Inter-simple Sequence Repeat Primers Research Articles

Inter simple sequence repeats and morphological traits to identify cultivated cotton varieties (Gossypium barbadense L.) in Egypt

Egyptian cultivated Cotton significantly impacted Egypt's economy, as it is well-known worldwide. This study aims to determine how much genetic and phenotypic variation exists in five different varieties of Egyptian Cotton using Inter Simple Sequence Repeats (ISSR) as a molecular marker and twenty-one quantitative and qualitative morphological traits as a taxonomic source in the development and evolution of this plant. Eleven ISSR primers were used, producing a total of 134 bands with a polymorphism percentage of 67%. Positive and negative significant Pearson correlations were found among the studied morphological traits in line with the phenotypic correlations in some characteristics. The genotypic correlation coefficient was higher in magnitude than that of phenotypic correlation. The five varieties were grouped into two major clusters using the UPGMA method based on morphological and ISSR analysis. The first one included G86 and G89 varieties, while the second cluster included G80 and G95; the G90 was separated from the other four varieties. This genetic relationship may be attributed to their similar ancestors. The information from this study should help with cotton breeding efforts to attain a high level of germplasm diversity and develop new high-yielding types to enhance cotton production and quality.

GENETIC DIVERSITY OF BAK-KALA (ETLINGERA ELATIOR (JACK) R.M. SM.) IN ACEH PROVINCE, INDONESIA

Torch ginger – bak-kala (Etlingera elatior (Jack) R.M.SM.) already has been proven an effective traditional medicine by tribes in Aceh Province, Indonesia. Solid primary evidence for the torch ginger, bak-kala’s medicinal properties include the healing effects experienced by the traditional tribes in Aceh, when treated for illnesses, such as, cough, fever, and sprains. Likewise, it has been proven to be a use for food ingredients, i.e., vegetables and spices to enhance the taste of food. Much of the available documented information focused on its biochemical and pharmacological aspects. Even though the utilization of bak-kala resources is mainly at the level of diversity, there is no information on its genetic diversity in Aceh. The high intensity of bak-kala utilization is not simultaneous with information on its genetic diversity. On the other hand, many uses of bak-kala are specific for certain accessions with particular benefits. The study aimed to create a classification system for bak-kala variations based on inter-simple sequence repeats (ISSR) markers and to provide information on the genetic diversity of bak-kala in Aceh Province. The 35 accessions were divided into eight populations based on the geographical locations from which they were collected. Data were recorded and analyzed using 10 ISSR primers with 77 total bands. The molecular characters divided the accessions into four major groups. As revealed by expected heterozygosity (He), genetic variability among the population showed that the Simeulu population possessed a greater level of variability than other populations. The results of the analysis of molecular variation (AMOVA) showed that the genetic variation within the population was higher (60%) than the genetic variation among populations (40%). The studies can be used to plan conservation strategies, optimal utilization of the species, and crop improvement programs in the future.

Morphological and Molecular Characterizations of Musa (ABB) 'Mali-Ong' in Thailand.

