GENETIC ENGINEERING OF TURFGRASS FOR ENHANCED PERFORMANCE UNDER ENVIRONMENTAL STRESS

Turfgrass species are agriculturally and economically important perennial crops that are susceptible to biotic stress (e.g. fungal pathogens) and abiotic stress (e.g. salinity and drought). Every year, environmental stress significantly influences turfgrass quality and production causing economic loss globally. My research explores the feasibility of using two novel transgenes - Penaeidin4-1 (Pen4-1) from the shrimp, Litopenaeus setiferus, and microRNA319a (miR319a) from the rice, Oryza sativa, to genetically engineer turfgrass for enhanced tolerance to environmental stress. The antimicrobial peptide - Pen4-1 has been reported to possess in vitro antifungal and antibacterial activities against various economically important pathogens. In this study, two DNA constructs were prepared containing either the coding sequence of a single peptide, Pen4-1 or the DNA sequence coding for the transit signal peptide of the secreted tobacco AP24 protein translationally fused to Pen4-1 coding sequence. Transgenic turfgrass plants containing different DNA constructs exhibited significantly enhanced resistance to dollar spot and brown patch, the two major fungal diseases in turfgrass. My results demonstrated the effectiveness of Pen4-1 in a perennial species against fungal pathogens and may suggest a potential strategy for engineering broad-spectrum fungal disease resistance in crop species. The miR319 family is one of the first characterized miRNA families in plants and it has been demonstrated to target TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) genes encoding plant-specific transcription factors. Transgenic plants overexpressing the rice miR319 gene, Osa-miR319a, exhibited dramatic morphological changes, including significantly decreased tiller numbers, wider and thicker leaves, larger stems, larger weight:area ratio and more total wax coverage. Overexpression of miR319 also led to enhanced drought and salt tolerance in transgenics, which might be attributed to the increased weight:area ratio and total wax coverage as well as less sodium uptake. Gene expression analysis in both wild-type and transgenic plants indicated that at least four putative miR319 target genes in turfgrass AsPCF5, AsPCF6, AsPCF8 and AsTCP14 were down-regulated in transgenic plants. These results provide important information leading to the development of novel molecular strategies to genetically engineer crop species for enhanced performance under unfavorable environmental conditions, contributing to agriculture production

Molecular responses and expression analysis of genes in a xerophytic desert shrub Haloxylon ammodendron (Chenopodiaceae) to environmental stresses

Haloxylon ammodendron (C.A Mey.) Bunge is a xero-halophytic desert shrub with excellent drought resistance and salt tolerance. To decipher the molecular responses involved in its drought resistance, the cDNA-AFLP (amplified fragment length polymorphism) technique was employed to identify genes expressed differentially in the leaves following drought treatment in the seedlings of H. ammodendron.Eighty-six non-redundant TDFs (transcript-derived fragments) were identified as drought responsive after verified by reverse northern. Of these, 49 TDFs showed significant homology to genes with knownor predicted function; 6 TDFs were homologous to unknown genes, while 31 TDFs did not show any significant matches. 10 TDFs were selected to further validate the cDNA-AFLP expression patterns bythe semi-quantitative RT-PCR. 57% of TDFs corresponding to proteins of known or putative functions are likely to participate in signal transduction, transcription regulation, protein synthesis, senescence,transport, cell wall synthesis, stress and defense response, development and growth, photosynthesis, and so on. Moreover, not many functions of these genes have been reported in plants adaptation to unfavorable conditions. The spatial and temporal expression patterns of the tested genes displayed several distinct patterns in response to osmotic stress, desiccation stress and application of exogenous ABA. The results provided general insights into the molecular adaptation mechanisms involved in this desert shrub’s response to desert conditions

Respuesta fisiológica y bioquímica de cuatro variedades de papa criolla (Solanum tuberosum L. Grupo Phureja) a condiciones de sequía

