Phenomic profiling to reveal tolerance mechanisms and regulation of ascorbate-glutathione cycle in wheat varieties (Triticum aestivum L.) under arsenic stress.

The potential of arsenic (As) tolerant and sensitive varieties of wheat (Triticum aestivum L.) has yet to be explored despite of alarming situation of arsenic toxicity. To fill this gap, the study aimed to explore the role of antioxidants, phytochelatins, and ascorbate-glutathione for As tolerance in wheat. A total of eight varieties were exposed to different arsenate treatments (0, 1, 5, 10, 50, 100, 200, 500, 1000, 2000, and 10,000 µM) initially to screen effective treatment as well as contrasting varieties via Weibull distribution frequency for further analysis. The Weibull analysis found 200 µM as the most effective treatment in the present study. Selected varieties were analyzed for accumulation of total As and As speciation, oxidative stress (malondialdehyde, hydrogen peroxide), antioxidants (superoxide dismutase, catalase, peroxidase), phytochelatins, and ascorbate-glutathione cycle (glutathione-S-transferase, glutathione reductase, glutathione peroxidase, ascorbate peroxidase). Tolerant varieties showed less accumulation and translocation of total As, arsenate, and arsenite to the shoots compared with sensitive varieties under 200 µM treatment. Low concentration in tolerant varieties correlated with better growth and development response. Tolerant varieties showed higher induction of metabolites (glutathione, phytochelatins) compared to sensitive ones. Furthermore, tolerant varieties showed better performance of antioxidant and ascorbate-glutathione cycle enzymes in response to As exposure. The findings of the present study provided great insight into the wheat tolerance mechanism upon As exposure between contrasting varieties.

Integrated Application of Salicylic Acid and PGPRs to Control Fusarium Wilt of Chickpea.

BACKGROUND: Fusarium wilt and Ascochyta blight are the most important diseases of chickpea. The current study was designed to investigate the individual and combined effect of salicylic acid (SA) with Pseudomonas stutzeri and Pseudomonas putida to suppress Fusarium wilt and promote growth of chickpea varieties: Thal-2006 and Punjab-2008. METHODS: At the time of sowing, inoculum of Fusarium oxysporum was applied to the soil and the incidence of Fusarium wilt was recorded after 60 days. The seeds were inoculated with Pseudomonas stutzeri and Pseudomonas putida prior to sowing. Chickpea plants were treated with salicylic acid at seedling stage. RESULTS: The combination of P. stutzeri and SA significantly increased root length (166% and 145%), shoot height (50% and 47%) and shoot biomass (300% and 233%) in cv. Thal-2006 and cv. Punjab-2008, respectively, in infected plants. Similarly, the combined treatment of P. putida + SA, also enhanced the plant growth parameters of chickpea varieties. Maximum reduction in disease severity was observed in both P. stutzeri + SA (90% and 84%) and P. putida + SA (79% and 77%) treatments in cv. Thal-2006 and Punjab-2008, respectively. Both P. putida + SA and P. stutzeri + SA treatments resulted in increased leaf relative water and total protein content, peroxidase, superoxide dismutase, phenylalanine ammonia-lyase and polyphenol oxidase activities in both resistant (cv. Thal-2006) and susceptible (cv. Punjab-2008) cultivars. Both treatments also significantly reduced malondialdehyde (MDA) and proline content in cv. Thal-2006 and Punjab-2008. Cultivar Thal-2006 was more effective than cv. Punjab-2008. CONCLUSIONS: The results suggested that, in combination, salicylic acid and P. stutzeri may play an important role in controlling Fusarium wilt diseases by inducing systemic resistance in chickpea.

Genome-Wide Association and Genomic Prediction for Stripe Rust Resistance in Synthetic-Derived Wheats.

