Type 2 diabetes linked FTO gene variant rs8050136 is significantly associated with gravidity in gestational diabetes in a sample of Bangladeshi women: Meta-analysis and case-control study.

OBJECTIVE: Gestational diabetes mellitus (GDM) is a growing public health concern that has not been extensively studied. Numerous studies have indicated that a variant (rs8050136) of the fat mass-associated gene, FTO, is associated with both GDM and Type 2 diabetes mellitus(T2DM). We conducted a meta-analysis on the association between the FTO single nucleotide polymorphism (SNP) rs8050136 and T2DM, followed by a case-control study on the association of the said SNP and GDM in a sample of Bangladeshi women. METHOD: A total of 25 studies were selected after exploring various databases and search engines, which were assessed using the Newcastle-Ottawa Scale (NOS). The MetaGenyo web tool was used to conduct this meta-analysis. A case-control study was performed on 218 GDM patients and 284 controls to observe any association between FTO rs8050136 and GDM. Genotyping was performed using the tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS) method, and statistical analyses were performed using various statistical softwares. RESULTS: In the meta-analysis 26231 cases and 43839 controls were examined. Pooled association analyses revealed a statistically significant relationship between the FTO rs8050136 polymorphism and an elevated risk of T2DM under all genetic models (P<0.05). In the case-control study, synergistic analyses of the SNP and gravida with GDM revealed a significant (P<0.01) association with an increase in odds by 1.6 to 2.4 folds in multigravida and decrease in odds by 2 folds in primigravida. A positive family history of diabetes and the minor allele of this SNP collectively increased the risk of developing GDM by many-fold (1.8 to 2.7 folds). However, after accounting for family history of diabetes and gravidity, analyses showed no significant association with GDM. CONCLUSION: Our meta-analysis revealed a significant association between SNP rs8050136 of FTO with T2DM, and this variant was substantially associated with an increased risk of GDM in a sample of Bangladeshi multigravida women.

A high-quality chromosome-level wild rice genome of Oryza coarctata.

Oryza coarctata (2n = 4X = 48, KKLL) is an allotetraploid, undomesticated relative of rice and the only species in the genus Oryza with tolerance to high salinity and submergence. Therefore, it contains important stress and tolerance genes/factors for rice. The initial draft genome published was limited by data and technical restrictions, leading to an incomplete and highly fragmented assembly. This study reports a new, highly contiguous chromosome-level genome assembly and annotation of O. coarctata. PacBio high-quality HiFi reads generated 460 contigs with a total length of 573.4 Mb and an N50 of 23.1 Mb, which were assembled into scaffolds with Hi-C data, anchoring 96.99% of the assembly onto 24 chromosomes. The genome assembly comprises 45,571 genes, and repetitive content contributes 25.5% of the genome. This study provides the novel identification of the KK and LL genome types of the genus Oryza, leading to valuable insights into rice genome evolution. The chromosome-level genome assembly of O. coarctata is a valuable resource for rice research and molecular breeding.

Paired growth of cultivated and halophytic wild rice under salt stress induces bacterial endophytes and gene expression responses.

Introduction: Utilizing salt-affected marginal lands in coastal regions can help meet the growing demand for rice. We explored a nature-based solution involving wild halophytic rice (O. coarctata, Oc) and commercial rice BRRI Dhan 67 (O. sativa, Os) grown in close proximity to each other under salt stress. Methods: This was to investigate whether a paired planting strategy could help complement rice growth and yield under stress. We also investigated the gene expression and endophytic bacterial profiles of both Os and Oc in unpaired and paired conditions without and with salt. Results: Paired plants exhibited lower salt damage indicators such as smaller reduction in plant height, electrolyte leakage and chlorophyll loss, as well as higher K+/Na+ ratio under saline stress. Some of the 39 endophytic bacteria in the mutualism experiment were unique to Oc and transferred to Os when paired. Differentially expressed genes in leaves of paired Os versus unpaired Os were 1097 (994 up-regulated, 101 down-regulated) without salt and 893 (763 up-regulated, 130 down-regulated) under salt stress. The presence of Oc plants under salt stress influenced major biological processes in Os, including oxidative stress; chitinase activity; phenylalanine catabolic process and response to ABA. Protein binding and serine/threonine kinase activity were primarily affected in molecular function. The downregulated WRKY transcription factor 22 in paired conditions under salt stress played a role in the MAPK signaling pathway, reducing respiratory cell death. The upregulated auxin-responsive protein IAA18 gene, involved in hormone signaling and cell enlargement, was present only in paired plants. Discussion: Our findings therefore, offer insights into developing more effective cultivation strategies for sustainable rice production.

