Genome survey and genetic characterization of Acacia pachyceras O. Schwartz.

Acacia pachyceras O. Schwartz (Leguminoseae), a woody tree growing in Kuwait is critically endangered. High throughput genomic research is immediately needed to formulate effective conservation strategies for its rehabilitation. We therefore, performed a genome survey analysis of the species. Whole genome sequencing generated ~97 Gb of raw reads (92x coverage) with a per base quality score above Q30. The k-mer analysis (17 mer) revealed its genome to be 720Mb in size with an average guanine-cytosine (GC) ratio of 35%. The assembled genome was analyzed for repeat regions (45.4%-interspersed repeats; 9%-retroelements; 2%-DNA transposons). BUSCO assessment of completeness of genome identified 93% of assembly to be complete. Gene alignments in BRAKER2 yielded 34,374 transcripts corresponding to 33,650 genes. Average length of coding sequences and protein sequences were recorded as 1,027nts and 342aa, respectively. GMATA software filtered a total of 901,755 simple sequence repeats (SSRs) regions against which 11,181 unique primers were designed. A subset of 110 SSR primers were PCR validated and demonstrated for its application in genetic diversity analysis of Acacia. The SSR primers successfully amplified A. gerrardii seedlings DNA depicting cross transferability among species. The principal coordinate analysis and the split decomposition tree (bootstrapping runs of 1000 replicates) distributed the Acacia genotypes into two clusters. The flow cytometry analysis revealed the A. pachyceras genome to be polyploid (6x). The DNA content was predicted as 2.46 pg, 1.23 pg, and 0.41 pg corresponding to 2C DNA, 1C DNA and 1Cx DNA, respectively. The results provide a base for further high throughput genomic studies and molecular breeding for its conservation.

Bacterial and fungal communities in indoor aerosols from two Kuwaiti hospitals.

The airborne transmission of COVID-19 has drawn immense attention to bioaerosols. The topic is highly relevant in the indoor hospital environment where vulnerable patients are treated and healthcare workers are exposed to various pathogenic and non-pathogenic microbes. Knowledge of the microbial communities in such settings will enable precautionary measures to prevent any hospital-mediated outbreak and better assess occupational exposure of the healthcare workers. This study presents a baseline of the bacterial and fungal population of two major hospitals in Kuwait dealing with COVID patients, and in a non-hospital setting through targeted amplicon sequencing. The predominant bacteria of bioaerosols were Variovorax (9.44%), Parvibaculum (8.27%), Pseudonocardia (8.04%), Taonella (5.74%), Arthrospira (4.58%), Comamonas (3.84%), Methylibium (3.13%), Sphingobium (4.46%), Zoogloea (2.20%), and Sphingopyxis (2.56%). ESKAPEE pathogens, such as Pseudomonas, Acinetobacter, Staphylococcus, Enterococcus, and Escherichia, were also found in lower abundances. The fungi were represented by Wilcoxinia rehmii (64.38%), Aspergillus ruber (9.11%), Penicillium desertorum (3.89%), Leptobacillium leptobactrum (3.20%), Humicola grisea (2.99%), Ganoderma sichuanense (1.42%), Malassezia restricta (0.74%), Heterophoma sylvatica (0.49%), Fusarium proliferatum (0.46%), and Saccharomyces cerevisiae (0.23%). Some common and unique operational taxonomic units (OTUs) of bacteria and fungi were also recorded at each site; this inter-site variability shows that exhaled air can be a source of this variation. The alpha-diversity indices suggested variance in species richness and abundance in hospitals than in non-hospital sites. The community structure of bacteria varied spatially (ANOSIM r 2 = 0.181-0.243; p < 0.05) between the hospital and non-hospital sites, whereas fungi were more or less homogenous. Key taxa specific to the hospitals were Defluvicoccales, fungi, Ganodermataceae, Heterophoma, and H. sylvatica compared to Actinobacteria, Leptobacillium, L. leptobacillium, and Cordycipitaceae at the non-hospital site (LefSe, FDR q ≤ 0.05). The hospital/non-hospital MD index > 1 indicated shifts in the microbial communities of indoor air in hospitals. These findings highlight the need for regular surveillance of indoor hospital environments to prevent future outbreaks.

Comparison and optimization of DNA Isolation protocols for high throughput genomic studies of Acacia pachyceras Schwartz.