Stripe rust caused by Puccnina striiformis (Pst) is an economically important disease attacking wheat all over the world. Identifying and deploying new genes for Pst resistance is an economical and long-term strategy for controlling Pst. A genome-wide association study (GWAS) using single nucleotide polymorphisms (SNPs) and functional haplotypes were used to identify loci associated with stripe rust resistance in synthetic-derived (SYN-DER) wheats in four environments. In total, 92 quantitative trait nucleotides (QTNs) distributed over 65 different loci were associated with resistance to Pst at seedling and adult plant stages. Nine additional loci were discovered by the linkage disequilibrium-based haplotype-GWAS approach. The durable rust-resistant gene Lr34/Yr18 provided resistance in all four environments, and against all the five Pst races used in this study. The analysis identified several SYN-DER accessions that carried major genes: either Yr24/Yr26 or Yr32. New loci were also identified on chr2B, chr5B, and chr7D, and 14 QTNs and three haplotypes identified on the D-genome possibly carry new alleles of the known genes contributed by the Ae. tauschii founders. We also evaluated eleven different models for genomic prediction of Pst resistance, and a prediction accuracy up to 0.85 was achieved for an adult plant resistance, however, genomic prediction for seedling resistance remained very low. A meta-analysis based on a large number of existing GWAS would enhance the identification of new genes and loci for stripe rust resistance in wheat. The genetic framework elucidated here for stripe rust resistance in SYN-DER identified the novel loci for resistance to Pst assembled in adapted genetic backgrounds.

Green Synthesis of Nickel Oxide Nanoparticles from Berberis balochistanica Stem for Investigating Bioactivities.

Green synthesis of nanomaterials is advancing due to its ease of synthesis, inexpensiveness, nontoxicity and renewability. In the present study, an eco-friendly biogenic method was developed for the green synthesis of nickel oxide nanoparticles (NiONPs) using phytochemically rich Berberis balochistanica stem (BBS) extract. The BBS extract was rich in phenolics, flavonoids and berberine. These phytochemicals successfully reduced and stabilised the NiNO3 (green) into NiONPs (greenish-gray). BBS-NiONPs were confirmed by using UV-visible spectroscopy (peak at 305 nm), X-ray diffraction (size of 31.44 nm), Fourier transform infrared spectroscopy (identified -OH group and Ni-O formation), energy dispersive spectroscopy (showed specified elemental nature) and scanning electron microscopy (showed rhombohedral agglomerated shape). BBS-NiONPs were exposed to multiple in vitro bioactivities to ascertain their beneficial biological applications. They exhibited strong antioxidant activities: total antioxidant capacity (64.77%) and 2, 2-diphenyl-1-picrylhydrazyl (71.48%); and cytotoxic potential: Brine shrimp cytotoxicity assay with IC50 (10.40 µg/mL). BBS-NiONPs restricted the bacterial and fungal pathogenic growths at 1000, 500 and 100 µg/mL. Additionally, BBS-NiONPs showed stimulatory efficacy by enhancing seed germination rate and seedling growth at 31.25 and 62.5 µg/mL. In aggregate, BBS extract has a potent antioxidant activity which makes the green biosynthesis of NiONPs easy, economical and safe. The biochemical potential of BBS-NiONPs can be useful in various biomedical and agricultural fields.

Green synthesis of nickel oxide nanoparticles using leaf extract of Berberis balochistanica: Characterization, and diverse biological applications.

In current report, nickel oxide nanoparticles (NiONPs) were synthesized using leaf extract of Berberis balochistanica (BB) an endemic medicinal plant. The BB leaves extract act as a strong reducing, stabilizing, and capping agent in the synthesis of BB@NiONPs. Further, BB@NiONPs were characterized using Uv-visible spectroscopy (UV-vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Energy dispersive spectroscopy (EDS), scanning electron microscopy (SEM), and average size was calculated ~21.7 nm). Multiple in vitro biological activities were performed to determine their therapeutic potentials. The BB@NiONPs showed strong antioxidant activities in term of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and total antioxidant capacity (TAC) with scavenging potential of 69.98 and 59.59% at 200 µg/ml, respectively. The antibacterial and antifungal testes were examined using different bacterial and fungal strains and dose-dependent inhibition response was reported. Laterally, cytotoxic and phytotoxic activities were studied using brine shrimp and radish seeds. The result determined potential cytotoxic activity with LD50 value (49.10 µg/ml) and outstanding stimulatory effect of BB@NiONPs on seed germination at lower concentrations as compared to control. Overall, result concluded that biosynthesis of NiONPs using leaf extracts of Berberis balochistanica is cheap, easy, and safe method and could be used in biomedical and agriculture field as nanomedicine and nano fertilizer.