ilustraciones, gráficas, tablasLa papa es un cultivo cuyo rendimiento se ve significativamente afectado en condiciones de sequía por lo que es importante caracterizar los materiales genéticos en cuanto a su tolerancia y determinar la base de dicha tolerancia. El objeto de este estudio fue determinar las respuestas a nivel fisiológico, bioquímico y de rendimiento de cuatro genotipos de S. tuberosum Grupo Phureja en condiciones de déficit hídrico. Plantas de las variedades. Colombia, Milagros, Paola y Violeta fueron sometidas a dos tratamientos de suministro hídrico, déficit hídrico aplicado en inicio de tuberización durante 16 días (WD) y riego continuo (WW). Todas las variedades en WD a partir de los 4 días de tratamiento (ddt) presentaron una disminución significativa en el contenido volumétrico del agua en el suelo, el potenciál hídrico foliar, el contenido relativo de agua y la conductancia estomática. En rendimiento cuántico máximo del PSII dimisminuyó desde los 4 ddt para la variedad Colombia y partir de los 12 ddt para todas las variedades. El contenido de clorofilas aumentó significativamente en todas las variedades en WD, mientras que el contenido de carotenoides solo aumentó en la variedad Colombia (0.21 mg g-1 FM – 0.41 mg g-1 FM). El aumento de las clorofilas se asocia a la disminución en el crecimiento observada y determinada a partir del área foliar específica. En la perdida de electrolitos se presentó un aumento en las plantas WD a partir de los 8 ddt para las variedades Colombia, Milagros y Violeta y a partir de los 12 ddt para todas las variedades, siendo mayor en la variedad Colombia (45.2 %). El contenido de prolina aumentó significativamente para todas las variedades WD desde los 4 ddt, siendo a los 4 ddt y 8ddt mayor para Milagros (1965 μg g-1 FM) y Violeta (1438 μg g-1 FM). La variedad que presentó menor acumulación de prolina fue Colombia (638 μg g-1 FM) y Paola (270 μg g-1 FM). En el contenido de proteína en WD se observó a los 4 ddt y 8 ddt una disminución en Paola (3.09 mg g-1 FM – 2.83 mg g-1 FM) y a los 12 ddt un aumento en violeta (2.43 mg g-1 FM – 2.70 mg g-1 FM). En la variedad Colombia el contenido de proteína disminuyó desde los 4 ddt mientras que en la variedad milagros no hubo diferencias con las plantas WW. El contenido de azúcares fue mayor en las variedades Colombia, Milagros y Paola desde los 8 ddt. La actividad de la enzima antioxidante CAT aumentó singnificativamente para todas las variedades WD a partir de los 4 ddt siendo mayor en milagros (37.8 UACAT – 220.9 UACAT) y menor en Colombia (23.5 UACAT – 81.8 UACAT). En el rendimiento la varidad Milagros WD (180.6 g/planta) no presentó diferencias significativas con las WW, mientras que en las demás variedades WD hubo una disminución significativa, la cual fue mayor para Colombia (47.2 %), seguida de Paola (27.0 %) y Violeta (19.4 %). El índice de susceptibilidad fue mayor para Colombia (1.58) y menor para Milagros (0.26). Los datos sugieren que la variedad Milagros es la mas tolerante lo que se atribuye al aumento temprano en el contenido de prolina que le permitió hacer ajuste osmótico y tomar mas agua y así disminuir de forma gradual la conductancia estomática. Igualmente al presentar una mayor respuesta antioxidante presentó menos daño a nivel de las membranas y del metabolismo celular. (Texto tomado de la fuente).Potato is a crop whose yield is affected by drought conditions, so it is important to characterize the genetic material as to its tolerance and to determine the basis of such tolerance. The objective of this study was the responses to a physiological, biochemical and yield level of four genotypes of S. tuberosum group Phureja under condi tions of water deficit. Plants of the varied. Colombia, Milagros, Paola and Violeta were submitted to two treatments of water supply, water deficit applied at the beginning of the tuberization for 16 days (WD) and continuou s irrigation (WW). All WD varieties from 4 days of treatment (ddt) showed a significant decrease in the volumetric content of water in the soil, water potential, r elative water content and stomatal conductance . In the maximum possible yield of PSII decreased from 4 ddt for the Colo mbia variety and from 12 ddt for all varieties. Chlorophyll content increased significantly in all varieties in WD, while carotene content increased only in the Colombia variety (0.21 mg g - 1 FM - 0.41 mg g - 1 FM). The increase of the chlorophylls is associa ted to the decrease in the observed growth and determined from the specific leaf area. In the loss of electrolytes, an increase in WD plants was observed from 8 ddt for the varieties Colombia, Milagros and Violeta and from 12 ddt for all varieties, being h igher in the Colombia variety (45.2%). Proline content increased significantly for all WD varieties from 4 ddt, with 4 dt and 8 ddt higher for Milagros (1965 μg g - 1 FM) and Violet (1438 μg g - 1 FM). The variety with the lowest accumulation of proline was Co lombia (638 μg g - 1 FM) and Paola (270 μg g - 1 FM). In the WD protein content at 4 ddt and 8 ddt, a decrease in Paola (3.09 mg g - 1 FM - 2.83 mg g - 1 FM) was observed at 12 dt and an increase in violet (2.43 mg g - 1 FM 2.70 mg g - 1 FM). In the Colombia variety t he protein content decreased from the 4 ddt while in the variety miracles there were no differences with the WW plants. The sugar content was higher in the varieties Colombia, Milagros and Paola from the 8 ddt. The activity of the antioxidant enzyme CAT in creased significantly for all WD varieties from the 4 ddt being higher in miracles (37.8 UACAT - 220.9 UACAT) and lowest in Colombia (23.5 UACAT - 81.8 UACAT). In the yield the Milagros WD variance (180.6 g / plant) did not present significant differences with the WW, while in the other WD varieties there was a significant decrease, which was higher for Colombia (4 7.2%), followed by Paola (27.0% ) and Violet (19.4%). The susceptibility index was higher for Colombia (1.58) and lower for Milagros (0.26). The d ata suggest that the Milagros variety is the most tolerant which is attributed to the early increase in proline content that allowed it to do osmotic adjustment and take more water and thus gradually decrease stomatal conductance. Likewise presenting a hig her antioxidant response presented less damage to the membranes and cellular metabolism.MaestríaMagíster en Ciencias AgrariasFisiología de cultivosCiencias Agronómica