Simple SummaryThe banana cultivar Musa (ABB) ‘Mali-Ong’ is widely used in the processed food industry in Thailand, where it determines the quality of the products. However, the sub-cultivars of ‘Nam Wa Mali-Ong’ are almost indistinguishable, with few morphological differences and minimal genetic variation. This study used 77 morphological characteristics and two types of molecular markers to distinguish Nam Wa Mali-Ong from other cultivars. The study also assessed the genetic variation of nine ‘Nam Wa Mali-Ong’ clones and compared them with 10 other samples of bananas with different genomes or chromosome sets. The molecular markers grouped the ‘Nam Wa Mali-Ong’ samples. Four clones (A, B, D, and I) were superior and had higher bunch weights. This study will be useful for germplasm evaluation and future ‘Nam Wa Mali-Ong’ improvements.Musa (ABB) ‘Mali-Ong’ is an economically important banana cultivar in Thailand. We morphologically and molecularly characterized ‘Nam Wa Mali-Ong’. Leaf blade width was the only statistically different morphological character among the clones. To determine genetic variation, nine ‘Nam Wa Mali-Ong’ clones were compared with 10 samples of Musa ABB, AA, and BB cultivars by fingerprinting using seven pairs of sequence-related amplified polymorphism (SRAP) and eight inter simple sequence repeat (ISSR) markers. The SRAP and ISSR primers generated 65 and 76 amplicons, respectively, of which 57 (87.7%) and 62 (81.6%) amplicons, respectively, were polymorphic; the polymorphic information content was 0.28–0.49. The SRAP data revealed two distinct groups: Group I, comprising two subgroups (one including all ABB samples and the other containing the BB genome accessions), and Group II, comprising the AA genome accessions. The ISSR data revealed two groups: Group I, which incorporated the AA (Hom Champa) genome, and Group II, consisting of two subgroups: Subgroup A, comprising only the AA (Hom Chan) accessions, and subgroup B, comprising all the ABB accessions and wild banana M. balbisiana (BB genome). The ‘Nam Wa Mali-Ong’ samples clustered together, regardless of the markers used. SRAP and ISSR markers will be useful for germplasm evaluation and future Musa (ABB) improvements.

Analysis of genetic diversity among Chinese Cyclocybe chaxingu strains using ISSR and SRAP markers.

BackgroundCyclocybe chaxingu is an edible and medicinal fungal species commonly cultivated in China. The major problems currently facing by growers of C. chaxingu is the random labeling of strains and strains aging and degeneration. Therefore, an evaluation of genetic diversity is essential for the conservation and reproducing programs of this species.MethodsIn the present study, 24 widely cultivated strains were collected from the main producing areas of China, and the genetic diversity analysis was performed. DNA polymorphism among these Chinese C. chaxingu strains was analyzed using inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers.ResultsEight ISSR primers amplified a total of 75 DNA fragments of which 61 (81.33%) were polymorphic. Fifteen SRAP primer combinations amplified 166 fragments of which 132 (79.52%) were polymorphic. Cluster analysis showed that the C. chaxnigu strains fall into five groups with a genetic distance values ranging from 0.06 to 0.60 by ISSR analysis, while the SRAP analysis divided the test strains into four groups within the range of genetic distance from 0.03 to 0.57. The results of the present study reveal a high level of genetic diversity among the widely cultivated C. chaxingu strains.

Efficient shoot regeneration of medicinal plant Haplophyllum tuberculatum by direct and indirect organogenesis and genetic fidelity assessment using Inter Simple Sequence Repeats markers.

In vitro plant cell and tissue cultures are potent tools to propagating germplasm resources in conserving and managing plant genetic resources. A reliable micropropagation protocol was developed for efficient callus proliferation and direct and indirect shoot regeneration of Meseika (Haplophyllum tuberculatum). With the applied sterilization procedure, immature, unopened H. tuberculatum seed pods can be identified as a potent explant with high viability and low contamination percentage. Multiple shoots were regenerated from leaf and stem explants through direct organogenesis on Murashige and Skoog’s (MS) + 3% sucrose medium amended with BAP. Indirect regeneration of several shoots was achieved on 1/2 MS + 1% sucrose media amended with 2 and 4 mg/l BAP. An efficient callus proliferation from both explants can be achieved by supplementing the MS media with NAA and BAP. All the cultures were incubated in a controlled growth chamber under 5/19 h light/dark photoperiod, temperature (25 ± 2°C), and 60% relative humidity (RH).10 ISSR (Inter Simple Sequence Repeat) markers were screened to test the genetic fidelity of regenerated H. tuberculatum shoots. Callus development was observed after 15 days and shoot regeneration was occurred after 30 days after callus initiation. 10 ISSR primers produced a total of 39 clear, distinct amplicons. 75, 60, 40, and 16% polymorphism percentages were recorded by the ISSR primer 11, 7, 5, and 4, respectively. The developed micropropagation protocol is appropriate for rapid in-vitro multiplication of H. tuberculatum shoots and callus.

