Einkorn genomics sheds light on history of the oldest domesticated wheat.

Einkorn (Triticum monococcum) was the first domesticated wheat species, and was central to the birth of agriculture and the Neolithic Revolution in the Fertile Crescent around 10,000 years ago1,2. Here we generate and analyse 5.2-Gb genome assemblies for wild and domesticated einkorn, including completely assembled centromeres. Einkorn centromeres are highly dynamic, showing evidence of ancient and recent centromere shifts caused by structural rearrangements. Whole-genome sequencing analysis of a diversity panel uncovered the population structure and evolutionary history of einkorn, revealing complex patterns of hybridizations and introgressions after the dispersal of domesticated einkorn from the Fertile Crescent. We also show that around 1% of the modern bread wheat (Triticum aestivum) A subgenome originates from einkorn. These resources and findings highlight the history of einkorn evolution and provide a basis to accelerate the genomics-assisted improvement of einkorn and bread wheat.

Precision mapping and expression analysis of recessive bacterial blight resistance gene xa-45(t) from Oryza glaberrima.

BACKGROUND: Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice leading to huge yield losses in Southeast Asia. The recessive resistance gene xa-45(t) from Oryza glaberrima IRGC102600B, mapped on rice chromosome 8, spans 80 Kb with 9 candidate genes on Nipponbare reference genome IRGSP-1.0. The xa-45(t) gene provides durable resistance against all the ten Xanthomonas pathotypes of Northern India, thus aiding in the expansion of recessive bacterial blight resistance gene pool. Punjab Rice PR127, carrying xa-45(t), was released for wider use in breeding programs. This study aims to precisely locate the target gene among the 9 candidates conferring resistance to bacterial blight disease. METHODS AND RESULTS: Sanger sequencing of all nine candidate genes revealed seven SNPs and an Indel between the susceptible parent Pusa 44 and the resistant introgression line IL274. The genotyping with polymorphic markers identified three recombinant breakpoints for LOC_Os08g42370, and LOC_Os08g42400, 15 recombinants for LOC_Os08g423420 and 26 for LOC_Os08g42440 out of 190 individuals. Relative expression analysis across six time intervals (0, 8, 24, 48, 72, and 96 h) after bacterial blight infection showed over expression of LOC_Os08g42410-specific transcripts in IL274 compared to Pusa 44, with a significant 4.46-fold increase observed at 72 h post-inoculation. CONCLUSIONS: The Indel marker at the locus LOC_Os08g42410 was found co-segregating with the phenotype, suggesting its candidacy towards xa-45(t). The transcript abundance assay provides strong evidence for the involvement of LOC_Os08g42410 in the resistance conferred by the bacterial blight gene xa-45(t).

Wheat Pore-Forming Toxin-Like Protein Confers Broad-Spectrum Resistance to Fungal Pathogens in Arabidopsis.

Fusarium head blight (FHB), caused by the hemibiotrophic fungus Fusarium graminearum, is one of the major threats to global wheat productivity. A wheat pore-forming toxin-like (PFT) protein was previously reported to underlie Fhb1, the most widely used quantitative trait locus in FHB breeding programs worldwide. In the present work, wheat PFT was ectopically expressed in the model dicot plant Arabidopsis. Heterologous expression of wheat PFT in Arabidopsis provided a broad-spectrum quantitative resistance to fungal pathogens including F. graminearum, Colletotrichum higginsianum, Sclerotinia sclerotiorum, and Botrytis cinerea. However, there was no resistance to bacterial or oomycete pathogens Pseudomonas syringae and Phytophthora capsici, respectively in the transgenic Arabidopsis plants. To explore the reason for the resistance response to, exclusively, the fungal pathogens, purified PFT protein was hybridized to a glycan microarray having 300 different types of carbohydrate monomers and oligomers. It was found that PFT specifically hybridized with chitin monomer, N-acetyl glucosamine (GlcNAc), which is present in fungal cell walls but not in bacteria or oomycete species. This exclusive recognition of chitin may be responsible for the specificity of PFT-mediated resistance to fungal pathogens. Transfer of the atypical quantitative resistance of wheat PFT to a dicot system highlights its potential utility in designing broad-spectrum resistance in diverse host plants. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification and high-resolution mapping of a novel tiller number gene (tin6) by combining forward genetics screen and MutMap approach in bread wheat.

