The PIC of the SRAP marker (0.470) has a higher value than the RAPD marker (0.264). The highest similarity coefficients within genus Boesenbergia of 1.000 and 0.952 were obtained from SRAP and RAPD markers, respectively. The UPGMA dendrogram of SRAP and RAPD information among krachai presented 2 and 4 groups, respectively. The cluster of Boesenbergia was separated from K. parviflora and other species in same family. Furthermore, the results also pointed that Boesenbergia sp. from Phayao is in correct genus and is B. pandurata (Roxb.) Schltr. because of having red leaf while Boesenbergia sp. from Chiang Rai showed confusion between SRAP and RAPD data. It was concluded that SRAP and RAPD have great potential for the study of genetic diversity of Boesenbergia and other species in family Zingiberaceae.

Dye decolorization of magnetically retrievable formulation of laccase with amine-functionalized microparticles

Laccase is an important enzyme used in many industries because of its multi-substrate catalyst. New immobilization agents are excellent tools for enhancing the abilities of this enzyme. In this study, immobilization of laccase on silica microparticles with NH2 (S-NH2) surface modification to use in dye removal applications was aimed. The yield of immobilization by this method was found to be 93.93 ± 2.86% under optimum conditions. In addition, this newly created immobilized enzyme was adapted to a decolorization application with 87.56 ± 1.60% efficiency. Silica microparticles with NH2 (S-NH2) surface modification were used for laccase immobilization and this immobilized laccase had quite good potential. Besides, Random Amplified Polymorphic DNA (RAPD) analysis in evaluating the toxicity of the decolorization process was utilized. After amplification with two RAPD primers, decreased toxicity of dye in this study was observed. This study showed that RAPD analysis in toxicity testing could be accepted as an alternative and practical method that this approach will contribute to the literature in terms of providing fast and reliable results. The use of amine-modified surface silica microparticles for laccase immobilization and RAPD for toxicity testing is a crucial aspect of our investigation.

Molecular cloning and development of RAPD-SCAR markers for the selection of thermo-tolerant line of tropical tasar silkworm

Aim: To develop random amplified polymorphic DNA (RAPD) -sequence-characterized amplified region (SCAR) markers for selecting thermo-tolerant line of tropical tasar silkworm. Methodology: In the environmental chamber, Daba BV cocoons of A. mylitta were exposed to high temperature at 46°C/4 hr for 3 days. After the emergence, genomic DNA was extracted from thermo-tolerant moths and thermo-susceptible pupae. The genomic DNA was amplified using 30 RAPD random decamer primers. The improved RAPD fragments that can differentiate thermo-tolerant and susceptible line of A. mylitta were eluted, cloned in pJET1.2 vector and sequenced. SCAR markers were developed based on the sequence and validated in the subsequent generations. Results: Among 30 RAPD primers, OPK04, OPAJ15 and OPA17 generated polymorphic bands to differentiate thermo-tolerant and susceptible line of A. mylitta. These polymorphic bands were eluted, cloned and sequenced. Sequencing of three cloned fragments revealed that clone PB1 comprised of 1412 bp, clone PB2 comprised of 704 bp and clone PB3 comprised of 931 bp. Sequence specific stable multiplex SCAR markers TT-PB1, TT-PB2 and TT-PB3 were designed and synthesized. PCR amplification was performed using DNA templates of 10 thermo-tolerant and 10 thermo-susceptible samples. SCAR marker TT-PB1 was observed to be more specific to thermo-tolerant line of tropical tasar silkworm and validation with 25 samples each in next generation also supported the specificity of TT-PB1. Interpretation: Among three SCAR markers, TT-PB1 showed more specificity for selecting the thermo-tolerant line of A. mylitta. Therefore, the study provides an effective and precise PCR-based molecular marker system for selecting thermo-tolerant lines in tropical tasar silkworm to overcome seed crop loss due to high temperature stress in tasar rearing hotter zones. Key words: Antheraea mylitta, cloning, RAPD, SCAR m­­­arker, Thermo-tolerance

Intra Specific Relationships and Biochemical Composition of Laportea Aestuans (L.) Chew, Sida Rhombifolia L. And Commelina Verginica L. in Lagos State

Laportea aestuans L. (Chew), Commelina virginica (L.) and Sida rhombifolia (L.) are common wild plants used in treating several ailments including diarrhea, dysentery, hernia, oedema, ulcers and many more in traditional African medicine especially, Nigeria. The potentials of Random Amplified polymorphic DNA (RAPD) primers in delimiting intra-specific variation in L. aestuans, C. virginica and S. rhombifolia was assessed using three RAPD primers. Plant and soil samples were collected from 19 local government areas in Lagos State and assessed for genetic and biochemical relationships. A total of 56 bands were produced of which 44 were polymorphic. Maximum number of bands (21) was produced by OPY20 while OPC04 produced minimum number of bands (14). The maximum and minimum percentage polymorphisms were 100% (OPC04) and 50% (OPA12), The dendrogram of genetic diversity had a genetic distance range of 0.57 to 1.00, 0.58 to 1.00 and 0.52 to 1.00 and clustered at 0.57, 0.58, 0.52 implying 57%, 58%, 52% similarity and 43%, 42% and 48% variability for L. aestuans, C. virginica and S. rhombifolia respectively. Results of phytochemical analysis show that S. rhombifolia is particularly high in steroids (I134.19±0.23), flavonoids (118.65±0.18), phenolics (125.25±1.43), alkaloids (63.56±0.11) and triterpenes (72.44±0.12) but low in saponins (23.29±0.05), glycosides (6.79±0.01) and tannins (7.10±0.02).

