A simple and cost-effective method for DNA extraction suitable for PCR in “sucupira branca”

“Sucupira branca” is a plant found in the Brazilian Cerrado and is adapted to low fertility soils, and its fruit extract has anti-inflammatory, healing, antiulcerogenic, antimicrobial, cercaricidal, leishmanicidal and antioxidant activities. Furthermore, it provides protection against oxidative stress, is a natural biocontrol agent of Aedes aegypti, has very resistant wood, is a melliferous plant and has been used in reforestation programs. The development of conservation strategies is important for maintaining diversity in natural populations of “sucupira branca” since these populations are in the process of genetic erosion. Molecular biology techniques, which are important for characterizing the genetic diversity of plants to develop conservation strategies, require sufficient high-quality genomic deoxyribonucleic acid (DNA). This study aimed to compare five methods to extract DNA from “sucupira branca”. The quality and concentration of DNA were revealed by agarose gel electrophoresis, and only the protocols of Dellaporta, Wood and Hicks et al. (1983) and Khanuja et al. (1999) did not result in satisfactory quantities of DNA. When PCR (Polymerase Chain Reaction) was performed with three inter-simple sequence repeat (ISSR) primers, DNA was successfully amplified from extractions performed with the protocols proposed by Doyle and Doyle (1987), Romano and Brasileiro (1998) and Ferreira and Grattapaglia (1995), which are less expensive than commercial purification kits. These protocols resulted in DNA of sufficient quality and quantity after the amplification reactions were performed.

Analysis of genetic diversity in selected sugarcane (Saccharum officinarum L.) accessions using inter simple sequence repeat (ISSR) markers

<p class="042abstractstekst"><span lang="EN-US">Genetic diversity information among a population is important in exploiting heterozygosity for the improvement of crop species through breeding programmes. This study was therefore, conducted to assess genetic diversity and establish molecular relationships among 20 selected exotic sugarcane accessions from the Unilorin Sugar Research Institute germplasm using Inter Simple Sequence Repeat (ISSR) molecular markers. Genomic DNA was extracted from the sugarcane leaf. Fragments amplification was then performed by polymerase chain reaction (PCR) with ISSR markers and the data obtained were analyzed using MEGA 4 software. Analysis of the electropherogram showed a total of 39 loci consisting of 369 bands, out of which 95.8% were polymorphic. The biplot analysis showed all the markers contributed to the observed diversity with the least achieved with ISSR6. The principal co-ordinate analysis grouped the accessions into four clusters, comprising mixtures of all the six collection sites. The polymorphism obtained in the present study showed that the ISSR markers are effective for assessment of genetic diversity of the sugarcane accessions as it reveals the genetic similarity or divergence of the accessions regardless their place of origin or cultivation.</span></p>

Functional Characteristics and Molecular Identification of Interspecific Hybrids from Genus Capsicum

The taxonomy of the genus Capsicum has distinguished more than twenty species of peppers with only C. annuum L. being a vegetable of economic importance. The remaining species can, however, be used in breeding via interspecies hybridization as a source of valuable functional traits. The aim of the study was to obtain interspecific hybrids of peppers with increased agronomic potential and their molecular identification. Based on their agromorphological characteristics, the obtained hybrids (‘Anchi’ × C. frutescens L.)F1 and (‘Anchi’ × C. baccatum var. pendulum)F1 formed fruits that had the combined functional traits typical of the cultivar ‘Anchi’, with a high content of soluble solids, and the wild species, with dry matter. To identify hybrids and their parental forms, the random amplified polymorphic DNA polymerase chain reaction (PCR-RAPD) and inter simple sequence repeat polymerase chain reaction (PCR-ISSR) techniques were used. The polymorphic products obtained in both methods totalled 65% and 59%, respectively, and they allowed for the identification of all tested genotypes, excluded the possibility of self-pollination of ‘Anchi’ plants, and confirmed the hybrid characteristics of the crossbreed (‘Anchi’ × C. frutescens L.)F1.