Wheat (Triticum aestivum) is one of the most important food crops worldwide, providing up to 20% of the caloric intake per day. Developing high-yielding wheat cultivars with tolerance against abiotic and biotic stresses is important to keep up with the increasing human population. Tiller number is one of the major yield-related traits, directly affecting the number of grains produced per plant; however, only a small number of QTL and underlining genes have been identified for this important factor. Identification of novel genetic variation underlying contrasting traits and their precise genetic mapping in wheat is considered difficult due to the complexity and size of the genome; however, advancements in genomic resources have made efficient gene localization more possible. In this study, we report the characterization of a novel tillering number gene using a mutant identified in the forward genetic screen of an ethyl methane sulfonate (EMS)-treated population of cv. "Jagger." By crossing the low tillering mutant with the Jagger wild-type plant, we generated an F2 population and used the MutMap approach to identify a novel physical interval on 11 Mb on chromosome 2DS. Using an F2 population of 442 gametes and polymorphic SNP markers, we were able to delineate the tin6 locus to a 2.1 Mb region containing 22 candidate genes.

Physical mapping of the wheat genes in low-recombination regions: radiation hybrid mapping of the C-locus.

KEY MESSAGE: This work reports the physical mapping of an important gene affecting spike compactness located in a low-recombination region of hexaploid wheat. This work paves the way for the eventual isolation and characterization of the factor involved but also opens up possibilities to use this approach to precisely map other wheat genes located on proximal parts of wheat chromosomes that show highly reduced recombination. Mapping wheat genes, in the centromeric and pericentromeric regions (~ 2/3rd of a given chromosome), poses a formidable challenge due to highly suppressed recombination. Using an example of compact spike locus (C-locus), this study provides an approach to precisely map wheat genes in the pericentromeric and centromeric regions that house ~ 30% of wheat genes. In club-wheat, spike compactness is controlled by the dominant C-locus, but previous efforts have failed to localize it, on a particular arm of chromosome 2D. We integrated radiation hybrid (RH) and high-resolution genetic mapping to locate C-locus on the short arm of chromosome 2D. Flanking markers of the C-locus span a physical distance of 11.0 Mb (231.0-242 Mb interval) and contain only 11 high-confidence annotated genes. This work demonstrates the value of this integrated strategy in mapping dominant genes in the low-recombination regions of the wheat genome. A comparison of the mapping resolutions of the RH and genetic maps using common anchored markers indicated that the RH map provides ~ 9 times better resolution that the genetic map even with much smaller population size. This study provides a broadly applicable approach to fine map wheat genes in regions of suppressed recombination.

Micro-characteristics of a naturally ventilated classroom air quality under varying air purifier placements.

A naturally-ventilated operational classroom was instrumented at 18 locations to assess spatial variations of classroom air pollution (CRAP), thermal comfort and ventilation indicators under 10 different scenarios (base scenario without air purifier (AP); three single AP scenarios; three scenarios with two APs at same locations; three scenarios with two APs at different locations). Unlike PM2.5, monitored PM10 and CO2 concentrations followed the diurnal occupancy profile. Highest vertical variation (38%) in CO2 was at the classroom entry zone at 40-300 cm height. CO2 increased until 225 cm before stratifying further. PM10 increased to highest levels at children sitting height (100 cm) before decreasing to adult breathing height (150 cm). Highest horizontal variations in CO2 (PM10) were 29% (22%) at 40 cm height between the entry and occupied zones. Teachers' exposure to CO2 (PM10) in breathing zone varied by up to 6% (3%); the corresponding variations across monitored locations were up to 14% (19%). Teachers' exposure to CO2 was up to 13% higher than that of children and 18% lower for PM10. Traffic emissions (PM2.5 and NOx), secondary pollutants (VOCs and O3), thermal comfort parameters and noise level in the classroom varied insignificantly among scenarios. PM10 reduction was not doubled by using two air purifiers, which were most effective when placed within the highest PM concentration zone. Cross-comparisons of scenarios showed: use of AP reduced classroom's spatial average PM10 up to 14%; PM10 was reduced by increasing the AP's filtration capacity; and AP had insignificant impact on spatial average CO2. PM10 showed a maximum reduction of 46% (teacher zone), 62% (occupied zone) and 50% (entry zone) at children's breathing height, depending on usage scenario. This study produced high-resolution data for validating the detailed numerical models for classrooms and informing decision-making on AP's placement to minimise children's exposure to CRAP and re-breathed CO2.