Eco-physiological response and genotoxicity induced by crude petroleum oil in the potential phytoremediator Vinca rosea L

BackgroundPhytoremediation is determined as an emerging green technology suitable for the safe remediation and restoration of polluted terrestrial and aquatic environments. In this study, the assessment of an ornamental plant, Vinca rosea L., as a phytoremediator of crude oil in polluted soils was conducted. In an open greenhouse experiment, plants were raised in sandy-clayey soils treated with 1, 3, 5, and 7% oil by weight. The experiment was conducted over 5 months. ResultsTotal petroleum hydrocarbon (TPH) degradation percentage by V. rosea after a 5-month growth period ranged from 86.83 ± 0.44% to 59.05% ± 0.45% in soil treated with 1 and 7%, respectively. Plants raised in polluted soils demonstrated a dramatic reduction in germination rates, in addition to growth inhibition outcomes shown from decreased plant height. An increase in branching was observed with an increase in oil pollution percentages. Moreover, the phytomass allocated to the leaves was higher, while the phytomass witnessed lower values for fine roots, flowering and fruiting when compared to the controls. Apart from the apparent morphological changes, there was a decrease in chlorophyll a/b ratio, which was inversely proportional to the oil pollution level. The contents of carotenoids, tannins, phenolics, flavonoids, and antioxidant capacity were elevated directly with an increase in oil pollution level. The start codon-targeted (SCoT) polymorphisms and inter-simple sequence repeat (ISSR) primers showed the molecular variations between the control and plants raised in polluted soils. The genetic similarity and genomic DNA stability were negatively affected by increased levels of crude oil pollution. ConclusionsThe ability of V. rosea to degrade TPH and balance the increased or decreased plant functional traits at the macro and micro levels of plant structure in response to crude oil pollution supports the use of the species for phytoremediation of crude oil-polluted sites. The genotoxic effects of crude oil on V. rosea still require further investigation. Further studies are required to demonstrate the mechanism of phenolic, flavonoid, and antioxidant compounds in the protection of plants against crude oil pollution stress. Testing different molecular markers and studying the differentially expressed genes will help understand the behavior of genetic polymorphism and stress-resistant genes in response to crude oil pollution.

Genetic variation and molecular characterization of Zygophyllum coccineum L. ecotypes of the iron mining area of El-Wahat El-Bahariya in Egypt

Remediation and mitigation processes can recover the ecosystems affected by mining operations. Zygophyllum coccineum L. is a native indigenous xerophyte that grows in Egypt's Western Desert, particularly around the iron mining ore deposits, and accumulates high rates of potentially toxic elements (PTEs) in its succulent leaves. The present study evaluated the genetic variation and molecular responses of Z. coccineum to heavy metal stressful conditions in three sites. Results revealed that Z. coccineum bioaccumulation capacity was greater than unity and varied amongst the three locations. In response to heavy metal toxicity, Z. coccineum plants boosted their antioxidative enzymes activity and glutathione levels as a tolerance strategy. Anatomically, a compact epidermis, a thick spongy mesophyll with water storage cells, and a thicker vascular system were observed. Protein electrophoretic analysis yielded 20 fragments with a polymorphism rate of 85%. The antioxidant genes (CAT: catalase, POD: peroxidase and GST: polyphenol oxidase) showed greater levels of expression. In addition, DNA-based molecular genetic diversity analyses using Start Codon Targeted (SCoT) and Inter Simple Sequence Repeat (ISSR) markers yielded 54 amplified fragments (i.e. 24 monomorphic and 30 polymorphic), with 12 unique fragments and a polymorphism rate of 55.5%. The greatest PIC values were recorded for SCoT-6 (0.36) and for both of the 14 A and 44 B ISSR primers (0.25). Diversity index (DI) of all SCoT and ISSR amplified primers was 0.23. The present findings reveal the distinct heavy metal's adaption attributes of Z. coccineum, indicating its improved survival in severely arid mining environments.