Metal accumulation potential, human health risks, and yield attributes of hundred bread wheat genotypes on irrigation with municipal and remediated wastewater.

This study was carried out to screen historical diversity panel of bread wheat against municipal wastewater (MW) and remediated wastewater (RW) irrigation to find tolerant and sensitive genotypes and their impact on yield attributes. The experiment was conducted in randomized complete block design (RCBD) with three water treatments, i.e., tap water (TW), RW, and MW. Yield attributes, health risk assessment, water and soil chemistry were recorded. Principal component analysis (PCA) was used to identify tolerant and sensitive genotypes of wheat on the basis of metal accumulation. Metal accumulation in grains increased in pattern K > Fe > Zn in all irrigation treatments. Tolerant genotypes in MW showed lowest hazard quotient (HQ) and hazard index (HI) values (adults 0.62; children 0.67) for Fe and Zn as compared to sensitive genotypes (adults 1.53; children 1.70). However, HI values in sensitive and tolerant genotypes of RW were recorded < 1. Mean values of yield attributes, i.e., plant height, spike length, spikelet per spike, grains per plant, biological yield, grain yield, and thousand kernel weight, were recorded in pattern, i.e., MW > RW > TW. In this study, yield attributes and human health are affected in both cases of higher and lower concentration of Fe and Zn metal. It is suggested that tolerant genotypes can prove useful for cultivation in areas receiving MW and also provide molecular breeding opportunities for seeking tolerance against metal stresses.

Identification of genetic factors controlling phosphorus utilization efficiency in wheat by genome-wide association study with principal component analysis.

Despite the economic importance of P utilization efficiency, information on genetic factors underlying this trait remains elusive. To address that, we performed a genome-wide association study in a spring wheat diversity panel ranging from landraces to elite varieties. We evaluated the phenotype variation for P utilization efficiency in controlled conditions and genotype variation using wheat 90 K SNP array. Phenotype variables were transformed into a smaller set of uncorrelated principal components that captured the most important variation data. We identified two significant loci associated with both P utilization efficiency and the 1st principal component on chromosomes 3A and 4A: qPE1-3A and qPE2-4A. Annotation of genes at these loci revealed 53 wheat genes, among which 6 were identified in significantly enriched pathways. The expression pattern of these 6 genes indicated that TraesCS4A02G481800, involved in pyruvate metabolism and TCA cycle, had a significantly higher expression in the P efficient variety under limited P conditions. Further characterization of these loci and candidate genes can help stimulate P utilization efficiency in wheat.

Heavy metal phyto-accretion, biochemical responses and non-carcinogenic human health risks of genetically diverse wheat genotypes cultivated with sewage of municipal origin.

Current study explored the effects of municipal sewage (MS) irrigation on heavy metal phyto-accretion, biochemical responses and human health risks of diverse wheat genotypes along with recycled municipal sewage (RMS). Mean concentrations of PO43-, NO3--N, chemical oxygen demand, biological oxygen demand, K, Co, Cu, Cd, Cr and Ni were found higher in MS than irrigation criteria. This led to significant increase in heavy metal contents in roots, stem and grains of MS irrigated wheat genotypes compared to RMS and control treatments. No adverse health risk effects for individual or multiple metals were recorded in RMS irrigated wheat genotypes on grounds of lowest heavy metal accumulation. Multivariate techniques i.e. principal component analyses (PCA) and hierarchical agglomerative cluster analyses (HACA) identified tolerant (inefficient metal accumulators) and sensitive (efficient metal accumulators) wheat genotypes in MS and RMS. Tolerant wheat genotypes showed lowest accumulation of heavy metals, efficient biochemical mechanisms to combat oxidative stress and lower health risks to adults/children. Cultivation of identified tolerant wheat genotypes is recommended in areas receiving municipal wastes to reduce human and environmental health risks. Moreover, genetic potential of identified tolerant wheat genotypes from MS and RMS can be utilized in breeding heavy metal tolerant wheat germplasm worldwide.