Menstrual changes after spinal cord injury.

STUDY DESIGN: This was a single-centre, prospective, descriptive, hospital-based study in females with spinal cord injuries (SCI). OBJECTIVES: To study menstrual changes after SCI. SETTING: The in-patient and out-patient services of the Department of Physical Medicine and Rehabilitation of a tertiary care institute in India between October 2018 and October 2020. METHODS: SCI females who were included in the study answered a questionnaire regarding amenorrhea after injury, menstrual cycle regularity, frequency, duration, flow, dysmenorrhoea and presence of autonomic dysreflexia during menstruation. All the study related data was analysed using SPSS version 24. A p value < 0.05 was considered as statistically significant. RESULTS: 40 females were included. 31 (77.5%) had amenorrhea. The mean duration of return of menstruation was 2.65 months. There was significant reduction in the duration of menstrual flow (p value < 0.001), amount of flow (p value = 0.041) and dysmenorrhea (p value < 0.001) after SCI. CONCLUSIONS: Amenorrhea was seen in 77.5% females. Most of them resumed their menstrual cycle. The menstruation duration and flow were reduced significantly. There is a need to address concerns and reassure females regarding resumption of menstruation after SCI.

A chromosome-scale reference genome of trifoliate orange (Poncirus trifoliata) provides insights into disease resistance, cold tolerance and genome evolution in Citrus.

Trifoliate orange (Poncirus trifoliata), a deciduous close relative of evergreen Citrus, has important traits for citrus production, including tolerance/resistance to citrus greening disease (Huanglongbing, HLB) and other major diseases, and cold tolerance. It has been one of the most important rootstocks, and one of the most valuable sources of resistance and tolerance genes for citrus. Here we present a high-quality, chromosome-scale genome assembly of P. trifoliata. The 264.9-Mb assembly contains nine chromosomal pseudomolecules with 25 538 protein-coding genes, covering 97.2% of the estimated gene space. Comparative analyses of P. trifoliata and nine Citrus genomes revealed 605 species-specific genes and six rapidly evolving gene families in the P. trifoliata genome. Poncirus trifoliata has evolved specific adaptation in the C-repeat/DREB binding factor (CBF)-dependent and CBF-independent cold signaling pathways to tolerate cold. We identified candidate genes within quantitative trait loci for HLB tolerance, and at the loci for resistance to citrus tristeza virus and citrus nematode. Genetic diversity analysis of Poncirus accessions and Poncirus/Citrus hybrids shows a narrow genetic base in the US germplasm collection, and points to the importance of collecting and preserving more natural genetic variation. Two phenotypically divergent Poncirus accessions are found to be clonally related, supporting a previous conjecture that dwarf Flying Dragon originated as a mutant of a non-dwarfing type. The high-quality genome reveals features and evolutionary insights of Poncirus, and it will serve as a valuable resource for genetic, genomic and molecular research and manipulation in citrus.

Triple null mutations in starch synthase SSIIa gene homoeologs lead to high amylose and resistant starch in hexaploid wheat.

BACKGROUND: Lack of nutritionally appropriate foods is one of the leading causes of obesity in the US and worldwide. Wheat (Triticum aestivum) provides 20% of the calories consumed daily across the globe. The nutrients in the wheat grain come primarily from the starch composed of amylose and amylopectin. Resistant starch content, which is known to have significant human health benefits, can be increased by modifying starch synthesis pathways. Starch synthase enzyme SSIIa, also known as starch granule protein isoform-1 (SGP-1), is integral to the biosynthesis of the branched and readily digestible glucose polymer amylopectin. The goal of this work was to develop a triple null mutant genotype for SSIIa locus in the elite hard red winter wheat variety 'Jagger' and evaluate the effect of the knock-out mutations on resistant starch content in grains with respect to wild type. RESULTS: Knock-out mutations in SSIIa in the three genomes of wheat variety 'Jagger' were identified using TILLING. Subsequently, these loss-of function mutations on A, B, and D genomes were combined by crossing to generate a triple knockout mutant genotype Jag-ssiia-∆ABD. The Jag-ssiia-∆ABD had an amylose content of 35.70% compared to 31.15% in Jagger, leading to ~ 118% increase in resistant starch in the Jag-ssiia-∆ABD genotype of Jagger wheat. The single individual genome mutations also had various effects on starch composition. CONCLUSIONS: Our full null Jag-ssiia-∆ABD mutant showed a significant increase in RS without the shriveled grain phenotype seen in other ssiia knockouts in elite wheat cultivars. Moreover, this study shows the potential for developing nutritionally improved foods in a non-GM approach. Since all the mutants have been developed in an elite wheat cultivar, their adoption in production and supply will be feasible in future.