Population Genetic Structure of Feral and Cultured African Catfish (Clarias gariepinus) inferred from Random Amplified Polymorphic DNA in Kano, Nigeria

Information on the genetic structure of fish is a useful means for optimizing identification of stocks, stock enhancement, breeding programs, management of sustainable yield and preservation genetic diversity. The population genetic structure of African Catfish, Clarias gariepinus (Burchell, 1822) in cultured population and feral populations from Rivers Guzuguzu, Fada and Magaga (Kano State) were investigated using RAPD (Random Amplified Polymorphic DNA). Using a CTAB protocol, genomic DNA was extracted from the caudal peduncle of 157 samples of live specimen collected from each population. Five RAPD primers were used to amplify different loci on the extracted genomic DNA by Polymerase Chain Reaction (PCR) and the resultant DNA fragments were analyzed on agarose gel. A total of 406 reproducible bands were obtained in four populations for five primers. The dendrogram separated the four C. gariepinus populations into two distinct clades, Guzuguzu and Fada populations being in one clade, while Magaga and Cultured populations belonged to the other clade. The results based on RAPD-PCR profile ranged from 0.012 in Magaga/Cultured to 0.089 in Guzuguzu/Cultured. The genetic identity of C. gariepinus from four populations also ranged between 0.885 in Magaga/Guzguzu to 0.998 in Magaga/Cultured. The Nei’s genetic distance and identity also confirmed the above information with the following ranges: 0.002 in Fada/Cultured to 0.102 in Magaga/Guzuguzu, and 0.903 in Magaga/Guzuguzu to 1.000 in Magaga/Cultured respectively. In conclusion, the genetic diversity and allele richness of the feral and domestic fish populations were comparable.

Genetic differentiation among Batak fish populations (Neolissochilus sumatranus, Tor douronensis, and Tor soro) in North Sumatra, Indonesia revealed by RAPD markers

Abstract. Barus TA, Wahyuningsih H, Simanjuntak BN, Ginting RH, Batubara AS, Hartanto A. 2023. Genetic differentiation among Batak fish populations (Neolissochilus sumatranus, Tor douronensis, and Tor soro) in North Sumatra, Indonesia was revealed by RAPD markers. Biodiversitas 24: 1327-1332. Mahseer or Batak fish species within the genera of Neolissochilus and Tor are highly valued as a source of food for local communities, especially in North Sumatra. Assessment of genetic differentiation of the Batak fish population, namely Tor soro, Tor douronensis, and Neolissochilus sumatranus in the North Sumatra Rivers, has been conducted. The objective of this study was to determine the possible genetic divergence among congeneric fish and supply the genetic information for the design of conservation strategies. Fish specimens were collected from three rivers in North Sumatra Bahorok River (Langkat District), Asahan River (Toba Samosir District), and Batangtoru River (South Tapanuli District). The genetic analysis employed the Random Amplified Polymorphism DNA (RAPD) markers using a set of RAPD primers: OPA-2, OPA-3, OPA-6, OPA-7, OPA-9, OPA-11, and OPA-17. The amplified fragments were analyzed using the Numerical Taxonomy and Multivariate Analysis System (NTSYS) 2.00 program to construct the dendrogram of relationship among accessions. The genetic similarity coefficient among species of T. soro, T. douronensis, and N. sumatranus reached 0.44-0.86, with the lowest similarity at 44%. The cluster analysis indicated that T. douronensis is more closely related to N. sumatranus than T. soro based on the grouping of accessions. There is an indication of natural hybrids occurrence between N. sumatranus and T. soro populations despite the different habitats and locations of sampling. Our study revealed that using RAPD markers may discriminate inter- and intraspecific Batak fish populations in North Sumatra.

Molecular identification of mother trees of four matoa cultivars (Pometia pinnata Forst & Forst) from Pekanbaru City, Indonesia using RAPD markers

Abstract. Zulfahmi, Pertiwi SA, Rosmaina, Elfianis R, Gulnar Z, Zhaxybay T, Bekzat M, Zhaparkulova G. 2023. Molecular identification of mother trees of four matoa cultivars (Pometia pinnata Forst & Forst) from Pekanbaru City, Indonesia using RAPD markers. Biodiversitas 24: 1524-1530. Pekanbaru city has four matoa (Pometia pinnata Forst & Forst) cultivars, i.e. red matoa, yellow matoa, green matoa, and black matoa, but the knowledge of the genetic variation among matoa cultivars is not available. The objective of this study was to assess the genetic variation among matoa cultivars using Random amplified polymorphism DNA (RAPD) markers and to identify the specific marker to distinguish among matoa cultivars. Eighteen primers were initially screened and four matoa cultivars were analyzed with twelve primers using the RAPD marker. The results of this study found that the twelve selected RAPD primers generated 39 fragments, with fragment sizes ranging from 200 to 1500 bp. The percentage of fragment polymorphic was 80.41%, indicating the high genetic variation of the matoa cultivar. The high genetic variation of matoa in this study is caused by cross-pollinated among matoa cultivars in the field. Eleven primers can differentiate four matoa cultivars with specific bands. The obtaining unique bands can be utilized by breeders and farmers as a basis to select the parents for the genetic improvement of Matoa, cultivars or clones protection, detection of seedling purity in a nursery, and to verify the originality of the seedlings that will be cultivated.