Genetics and Genomics of Forest Trees

Forest tree genetics and genomics are advancing at an accelerated rate, thanks to recent developments in high-throughput, next-generation sequencing capabilities, and novel biostatistical tools. Population and landscape genetics and genomics have seen the rise of new approaches implemented in large-scale studies that employ the use of genome-wide sampling. Such studies have started to discern the dynamics of neutral and adaptive variation in nature and the processes that underlie spatially explicit patterns of genetic and genomic variation in nature. The continuous development of genetic maps in forest trees and the expansion of QTL and association mapping approaches contribute to the unravelling of the genotype-phenotype relationship and lead to marker-assisted and genome-wide selection. However, major challenges lie ahead. Recent literature suggests that species demography and genetic diversity have been affected both by climatic oscillations and anthropogenically induced stresses in a way calls into question the possibility of future adaptation. Moreover, the pace of contemporary environmental change presents a great challenge to forest tree populations and their ability to adapt, taking into consideration their life history characteristics. Several questions emerge that include, but are not limited to, the interpretation of forest tree genome surveillance and their structural/functional properties, the adaptive and neutral processes that have shaped forest tree genomes, the analysis of phenotypic traits relevant to adaptation (especially adaptation under contemporary climate change), the link between epigenetics/epigenomics and phenotype/genotype, and the use of genetics/genomics as well as genetic monitoring to advance conservation priorities

