Molecular Characterization of Dehydrin in Azraq Saltbush among Related Atriplex Species.

Atriplex spp. (saltbush) is known to survive extremely harsh environmental stresses such as salinity and drought. It mitigates such conditions based on specialized physiological and biochemical characteristics. Dehydrin genes (DHNs) are considered major players in this adaptation. In this study, a novel DHN gene from Azrak (Jordan) saltbush was characterized along with other Atriplex species from diverse habitats. Intronless DHN-expressed sequence tags (495-761 bp) were successfully cloned and sequenced. Saltbush dehydrins contain one S-segment followed by three K-segments: an arrangement called SK3-type. Two substantial insertions were detected including three copies of the K2-segemnet in A. canescens. New motif variants other than the six-serine standard were evident in the S-segment. AhaDHN1 (A. halimus) has a cysteine residue (SSCSSS), while AgaDHN1 (A. gardneri var. utahensis) has an isoleucine residue (SISSSS). In contrast to the conserved K1-segment, both the K2- and K3-segment showed several substitutions, particularly in AnuDHN1 (A. nummularia). In addition, a parsimony phylogenetic tree based on homologs from related genera was constructed. The phylogenetic tree resolved DHNs for all of the investigated Atriplex species in a superclade with an 85% bootstrap value. Nonetheless, the DHN isolated from Azraq saltbush was uniquely subclustred with a related genera Halimione portulacoides. The characterized DHNs revealed tremendous diversification among the Atriplex species, which opens a new venue for their functional analysis.

Dehydration Stress Memory Genes in Triticum turgidum L. ssp. durum (Desf.).

Exposure to successive stress cycles can result in a variety of memory response patterns in several plant species. We have investigated a group of these patterns at both the transcriptional and physiological memory levels in durum wheat. The data revealed huge discrepancies between investigated durum wheat cultivars, which presumably are all drought tolerant. It was possible to generate a consensus memory response pattern for each cultivar, where Hourani 27 was the most tolerant followed by Balikh 2 and then Omrabi 5. When durum wheat homologs from rice and maize were compared, only 18% gave similar memory response patterns. The data would indicate the presence of potentially divergent memory mechanisms in different plant species and genotypes. Ultimately, a thorough examination is required for each genotype before giving solid memory-based conclusions that can be applied in plant breeding and agricultural management practices.

Development of SSR Markers Linked to Stress Responsive Genes along Tomato Chromosome 3 (Solanum lycopersicum L.).

This study aimed to develop novel SSR markers in tomato. Several BAC clones along chromosome 3 in tomato were selected based on their content. The criteria was the availability of genes, either directly or indirectly related to stress response (drought, salinity, and heat) in tomato. A total of 20 novel in silico SSR markers were developed and 96 important nearby genes were identified. The identified nearby genes represent different tomato genes involved in plant growth and development and biotic and abiotic stress tolerance. The developed SSR markers were assessed using tomato landraces. A total of 29 determinate and semi-determinate local tomato landraces collected from diverse environments were utilized. A total of 33 alleles with mean of 1.65 alleles per locus were scored, showing 100% polymorphic patterns, with a mean of 0.18 polymorphism information content (PIC) values. The mean of observed and expected heterozygosity were 0.19 and 0.24, respectively. The mean value of the Jaccard similarity index was used for clustering the landraces. The developed microsatellite markers showed potential to assess genetic variability among tomato landraces. The genetic distance information reported in this study can be used by breeders in future genetic improvement of tomato for tolerance against diverse stresses.

Complete mitochondrial genome sequence of Awassi-Jo sheep breed (Ovis aries) in Jordan.

Using high-throughput sequencing technology, the complete mitochondrial genome of Awassi-Jo breed (Ovis aries) was decoded. Mitochondrial genome was 16,617 bp in length. The genome contained 37 genes (13 protein-coding, 22 tRNA, and 2 rRNA) and a control region (D-loop region). The genes were encoded on the H-strand, except for the ND6 gene and 8 tRNA genes, which were encoded on the L-strand. The GC content is 38.9%. Phylogenetic analysis was performed to compare Awassi-Jo with other sheep breeds. The phylogenetic tree showed that Awassi-Jo diverged earlier than related breeds (Turkey, Italy, Germany, and Netherland) with a common ancestor in haplogroup HB. The results revealed the importance of mitochondrial data in studying sheep evolution and domestication.

Physiological and molecular responses for long term salinity stress in common fig (Ficus carica L.).

Salinity stress in increasingly becoming a major challenge in current and expanding agricultural ecosystems. Unlike temporal abiotic stresses, plants are usually exposed to salinity stress for an entire lifespan. Therefore, a long term effect (10 weeks) of continuous salinity exposure was investigated for three common fig landraces (Zraki, Mwazi, and Khdari). Both relative water content and chlorophyll content decreased with elevated salinity stress, while stem length barely changed. The most prominent decline was observed in root biomass. The data would align common fig to moderately tolerant threshold slop with a C50 range of 100 to 150 mM NaCl. A high and significant correlation was evident between root biomass and chlorophyll content (85%). Concurrently, differential expression of putative salinity responsive genes in common fig were determined; signal peptide peptidase-like 2B (FcSPPL2B), dehydration responsive element binding protein (FcDREB), calcineurin B-like protein (CBL)-CBL-interacting serine/threonine-protein kinase 11 (FcCIPK11), sorbitol dehydrogenase (FcSORD) and dehydrin (FcDHN). The data were discussed for each gene in respect of its potential role in salinity stress mitigation. The combined physiological and molecular data would conclude Zraki as the most salinity tolerant genotype. The major implication of the data emphasizes the tremendous genotype by environment (salinity stress) interaction in common fig. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s12298-020-00921-z).