Discovery of a susceptibility factor for Fusarium head blight on chromosome 7A of wheat.

KEY MESSAGE: Discovery and mapping of a susceptibility factor located on the short arm of wheat chromosome 7A whose deletion makes plants resistant to Fusarium head blight. Fusarium head blight (FHB) disease of wheat caused by Fusarium spp. deteriorates both quantity and quality of the crop. Manipulation of susceptibility factors, the plant genes facilitating disease development, offers a novel and alternative strategy for enhancing FHB resistance in plants. In this study, a major effect susceptibility gene for FHB was identified on the short arm of chromosome 7A (7AS). Nullisomic-tetrasomic lines for homoeologous group-7 of wheat revealed dosage effect of the gene, with tetrasomic 7A being more susceptible than control Chinese Spring wheat, qualifying it as a genuine susceptibility factor. Five chromosome 7A inter-varietal substitution lines and a tetraploid Triticum dicoccoides 7A substitution line showed similar susceptibility as that of Chinese Spring, indicating toward the commonality of the susceptibility factor among these diverse genotypes. The susceptibility factor was named as Sf-Fhb-7AS and mapped on chromosome 7AS to a 48.5-50.5 Mb peri-centromeric region between del7AS-3 and del7AS-8. Our results showed that deletion of Sf-Fhb-7AS imparts 50-60% type 2 FHB resistance and its manipulation can be used to enhance resistance against FHB in wheat.

An approach for high-resolution genetic mapping of distant wild relatives of bread wheat: example of fine mapping of Lr57 and Yr40 genes.

KEY MESSAGE: The article reports a powerful but simple approach for high-resolution mapping and eventual map-based cloning of agronomically important genes from distant relatives of wheat, using the already existing germplasm resources. Wild relatives of wheat are a rich reservoir of genetic diversity for its improvement. The effective utilization of distant wild relatives in isolation of agronomically important genes is hindered by the lack of recombination between the homoeologous chromosomes. In this study, we propose a simple yet powerful approach that can be applied for high-resolution mapping of a targeted gene from wheat's distant gene pool members. A wheat-Aegilops geniculata translocation line TA5602 with a small terminal segment from chromosome 5 Mg of Ae. geniculata translocated to 5D of wheat contains genes Lr57 and Yr40 for leaf rust and stripe rust resistance, respectively. To map these genes, TA5602 was crossed with a susceptible Ae. geniculata 5 Mg addition line. Chromosome pairing between the 5 Mg chromosomes of susceptible and resistant parents resulted in the development of a high-resolution mapping panel for the targeted genes. Next-generation-sequencing data from flow-sorted 5 Mg chromosome of Ae. geniculata allowed us to generate 5 Mg-specific markers. These markers were used to delineate Lr57 and Yr40 genes each to distinct ~ 1.5 Mb physical intervals flanked by gene markers on 5 Mg. The method presented here will allow researchers worldwide to utilize existing germplasm resources in genebanks and seed repositories toward routinely performing map-based cloning of important genes from tertiary gene pools of wheat.

High-resolution mapping of the Mov-1 locus in wheat by combining radiation hybrid (RH) and recombination-based mapping approaches.

KEY MESSAGE: This work reports a quick method that integrates RH mapping and genetic mapping to map the dominant Mov-1 locus to a 1.1-Mb physical interval with a small number of candidate genes. Bread wheat is an important crop for global human population. Identification of genes and alleles controlling agronomic traits is essential toward sustainably increasing crop production. The unique multi-ovary (MOV) trait in wheat holds potential for improving yields and is characterized by the formation of 2-3 grains per spikelet. The genetic basis of the multi-ovary trait is known to be monogenic and dominant in nature. Its precise mapping and functional characterization is critical to utilizing this trait in a feasible manner. Previous mapping efforts of the locus controlling multiple ovary/pistil formation in the hexaploid wheat have failed to produce a consensus for a particular chromosome. We describe a mapping strategy integrating radiation hybrid mapping and high-resolution genetic mapping to locate the chromosomal position of the Mov-1 locus in hexaploid wheat. We used RH mapping approach using a panel of 188 lines to map the Mov-1 locus in the terminal part of long arm of wheat chromosome 2D with a map resolution of 1.67 Mb/cR1500. Then using a genetic population of MOV × Synthetic wheat of F2 lines, we delineated the Mov-1 locus to a 1.1-Mb physical region with a small number of candidate genes. This demonstrates the value of this integrated strategy to mapping dominant genes in wheat.