Characterization of Fusarium Spp. Inciting Vascular Wilt of Tomato and Its Management by a Chaetomium-Based Biocontrol Consortium

Though the vascular wilt of tomato caused by the species of Fusarium is globally reported to be a complex disease in certain countries, for example, India, our studies indicated that the disease is caused by either Fusarium oxysporum f. spp. lycopersici (Fol) or Fusarium solani (FS) with the Fol being widely prevalent. In assessing the genetic diversity of 14 Fol strains representing the four Indian states by the unweighted pair group method with arithmetic averaging using Inter Simple Sequence Repeat (ISSR) amplicons, the strains distinguished themselves into two major clusters showing no correlation with their geographic origin. In pot experiments under polyhouse conditions, the seed dressing and soil application of a talc-based formulation of a biocontrol treatment, TEPF-Sungal-1 (Pseudomonas putida) + S17TH (Trichoderma harzianum) + CG-A (Chaetomium globosum), which inhibited Fol, was equally effective like the cell suspensions and was even better than the fungicidal mixture (copper oxychloride-0.25% + carbendazim-0.1%) in promoting the crop growth (52.3%) and reducing vascular wilt incidence (75%) over the control treatment, despite the challenge of inoculation with a highly pathogenic TOFU-IHBT strain. This was associated with significant expressions of the defense genes, indicating the induction of host resistance by a biocontrol consortium. In field experiments on two locations, the bioconsortium was highly effective in recording maximum mean fruit yields (54.5 and 60%) and a minimum mean vascular wilt incidence (37.5%) in comparison to the untreated control. Thus, Chaetomium-based bioconsortium demonstrated consistency in its performance across the two experiments in 2 years under the two field conditions.

An investigation of the genotoxic and cytotoxic effects of myclobutanil fungicide on plants

Myclobutanil is a chemical pesticide commonly used in the production of some vegetables and fruits like greenhouse peppers, grapes, and apples. The aim of this study was to investigate the genotoxic and cytotoxic effects of myclobutanil fungicide on the Allium cepa plant, the model organism. Randomly amplified polymorphic DNA and inter simple sequence repeat-PCR techniques were performed on the DNA of A. cepa exposed to the different myclobutanil doses and time periods. The nucleus anomalies and abnormal anaphases were investigated using a light microscope. PCR analyses showed that myclobutanil causes some DNA sequence changes on the onion genome depending on the increase in the fungicide dose and exposure time. It was determined that myclobutanil has a serious genotoxic effect, even in low doses like 25–50 ppm.

Molecular Characterization and Population Genetic Structure of Fagopyrum Species Cultivated in Himalayan Regions

Fagopyrum spp. (buckwheat) is a dicotyledonous pseudocereal crop mainly cultivated in the north-western Himalayan regions for its highly nutritional, antioxidant and therapeutic values. In the present investigation, molecular characterization was performed by using ISSR (inter simple sequence repeat) markers on 42 accessions of four buckwheat species (Fagopyrum esculentum, F. sagittatum, F. tataricum and F. kashmirianum). The 12 pre-screened ISSR primers amplified 102 bands, and amongst them 85 bands exhibited polymorphism with an average polymorphism of 82.73%. The results revealed that Shannon’s information indices (I) and Nei’s genetic diversity (H) were low for F. tataricum (I = 0.1028 ± 0.2307; H = 0.0707 ± 0.1617) and high for F. esculentum (I = 0.1715 ± 0.2622; H = 0.1164 ± 0.1796). It was estimated that within the accessions of Fagopyrum species, the species diversity (HT) and mean diversity (HS) were 0.3200 and 0.1041, respectively. Molecular variance partitioning by AMOVA also indicated a significant genetic differentiation accounting for 73% among and 27% within the accessions of Fagopyrum species. Overall, accessions of F. esculentum had the greatest distance from the other accessions of buckwheat species, which includes F. sagittatum, F. tataricum and F. kashmirianum as revealed by FST distance and Nei’s unbiased genetic distance. The dendograms based on UPGMA and PCoA segregated 42 accessions of four buckwheat species into three major groups. This study clearly reveals a considerable amount of genetic diversity at the intra-specific level in F. esculentum, F. sagittatum and F. kashmirianum accessions. The factors responsible for it are diverse geographical conditions, pollinating behavior and cultivation practices adapted in these regions. The study also indicated a close phylogenetic relationship between F. tataricum and F. kashmirianum.