The role of small RNAs in susceptibility and tolerance to cassava mosaic disease

A dissertation presented by Sarah Jane Rogans to The Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Cell Biology. 2016Cassava (Manihot esculenta, Crantz) is considered to be an important food security crop consumed by over a billion peoples globally, many who subsist on it. Cassava mosaic disease (CMD) is one of the main biotic and economically important constraints to cassava cultivation in sub-Saharan Africa. Geminiviruses are the casual agents of CMD and cause disease to many staple food and cash crops of great economic importance worldwide. There are currently 11 species of Begomoviruses that belong to the Geminiviridae family. South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite (DNA A and DNA B components) begomovirus belonging to the family Geminiviridae, and is one of the causal agents of cassava mosaic disease (CMD) endemic to southern Africa. Various strategies to control CMD are currently being investigated, one of which is cis-genics, which involves manipulation of endogenous host genes to combat viral pathogens. In order to achieve this, it is imperative to elucidate molecular mechanisms involved in host-virus interactions. Endogenous small RNAs (sRNAs), including microRNAs (miRNAs), have been found associated with gene regulatory mechanisms in response to virus infection. Amongst the non-coding host sRNAs targeting viruses are small interfering RNAs (siRNAs) associated with posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS), which are involved in the host RNA silencing pathway. The RNA silencing pathway is a highly conserved basal immunity pathway involved in host defence against plant viruses. The aim of this study was to identify siRNAs and miRNAs associated with gene regulatory mechanism in response to SACMV infection and to determine if they a play a role in the susceptible or recovery phenotype observed in SACMV tolerant cassava landrace TME3 or T200, respectively. Furthermore, virus-derived siRNA (vsRNA) populations targeting the DNA A and B components of SACMV were also investigated. MicroRNAs (miRNAs) are an important class of endogenous non-coding single-stranded small RNAs (21-24 nt in length), which serve as post-transcriptional negative regulators of gene expression in plants. Despite the economic importance of Manihot esculenta Crantz (cassava) only 153 putative cassava miRNAs (from multiple germplasm) are available to date in miRBase (V.21). Therefore, both conserved and novel miRNAs needed to be identified in cassava before we could determine what association they had with SACMV infection. In this part of the study, mature sequences of all known plant miRNAs were used as a query for homologous searches against cassava EST and GSS databases, and additional identification of novel and conserved miRNAs were gleaned from next generation sequencing (NGS) of two cassava landraces (T200 from southern Africa and TME3 from West Africa) at three different growth stages post explant transplantation and acclimatization. EST and GSS derived data revealed 259 and 32 conserved miRNAs in cassava, and one of the miRNA families (miR2118) from previous studies has not been reported in cassava. NGS data collectively displayed expression of 289 conserved miRNAs in leaf tissue, of which 230 had not been reported previously. Of the 289 conserved miRNAs identified in T200 and TME3, 208 were isomiRs. Thirty-nine novel cassava-specific miRNAs of low abundance, belonging to 29 families, were identified. Thirty-eight (98.6%) of the putative new miRNAs identified by NGS have not been previously reported in cassava. Several miRNA targets were identified in T200 and TME3, highlighting differential temporal miRNA expression between the two cassava landraces. This study contributes to the expanding knowledge base of the micronome of this important crop. MicroRNAs play a crucial role in stress response in plants, including biotic stress caused by viral infection. Viruses however can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of miRNAs. This study aimed to understand the regulation of miRNAs in tolerant (TME3) and susceptible (T200) cassava landraces infected with SACMV. Next-generation sequencing was used for analysing small RNA libraries from infected and mock-inoculated cassava leaf tissue collected at 12, 32 and 67 dpi (days post-inoculation). The total number of differentially expressed miRNAs (normalized against mock-inoculated samples) across all three time points was 204 and 209 miRNAs, in TME3 and T200 infected plants, respectively, but the patterns of log2fold changes in miRNA families over the course of infection differed between the two landraces. A high number were significantly altered at 32 dpi when T200 and TME3 plants showed severe symptoms. Notably, in T200 69% and 28 (100%) of miRNA families were upregulated at 12 and 32 dpi, respectively. In contrast, TME3 showed an early pre-symptomatic response at 12 dpi where a high number (87%) of miRNAs showed a significant log2fold downregulation. Endogenous targets were predicted in the cassava genome for many of the identified miRNA families including transcription factors, disease resistance (R)-genes and transposable elements. Interestingly, some of the miRNA families (miR162, miR168 and miR403) that were significantly affected in both T200 and TME3 upon SACMV infection were shown to target proteins (DCL1, AGO1 and AGO2) that play important roles in the RNA silencing pathway. From results, we suggest that the early (12 dpi) miRNA response to SACMV in TME3 appears to involve PTGS-associated AGO1, DCL2 and a cohort of R genes belonging to the miR395 family which may prime the plant for tolerance and recovery downstream, while in T200, SACMV suppresses AGO1, AGO2 (at 32 and 67 dpi), and DCL2 (32 dpi) mediated RNA silencing, leading to severe persistent disease symptoms. This study provides insights into miRNA-mediated SACMV cassava interactions and may provide novel targets for control strategies aimed at developing CMD-resistance cassava varieties Endogenous small RNAs (sRNAs) associated with gene regulatory mechanisms respond to virus infection, and virus-derived small interfering RNAs (vsRNAs) have been implicated in recovery or symptom remission in some geminivirus-host interactions. Transcriptional gene silencing (TGS) (24 nt vsRNAs) and post transcriptional gene silencing (PTGS) (21-23 nt vsRNAs) have been associated with geminivirus intergenic (IR) and coding regions, respectively. In this Illumina deep sequencing study, we compared for the first time, the small RNA response to South African cassava mosaic virus (SACMV) of cassava landrace TME3 which shows a recovery and tolerant phenotype, and T200, a highly susceptible landrace. Interestingly, different patterns in the percentage of SACMV-induced normalized total endogenous sRNA reads were observed between T200 and TME3. Notably, in T200 there was a significant increase in 21 nt sRNAs during the early pre-symptomatic response (12 dpi) to SACMV compared to mock, while in TME3, the 22 nt size class increased significantly at 32 dpi. While vsRNAs of 21 to 24 nt size classes covered the entire SACMV DNA- A and DNA-B genome components in T200 and TME3, vsRNA population counts were significantly lower at 32 (symptomatic stage) and 67 dpi in tolerant TME3 compared with T200 (non-recovery). It is suggested that the high accumulation of primary vsRNAs, which correlated with high virus titres and severe symptoms in susceptible T200, may be due to failure to target SACMV-derived mRNA. In contrast, in TME3 low vsRNA counts may represent efficient PTGS of viral mRNA, leading to a depletion/sequestration of vsRNA populations, supporting a role for PTGS in tolerance/recovery in TME3. Notably, in TME3 at recovery (67 dpi) the percentage (expressed as a percentage of total vsRNA counts) of redundant and non-redundant (unique) 24 nt vsRNAs increased significantly. Since methylation of the SACMV genome was not detected by bisulfite sequencing, and vsRNA counts targeting the IR (where the promoters reside) were very low in both the tolerant or susceptible landraces, we conclude that 24 nt vsRNA-mediated RNA directed genome methylation does not play a central role in disease phenotype in these landraces, notwithstanding recognition for a possible role in histone modification in TME3. This work represents an important step toward understanding variable roles of sRNAs in different cassava genotype-geminivirus interactions. Also, by comparing the differences between a tolerant and susceptible host the aim is to achieve better understanding of the effect of pathogens on host sRNAome, an area that is deserving of me attention in plant systems. The expectation is that these findings presented in the PhD will contribute to the long-term goals of devising new methods of disease control against SACMV and understanding the complex interconnected mechanisms involved in virus-host interactome.LG201