Cryobanking of primary somatic cells of elite farm animals - A pilot study in domesticated water buffalo (Bubalus bubalis).

Buffalo is an important farm animal species in South and South-east Asian countries. Cryopreservation allows long-term storage of somatic cells, which can be made available to research communities. This study aimed to 1) establish and cryopreserve somatic cells from elite buffaloes, and 2) share stored somatic cells and their associated data with researchers. To achieve these targets, somatic cells were established successfully from tail-skin biopsies of 17 buffaloes. The informative data such as buffalo details (breed, date of birth, sex, and age at the time of tissue biopsy collection, and production traits), the number of cryovials stored, and freezing dates were recorded in an electronic file and a printed inventory record. The established somatic cells were flat, spindle-shaped morphology, and expressed vimentin (a fibroblast-like cell type marker) and the negative expression of cytokeratin-18 (an epithelial cell type marker). Altogether, we cryopreserved 970 cryovials (0.1 million cells per vial) from two buffalo breeds, namely Murrah and Nili-Ravi (at least 45 cryovials per animal), for cryobanking. Somatic cell nuclear transfer (SCNT) experiments demonstrated the utility of cryopreserved cells to produce cloned buffaloes. Importantly, these cryopreserved somatic cells are made available to scientific communities. This study encourages the cryopreservation of somatic cells of elite farm animals for their utilization in cell-based research.

Media switching at different time periods affects the reprogramming efficiency of buffalo fetal fibroblasts.

Many contrasting reports are available on generation of bovine induced pluripotent stem cells (iPSCs) employing different timelines and culture conditions which signifies reprogramming process varies between species and cell types. The present study determines an optimum time period required to re-initiate reprogramming events in buffalo fibroblasts after introduction of exogenous genes (OCT4, SOX2, KLF4 and c-MYC) by lentiviral vector. The reprogramming efficiency is cumulative result of many factors including culture conditions and addition of growth factors in culture media. In our study, we observed when stem cell culture conditions were provided Day 5 post-transduction, it results in maximum reprogramming efficiency in comparison when same conditions were provided too early or on later days. The putative iPSCs were expanded on feeder layer for 15 passages and found positive for alkaline phosphatase and pluripotency markers (OCT4, SOX2, KLF4, c-MYC, UTF, TELOMERASE, FOXD3, REX1, STAT3, NUCLEOSTAMIN and TRA1-81). Also, they produced embryoid bodies showing expression for ectodermal (NF68, MOBP), mesodermal (ASA, BMP4) and endodermal (GATA4, AFP) markers to confirm their pluripotent nature. Our results suggest that reprogramming is accompanied by time dependent events and providing stem cell culture conditions at definite time during reprogramming can help in generation of iPSCs with greater efficiency.

Screening of an Ethyl Methane Sulfonate Mutagenized Population of a Wheat Cultivar Susceptible to Fusarium Head Blight Identifies Resistant Variants.

Fusarium head blight (FHB) primarily caused by Fusarium graminearum is a key disease of small grains. Diseased spikes show symptoms of premature bleaching shortly after infection and have aborted or shriveled seeds, resulting in reduced yields. The fungus also deteriorates quality and safety of the grain because of production of mycotoxins, especially deoxynivalenol (DON), which can result in grain being docked or rejected at the point of sale. Genetic host resistance to FHB is quantitative, and no complete genetic resistance against this devastating disease is available. Alternative approaches to develop new sources of FHB resistance are needed. In this study, we performed extensive forward genetic screening of the M4 generation of an ethyl methane sulfonate-induced mutagenized population of cultivar Jagger to isolate variants with FHB resistance. In field testing, 74 mutant lines were found to have resistance against FHB spread, and 30 of these lines also had low DON content. Subsequent testing over 2 years in controlled greenhouse conditions revealed 10 M6 lines showing significantly lower FHB spread. Seven and 6 of those 10 lines also had reduced DON content and fewer Fusarium-damaged kernels, respectively. Future endeavors will include identification of the mutations that led to resistance in these variants.

