Delineating Marker-Trait Associations for Fusarium Wilt in Chickpea Using the Axiom® CicerSNP Array.

Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. ciceri is a devastating disease of chickpea (Cicer arietinum). To identify promising resistant genotypes and genomic loci for FW resistance, a core set of 179 genotypes of chickpea was tested for FW reactions at the seedling and reproductive stages under field conditions and controlled conditions in the greenhouse. Our results revealed that at the seedling stage, most of the genotypes were resistant, whereas at the reproductive stage, most of the genotypes were susceptible. Genotyping using a 50K Axiom® CicerSNP Array and trait data of FW together led to the identification of 26 significant (P ≤ E-05) marker-trait associations (MTAs) for FW resistance. Among the 26 MTAs, 12 were identified using trait data recorded in the field (three at the seedling and nine at the reproductive stage), and 14 were identified using trait data recorded under controlled conditions in the greenhouse (six at the seedling and eight at the reproductive stage). The phenotypic variation explained by these MTAs varied from 11.75 to 15.86%, with an average of 13.77%. Five MTAs were classified as major, explaining more than 15% of the phenotypic variation for FW, and two were declared stable, being identified in two environments. One of the promising stable and major MTAs (Affx_123280060) detected in field conditions at the reproductive stage was also detected in greenhouse conditions at the seedling and reproductive stages. The stable and major (>15% PVE) MTAs can be used in chickpea breeding programs.

Genome-wide characterization and identification of cyclophilin genes associated with leaf rust resistance in bread wheat (Triticum aestivum L.).

Cyclophilins (CYPs) are a group of highly conserved proteins involved in host-pathogen interactions in diverse plant species. However, the role of CYPs during disease resistance in wheat remains largely elusive. In the present study, the systematic genome-wide survey revealed a set of 81 TaCYP genes from three subfamilies (GI, GII, and GIII) distributed on all 21 wheat chromosomes. The gene structures of TaCYP members were found to be highly variable, with 1-14 exons/introns and 15 conserved motifs. A network of miRNA targets with TaCYPs demonstrated that TaCYPs were targeted by multiple miRNAs and vice versa. Expression profiling was done in leaf rust susceptible Chinese spring (CS) and the CS-Ae. Umbellulata derived resistant IL "Transfer (TR). Three homoeologous TaCYP genes (TaCYP24, TaCYP31, and TaCYP36) showed high expression and three homoeologous TaCYP genes (TaCYP44, TaCYP49, and TaCYP54) showed low expression in TR relative to Chinese Spring. Most of the other TaCYPs showed comparable expression changes (down- or upregulation) in both contrasting TR and CS. Expression of 16 TaCYPs showed significant association (p < 0.05) with superoxide radical and hydrogen peroxide abundance, suggesting the role of TaCYPs in downstream signaling processes during wheat-leaf rust interaction. The differentially expressing TaCYPs may be potential targets for future validation using transgenic (overexpression, RNAi or CRISPR-CAS) approaches and for the development of leaf rust-resistant wheat genotypes.

Genome-wide association analysis to delineate high-quality SNPs for seed micronutrient density in chickpea (Cicer arietinum L.).

Chickpea is the most important nutrient-rich grain legume crop in the world. A diverse core set of 147 chickpea genotypes was genotyped with a Axiom(®)50K CicerSNP array and trait phenotyped in two different environments for four seed micronutrients (Zn, Cu, Fe and Mn). The trait data and high-throughput 50K SNP genotypic data were used for the genome-wide association study (GWAS). The study led to the discovery of genes/QTLs for seed Zn, Cu, Fe and Mn, concentrations in chickpea. The analysis of seed micronutrient data revealed significant differences for all four micronutrient concentrations (P ≤ 0.05). The mean concentrations of seed Zn, Cu, Fe and Mn pooled over the 2 years were 45.9 ppm, 63.8 ppm 146.1 ppm, and 27.0 ppm, respectively. The analysis of results led to the identification of 35 SNPs significantly associated with seed Zn, Cu, Fe and Mn concentrations. Among these 35 marker-trait associations (MTAs), 5 were stable (consistently identified in different environments), 6 were major (explaining more than 15% of the phenotypic variation for an individual trait) and 3 were both major and stable MTAs. A set of 6 MTAs, MTAs (3 for Mn, 2 for Fe, and 1 for Cu) reported by us during the present study have been also reported in the same/almost same genomic regions in earlier studies and therefore declared as validated MTAs. The stable, major and validated MTAs identified during the present study will prove useful in future chickpea molecular breeding programs aimed at enhancing the seed nutrient density of chickpea.