We describe the optimization and validation of six DNA isolation protocols from fresh leaves of the rare tree Acacia pachyceras. The first four protocols employed three commercial kits (Sigma, Nucleospin1, Nucleospin 2, Promega) whereas the remaining two were based on the traditional sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide CTAB methods. Each protocol provided significantly different results concerning DNA concentration (p < 0.032), yield (p < 0.000), contaminant carry over, protocol duration, cost per sample, and comprehensive cost. We demonstrated the applicability of all the tested protocols in DNA barcoding. The protocol yielded maximum amounts (92.85 µg) of DNA in a rapid turnaround time (8 h). The quantity and purity surpassed all the other tested methods. DNA extracted by the CTAB method was the best for NGS (Phred score >Q30). These protocols will be useful tools for molecular research of Acacia pachyceras and other closely related tree species.

Genetic Diversity of Rhanterium eppaposum Oliv. Populations in Kuwait as Revealed by GBS.

Natural populations of Rhanterium eppaposum Oliv. (Arfaj), a perennial forage shrub, have depleted due to unethical human interventions and climate change in Kuwait. Therefore, there is an urgent need to conserve this native plant through the assessment of its genetic diversity and population structure. Genotyping by sequencing (GBS) has recently emerged as a powerful tool for the molecular diversity analysis of higher plants without prior knowledge of their genome. This study represents the first effort in using GBS to discover genome-wide single nucleotide polymorphisms (SNPs) of local Rhanterium plants to assess the genetic diversity present in landraces collected from six different locations in Kuwait. The study generated a novel set of 11,231 single nucleotide polymorphisms (SNPs) and indels (insertions and deletions) in 98 genotypes of Rhanterium. The analysis of molecular variance (AMOVA) revealed ~1.5% variation residing among the six populations, ~5% among the individuals within the population and 93% variation present within the populations (FST = 0.029; p = 0.0). Bayesian and UPGMA analyses identified two admixed clusters of the tested samples; however, the principal coordinates analysis returned the complete population as a single group. Mantel's test returned a very weak correlation coefficient of r2 = 0.101 (p = 0.00) between the geographic and genetic distance. These findings are useful for the native species to formulate conservation strategies for its sustainable management and desert rehabilitation.

Benthic foraminifera as proxies for the environmental quality assessment of the Kuwait Bay (Kuwait, Arabian Gulf): Morphological and metabarcoding approaches.

The rapid urbanization and industrialization of Kuwait and the consequent effluent discharges into marine environments have resulted in a degradation of water and sediment quality in the coastal marine ecosystems such as in the Kuwait Bay. This study investigates the ecological response of benthic foraminifera (protists) to environmental stress in the Kuwait Bay. The traditional morphological approach was compared to the innovative environmental DNA (eDNA) metabarcoding to evaluate the ecological quality status (EcoQS). Forty-six surface sediment samples were collected from selected stations in the Kuwait Bay. To detect the pollution gradient, environmental parameters from water (e.g., salinity, pH, dissolved oxygen) and sediment (e.g., grain-size, trace metals, total organic carbon, total petroleum hydrocarbons) were measured at each station. Although the foraminiferal assemblages were different in the morphological and molecular datasets, the species turnover was congruent and statistically significant. Diversity-based biotic indices derived from both morphological and metabarcoding approaches, reflect the environmental stress gradient (i.e., organic and metal contaminations) in the Kuwait Bay. The lowest values of EcoQS (i.e., bad to poor) are found in the innermost part (i.e., Sulaibikhat Bay and Ras Kazmah), while higher EcoQS values occur in the outer part of the bay. This study constitutes the first attempt to apply the foraminiferal metabarcoding to assess the EcoQS within the Arabian Gulf and presents its advantages compared to the conventional morphological approach.

Draft genome sequence and SSR mining data of Acacia pachyceras Schwartz.

Acacia tree population is declining in several countries of the world especially in the Arabian peninsula due to human-induced activities. The tree has potential medicinal and economic benefits as a source of fuel and timber. It can fix nitrogen, a significant property that assists in desert rehabilitation. However, the lack of genomic information of Acacia pachyceras hampers its genetic study and breeding process. We performed paired-end sequencing of A. pachyceras at a depth of 120X to obtain raw sequences of 108.9 GB with a per base quality >Q30. Filtered raw data was assembled into a fasta file of 4 GB. The assembled genomic sequences consisted of 901,755 single sequence repeats (SSRs). In total 11,596 primer pairs were designed against these SSR motifs. The data generated provides baseline genomic information about the species and formulates a base for further sequencing of A. pachyceras through PACBio and HiC technologies. The novel developed SSR markers will facilitate genetic diversity and conservation studies for Acacia species.