Genome-wide association study identifies five new cadmium uptake loci in wheat.

Cadmium (Cd) toxicity is a serious threat to future food security and health safety. To identify genetic factors contributing to Cd uptake in wheat, we conducted a genome-wide association study with genotyping from 90K SNP array. A spring wheat diversity panel was planted under normal conditions and Cd stress (50 mg Cd/kg soil). The impact of Cd stress on agronomic traits ranged from a reduction of 16% in plant height to 93% in grain iron content. Individual genotypes showed a considerable variation for Cd uptake and translocation subdividing the panel into three groups: (1) hyper-accumulators (i.e. high Leaf_Cd and low Seed_Cd ), (2) hyper-translocators (i.e. low Leaf_Cd and high Seed_Cd ), and (3) moderate lines (i.e. low Leaf_Cd and low Seed_Cd ). Two lines (SKD-1 and TD-1) maintained an optimum grain yield under Cd stress and were therefore considered as Cd resistant lines. Genome-wide association identified 179 SNP-trait associations for various traits including 16 for Cd uptake at a significance level of P < .001. However, only five SNPs were significant after applying multiple testing correction. These loci were associated with seed-cadmium, grain-iron, and grain-zinc: qSCd-1A, qSCd-1D, qZn-2B1, qZn-2B2, and qFe-6D. These five loci had not been identified in the previously reported studies for Cd uptake in wheat. These loci and the underlying genes should be further investigated using molecular biology techniques to identify Cd resistant genes in wheat.

Genome-Wide Association Study and QTL Meta-Analysis Identified Novel Genomic Loci Controlling Potassium Use Efficiency and Agronomic Traits in Bread Wheat.

Potassium use efficiency, a complex trait, directly impacts the yield potential of crop plants. Low potassium efficiency leads to a high use of fertilizers, which is not only farmer unfriendly but also deteriorates the environment. Genome-wide association studies (GWAS) are widely used to dissect complex traits. However, most studies use single-locus one-dimensional GWAS models which do not provide true information about complex traits that are controlled by multiple loci. Here, both single-locus GWAS (MLM) and multi-locus GWAS (pLARmEB, FASTmrMLM, mrMLM, FASTmrEMMA) models were used with genotyping from 90 K Infinium SNP array and phenotype derived from four normal and potassium-stress environments, which identified 534 significant marker-trait associations (MTA) for agronomic and potassium related traits: pLARmEB = 279, FASTmrMLM = 213, mrMLM = 35, MLM = 6, FASTmrEMMA = 1. Further screening of these MTA led to the detection of eleven stable loci: q1A, q1D, q2B-1, q2B-2, q2D, q4D, q5B-1, q5B-2, q5B-3, q6D, and q7A. Moreover, Meta-QTL (MQTL) analysis of four independent QTL studies for potassium deficiency in bread wheat located 16 MQTL on 13 chromosomes. One locus identified in this study (q5B-1) colocalized with an MQTL (MQTL_11 ), while the other ten loci were novel associations. Gene ontology of these loci identified 20 putative candidate genes encoding functional proteins involved in key pathways related to stress tolerance, sugar metabolism, and nutrient transport. These findings provide potential targets for breeding potassium stress resistant wheat cultivars and advocate the advantages of multi-locus GWAS models for studying complex traits.

Resistance associated metabolite profiling of Aspergillus leaf spot in cotton through non-targeted metabolomics.