Identification of Novel Broad-Spectrum Leaf Rust Resistance Sources from Khapli Wheat Landraces.

Wheat leaf rust caused by Puccinia triticina Eriks is an important disease that causes yield losses of up to 40% in susceptible varieties. Tetraploid emmer wheat (T. turgidum ssp. Dicoccum), commonly called Khapli wheat in India, is known to have evolved from wild emmer (Triticum turgidum var. dicoccoides), and harbors a good number of leaf rust resistance genes. In the present study, we are reporting on the screening of one hundred and twenty-three dicoccum wheat germplasm accessions against the leaf rust pathotype 77-5. Among these, an average of 45.50% of the germplasms were resistant, 46.74% were susceptible, and 8.53% had mesothetic reactions. Further, selected germplasm lines with accession numbers IC138898, IC47022, IC535116, IC535133, IC535139, IC551396, and IC534144 showed high level of resistance against the eighteen prevalent pathotypes. The infection type varied from ";", ";N", ";N1" to ";NC". PCR-based analysis of the resistant dicoccum lines with SSR marker gwm508 linked to the Lr53 gene, a leaf rust resistance gene effective against all the prevalent pathotypes of leaf rust in India and identified from a T. turgidum var. dicoccoides germplasm, indicated that Lr53 is not present in the selected accessions. Moreover, we have also generated 35K SNP genotyping data of seven lines and the susceptible control, Mandsaur Local, to study their relationships. The GDIRT tool based on homozygous genotypic differences revealed that the seven genotypes are unique to each other and may carry different resistance genes for leaf rust.

Genome-Wide Identification and Expression Analysis of the Thioredoxin (Trx) Gene Family Reveals Its Role in Leaf Rust Resistance in Wheat (Triticum aestivum L.).

Bread wheat (Triticum aestivum L.; Ta) is the staple cereal crop for the majority of the world's population. Leaf rust disease caused by the obligate fungal pathogen, Puccinia triticina L., is a biotrophic pathogen causing significant economic yield damage. The alteration in the redox homeostasis of the cell caused by various kinds of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to pathogenic infections is controlled by redox regulators. Thioredoxin (Trx) is one of the redox regulators with low molecular weight and is thermostable. Through a genome-wide approach, forty-two (42) wheat Trx genes (TaTrx) were identified across the wheat chromosome groups A, B, and D genomes containing 12, 16, and 14 Trx genes, respectively. Based on in silico expression analysis, 15 TaTrx genes were selected and utilized for further experimentation. These 15 genes were clustered into six groups by phylogenetic analysis. MicroRNA (miRNA) target analysis revealed eight different miRNA-targeted TaTrx genes. Protein-protein interaction (PPI) analysis showed TaTrx proteins interact with thioredoxin reductase, peroxiredoxin, and uncharacterized proteins. Expression profiles resulting from quantitative real-time PCR (qRT-PCR) revealed four TaTrx genes (TaTrx11-5A, TaTrx13-5B, TaTrx14-5D, and TaTrx15-3B) were significantly induced in response to leaf rust infection. Localization of ROS and its content estimation and an assay of antioxidant enzymes and expression analysis suggested that Trx have been involved in ROS homeostasis at span 24HAI-72HAI during the leaf rust resistance.

Analysis of NIA and GSNOR family genes and nitric oxide homeostasis in response to wheat-leaf rust interaction.