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest

The Phenotypic Effects and Transcript Response of Salt Stress, the Impact of Viral Infection on Salt Stress Symptoms, and the Effect of Salt Stress on Soybean Virus Vector Activity in Soybean Varieties That Vary in Chloride Uptake

With the increase in saline soils worldwide, understanding the mechanisms for salt tolerance in plants is important to reduce yield loss due to salt stress. Soybean, Glycine max (L.) Merr., genotypes differ in chloride uptake with genotypes that take up chloride into foliar tissues tending to be salt-sensitive whereas those that partially exclude chloride from the leaves are more salt-tolerant. Transcriptional and physiological responses were measured in two soybean cultivars, Clark and Manokin, which differ in chloride uptake in response to salt stress and in combination with Soybean mosaic virus (SMV) and its aphid vector, Aphis glycines . The interaction of cultivar and salt treatment revealed using Gene ChipTM analysis a total of 386 genes were differentially regulated. These results were validated with RT-PCR and RT-qPCR. NaCl stress caused damage in Clark and to a lesser extent in Manokin. Likewise, the photosynthesis rate in Clark was greatly reduced by salt stress and was reduced to a lesser extent in Manokin. Reciprocal grafting demonstrated that soybean roots were responsible for salt tolerance and should be the focus of future studies on the genetic basis of salt tolerance. To study the interaction between SMV infection and salt stress, soybeans were treated with salts either prior to or after inoculation. SMV levels and gene expression levels were measured using RT-PCR, RT-qPCR, and ELISA. The SMV level in both treatment regimens was reduced in Manokin in response to the sub-lethal NaCl and CaCl2 levels and in Clark in response to CaCl2. The rate of photosynthesis was not decreased in CaCl2-treated soybeans that were infected with SMV; however, SMV infection did not alter the salt damage phenotype and salt stress did not impact SMV symptoms. Aphid populations were reduced by salt stress in both cultivars. The production of aphid-induced volatile terpenes were suppressed with salt stress and SMV infection. SMV levels were reduced in salt stressed Manokin with aphid feeding, but not in Clark or any other treatment combination. The results show a potential synergistic interaction between SMV, salt stress, and the soybean aphid with implications for growers facing biotic and abiotics stresses in the field