Bioinformatics analysis of ubiquitin expression protein gene from Heterodera latipons.

Ubiquitin expression protein DNA clone (Hl-UBI) was isolated from Heterodera latipons collected from North Jordan. Its sequence of 285 nucleotides was also determined and deposited in the GeneBank. The 285-bp open reading frame coded for 76-amino acid protein having two domains; the ubiquitin domain and the C terminal extension. The first 59 amino acids were predicted with the ubiquitin domain with identity percentages of 78% to ubiquitin of H. schachtii, 77% to polyubiquitin of Globodera pallida, 74% to ubiquitin of Globodera rostochiensis and 72% to ubiquitin of Heterodera glycines. The other domain at the C-terminus, containing 17 amino acids, showed no homology to any known protein. Sequence analysis showed a calculated encoding amino acids molecular weight of 8.77 kDa, theoretical isoelectric point = 4.76, negatively charged residues = 12, positively charged residues = 9, extinction coefficient = 1490, estimated half-life = 30 h, instability index = 32.51 and grand average of hydropathicity = -0.537. The demonstrated subcellular localization analysis of cytoplasm, cell nucleus, mitochondrion, cell skeleton and plasma membrane of Hl-UBI protein occupied about 52.20, 21.70, 17.40, 4.30 and 4.30%, respectively. Sequence, homology and structural analysis confirmed that Hl-UBI gene was highly conserved during evolution and belonged to ubiquitin gene family.

Morphological and morphometrical analysis of Heterodera spp. populations in Jordan.

Phenotypic diversity of five Jordanian populations of cyst nematodes, Heterodera spp. collected from five regions from Jordan (Ar-Ramtha, Madaba, Dana, Al-Karak, and Jerash) was investigated. Soil samples were collected from one representative field in each region. Morphological and morphometrical characteristics revealed that Heterodera latipons is dominated in cereal fields at Ar-Ramtha, Madaba, Dana and Al-Karak regions and Heterodera schachtii in Jerash. Cysts populations from all cereal fields had bifenestrate vulval cone and a strong underbridge. Wherever, cysts of the cabbage population had ambifenestrate vulval cone with long vulval slit. The bullae were absent in Ar-Ramtha, Madaba and Dana populations, but present in Al-Karak and Jerash. Based on 12 morphometrical characters, the first three functions in canonical discriminant analysis accounted 99.3% of the total variation. Distance from dorsal gland duct opening to stylet base, underbridge length, a = L/W (body length/midbody width) and length of hyaline tail tip had strong and significant contributions in the first function. While the second function was strongly influenced by length of hyaline tail, fenestral length, fenestral width and tail length. However, the third canonical discriminate function was found to be influenced by stylet length, fenestral length, a = L/W (body length/midbody width) and underbridge width. The graphical representation of the distribution of the samples showed that the first canonical discriminant function clearly separated H. schachtii from Jerash from other populations. Whereas, H. latipons collected from Madaba and Dana were clearly separated in the second function. The results indicated that differences at morphological and morphometrical levels revealed diverse populations of Heterodera spp. in Jordan.

Cloning and Expression Profiling of the Polycomb Gene, Retinoblastoma-related Protein from Tomato Solanum lycopersicum L.

Cell cycle regulation mechanisms appear to be conserved throughout eukaryotic evolution. One of the important proteins involved in the regulation of cell cycle processes is retinoblastoma-related protein (RBR), which is a negative regulator of cell cycle progression, controlling the G1/S transition in plants and animals. In this study, we present the cloning and genomic structure of a putative SlRBR gene in the tomato Solanum lycopersicum L. by isolating cDNA clones that correspond to the SlRBR gene from tomato using primers that were designed from available Solanaceae ESTs based on conserved sequences between the PcG genes in Arabidopsis thaliana and tomato. The SlRBR cDNAs were cloned into the pBS plasmid and sequenced. Both 5'- and 3'-RACE were generated and sequenced. FlcDNA of the SlRBR gene of 3,554 bp was composed of a 5'-UTR of 140 bp, an ORF of 3,054 bp, and a 3'-UTR of 360 bp. The translated ORF encodes a polypeptide of 1,018 amino acids. An alignment of the deduced amino acids indicates that there are highly conserved regions between the tomato SlRBR predicted protein and plant hypothetical RBR gene family members. Both of the unrooted phylogenetic trees, which were constructed using maximum parsimony and maximum likelihood methods, indicate a close relationship between the SlRBR predicted protein and the RBR protein of Nicotiana benthamiana. QRT-PCR indicates that SlRBR gene is expressed in closed floral bud tissues 1.7 times higher than in flower tissues, whereas the expression level in unripe fruit tissue is lower by about three times than in flower tissues.

Development of a Cucumis sativus TILLinG platform for forward and reverse genetics.

BACKGROUND: Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics. PRINCIPAL FINDINGS: A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147 Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation. CONCLUSIONS: We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.