Aspergillus tubingensis is an important pathogen of economically important crops. Different biotic stresses strongly influence the balance of metabolites in plants. The aim of this study was to understand the function and response of resistance associated metabolites which, in turn are involved in many secondary metabolomics pathways to influence defense mechanism of cotton plant. Analysis of non-targeted metabolomics using ultra high performance liquid chromatography-mass spectrometry (UPLC-MS) revealed abundant accumulation of key metabolites including flavonoids, phenylpropanoids, terpenoids, fatty acids and carbohydrates, in response to leaf spot of cotton. The principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and partial least squares discriminant analysis (PLS-DA) score plots illustrated the evidences of variation between two varieties of cotton under mock and pathogen inoculated treatments. Primary metabolism was affected by the up regulation of pyruvate and malate and by the accumulation of carbohydrates like cellobiose and inulobiose. Among 241 resistance related (RR) metabolites, 18 were identified as resistance related constitutive (RRC) and 223 as resistance related induced (RRI) metabolites. Several RRI metabolites, identified in the present study were the precursors for many secondary metabolic pathways. These included phenylpropanoids (stilbenes and furanocoumarin), flavonoids (phlorizin and kaempferol), alkaloids (indolizine and acetylcorynoline) and terpenoids (azelaic acid and oleanolic acid). Our results demonstrated that secondary metabolism, primary metabolism and energy metabolism were more active in resistant cultivar, as compared to sensitive cultivar. Differential protein and fatty acid metabolism was also depicted in both cultivars. Accumulation of these defense related metabolites in resistant cotton cultivar and their suppression in susceptible cotton cultivar revealed the reason of their respective tolerance and susceptibility against A. tubingensis.

Treatment efficiency of a hybrid constructed wetland system for municipal wastewater and its suitability for crop irrigation.

Design and implementation of wastewater treatment is inevitable due to toxic effects of wastewater irrigation on crops, soil and human health. Current investigation is the pioneer attempt on full-scale hybrid constructed wetland system (HCWS) built for municipal wastewater treatment from Pakistan. HCWS was comprised of vertical sub-surface flow constructed wetland (VSSF-CW) and five phyto-treatment ponds connected in series. Higher environmental risk was associated with untreated municipal wastewater usage in irrigation as estimated through discharge of metals to recipient soils. Treatment efficiency percentages recorded for HCWS reclaimed water quality parameters were, i.e., EC (56.68), TDS (56.86), alkalinity (39.67), chloride (39.68), sulfate (46.73), Na (28.80), Mn (65.24), Cr (78.07), Ni (81.02), BOD (68.74), total hardness (19.56), Fe (70.09), phosphate (55.40), Pb (80.48), COD (63.64), Mg (17.24), K (60.05), Co (100), Cu (67.73), Zn (59.97), Cd (100), and Ca (21.47) respectively. Wastewater treatment in HCWS was due to aquatic plants [Phragmites australis Cav. Trin. ex Steud., Canna indica L. Typha latifolia L., and Hydrocotyle umbellata L.], microbial activities and substrate based wetland processes. The HCWS treated water was well under irrigation standards and recommended for safer crop production in water scarce regions.

Deciphering adverse effects of heavy metals on diverse wheat germplasm on irrigation with urban wastewater of mixed municipal-industrial origin.

The current study provides one of the first attempts to identify tolerant, moderately sensitive, and highly sensitive wheat genotypes on the basis of heavy metal accumulation, biochemical attributes, and human health risk assessments on urban wastewater (UW) irrigation. Mean heavy metals (Fe, Co, Ni, Cu, Zn, Pb, Cd, Cr, Mn) and macro-nutrients (Na, K, Ca, Mg) levels increased in the roots, stem, and grains of studied genotypes. Except K (stem > root > grain), all metals were accumulated in highest concentrations in roots followed by stem and grains. Principal component analyses (PCA) identified three groups of UW-irrigated genotypes which were confirmed by hierarchical agglomerative cluster analyses (HACA). Wheat genotypes with the lowest metal accumulation were regarded as tolerant, whereas those with maximum accumulation were considered highly sensitive. Tolerant genotypes showed the lowest hazard quotient for heavy metals, i.e., Co, Mn, Cd, Cu, Fe, Pb, and Cr, and hazard index (HI) values (adults, 2.04; children, 2.27) than moderately and highly sensitive genotypes. Higher health risks (HI) associated with moderate (adults 2.26; children 2.53) and highly sensitive (adults 2.52; children 2.82) genotypes revealed maximum uptake of heavy metals. The heatmap showed higher mean biochemical levels of chlorophyll, carotenoids, membrane stability index (MSI%), sugars, proteins, proline, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in tolerant genotypes than remaining genotypes. With the lowest metal accumulation and advanced biochemical mechanisms to cope with the adverse effects of heavy metals in their plant bodies, tolerant genotypes present a better option for cultivation in areas receiving UW or similar type of wastewater.