Salt tolerance QTLs of an endemic rice landrace, Horkuch at seedling and reproductive stages.

Salinity has a significant negative impact on production of rice. To cope with the increased soil salinity due to climate change, we need to develop salt tolerant rice varieties that can maintain their high yield. Rice landraces indigenous to coastal Bangladesh can be a great resource to study the genetic basis of salt adaptation. In this study, we implemented a QTL analysis framework with a reciprocal mapping population developed from a salt tolerant landrace Horkuch and a high yielding rice variety IR29. Our aim was to detect genetic loci that contributes to the salt adaptive responses of the two different developmental stages of rice which are very sensitive to salinity stress. We identified 14 QTLs for 9 traits and found that most are unique to specific developmental stages. In addition, we detected a significant effect of the cytoplasmic genome on the QTL model for some traits such as leaf total potassium and filled grain weight. This underscores the importance of considering cytoplasm-nuclear interaction for breeding programs. Finally, we identified QTLs co-localization for multiple traits that highlights the possible constraint of multiple QTL selection for breeding programs due to different contributions of a donor allele for different traits.

CDKAL1 gene rs7756992 A/G and rs7754840 G/C polymorphisms are associated with gestational diabetes mellitus in a sample of Bangladeshi population: implication for future T2DM prophylaxis.

BACKGROUND: Association of single nucleotide polymorphisms (SNP) rs7756992 A/G and rs7754840 G/C of cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1) gene with the susceptibility of gestational diabetes mellitus (GDM) has been studied in a group of Bangladeshi women. METHODS: In this case-control study, 212 GDM patients and 256 control subjects were genotyped for rs7756992 and rs7754840 by PCR-RFLP and TaqMan™ allelic discrimination assay method respectively. Genotyping results were confirmed by DNA sequencing and replicated TaqMan™ assay. The odds ratios and their 95% confidence interval were calculated by logistic regression to determine the associations between genotypes and GDM. RESULTS: The genotype frequencies of rs7756992-AA/AG/GG in the GDM group and the control group were 37%/48%, 53%/45%, 10%/7% and those of rs7754840-CC/CG/GG were 51%/55%, 40.1%/39.8%, 9%/5% respectively. Under dominant and log additive models rs7756992 was revealed significantly associated with GDM after being adjusted for family history of diabetes (FHD) and gravidity. Conversely, rs7754840 was significantly associated (P = 0.047) with GDM only under the recessive model after the same adjustment. The risk allele frequency of both SNPs was higher in the GDM group but significantly (P = 0.029) increased prevalence was observed in the rs7756992 G allele. When positive FHD and risk alleles of these SNPs were synergistically present in any pregnant woman, the chance of developing GDM was augmented by many folds. The codominant model revealed 2.5 and 2.1 folds increase in odds by AG (rs7756992) and GC (rs7754840) genotypes and 3.7 and 4.5 folds by GG (rs7756992) and CC (rs7754840) genotypes respectively. A significant 2.7 folds (P = 0.038) increase in odds of GDM resulted from the interaction of rs7756992 and family history of diabetes under the dominant model. The cumulative effect of multigravidity and risk alleles of these SNPs increased the odds of GDM more than 1.5 folds in different genotypes. CONCLUSION: This study not only revealed a significant association between rs7756992 and rs7754840 with GDM but also provided the possibility as potential markers for foretelling about GDM and type 2 diabetes mellitus in Bangladeshi women.