SCREENING AND IDENTIFICATION OF GENETIC PURITY OF OKRA HYBRID [RANI] AND ITS PARENTAL LINES BASED ON MOLECULAR MARKERS

The present investigation was carried out to identify the genetic purity of okra hybrid and its parental line based on molecular markers. For the evaluation of genetic purity and varietal identification from collected Okra samples, molecular markers and polymerase chain reaction, as well as DNA fingerprinting approaches, were advocated. These samples were accumulated from field and nursery Adithya seeds Pvt. Ltd, Raipur (C.G) within the duration of study and research from January to July 2018. Genetic purity test over the conventional Grow-Out-Test (GOT) was achieved by using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) primers for identifying and emphasizing of hybrid. Using 3 lines of Okra cultivars, we analysed the products of PCR with RAPD and ISSR primers and discussed the design for constructing their Genetic Purity. Towards this study 64 random primers were screened between okra hybrid (Rani) and its parental lines [male line (206) and female line (189)]. Out of 64 primers only one primer (ISSR-B17898) showed polymorphic banding pattern between hybrid and its parental lines which helped in the identification and confirmation of okra hybrid. Hence, concluded that Genetic purity test is necessary for seed certification of different categories in seed multiplication chain both from agronomic as well as breeding point of view and proved to be explicit technology for researchers.

Identification of purple nutsedge (Cyperus rotundus L.) ecotypes and the effect of environmental factors on tuber sprouting in China

AbstractPurple nutsedge (Cyperus rotundus L.) is a widely distributed weed. To evaluate the effect of environmental factors on tuber sprouting in different ecotypes, an inter‐simple sequence repeat (ISSR)‐based genetic analysis approach was used to assess the genetic relatedness among 24 purple nutsedge populations collected in China. Twenty‐one ISSR primers were selected to generate 167 scorable bands. The percentage of polymorphic bands (PPB) was 97%, indicating a high level of genetic diversity, and the cluster results largely coincided with the geographical locations. Three ecotypes, HN‐1, HB‐1 and SD‐1 (collected from Hai'nan Island, central China and northern China, respectively), with large genetic and geographic distances from each other, were selected to assess variation in the purple nutsedge sprouting trait. Compared with the other ecotypes, ecotype SD‐1 showed the highest sprouting capacity at constant 15°C and at 10/20°C, and the shortest time to reach 80% sprouting (t80) at constant 20°C and at 15/25 and 20/30°C. The sprouting percentage was very high (95%–98.8%) and without significant differences between the three ecotypes over a pH range between 5 and 9. The osmotic potentials that caused 50% inhibition of the maximum sprouting percentages of HN‐1, HB‐1 and SD‐1 were −0.37, −0.43 and −0.44 MPa respectively. Ecotype SD‐1 appeared to maintain a slightly higher sprouting percentage under osmotic stress, but the difference was not significant. These findings suggest that the higher tolerance to lower temperatures and drought of ecotype SD‐1 might be an adaptation to lower winter temperature and drier climatic conditions in North China. Abundant genetic diversity may be a potential factor that endows purple nutsedge with strong environmental adaptability.

Research on germplasm diversity of Amomum villosum. Lour in genuine producing area.