Wheat Fermentation With Enterococcus mundtii QAUSD01 and Wickerhamomyces anomalus QAUWA03 Consortia Induces Concurrent Gliadin and Phytic Acid Degradation and Inhibits Gliadin Toxicity in Caco-2 Monolayers.

Foods containing high amounts of either phytic acid or gliadin can pose a risk for development of iron deficiency and celiac disease, respectively. The present study was conducted to evaluate the effects of preselected gliadin degrading strains, Enterococcus mundtii QAUSD01 and Wickerhamomyces anomalus QAUWA03, on phytic acid and gliadin degradation in six wheat cultivars (Lasani 2008, Seher 2006, Chakwal 97, Shafaq 2006, Bars 2009, Barani 83). Tight junction proteins, trans-epithelial resistance (TER) and ruffle formation in Caco-2 cells were evaluated relative to Saccharomyces cerevisiae-mediated fermented and unfermented controls. Phytic acid degradation was demonstrated in all six cultivars fermented with E. mundtii QAUSD01 and W. anomalus QAUWA03 consortia. Among the six fermented cultivars, Shafaq 2006 showed relatively higher degradation of gliadin. In comparison to the other tested wheat varieties, fermentation of Lasani 2006 was associated with minimal toxic effects on Caco-2 cells in terms of ruffle formation, tight junction proteins and TER, which can be attributed to extensive degradation of toxic gliadin fragments.

Wheat syntenome unveils new evidences of contrasted evolutionary plasticity between paleo- and neoduplicated subgenomes.

Bread wheat derives from a grass ancestor structured in seven protochromosomes followed by a paleotetraploidization to reach a 12 chromosomes intermediate and a neohexaploidization (involving subgenomes A, B and D) event that finally shaped the 21 modern chromosomes. Insights into wheat syntenome in sequencing conserved orthologous set (COS) genes unravelled differences in genomic structure (such as gene conservation and diversity) and genetical landscape (such as recombination pattern) between ancestral as well as recent duplicated blocks. Contrasted evolutionary plasticity is observed where the B subgenome appears more sensitive (i.e. plastic) in contrast to A as dominant (i.e. stable) in response to the neotetraploidization and D subgenome as supra-dominant (i.e. pivotal) in response to the neohexaploidization event. Finally, the wheat syntenome, delivered through a public web interface PlantSyntenyViewer at http://urgi.versailles.inra.fr/synteny-wheat, can be considered as a guide for accelerated dissection of major agronomical traits in wheat.

Cross-genome map based dissection of a nitrogen use efficiency ortho-metaQTL in bread wheat unravels concerted cereal genome evolution.

Monitoring nitrogen use efficiency (NUE) in plants is becoming essential to maintain yield while reducing fertilizer usage. Optimized NUE application in major crops is essential for long-term sustainability of agriculture production. Here, we report the precise identification of 11 major chromosomal regions controlling NUE in wheat that co-localise with key developmental genes such as Ppd (photoperiod sensitivity), Vrn (vernalization requirement), Rht (reduced height) and can be considered as robust markers from a molecular breeding perspective. Physical mapping, sequencing, annotation and candidate gene validation of an NUE metaQTL on wheat chromosome 3B allowed us to propose that a glutamate synthase (GoGAT) gene that is conserved structurally and functionally at orthologous positions in rice, sorghum and maize genomes may contribute to NUE in wheat and other cereals. We propose an evolutionary model for the NUE locus in cereals from a common ancestral region, involving species specific shuffling events such as gene deletion, inversion, transposition and the invasion of repetitive elements.

Combined meta-genomics analyses unravel candidate genes for the grain dietary fiber content in bread wheat (Triticum aestivum L.).

