Overview of the potential of microRNAs and their target gene detection for cassava (Manihot esculenta) improvement

Production and utilization of cassava (Manihot esculenta) is significantly constrained by pests, diseases, poor yields and low nutritional content. Various approaches are currently being applied to mitigate these constraints. However, an aspect of plant developmental genetics little known in cassava is the role that microRNAs (miRNAs) play in gene regulation. miRNAs are 20 - 24 nucleotide long nonprotein-coding RNAs that play important roles in post-transcriptional gene silencing in many organisms. Thorough understanding of the mechanisms involved in miRNAs mediated posttranscriptional gene regulation will have implications for crop production improvement. The potential of miRNAs for cassava improvement and also some data obtained on cassava miRNAs using comparative genomics analysis have been briefly discussed. 17 miRNA families and target genes in cassava that are also conserved in other plant species have been revealed. However, the ESTs representing seven of these miRNA families produced foldbacks that show more than 3 nucleotides not involved in canonical base pairing within a loop or bulge in the mature miRNA: RNA* dimer, thus were not considered miRNA secondary structures. Consistent with previous reports, majority of the target genes identified are transcription factors. Other targets appear to play roles in diverse physiological processes. Furthermore, a detailed description and insight into some of the current bioinformatic resources and approaches applicable to cassava have been discussed. Such information will further enhance the rapid discovery and analysis of more novel miRNAs in cassava towards its improvement.Keywords: Cassava, microRNAs, target genes, improvement, characterizatio

Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera

Abstract Background Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX) derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates. Results Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18), Efalpha1 (Elongation factor 1 alpha), ACT 2 (Actin2), UBQ (polyubiquitin), PEX4 (Peroxisomal ubiquitin conjugating enzyme) and At1g09770.1 (Arabidopsis thaliana cell division cycle 5). Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues) in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEfα1 or BoechUBQ and BoechEfα1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed, different optimal combinations were identified in sexual ovules (BoechUBQ and BoechEfα1), in anthers from both reproductive systems (BoechACT2 and BoechEfα1), in apomictic vegetative tissues (BoechEfα1 and BoechACT2) and sexual vegetative tissues (BoechRPS18 and BoechEfα1). NormFinder ranked BoechACT2 as the most stable in the apomictic plant, while BoechRPS18 was the best in the sexual plant. The subgroups analysis identified the best gene for both apomictic and sexual ovules (BoechRPS18), for anthers from both reproductive system (BoechEfα1) and for apomictic and vegetative tissues (BoechACT2 and BoechRPS18 respectively) Conclusions From a total of six tested genes, BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ showed the best stability values. We furthermore provide detailed information for the accurate normalization of specific tissue gene expression analyses of apomictic and sexual Boechera.</p

Ex Vitro Propagation of Rubber Tree (Hevea Brasiliensis) using Stem Cuttings

Stem cutting propagation preserves the genetic traits and leads to transfer of superior and genetically similar traits of parent plants to progenies. This method is also used to propagate recalcitrant, nonviable and difficult to germinateseeds. Stem cutting in tree species is used to address phenological and intraclonal problems. The use of rubber cuttings as planting material is a feasible option, worthy of investigation. There has been little or no research studies into the USAge of Hevea brasiliensis stem cuttings as an alternative vegetative propagation method for an in vivo propagation of rubber tree in Ghana. Propagation of H. brasiliensis by stem cutting techniques was used to study alternative procedures for mass production of rubber planting materials. Brown and green rubber stem cuttings of Clone I and Clone II were soaked for 6 hours in 0.0-22.5g/L Naphthalene Acetic Acid (NAA) followed by propagation in a nursery bag filled with nutrient-rich soil. Only the brown stem cuttings of H. brasiliensis survived. The percent survival, length of shoots, number of roots as well as length of roots of Clone II was significantly (

Controlled transmission of African cassava mosaic virus (ACMV) by Bemisia tabaci from cassava (Manihot esculenta Crantz) to seedlings of physic nut (Jatropha curcas L.)

Jatropha curcas, a plant with great biodiesel potential is also used to reduce the population of whiteflies, Bemisia tabaci on cassava fields when planted as a hedge. We therefore, investigated the transmission of African cassava mosaic virus (ACMV) by the whitefly vector from cassava to seedlings of 10 accessions of J. curcas as part of a wider investigation on the possible role of J. curcas as an alternative host of ACMV. Transmission tests were conducted in insect-proof cages using adult B. tabaci collected from ACMV-infected cassava in the field, at a rate of three adult whiteflies per J. curcas seedling and a transmission feeding period of four days. Twenty one (21) days after the infestation, leaf samples from individual plants of the 10 J. curcas accessions were tested for the presence of ACMV by the polymerase chain reaction (PCR) and the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using the monoclonal antibodies SCRI 33. DAS-ELISA detected ACMV in five out of the ten J. curcas accessions while PCR detected it in eight of the 10 accessions. Furthermore, 18 out of the 35 Nicotiana benthamiana indicator plants mechanically inoculated with sap from symptomatic J. curcas seedlings produced symptoms typical of ACMV infection. This indicates that J. curcas is a likely host of ACMV and it may in turn, be able to infect cassava, and presumably other ACMV-susceptible hosts, in the presence of the vector.Keywords: Jatropha curcas accessions, controlled transmission, mechanical inoculation, enzyme-linked immunosorbent assay, African cassava mosaic virus (ACMV)-susceptible hosts.African Journal of Biotechnology Vol. 12(28), pp. 4465-447