Genuine Chinese medicine is produced from medicinal plant cultivated in a specific region and is of better quality and efficacy, more consistently qualified and famous than that from the same medicinal plant cultivated in other regions. The cultivating region of genuine medicinal plant is known as the genuine producing area. Yangchun City, which is in Guangdong Province of China, is a genuine producing area for the famous Chinese medicine Amomi Fructus (also called Sharen). Amomi Fructus is the ripe and dry fruit of the Zingiberaceae plant A. villosum Lour.. A. villosum was introduced from the Persian Gulf region and has been cultivated in China for over 1000 years. Until now there are no reports on screening for good germplasm of A. villosum. The contents of volatile oil and bornyl acetate of Amomi Fructus from 14 populations were determined with GC method, and the relative contents of the main chemical components in the volatile oils were determined with GC-MS method. Evaluation and variance analysis of the comprehensive quality of the 14 samples were conducted by means of a multi-indicator entropy-weight TOPSIS model (Technique for Order Preference by Similarity to an Ideal Solution) combined with OPLS-DA (Orthogonal Partial Least Squares Discrimination Analysis) and HCA (Hierarchical Clustering Analysis). The ISSR (Inter-Simple Sequence Repeat) molecular marker technique and the UPGMA (unweighted pair-group method with arithmetic means) were employed to analyze the genetic relationship among A. villosum populations. The contents of volatile oil and bornyl acetate differed significantly among the different populations, but the main chemical component in the volatile oil was the same in all the samples, which was bornyl acetate. OPLS-DA results showed that 9 indicators were the main factors influencing the quality differences among the 14 populations. The entropy-weight TOPSIS results showed that there were significant differences in the comprehensive qualities of the 12 populations from the genuine producing area. The best quality of fruit was found in the genuine producing area of Chunwan Town; the qualities of 33% of genuine fruits were lower than that of non-genuine fruits. Twenty-three DNA fragments were obtained by ISSR-PCR amplification using four ISSR primers, eleven of which were polymorphic loci, which accounted for 47.8%. The similarity coefficients (GS) of different populations of A. villosum ranged from 0.6087 to 0.9565. There are significant differences among different populations of A. villosum in terms of the kinds of major chemical components and their contents, comprehensive quality and genetic diversity. The germplasm resources of A. villosum are rich in the genuine producing area. It means superior germplasm could be selected in the area. The comprehensive quality of the fruit of A. villosum from the non-genuine producing area is better than some of that from genuine producing area, proving that the non-genuine producing area can also produce Amomi Fructus with excellent quality.

GENERAL CHARACTERISTICS OF CANISTEL (POUTERIA CAMPECHIANA) IN TRA VINH PROVINCE BASED ON MORPHOLOGICAL FEATURES

Canistel is a high-value fruit tree whose flesh is rich in nutrients and flavor. However, the research has not been focused on the management and conservation of the genetic resources of this crop. Molecular markers have been used in many studies to assess the diversity of different crops. However, studies evaluating the genetic diversity of canistel plants are still limited. The study aimed to evaluate the genetic diversity of canistel samples with morphological characteristics and molecular markers (inter-simple sequence repeat marker and random amplified polymorphic DNA marker). The diversity of morphological characteristics, in terms of Shannon’s information index was 0.83, suggesting high genetic diversity of 30 canistel genotypes. Four inter-simple sequence repeat (ISSR) primers generated 25 polymorphic bands. Four RAPD primers revealed 55 random amplified polymorphic DNA (RAPD) fragments. Both ISSR and RAPD primers reached 100% polymorphism. The obtained results indicate the high genetic diversity between the individuals of Pouteria campechiana in Tra Vinh, Vietnam.

The first study of genetic diversity and population structure of Indo-Pacific bottlenose dolphin (Tursiops aduncus) and pantropical spotted dolphin (Stenella attenuata) in the Thai Andaman Sea based on ISSR.

Background and Aim:The Indo-Pacific bottlenose dolphin, Tursiops aduncus, and the pantropical spotted dolphin, Stenella attenuata, are protected marine mammals in Thailand; however, knowledge regarding the populations of both species in Thai seas is minimal. We aimed to reveal the genetic diversity and population structure of two species, T. aduncus, and S. attenuata, based on inter-simple sequence repeats (ISSRs).Materials and Methods:Samples of stranded T. aduncus (n = 30) and S. attenuata (n = 23) found along Thai Andaman Sea coasts from 1998 to 2018 were used in this study. A total of 17 and 16 ISSR primers that produced clear and polymorphic bands were selected for T. aduncus and S. attenuata, respectively.Results:The highest percentages of polymorphic bands for T. aduncus and S. attenuata were 93.750% and 92.857%, respectively. Phylogenetic dendrograms indicated that the population of each species was clustered into three groups. This outcome was consistent with the genetic population structure, as both suggested three genetic clusters (DK = 3). Genetic diversity analysis revealed that the average Shannon’s information index (I) was 1.926 ± 0.066 for T. aduncus and 1.714 ± 0.090 for S. attenuata, which indicate a high level of genetic variation. Further, low fixation index (F) values were observed for T. aduncus and S. attenuata at −0.231 ± 0.024 and −0.312 ± 0.042, respectively, suggesting that inbreeding is unlikely to have occurred for both species over the past decades.Conclusion:At least three genetic clusters of both species were found in the Thai Andaman Sea, and the diversity indices of each species indicated that these species are not at a critical level for extinction. However, monitoring their population status should be prioritized to observe any future changes in the level of diversity.