GSP: a web-based platform for designing genome-specific primers in polyploids.

MOTIVATION: The sequences among subgenomes in a polyploid species have high similarity, making it difficult to design genome-specific primers for sequence analysis. RESULTS: We present GSP, a web-based platform to design genome-specific primers that distinguish subgenome sequences in a polyploid genome. GSP uses BLAST to extract homeologous sequences of the subgenomes in existing databases, performs a multiple sequence alignment, and design primers based on sequence variants in the alignment. An interactive primers diagram, a sequence alignment viewer and a virtual electrophoresis are displayed as parts of the primer design result. GSP also designs specific primers from multiple sequences uploaded by users. AVAILABILITY AND IMPLEMENTATION: GSP is a user-friendly and efficient web platform freely accessible at http://probes.pw.usda.gov/GSP Source code and command-line application are available at https://github.com/bioinfogenome/GSP CONTACTS: yong.gu@ars.usda.gov or devin.coleman-derr@ars.usda.gov SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparative in vivo toxicity assessment places multiwalled carbon nanotubes at a higher level than mesoporous silica nanoparticles.

In the present work, we took two nanomaterials (NMs), mesoporous silica nanoparticles (MSNs) and multiwalled carbon nanotubes (MWCNTs), and compared their in vivo toxicity taking albino mice as a test animal model. Presently, conflicting data persist regarding behavior of these NMs with macromolecules like protein and lipid at the cellular level in cell lines as well as in animal models and this generated the interest to study them. The mice were treated orally with a single dose of 50 ppm MWCNTs and intraperitoneally with 10, 25, and 50 mg kg-1 body weight (BW) of MSNs and 1.5, 2.0, and 2.5 mg kg-1 BW of MWCNTs. Liver enzyme markers serum aspartate aminotransferase (AST), alanine aminotransferase, and alkaline phosphatase along with total protein (TP) levels were evaluated 7 days postexposure. No significant differences in organ weight indices or enzyme levels were observed between different treatment doses but there were significant differences between the treatment groups and the controls. Of the three enzymes assayed, AST displayed a peculiar pattern, especially in the MWCNTs intraperitoneally treated group. TP level was significantly increased in the orally treated MWCNTs group. The results showed that MWCNTs even at much smaller doses than MSNs displayed similar toxicity levels, suggesting that toxicity of MWCNTs is greater than MSNs.

Exploring the tertiary gene pool of bread wheat: sequence assembly and analysis of chromosome 5M(g) of Aegilops geniculata.

Next-generation sequencing (NGS) provides a powerful tool for the discovery of important genes and alleles in crop plants and their wild relatives. Despite great advances in NGS technologies, whole-genome shotgun sequencing is cost-prohibitive for species with complex genomes. An attractive option is to reduce genome complexity to a single chromosome prior to sequencing. This work describes a strategy for studying the genomes of distant wild relatives of wheat by isolating single chromosomes from addition or substitution lines, followed by chromosome sorting using flow cytometry and sequencing of chromosomal DNA by NGS technology. We flow-sorted chromosome 5M(g) from a wheat/Aegilops geniculata disomic substitution line [DS5M(g) (5D)] and sequenced it using an Illumina HiSeq 2000 system at approximately 50 × coverage. Paired-end sequences were assembled and used for structural and functional annotation. A total of 4236 genes were annotated on 5M(g) , in close agreement with the predicted number of genes on wheat chromosome 5D (4286). Single-gene FISH indicated no major chromosomal rearrangements between chromosomes 5M(g) and 5D. Comparing chromosome 5M(g) with model grass genomes identified synteny blocks in Brachypodium distachyon, rice (Oryza sativa), sorghum (Sorghum bicolor) and barley (Hordeum vulgare). Chromosome 5M(g) -specific SNPs and cytogenetic probe-based resources were developed and validated. Deletion bin-mapped and ordered 5M(g) SNP markers will be useful to track 5M-specific introgressions and translocations. This study provides a detailed sequence-based analysis of the composition of a chromosome from a distant wild relative of bread wheat, and opens up opportunities to develop genomic resources for wild germplasm to facilitate crop improvement.

Comprehensive meta-analysis, co-expression, and miRNA nested network analysis identifies gene candidates in citrus against Huanglongbing disease.