Nitric oxide (NO) modulates plant response to biotic and abiotic stresses by S-nitrosylation-mediated protein post-translational modification. Nitrate reductase (NR) and S-nitrosoglutathione reductase (GSNOR) enzymes are essential for NO synthesis and the maintenance of Nitric oxide/S-nitroso glutathione (NO/GSNO) homeostasis, respectively. S-nitrosoglutathione, formed by the S-nitrosylation reaction of NO with glutathione, plays a significant physiological role as the mobile reservoir of NO. The genome-wide analysis identified nine NR (NIA) and three GSNOR genes in the wheat genome. Phylogenic analysis revealed that the nine NIA genes +were clustered into four groups and the 3 GSNORs into two groups. qRT-PCR expression profiling of NIAs and GSNORs was done in Chinese spring (CS), a leaf rust susceptible wheat line showing compatible interaction, and Transfer (TR), leaf rust-resistant wheat line showing incompatible interaction, post-inoculation with leaf rust pathotype 77-5 (121-R-63). All the NIA genes showed upregulation during incompatible interaction in comparison with the compatible reaction. The GSNOR genes showed a variable pattern of expression: the TaGSNOR1 showed little change, whereas TaGSNOR2 showed higher expression during the incompatible response. TaGSNOR3 showed a rise of expression both in compatible and incompatible reactions. Before inoculation and after 72 h of pathogen inoculation, NO localization was studied in both compatible and incompatible reactions. The S-nitrosothiol accumulation, NR, and glutathione reductase activity showed a consistent increase in the incompatible interactions. The results demonstrate that both NR and GSNOR plays significant role in defence against the leaf rust pathogen in wheat by modulating NO homeostasis or signalling.

Genetic diversity may help evolutionary rescue in a clonal endemic plant species of Western Himalaya.

Habitat loss due to climate change may cause the extinction of the clonal species with a limited distribution range. Thus, determining the genetic diversity required for adaptability by these species in sensitive ecosystems can help infer the chances of their survival and spread in changing climate. We studied the genetic diversity and population structure of Sambucus wightiana-a clonal endemic plant species of the Himalayan region for understanding its possible survival chances in anticipated climate change. Eight polymorphic microsatellite markers were used to study the allelic/genetic diversity and population structure. In addition, ITS1-ITS4 Sanger sequencing was used for phylogeny and SNP detection. A total number of 73 alleles were scored for 37 genotypes at 17 loci for 8 SSRs markers. The population structural analysis using the SSR marker data led to identifying two sub-populations in our collection of 37 S. wightiana genotypes, with 11 genotypes having mixed ancestry. The ITS sequence data show a specific allele in higher frequency in a particular sub-population, indicating variation in different S. wightiana accessions at the sequence level. The genotypic data of SSR markers and trait data of 11 traits of S. wightiana, when analyzed together, revealed five significant Marker-Trait Associations (MTAs) through Single Marker Analysis (SMA) or regression analysis. Most of the SSR markers were found to be associated with more than one trait, indicating the usefulness of these markers for working out marker-trait associations. Moderate to high genetic diversity observed in the present study may provide insurance against climate change to S. wightiana and help its further spread.

Development and use of miRNA-derived SSR markers for the study of genetic diversity, population structure, and characterization of genotypes for breeding heat tolerant wheat varieties.

Heat stress is an important abiotic factor that limits wheat production globally, including south-east Asia. The importance of micro (mi) RNAs in gene expression under various biotic and abiotic stresses is well documented. Molecular markers, specifically simple sequence repeats (SSRs), play an important role in the wheat improvement breeding programs. Given the role of miRNAs in heat stress-induced transcriptional regulation and acclimatization, the development of miRNA-derived SSRs would prove useful in studying the allelic diversity at the heat-responsive miRNA-genes in wheat. In the present study, efforts have been made to identify SSRs from 96 wheat heat-responsive miRNA-genes and their characterization using a panel of wheat genotypes with contrasting reactions (tolerance/susceptible) to heat stress. A set of 13 miRNA-derived SSR markers were successfully developed as an outcome. These miRNA-SSRs are located on 11 different common wheat chromosomes (2A, 3A, 3B, 3D, 4D, 5A, 5B, 5D, 6A, 6D, and 7A). Among 13 miRNA-SSRs, seven were polymorphic on a set of 37 selected wheat genotypes. Within these polymorphic SSRs, three makers, namely HT-169j, HT-160a, and HT-160b, were found promising as they could discriminate heat-tolerant and heat-susceptible genotypes. This is the first report of miRNA-SSR development in wheat and their deployment in genetic diversity and population structure studies and characterization of trait-specific germplasm. The study suggests that this new class of molecular makers has great potential in the marker-assisted breeding (MAB) programs targeted at improving heat tolerance and other adaptability or developmental traits in wheat and other crops.