Characterization of indigenous phalsa (Grewia subinequalis) genotypes using morphological traits and ISSR markers

BackgroundPhalsa (Grewia subinequalis L.) is a commercial and nutritionally important berry fruit. It is cultivated in Pakistan as a minor fruit crop without any variety characterization. Therefore, the objective of to assess genetic diversity through ISSR markers and morphological features of wild phalsa genotypes collected from different parts of Punjab, Pakistan. MethodsMorphological characteristics such as plant height, stem circumference, growth habit, leaf length, leaf width, leaf area, leaf color and leaf apex shape showed high variation among genotypes. ResultsAmong the twenty inter-simple sequence repeats (ISSR) primers, UBC-812 exhibited the highest PIC values of (0.485) and Dj (0.389) compared to other primers, which considered it better for the identification of phalsa genotypes and prediction of diversity. Moreover, the unweighted pair group method with arithmetic mean cluster analysis divided the sampled genotypes into five clusters (clusters A-E) based on morphological analysis, while molecular data divided the genotypes into eight clusters (clusters A-H). ConclusionThis study confirmed the high diversity in wild populations especially 'O1P2” and “O7P3” genotypes, with both DNA-based and morphological descriptors that depict their potential use for future phalsa breeding programs.

Characterization and selection of inter simple sequence repeat markers for genetic studies of mesquite

Since characterization and selection of molecular markers are crucial steps in genetic research for plant conservation and/or (pre) breeding, this study aimed to describe the amplification profile of Inter Simple Sequence Repeat (ISSR) markers in Prosopis juliflora (Sw.) DC. so that the most suitable ones could be selected. To do so, the genomic DNA of 16 individuals collected at the Federal Institute - Campus Itapetinga, Bahia State, were extracted at the Applied Molecular Genetics Laboratory of the State University of Southwest Bahia, in Itapetinga-BA (Brazil). The amplification profile was obtained using 23 ISSR primers, visualized after electrophoresis in 2% agarose gels, and photo-documented under ultraviolet light; then, genotyping was performed. In total, amplification of 206 markers was observed, with an average of nine tags per primer, a polymorphic percentage equivalent to 90% of the markers generated; and mean polymorphic information content of 0.63. Expected and observed heterozygosity means were similar (0.28 and 0.29, respectively). Our findings confirm that ISSR primers are suitable for molecular-genetic studies of P. juliflora. Among the 23 analyzed primers, 13 can be prioritized (DiGA3’G, DiCA 3'RG, DiCA 3'YG, DiGA 3'C, DiGA 3'RC, DiGA 3'T, TriCAG 3'RC, TriTGT 3'YC, TriAAC 3'RC, TriAAG 3'RC, TriCGC 3'RC, TriGAC 3'RC, and TriGGA 3'RC) for genetic studies of P. juliflora.

Molecular analyses of genetic diversity in thepopulation of Calligonum Comosum L. ‘Her in Hail region in the Kingdom of Saudi Arabia

This paper presents the first molecular study on Calligonum comosum L. ‘Her (Polygonaceae) in the Kingdom of Saudi Arabia, due it’s known to be part of Saudi Arabia’s flora, as a famous medicinal plant, it has several other uses, as it is palatable as animal feed, and dune stabilization, and to increase soil organic matter content. And it has been included on the endangered species list due to its frequent use. Twenty-five C. comosum samples were collected from five different populations from their natural environment and screened using Inter-Simple Sequence Repeat (ISSR) markers. Eleven ISSR primers amplified 975 reproducible bands of which 495 were polymorphic, and polymorphism of genetic variation between species was 50.8%. This ratio is considered high Among C. comosummembers from multiple populations, this ratio is an indication of the quality of genetic diversity within and between C. comosum populations, which is of great importance for the preservation of the plant. As considered ISSR markers are an effective tool to provide good information for the genetic diversity available in C. comosum inputs from different populations, and the results of this study will help to understand genetic variation and evolutionary dynamics in C. comosum and expand the genetic base for breeding wild species. That additional selection of inputs in each species to assess genetic diversity leads to more useful and reliable results.