BACKGROUND: Huanglongbing (HLB), the most devastating disease of citrus, is associated with infection by Candidatus Liberibacter asiaticus (CaLas) and is vectored by the Asian citrus psyllid (ACP). Recently, the molecular basis of citrus-HLB interactions has been examined using transcriptome analyses, and these analyses have identified many probe sets and pathways modulated by CaLas infection among different citrus cultivars. However, lack of consistency among reported findings indicates that an integrative approach is needed. This study was designed to identify the candidate probe sets in citrus-HLB interactions using meta-analysis and gene co-expression network modelling. RESULTS: Twenty-two publically available transcriptome studies on citrus-HLB interactions, comprising 18 susceptible (S) datasets and four resistant (R) datasets, were investigated using Limma and RankProd methods of meta-analysis. A combined list of 7,412 differentially expressed probe sets was generated using a Teradata in-house Structured Query Language (SQL) script. We identified the 65 most common probe sets modulated in HLB disease among different tissues from the S and R datasets. Gene ontology analysis of these probe sets suggested that carbohydrate metabolism, nutrient transport, and biotic stress were the core pathways that were modulated in citrus by CaLas infection and HLB development. We also identified R-specific probe sets, which encoded leucine-rich repeat proteins, chitinase, constitutive disease resistance (CDR), miraculins, and lectins. Weighted gene co-expression network analysis (WGCNA) was conducted on 3,499 probe sets, and 21 modules with major hub probe sets were identified. Further, a miRNA nested network was created to examine gene regulation of the 3,499 target probe sets. Results suggest that csi-miR167 and csi-miR396 could affect ion transporters and defence response pathways, respectively. CONCLUSION: Most of the potential candidate hub probe sets were co-expressed with gibberellin pathway (GA)-related probe sets, implying the role of GA signalling in HLB resistance. Our findings contribute to the integration of existing citrus-HLB transcriptome data that will help to elucidate the holistic picture of the citrus-HLB interaction. The citrus probe sets identified in this analysis signify a robust set of HLB-responsive candidates that are useful for further validation.

A putative candidate for the recessive gall midge resistance gene gm3 in rice identified and validated.

KEY MESSAGE: We report here tagging and fine-mapping of gm3 gene, development of a functional marker for it and its use in marker-assisted selection. The recessive rice gall midge resistance gene, gm3 identified in the rice breeding line RP2068-18-3-5 confers resistance against five of the seven Indian biotypes of the Asian rice gall midge Orseolia oryzae. We report here tagging and fine-mapping of gm3 gene, development of a functional marker for it and demonstrated its use in marker-assisted selection (MAS). A mapping population consisting of 302 F10 recombinant inbred lines derived from the cross TN1 (susceptible)/RP2068-18-3-5, was screened against gall midge biotype 4 (GMB4) and analyzed with a set of 89 polymorphic SSR markers distributed uniformly across the rice genome. Two SSR markers, RM17480 and gm3SSR4, located on chromosome 4L displayed high degree of co-segregation with the trait phenotype and flanked the gene. In silico analysis of the genomic region spanning these two markers contained 62 putatively expressed genes, including a gene encoding an NB-ARC (NBS-LRR) domain containing protein. A fragment of this gene was amplified with the designed marker, NBcloning 0.9 Kb from the two susceptible TN1, Improved Samba Mahsuri (B95-1) and two resistant cultivars, RP 2068-18-3-5 and Phalguna (with Gm2 gene). The amplicons were observed to be polymorphic between the susceptible and resistant genotypes and hence were cloned and sequenced. A new primer, gm3del3, which was designed based on sequence polymorphism, amplified fragments with distinct size polymorphism among RP2068-18-3-5, Phalguna and TN1 and B95-1 and displayed no recombination in the entire mapping population. Expression of the candidate NB-ARC gene in the susceptible TN1 and the resistant RP2068-18-3-5 plants following infestation with GMB4 was analyzed, through real-time reverse transcription PCR. Results showed twofold enhanced expression in RP2068-18-3-5 plants, but not in TN1 plants, 120 h after infestation. Amino acid sequence and structure analysis of the proteins coded by different alleles of gm3 gene showed deletion of eight amino acids due to an early stop codon in RP2068-18-3-5 resulting in a change in the functional domain of the protein. The gm3del3 was used as a functional marker for introgression of gm3 gene into the genetic background of the elite bacterial blight resistant cultivar Improved Samba Mahsuri (B95-1) through MAS.