Development of white-grained PHS-tolerant wheats with high grain protein and leaf rust resistance.

The present study involved incorporation of two major QTLs for pre-harvest sprouting tolerance (PHST) in an Indian wheat cultivar named Lok1, which happens to be PHS susceptible. For transfer of two QTLs, two independent programmes with two different donors (AUS1408, CN19055) were utilized. The recipient cv. Lok1 was crossed with each of the two donors, followed by a number of backcrosses. Each backcross progeny was subjected to foreground and background selections. KASP assay was also used for confirming the presence of PHST QTL. In one case, PHST QTL was later also pyramided with a gene for high grain protein content (Gpc-B1) and a gene for leaf rust resistance (Lr24). The MAS derived lines were screened for PHS using simulated rain chambers leading to selection of 10 PHST lines. Four of these advanced lines carried all the three QTL/genes and exhibited high level of PHST (PHS score 2-3) associated with significant improvement in GPC and resistance against leaf rust. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01234-z.

Plant microRNAs: biogenesis, gene silencing, web-based analysis tools and their use as molecular markers.

MicroRNAs (miRNAs) are tiny (20-24 nt bp) regulatory non-protein-coding RNA molecules that have been extensively characterized and found important for many physiological and developmental processes. The miss-expression of miRNAs leads to various defects in plants. MicroRNAs repress gene expression by directing mRNA degradation or translational arrest. Several proteins such as PP43A, HYL1, DCL, HST are indispensable role players in promoting miRNA biogenesis in plants. During miRNA biogenesis, lariat RNAs are produced as by-products of pre-mRNA splicing which have a negative role in regulation of miRNA homeostasis. By acting as a decoy and by sequestering to the dicing complex, lariat RNA can prevent the processing of miRNAs. A number of bioinformatic tools with different methodologies are available to identify and validate miRNAs and their targets. Many miRNAs have been reported in different crops for different traits; however, no reports are available on their use in plant breeding. Recently, researchers have developed trait specific miRNA-based molecular markers (miRNA-SSRs/SNP) for many quantitative traits in different plant species. In the future, these molecular markers can be used for plant breeding programs. In this review, a comprehensive up-to-date information is provided on the bioinformatic tools used for analysis of plant miRNAs and their targets, the number of miRNAs, their biogenesis, gene silencing mechanism and miRNA-based molecular markers.

A study of CCD8 genes/proteins in seven monocots and eight dicots.

In plants, the enzyme CCD8 (carotenoid cleavage dioxygenase 8) is involved in the synthesis of an important hormone, strigolactone, and therefore, plays an important role in controlling growth and development. Using cDNA and protein sequence derived from the gene ZmCCD8 from maize, we identified putative orthologs of the gene encoding CCD8 in six other monocots and eight dicots; the sequence similarity ranged from 52-75.9% at the gene level and 60.9-93.7% at the protein level. The average length of the gene was ~3.3 kb (range: 2.08 to 3.98 kb), although the number of introns within the genes differed (4 or 5 in dicots and 3 or 4 in monocots, except in T. urartu with 6 introns). Several cis-acting regulatory elements were identified in the promoters of CCD8 genes, which are known to respond to biotic and abiotic stresses. The N-terminal end (up to ~70 amino acids) of CCD8 proteins was highly variable due to insertions, deletions and mismatches. The variation in genes and proteins were particularly conspicuous in T. urartu and Ae. tauschii among the monocots and A. thaliana and P. persica among the dicots. In CCD8 proteins, 12 motifs were also identified, of which 6 were novel; 4 of these novel motifs occurred in all the 15 species. The 3D structures of proteins had the characteristic features of the related enzyme apocarotenoid oxygenase (ACO) of Synechocystis (a representative of cyanobacteria). The results of qRT-PCR in wheat revealed that under phosphorous (P)-starved condition (relative to expression under optimum P used as control), the expression of TaCCD8 genes increased ~37 fold in root tissue of the cultivar C306 and ~33 fold in shoot tissue of the cultivar HUW468 (the two cultivars differed in their P-use efficiency). This suggested that expression of TaCCD8 genes is genotype-dependent and tissue-specific and is regulated under different levels of P supply.