Assessment of Vegetative Growth and Genetic Integrity of Vanilla planifolia Regenerants after Cryopreservation.

Vanilla planifolia Jacks. ex Andrews is the vanilla species with the most commercial and greatest economic importance. It has been used as a case study in different cryopreservation studies that involve three vitrification-based approaches: droplet-vitrification (D-V), V-cryoplate (V-Cp) and D-cryoplate (D-Cp). The aim of this study was to compare the impact of these cryogenic techniques on vegetative growth (survival, stem length and leaf number) between cryo-derived plants and in vitro-derived controls during 12 months of greenhouse growth. Genetic stability was also assessed using the inter-simple sequence repeat (ISSR) markers. There were no significant differences found in the survival and stem lengths of the in vitro-derived regenerants and cryo-derived plants. A significant increase in the number of leaves was only detected in cryo-derived plants when using the V-Cp method. The electrophoretic profiles, based on seven ISSR primers, detected low variability: 81 total bands and 27% polymorphism. This is the first report on the assessment of vegetative growth and genetic integrity in cryo-derived V. planifolia plants recovered under greenhouse conditions. Of the three cryogenic approaches, D-Cp appears to yield V. planifolia regenerants plants with more vigorous vegetative growth and a lower level of polymorphism. Future research should focus on the reproductive growth of vanilla regenerants.

Gamma Radiation (60Co) Induces Mutation during In Vitro Multiplication of Vanilla (Vanilla planifolia Jacks. ex Andrews)

In vitro mutagenesis is an alternative to induce genetic variation in vanilla (Vanilla planifolia Jacks. ex Andrews), which is characterized by low genetic diversity. The objective of this study was to induce somaclonal variation in V. planifolia by gamma radiation and detect it using inter-simple sequence repeat (ISSR) molecular markers. Shoots previously established in vitro were multiplied in Murashige and Skoog culture medium supplemented with 2 mg·L−1 BAP (6-benzylaminopurine). Explants were irradiated with different doses (0, 20, 40, 60, 80 and 100 Gy) of 60Co gamma rays. Survival percentage, number of shoots per explant, shoot length, number of leaves per shoot, and lethal dose (LD50) were recorded after 60 d of culture. For molecular analysis, ten shoots were used for each dose and the donor plant as a control. Eight ISSR primers were selected, and 43 fragments were obtained. The percentage of polymorphism (% P) was estimated. A dendrogram based on Jaccard’s coefficient and the neighbor joining clustering method was obtained. Results showed a hormetic effect on the explants, promoting development at low dose (20 Gy) and inhibition and death at high doses (60–100 Gy). The LD50 was observed at the 60 Gy. Primers UBC-808, UBC-836 and UBC-840 showed the highest % P, with 42.6%, 34.7% and 28.7%, respectively. Genetic distance analysis showed that treatments without irradiation and with irradiation presented somaclonal variation. The use of gamma rays during in vitro culture is an alternative to broaden genetic diversity for vanilla breeding.