Data on draft genome assembly and annotation of Haloxylon salicornicum Moq.

Haloxylon salicornicum Moq. Bunge ex Boiss (Rimth) is one of the main structural elements in Eastern Arabian vegetation associations. The plant is utilized as a food source for domestic stock, stabilizes the soil surface besides providing suitable microclimates for exotic species. It is considered one of the most promising species for re-vegetation. H. salicornicum community is under threat from overgrazing leading to a reduction in the percentage of distribution from 22.7% to 2.2% in Kuwait. Therefore, genome characterization of this important Kuwaiti plant is required to formulate strategies for its conservation. Here we report the draft of the H. salicornicum genome, which was sequenced on an Illumina HiSeq 2500 platform. BUSCO assessment revealed 69% of the genome was to be complete. Overall, 12960 gene structures, 11280 protein-coding genes, 11309 mRNAs (protein-coding), 51265 exons and 48100 CDSs were predicted. Functional annotation was carried out by interproscan-5.29-68.0. A total of 7222 protein-coding sequences were, annotated out of 11309 by at least one ontology term. All these genes were associated with 11 major biological processes branched into 60 child processes.

Spatio-temporal variations in bacterial and fungal community associated with dust aerosol in Kuwait.

Kuwait is a country with a very high dust loading; in fact it bears the world's highest particulate matter concentration in the outdoor air. The airborne dust often has associated biological materials, including pathogenic microbes that pose a serious risk to the urban ecosystem and public health. This study has established the baseline taxonomic characterization of microbes associated with dust transported into Kuwait from different trajectories. A high volume air sampler with six-stage cascade impactor was deployed for sample collection at a remote as well as an urban site. Samples from three different seasons (autumn, spring and summer) were subjected to targeted amplicon sequencing. A set of ~ 50 and 60 bacterial and fungal genera, respectively, established the core air microbiome. The predominant bacterial genera (relative abundance ≥ 1%) were Brevundimonas (12.5%), Sphingobium (3.3%), Sphingopyxis (2.7%), Pseudomonas (2.5%), Sphingomonas (2.4%), Massilia (2.3%), Acidovorax (2.0%), Allorhizobium (1.8%), Halomonas (1.3%), and Mesorhizobium (1.1%), and the fungal taxa were Cryptococcus (12%) followed by Alternaria (9%), Aspergillus (7%), Candida (3%), Cladosporium (2.9%), Schizophyllum (1.6%), Fusarium (1.4%), Gleotinia (1.3%) and Penicillium (1.15%). Significant spatio-temporal variations were recorded in terms of relative abundances, α-diversities, and ß-diversities of bacterial communities. The dissimilarities were less pronounced and instead the communities were fairly homogenous. Linear discrimant analysis revealed three fungal genera known to be significantly differentially abundant with respect to different size fractions of dust. Our results shed light on the spatio-temporal distribution of airborne microbes and their implications in general health.

Genetic diversity and population structure of Haloxylon salicornicum moq. in Kuwait by ISSR markers.

Haloxylon salicornicum moq. Bunge ex Boiss (Rimth) is one of the native plants of Kuwait, extensively depleting through the anthropogenic activities. It is important to conserve Haloxylon community in Kuwait as it can tolerate extreme adverse conditions of drought and salinity to be potentially used in the desert and urban revegetation and greenery national programs. Therefore, a set of 16 inter simple sequence repeat (ISSR) markers were used to assess genetic diversity and population structure of 108 genotypes from six locations in Kuwait. The ISSR primers produced 195 unambiguous and reproducible bands out of which 167 bands were polymorphic (86.5%) with a mean PIC value of 0.31. The overall average values of Nei's gene diversity (h') and Shannon's diversity index (I) were 0.254 and 0.375, respectively. Results of AMOVA revealed high genetic variations within populations (77.8%) and low among populations (22%). The values of Fixation index (FST = 0.22; P = 0.0), Genetic differentiation (GST = 0.262; G'ST = 0.327; D = 0.335 and Gene flow (NM = 0.880) were indicative of heterozygous populations. The results of STRUCTURE and split decomposition analysis suggested that the Rimth accessions of Kuwait can be grouped into five and six subpopulations, respectively. Principal coordinate analysis (PCoA) grouped them into three clusters. The pairwise Nei's genetic distances (DS) among populations demonstrated a narrow range from 0.047 to 0.187 (Scale-0.0 to 1.0). The Mantel's test revealed a weak correlation (r2- 0.188; P-0.013) between the genetic distance and geographic distances. Our results suggest that the narrowly distributed Haloxylon community in Kuwait demonstrated a high genetic diversity within the populations however the overall population structure was weak.