Functional genomic analysis of K+ related salt-responsive transporters in tolerant and sensitive genotypes of rice.

Introduction: Salinity is a complex environmental stress that affects the growth and production of rice worldwide. But there are some rice landraces in coastal regions that can survive in presence of highly saline conditions. An understanding of the molecular attributes contributing to the salinity tolerance of these genotypes is important for developing salt-tolerant high yielding modern genotypes to ensure food security. Therefore, we investigated the role and functional differences of two K+ salt-responsive transporters. These are OsTPKa or Vacuolar two-pore potassium channel and OsHAK_like or a hypothetical protein of the HAK family. These transporters were selected from previously identified QTLs from the tolerant rice landrace genotype (Horkuch) and sensitive genotype (IR29). Methods: In silico comparative sequence analysis of the promoter sequences of two these genes between Horkuch and IR29 was done. Real-Time expression of the selected genes in leaves and roots of IR29 (salt-sensitive), I-14 and I-71 (Recombinant Inbred Lines of IR29(♀)× Horkuch), Horkuch and Pokkali (salt-tolerant) under salt-stress at different time points was analyzed. For further insight, OsTPKa and OsHAK_like were chosen for loss-of-function genomic analysis in Horkuch using the CRISPR/Cas9 tool. Furthermore, OsTPKa was chosen for cloning into a sensitive variety by Gateway technology to observe the effect of gain-of-function. Results: The promoter sequences of the OsTPKa and OsHAK_like genes showed some significant differences in promoter sequences which may give a survival advantage to Horkuch under salt-stress. These two genes were also found to be overexpressed in tolerant varieties (Horkuch and Pokkali). Moreover, a coordinated expression pattern between these two genes was observed in tolerant Horkuch under salt-stress. Independently transformed plants where the expression of these genes was significantly lowered, performed poorly in physiological tests for salinity tolerance. On the other hand, positively transformed T0 plants with the OsTPKa gene from Horkuch consistently showed growth advantage under both control and salt stress. Discussion: The poor performance of the transgenic plants with the down-regulated genes OsTPKa and OsHAK_like under salt stress supports the assumption that OsTPKa and OsHAK_like play important roles in defending the rice landrace Horkuch against salt stress, minimizing salt injury, and maintaining plant growth. Moreover, the growth advantage provided by overexpression of the vacuolar OsTPKa K+ transporter, particularly under salt stress reconfirms its important role in providing salt tolerance. The QTL locus from Horkuch containing these two transporters maybe bred into commercial rice to produce high-yielding salt tolerant rice.

Novel QTLs for salinity tolerance revealed by genome-wide association studies of biomass, chlorophyll and tissue ion content in 176 rice landraces from Bangladesh.

Farmland is on the decline and worldwide food security is at risk. Rice is the staple of choice for over half the Earth's people. To sustain current demands and ascertain a food secure future, substandard farmland affected by abiotic stresses must be utilized. For rapid crop improvement, a broader understanding of polygenic traits like stress tolerance and crop yield is indispensable. To this end, the hidden diversity of resilient and neglected wild varieties must be traced back to their genetic roots. In this study, we separately assayed 11 phenotypes in a panel of 176 diverse accessions predominantly comprised of local landraces from Bangladesh. We compiled high resolution sequence data for these accessions. We collectively studied the ties between the observed phenotypic differences and the examined additive genetic effects underlying these variations. We applied a fixed effect model to associate phenotypes with genotypes on a genomic scale. Discovered QTLs were mapped to known genes. Our explorations yielded 13 QTLs related to various traits in multiple trait classes. 10 identified QTLs were equivalent to findings from previous studies. Integrative analysis assumes potential novel functionality for a number of candidate genes. These findings will usher novel avenues for the bioengineering of high yielding crops of the future fortified with genetic defenses against abiotic stressors.

HKT1;5 Transporter Gene Expression and Association of Amino Acid Substitutions With Salt Tolerance Across Rice Genotypes.