Grain dietary fiber content in wheat not only affects its end use and technological properties including milling, baking and animal feed but is also of great importance for health benefits. In this study, integration of association genetics (seven detected loci on chromosomes 1B, 3A, 3D, 5B, 6B, 7A, 7B) and meta-QTL (three consensus QTL on chromosomes 1B, 3D and 6B) analyses allowed the identification of seven chromosomal regions underlying grain dietary fiber content in bread wheat. Based either on a diversity panel or on bi-parental populations, we clearly demonstrate that this trait is mainly driven by a major locus located on chromosome 1B associated with a log of p value >13 and a LOD score >8, respectively. In parallel, we identified 73 genes differentially expressed during the grain development and between genotypes with contrasting grain fiber contents. Integration of quantitative genetics and transcriptomic data allowed us to propose a short list of candidate genes that are conserved in the rice, sorghum and Brachypodium chromosome regions orthologous to the seven wheat grain fiber content QTL and that can be considered as major candidate genes for future improvement of the grain dietary fiber content in bread wheat breeding programs.

Improved criteria and comparative genomics tool provide new insights into grass paleogenomics.

In the past decade, a number of bioinformatics tools have been developed to perform comparative genomics studies in plants and animals. However, most of the publicly available and user friendly tools lack common standards for the identification of robust orthologous relationships between genomes leading non-specialists to often over interpret the results of large scale comparative sequence analyses. Recently, we have established a number of improved parameters and tools to define significant relationships between genomes as a basis to develop paleogenomics studies in grasses. Here, we describe our approaches and propose the development of community-based standards that can be used in comparative genomic studies to (i) identify robust sets of orthologous gene pairs, (ii) derive complete sets of chromosome to chromosome relationships within and between genomes and (iii) model common paleo-ancestor genome structures. The rice and sorghum genome sequences are used to exemplify step-by-step a methodology that should allow users to perform accurate comparative genome analyses in their favourite species. Finally, we describe two applications for accurate gene annotation and synteny-based cloning of agronomically important traits.

Genomics in cereals: from genome-wide conserved orthologous set (COS) sequences to candidate genes for trait dissection.

Recent updates in comparative genomics among cereals have provided the opportunity to identify conserved orthologous set (COS) DNA sequences for cross-genome map-based cloning of candidate genes underpinning quantitative traits. New tools are described that are applicable to any cereal genome of interest, namely, alignment criterion for orthologous couples identification, as well as the Intron Spanning Marker software to automatically select intron-spanning primer pairs. In order to test the software, it was applied to the bread wheat genome, and 695 COS markers were assigned to 1,535 wheat loci (on average one marker/2.6 cM) based on 827 robust rice-wheat orthologs. Furthermore, 31 of the 695 COS markers were selected to fine map a pentosan viscosity quantitative trait loci (QTL) on wheat chromosome 7A. Among the 31 COS markers, 14 (45%) were polymorphic between the parental lines and 12 were mapped within the QTL confidence interval with one marker every 0.6 cM defining candidate genes among the rice orthologous region.

Structure and expression analysis of rice paleo duplications.

Having a well-known history of genome duplication, rice is a good model for studying structural and functional evolution of paleo duplications. Improved sequence alignment criteria were used to characterize 10 major chromosome-to-chromosome duplication relationships associated with 1440 paralogous pairs, covering 47.8% of the rice genome, with 12.6% of genes that are conserved within sister blocks. Using a micro-array experiment, a genome-wide expression map has been produced, in which 2382 genes show significant differences of expression in root, leaf and grain. By integrating both structural (1440 paralogous pairs) and functional information (2382 differentially expressed genes), we identified 115 paralogous gene pairs for which at least one copy is differentially expressed in one of the three tissues. A vast majority of the 115 paralogous gene pairs have been neofunctionalized or subfunctionalized as 88%, 89% and 96% of duplicates, respectively, expressed in grain, leaf and root show distinct expression patterns. On the basis of a Gene Ontology analysis, we have identified and characterized the gene families that have been structurally and functionally preferentially retained in the duplication showing that the vast majority (>85%) of duplicated have been either lost or have been subfunctionalized or neofunctionalized during 50-70 million years of evolution.