Comparative assessment of SCoT and ISSR markers for analysis of genetic diversity and population structure in some Aegilops tauschii Coss. accessions

Aegilops tauschii, the diploid progenitor of the wheat D-genome, is a valuable genetic resource for wheat breeders. In this study, we compared the efficiency of inter-simple sequence repeat (ISSR) (as an arbitrary technique) and start codon targeted (SCoT) (as a gene-targeting technique) markers in determining the genetic diversity and population structure of 90 accessions of Ae. tauschii. SCoT markers indicated the highest values for polymorphism information content, marker index and effective multiplex ratio compared to ISSR markers. The total genetic diversity (Ht) and genetic diversity within populations (Hs) parameters were comparably modest for the two marker systems. The results of the analysis of molecular variance showed that the genetic variation within populations was significantly higher than among them (ISSR: 92 versus 8%; SCoT: 88 versus 12%). Furthermore, SCoT markers discovered a high level of genetic differentiation among populations than ISSRs (0.19 versus 0.05), while the amount of gene flow detected by ISSR was higher than SCoT (2.13 versus 8.62). Cluster analysis and population structure of SCoT and ISSR data divided all investigated accessions into two and four main clusters, respectively. Our results revealed that SCoT and ISSR fingerprinting could be used to further molecular analysis in Ae. tauschii and other wild species. The high-genetic variability found in this study also indicates the valuable genetic potential present in the investigated Ae. tauschii germplasm, which could be utilized for future genetic analysis and linkage mapping in breeding programmes.

A novel temporary immersion bioreactor system for large scale multiplication of banana (Rasthali AAB—Silk)

Musa sp. cultivar Rasthali (Silk AAB) is a choice variety of the Asian sub-continent. Its production and sustenance are threatened by Fusarium wilt, which affects the livelihoods of small and marginal farmers. The use of quality planting material is one of the strategies to manage the disease. Availability of quality planting material for varieties other than Grand Naine is limited. Large-scale micropropagation using existing technologies is laborious and expensive. Temporary immersion bioreactor system is emerging as a potential advancement in the micropropagation industry. In this study, a cost-effective temporary immersion bioreactor (TIB) system has been developed and an efficient micropropagation method has been standardized. Explants cultured in TIB with 250 ml of culture medium in a 2-min immersion frequency of 6 h were found to be efficient for shoot proliferation and rooting. Its efficacy has been compared with the semisolid culture method. At the end of the 6th subculture, 1496 ± 110 shoots per explant were obtained in TIB. Chlorophyll, carotenoid, stomatal index, and the number of closed stomata were examined to determine the physiological functions of the plants grown in TIB and compared with semisolid grown plantlets. Plantlets grown in TIB were genetically stable and were confirmed using inter-simple sequence repeat (ISSR) markers. The multiplication of shoots in TIB was 2.7-fold higher than the semisolid culture method, which is suitable for large-scale production of planting material for commercial applications.

Inter-Simple Sequence Repeat (ISSR) Markers Revealed Genetic Diversity and Population Structure Among 9 Wild Species of Clematis L.

To analyze the genetic diversity of 9 species of Clematis from 31 different populations, we extracted DNA by the improved CTAB method, used ISSR-PCR for amplification, and then selected 9 primers with clear amplified bands from amongst 220 primers. A total of 127 clear bands were amplified, of which 126 were polymorphic bands, yielding a ratio of 99.2%. The polymorphism information index (PIC) of the primers ranged from 0.9326 to 0.9649. The Nei’s genetic diversity index (H) was 0.2750, the total gene diversity (Ht) was 0.2845, and the genetic differentiation coefficient (Gst) was 0.6696, indicating high genetic differentiation among populations of Clematis. After cluster analysis, the 31 Clematis populations were divided into 3 categories. Principal coordination analysis (PCoA) of 9 Clematis species then showed that the genetic relationship between samples of the same Clematis germplasms was closer than that of samples from the same region. The mantel test revealed a significant positive correlation between genetic distance and geographical distance among the populations. The population clustering results are broadly consistent with the clustering graphs of UPGMA and PCoA. We can conclude the polymorphism of the 9 primers is good, and that the genetic diversity of 31 Clematis populations is rich. Individual Clematis germplasms are closely related and will gather together preferentially.