Plants need to maintain a low Na+/K+ ratio for their survival and growth when there is high sodium concentration in soil. Under these circumstances, the high affinity K+ transporter (HKT) and its homologs are known to perform a critical role with HKT1;5 as a major player in maintaining Na+ concentration. Preferential expression of HKT1;5 in roots compared to shoots was observed in rice and rice-like genotypes from real time PCR, microarray, and RNAseq experiments and data. Its expression trend was generally higher under increasing salt stress in sensitive IR29, tolerant Pokkali, both glycophytes; as well as the distant wild rice halophyte, Porteresia coarctata, indicative of its importance during salt stress. These results were supported by a low Na+/K+ ratio in Pokkali, but a much lower one in P. coarctata. HKT1;5 has functional variability among salt sensitive and tolerant varieties and multiple sequence alignment of sequences of HKT1;5 from Oryza species and P. coarctata showed 4 major amino acid substitutions (140 P/A/T/I, 184 H/R, D332H, V395L), with similarity amongst the tolerant genotypes and the halophyte but in variance with sensitive ones. The best predicted 3D structure of HKT1;5 was generated using Ktrab potassium transporter as template. Among the four substitutions, conserved presence of aspartate (332) and valine (395) in opposite faces of the membrane along the Na+/K+ channel was observed only for the tolerant and halophytic genotypes. A model based on above, as well as molecular dynamics simulation study showed that valine is unable to generate strong hydrophobic network with its surroundings in comparison to leucine due to reduced side chain length. The resultant alteration in pore rigidity increases the likelihood of Na+ transport from xylem sap to parenchyma and further to soil. The model also proposes that the presence of aspartate at the 332 position possibly leads to frequent polar interactions with the extracellular loop polar residues which may shift the loop away from the opening of the constriction at the pore and therefore permit easy efflux of the Na+. These two substitutions of the HKT1;5 transporter probably help tolerant varieties maintain better Na+/K+ ratio for survival under salt stress.

Overexpression of heterotrimeric G protein beta subunit gene (OsRGB1) confers both heat and salinity stress tolerance in rice.

Constitutive overexpression of the rice heterotrimeric G protein beta subunit gene (RGB1) in the commercial rice cultivar BRRI Dhan 55 resulted in improved tolerance to heat or salinity or their combination. Two independently in planta transformed plants with the gene confirmed to be integrated at T2 by Southern hybridization and showing high expression at the T3 seedling stage showed better physiological performance after 8 days in 120 mM salt stress than the wild type. The plants had significantly lower electrolyte leakage and malondialdehyde production, while showing higher levels of chlorophyll. Significantly higher germination at 48 °C or with combined stresses of 42/40 °C day/night stress in the presence of 120 mM salt for 2 days was also observed. Stress responsive genes such as OsAPX1, OsSOD, OsHKT1, OsHSP1, OsHSP2 and OsCOR47 showed higher expression in the RGB1 positive plants. These RGB1 transgenic plants can likely provide a strong defense against climate change.

Gene Expression analysis associated with salt stress in a reciprocally crossed rice population.

The rice landrace Horkuch, endemic to the southern saline coast of Bangladesh, is known to have salt tolerance traits and can therefore contribute to a high yielding recipient for breeding purposes. In this study, we reciprocally crossed Horkuch with high yielding but salt sensitive IR29 to detect the complement of genes that were responsible for conferring salt tolerance versus sensitivity at the seedling developmental stage. We looked at tolerant and sensitive F3 families from individual F2 segregating plants and analyzed them for differential gene expressions using RNAseq. In general, we observed higher numbers of genes differentially expressed in leaves compared to root tissues. This included both upregulation and downregulation of gene expression across our experimental factors. Gene expression decreased in sensitive leaf after stress exposure where tolerant plants showed the opposite trend. In root, tolerant plants expression decreased at higher time points of stress exposure. We also observed a strong maternal cytoplasmic effect on gene expression and this was most evident in roots where there was upregulation in functional enrichments related to phosphorylation, electron carriers, transporter and cation transmembrane activities. Stress groups (tolerant and sensitive) response in F3 families were distinctive in both cytoplasmic backgrounds and involved uniquely upregulated genes in tolerant progenies including membrane sensor proteins, enzymes involved with signaling pathways, such as those producing trehalose and G-protein coupled receptor proteins, photosynthesis-related enzymes and golgi body recycling as well as prolamin precursor proteins involved in refolding of proteins. On the other hand, sensitivity was found to be associated with differential upregulation of only a few redox proteins and higher number of apoptosis related genes compared to the tolerant response. Overall, our highly replicated experimental design was powerful and allowed the detection of relatively subtle differential expression. Our future goal is to correlate these expression differences with QTLs in this population, which would help identify the relative importance of specific genetic loci and provide a direct avenue for combining higher levels of salt tolerance with better agronomic traits in rice.