Analysis of conserved microRNAs in floral tissues of sexual and apomictic Boechera species

<p>Abstract</p> <p>Background</p> <p>Apomixis or asexual seed formation represents a potentially important agronomic trait whose introduction into crop plants could be an effective way to fix and perpetuate a desirable genotype through successive seed generations. However, the gene regulatory pathways underlying apomixis remain unknown. In particular, the potential function of microRNAs, which are known to play crucial roles in many aspects of plant growth and development, remains to be determined with regards to the switch from sexual to apomictic reproduction.</p> <p>Results</p> <p>Using bioinformatics and microarray validation procedures, 51 miRNA families conserved among angiosperms were identified in <it>Boechera</it>. Microarray assay confirmed 15 of the miRNA families that were identified by bioinformatics techniques. 30 cDNA sequences representing 26 miRNAs could fold back into stable pre-miRNAs. 19 of these pre-miRNAs had miRNAs with <it>Boechera</it>-specific nucleotide substitutions (NSs). Analysis of the Gibbs free energy (ΔG) of these pre-miRNA stem-loops with NSs showed that the <it>Boechera</it>-specific miRNA NSs significantly (p ≤ 0.05) enhance the stability of stem-loops. Furthermore, six transcription factors, the Squamosa promoter binding protein like SPL6, SPL11 and SPL15, Myb domain protein 120 (MYB120), RELATED TO AP2.7 DNA binding (RAP2.7, TOE1 RAP2.7) and TCP family transcription factor 10 (TCP10) were found to be expressed in sexual or apomictic ovules. However, only SPL11 showed differential expression with significant (p ≤ 0.05) up-regulation at the megaspore mother cell (MMC) stage of ovule development in apomictic genotypes.</p> <p>Conclusions</p> <p>This study constitutes the first extensive insight into the conservation and expression of microRNAs in <it>Boechera </it>sexual and apomictic species. The miR156/157 target squamosa promoter binding protein-like 11 (SPL11) was found differentially expressed with significant (p ≤ 0.05) up-regulation at the MMC stage of ovule development in apomictic genotypes. The results also demonstrate that nucleotide changes in mature miRNAs significantly (p ≤ 0.05) enhance the thermodynamic stability of pre-miRNA stem-loops.</p

Bacillus subtilis Diacylglycerol Kinase (DgkA) Enhances Efficient Sporulation

The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function

In Vitro Propagation of Rubber Tree (Hevea Brasiliensis) Using Shoot-Tip and Nodal Cutting Explants

Hevea brasiliensis which belongs to the Euphorbiaceae family is the primary source of natural rubber. Propagation of rubber tree by grafting on to unselected seedlings sustains intraclonal heterogeneity for vigour and productivity which could be improved via in vitro techniques. Micropropagation from rubber nodal and shoot tip explants is possible. In vitro technique is needful to mass propagate disease-free and genetically similar rubber plantlets. In vitro results in increased growth and vigour of rubber tree, However, in vitro techniques of rubber tree have not been given much critical research attention in Ghana. Propagation of H. brasiliensis by in vitro techniques was used to study alternative procedures for mass production of rubber planting materials. Murashige and Skoog (MS) basal medium amended with 30.0g/L sucrose, 100.0mg/L myo-inositol, 2.0g/L activated charcoal, 1.0mg/L silver nitrate AgNO3, 2.0mg/L GA3 and control, 2.5, 5.0, 7.5 or 10.0mg/L kinetin was used to culture both H. brasiliensis shoottip and nodal explants. The MS medium with 5.0mg/L kinetin significantly (P&lt;0.05) enhanced higher shoot development (84.00%), number of shoots (3.60) and leaves (23.40) of the shoot-tip explants compared to other treatments. In nodal explants, the control medium developed higher shoots (94.00%), the height of shoot (4.80cm), number of leaves (19.20), number of shoots (6.00) and number of roots (7.00) than those with kinetin treatments. Conversely, 7.5mg/L kinetin of the nodal culture also performed significantly after the controls. Successful in vitro regeneration of plantlets was achieved using Hevea brasiliensis shoot-tip and nodal cutting explants cultured on an MS medium supplemented with kinetin