Metagenomic analysis of rhizosphere microflora of oil-contaminated soil planted with barley and alfalfa.

The role of rhizosphere microbial communities in the degradation of hydrocarbons remains poorly understood and is a field of active study. We used high throughput sequencing to explore the rhizosphere microbial diversity in the alfalfa and barley planted oil contaminated soil samples. The analysis of 16s rRNA sequences showed Proteobacteria to be the most enriched (45.9%) followed by Bacteriodetes (21.4%) and Actinobacteria (10.4%) phyla. The results also indicated differences in the microbial diversity among the oil contaminated planted soil samples. The oil contaminated planted soil samples showed a higher richness in the microbial flora when compared to that of untreated samples, as indicated by the Chao1 indices. However, the trend was different for the diversity measure, where oil contaminated barley planted soil samples showed slightly lower diversity indices. While the clustering of soil samples grouped the oil contaminated samples within and across the plant types, the clean sandy soil samples formed a separate group. The oil contaminated rhizosphere soil showed an enrichment of known oil-degrading genera, such as Alcanivorax and Aequorivita, later being specifically enriched in the contaminated soil samples planted with barley. Overall, we found a few well known oil-degrading bacterial groups to be enriched in the oil contaminated planted soil samples compared to the untreated samples. Further, phyla such as Thermi and Gemmatimonadetes showed an enrichment in the oil contaminated soil samples, indicating their potential role in hydrocarbon degradation. The findings of the current study will be useful in understanding the rhizosphere microflora responsible for oil degradation and thus can help in designing appropriate phytoremediation strategies for oil contaminated lands.

Legionella detection and subgrouping in water air-conditioning cooling tower systems in Kuwait.

The main aim of the study was to test for the presence of Legionnaires' disease-causing microorganisms in air-conditioned buildings in Kuwait using molecular technologies. For this purpose, 547 samples were collected from 38 cooling towers for the analysis of Legionella pneumophila. These samples included those from water (n = 178), air (n = 231), and swabs (n = 138). Out of the 547 samples, 226 (41%) samples were presumptive positive for L. pneumophila, with L. pneumophila viable counts in the positive water samples ranging from 1 to 88 CFU/ml. Of the Legionella culture-positive samples, 204 isolates were examined by latex agglutination. These isolates were predominately identified as L. pneumophila serogroup (sg) 2-14. Using the Dresden panel of monoclonal antibodies, 74 representatives isolates were further serogrouped. Results showed that 51% of the isolates belonged to serogroup 7 followed by 1 (18%) and 3 (18%). Serogroups 4 (4%) and 10 (7%) were isolated at a lower frequency, and two isolates could not be assigned to a serogroup. These results indicate the wide prevalence of L. pneumophila serogroup 7 as the predominant serogroup at the selected sampling sites. Furthermore, the 74 L. pneumophila (sg1 = 13; sg3 = 13; sg4 = 3; sg7 = 38; sg10 = 5; sgX = 2) isolates were genotyped using the seven gene protocol sequence-based typing (SBT) scheme developed by the European Working Group for Legionella Infections (EWGLI). The results show that Legionella isolates were discriminated into nine distinct sequence typing (ST) profiles, five of which were new to the SBT database of EWGLI. Additionally, all of the ST1 serogroup 1 isolates were of the OLDA/Oxford subgroup. These baseline data will form the basis for the development of a Legionella environmental surveillance program and used for future epidemiological investigations.

Detection of genetically modified DNA in fresh and processed foods sold in Kuwait.

Developments in genetic engineering technology have led to an increase in number of food products that contain genetically engineered crops in the global market. However, due to lack of scientific studies, the presence of genetically modified organisms (GMOs) in the Kuwaiti food market is currently ambiguous. Foods both for human and animal consumption are being imported from countries that are known to produce GM food. Therefore, an attempt has been made to screen foods sold in the Kuwaiti market to detect GMOs in the food. For this purpose, samples collected from various markets in Kuwait have been screened by SYBR green-based real time polymerase chain reaction (RT-PCR) method. Further confirmation and GMO quantification was performed by TaqMan-based RT-PCR. Results indicated that a significant number of food commodities sold in Kuwait were tested positive for the presence of GMO. Interestingly, certain processed foods were tested positive for more than one transgenic events showing complex nature of GMOs in food samples. Results of this study clearly indicate the need for well-defined legislations and regulations on the marketing of approved GM food and its labeling to protect consumer's rights.