Introgression, Generational Expression and Salinity Tolerance Conferred by the Pea DNA Helicase 45 Transgene into Two Commercial Rice Genotypes, BR28 and BR47.

DNA helicase (PDH45) from the pea plant (Pisum sativum) is a member of the DEAD box protein family and plays a vital regulatory role in saline stress tolerance in plants. We previously reported that over-expression of PDH45 gene confers both seedling and reproductive stage salinity tolerance to a Bangladeshi rice landrace, Binnatoa (BA). In this study, transgenic BA-containing PDH45 (♂) was crossed with two different farmer-popular BRRI rice varieties (♀), BR28 and BR47, in a contained net house. F1 plants positive for the transgene and having recipient phenotype were advanced from F1 to F5. Expression of the PDH45 gene was detected in all generations. The expression level of PDH45 was 200-fold higher in the donor compared to the two recipient genotypes but without any effect on their salt stress tolerance ability in various assays. Under 120 mM NaCl stress at seedling stage, all rice genotypes showed vigorous growth, higher chlorophyll content, lower electrolyte leakage and lower LDS (Leaf Damage Score) compared to their corresponding wild types. At the reproductive stage under continuous salinity stress at 80 mM NaCl, the cross-bred lines BR28 and BR47 showed significantly better spikelet fertility and yield per plant, which were two- and 2.5-folds, respectively, than their corresponding wild types. The PDH45 transgene was observed to increase the expression of 6 salt stress-related downstream genes at 150 mM NaCl stress to similar differential degrees in the donor and recipient genotypes. However, the expression of OsLEA was significantly higher in transgenic BR28 compared to transgenic BR47, where the latter shows comparatively higher salt tolerance. The study shows stability of transgene expression across generations. It also demonstrates that there may be an effect of background genotype on transgene expression. Moreover, some downstream effects of the transgene may also be genotype-specific.

Reproductive stage physiological and transcriptional responses to salinity stress in reciprocal populations derived from tolerant (Horkuch) and susceptible (IR29) rice.

Global increase in salinity levels has made it imperative to identify novel sources of genetic variation for tolerance traits, especially in rice. The rice landrace Horkuch, endemic to the saline coastal area of Bangladesh, was used in this study as the source of tolerance in reciprocal crosses with the sensitive but high-yielding IR29 variety for discovering transcriptional variation associated with salt tolerance in the resulting populations. The cytoplasmic effect of the Horkuch background in leaves under stress showed functional enrichment for signal transduction, DNA-dependent regulation and transport activities. In roots the enrichment was for cell wall organization and macromolecule biosynthesis. In contrast, the cytoplasmic effect of IR29 showed upregulation of apoptosis and downregulation of phosphorylation across tissues relative to Horkuch. Differential gene expression in leaves of the sensitive population showed downregulation of GO processes like photosynthesis, ATP biosynthesis and ion transport. Roots of the tolerant plants conversely showed upregulation of GO terms like G-protein coupled receptor pathway, membrane potential and cation transport. Furthermore, genes involved in regulating membrane potentials were constitutively expressed only in the roots of tolerant individuals. Overall our work has developed genetic resources and elucidated the likely mechanisms associated with the tolerance response of the Horkuch genotype.