Genome-wide identification and characterization of gene family for RWP-RK transcription factors in wheat (Triticum aestivum L.).

RWP-RKs represent a small family of transcription factors (TFs) that are unique to plants and function particularly under conditions of nitrogen starvation. These RWP-RKs have been classified in two sub-families, NLPs (NIN-like proteins) and RKDs (RWP-RK domain proteins). NLPs regulate tissue-specific expression of genes involved in nitrogen use efficiency (NUE) and RKDs regulate expression of genes involved in gametogenesis/embryogenesis. During the present study, using in silico approach, 37 wheat RWP-RK genes were identified, which included 18 TaNLPs (2865 to 7340 bp with 4/5 exons), distributed on 15 chromosomes from 5 homoeologous groups (with two genes each on 4B,4D and 5A) and 19 TaRKDs (1064 to 5768 bp with 1 to 6 exons) distributed on 12 chromosomes from 4 homoeologous groups (except groups 1, 4 and 5); 2-3 splice variants were also available in 9 of the 37 genes. Sixteen (16) of these genes also carried 24 SSRs (simple sequence repeats), while 11 genes had targets for 13 different miRNAs. At the protein level, MD simulation analysis suggested their interaction with nitrate-ions. Significant differences were observed in the expression of only two (TaNLP1 and TaNLP2) of the nine representative genes that were used for in silico expression analysis under varying levels of N at post-anthesis stage (data for other genes was not available for in silico expression analysis). Differences in expression were also observed during qRT-PCR, when expression of four representative genes (TaNLP2, TaNLP7, TaRKD6 and TaRKD9) was examined in roots and shoots of seedlings (under different conditions of N supply) in two contrasting genotypes which differed in NUE (C306 with low NUE and HUW468 with high NUE). These four genes for qRT-PCR were selected on the basis of previous literature, level of homology and the level of expression (in silico study). In particular, the TaNLP7 gene showed significant up-regulation in the roots and shoots of HUW468 (with higher NUE) during N-starvation; this gene has already been characterized in Arabidopsis and tobacco, and is known to be involved in nitrate-signal transduction pathway.

Correction: Evaluation of 19,460 Wheat Accessions Conserved in the Indian National Genebank to Identify New Sources of Resistance to Rust and Spot Blotch Diseases.

[This corrects the article DOI: 10.1371/journal.pone.0167702.].

Evaluation of 19,460 Wheat Accessions Conserved in the Indian National Genebank to Identify New Sources of Resistance to Rust and Spot Blotch Diseases.

A comprehensive germplasm evaluation study of wheat accessions conserved in the Indian National Genebank was conducted to identify sources of rust and spot blotch resistance. Genebank accessions comprising three species of wheat-Triticum aestivum, T. durum and T. dicoccum were screened sequentially at multiple disease hotspots, during the 2011-14 crop seasons, carrying only resistant accessions to the next step of evaluation. Wheat accessions which were found to be resistant in the field were then assayed for seedling resistance and profiled using molecular markers. In the primary evaluation, 19,460 accessions were screened at Wellington (Tamil Nadu), a hotspot for wheat rusts. We identified 4925 accessions to be resistant and these were further evaluated at Gurdaspur (Punjab), a hotspot for stripe rust and at Cooch Behar (West Bengal), a hotspot for spot blotch. The second round evaluation identified 498 accessions potentially resistant to multiple rusts and 868 accessions potentially resistant to spot blotch. Evaluation of rust resistant accessions for seedling resistance against seven virulent pathotypes of three rusts under artificial epiphytotic conditions identified 137 accessions potentially resistant to multiple rusts. Molecular analysis to identify different combinations of genetic loci imparting resistance to leaf rust, stem rust, stripe rust and spot blotch using linked molecular markers, identified 45 wheat accessions containing known resistance genes against all three rusts as well as a QTL for spot blotch resistance. The resistant germplasm accessions, particularly against stripe rust, identified in this study can be excellent potential candidates to be employed for breeding resistance into the background of high yielding wheat cultivars through conventional or molecular breeding approaches, and are expected to contribute toward food security at national and global levels.