Genetic diversity of avocado (Persea americana Mill.) germplasm in Vietnam using RAPD and ISSR molecular markers

Avocado is a significant cash crop in Vietnam, while little is known about its genetic diversification. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were performed to investigate the genetic diversity of twenty-eight imported and domestic avocado cultivars being maintained at Western Highlands Agriculture and Forestry Science Institute, Dak Lak, Vietnam. 18 RAPD and 15 ISSR primers produced 3183 and 2807 scorable bands, of which 83.92% and 71.72% were polymorphic, respectively. The RAPD markers exhibited an average PIC of 0.27 and Rp index of 12.63 whereas the mean PIC and Rp values of ISSR primers were 0.21 and 13.37, respectively. The correlation between RAPD and ISSR markers was low (r = 0.338), suggesting that the ability to resolve genetic variation among varieties may relate to the number of detected polymorphisms rather than the marker employed. Nevertheless, the correlation between ISSR and combined data was high (r = 0.791) and the correlation between RAPD and combined matrices was even higher (r = 0.897). This indicates that RAPD markers have slightly higher efficiency over ISSR to resolve genetic variation among 28 avocado cultivars. High genetic diversity among 28 avocado cultivars was revealed with the Jaccard’s similarity coefficient ranging from 0.623 to 0.913 based on combined data analysis. The UPGMA dendrogram generated from combined RAPD and ISSR data grouped Vietnamese domestic and imported avocado cultivars into three different groups at 72% similarity. These results could be applied to the avocado conservation and breeding programs

Genetic diversity in populations of Girardinia diversifolia from Nepal Himalaya using ISSR markers

Girardinia diversifolia (Link) Friis from Urticaceae family is a perennial herb, commonly known as the Himalayan giant nettle. It has cultural, medicinal, and economic values among the indigenous peoples and local communities living in the mountains of Nepal and India. Prior knowledge of genetic diversity in plant species may contribute to conservation and sustainable utilization of important genotype. The aim of this study is to assess the genetic diversity within and among different populations of the 45 accessions of G. diversifolia collected from Far-western, Central, and Eastern regions of Nepal. The amplification of genomic deoxyribonucleic acid (DNA) with ten inter-simple sequence repeat (ISSR) primers yielded 131 clear DNA bands, of which 98.09% were found to be polymorphic. The mean effective number of alleles (ne), Nei's gene diversity (h) and Shannon's information index (I) were 1.598, 0.349, and 0.523, respectively. The total genetic diversity Ht (0.350 ± 0.04), low intrapopulation genetic diversity Hs (0.141 ± 0.03) and low estimated gene flow Nm (0.355 ± 0.11) reflected high genetic differentiation among population Gst (0.594 ± 0.08). The analysis of molecular variance among and within five populations of G. diversifolia showed that the value of genetic variation among the population was 60% whereas the value of genetic variation within the population was 40%. The results show that the ISSR markers are informative for the study of genetic diversity, which may help in the conservation and sustainable utilization of G. diversifolia and allied taxa.

Genetic diversity of Sterculia quadrifida from Kupang based on ISSR profiles, stomatal density, and chlorophyll content

Abstract. Jannah N, Pharmawati M, Uslan. 2022. Genetic diversity of Sterculia quadrifida from Kupang based on ISSR profiles, stomatal density, and chlorophyll content. Biodiversitas 23: 2690-2698. This study investigated the genetic diversity of Sterculia quadrifida R.Br.or faloak based on Inter Simple Sequence Repeats (ISSR) molecular markers and determined stomatal density and chlorophyll content from several localities in Kupang District, East Nusa Tenggara, Indonesia. For genetic diversity analysis, samples were collected from the Oelsonbai forest area of Maulafa Sub-district, Kupang City, whereas for stomatal density and chlorophyll content analysis, samples were collected from areas with various degrees of anthropogenic activity from Kupang city. DNA was isolated using Cetyl Trimethyl Ammonium Bromide (CTAB) method and amplified by three ISSR primers (UBC 811, UBC 812, and UBC 815), whereas stomatal density was observed using a microscope and chlorophyll content was determined using a spectrophotometer. Genetic data were analyzed using the unweighted pair group method with arithmetic mean (UPGMA) method and Nei & Li coefficient similarity in the MVSP software. A total of 78 ISSR bands ranging from 320-3200 bp were obtained, where populations were divided into four main clusters and six sub-clusters, similarly having moderate heterozygosity (He=0.50) not clustered based on their geographical distribution. There was a difference in stomatal density and chlorophyll content, probably influenced by anthropogenic disturbances. A conservation action plan is required to ensure the S. quadrifida's survival in the future.