Enhanced Salt Tolerance Conferred by the Complete 2.3 kb cDNA of the Rice Vacuolar Na(+)/H(+) Antiporter Gene Compared to 1.9 kb Coding Region with 5' UTR in Transgenic Lines of Rice.

Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na(+)/H(+) antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions (UTRs) of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5' UTR with CDS and the other of 2.3 kb, including 5' UTR, CDS, and the 3' UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5' and 3' UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3' UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P < 0.05) and the electrolyte leakage significantly lower (P < 0.05) in the order 2.3 kb > 1.9 kb > and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P < 0.01) compared, respectively, to the best 1.9 kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance.

Unique Genotypic Differences Discovered among Indigenous Bangladeshi Rice Landraces.

Bangladesh is a reservoir of diverse rice germplasm and is home to many landraces with unique, important traits. Molecular characterization of these landraces is of value for their identification, preservation, and potential use in breeding programs. Thirty-eight rice landraces from different regions of Bangladesh including some high yielding BRRI varieties were analyzed by 34 polymorphic microsatellite markers yielding a total of 258 reproducible alleles. The analysis could locate 34 unique identifiers for 21 genotypes, making the latter potentially amenable to identity verification. An identity map for these genotypes was constructed with all the 12 chromosomes of the rice genome. Polymorphism information content (PIC) scores of the 34 SSR markers were 0.098 to 0.89 where on average 7.5 alleles were observed. A dendogram constructed using UPGMA clustered the varieties into two major groups and five subgroups. In some cases, the clustering matched with properties like aromaticity, stickiness, salt tolerance, and photoperiod insensitivity. The results will help breeders to work towards the proper utilization of these landraces for parental selection and linkage map construction for discovery of useful alleles.

In silico predicted mycobacterial epitope elicits in vitro T-cell responses.

Epitope-based vaccines permit the selection of only a specific subset of epitopes to induce the necessary immune response, thus providing a rational alternative to conventional design approaches. Using a range of immunoinformatics tools, we identified a novel, contiguous 28 amino acid multi-epitope cluster within the highly conserved secretory protein Ag85B of Mycobacterium tuberculosis, the causative agent of TB. This cluster, named Ep85B, is composed of epitopes which bind to three HLA Class I and 15 Class II molecules, and harbors the potential to generate 99% population coverage in TB-endemic regions. We experimentally evaluated the capacity of Ep85B to elicit T-cell immune responses using whole blood cells and, as predicted, observed significant increases in populations of both CD4+ and memory CD4+ CD45RO+ T-cells. Our results demonstrate the practical utility of an epitope-based design methodology - a strategy that, following further evaluation, may serve as an additional tool for the development of novel vaccine candidates against TB and other diseases.

Physiology and gene expression of the rice landrace Horkuch under salt stress.

Good donors in breeding for salt tolerance are a prerequisite for food security under changing climatic conditions. Horkuch, a farmer-popular salt tolerant rice (Oryza sativa L.) variety from the south-west coast of Bangladesh was characterised up to maturity under NaCl stress, together with a modern variety (BRRI dhan41), a sensitive control (BRRI dhan29) and Pokkali, the salt-tolerant benchmark for rice. Horkuch had low reduction in shoot biomass, a low Na:K ratio in flag leaves, a low percent reduction in yield and good partitioning of Na in the older leaves, and maintained high levels of Ca and Mg in the flag leaves. In order to understand the physiology at the molecular level, the expression of salt-responsive genes was investigated using microarray analysis. Salt-stressed cDNA of Horkuch seedlings were hybridised with cDNA probes synthesised mainly from database sequences of Arabidopsis thaliana (L.) Heynh. The upregulated genes included transcription factors, signal transducers, metabolic enzymes, reactive oxygen species (ROS) scavengers, osmoprotectants and some specific salt-induced transcripts. An increase in expression of photosynthesis-related genes as well ROS scavengers suggested that this could be the reason for the better yield performance of Horkuch. The data therefore indicate Horkuch as a potential donor alternative to Pokkali in breeding programs for salt tolerance.