Safety on elective: a survey on safety advice and adverse events during electives.

The risk of students contracting HIV on electives has received much coverage. Few data exist, however, on risks of other adverse events. Medical schools gave comprehensive advice on infectious disease but little on personal safety. There were no reported parenteral infections or deaths from infection, despite cases of malaria and one needle-stick injury. Accidents were responsible for six deaths and three serious injuries from just nine medical schools. A further student committed suicide after return to the UK. Personal violence and events related to the political situation of the elected country were also reported. This paper is based on a retrospective survey of elective convenors from UK medical schools examining advice given and adverse events. Accidents appear to pose significant risk to medical students on elective and there appears to be a lack of advice on personal safety issues. Further research should examine behaviours that put students at risk of accidents, and strategies that could prevent such events.

Inhibition of phosphotyrosine phosphatase 1B causes resistance in BCR-ABL-positive leukemia cells to the ABL kinase inhibitor STI571.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of BCR-ABL-mediated transformation in vitro and in vivo. To investigate whether PTP1B modulates the biological effects of the abl kinase inhibitor STI571 in BCR-ABL-positive cells, we transfected Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia cell-derived K562 cells with either wild-type PTP1B (K562/PTP1B), a substrate-trapping dominant-negative mutant PTP1B (K562/D181A), or empty vector (K562/mock). Cells were cultured with or without STI571 and analyzed for its effects on proliferation, differentiation, and apoptosis. In both K562/mock and K562/PTP1B cells, 0.25 to 1 mumol/L STI571 induced dose-dependent growth arrest and apoptosis, as measured by a decrease of cell proliferation and an increase of Annexin V-positive cells and/or of cells in the sub-G(1) apoptotic phase. Western blot analysis showed increased protein levels of activated caspase-3 and caspase-8 and induction of poly(ADP-ribose) polymerase cleavage. Low concentrations of STI571 promoted erythroid differentiation of these cells. Conversely, K562/D181A cells displayed significantly lower PTP1B-specific tyrosine phosphatase activity and were significantly less sensitive to STI571-induced growth arrest, apoptosis, and erythroid differentiation. Pharmacologic inhibition of PTP1B activity in wild-type K562 cells, using bis(N,N-dimethylhydroxamido)hydroxooxovanadate, attenuated STI571-induced apoptosis. Lastly, comparison of the STI571-sensitive Ph+ acute lymphoblastic leukemia cell line SupB15 with a STI571-resistant subline revealed significantly decreased PTP1B activity and enhanced BCR-ABL phosphorylation in the STI571-resistant SupB15 cells. In conclusion, functional PTP1B is involved in STI571-induced growth and cell cycle arrest, apoptosis, and differentiation, and attenuation of PTP1B function may contribute to resistance towards STI571.

Differential effects of histone deacetylase inhibitors on interleukin-18 gene expression in myeloid cells.

Histone deacetyrase (HDAC) inhibitors induce growth arrest and differentiation of leukemia cell lines and tumor cells derived from a large variety of human tissues. Here we showed that HDAC inhibitors sodium butyrate, TSA, and valproate regulated the expression of Interleukin-18 (IL-18), a cytokine with antitumor and proinflammatory properties, in human acute myeloid leukemia cell lines U937 and HEL. Sodium butyrate increased expression of IL-18 protein and mRNA and activated 1357bp IL-18 gene promoter construct. IL-18 mRNA level was up-regulated by TSA or valproate, which also activated IL-18 full-length promoter. While sodium butyrate or TSA stimulated the 108-bp IL-18 minimal promoter, valproate failed to activate it, indicating that valproate may use a distinct mechanism from sodium butyrate and TSA to activate IL-18 gene expression.